Инструкция для OLYMPUS XCELLENCE

(скачивание инструкции бесплатно)
Формат файла: PDF
Доступность: Бесплатно как и все руководства на сайте. Без регистрации и SMS.
Дополнительно: Чтение инструкции онлайн
background image

 

 

 

 

User Manual 

Imaging Software for 
Life Science Microscopy

  

 

Imaging Software 

 

Life Science Microscopy 

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
125
126
127
128
129
130
131
132
133
134
135
136
137
138
139
140
141
142
143
144
145
146
147
148
149
150
151
152
153
154
155
156
157
158
159
160
161
162
163
164
165
166
167
168
169
170
171
172
173
174
175
176
177
178
179
180
181
182
183
184
185
186
187
188
189
190
191
192
193
194
195
196
197
198
199
200
201
202
203
204
205
206
207
208
209
210
211
212
213
214
215
216
217
218
219
220
221
222
223
224
225
226
227
228
229
230
231
232
233
234
235
236
237
238
239
240
241
242
243
244
245
246
247
248
249
250
251
252
253
254
255
256
257
258
259
260
261
262
263
264
265
266
267
268
269
270
271
272
273
274
275
276
277
278
279
280
281
282
283
284
285
286
287
288
289
290
291
292
293
294
295
296
297
298
299
300
301
302
303
304
305
306
307
308
309
310
311
312
313
314
315
316
317
318
319
320
321
322
323
324
325
326
327
328
329
330
331
332
333
334
335
336
337
338
339
340
341
342
343
344
345
346
347
348
349
350
351
352
353
354
355
356
357
358
359
360
361
362
363
364
365
366
367
368
369
370
371
372
373
374
375
376
377
378
379
380
381
382
383
384
385
386
387
388
389
390
391
392
393
394
395
396
397
398
399
400
401
402
Оглавление инструкции
  • Страница 1 из 403
    Imaging Software Life Science Microscopy User Manual Imaging Software for Life Science Microscopy
  • Страница 2 из 403
  • Страница 3 из 403
    Imaging Software for Life Science Microscopy Software Manual for Version 1.1 Imaging Stations
  • Страница 4 из 403
  • Страница 5 из 403
    Imaging Excellence We at Olympus Soft Imaging Solutions GmbH have tried to make the information in this manual as accurate and reliable as possible. Nevertheless, Olympus Soft Imaging Solutions GmbH disclaims any warranty of any kind, whether expressed or implied, as to any matter whatsoever
  • Страница 6 из 403
    OLYMPUS SOFT IMAGING SOLUTIONS GMBH Rupert-Mayer-Strasse 44 D-81379 München Tel: +49 89 - 89 55 805 660 Fax +49 89 - 89 55 805 6606 Email: info@olympus-sis.com www.olympus-sis.com
  • Страница 7 из 403
    LICENSE AGREEMENT between END USER and OLYMPUS SOFT SOFTWARE IMAGING SOLUTIONS regarding the OSIS PRODUCT. IMPORTANT-READ CAREFULLY: Below you will find the contractual agreements governing the use of the OSIS SOFTWARE PRODUCT. These conditions apply to you, the user, and to OLYMPUS SOFT IMAGING
  • Страница 8 из 403
    (2) In case of a contract violation the User has to return the original storage devices and all copies thereof including all modified copies, all printed and written documentation, and the software protection key to OLYMPUS SOFT IMAGING SOLUTIONS, or the User has to destroy these items. (3) In
  • Страница 9 из 403
    Software Manual Contents Contents Chapter 1 Imaging stations for life science experiments Chapter 2 A system chart and a list of all components 1 Introduction .................................................................................. 1 2 System
  • Страница 10 из 403
    Contents 5.3.3 5.4 5.4.1 5.4.2 5.4.3 Chapter 6 Touching up the image display by brightness, contrast and color adjustment and navigation through multidimensional image sets 6 Chapter 7 Basic processing routine like size calibration, overlay drawings, extraction and conversion of image sets 7 6.1
  • Страница 11 из 403
    Software Manual 7.5.1 7.5.2 7.5.3 7.6 7.7 7.8 7.8.1 7.8.2 7.9 7.9.1 7.9.2 7.9.3 7.9.4 7.9.5 7.10 7.10.1 7.10.2 7.11 Chapter 8 Data changing tools to improve the image quality, for example, by increasing the contrast or reducing the noise 8 8.1 8.1.1 8.1.2 8.1.3 8.2 8.3 8.3.1 8.3.2 8.3.3 8.4 8.4.1
  • Страница 12 из 403
    Contents 8.4.18 8.4.19 8.4.20 8.4.21 8.5 8.5.1 8.5.2 8.5.3 8.5.4 8.5.5 8.5.6 8.5.7 8.5.8 8.5.9 8.5.10 8.5.11 8.5.12 8.5.13 8.5.14 8.6 8.7 8.7.1 8.7.2 8.7.3 8.7.4 8.7.5 8.7.6 8.8 8.8.1 8.8.2 8.8.3 8.8.4 8.8.5 8.8.6 Chapter 9 The software contains a host of measuring tools in a special measurement
  • Страница 13 из 403
    Software Manual Contents 9.4.2 9.4.3 9.4.4 9.4.5 9.4.6 9.4.7 9.4.8 9.4.9 Chapter 10 Sophisticated analyses of fluorescence intensities, mostly for time sequences and Z-stacks 10 Create Measurement Sheet.......................................................... 203 Deleting Measurement
  • Страница 14 из 403
    Contents 10.12.3 FRET Analysis ...............................................................................245 10.13 Kymogram.................................................................................... 251 Chapter 11 Analyses of intensity kinetics of fluorescence image time series 11
  • Страница 15 из 403
    Software Manual Contents 13.5.1 13.5.2 13.5.3 13.5.4 13.5.5 13.5.6 13.5.7 13.6 13.6.1 13.6.2 13.6.3 13.6.4 13.6.5 13.6.6 13.6.7 Chapter 14 A complete, integrated development environment for macros based on the programming language Imaging C 14 Chapter 15 Telling the software which hardware modules
  • Страница 16 из 403
    Contents 15.2.6 15.3 15.4 15.5 15.6 15.7 15.8 15.9 15.9.1 15.9.2 Chapter 16 The details to take care of when installing a new software version 16 16.1 16.2 16.3 16.4 16.5 16.6 Configuration of the Filters of a Filter Wheel.................................357 Definition of Image Types
  • Страница 17 из 403
    Software Manual 1 Chapter 1 – Introduction Introduction Thank you very much for purchasing Olympus' state of art Imaging Station and for your confidence in our products and service. It is Olympus' main objective to provide you with solutions able to meet your experimental demands and thus pave the
  • Страница 18 из 403
    2 Chapter 1 – Introduction Introduction The concept and technology of introduces the next generation of imaging workstations. is designed as a modular imaging system for a broad range of life science experiments that supports the Olympus microscopes of the IX and BX series. 1.1 is the immediate
  • Страница 19 из 403
    Software Manual 2 Chapter 2 – System Overview System Overview The following chapter gives you a short overview of the basic system components (without additional peripherals). The system consists of different hardware and software devices, which are full, integrated. 3
  • Страница 20 из 403
    4 Chapter 2 – System Overview 2.1 System Chart The system chart shown exemplifies the different components of a standard how they are interconnected. system and
  • Страница 21 из 403
    Software Manual 2.2 Chapter 2 – System Overview Hardware The hardware devices of the imaging station are listed below: • Olympus microscope of the IX or BX series, according to your specification. • CCD camera (Olympus XM10T, Hamamatsu ORCA-R2 or other) or EMCCD camera (Hamamatsu C9100-13, Andor
  • Страница 22 из 403
    6 Chapter 2 – System Overview 2.3 Software • ObsConfig Configuration Software to configure the Illumination System MT10 / MT20. Imaging Software integrating the Experiment Manager for planning and executing • experiments, Viewport, Viewport Manager, and Image Manager for managing and displaying the
  • Страница 23 из 403
    Software Manual 3 Chapter 3 – Brief Introduction and First Steps Brief Introduction to the Software and First Steps The following sections explain the basic features and functions. They also introduce the most important terms used in the software and in this manual and thus should help you to get
  • Страница 24 из 403
    8 Chapter 3 – Brief Introduction and First Steps 3.1 The User Interface Viewport Manager Viewport Operands Box Image Manager Image Buffer Box Document Area Double-click the icon on your desktop to start the software or open it via Start Programs . The screenshot below shows the program's graphical
  • Страница 25 из 403
    Software Manual Chapter 3 – Brief Introduction and First Steps c Status bar: shows the connected CCD camera and displays information depending on the active functions. d Viewport Manager (top left below button bars): shows a thumbnail of the active image; a red rectangle indicates the current
  • Страница 26 из 403
    10 Chapter 3 – Brief Introduction and First Steps 3.1.2 The Viewport Manager The image in the Viewport Manager in the top left corner of the window shows a red rectangle. It represents the region of the image currently displayed in the Viewport – if the image is zoomed to an extent that is larger
  • Страница 27 из 403
    Software Manual Chapter 3 – Brief Introduction and First Steps 11
  • Страница 28 из 403
    12 Chapter 3 – Brief Introduction and First Steps 3.2 Simple Image Acquisition Live View, Snapshot, Camera Control, Illumination Settings The most important tools for simple image acquisition are the Live View and Snapshot buttons and the control boxes that are opened by the Camera Control and
  • Страница 29 из 403
    Software Manual Chapter 3 – Brief Introduction and First Steps – The camera starts acquiring images at maximal speed. None is stored; instead, the newest image overwrites the previous one in the temporary buffer. – The current image is displayed in the active Viewport. – A single image icon appears
  • Страница 30 из 403
    14 Chapter 3 – Brief Introduction and First Steps The command Open Database… to load an existing one can be found redundantly in the menus File, Acquisition and Database. Database files carry the extension *.apl. New databases can be created with the command New Database…, to be found in the same
  • Страница 31 из 403
    Software Manual Chapter 3 – Brief Introduction and First Steps most complex experiments without any programming knowledge. The Experiment Manager is explained in every detail in chapter 5. In the following only a brief introduction will be given. To open the Experiment Manager click the Experiment
  • Страница 32 из 403
    16 Chapter 3 – Brief Introduction and First Steps 3. The experiment itself is started, paused and stopped with Start!, Pause, and Stop. Any image or image sequence acquired with the Experiment Manager is automatically stored on the hard disk in a database. 3.6 Displaying Multi-Color Images Select
  • Страница 33 из 403
    Software Manual 3.7 Chapter 3 – Brief Introduction and First Steps Displaying Sequences Navigation Time-Lapse experiments and Z-stack acquisitions generate series of images that are all stored together within one file. The individual images can be accessed with the navigation buttons (First,
  • Страница 34 из 403
    18 Chapter 3 – Brief Introduction and First Steps
  • Страница 35 из 403
    Software Manual 4 Chapter 4 – Image Acquisition and Hardware Control Image Acquisition and Hardware Control The following chapters explain how to take snapshots or live images, what camera parameters have to be adjusted for good quality and how to use the illumination system with the excitation
  • Страница 36 из 403
    20 Chapter 4 – Image Acquisition and Hardware Control 4.1 Simple Image Acquisition 4.1.1 Snapshot and Live View Snapshot, Live View There are two ways to achieve simple image acquisition. The first one is the acquisition of a single image with the Snapshot button in the Acquisition toolbar or the
  • Страница 37 из 403
    Software Manual 4.1.2 Chapter 4 – Image Acquisition and Hardware Control AVI Recorder This function is not active after a standard installation of the software. Execute Special Add-In Manger. Click Add in the corresponding window and navigate to the program folder. Open AviRec.dlx. Close the Add-In
  • Страница 38 из 403
    22 Chapter 4 – Image Acquisition and Hardware Control Snapshot, Live View Binning. The CCD chip is composed of many light-sensitive units (pixels). These pixels can be read out individually (binning = 1x1) or the signal of neighboring pixels can be combined electronically on the CCD chip (binning >
  • Страница 39 из 403
    Software Manual Chapter 4 – Image Acquisition and Hardware Control either the slider or the mouse scroll wheel or by directly typing the value into the Exposure time box. The slider limit can be selected via its context menu (right click). Adjust with binning. This feature automatically adapts the
  • Страница 40 из 403
    24 Chapter 4 – Image Acquisition and Hardware Control Define. It is very convenient to define the sub-frame via a region-of-interest either in snapshots or live images. When the button is clicked, a rectangle-drawing tool appears in the Viewport. It can be moved freely and the size can be adjusted
  • Страница 41 из 403
    Software Manual Chapter 4 – Image Acquisition and Hardware Control ture in case of light sensitive specimens because the overall photo-exposure is reduced correspondingly. 4.3 Illumination Control Illumination Settings To adjust the parameters of the illumination open the control window with the
  • Страница 42 из 403
    26 Chapter 4 – Image Acquisition and Hardware Control Main switch: In this control window you find the Main switch Burner ON / OFF button to ignite the arc burner or switch it off again. At the front of the Illumination System MT20 / MT10 housing the green LED labeled BURNER ON indicates that the
  • Страница 43 из 403
    Software Manual Chapter 4 – Image Acquisition and Hardware Control The Microscope dialog window features only those modules that have been configured with the ObsConfig software, see Chapter 15.3, Configuring the Microscope. Thus, the contents of the dialog window may differ from setup to setup.
