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Dear listserv,

latest Brainbow paper has some useful imaging advice

http://www.nature.com/nmeth/journal/v10/n6/abs/nmeth.2450.html


        Improved tools for the Brainbow toolbox -pp540 - 547

Dawen Cai, Kimberly B Cohen, Tuanlian Luo, Jeff W Lichtman & Joshua R Sanes

doi:10.1038/nmeth.2450


I disagree with a couple of the authors imaging advice on page 544 (most 
of the advice is very good):

Authors: "Fluorophores with overlap in the excitation or emission 
spectra should be imaged sequentially rather than simultaneously to 
minimize fluorescence cross-talk and thereby optimize color separation".

On a typical confocal microscope (ex. Leica SP5, SP8 or Zeiss LSM710, 
780) there are several detectors (ex. 5 on SP5 or SP8) or detector array 
(ex. LSM710 or 780). Therefore, fluorophores that excite well at a given 
excitation wavelength should be imaged simultaneously. I also recommend 
the latest detectors, ex., HyD for leica, GaAsP for Zeiss, photon 
counting mode if available (and TCSPC lifetime if available).

In the "Image processing" paragraph on page 544, the authors suggest 
(for confocal) "slower scanning or averaging of multiple scans".

Slower scanning is more likely to lead to photobleaching (though the 
authors somewhat mitigate this by recommending low laser power). I 
recommend resonant mode (confocal or multiphoton), for users who have a 
resonant scanner.
I have previously suggested here on the listserv that acquiring X number 
of confocal frames without averaging, and then calculating the median 
for each pixel, is a much better approach than averaging. I encourage 
the confocal vendors to implement this, or even better "result value 
selection", based on the noise distribution of the instrument 
(especially the detectors). Even better would be to acquire the raw 
(R.S. mode) frames and clean up in the deconvolution algorithm.
       Speaking of detectors (and tto not leave out cameras): F. Huang, 
... J. Bewersdorf have a nice article showing how well sCMOS can work in 
single molecule localization microscopy ... doi:10.1038/nmeth.2488 
current abstract link is 
http://www.nature.com/nmeth/journal/vaop/ncurrent/abs/nmeth.2488.html

A lot of confocal microscope users are still unaware of simple (and 
free) image processing methods to improve imaging data, such as PiMP, 
http://jcs.biologists.org/content/125/9/2257.long  (I recommend for 
63x/1.4 NA confocal, 30 frame acquisition [current plugin is for single 
plane], filter 1.6, 16-bit output). Contact the authors for the ImageJ 
plugin.



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The Brainbow 3.x's "author's file" feature is at
http://www.nature.com/nmeth/journal/v10/n6/abs/nmeth.2487.html

Zebrafish fans and developmental biologists of all hues should find of 
interest the same group's Zebrabow paper, PMID:

    23757414



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The same issue of Nature Methods has several other microscope imaging 
articles, including:

http://www.nature.com/nmeth/journal/v10/n6/abs/nmeth.2481.html
CLARITY for mapping the nervous system

http://www.nature.com/nmeth/journal/v10/n6/abs/nmeth.2477.html
Mapping brain circuitry with a light microscope


Imaging human connectomes at the macroscale
http://www.nature.com/nmeth/journal/v10/n6/abs/nmeth.2482.html




-- 



George McNamara, Ph.D.
Single Cells Analyst
L.J.N. Cooper Lab
University of Texas M.D. Anderson Cancer Center
Houston, TX 77054