Инструкция для OLYMPUS SCAN R ANALYSIS 2.1

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INSTRUCTIONS 

 

AUTOMATED IMAGE AND DATA 

ANALYSIS SOFTWARE

 

This instruction manual describes the Olympus 

 Automated Image and Data 

Analysis Software for Life Science. To ensure safety, obtain optimum performance 
and familiarize yourself fully with the use of these products, we recommend that you 
study this manual thoroughly before operation. Together with this manual, please also 
read the 

 Automated Image Acquisition Software and Hardware manuals as 

well as the manuals of all other devices controlled by that software in order to under-
stand general operation methods. Retain this manual in an easily accessible place 
near a system for future reference.

 

® 

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Оглавление инструкции
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    ® INSTRUCTIONS AUTOMATED IMAGE AND DATA ANALYSIS SOFTWARE This instruction manual describes the Olympus Automated Image and Data Analysis Software for Life Science. To ensure safety, obtain optimum performance and familiarize yourself fully with the use of these products, we recommend that you
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    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
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    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
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    LICENSE AGREEMENT Preamble OLYMPUS SOFT IMAGING SOLUTIONS GMBH is manufacturer of the scan^R software and drivers/interfacing software to Olympus Soft Imaging Solutions manufactured hardware. These components are referred to as OSIS scan^R SOFTWARE PRODUCT. LICENSE AGREEMENT between END USER and
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    Analysis Software Manual Contents 1 Introduction............................................................................................ 1 1.1 Abstract.............................................................................................. 2 1.2 Technical Support
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    Analysis Software Manual 4.2.3 Color Gating................................................................................... 49 4.3 Well Results..................................................................................... 52 4.3.1 Measurement
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    Analysis Software Manual 1 Introduction Chapter 1 – Introduction 1
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    2 Chapter 1 – Introduction Thank you very much for purchasing Olympus’ Screening Station for Life Science and for your confidence in our products and services. The Analysis Software is designed for the automated analysis of images that were acquired by the Olympus Screening Station and with the
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    Analysis Software Manual Chapter 2 – Main User Interface 2 Main User Interface This chapter explains the features of the image displays and briefly introduces the different menu points and buttons accessible from the main user interface. 2.1 2.2 2.3 2.4 2.4.1 2.4.2 2.5 2.6 2.7 General
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    4 Chapter 2 – Main User Interface 2.1 General The scan^R Analysis main graphical user interface contains four histograms and an image viewer window. The functions of these histograms are explained in Chapter 2.4, Managing Histograms. The image viewer window shows the image with the object
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    Analysis Software Manual Analysis Open: opens a previous analysis (*.sca-file) Analysis Chapter 2 – Main User Interface Save/Save as: saves the current analysis (*.sca-file) Analysis Export Table: exports the values determined for each detected object to a spread sheet. The values exported depend
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    6 Chapter 2 – Main User Interface 2.2 General Settings The general preferences can be defined in the Settings menu. Max Gallery Objects. Sets the number of objects that are displayed in a gallery. Sort mode. The options are Typical, Random, yx and –yx. When Typical is set, the galleries display the
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    Analysis Software Manual Chapter 2 – Main User Interface Export2Txt. The results can be exported as .txt files or as .fcs files. (For export to .fcs format, see Chapter 6.2) Repositioning & Reclassification. Set the Port and the Address for communication with the scan^R screening system for
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    8 Chapter 2 – Main User Interface underneath the histogram. The X and Y pull down menus are then automatically updated to correspond to the measured parameters of the chosen object type (see assay definition). The X and Y pull down menus are used to change the parameters displayed in a histogram.
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    Analysis Software Manual 2.4.1 Chapter 2 – Main User Interface The Histogram Context Menu The histogram can be managed through the context menu accessible via right-click into the histogram. (Note that you will get the region context menu, if you right-click on a region border as described below).
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    10 Chapter 2 – Main User Interface Selector Color. A click on the colored field opens a window that allows selecting the cross-hair color. Region Color: A click on the colored field opens a window that allows selecting the color of the region outline. Color scheme. This color palette defines the
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    Analysis Software Manual 2.5 Chapter 2 – Main User Interface Using the Image Viewer Each analysis data point is directly linked to the image object it is generated from. These objects can be displayed in a close-up view in the image viewer. Browsing in the data set by clicking on data points with