  • Страница 44 из 403
    28 Chapter 4 – Image Acquisition and Hardware Control Note. If a piezo-electric objective mover PIFOC is installed the nosepiece might not necessarily use the shortest way to move from one objective to the next in order to avoid the winding up of the PIFOC cables. Magnification Changer. The default
  • Страница 45 из 403
    Software Manual 4.5 Chapter 4 – Image Acquisition and Hardware Control Motorized Stage Control The Imaging Software features a module for the easy control and efficient usage of motorized microscopes stages by Märzhäuser GmbH and Prior Scientific Instruments Ltd in imaging experiments via the
  • Страница 46 из 403
    30 Chapter 4 – Image Acquisition and Hardware Control 4.5.2 Correcting a Positions List It is possible to automatically correct all positions of an existing positions list. This may be convenient if a focus drift is observed, if the microscope was shaken causing the sample to move or if a sample
  • Страница 47 из 403
    Software Manual Chapter 4 – Image Acquisition and Hardware Control Focus List Correction. If you observe a focus drift, do the following: 1. Go to any list position and correct the focus. 2. Right-click the position in the list to open the context menu and click Set Current Position As Reference. A
  • Страница 48 из 403
    32 Chapter 4 – Image Acquisition and Hardware Control 2. Right-click the position in the list to open the context menu and click Set Current Position As Reference. A green letter R will appear to the left of the position number in the list. 3. Define two more reference positions by repeating steps
  • Страница 49 из 403
    Software Manual Chapter 4 – Image Acquisition and Hardware Control Auto Calibrate. Upon clicking this button the stage will always move to a certain corner position and set this as origin of the coordinate system. The red status box Not calibrated turns green and states Calibrated. Set Limits. This
  • Страница 50 из 403
    34 Chapter 4 – Image Acquisition and Hardware Control Once all four limits are set, a green cross appears in the center of the four buttons. A red dot indicates the current stage position. Any position in the positions list outside the limits will be labeled with a red diamond symbol. A reference
  • Страница 51 из 403
    Software Manual Chapter 4 – Image Acquisition and Hardware Control The focal position may not be found if the signal-to-noise ratio is too low, in other words, the camera settings Exposure time and Binning are not suitable, see Chapter 4.2, Camera Control. Also, quite obviously, the focus cannot be
  • Страница 52 из 403
    36 Chapter 4 – Image Acquisition and Hardware Control Use entire range. If this box is NOT checked, the following will be done: if the focality initially increases during an Autofocus scan and then worsens again, the process will be terminated and the found focality maximum will be set as reference
  • Страница 53 из 403
    Software Manual 5 Chapter 5 – Experiment Manager Experiment Manager The Experiment Manager is a universal and easy-to-use tool for planning, preparing, and executing experiments with the Imaging Station. This chapter explains the general concept of setting up experiments, lines out how the
  • Страница 54 из 403
    38 Chapter 5 – Experiment Manager 5.1 A Graphical Tool The Experiment Manager is a universal and easy-to-use tool for planning, preparing, and executing experiments with the Imaging Station . Planning an experiment: Your experiment may, for example, require capturing fluorescence images of a
  • Страница 55 из 403
    Software Manual 5.2 Chapter 5 – Experiment Manager Concept of Usage Experiment Manager Open the Experiment Manager with the Experiment Manager button in the toolbar or via the menu Acquisition Experiment Manager. Close the Experiment Manager in the same way or with the Close button or via the menu
  • Страница 56 из 403
    40 Chapter 5 – Experiment Manager The Experiment Manager consists of different components used to configure the different stages of an experiment (planning, preparing, executing). In the default configuration of the Experiment Manager these components are arranged in the following way: The
  • Страница 57 из 403
    Software Manual Chapter 5 – Experiment Manager position. Afterwards the size of the new independent toolbar window or properties window has to be adjusted via mouse drag. To reposition a detached component to the original location double-click on the title bar or drag&drop the window back into
  • Страница 58 из 403
    42 Chapter 5 – Experiment Manager 5.3 Setting Up Experiment Plans To set up new experiments you can either change an existing Experiment Plan or start from scratch with an "empty“ editor surface. To obtain an empty editor surface use the Experiment Manager menu File New. If the current Experiment
  • Страница 59 из 403
    Software Manual Chapter 5 – Experiment Manager Image Acquisition, deactivated (left) and active (right) Icons can simply be moved by drag&drop. Frames can be enlarged or diminished by dragging the black knots at the corners and sides that appear upon activation. Both can be deleted by activation
  • Страница 60 из 403
    44 Chapter 5 – Experiment Manager Use for the acquisition of an image. Execution of this command in an experiment will cause the following actions: 1. The illumination of the sample is switched on by opening the shutter. 2. Immediately afterwards image acquisition starts (upon a trigger pulse to
  • Страница 61 из 403
    Software Manual Chapter 5 – Experiment Manager Objective. This function appears only if a motorized microscope is configured. Select an objective from the pick list. Contrast Insert. This function appears only if a motorized microscope is configured. Select an insert from the pick list. Overlapping
  • Страница 62 из 403
    46 Chapter 5 – Experiment Manager On-line display is only possible in time-lapse or Z-stack experiments. If the option is activated in single image experiments, an error message is generated prior to the experiment execution. Brightness. This mapping function is mainly used to adjust the brightness
  • Страница 63 из 403
    Software Manual Chapter 5 – Experiment Manager In order to have this option activated be default open Options Preferences periment manager and check Display is enabled by default for: Channels. Display in the Ex- The multi-color frame can only be used if all Image Acquisition commands inside the
  • Страница 64 из 403
    48 Chapter 5 – Experiment Manager Height. This read-only information lists the total height of the Z-stack to be acquired. Layers. The total number of layers to be acquired can be typed in or changed by clicking on the arrow buttons next to the box. Step Width. The distance between successive image
  • Страница 65 из 403
    Software Manual Chapter 5 – Experiment Manager (caused by the step size limits of the selected Z-device a fit of the bottom and top positions will be done automatically with minimal possible changes. This is a convenient way to set the top and bottom position: 1. Start a Live View acquisition (see
  • Страница 66 из 403
    50 Chapter 5 – Experiment Manager Approximate minimal cycle time. This read-only parameter informs you about the minimal possible duration of a single cycle. The minimal cycle time depends on the time requirements of all commands in the Time-loop frame. Enable interaction. If this option is active
  • Страница 67 из 403
    Software Manual Chapter 5 – Experiment Manager All positions in one display. Select this option if you want to have just one online Viewport that always shows the most recently acquired image of the respective image type. If a stage loop contains more than one Image Acquisition command, each of
  • Страница 68 из 403
    52 Chapter 5 – Experiment Manager Thresholds and Output Scaling. See Chapter 10.8.2, Generating a Ratio Sequence for details. Properties, Calibration. See Chapter 10.8.2, Generating a Ratio Sequence for details. Properties, Store If the option Store is marked – this is indicated by the storage
  • Страница 69 из 403
    Software Manual 5.3.2.7 Chapter 5 – Experiment Manager Online kinetics Kinetics, command icon (with store option activated) Use for the online quantification of fluorescence intensity changes within ROIs over time and their display as a graph. See also Chapter 10.6, Intensity Kinetics in Time and
  • Страница 70 из 403
    54 Chapter 5 – Experiment Manager Stage Position. An online kinetics analysis is possible only for one of the stage positions in an experiment to be carried out at different positions. Set this position here. Properties, Store If the option Store is marked – this is indicated by the storage symbol
  • Страница 71 из 403
    Software Manual Chapter 5 – Experiment Manager Multi Digital Port Port 1 / 2 / 3 / 4. Select one or more of the four BNC ports labeled 1, 2, 3 and 4 at the front of the imaging computer. High level. This command sets a voltage of >2.4 V to the selected port. Low level. This command sets the voltage
  • Страница 72 из 403
    56 Chapter 5 – Experiment Manager Wait for time, Delay time. This parameter sets the delay before the execution of the subsequent command. Wait for trigger, Trigger Signal. Select here if the incoming pulse is to be TTL high or TTL low. Wait for trigger, Trigger channel. Select the port at the
  • Страница 73 из 403
    Software Manual Chapter 5 – Experiment Manager Range. Set here the range of Z-positions to be scanned during the Autofocus process. The range is centered on the current Z-position or – in case the Experiment Plan features a Stage loop– the Z position defined in the positions list. The narrower the
  • Страница 74 из 403
    58 Chapter 5 – Experiment Manager Properties Range. Set here the scan range the ZDC may use when searching for the interface. The larger it is, the longer will the detection take. Make sure, however, that the range is larger than any expected Z-drift. Sample offset. This is the absolute difference
  • Страница 75 из 403
    Software Manual Chapter 5 – Experiment Manager switch filter filter switch filter cube filter cube open or close shutter status switch transmission contrast insert contrast insert (selection displayed in icon) switch transmission lamp on/off intensity (voltage displayed in icon) switch camera port
  • Страница 76 из 403
    60 Chapter 5 – Experiment Manager 5.3.2.13 Additional "atomic" commands – the MT20 and CCD Cameras Commands MT20 Commands, CCD Cameras Additional "atomic" command icons Command icon Action Parameter to set change attenuation light intensity switch excitation filter filter cube open or close MT20 /
  • Страница 77 из 403
    Software Manual 5.3.3 Types of Experiments 5.3.3.1 Chapter 5 – Experiment Manager Single images – monochromatic or in multiple colors The simplest possible Experiment Plan consists of a single Image Acquisition icon. Obviously, the result would be a single image file taken with a certain excitation
  • Страница 78 из 403
    62 Chapter 5 – Experiment Manager 5. In parallel ( real-time only): – camera reads out the DAPI image data – filter wheel moves into FITC filter into position – attenuator sets intensity 6. Shutter opens. 7. Camera gets exposed and acquires the image. 8. Shutter closes. 9. In parallel ( real-time
  • Страница 79 из 403
    Software Manual Chapter 5 – Experiment Manager Once an Image Acquisition command has been placed and defined on the editor surface outside the Multi-Color Frame it cannot be subsequently moved into the frame and connected with another command placed in there. It would be necessary to delete the
  • Страница 80 из 403
    64 Chapter 5 – Experiment Manager Texas Red image. Afterwards, while the TxRed image data are still readout from the camera the PIFOC or motorized Z-drive moves to the second lowest position. Once the system is ready again a DAPI image is taken first, then a FITC and finally a TxRed image. This
  • Страница 81 из 403
    Software Manual Chapter 5 – Experiment Manager The Image Manager a time sequences acquired with a single excitation wavelength with a film stripe icon containing a "black&white" cell. Monochromatic time sequence In a multi-color time sequence the system will perform the following. Images will be
  • Страница 82 из 403
    66 Chapter 5 – Experiment Manager 5.3.3.5 3-D Time-lapse experiments – acquisition of series of Z-stacks In so-called 3-D time-lapse experiments the acquisition of Z-stacks is repeatedly carried out. To set this up in an Experiment Plan, a Time Loop Frame has to be drawn around a Z-Stack Frame. The
  • Страница 83 из 403
    Software Manual 5.3.3.7 Chapter 5 – Experiment Manager Complex experiments with trigger pulses and several time loops The Experiment Manager is powerful and flexible enough to set up much more complex experiments than in the examples above. As an example, let's imagine the following ratiometric
  • Страница 84 из 403
    68 Chapter 5 – Experiment Manager It is not necessary to draw the second time-lapse acquisition part of the Experiment Plan anew. Pressing the <shift> key and simultaneously clicking with the mouse into the frame can entirely mark the first part. Then it can be copied and pasted (<Ctl+c> and
  • Страница 85 из 403
    Software Manual Chapter 5 – Experiment Manager A Stage frame can also be drawn around a Time Loop frame. In that case, a time-lapse sequence is first acquired entirely at the first stage position, then the stage moves to the second position where another time-lapse sequence is acquired and so on.
  • Страница 86 из 403
    70 Chapter 5 – Experiment Manager 5.4 Conducting Experiments / Data Acquisition 5.4.1 Opening a Database Before the start of image acquisition with the Experiment Manager, it is necessary to open a database. There are two possibilities: to open an already existing database or to create a new one.
