Analysis Software

Fluorescence Microscopy & Imaging Center

Since the acquisition software used in the FMIC is all Windows-based, and the images are usually saved in a proprietary format of the equipment manufacturer, for analysis the Center has standardized on Windows-based software that is friendly to these formats. However, there are many choices, including options for those of you who use Mac computers back in your lab.

No matter what you do to your images while analyzing, you should keep the raw data unchanged in its original format, and keep copies of it, along with any image manipulations you like, on some other device besides the primary storage medium (e.g. file server "Spot").

Click on one of these links to scroll down to the software used for that application.

Software Applications

Note: Except for the free LSM Image Browser and ImageJ programs, the software applications listed in this section are licensed copies available only on the workstation called "keymaker(http://www.niehs.nih.gov/research/atniehs/core/fmic/equipment.cfm#keymaker)".

General Purpose: Viewing images; contrast adjustment; cropping; exporting to JPG, TIF, or AVI

Windows users

Since most of the data is in Zeiss's proprietary "lsm" format, and the free Zeiss LSM Image Browser(http://www.zeiss.com/C12567BE0045ACF1/ContentsWWWIntern/CAA2EF638EC5F0D3C1256ADF0050E2F1) is relatively useful and easy to use, the FMIC recommends that you have it installed on your computer. You will have to ask your CSP (Computer Support Person) to give "temporary admin privileges" to either you or "FMIC's Jeff(http://www.niehs.nih.gov/research/atniehs/core/fmic/staff.cfm), and then schedule with Jeff to install Image Browser on your computer.

Mac users

Option 1: If you have System X, you can read the Zeiss lsm image files, with all the image acquisition parameters, using the free software ImageJ and the associated LSM Reader plugin(http://rsb.info.nih.gov/ij/plugins/lsm-reader.html) . Your Mac CSP (Computer Support Person) may need to give you "temporary admin privileges" to your Mac so you can install this software, with help from FMIC's Jeff if needed.

Option 2: If you don't have System X, or you are already comfortable with another image manipulation program on your Mac and don't have the need to learn ImageJ, you can use any Windows computer that has Zeiss's LSM Image Browser installed, including the keymaker workstation in F220, to export the images to TIFF format.

Option 2a: If you like option 2 except you would rather be sitting at your Mac instead of a Windows box, you can use "Remote Desktop for Mac" to log on to "inky", the Windows print server in F220, which has the free Zeiss LSM Image Browser installed. Setup of this connection requires help from both your Mac CSP and FMIC's Jeff. (Note: Please use inky only for simple exporting of files, and make sure you log out as soon as you finish with that task!)

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Colocalization

The Zeiss LSM Image Examiner software provides several measures of colocalization(http://www.niehs.nih.gov/research/atniehs/core/fmic/resources.cfm#colocalization) between two (thresholded) fluorescence images:

• Colocalization coefficent, which is the fraction of threshholded pixels in one image that is colocalized with the threshholded pixels of the other image
• Weighted colocalization coefficent, which is the same as the colocalization coefficent but weighted linearly by pixel value, so brighter pixels contribute more to the coefficient
• Overlap coefficient (Manders-derived), which is like the weighted colocalization coefficent, but defined so that it is isotropic with respect to the two images
• Pearson's correlation coefficient

3D deconvolution of Z-stacks

Deconvolution(http://www.niehs.nih.govresearch/atniehs/core/fmic/resources.cfm#deconvolution) can be extremely useful in improving the spatial resolution of microscope images. The FMIC offers two choices:

• The Center has licenses for the Huygens(http://www.svi.nl/) software to work with z-stacks from widefield, single-point confocal, Nipkow disk, and multi-photon excitation microscopes
• Zeiss' LSM Image Examiner will work with confocal and 2-photon only

3D reconstruction of Z-stacks

The Center has a few software options for those that need 3D reconstructions of their z-stacks, usually as rotating animations for PowerPoint.

• Zeiss' LSM Image Examiner
• Molecular Devices' Metamorph
• ImageJ with plugins (free)
• SVI's (Scientific Visualization, Inc.) Huygens -- single frame only; no animation

3D volume measurement

• Zeiss' 3D for LSM

Ratio imaging

Ratio imaging is useful for semi-quantitative measurement of ion concentrations, pH, FRET(http://www.niehs.nih.gov/research/atniehs/core/fmic/resources.cfm#fret), and FAIM(http://www.niehs.nih.gov/research/atniehs/core/fmic/resources.cfm#faim) in microscope images. The FMIC has developed a preferred protocol where the images are optionally smoothed and cropped in LSM Image Examiner, before being processed in Molecular Devices' MetaFluor.

FRET

Typically people are interested in relative changes in FRET, in which case the standard technique for ratio imaging (immediately above) will normally work. When quantitative FRET is required, i.e. when it is desired to calibrate the ratio data to obtain estimates of the fraction of molecules that are FRETting, the FMIC offers these choices:

• Zeiss' LSM Image Examiner
• Molecular Devices' Metamorph
• ImageJ(http://rsb.info.nih.gov/ij/) with plugins (free)

Other advanced image processing

• Molecular Devices' Metamorph
• ImageJ with plugins (free)

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