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All of our Carl Zeiss confocal microscopes and the fluorescence correlation spectrometer use the Carl Zeiss AIM software. The software controls the hardware and has powerful image processing capabilities, including 3D visualization. The functionality can be extended using macros.
All images are saved as *.lsm files and are typically accessed through a *mdb Microsoft Access database. The imaging parameters are stored within the image file as well as in the database. Images not associated with a database can be opened using the "File → Import" command. The images can be exported in a variety of file formats, including *.tiff, *.jpg and *.avi. Because exported images lose all the imaging specific information, one should always retain original files in addition to exported images.
The software can be used in online mode at the microscopes and offline at any Windows 2000 computer. To run the software a licence is required. A free version called "Image Browser" with reduced functionality is available (see links). Although the Carl Zeiss Axiovision software uses the different *.zvi file format, confocal images can be imported or exported into AxioVision and vice versa. The free AxioVision software package is called "AxioVision LE".
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Stowers Links:
Adv. Instr. & Physics: Confocal Microscopy
Adv. Instr. & Physics: Two-Photon Microscopy
Adv. Instr. & Physics: Fluorescence Correlation Spectroscopy
Image Center: Instrumentation
Download Carl Zeiss LSM Image Browser
Download AxioVision LE
Literature:
Manual Carl Zeiss LSM 510 META Rel. 3.5
Manual Carl Zeiss LSM 5 PASCAL Rel. 3.5
Manual Carl Zeiss ConfoCor 3
Tutorial Colocalization
Description *.lsm File Format Rel. 3.2
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The functionality of the AIM software can be extended using macros. They can be accessed using the "Macro" menu. The assignment is user specific, thus macros might be installed at the system but not be visible for the individual users. Use the "Macro → Macro..." command to add specific macros to your login. Usually macros can be found in the folder C:AIM/Macros. Make sure to use macros with the same version number as the AIM software you are using. Additional macros can be downloaded from the Carl Zeiss Web page or the Carl Zeiss ftp server. For ftp access the username is the serial number of a Carl Zeiss confocal microscope and the password is the number of the associated dongle.
The easiest way to write macros is to use the "Visual Macro Editor" at "Macro → Visual Macro". A macro is assembled by dragging building blocks into "Program flow" or "Data flow" cells. The blocks are connected by red program flow or blue data flow connections. After the selection of a block, function specific parameters are accessible. Often they can be read back (copied) from associated control windows. Most of the parameters can be associated with variables and mathematical expressions.
More sophisticated macros are written in Visual Basics for Applications (VBA).
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Web Links:
Carl Zeiss: Macros
Carl Zeiss: LSM FTP Server (password required)
Wikipedia: Dongle
Literature:
Manual Macros Download PDF
Instruction "How to access FTP server"
Description LSM 5 Macro Interface Rel. 3.2
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Our fluorescence correlation spectrometer Carl Zeiss ConfoCor 3 uses software based on the AIM confocal microscopy software with FCS specific additions. It controls the hardware and allows fitting of correlation functions.
FCS Viewer for Raw-Data Files
The software can save raw data files describing the arrival time of each detected photon. The file format was changed when upgrading from software version 3.2 to 4.0. We have written a program to inspect the raw data software version 3.2.
Bug Fix for FCS Data Copy
Within the Carl Zeiss ConfoCor 3 software, measurement results can be copied between different windows. After saving and opening the data file, a bug in the software prevents the display of the data. This small piece of software fixes the saved data file.
ImageJ Plugins for Photon Counting Histogram and Correlation Analysis
These plugins and libraries allow for fitting of one and two dimensional photon counting histograms with models of molecular brightness. In addition there are plugins for simulation of fluorescence fluctuation data and global fitting of correlation curves. The plugins are zipped with documentation for installation and use here.
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Stowers Links:
Adv. Instr. & Physics: Fluorescence Correlation Spectroscopy
Image Center: LSM-510-DEV (FCS ConfoCor 3)
BioInformatics:Earl Glynn
FCS Viewer Utility: Description
FCS Viewer Utility: Download
FCSFIX: Description
FCSFIX: Download
Literature:
Manual Carl Zeiss ConfoCor 3
Description ConfoCor Raw Data Format Rel. 3.2
Description ConfoCor Raw Data Format Rel. 4.0
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Mathematica is a powerful tool for symbolic and numeric computation. At the Stowers Institute we have licenses for Mathematica and webMathematica.
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Stowers Links:
Stowers Research Wiki: Mathematica
WebMathematica: Start Page
Stowers Research Wiki: WebMathematica
Web Links:
Wolfram Research: Mathematica
Wolfram Research: webMathematica
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ImageJ is the Java version of the popular free NIH Image software. It is platform independent. The source code is available. Its functionality can be extended using easy to write macros and plugins. There is a huge user community and for almost all microscope image processing needs solutions are available.