  • Страница 87 из 403
    Software Manual Chapter 5 – Experiment Manager To create a new database, select New Database... in the Database menu. Type a name in the Database name field of the New Database window and click on the (now enabled) Next > button. In the following dialog box select Finish to create the new database
  • Страница 88 из 403
    72 Chapter 5 – Experiment Manager 5.4.2.1 Check Check Before an Experiment Plan can be executed, the Experiment Plan needs to be validated. The system has to check whether all interconnections of the commands are consistent, the parameter settings reasonable, etc. If the experiment plan passes the
  • Страница 89 из 403
    Software Manual 5.4.2.3 Chapter 5 – Experiment Manager Experiment start Start Pressing the Start button effects the immediate start of the experiment. In order not to slow down the data transfer, only a limited number of user interactions, which might be required for online data inspection, are
  • Страница 90 из 403
    74 Chapter 5 – Experiment Manager The experiment time will continue to elapse during an interruption, meaning that: • The total experiment time will increase by the duration of the interruption. If the experiment is interrupted during acquisition of an image sequence (Time-Lapse command) the cycle
  • Страница 91 из 403
    Software Manual 5.4.3 Chapter 5 – Experiment Manager Data Storage and Preferences Images acquired in experiments via the Experiment Manager – unlike snapshots or live images – are stored automatically in the active database in a folder that is generated automatically and carries the name of the
  • Страница 92 из 403
    76 Chapter 5 – Experiment Manager Database: Database name. Type in the path and name of a database to be used automatically for storage. You may also select it via use of the "…" browse button. If a default database is given it is not necessary to open a database prior to the experiment execution
  • Страница 93 из 403
    Software Manual 6 Chapter 6 – Image Display and Navigation Image Display and Navigation The software interface features a special window, the Viewport, to display up to 16 images. Raw images usually need a little touching up in the display to look appealing; contrast, brightness and color can be
  • Страница 94 из 403
    78 Chapter 6 – Image Display and Navigation 6.4.2 6.5 6.6 6.1 EFI – Extended Focal Imaging. .....................................................100 Fluorescence and Transmission Image Overlay .......................... 101 Intensity Modulated
  • Страница 95 из 403
    Software Manual Chapter 6 – Image Display and Navigation Zoom Factor From the shortlist you can adjust the size of the displayed images, given as % of the original image size. If you select Auto the image size is automatically adjusted to fit into the size of the Viewport. Adjust Zoom This allows
  • Страница 96 из 403
    80 Chapter 6 – Image Display and Navigation Close Full Screen: returns to standard view. Select previous buffer: displays the image in the Image Manager buffer atop the current image. Select next buffer: displays the image in the Image Manager buffer below the current image. Select a document:
  • Страница 97 из 403
    Software Manual Chapter 6 – Image Display and Navigation Most monitors can only display 256 gray values (8 bit), which is already far beyond the number of gray levels the human eye can differentiate. Color monitors generate colors by mixing the three existing color channels, red, green, and blue,
  • Страница 98 из 403
    82 Chapter 6 – Image Display and Navigation Display Channel (colored squares) Adjust Channel (bars below) Load LUT, Edit Fluorescence Color Adjust Channel buttons. These buttons select the corresponding channel for individual adjustment when more than one channel is being displayed. The histogram
  • Страница 99 из 403
    Software Manual Chapter 6 – Image Display and Navigation Brightness, Contrast and Gamma Correction These bars become active if one channel is activated. The values can be increased or decreased by increments of 0.1 by respectively clicking into the bar above or below the sliders. Brightness. Values
  • Страница 100 из 403
    84 Chapter 6 – Image Display and Navigation 6.2.3 Auto Adjust Auto Adjust Display, Auto Adjust Framewise Auto Adjust Display. This is a quick way to adjust the channel intensities of multi-dimensional image sets. The entire data set – not just the current image – is checked for the brightest and
  • Страница 101 из 403
    Software Manual Chapter 6 – Image Display and Navigation 3. Position this circle with the mouse. To resize it, keep the left mouse button pressed as you move the mouse. 4. Right-click to confirm the circle and apply the white-balance. 6.2.5 Black Balance This function is similar to the white
  • Страница 102 из 403
    86 Chapter 6 – Image Display and Navigation 6.2.7 Fluorescence Color This command is used for displaying single-channel images or individual channels of a multiConfiguration Software channel image with the monochrome palette defined in the (see Chapter 15, Configuration) for the corresponding
  • Страница 103 из 403
    Software Manual 6.2.9 Chapter 6 – Image Display and Navigation False-Color… False-Color button Use this command (or Image Image Display Load LUT…) to select a color palette from the list in the dialog box RGB Lookup Tables of Color Channel '<...>'. The library of palette files provided with is
  • Страница 104 из 403
    88 Chapter 6 – Image Display and Navigation The intensity values (counts) of monochrome images’ pixels comprise the palette’s input – e.g., 0 – 255 for 8 bit images. These intensity values are listed according to their respective line (number) – (see the example table below). Each intensity value
  • Страница 105 из 403
    Software Manual 6.2.9.1 Chapter 6 – Image Display and Navigation The palette bar – displaying the current LUT It is possible to have the current lookup table (LUT) of displayed in the Viewport. To do so open the Scale Bar Properties window via Image Scale Bar Properties... and select the Palette
  • Страница 106 из 403
    90 Chapter 6 – Image Display and Navigation • Generate palettes interactively by defining polygons. • Use formulae to have the palette computed. The Edit False-Color... command opens a dialog box, which provides tabs for each of the abovementioned methods. Each of the three methods generates a
  • Страница 107 из 403
    Software Manual Chapter 6 – Image Display and Navigation Interval... When you click this button, the Set Thresholds dialog box will open. Here’s where you can define a gray value interval for the definition of LUT values. When you exit the Set Threshold dialog box via OK, the corresponding interval
  • Страница 108 из 403
    92 Chapter 6 – Image Display and Navigation 6.2.10.2 The Polygon tab Diagram. Within the diagram, lines (or "polygons") of the respective colors represent the three primary colors of the LUT. The horizontal axis represents gray values from 0 – 255. The vertical axis represents LUT entries, i.e.,
  • Страница 109 из 403
    Software Manual Chapter 6 – Image Display and Navigation Monochrome. If you select the Monochrome check box, the polygon will become yellow. All changes will be applied to all three colors simultaneously, thus enabling you to define a monochrome gray value LUT. File.... Click on the File... button
  • Страница 110 из 403
    94 Chapter 6 – Image Display and Navigation • • • • • provided with a point (e.g., 100. or 100.0) to enable calculations with the precision of floating decimal point operations. Multiplication and division operations will be performed before addition and subtraction. If necessary, use brackets. n
  • Страница 111 из 403
    Software Manual 6.3 Image Navigation 6.3.1 Chapter 6 – Image Display and Navigation General In most cases the acquired images are multi-dimensional, i.e., they consist of image stacks of different color channels, time sequences or z-stacks. With the commands in the Navigation toolbar you can easily
  • Страница 112 из 403
    96 Chapter 6 – Image Display and Navigation 6.3.3 Displaying Different Color Bands in the Tile View Mode It is possible to individually display all color bands of a multi-color image at the same time in different Viewports. To do so a suitable Viewport arrangement has to be chosen with the Arrange
  • Страница 113 из 403
    Software Manual Chapter 6 – Image Display and Navigation Animate An additional feature enables the user to animate the acquired image sequence. Pressing the Animate button opens the Animate Image Stack window. Reverse, Stop and Play Here you find the buttons to start (Play) the animation, to Stop
  • Страница 114 из 403
    98 Chapter 6 – Image Display and Navigation Direction. In this field you can select the direction of the animation. Choose here whether the stack ) or meandering back and forth ( ). is to be animated uni-directionally ( 6.3.5 Z-Stacks Navigate Z In Z (3D) experiments stacks of images are acquired
  • Страница 115 из 403
    Software Manual Chapter 6 – Image Display and Navigation Navigation toolbar allows selecting through which dimension to navigate: either time or Z. If the Navigate Time button is activated, the navigation tools work with respect to the time axis. If Navigate z button is activated, they work with
  • Страница 116 из 403
    100 Chapter 6 – Image Display and Navigation The projection functions in the pick lists of the Navigate Z and Navigate Time buttons do not create a new image. The projections are only displayed temporarily. In order to generate new projection image data choose the projection commands from the
  • Страница 117 из 403
    Software Manual 6.5 Chapter 6 – Image Display and Navigation Fluorescence and Transmission Image Overlay The selective fluorescence labeling of sub-cellular structures causes – consequently – that often a cell is not visible entirely. To see where the fluorescent structures are located in the cell,
  • Страница 118 из 403
    102 Chapter 6 – Image Display and Navigation 6.6 Intensity Modulated Display Images sets that are created by pixel-by-pixel division of source images or color channels, most significantly those resulting from Delta F / F or Ratio calculations (Chapter 10.7, Delta F / F (∆F/F) Analysis, and 10.8,
  • Страница 119 из 403
    Software Manual 7 Chapter 7 – Image Data Handling Image Data Handling If the system is configured thoroughly all images will have the correct spatial calibration automatically. Nevertheless, here it is explained how to adjust the calibration. The software offers the possibility to show a scale bar
  • Страница 120 из 403
    104 Chapter 7 – Image Data Handling 7.9.2 7.9.3 7.9.4 7.9.5 7.10 7.10.1 7.10.2 7.11 To 8-Bit .........................................................................................121 To 16-Bit .......................................................................................122 To RGB
  • Страница 121 из 403
    Software Manual 1. Open the XY Calibration tab in the Acquisition Chapter 7 – Image Data Handling Camera Configuration dialog window. 2. Click the Unit button to open the Set Unit dialog window and select m as Basic unit and µ as Scale and confirm with OK. 3. Back in the Configure Input dialog
  • Страница 122 из 403
    106 Chapter 7 – Image Data Handling 5. Click Add to load the new settings. The following message will appear; it refers to the default settings. Click Yes to accept. 6. Click OK to confirm the new Magnification Table. 7. Click OK to confirm the new XY Calibration settings. 7.1.3 XY-Calibration
  • Страница 123 из 403
    Software Manual Chapter 7 – Image Data Handling How to calibrate the active image with a fixed X/Y ratio and unknown pixel size: 1. Select the image. 2. Execute the Image Calibrate Image... command to open the corresponding window. 3. Set the magnification in the Magnification box, if known. 4.
  • Страница 124 из 403
    108 Chapter 7 – Image Data Handling 6. If the calculated value (CCD pixel size/magnification) is sufficiently accurate, type it into the X calibration box. The Y calibration value will be automatically set. Exit with Ok. 7. Otherwise enter the Calibration length for the length of the calibration
  • Страница 125 из 403
    Software Manual Chapter 7 – Image Data Handling Z-Spacing. This is the step width between two neighboring layers of a stack. It is read in automatidata. cally when loading Unit… This function opens the same Set Unit dialog box as in XY Calibration tab. 7.2 Scale Bar 7.2.1 General In all scientific
  • Страница 126 из 403
    110 Chapter 7 – Image Data Handling shown. A Palette bar depicts the color/gray scale coding of the light intensities and is especially useful for images displayed with a pseudo-color palette. Show scale bar for field. In case images are to be printed or exported to the clipboard with the scale the
  • Страница 127 из 403
    Software Manual 7.3 Chapter 7 – Image Data Handling Show Markers, Time and Z Information The Experiment Manager allows the user to set markers by hand (mouse click) during the execution of an experiment. This is useful to mark certain actions that are carried out or events that happen during an
  • Страница 128 из 403
    112 Chapter 7 – Image Data Handling Options group. Use starting point. Select the Use starting point check box – along with the Interactive button – to determine grid frame position in the image. If you clear the check box, the grid will always start in the upper-left corner of the image at the
  • Страница 129 из 403
    Software Manual Chapter 7 – Image Data Handling ing the dialog box settings. If a grid has been written into the overlay it will be deleted when you click Interactive. Keeping the left mouse button pressed, move the mouse to enlarge or diminish the rectangle to set the size of the grid frames. If
  • Страница 130 из 403
    114 Chapter 7 – Image Data Handling 7.5.3 Creating and Editing Overlays Edit Overlay This button allows you to select an existing object in an overlay. Upon clicking the button the cursor automatically moves into the image and is confined to this area until a right mouse click terminates the Edit
  • Страница 131 из 403
    Software Manual Chapter 7 – Image Data Handling Layer This button opens a pull down menu with the following commands: Image And Overlay. Use this command to display the image and the overlay simultaneously. Warning: This is a global setting, i.e., this flag determines the display of the image
  • Страница 132 из 403
    116 Chapter 7 – Image Data Handling Load Objects This command enables you to load a previously stored overlay file (*.ovl) from a storage medium. Other object types that can be loaded are bitmaps (*.bmp), icons (*.ico) and enhanced meta files (*.emf). Save Objects This command stores all objects
  • Страница 133 из 403
    Software Manual Chapter 7 – Image Data Handling Ellipse Enables ellipses to be drawn. Position the top left corner of the rectangle that describes the ellipse as desired, then depress the left mouse button to create an ellipse of the desired size. Right-click to superimpose the box. Line Enables
  • Страница 134 из 403
    118 Chapter 7 – Image Data Handling 7.6 Separate The Image Separate… commands allow dividing a multi-dimensional data set into several independent subsets within one dimension (time, Z or color). Available. The commands for dimensions not contained in the data set are disabled, like Z-Layers in the
  • Страница 135 из 403
    Software Manual Chapter 7 – Image Data Handling To enlarge the cropping area by a number of pixels in the ±X and ±Y directions type a value into the Border [pixel] box. Execute the cropping by clicking OK. The Dimensions tab Here you have to define the Color Channels and range (From / to) of
  • Страница 136 из 403
    120 Chapter 7 – Image Data Handling contain the same number of color channels. On the other hand – let's assume the two series are monochrome – a combined time series of 50 dual color images could likewise be generated. 7.8.2 Combining Data Sets The Combine window is opened with the Combine button
  • Страница 137 из 403
    Software Manual 7.9 Convert Image 7.9.1 Chapter 7 – Image Data Handling General The Image Convert… command opens a submenu containing the commands for increasing or reducing the number of bits per pixel – i.e., bit depth. This might be necessary if you wish to use images in other application
  • Страница 138 из 403
    122 Chapter 7 – Image Data Handling 7.9.3 To 16-Bit Use this command to convert (nx8) bit multi-color images into (nx16) bit multi-color images or 8 bit gray-value images into 16 bit gray-value images. This might be important if you want to carry out certain arithmetic operations. This conversion
  • Страница 139 из 403
    Software Manual Chapter 7 – Image Data Handling Transfer function. If the greatest number for gray value display is Gmax and Go stands for a pixel’s gray value in the resulting image, then, this pixel’s gray value will have the following value in the resulting image: G = Gmax – Go What will
  • Страница 140 из 403
    124 Chapter 7 – Image Data Handling Image Manager context menu. Right-click on any image buffer to open the corresponding context menu and then click on Image Information… The information then displayed refers to the image you right-clicked on – it doesn't have to be the active one. Viewport
  • Страница 141 из 403
    Software Manual Chapter 7 – Image Data Handling Memory size displays the size of the computer memory used by the image. File size gives the information about the file size if the image is stored as a file. Frame. The Frame field displays the number of the current image and the total number of
  • Страница 142 из 403
    126 Chapter 7 – Image Data Handling 7.11 Image Statistics Right-click on an image to open the context menu and select Image Statistics. Immediately a table will be generated with statistical data for each color channel of each image of a multidimensional data set. The parameters to be analyzed
  • Страница 143 из 403
    Software Manual 8 Chapter 8 – Image Processing Image Processing The following sections explain how images can be improved by data manipulation. This is in difference to the display adjustments explained in the previous chapter. The shading correction corrects for regular intensity gradients. A host
  • Страница 144 из 403
    128 Chapter 8 – Image Processing 8.4.15 8.4.16 8.4.17 8.4.18 8.4.19 8.4.20 8.4.21 8.5 8.5.1 8.5.2 8.5.3 8.5.4 8.5.5 8.5.6 8.5.7 8.5.8 8.5.9 8.5.10 8.5.11 8.5.12 8.5.13 8.5.14 8.6 8.7 8.7.1 8.7.2 8.7.3 8.7.4 8.7.5 8.7.6 8.8 8.8.1 8.8.2 8.8.3 8.8.4 8.8.5 8.8.6 NxN
  • Страница 145 из 403
    Software Manual 8.1 Shading Correction... 8.1.1 Chapter 8 – Image Processing General Gradual noise and sensitivity variations across the CCD chip and the corresponding inherent intensity gradients in the images are termed shading. The cause is a gradual mismatch of photodiodes and on-chip
  • Страница 146 из 403
    130 Chapter 8 – Image Processing Multiplicative. Check here if it is assumed that the shading results from a multiplicative effect. The shading correction will divide the current image by a correction image, the Source for shading image. Additive. Check here if it is assumed that the shading
  • Страница 147 из 403
    Software Manual Chapter 8 – Image Processing NxN average filter. The NxN filter is a smoothing filter to eliminate noise as explained in Chapter 8.4.15. It averages the pixel values in a square area – defined by the value in the Size field – around each pixel and assigns the result to it. This
  • Страница 148 из 403
    132 Chapter 8 – Image Processing Select the Polynomial fit option to have the correction image calculated based on a twodimensional fit of the original image ("least-square fit"). If this option has been selected, a new group will appear in the lower left corner of the dialog box in the Polynomial
  • Страница 149 из 403
    Software Manual 8.2 Chapter 8 – Image Processing Bleaching Correction All fluorophores undergo photodegradation to greater or lesser extent upon exposure to the usually very bright fluorescence illumination. Correspondingly the intensity of the signal decays, which has immediate influence on any
  • Страница 150 из 403
    134 Chapter 8 – Image Processing Dimension Time, Z Select the dimension in which to carry out the bleaching correction in case of 3-d TimeLapse data the dimension. The selection is set automatically in case of simple time-lapse data and Z-stacks. Reference Whole image. The entire image is used to
  • Страница 151 из 403
    Software Manual 8.3 Thresholds and Binarization 8.3.1 Chapter 8 – Image Processing Set Thresholds This command sets thresholds of one or more phases for the images. A number of image-processing functions are only available for use on binary images. If your image isn’t binary already, you will need
  • Страница 152 из 403
    136 Chapter 8 – Image Processing The thresholds you define represent the upper and lower ‘limits’ of a phase. A phase having its thresholds at ‘0’ and ‘100’ is comprised of all gray values (G) between ‘0’and ‘100’: this can be expressed in the following way: 0<=G<=100. Click the New button to
  • Страница 153 из 403
    Software Manual Chapter 8 – Image Processing Possible spread factors for 8-bit images are 2x and 4x. 16-bit images can in addition be spread to 8x, 16x, 32x, 64x, 128x and 256x. The current spread factor is displayed on the left of the dialog box - next to the scroll bar. Use the X scroll bar to
  • Страница 154 из 403
    138 Chapter 8 – Image Processing This check box is only available in conjunction with the Current and Background check boxes. Click the File... button to open the standard dialog box for the opening and loading of files. This is where you can save threshold settings – or load threshold settings you
  • Страница 155 из 403
    Software Manual Chapter 8 – Image Processing Select the None option if you wish the whole gray value range to be divided up into n phases (n= the number of phases desired). Select the Preview check box to have the phases displayed in color. The phases will be displayed in the standard colors as
  • Страница 156 из 403
    140 Chapter 8 – Image Processing 8.3.2 Set Color Thresholds Sets color thresholds using the RGB or HSI color space. Thresholds cannot be set for a single image or image buffer. They apply to all images you currently have loaded. To convert a color image into a binary one, you must define a range
  • Страница 157 из 403
    Software Manual Chapter 8 – Image Processing anywhere between the values 0 and 255. The easiest way to define color ranges is to go right into the image and select a portion of the picture containing the color desired. To do this, first click on the New button (located in the Include pixel group).
