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Title: How to use STEREOLOGY (optical fractionator) in image stacks (confocal microsc.) Post by: anikapl on Mar 9, 2015 Hi! I'm using stack images after confocal microscopy to measure 2 types of cells in mouse brain after status epilepticus. PV cells (red channel) and VVA cells (green channel). I also want to measure their co-localization. Which is the best way to do the measurement in stereology? Should I use the stacks with all the channels and do all the three measurements there? Or should I use each channel separately?
I really need some advice here. Thank you in advance. Title: Re: How to use STEREOLOGY (optical fractionator) in image stacks (confocal microsc.) Post by: kconnors on Mar 16, 2015 To perform the optical fractionator method of stereology, you should first ensure that you collect your confocal stacks in a systematic random fashion (SRS imaging) within your region of interest. Once the systematic-random stacks are acquired, you can analyze the stacks by either viewing & marking cells that meet the rules to be counted (with an appropriately sized 3D disector) on each channel separately or you can view a merged image at each Z plane. The amount of colocalization, I think, will determine whether it is easier to look at each channel separately or merged. Stereo Investigator is a stereology program that can control confocal microscopes for proper acquisition of image data for stereologic quantification. For full disclosure, I work for MBF Bioscience, but the advice is applicable to whatever stereology software you use. However, the control of confocal microscopes for acquisition of image stacks for stereology is unique to Stereo Investigator.
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