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    12 Chapter 2 – Main User Interface Row/Column/Position/Time. Use these entries to select a specific image to be displayed. The up/down arrows allow fast navigation though your image data set by incrementing or decrementing the Time, Position and/or Well number. Interactive objects. Select the
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    Analysis Software Manual Chapter 2 – Main User Interface Dynamic [%]. Define here how many pixels (as a percentage of the total number of pixels) will be displayed with maximum and minimum brightness. The intensity of the remaining range of pixels will then be scaled linearly in between. The higher
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    14 Chapter 2 – Main User Interface Right-click on one of the scanned wells gives you the option Well Overview. For a time series you will have to set the time point first. The Well Overview will display all the positions you recorded for one well. Selecting one of the images will load the image in
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    Analysis Software Manual Chapter 3 – Assays 3 Assays Assays are the recipes to extract the data of interest from the images of a scan. They define which objects are to be recognized and how and which measurements are to be performed on the found objects. This chapter explains in detail how assays
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    16 Chapter 3 – Assays 3.1 General An assay defines all steps necessary to extract quantitative data from the acquired images. It usually starts with some sort of image processing like background correction. Secondly, objects have to be detected in the images. The analysis, for example different
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    Analysis Software Manual Chapter 3 – Assays Settings list. Each Object Finder Module has a list of preset parameters. The lists can be modified and stored at will. Individual modifications of these settings are marked as Modified. Select the settings of choice from the shortlist. Adjust. This
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    18 Chapter 3 – Assays View button opens the View Segmentation window that contains on the left a display of the objectcircumscribing rectangles and on the right a display of the segments. 3.3 Sub-object Finder: Detecting Sub-objects Sub-objects are structures that are directly linked to individual
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    Analysis Software Manual Chapter 3 – Assays Width. Extension of the sub-object mask The examples below illustrate the effects of these parameters. Overlap treatment. Options are Segment, Segment (slow) and remove. Main object mask Distance 0, Width 1 Distance -5, Width 5 Distance 8, Width 8 When
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    20 Chapter 3 – Assays 3.4 3.4.1 Object Finder Modules Entire Image This is a very simple object definition: the entire image is used as object. You may use this for measurements of integral intensities of your sample, i.e., for each image and each parameter (see. Chapter 3.5, Measurement
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    Analysis Software Manual Chapter 3 – Assays Fill holes within objects. Check this box to fill the object mask in case it contains holes. Minimum/Maximum object size. Check these boxes and adjust the values to apply minimum and maximum size filters to the objects (in order to ignore objects that are
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    22 Chapter 3 – Assays To reduce complexity, the process of finding the right settings is split up into three independent steps. The three settings clusters in the Object Finder menu reflect these three steps. They are traversed from left to right, but you can always jump back and adjust the
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    Analysis Software Manual Chapter 3 – Assays low circle appearing in the images. 3. Play around with the Selectivity slider to get just the strong edges (up) or also the weak edges (down). Try to increase the selectivity as much as possible, thereby removing edges due to noise and artifacts, without
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    24 Chapter 3 – Assays 5. First decide if there may have already been sufficient particles detected in step two (the green ones). If you think so you can skip the closing process by unchecking Close edges, thereby reducing processing time dramatically. All unclosed edges (red) are discarded then.
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    Analysis Software Manual 3.5 Chapter 3 – Assays Measurement Parameters The scan^R Object Analyzer modules in the standard configuration offer a large number of individual parameters that may be measured for each recognized object. To limit memory utilization and save CPU time, only the values for
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    26 Chapter 3 – Assays 3.6 Derived Parameters The Assay Settings Derived Parameters tab allows performing calculations on the Parameters defined in the Measurements list by the use of algebraic expressions (+, -, ×, ÷, sqrt, ^ etc. For a complete list see Appendix 6.4). This is especially useful for
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    Analysis Software Manual Chapter 3 – Assays • MEAN(ID). This operator averages all values of the given parameter ID of the Sub-objects belonging to each individual Main Object that belong to the same Sub-object type. • STDV(ID). This operator calculates the standard deviation of all values of the
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    28 Chapter 3 – Assays execution. Moving the active entry up or down the list by using the arrow buttons changes the order of execution. Module. Select an Image Processor Module from this shortlist. Color channel. Assign a Color channel from the shortlist to the active Image Processor Module.