  • Страница 158 из 403
    142 Chapter 8 – Image Processing Use the options located in the Preview group to see whether all the image structures you’re interested in have been included. If need be, simply correct the thresholds by adjusting them directly in the histogram. When you click the New button, the color thresholds
  • Страница 159 из 403
    Software Manual Chapter 8 – Image Processing The Redo button will only become available once you’ve ‘undone’ a threshold setting. Click this button to reproduce a threshold setting you had undone. The HIS tab… … sets thresholds based on the HSI (Hue, Saturation, Intensity) model. The RGB and HSI
  • Страница 160 из 403
    144 Chapter 8 – Image Processing same color as in the Preview. Activate any of these color channels for a particular phase by simply left-clicking on the color bar of that channel. If you switch back and forth between channels the curve’s progression displayed in the diagram will, of course change
  • Страница 161 из 403
    Software Manual Chapter 8 – Image Processing Binarize If only one phase has been defined currently, the Binarize command will automatically use the phase’s left and right threshold to generate a binary image. If several phases are currently defined, the Binarize command will open the dialog box of
  • Страница 162 из 403
    146 Chapter 8 – Image Processing • reduction of acquisition errors, e.g., CCD cameras sometimes provide erroneous gray values for individual pixels, • restoration of poorly-focused images, • suppression or accentuation of minute image detail, • accentuation of basic structures such as gray value
  • Страница 163 из 403
    Software Manual Chapter 8 – Image Processing Derivative filters. Negative weight factors are admitted in the filter matrix of derivative filters and the sum of the weight factors equals zero. The result for homogeneous gray value areas is 0: a black area. On the other hand side, gray-value edges –
  • Страница 164 из 403
    148 Chapter 8 – Image Processing Filter parameter. There is a distinction between predefined and (user-) definable filters. Parameters of definable filters can be set, thus allowing you to adjust the filter’s effect. In the following chapters the available filters will be explained in detail.
  • Страница 165 из 403
    Software Manual 8.4.3 Chapter 8 – Image Processing Sharpen II Application. Detail emphasis Matrix. -1 -1 -1 -1 9 -1 -1 -1 -1 Comparison. This filter is similar to the Sharpen I filter but has a more pronounced effect. 8.4.4 Differentiate X Application. Extraction of gray value edges in the Y
  • Страница 166 из 403
    150 Chapter 8 – Image Processing Comparison. This filter is analogous to the Differentiate X filter but enhances edges parallel to the X-axis. 8.4.6 Laplace I Application. Extraction of gray value edges The Laplace I derivative filter accentuates edges and smaller image structures in both the X and
  • Страница 167 из 403
    Software Manual 8.4.8 Chapter 8 – Image Processing Mean Application. Smoothing The Mean filter replaces every pixel with the arithmetical mean of that pixel and its eight neighboring pixels. This results in noise reduction. Abrupt gray-value transitions will also be smoothed over however, thus
  • Страница 168 из 403
    152 Chapter 8 – Image Processing 8.4.10 Pseudo Filter This filter is based on the settings in the Processing Define Filter Define Pseudo window. Application. Relief-like appearance with "shadows" Filtered images will appear topographical as if a relief-like surface is illuminated by an oblique
  • Страница 169 из 403
    Software Manual 8.4.11 Chapter 8 – Image Processing Sobel Application. Edge detection The Sobel filter is a genuine edge detector and does not react so sensitively to noise effects. This is because differentiation is conducted using the lines and columns beyond the next-immediate ones. Thus any
  • Страница 170 из 403
    154 Chapter 8 – Image Processing 8.4.13 Reimer This filter is based on the settings in the Processing Define Filter Define Reimer window. What will happen? The Reimer filter subdivides the entire image into areas of pixels with the same intensity within a certain tolerance range. The borderlines
  • Страница 171 из 403
    Software Manual Chapter 8 – Image Processing ing the weight factors of the individual factors. Another application is modification of existing filters. This is done by increasing the mean image brightness by adding an offset. What will happen? If you apply the user filter the following operation
  • Страница 172 из 403
    156 Chapter 8 – Image Processing 8.4.15 NxN This filter is based on the settings in the Processing Define Filter Define NxN window. Application. Reduction of statistical noise What will happen? The NxN filter averages the gray values of all pixels surrounding a central pixel and assigns them to
  • Страница 173 из 403
    Software Manual Chapter 8 – Image Processing The number of iterations and matrix size also determine the time required to complete the filter operation. 8.4.16 Lowpass Application. A lowpass filter transmits space frequency in a highly unadulterated form. High space frequencies – generally image
  • Страница 174 из 403
    158 Chapter 8 – Image Processing values of the original image will predominate while a higher percentage will result in the predomination of lighter gray values. The preset percentage is 50%. 8.4.18 Rank This filter is based on the settings in the Processing Define Filter Define Rank window.
  • Страница 175 из 403
    Software Manual 8.4.19 Chapter 8 – Image Processing Sigma This filter is based on the settings in the Processing Define Filter Define Sigma window. Application: statistical noise filtering Assumption. Local noise in an image follows a Gaussian distribution. The Gaussian curve can be described by
  • Страница 176 из 403
    160 Chapter 8 – Image Processing 8.4.20 DCE – Differential Contrast Enhancement This filter is based on the settings in the Processing Define Filter Define DCE window. Application: selective enhancement of weak differences in contrast What will happen? The DCE filter renders image structures
  • Страница 177 из 403
    Software Manual Chapter 8 – Image Processing The result of the DCE filter depends on the size of the image area in which you apply it. This means that the image area in the smaller Preview window may look different than the completely filtered resulting image. This difference will be even greater
  • Страница 178 из 403
    162 Chapter 8 – Image Processing Boundary shape The separator affects light or dark lines dividing objects of the same color – or affects objects distinguishable from the background due to their intensity value. Dark. Select this option if bright objects have a dark outline. Bright. Select this
  • Страница 179 из 403
    Software Manual Chapter 8 – Image Processing (255 intensity) or black (0 intensity). You can decide whether the image background is either black or white accordingly, meaning the resulting image will be a binary image. Or you may take the image background from the original image. In this case, the
  • Страница 180 из 403
    164 Chapter 8 – Image Processing Erase All. Click this button to have all lines of separation erased from the overlay. Set All. Click this button to reconstruct all lines of separation that you’d erased since opening the Edit Lines dialog box. All lines either computed by the separator and/or drawn
  • Страница 181 из 403
    Software Manual Chapter 8 – Image Processing to false-color images as well, select the Allow operations on false-color images option in the Special Preferences Image tab. Filters analyze gray values, i.e., intensities. The filters of the second and third group can also be applied to all image
  • Страница 182 из 403
    166 Chapter 8 – Image Processing in the small object denoted by the arrow. Two black pixels are in its square, 3x3 neighborhood. This means that the central pixel will become black due to the erosion. In a cross-shaped neighborhood, the central pixel is surrounded entirely by white pixels so that
  • Страница 183 из 403
    Software Manual Chapter 8 – Image Processing Bright. Edge pixels will be set white (gray value 255). Use this option for bright objects. Dark. Edge pixels will be set black (gray value 0). Use this option for dark objects. Set. Click here to select all pixels of the neighborhood. Delete. Click here
  • Страница 184 из 403
    168 Chapter 8 – Image Processing Morphological operations are not linear. Thus, erosion does not reverse the effect of prior dilatation. Erosion is designed for binary images, it can however be used for gray-value images as well. The use of the result may be questionable. 8.5.3 Dilation What will
  • Страница 185 из 403
    Software Manual 8.5.4 Chapter 8 – Image Processing Morph. Open What will happen... This filter executes both the Erosion and Dilation filter operations one after the other. (See previous chapters.) First, the outer edge of objects is removed, noise and thin lines vanish. Then the object will be
  • Страница 186 из 403
    170 Chapter 8 – Image Processing 8.5.6 Gradient What will happen... This filter is the subtractive combination of the Erosion and Dilation filters. (See previous chapters.) It performs the subtraction of the eroded image from the dilated image. As a result only the smoothed edges of the objects
  • Страница 187 из 403
    Software Manual 8.5.8 Chapter 8 – Image Processing Top Hat Dark What will happen... This filter performs a Morph. Close filtering (see chapter 8.5.5) and subtracts the result from the original image. If the source is a binary image the resulting image will be dark and show only those pixels added
  • Страница 188 из 403
    172 Chapter 8 – Image Processing 8.5.10 Distance Dark Application. Use this filter for displaying the distance of each non-object pixel to the object edge. These distances will be coded in gray values with pixels far away from the next object being bright and those close to an object dark. The
  • Страница 189 из 403
    Software Manual 8.5.12 Chapter 8 – Image Processing Ultimate Erode Dark Application. Use this filter to erode dark objects to a single bright pixel or to a small, bright line. This reduced information can be used for counting dark particles, for example. The result of this filter is critically
  • Страница 190 из 403
    174 Chapter 8 – Image Processing Process Morphological Filter Define Skeleton. Use this command to open the respective window. Here the parameters for the skeleton filter are defined. General settings for morphological filters as set in the Define Morphological Filter window are ignored in the
  • Страница 191 из 403
    Software Manual Chapter 8 – Image Processing separated correctly (right). The width of the lines separating the pixels is 2 pixels. The arrow indicates where a fairly typical separation error will occur if the object in the original image is not a fully closed structure. Comparison. The Process
  • Страница 192 из 403
    176 Chapter 8 – Image Processing What happens if there’s a pixel value overflow? Commands execute the arithmetical operations one pixel at a time, thus processing all gray values. 8-bit channels have values between 0 and 255; 16-bit channels have values between 0 and 65535. When applying addition,
  • Страница 193 из 403
    Software Manual 8.7 Image Geometry 8.7.1 Chapter 8 – Image Processing Resize The function Process Image Geometry Resize creates a copy of different size of the active image without cutting of parts like the Extract ROI cropping function does (see Chapter 7.7, Extract…). The original data remain
  • Страница 194 из 403
    178 Chapter 8 – Image Processing Zoom axes independently. If this option is selected, it is possible to set, for example, different Factors for X and Y. Doing so leads to a distortion of the resulting image. Zoom axes homogeneously. This option is available for Z-stacks only. Here all three Factors
  • Страница 195 из 403
    Software Manual Chapter 8 – Image Processing 5. The Rotate window will open again with the interactively defined angle set in the Angle box. 6. Continue with setting the Direction and the Options. 7. Execute the rotation by clicking OK. Direction. Chose between a left (counterclockwise) and right
  • Страница 196 из 403
    180 Chapter 8 – Image Processing 8.7.3 Mirror The function Process Image Geometry Mirror allows reflecting single images and entire data sets at four predefined axes of choice: the central Vertical, the central Horizontal or any of the two Diagonal axes. Check the radio button of the desired axes
  • Страница 197 из 403
    Software Manual 8.7.5 Chapter 8 – Image Processing Auto Align Z Especially in long-term time-lapse experiments it may happen that the sample is moved in the XY plane. This may be caused by an accidental push of the microscope or the table or upon intentionally manipulating the sample, may be after
  • Страница 198 из 403
    182 Chapter 8 – Image Processing Horizontal / Vertical Shift. Use the arrow buttons to shift the selected channel relative to the others. Center. Use this button to move the active channel back to the original position. Save values for other uses. If this option is selected, the values of the
  • Страница 199 из 403
    Software Manual Chapter 8 – Image Processing Requirements. The images need to be calibrated in the X, Y and Z dimensions, see Chapter 7.1, Calibrate Image; otherwise an error message will be generated. Click the Deblurring button (or select Process 3D-Images Deblurring). If deblurring was not
  • Страница 200 из 403
    184 Chapter 8 – Image Processing Apply changes of numerical aperture and refractive index to all channels. It is useful to keep this option activated. OK. Confirm the settings and return to the Deblurring dialog window. 8.8.3 No Neighbor The No Neighbor algorithm is the faster algorithm as compared
  • Страница 201 из 403
    Software Manual Chapter 8 – Image Processing Reset filter parameters Reset filter parameters. Click here to set the parameters back to default Transmitted Brightfield. Mark the check box if you use a brightfield setup. The check box Phase Object becomes available. Phase Object. Mark the check box
  • Страница 202 из 403
    186 Chapter 8 – Image Processing 8.8.5 Wiener Filter The Wiener Filter is used in signal processing to reduce the amount of noise by comparison with an estimation of the desired noiseless signal based on a statistical approach. It is used here as a one step non-iterative deconvolution method and is
  • Страница 203 из 403
    Software Manual 8.8.6 Chapter 8 – Image Processing 3-D AMLE Deconvolution: Advanced Maximum Likelihood This feature is part of the different deconvolution modules C-MOD-XRID* of the software and not available in the basic software packages. This is a true, intensity restoring deconvolution method.