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Web Links:
NIH: ImageJ download and documentation
Gabriel Landini: a collection of very useful plugins
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IDL is a programming language optimized for image processing. Typically programs run in a programming environment, but it is possible to write stand-alone applications.
Users of IDL at the Stowers Institute can discuss programming questions with IDL specialists using the Stowers Portal at ITT (the producer of IDL). To get access please contact Earl Glynn.
At the Stowers Institute we have several licenses.
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Stowers Links:
Stowers Research Wiki: IDL
Bioinformatics: IDL resources
Earl Glynn's IDL resources
Web Links:
ITT: IDL Home Page
ITT: Stowers IDL Portal
David Fanning : Coyote's Guide to IDL Programming
Markwardt: IDL Library
Literature:
Getting Started with IDL (from here you can link to other IDL manuals)
Using IDL (from here you can link to other IDL manuals)
Image Processing in IDL (from here you can link to other IDL manuals)
IDL Quick Reference (from here you can link to other IDL manuals)
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Imaris allows visualization of 3D data sets in a real time interactive manner. This allows you to quickly make visual assessments of your experiments in 3D and 4D and discover relationships otherwise hidden by the limits of 2D analysis. In addition to unrivalled rendering quality, speed, precision and interactivity, Imaris also has a large variety of segmentation options. Imaris provides you with effective tools to segment challenging datasets to identify, separate, and visualize individual objects their associated statistics.
The “Snapshot” command is a convenient way to export any image view in a standard *.tif format that can be used for presentations and publications. The “Key Frame Animator” allows for creation of even the most complex movies from 3D and 4D scenes and allows those movies to be saved as a QuickTime or *.avi file.
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Stowers Links:
Stowers Institute Home Page
Stowers Institute Core Facilities
Web Links:
Imaris on the Bitplane website
Imaris Webinar Training
Imaris Batch
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Assay Builder is a software package for analyzing high content fluorescence assays. The full power of the software is available when experiments use multiple color labels that can be used to segment different regions of cells.
There are 5 unique analysis modes within Assay Builder ranging from general to specific cell events:
- Physiology Analyst tracks all membrane events except membrane trafficking, cell cycle, and colocalization.
- Morphology Analyst tracks all morphology events except cell motility.
- Membrane Analyst tracks colocalization and membrane trafficking.
- Cell Cycle Analyst tracks cell cycle and cell regulation.
- Motility Analyst tracks cell motility.
Once an assay has been setup using one of the above analysts, data can be grouped within three hierarchical levels: well level, field level, and object level. Assay Builder provides interactive graphs of data within each data level. All graphs provide the ability to add custom fields and drill down to individual data points. Filters can be set to either include or exclude specific regions of data, ensuring that the analysis includes only those objects of interest.
Assay builder is a powerful tool for high content fluorescence assays, but it should only be used for that purpose. It will become quite cumbersome if there are only a few images to analyze. But if there are hundreds of images to process, Assay Builder could be a great help in the analysis process.
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Stowers Links:
Stowers Institute Home Page
Stowers Institute Core Facilities
Web Links:
Carl Zeiss: Assay Builder
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AxioVision is a powerful and flexible software package for capturing, archiving and preparing images in microscope systems. In particular,
AxioVision administers the imaging of wide field motorized microscopes from Zeiss. Apart from several features that controls the microscope hardware, AxioVision also has built-in packages for microscope image processing and image analysis. One of the main advantage in using AxioVision for microscope imaging is that we can store the desired microscope parameters, ensuring the repeatability of the experiments and reusability of the images obtained. Further, scaling factors can be recalled during the analysis, thus increasing the speed at which measurements can be performed. On the image processing front, AxioVision offers a collection of features such as,
1. contrast, brightness and color control
2. noise suppression, smoothing, and contour enhancement
3. improved sharpness and detail emphasis
4. correction of illumination conditions and white balance control.
Further, one can perform interactive measurements, such as length, area, and angles with the option of optimizing the workflow with the help of measurement wizard. Also, the measurement data can be exported to spreadsheets such as Microsoft Excel. In addition, AxioVision can perform many complex tasks and it can be customized so that we only see the information and controls that are needed. Special macros in VBA (Visual Basic) can be written to perform additional tasks.
For further information on the features available, please download and view the AxioVision brochure available at:
http://www.zeiss.com/4125681F004E2140/EmbedTitelIntern/AxioVisionforMaterials/$File/AxioVision_Materials.pdf
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Stowers Links:
Stowers Institute Home Page
Stowers Institute Core Facilities
Web Links:
Carl Zeiss: Axiovision
Literature:
Carl Zeiss: Axiovision Takeoff Guide
Quick Start Axiovision
Image Capture Procedure
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