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    Analysis Software Manual Chapter 3 – Assays Example for small Size filter for background correction. 3.7.2 XY Shift In certain cases it may occur, that image channels are shifted relative to each other in the channel overlay. This is rather often the case if an observation emission filter wheel is
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    30 Chapter 3 – Assays 3.7.4 Cut Image This module allows defining regions of interest inside an image and setting all image parts outside the region to the intensity 0. Different drawing tools are available. Rectangle Define a rectangle by mouse drag. Mouse dragging the center changes its position.
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    Analysis Software Manual 3.8 Chapter 3 – Assays Virtual Channels Virtual channels are image channels that are not created via image acquisition during the execution of a scan. Instead they are a result of post-acquisition image processing and added as new channels to the original image data. These
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    32 Chapter 3 – Assays Module. Select Simple Math from the Module pull-down list to set this module as active entry in the VC Process List. Input Channels list. All color channels of the images are possible Input Channels for the Simple Math module. The channel selected as first Input Channel is
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    Analysis Software Manual Chapter 3 – Assays In this example it is not possible to detect the cytoplasm on a single color channel because the area of the nucleus has very little cytoplasmatic staining: Problem. The detection is incomplete because staining is missing on the nucleus area. Solution.
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    34 Chapter 3 – Assays Show details. Displays the matrix created by the selection of the stains. This matrix is used for the processing of the images. The entries of the matrix can be changed manually. A graphical representation is displayed. Output channels. Select the stains to see the result of
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    Analysis Software Manual Chapter 3 – Assays 9. Click the Get from ROI button. 10. Repeat steps 4 – 7 for the other fluorophores (stains). In order to perform the spectral unmixing the software has to determine the contribution of the fluorescence of different fluorophores to the different color
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    36 Chapter 3 – Assays When clicking on one tracked object in the image now not only the object is highlighted with a green border but also the trace it covered during acquisition will be displayed. With time the object moves from the blue end of the line to the red end. Like in the Population View
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    Analysis Software Manual Chapter 3 – Assays Gallery. it displays a time-gallery of the selected object. Gallery display in time-lapse mode Movie. This opens the menu Trace Movie which allows you to export a movie of a single trace or a complete position. 3.9.1 Tracking Configuration The Tracking
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    38 Chapter 3 – Assays Range (Pixel). Set here the maximum difference that is allowed to change between two frames in order to maintain a track. If the difference exceeds the Range an object will NOT be related to similar objects in the previous and subsequent images of the time-series. OK. Click
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    Analysis Software Manual Chapter 3 – Assays Trace parameters. Well, Position, Trace. You can navigate through the set of curves using these functions and their arrow buttons. Add Range. Click here if you want to analyze just a part of the trace. Two vertical blue bars appear in the graph to define
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    40 Chapter 3 – Assays Parameter. Select the measurement parameter of which the time-curve is to be shown in the graph display. Derivative. Check this option to calculate and display the first derivative of the kinetics time-curve of the selected Parameter. Smoothing (Sigma). This function applies a
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    Analysis Software Manual Chapter 3 – Assays Define. This opens a menu to set the options for curve fitting. As objects might move in or out of the focal plane, or the value entered for Range might be set too small, there might be a large number of short traces. Therefore Lifetime is a useful
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    42 Chapter 3 – Assays 3.9.2.3 Derived Parameters The Trace Parameters Derived Parameters tab allows to perform calculations with the parameters listed in the Definitions list described above by the use of basic algebraic expressions (+, -, ×, ÷, sqrt, ^ etc. For a complete list, see Appendix 6.4).
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    Analysis Software Manual 3.9.3 Chapter 3 – Assays Trace Viewer Open the Trace Viewer by selecting Tracking Show Traces in the main menu. Alternatively you can right-click on one detected object in the image in the Trace View and select Show Trace to see the trace of this individual object. Set the
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    44 Chapter 3 – Assays Parameter. Select parameter of which the time-curve is to be shown in the graph display. Derivative. Check this option to display the first derivative of the curve of the selected Parameter. Smoothing (Sigma). This function applies a smoothing filter on the .time-curve. Set
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    Analysis Software Manual Chapter 4 – Analysis Results 4 Analysis Results This chapter describes how an analysis is executed, how the results can be grouped into object populations and how these can then be analyzed and statistically evaluated. 4.1 Running an Analysis