  • Страница 204 из 403
    188 Chapter 8 – Image Processing Spherical Aberration Detection. See Chapter 8.8.5, Wiener Filter, and Chapter 8.8.2, Edit Image Parameters. Microscope. The available options depend on the software module that is installed.
  • Страница 205 из 403
    Software Manual 9 Chapter 9 – Measurements Measurements For length and area measurements the software provides a designated environment for length and area measurements with its own button bar and the Measurement View in the Image Manager. All measurements become linked with the parent image and
  • Страница 206 из 403
    190 Chapter 9 – Measurements 9.1 Measurements Toolbar Measurements Click on the Measurements button at the bottom of the Image manager to open the Measurements View. Select Measurement Measurements Bar or press <Alt+4> to open the measurement button bar. The measurement functions of the button bar
  • Страница 207 из 403
    Software Manual Chapter 9 – Measurements If you do not want the results to be shown in the overlay select Show labels None in Special Preferences Measure (or Measurement Properties button in the Measurement View or key F8). 9.2 Drawing Tools for Length and Area Measurements 9.2.1 Point Point Use
  • Страница 208 из 403
    192 Chapter 9 – Measurements 9.2.3 Length 9.2.3.1 Vertical line Vertical line Use this command to measure a vertical distance between two horizontal lines. Measurement Results. The distance to be measured will be denoted by a vertical line labeled with the length by default. This vertical distance
  • Страница 209 из 403
    Software Manual Chapter 9 – Measurements How to define a vertical distance: A horizontal measurement is done using the same steps as for the vertical distance. 9.2.3.3 Arbitrary line Arbitrary line Use this command to measure distances between two parallel lines of arbitrary orientation.
  • Страница 210 из 403
    194 Chapter 9 – Measurements 3. In case corrections are necessary, keeping the <Shift> key pressed, left-click to delete previous figure segments one by one from the overlay. 4. Right-click to finish the line. Its length will be displayed in the overlay and written into the measurement display. 5.
  • Страница 211 из 403
    Software Manual 9.2.4.2 Chapter 9 – Measurements 4 Points Angle 4 Points Angle Use this command to measure the angle between two lines. It is called 4 points because each line has a separate starting and end point. The intersection divides each line into two segments. The angle is measured
  • Страница 212 из 403
    196 Chapter 9 – Measurements 2. Drag the mouse with the left button pressed to change the size and shape of the rectangle. 3. To fix the rectangle right-click, and a new rectangle will appear. Proceed as before. 4. To exit the measurement press <ESC>. The result will be displayed in the overlay and
  • Страница 213 из 403
    Software Manual 9.2.5.3 Chapter 9 – Measurements 3 Points Circle 3 Points Circle Use this command to measure a circular object by selecting 3 points on the arc of the circle. Default measurements are area and perimeter. Several measurement series can be conducted successively. 1. Mark a point at
  • Страница 214 из 403
    198 Chapter 9 – Measurements 9.2.5.5 Ellipse Ellipse Use this command to measure an elliptical region of arbitrary orientation. Default measurements are area and perimeter. Several measurement series can be conducted successively. As soon as the button is clicked a blue ellipse circumscribed by a
  • Страница 215 из 403
    Software Manual Chapter 9 – Measurements 2. Move the mouse and left-click at the corner positions of the area to draw a multisegment line. 3. Drag the mouse with the left button pressed to draw a freehand line or left-click at the corner positions to draw a multi-segment line. 4. In case
  • Страница 216 из 403
    200 Chapter 9 – Measurements 9.2.5.8 Fitted Polygon Fitted Polygon This command is identical to the Interpolated Polygon command with the difference that the segmented polygon will circumscribe the interpolated line instead of vice versa. A right-click automatically draws a line to close the
  • Страница 217 из 403
    Software Manual 9.3.1 Chapter 9 – Measurements Magic Wand Options Measurement Properties (in the Measurement View) The Magic Wand Options are part of the Measure Preferences, to be found on the Measure tab in Special Preferences. Shortcuts to these options are the F8 key and the Measurement
  • Страница 218 из 403
    202 Chapter 9 – Measurements o'clock (240°). With hue as similarity criterion artifacts may result in fluorescence images because the pixel intensity is not considered at all. Intensity Intensity. Here the similarity criterion is the overall pixel intensity added up from all color channels. In
  • Страница 219 из 403
    Software Manual 9.4.2 Chapter 9 – Measurements Create Measurement Sheet Create Measurement Sheet All executed measurements create an entry in the measurement display. These results remain linked to the image after the image has been saved. Additionally it is possible to list the measurement results
  • Страница 220 из 403
    204 Chapter 9 – Measurements 9.4.3 Deleting Measurement Results Delete Measurement from Image Use this command to delete individual objects and their measurements interactively. 1. Click the Delete Measurement from Image button and select the object to be them in the image overlay. 2. Delete
  • Страница 221 из 403
    Software Manual 9.4.4 Chapter 9 – Measurements Image Link Image Link Use this command to locate measurement values in the Measurement display by selecting them in the image overlay. This command is useful when many measurements have been taken. 1. Click the Image Link button located in the
  • Страница 222 из 403
    206 Chapter 9 – Measurements Select Measurements Tree View. It is located in the upper left hand part of the dialog box and lists the different types of measurement objects. Click on any type to list all measurements possible for this type in the Measurement Parameters list. Measurement Parameters
  • Страница 223 из 403
    Software Manual 9.4.7 Chapter 9 – Measurements Define Statistics Define Statistics This button opens the Define Statistics window. Available. All available statistics parameters that are not yet selected are listed here. Current. All statistics parameters to be shown in the Statistics field of the
  • Страница 224 из 403
    208 Chapter 9 – Measurements 9.4.8 The Preferences Measure Tab Measurement Properties Use this function to open the Preferences Measure Tab. For a detailed description refer to Chapter 13.5.4, The Preferences Measure Tab. The Magic Wand Options are described in Chapter 9.3.1, Magic Wand Options.
  • Страница 225 из 403
    Software Manual Chapter 9 – Measurements Select an entry from the list of current parameters. Click on the Up button to have this function moved up one position in this list. Click on the Down button to have this function moved down one position in this list. These buttons are only active if you
  • Страница 226 из 403
    210 Chapter 9 – Measurements
  • Страница 227 из 403
    Software Manual Chapter 10 – Intensity Analyses 10 Intensity Analyses The analysis features explained in this chapter are very important for cell biological applications. Line intensity profiles illustrate intensity changes along arbitrary lines within an image. Commonly regions of interest are
  • Страница 228 из 403
    212 Chapter 10 – Intensity Analyses 10.8.1 10.8.2 10.9 10.9.1 10.9.2 10.9.3 10.9.4 10.9.5 10.9.6 10.9.7 10.9.8 10.10 10.10.1 10.10.2 10.11 10.12 10.12.1 10.12.2 10.12.3 10.13 General..........................................................................................226 Generating a Ratio
  • Страница 229 из 403
    Software Manual Chapter 10 – Intensity Analyses Stop. You can terminate the action by opening the respective dialog with a right mouse click. Measurement Results. Mark the pixels whose coordinates and gray values you wish to determine. A cross in the overlay will denote each pixel marked. The
  • Страница 230 из 403
    214 Chapter 10 – Intensity Analyses 10.3 Line Profiles: Intensity 10.3.1 Horizontal Line Profile Use the Measure Intensity Profile along a horizontal line. Horizontal command to determine the gray value intensity How to measure a horizontal intensity profile: 1. Select the Horizontal command. A
  • Страница 231 из 403
    Software Manual Chapter 10 – Intensity Analyses The line profiles of all color channels will be drawn into a separate graph for each of the lines drawn into the image overlay. This command works currently only on the active frame, not on an entire multidimensional image set. 10.3.2 Vertical Line
  • Страница 232 из 403
    216 Chapter 10 – Intensity Analyses 5. Continue with drawing another line or terminate the command with a right click and confirm yes. This command works currently only on the active frame, not on an entire multidimensional image set. 10.3.4 Average Intensity of Neighboring Pixels Use this command
  • Страница 233 из 403
    Software Manual 10.4 Regions of Interest – ROIs 10.4.1 Chapter 10 – Intensity Analyses General Regions of Interest or ROIs are parts of images that are defined by the user for subsequent analyses. The ROIs do not become part of the images even if being displayed, that is, they do not change the
  • Страница 234 из 403
    218 Chapter 10 – Intensity Analyses Label: By default, i.e., if no name is typed into the box, the ROIs are labeled "ROI <consecutive number>". ROIs with identical names will likewise be numbered consecutively. Labels can be de leted or changed by clicking on the respective commands in the
  • Страница 235 из 403
    Software Manual Chapter 10 – Intensity Analyses Rectangle Rectangle. A rectangle opens, the size and shape of which can be adjusted by dragging with the left mouse button. Rotated rectangle Rotated rectangle. With this command rectangles of arbitrary orientation can be drawn. The left mouse button
  • Страница 236 из 403
    220 Chapter 10 – Intensity Analyses Delete Delete. This removes the ROI highlighted in the "Active ROI" list 10.4.3 ROI Measurements (2-D) If the ROIs are already drawn when any of the measurement commands is called you have to select the ROIs to be measured by mouse click. If no ROIs drawn the
  • Страница 237 из 403
    Software Manual 10.5 Background Subtraction... 10.5.1 Chapter 10 – Intensity Analyses General Background intensity is unavoidable in wide-field fluorescence spectroscopy. Working in a dark room can eliminate environmental light, but there will always be a certain amount of excitation light that
  • Страница 238 из 403
    222 Chapter 10 – Intensity Analyses This function will create a new data set. The original (source) data remain untouched. Background Settings tab Constant. This option subtracts a fixed value from all pixel intensities in all images of all channels. The constant has to be typed into the box or can
  • Страница 239 из 403
    Software Manual 10.6 Chapter 10 – Intensity Analyses Intensity Kinetics in Time and Z Kinetics Open the Kinetics dialog window with the Kinetics button or via Measure Kinetics… This tool is used to quantify the changes of fluorescence intensity within ROIs over time or within a Z-stack and display
  • Страница 240 из 403
    224 Chapter 10 – Intensity Analyses 10.7 DeltaF / F (∆F/F) Analysis 10.7.1 General While few fluorochromes allow the determination of absolute ion concentrations via ratiometric methods, the majority of ion sensitive dyes only allow the qualitative observation of changes in concentration. A
  • Страница 241 из 403
    Software Manual Chapter 10 – Intensity Analyses fixed background value is subtracted from all pixel intensities in all images. The constant is set in the box that is only visible if this option is chosen. ROI: The typical approach to background subtraction is to mark a ROI in an image area that
  • Страница 242 из 403
    226 Chapter 10 – Intensity Analyses Thresholds field. The thresholding is performed to prevent low-signal areas and background areas from resulting in areas of pronounced noise in the ∆F/F sequence (the reason being the division of small values by other small values). The threshold is set in the
  • Страница 243 из 403
    Software Manual 10.8.2 Chapter 10 – Intensity Analyses Generating a Ratio Sequence Ratio Open the Ratio dialog window with the Ratio button or via Measure Ratio… Ratio Settings tab Output field: The selection of the options Image, Kinetics and Calibrated determine if a ratio image sequence, a graph
  • Страница 244 из 403
    228 Chapter 10 – Intensity Analyses Value 2. ROI: The typical approach to background subtraction is to mark a ROI in an image area that contains only background intensity and use the average intensity in this ROI as background value. This value is determined individually in each image of each
  • Страница 245 из 403
    Software Manual Chapter 10 – Intensity Analyses copy offers the possibility to visualize the spatio-temporal behavior of cellular, sub-cellular, and molecular structures. In order to simultaneously visualize different structures, labels with different spectral properties have to be used. 10.9.2 The
  • Страница 246 из 403
    230 Chapter 10 – Intensity Analyses see a relatively strong, but undesired fluorescence of the nucleus upon YFP-excitation. This phenomenon, known as 'bleed-through', strongly reduces color resolution and constrains scientific conclusions. 10.9.3 The Solution is the first imaging system to
  • Страница 247 из 403
    Software Manual 10.9.4 Chapter 10 – Intensity Analyses How Does it Work? The principles of the method can easily be explained considering the example shown in Figure 2. To ‘unmix’ the spectral information of the fluorochromes with strongly overlapping emission spectra, it is necessary to determine
  • Страница 248 из 403
    232 Chapter 10 – Intensity Analyses Now the system is set to examine samples labeled with both fluorochromes. The intensity measured at 450–490 nm excitation is composed of fluorescence due to eGFP plus fluorescence due to eYFP. YFP exciter dichroic GFP exciter GFP exciter emitter observed emission
  • Страница 249 из 403
    Software Manual 10.9.6 Chapter 10 – Intensity Analyses Calibration The first step is to calibrate the contribution of the fluorochromes to the different color channels. Depending on the number of different fluorochromes to be analyzed, different reference samples stained with only one fluorochrome
  • Страница 250 из 403
    234 Chapter 10 – Intensity Analyses 4. Afterwards close the Define ROIs window. The ratio of the average intensities within in the first ROI after background subtraction (see chapter 10.5) is computed automatically and its value displayed in the dialog box. 5. Label the ratio (e.g., GFP) in the
  • Страница 251 из 403
    Software Manual Chapter 10 – Intensity Analyses 1. Acquire a multi-color image of a multi-labeled sample using the same filters that were used for the calibration described above. 2. Load the multi-color image of a multi-labeled sample into the active image buffer. Our example image is a
  • Страница 252 из 403
    236 Chapter 10 – Intensity Analyses 7. Press the OK button to start the unmixing algorithms. The processed image is automatically loaded into the current destination image buffer. Acknowledgement. The images were acquired and unmixed with a cell^R ( ) Imaging Station and kindly provided by Dr.
