Re: FISH measurement

Posted by John Oreopoulos on
URL: http://confocal-microscopy-list.275.s1.nabble.com/FISH-measurement-tp3480943p3481245.html

Correct me if I'm wrong, but in order to properly sample the image without aliasing (ie: proper Nyquist sampling), must you not set the lateral pixel and z-step size such that a single diffraction limited spot is sampled 2.3 times in xy and in z as well (bearing in mind that the resolution - counting just the voxels in that case would overestimate the volume, wouldn't it?

If you purposely set the voxel size to by 2 Airy units instead of 1, then you might avoid this problem, but you risk losing spatial resolution and creating aliasing artifacts, right? Perhaps this is okay if you don't care about the loss of spatial resolution?

John Oreopoulos

On 20-Aug-09, at 3:11 PM, JOEL B. SHEFFIELD wrote:

It seems to me that you first have to be sure that each z-slice is independent --that you are not detecting fluorescence from one slice in another.--
Joel

On Thu, Aug 20, 2009 at 2:31 PM, Carl Boswell <[hidden email]> wrote:

Hi all,
I have a user who wants to measure the volume, not intensity, of the FISH-labeled region in a z-series.

Any issues with the following proposal, besides using a histogram to get pixel numbers?

1. Threshold the FISH signals for control and experimental Z-Series
images with identical conditions.
2. Create a histogram for each thresholded image and use total pixel number as
the FISH signal volume.

Thanks,
C

Carl A. Boswell, Ph.D.
Molecular and Cellular Biology
University of Arizona
520-954-7053
FAX [phone]

--


Joel B. Sheffield, Ph.D
Department of Biology
Temple University
Philadelphia, PA 19122
Voice: 215 204 8839
e-mail: [hidden email]
URL:  http://astro.temple.edu/~jbs