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    46 Chapter 4 – Analysis Results 4.1 Running an Analysis 1. Execute Scan Open… and select the Experiment Descriptor.xml file in the storage folder of the scan. By doing so the experimental settings are loaded and the first image of the scan (taken at the first position of the first well) will be
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    Analysis Software Manual 4.2 Chapter 4 – Analysis Results Managing Gates scan^R Analysis detects objects in all images acquired during a scan and performs measurements and analyses on each object found. The data can be displayed in form of histograms where each colorcoded data point represents the
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    48 Chapter 4 – Analysis Results Remove (Region Box). This command removes the selected entry from the Regions list and deletes the defined Region. Gates. It lists all gates with their Names and Definitions. Name. Set the name of a Gate. Color. Click into the box to open the dialog window to select
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    Analysis Software Manual Chapter 4 – Analysis Results xCV% (yCV%). Coefficient of variation of the abscissa (ordinate) parameter of all objects in the histogram Applied Gate. This command allows changing the applied gate in the selected histogram. The pulldown menu lists all gates from the Gates
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    50 Chapter 4 – Analysis Results – Objects not belonging to a gate are shown in black. – If there is no majority Gate the Color order of the Gating list will be used (Analysis Assay Gat- ing…; see Chapter 4.2.2, The Gate Manager). The "ranking" can be changed with the arrow buttons in that window. –
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    Analysis Software Manual Chapter 4 – Analysis Results 2. In this example the situation is identical to the first example, but the order has been changed. All Pixels having a majority of G1 will now be shown in a green. All G1 objects also belong to Selected but when the same number of objects
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    52 Chapter 4 – Analysis Results 4.3 Well Results The Well Results window (via Analysis Assay Gating) shows the results of a complete scan analysis. Statistical results of different parameters are listed for gated populations of the individual wells or of groups of wells and are displayed
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    Analysis Software Manual Chapter 4 – Analysis Results CV%. Coefficient of variation of Mean. StdV. Standard deviation of Mean. It is shown in blue in the bar plot. Object Type. Select the object type from the shortlist (Main/Sub-objects). Gate. Select a gate from the shortlist in order to get the
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    54 Chapter 4 – Analysis Results Display Counts / %. This toggle button switches between the listing of total numbers and the percentage of objects. Object type. Select the object type from the pull-down list (Main/Sub-objects). Reference gate. The population of the selected Reference gate is set
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    Analysis Software Manual Chapter 4 – Analysis Results New. Creates a new tab-delimited .txt-file for export. Export file name. Enter the name for the txt file here. The default is Result0*.txt Remove. Removes the selected File and its selection Export. Allows setting set the folder where the
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    56 Chapter 4 – Analysis Results 4.3.4 Export results of individual objects Analysis Export Table: exports the values determined for each detected object to a spread sheet. The values exported depend on the active view. When also sub-objects are detected not only one file is exported but for every
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    Analysis Software Manual Chapter 5 – Example Assay – Step by Step 5 Example Assay – Step by Step This chapter guides you step by step through the setup and execution of an assay and the analysis of the results. 5.1 5.2 5.3 Setting-up and Executing an Assay
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    58 Chapter 5 – Example Assay – Step by Step 5.1 Setting-up and Executing an Assay The example scan which is used here to guide you through the setup and execution of an assay consists of images taken from cells labeled with DAPI to mark the nuclei and with FITC to mark the microtubules. A repair
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    Analysis Software Manual Chapter 5 – Example Assay – Step by Step 6. The first step in Analysis is to select the Background Correction Image processor Module on the Image Processing tab for each of the image channels. Press Adjust and observe in the new window the background in the image before
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    60 Chapter 5 – Example Assay – Step by Step DAPI-labeled nuclei Detected objects 10. In the Parameter tab you can choose which parameters will be calculated during the analysis, i.e. you need to select the measurements that are to be carried out. Typical measurements are area, mean and total
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    Analysis Software Manual Chapter 5 – Example Assay – Step by Step 12. Now you have set up the analysis. Press OK to exit the Assay Settings menu. You can save your assay with Analysis Save assay… 13. Start the analysis by clicking on the Run button. 14. Prepare the histograms in the main window if
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    62 Chapter 5 – Example Assay – Step by Step 2. For further analysis only consider those objects that do not differ significantly in size and shape from the bulk. To do so, use the Region tool and draw a polygon around the most densely populated part of the scatter plot. (Close the region by double