  • Страница 253 из 403
    Software Manual Chapter 10 – Intensity Analyses 10.10 Phase Color Coding and Analysis 10.10.1 Phase Color Coding This command colors image regions according to the threshold settings. This command is only available for 8- or 16-bit gray-value images, and for binary images, as well – not for 24-bit
  • Страница 254 из 403
    238 Chapter 10 – Intensity Analyses 10.10.2 Phase Analysis This command evaluates area fractions according to the threshold settings. This command is only available for 8- or 16-bit gray-value images, and for binary images, as well – not for 24-bit true-color images. It is available for 8-bit
  • Страница 255 из 403
    Software Manual Chapter 10 – Intensity Analyses Colocalization is of great interest in certain applications because it may, for example, indicate that the different molecules bind to the same target. Thus, qualitative and quantitative analysis is of importance. The analysis is available for
  • Страница 256 из 403
    240 Chapter 10 – Intensity Analyses 10.12 The FRET Software Module FRET (Foerster Resonance Energy Transfer) studies are based on the observation at different wavelengths, which can be performed conveniently with the fast switching filter wheel U-FFWO (see the U-FFWO Instruction Manual for
  • Страница 257 из 403
    Software Manual Chapter 10 – Intensity Analyses The Camera Control window features the option Enable image split (as long as the DV2/DualView™ Micro-Imager is configured in the ObsConfig software as described in Chapter 15.9, The Configuration of the DV2/Dual-View™ Micro-Imager). When this option
  • Страница 258 из 403
    242 Chapter 10 – Intensity Analyses 10.12.2 FRET Image Correction Factors FRET Correction The analysis of FRET image series becomes complicated by signal bleed through – or excitation cross talk – that is common for typical donor/acceptor pairs. The excitation characteristics of the fluorophores
  • Страница 259 из 403
    Software Manual Chapter 10 – Intensity Analyses 4. The ratio of the average intensities within the first ROI after background subtraction (see Chapter 10.5, Background Subtraction) is computed automatically and its value displayed in the dialog box. Manual ratios. Type in known intensity ratios
  • Страница 260 из 403
    244 Chapter 10 – Intensity Analyses If a DV2/Dual-View™ Micro-Imager is used, only the long wavelength channels of the two resulting split images are needed for the calculation. This means, the Ffret and Facc channels have to be selected from different source images. 6. Define two ROIs as described
  • Страница 261 из 403
    Software Manual Chapter 10 – Intensity Analyses 10.12.3 FRET Analysis FRET Analysis The FRET module offers four different methods for the calculation of FRET images. The simplest is the Ratio Method that does not correct for the channel bleed-through and relies exclusively on images acquired with
  • Страница 262 из 403
    246 Chapter 10 – Intensity Analyses 5. The Background Subtraction, Thresholds and Output Scaling functions are described in Chapter 10.8.2, Generating a Ratio Sequence. 6. Confirmation with OK creates an Ffret/Fdon ratio image. Note that such a ratio image itself has little meaning. Only an
  • Страница 263 из 403
    Software Manual 10.12.3.2 Chapter 10 – Intensity Analyses MicroFRET: Quantification According to Youvan The method described by Youvan is a subtractive method. The correction factors generated as described in Chapter 10.10.4, FRET Image Correction Factors, are used to estimate the contribution of
  • Страница 264 из 403
    248 Chapter 10 – Intensity Analyses 10.12.3.3 MicroFRET Experiments with an Observation Filter Wheel In case an observation filter wheel is used to monitor the two emission wavelengths, a standard triple-color image acquisition Experiment Plan has to be setup; see Chapter 5.3.3.4, Time-lapse
  • Страница 265 из 403
    Software Manual Chapter 10 – Intensity Analyses 3. Make sure that the Fdon, Facc and Ffret channels are selected correctly. 4. The Background Subtraction, Thresholds and Output Scaling functions are described in Chapter 10.8.2, Generating a Ratio Sequence. 5. Confirmation with OK creates a
  • Страница 266 из 403
    250 Chapter 10 – Intensity Analyses It is not possible to combine two split images with a multi-color frame with the intention to generate a four-channel image. The image acquired with the acceptor exciter (500 YFP in the example) contains a channel that is without use for the analysis: the short
  • Страница 267 из 403
    Software Manual Chapter 10 – Intensity Analyses 10.13 Kymogram A kymogram is a graphical representation of spatial position over time in which a spatial axis represents time. Kymograms of time-lapse series are a means to visualize the movement of particles, for example. The Kymogram command
  • Страница 268 из 403
    252 Chapter 10 – Intensity Analyses
  • Страница 269 из 403
    Software Manual Chapter 11 – Graph Display and Graph Analysis 11 Graph Display and Graph Analysis The previous chapter lined out how changes in intensity, for example in time-lapse experiments, can be quantified and plotted in graphs. In the following section you will means to modify and further
  • Страница 270 из 403
    254 Chapter 11 – Graph Display and Graph Analysis Several functions do not work on Kinetics graphs. 11.1 Graph Documents For displaying and processing one-dimensional data, contains an additional document format, the graph document. Similar to images, newly generated Graph documents or those loaded
  • Страница 271 из 403
    Software Manual 11.2 Chapter 11 – Graph Display and Graph Analysis The Graph Window Upon start of the Imaging Software the Graph window is minimized and located in the bottom left corner of the document area. If not minimized it might be hidden behind the Viewport but will be brought to the front
  • Страница 272 из 403
    256 Chapter 11 – Graph Display and Graph Analysis stretches or compresses the X scaling. Alternatively, the scaling buttons of the Graphs button bar can be used; this is explained in detail in Chapter 11.2.3, The Graphs Button Bar. Upon zooming in, scroll bars appear at the right side and at the
  • Страница 273 из 403
    Software Manual Chapter 11 – Graph Display and Graph Analysis Zoom Out Click the Zoom Out button to increase the displayed X range, i.e., to compress the graph in the X direction. Scale X Click the Scale X button to enlarge any X segment of the displayed graph to full axis size. Mark the left and
  • Страница 274 из 403
    258 Chapter 11 – Graph Display and Graph Analysis Auto. Max Y This is the dynamic analog of Max. Y: The scaling is automatically updated when moving the graph in the X direction by using the scroll bar at the bottom of the graph document. 11.2.3.3 Further zooming and scaling functions Define
  • Страница 275 из 403
    Software Manual Chapter 11 – Graph Display and Graph Analysis Show Legend Click the Show Legend button to display legends of the individual curves in a graph containing the kinetics of more than one image ROI or more than one line profile. 11.2.3.5 Overlay Functions These functions do not work on
  • Страница 276 из 403
    260 Chapter 11 – Graph Display and Graph Analysis 11.3 The Graph Menu The Graph menu contains special commands for graph processing; they do not work on images. Theses commands are described in the following. 11.3.1 Markers and Labels 11.3.1.1 Display markers Use Graph Markers and Labels
  • Страница 277 из 403
    Software Manual Chapter 11 – Graph Display and Graph Analysis frame can only be positioned in the visible part of the graph. You cannot scroll or zoom the graph while you set the labels. Anchor text. Check this box to position the text frame at a fixed position of the graph document. The position
  • Страница 278 из 403
    262 Chapter 11 – Graph Display and Graph Analysis 11.3.1.3 Modify... Use the Graph Markers and labels Modify... command to change the text and/or the position of the previously set annotations in the active graph. Alternatively, right-click within a graph document to open the context menu and then
  • Страница 279 из 403
    Software Manual Chapter 11 – Graph Display and Graph Analysis This command is deactivated for graphs resulting from a Kinetics calculation. 11.3.2 Protecting and Deleting a Graph Use Graph Analysis Protect Graph or click on the Protect Graph button in the Standard toolbar to protect the graph from
  • Страница 280 из 403
    264 Chapter 11 – Graph Display and Graph Analysis X-axis and Y-axis: Displays the title (legend) and the unit of the X and Y-axis, respectively. The parameters can be changed. The changes will be displayed in the graph document. All this graph information can be changed by the user and will be
  • Страница 281 из 403
    Software Manual 11.3.4.1 Chapter 11 – Graph Display and Graph Analysis Edit Column Header Use Sheet context menu Edit Column Header... to change the title/header of the highlighted sheet column. The header is limited to 31 characters. 11.3.4.2 Sort Ascending Use Sheet context menu ascending order.
  • Страница 282 из 403
    266 Chapter 11 – Graph Display and Graph Analysis User defined... Use this command to define the lower and upper end of an interval for the rows to be displayed. If Exclude rows is checked, the rows within the interval will be excluded. String... Use this command to define a string to display only
  • Страница 283 из 403
    Software Manual 11.3.4.7 Chapter 11 – Graph Display and Graph Analysis Convert to Sheet Use Graph Analysis new datasheet. Convert to Sheet to convert the coordinates of the displayed graph into a 11.3.4.8 and Excel ExcelDDE toolbar Use the Start Excel button (left) to open the spreadsheet program
  • Страница 284 из 403
    268 Chapter 11 – Graph Display and Graph Analysis
  • Страница 285 из 403
    Software Manual Chapter 12 – Database 12 Database The database is an integral part of the Imaging Software. It allows an organized storage of all acquired images, created graphs and documents like sheets, diagrams and texts. The design of the database facilitates an easy and fast access to even a
  • Страница 286 из 403
    270 Chapter 12 – Database 12.1 Directories for Data Storage How to specify the directories for data storage: 1. Select Special Preferences... from the menu bar or alternatively press <F8>. 2. Select the tab Database. 3. Specify a default directory to store all database files in the Database files
  • Страница 287 из 403
    Software Manual Chapter 12 – Database – When the system administrator stores the network database, the value for the backup volume capacity should be set to ‘0’. On principle it is possible to use network databases or write directly to CD. However, this is strongly not recommended, problems might
  • Страница 288 из 403
    272 Chapter 12 – Database The Exclusive option is only possible if the database is currently not opened by another user. On the other hand, if the database is opened in the Exclusive mode, no other user has access to it. 12.3 New Database... A database assistant makes it easy to create a new
  • Страница 289 из 403
    Software Manual 12.4 Chapter 12 – Database The Database Features Images acquired in experiments via the Experiment Manager– unlike snapshots or live images – are stored automatically in the active database in a folder carrying the name of the experiment. Likewise stored are online analysis results
  • Страница 290 из 403
    274 Chapter 12 – Database Organizational fields An organizational field is a database field associated to a database folder and thus to all data records within that database folder as well. The organizational field has the same value for all records created in a respective database folder. template
  • Страница 291 из 403
    Software Manual Chapter 12 – Database Preview. This area in the upper left corner shows a thumbnail of the currently active item in the Tree view. Tree view. The Tree view in the lower left corner resembles the structure known from file management programs like Windows Explorer. However, only the
  • Страница 292 из 403
    276 Chapter 12 – Database • the information table of the Form area • the Info Window • the optional Table View display of the database window • the Query by Example dialog box • the Insert dialog box in the currently active database. View Type lists all available views and data types, Current lists
  • Страница 293 из 403
    Software Manual Chapter 12 – Database In the Table View the thumbnails of the Gallery is converted into a table with icons instead of thumbnails. Additionally it lists the "cryptic" default names under which the files are stored and appear in disk management programs, see Chapter 12.4.1, General
  • Страница 294 из 403
    278 Chapter 12 – Database the information about the active item. The Info Window can be closed as usual with the Close button. 12.5 Working with the Database 12.5.1 Loading Documents Any document contained in a database can simply be loaded by a double-click on the thumbnail in the Gallery.