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    Analysis Software Manual Chapter 5 – Example Assay – Step by Step 6. You will observe that the distributions in G1 and G2 look different. In G1 there are only nuclei with a low mean intensity in TexasRed, whereas in G2 there are two populations: one with a lower mean intensity in TexasRed and one
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    64 Chapter 5 – Example Assay – Step by Step You will notice that in the first 8 wells the numbers of active and passive nuclei are almost identical. However in the last 4 wells the number of active cells in G2 is strongly decreased. 9. To export the results either select Export Table in the
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    Analysis Software Manual Chapter 5 – Example Assay – Step by Step 1. Execute Scan Open…, navigate to the folder where the demo data are stored, select the experiment_descriptor.xml file (
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    66 Chapter 5 – Example Assay – Step by Step 7. Confirm the Assay Settings with OK and Run the analysis. 8. After the analysis you can plot the Histograms and Scatterplots as described in Chapter 5.2, Analyzing the Data. 9. The next step after the image segmentation and the extraction of the
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    Analysis Software Manual Chapter 5 – Example Assay – Step by Step 11. Close the menu with OK. The analysis of the curves is performed. In the lower right of the front panel the status bar displays the progress of the analysis. 12. When the analysis is finished you can plot a lifetime histogram in
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    68 Chapter 5 – Example Assay – Step by Step 14. In the second histogram display, plot max(MeanIntensity(GFP)). Right-click in the histogram and select Set Gate Longtraces in order to include only the cells that were detected in all images into the analysis. Draw a rectangle around the datapoints on
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    Analysis Software Manual Chapter 5 – Example Assay – Step by Step 17. As you can see, the green curve shows no mitosis but has a high mean intensity in GFP all the time. (When you select the green curve and observe the corresponding object in the images you see that this is probably an apoptotic
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    70 Chapter 5 – Example Assay – Step by Step
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    Analysis Software Manual Chapter 6 – Appendix 6 Appendix 6.1 6.1.1 6.1.2 6.1.3 6.1.4 6.2 6.3 6.3.1 6.3.2 6.3.3 6.3.4 6.4 File Conversion ...........................................................................................72 Images
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    72 Chapter 6 – Appendix 6.1 File Conversion scan^R Analysis is able to analyze image data acquired with a third party imaging system as long as the data are stored as (monochromatic) 16-bit single TIFF images. The file names must allow the assignment of the images to individual positions, time
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    Analysis Software Manual Chapter 6 – Appendix Image list: Retrieve. This function reads the matching files into the Full file paths list. The arrow buttons allow easy browsing and verification of retrieved image data. 6.1.2 Image Channel Definition If the file names of the third party images
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    74 Chapter 6 – Appendix 6.1.3 Scan Settings Scan settings are used to reconstruct a minimal configuration file for the data conversion. scan^R needs information about the used multi-well plate and the arrangement of the images within the single wells in order to represent the data properly in the
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    Analysis Software Manual 6.2 Chapter 6 – Appendix FCS Export Functionality scan^R is able to export single cell data to ISAC FCS 3.0 Standard files. Please note, that this standard is not fully implemented by most of the programs that specify FCS 3.0 import support. Therefore some settings
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    76 Chapter 6 – Appendix Export Data Type. Here you can choose the data types exported (Integer 16 / Integer 32 / double (64bit floating point) according to FCS 3.0 standard. It is recommended to use the highest precision that is supported by the program intended to import the fcs files. Scale
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    Analysis Software Manual 6.3 6.3.1 Libraries Object Analyzers Library (OAL) Chapter 6 – Appendix 77
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    78 Chapter 6 – Appendix The password-protected Object Analyzers Library can be accessed through Modules ers Library. Object Analyz- It allows managing the set of parameters selectable in the Edit Assay tab Parameters; see Chapter 4.6, Measurement Parameters. Add. Click here to add a new entry to
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    Analysis Software Manual 6.3.4 Chapter 6 – Appendix Virtual Channel Library The password-protected Virtual Channel Library can be accessed through Modules VC Processors… 79
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    80 Chapter 6 – Appendix It allows managing the set of available Virtual Channel algorithms in the module selector on the Edit Assay Virtual Channels tab. Add Module. Click here to add a new entry to the list. Remove Module. Click here remove an entry from the list. 6.4 Valid functions for derived
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    Analysis Software Manual Chapter 6 – Appendix rand( ) Random Number (0 – 1) Produces a floating-point number between 0 and 1 exclusively. rem(x,y) Quotient & Remainder Computes the remainder of x/y, when the quotient is rounded to the nearest integer. si(x) Sine Integral Evaluates the sine integral
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    ® OLYMPUS LIFE SCIENCE EUROPA GMBH Postfach 10 49 08, 20034, Hamburg, Germany Printed in Germany 2008 12 M 1.1.0.6
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