  • Страница 295 из 403
    Software Manual Database Insert Chapter 12 – Database Image... This has the same function as the Insert Image button. Database Insert Images... This command allows inserting a series of images from the Image Manager into the currently opened database folder. Select the images to be added from the
  • Страница 296 из 403
    280 Chapter 12 – Database Database Insert Document File... The command allows to import stored document files, e.g. from MSWord, MSExcel, etc., into a database folder. 12.5.3 Query Query (by Example…) Database Query by Example… The database architecture of allows to search the contents of a
  • Страница 297 из 403
    Software Manual Chapter 12 – Database If the active entry in the database is a folder, the query will only scan the contents of this folder. Activate the database icon in the Tree view to search the entire database. A query can be further refined by marking data types in the Advanced >> view of the
  • Страница 298 из 403
    282 Chapter 12 – Database Organizational field. Mark this check box to identify a database field as an organizational field. The content of an organizational field identifies all of the data, files and documents that belong to this organizational element. Picklist Edit. Click here to open the Edit
  • Страница 299 из 403
    Software Manual Chapter 13 – The Special Menu and the Window Menu 13 The Special Menu and the Window Menu Macros are little programs that allow repeating series of processing and analysis steps upon a click of a button. Here we explain how such macros can be generated. Also you will find in detail
  • Страница 300 из 403
    284 Chapter 13 – The Special Menu and the Window Menu 13.5.7 13.6 13.6.1 13.6.2 13.6.3 13.6.4 13.6.5 13.6.6 13.6.7 13.1 Macros 13.1.1 The Preferences Database Tab ..................................................312 Window
  • Страница 301 из 403
    Software Manual Chapter 13 – The Special Menu and the Window Menu Application. Certain image analysis processes frequently require series of commands that are repeated over and over again. The macro recorder enables you to record these commands and subsequently have them all run automatically
  • Страница 302 из 403
    286 Chapter 13 – The Special Menu and the Window Menu and then compile your module using the Build Module command (in the Special C Module menu, see Chapter 14.7, Build Module). A new button that allows to execute the macro/module will appear in the main user interface to. Editing macros. If you
  • Страница 303 из 403
    Software Manual 13.1.4 Chapter 13 – The Special Menu and the Window Menu Stop Macro Recorder Click on the second button in the Recorder toolbar (or select Special Recorder Stop Macro Recorder) after you have executed all commands or image analysis steps to be included in the macro. 13.1.5 Run Macro
  • Страница 304 из 403
    288 Chapter 13 – The Special Menu and the Window Menu The Single Step and the Run Macro command are similar. Single Step, however, executes the macro one step at a time – under user control. Application. This command is very useful for running a systematic error search in a new macro. You can
  • Страница 305 из 403
    Software Manual Chapter 13 – The Special Menu and the Window Menu What will happen? All variables and functions defined in the interpreter mode will be deleted internally but not in the text document. 13.1.8 Set as Default-Macro Use this command to define the active text document as the default
  • Страница 306 из 403
    290 Chapter 13 – The Special Menu and the Window Menu All macros that are integrated into the Graphical User Interface using the Define Macros... command must be saved in a user configuration. Use the Special Configuration Save... command to save the configuration. Macros. Name your macro in the
  • Страница 307 из 403
    Software Manual 13.2 Chapter 13 – The Special Menu and the Window Menu Add-In Manager... Use this command to add/load optional extensions to the standard configuration. Definition. The range of functions provides can be extended as much as you like. An Add-In is an additional program developed for
  • Страница 308 из 403
    292 Chapter 13 – The Special Menu and the Window Menu Add-ins that have not yet been activated remain on offer. We recommend however, that you activate only those add-ins that you really need. Otherwise, you’ll have too many superfluous button bars or commands making the appearance of the Graphical
  • Страница 309 из 403
    Software Manual Chapter 13 – The Special Menu and the Window Menu Any alterations you make to menu definitions via the Define Menu Bar... command and integrate into the Graphical User Interface have to be saved in a user configuration. Use the Special Configuration Save... command to save the
  • Страница 310 из 403
    294 Chapter 13 – The Special Menu and the Window Menu Modify (Add). Click on the Modify button to alter the name of a menu or command. When you’ve selected an entry in the Menu list you can alter the name of the entry in the Menu field. As soon as you’ve entered the new name, the Add button’s
  • Страница 311 из 403
    Software Manual Chapter 13 – The Special Menu and the Window Menu • Create a sub-menu: 1. Select the Define Menu Bar... command in the Special menu. 2. Double-click on the name of the menu for which you wish to create a submenu. 3. Enter the name of the submenu into the Menu field. 4. Select the
  • Страница 312 из 403
    296 Chapter 13 – The Special Menu and the Window Menu • Macros generated using the Special Macros Define Macros... command. The configuration does not include the settings for the Viewport Manager or the Image Manager. Settings of individual dialog boxes are not part of the configuration either.
  • Страница 313 из 403
    Software Manual Chapter 13 – The Special Menu and the Window Menu and include functions defined in selected configuration option. In most cases, this option will get you the desired result. No update. If the configuration you wish to load is however a reduced version of an older standard
  • Страница 314 из 403
    298 Chapter 13 – The Special Menu and the Window Menu 13.5 Preferences... Use this command for setting preferences within ternatively be opened with <F8>. 13.5.1 The Preferences . The Preferences dialog box can al- Image Tab The settings on this tab apply the handling of images. Overview • You can
  • Страница 315 из 403
    Software Manual Chapter 13 – The Special Menu and the Window Menu • You can predefine the image name automatically set for image acquisition. Imaging functions The settings in this group are relevant for image processing operations, for altering gray or color values of an image. Available image
  • Страница 316 из 403
    300 Chapter 13 – The Special Menu and the Window Menu None. A newly taken snapshot will always be placed into the active buffer in the Image Manager overwriting the any image currently being place there. Image buffers (All). A newly taken snapshot will always be placed into the next buffer in the
  • Страница 317 из 403
    Software Manual Chapter 13 – The Special Menu and the Window Menu • Decide whether the mouse cursor is to be transformed into the shape of an arrowhead or of crosshairs during interactive measuring. • Decide whether the name of a function is to appear along with a brief description of the command
  • Страница 318 из 403
    302 Chapter 13 – The Special Menu and the Window Menu Auxiliary. Determine the temporary secondary color, for example, of the distance to be measured. After terminating the action the color switches to the designated (permanent) color. Navigator. Determine the color of the frame indicating the
  • Страница 319 из 403
    Software Manual the image itself. In comments and overlays. Chapter 13 – The Special Menu and the Window Menu this non-image information refers to, e.g., calibration data, image JPEG (JFIF, *.jpg), JPEG200 (*.jp2), LEAD Compression (*.cmp). In order to achieve a stronger data reduction you may
  • Страница 320 из 403
    304 Chapter 13 – The Special Menu and the Window Menu TIFF / Preprocessing. Before saving the image you can adjust whether the image should be Converted form 16-bit to 8-bit and whether the Burn overlay into image should be done, by clicking the respective check boxes. TIFF / Compression. The TIFF
  • Страница 321 из 403
    Software Manual Chapter 13 – The Special Menu and the Window Menu Sheet format Decide which file format the system will offer by default for your use when you save. This format is a pre-set file type – i.e., you are free to alter the actual format of a sheet anytime you like. To make an alteration
  • Страница 322 из 403
    306 Chapter 13 – The Special Menu and the Window Menu user-defined configuration saved automatically upon shut down. A configuration will only be saved if you’ve made alterations to it. The current configuration will be saved in the corresponding configuration file, thus overwriting the previous
  • Страница 323 из 403
    Software Manual Chapter 13 – The Special Menu and the Window Menu Number of entries in recent file list. Here you can select how many of the recently used files are displayed in the file list within the File and Database menu. 13.5.4 The Preferences Measure Tab The settings in this tab are mainly
  • Страница 324 из 403
    308 Chapter 13 – The Special Menu and the Window Menu Numbering. If selected, individual measurements conducted within a series of measurements will be numerated in the overlay. When you select this check box the Measurement results option will be cleared – you cannot display numbers and
  • Страница 325 из 403
    Software Manual 13.5.5 The Preferences Chapter 13 – The Special Menu and the Window Menu Module Tab The settings in this tab apply to users who wish to create their own macros or Imaging C modules . for use in Modules Auto save on build. When selected, the text document is automatically saved –
  • Страница 326 из 403
    310 Chapter 13 – The Special Menu and the Window Menu Maximum number of modules. Enter the maximum number of C modules you wish to have loaded simultaneously. The standard value here is 20. You should only increase this number if you really need all those modules – in order not to occupy more
  • Страница 327 из 403
    Software Manual 13.5.6 The Preferences Chapter 13 – The Special Menu and the Window Menu Graph Tab The settings on this tab apply to the handling of graphs. Overview • You can adjust the number of available graph buffers in the image manager. • You can determine whether the graphs are displayed in
  • Страница 328 из 403
    312 Chapter 13 – The Special Menu and the Window Menu 13.5.7 The Preferences Database Tab Use this tab to define presets for a database you wish to create. See also Chapter 12.1, Directories for Data Storage. Overview. On this tab you can decide • the current size of the thumbnail display for all
  • Страница 329 из 403
    Software Manual Chapter 13 – The Special Menu and the Window Menu We recommend you to use the TIF format as default for the reasons already described in Chapter 13.5.3, The File Tab. In the Location field you can determine the pathname for the Database files and the Temporary storage directory, as
  • Страница 330 из 403
    314 Chapter 13 – The Special Menu and the Window Menu Activate Activate. This button activates the window selected in the Document/Name list. The same command can be executed with a double-click on the window. Minimize, Maximize, Restore, Close Minimize, Maximize, Restore, Close. These buttons
  • Страница 331 из 403
    Software Manual Chapter 13 – The Special Menu and the Window Menu What will happen? All open documents will be displayed at the same level and in the same size, using up the whole documents area. Document windows will be enlarged or reduced as needed. If up to three documents are selected they will
  • Страница 332 из 403
    316 Chapter 13 – The Special Menu and the Window Menu What will happen? A tick will appear next to this command when the Image Manager is On. 13.6.6 Status Bar Use this command to turn the Status Bar On or Off. What is the Status Bar? The Status Bar is usually displayed at the bottom of the
  • Страница 333 из 403
    Software Manual Chapter 13 – The Special Menu and the Window Menu Saving Command Lines. In contrast to other document windows, the contents of the Command Window cannot be saved using the Save As command. However, you can circumvent this problem, therefore, open a new standard text document and
  • Страница 334 из 403
    318 Chapter 13 – The Special Menu and the Window Menu
  • Страница 335 из 403
    Software Manual Chapter 14 – Imaging C 14 Imaging C has its own macro or programming language: Imaging C. It is a variation of the ANSI C standard, which has been expanded by a number of functions. Why program in Imaging C? Imaging C offers the possibility of automating processes you use over and
  • Страница 336 из 403
    320 Chapter 14 – Imaging C 14.1 General The Special C-Module menu contains commands for the administration of Imaging C modules. A C module provides one or more C module functions. C modules are written using the macro language Imaging C, translated so that the computer is able to understand it,
  • Страница 337 из 403
    Software Manual 14.2 Chapter 14 – Imaging C New Module... Use Special C Module New Module... to create a new C module. What will happen? The New Module... command lightens your workload when you wish to generate a new directory, then set up a source file (SFM) there and then have this file
  • Страница 338 из 403
    322 Chapter 14 – Imaging C Adding source files to a new module If the Create sub-directory check box is the only check box that has been selected, the Edit Module dialog box will be opened. Here is where you can have files added to a C module. This allows you to integrate existing source and
  • Страница 339 из 403
    Software Manual Chapter 14 – Imaging C function description string. The first thing you enter into a MAPI function’s Function description is the MAPI constant. This function description is located between the ‘head’ and ‘foot’ of a function. It is enclosed by the following symbols: '<' and '>':
  • Страница 340 из 403
    324 Chapter 14 – Imaging C If you desire to change the name of the MAPI function SampleFunction after the C module has been generated, be sure not to forget to change the assignment of the SampleFunction’s ‘T’ button as well. String Table Select the String table check box if you wish the new C
  • Страница 341 из 403
    Software Manual Chapter 14 – Imaging C EnableDlgItem (hDlg, IDD_BEEP, bCheck); break; case WM_COMMAND: switch (LOWORD (wParam)) { case IDD_BEEP: MessageBeep (MB_ICONQUESTION); break; case IDD_CHECKBOX: bEnable = IsDlgButtonChecked (hDlg, IDD_CHECKBOX); EnableDlgItem (hDlg, IDD_BEEP, bEnable);
  • Страница 342 из 403
    326 Chapter 14 – Imaging C 4. Select the "New" C module from the Command groups list. The Commands list will now contain the SampleFunction entry as well as the name of the new function called "An_other". 5. Select the "T" button in the New Button field. 6. Select "An_other" in the Commands fields.
  • Страница 343 из 403
    Software Manual Chapter 14 – Imaging C If you change the function description of a MAPI function, the configuration file does not have to be adapted accordingly. All you need to do is have the C module recompiled using the Build Module (<F7>) command. 14.3 Open Module... Use Special C Module Open
  • Страница 344 из 403
    328 Chapter 14 – Imaging C What will happen? The module will be included in the Loaded list in the Module Manager. All global and static variables of the C module will be initiated as well as the C module’s main function. If this function results in a value of -1, the loading of the C module will
  • Страница 345 из 403
    Software Manual Chapter 14 – Imaging C Before you use this command. Activate the module in question in the module manager. Then you can check and see whether the module has a MKU make file using the Open... button in the Open Module dialog box. Then activate the text document that you wish to add.
  • Страница 346 из 403
    330 Chapter 14 – Imaging C • external programs. On the other hand, the configuration does not contain the settings of the Viewport Manager or the Image Manager. Individual dialog box settings and which modules are currently loaded are not part of the configuration. What’s it for? Use the command
  • Страница 347 из 403
    Software Manual Chapter 14 – Imaging C tension, it will be treated as a function source file and thus appended to the Source files list. If the list position of the appended file is to be changed, you can adjust it using the Up and Down buttons. Remove. To remove a file from the module, select that
  • Страница 348 из 403
    332 Chapter 14 – Imaging C You may also press the <F7> key. Before you use this command activate the module in question in the module manager. Then you can check and see whether the module has a MKU make file using the Open... button in the Open Module dialog box. What will happen? All files of the
  • Страница 349 из 403
    Software Manual Chapter 14 – Imaging C Before you use this command activate the module in the module manager. On close remove module functions from configuration. This check box is only available if the C module contains a configuration (file SCX) and there is a MKU make file. Select this check box
  • Страница 350 из 403
    334 Chapter 14 – Imaging C Most modules can also be loaded and closed in the Add-In Manager. Some modules will be automatically loaded: for example, when you load a configuration, use userdefined measurement or statistical parameters or activate an image import filter. These modules will be shown
  • Страница 351 из 403
    Software Manual Chapter 14 – Imaging C Load. Click on the Load button to load the module you’ve selected. The module’s functions will be currently available. The module will be moved from the Other list into the Loaded list. This button is only available if you have selected a module in the Other
  • Страница 352 из 403
    336 Chapter 14 – Imaging C Module. The Module field indicates the path of the module currently selected in either the Loaded or the Other lists. File. The File field indicates the path of the active text document. Using the Add button you can add this text document to the selected module. Active.
  • Страница 353 из 403
    Software Manual Chapter 14 – Imaging C Folder name. The Folder name field shows the directory, meaning the complete path of the directory you have selected in the Look in field. You can keep the directory or enter another directory. The complete path of this directory can be added to the Selected
  • Страница 354 из 403
    338 Chapter 14 – Imaging C Modules’). This list will then disappear and replaced by the German function names check box - if you’ve selected the ’Internal Function’ type of symbol. Definition. A Symbol can be a function, a structure, a union, an object, a variable, a dialog or a constant. Type. The
  • Страница 355 из 403
    Software Manual Chapter 14 – Imaging C What’s it for? This command is for use when you’re programming in Imaging C and you don’t know the precise name of a symbol – or the symbol’s name is extremely long. What you would do in this case would be to, e.g., enter the first few letters of the symbol
  • Страница 356 из 403
    340 Chapter 14 – Imaging C 14.14 Quick Watch... Use this command to determine the value of the selected expression or variable during an error search (i.e., debugging). Available. This command is available when a text document is open and active. You may also use the <Shift+F9> keys. Before you use
  • Страница 357 из 403
    Software Manual Chapter 14 – Imaging C Add... Use the Add... button to add the expression to the Expression list in the Watch Variables dialog box. The Watch Variables dialog box will be opened if it wasn’t already. This dialog box is non-modal, meaning that it can remain open during debugging.
  • Страница 358 из 403
    342 Chapter 14 – Imaging C This dialog box is non-modal, meaning it can be kept open during debugging. This enables you to track a variable’s current value during the debugging. Expression, Add, Expression list The Add button will evaluate the Imaging C expression entered into the Expression field
  • Страница 359 из 403
    Software Manual Chapter 14 – Imaging C In a text window, the symbol of a hand at the beginning of a line indicates a breakpoint. Definition. When a command, for which a breakpoint has been defined, is executed the execution of the Imaging C program or module will be interrupted. The definition at
  • Страница 360 из 403
    344 Chapter 14 – Imaging C Debugging must be terminated before you can close . If you cannot close the Imaging C program proper, then click on the Stop Execution button in the Debug button bar. You cannot terminate debugging by closing the source text’s document window, nor by turning ‘off’ the
  • Страница 361 из 403
    Software Manual Chapter 14 – Imaging C Status Status Meaning: breakpoint is + valid - invalid E enabled (active) D disabled (not active) Delete All. Click on the Delete All button to delete all breakpoints from the Breakpoint list and from source texts. Deletion is not reversible. Available. The
  • Страница 362 из 403
    346 Chapter 14 – Imaging C
  • Страница 363 из 403
    Software Manual 15 Chapter 15 – Configuration Configuration A special Configuration Software, ObsConfig, is provided for the configuration of the Imaging Station components: for the Illumination System MT20 / MT10, the motorized microscope components and for the optional PIFOC z-drives.
  • Страница 364 из 403
    348 Chapter 15 – Configuration 15.1 The Illumination System MT20 / MT10 15.1.1 Configuring the Excitation Filters The excitation: filters are mounted in the filter wheel of the Illumination System MT20 / MT10 which is accessible via the filter flap on the right side of the illumination system
  • Страница 365 из 403
    Software Manual Chapter 15 – Configuration In brief, start the configuration software by clicking the ObsConfig.exe button and go to the Illumination System Excitation filter page. A gray arrow in the list of program pages on the left hand side indicates the selection. With the vertical slider bar
  • Страница 366 из 403
    350 Chapter 15 – Configuration the conventions, 0 corresponds to red, 180 to cyan. Each value has a corresponding wavelength, indicated below the quadratic color field. The saturation and the intensity can be set between 0 and 1000 on an arbitrary scale. A Brightness of 0 results in the color black
  • Страница 367 из 403
    Software Manual 15.1.3 Chapter 15 – Configuration Using MT20 / MT10 without the Imaging Software You can operate the Illumination System MT10 / MT20 with the dialog box Illumination System Imaging Software, that is, you can switch the burner on Full control without using the and off, change the
  • Страница 368 из 403
    352 Chapter 15 – Configuration Shutter. If the transmission light path of an IX51/71/81 microscope is equipped with a shutter select Transmitted. This is not available for the upright microscopes BX51/61. does not feature a reflected light shutter at the microscope frame. The corresponding shutter
  • Страница 369 из 403
    Software Manual 15.2.2 Chapter 15 – Configuration Z-Drive Configuration Z-Drive available. Activate the motorized Z-drive by checking the box if the microscope is equipped with one. Position box and range sliders. The box displays the current Z-drive position. You can move the Z-drive by typing in
  • Страница 370 из 403
    354 Chapter 15 – Configuration By carefully setting the stage limits one can avoid driving the objective accidentally into the specimen. In case of an inverted microscope, for example, the topmost position of the Z-drive should not lift an oil-immersion objective with short working distance more
  • Страница 371 из 403
    Software Manual 15.2.4 Chapter 15 – Configuration Configuration of the Fluorescence Filter Turret Fluorescence Turret. If a motorized fluorescence turret is available, select it from the shortlist to be able to use it via the software. Shutter. This button is available only if the IX2-FRFACA is
  • Страница 372 из 403
    356 Chapter 15 – 15.2.5 Configuration Configuration of the Transmission Contrast Inserts Condenser. Check here if a Motorized condenser is available to be able to use it via the software. Contrast inserts slider. You can use the slider to move any of the mounted contrast inserts into software.
  • Страница 373 из 403
    Software Manual 15.2.6 Chapter 15 – Configuration Configuration of the Filters of a Filter Wheel Filterwheel. Select here if an additional filter wheel is available to be able to use it via the software. The illumination system MT20 / MT10 features an integrated filter wheel and no additional
  • Страница 374 из 403
    358 Chapter 15 – 15.3 Configuration Definition of Image Types The definition of image types corresponding to certain excitation wavelengths is important for the set-up of experiments with the Experiment Manager and for the display of images onscreen. Image Type. The Experiment Manager will use the
  • Страница 375 из 403
    Software Manual Chapter 15 – Configuration Add Illumination, Delete Illumination. The Add Illumination button allows to add a second or third (or even further) illumination source to a single Image Type. This can be useful, for example, for simultaneous multi-color TIRF with a cell^TIRF system. Use
  • Страница 376 из 403
    360 Chapter 15 – Configuration example a black-to-dark orange color palette will be used. If no color is selected a black&white (gray scale) display will be used. 15.4 Configuration of Additional Shutters Additional shutters, which are not integral parts of the IX81 or BX61 frames (those are
  • Страница 377 из 403
    Software Manual Chapter 15 – Configuration Transmission. Check here to make the shutter available for image types with Transmission as setting in the Illumination / Excitation Filter column in the Image Types page, see previous chapter. 15.5 Configuration of the PIFOC Use the PIFOC dialog for the
  • Страница 378 из 403
    362 Chapter 15 – Configuration Move. The scroll bar represents the traverse path of the PIFOC. You can move the PIFOC within these limits either stepwise by clicking on the arrows or via mouse scrolling. 15.6 Configuration of the Motorized Stage currently supports a number of motorized stages by
  • Страница 379 из 403
    Software Manual Chapter 15 – Configuration Speed. The maximum possible Velocity and Acceleration the selected stage can deliver will be set by default. However, for certain applications it might be advantageous to decrease these settings, for example, to minimize vibrations. This can be done by
  • Страница 380 из 403
    364 Chapter 15 – Configuration Please check the status on the light panel of the UCB in case of software problems for an appropriate diagnosis. 1. Exit the software and shut off the UCB controller. 2. Switch on the UCB controller box ONLY. 3. Check the status of the light panel. Any problems
  • Страница 381 из 403
    Software Manual Chapter 15 – Configuration 4. Click the next Objective button in the window Parfocality Correction (z). The nosepiece will move this objective into position. 5. Focus again. 6. Press the <<Read button. The new z-drive position will be set in the Position box of the chosen objective.
  • Страница 382 из 403
    366 Chapter 15 – 15.9 Configuration Configuration of the DV2/Dual-View™ Micro-Imager This optional device may not be part of your 15.9.1 Imaging Station. Configuring the Emission Filters Open the ObsConfig software and go to page Image Splitter. The empty square button gives the default setting for
  • Страница 383 из 403
    Software Manual Chapter 15 – Configuration Horizontal split. Click this button if the DV2/Dual-View™ Micro-Imager is aligned relative to the camera so that the image is split into an upper and a lower half. Filters. Select a filter from the shortlist or type in a filter name and select a color for
  • Страница 384 из 403
    368 Chapter 15 – Configuration
  • Страница 385 из 403
    Software Manual Chapter 16 – Installing the 16 Installing the ware Software Soft- The Imaging Station is delivered with the software installed. However, in case of software updates – for example of cell^M / cell^R Imaging Stations – a new installation is necessary. Several administrative
  • Страница 386 из 403
    370 Chapter 16 – Installing the Software Usually is installed on the imaging PC's larger second hard disk where the archive for the image data sets is located by default as well. In case you want to install a virus scan software on the imaging PC, be sure that tiff format files are not scanned.
  • Страница 387 из 403
    Software Manual Chapter 16 – Installing the Software If you select New Installation make a back-up copy of the software configuration file ObsConfig.xml that contains the information about excitation filters, image types etc. Thus you avoid having to go through parts of the configuration steps
  • Страница 388 из 403
    372 Chapter 16 – Installing the Software 10. In the next window, choose in which group program shortcuts for Start Programs are . It is most convenient to acto be added. The setup will suggest the folder cept this and simply continue with Next >. The installation will be executed. 16.2 Updating the
  • Страница 389 из 403
    Software Manual Chapter 16 – Installing the Software The update lasts few minutes. Once it is completed, a window with the confirmation appears. Switch off the MT10 / MT20 illumination system after the update. Switch it on again after about 5 seconds. This is necessary for a complete
  • Страница 390 из 403
    374 Chapter 16 – Installing the Software 3. In case of a F-ViewII-T camera select OBS MegaView. Otherwise select the corresponding camera type or manufacturer. 4. Exit with OK. 5. Calibrate the camera pixel size as described in Chapter 7.1, Calibrate Images. 16.4 Single-User Systems (Administrator
  • Страница 391 из 403
    Software Manual 16.5 Chapter 16 – Installing the Software Multi-User Systems Note the following if user accounts in addition to the Administrator are to be installed on the computer. There are two main differences between operating systems in this context: • MSWindows 2000 requires administrator
  • Страница 392 из 403
    376 Chapter 16 – Installing the Software 5. Check that the Environment variables for the user are set to D:\Temp by doing the following: a Open Settings Control Panel System from the Start menu. b Select the Advanced tab. c Check that the User variables Temp and tmp are set to D:\Temp. Otherwise
  • Страница 393 из 403
    Software Manual Chapter 16 – Installing the 8. Select Local Policies Software User Rights Assignment. 9. Select the item Increase scheduling priority on the list on the right side. 10. Select Action Properties. 11. Click Add user or group, write the name of the new user under Enter the object names
  • Страница 394 из 403
    378 Chapter 16 – Installing the Software 6. Return to the CTR Properties window with OK.
  • Страница 395 из 403
    Software Manual Chapter 16 – Installing the Software 7. Click on the Configure button and go to the Advanced tab of the Realtek window that opens. 8. Select the Property Link Speed/Duplex Mode and then the Value 10Mbps/Full Duplex from the shortlist. Confirm with OK. Additionally, it is important
  • Страница 396 из 403
    380 Chapter 16 – Installing the Software
  • Страница 397 из 403
    Software Manual Chapter 16 – Installing the Index Acquisition 12, 20, 43 3-D Time-lapse 66 FRET 240 Kinetics 53, 66 Multi-Color 46 Not synchronous 60 Ratio image 51, 66 Single image 61, 62 Time-lapse 49, 65 Z-Stack 47, 63 Add-Ins 291 Arithmetic Operations 175 Image types 358 Microscope 351
  • Страница 398 из 403
    Experiment 71 Marker 74 Illumination (system) 25, 60 Configuration 348 Experiment Manager 14, 38 Digital port, TTL 54, 67 Execution Center 41 Experiment Plan 42 Microscope 58 Preferences 75 Wait 55 Image Align 180 Animate 17 Filter 145 DCE 160 Differentiate 149 Edge enhance 157 Laplace 150 Lowpass
  • Страница 399 из 403
    Software Manual Microscope 26 Configuration 351 Module 321 Network connection 377 Parfocality correction 364 Phase Analysis 238 Color coding 237 Preferences Database 312 File 302 Graph 311 Image 298 Measure 307 Module 309 View 300 Chapter 16 – Installing the Scale bar 109 Shading correction 129
  • Страница 400 из 403
  • Страница 401 из 403
  • Страница 402 из 403
  • Страница 403 из 403