*****
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http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy*****
nice paper showing how to get the most out of sCMOS cameras (they used a
Hamamatsu FLASH4.0, mention using others):
*Huang F*, Hartwich TM, Rivera-Molina FE, Lin Y, Duim WC, Long JJ, Uchil
PD, Myers JR, Baird MA, Mothes W, Davidson MW, Toomre D, *Bewersdorf J*.
Video-rate nanoscopy using sCMOS camera-specific single-molecule
localization algorithms. </pubmed/23708387>Nat Methods. 2013 May 26.
doi: 10.1038/nmeth.2488. [Epub ahead of print] PMID: 23708387
http://www.nature.com/nmeth/journal/vaop/ncurrent/full/nmeth.2488.html Newly developed scientific complementary metal-oxide semiconductor
(sCMOS) cameras have the potential to dramatically accelerate data
acquisition, enlarge the field of view and increase the effective
quantum efficiency in single-molecule switching nanoscopy. However,
sCMOS-intrinsic pixel-dependent readout noise substantially lowers
the localization precision and introduces localization artifacts. We
present algorithms that overcome these limitations and that provide
unbiased, precise localization of single molecules at the
theoretical limit. Using these in combination with a multi-emitter
fitting algorithm, we demonstrate single-molecule localization
super-resolution imaging at rates of up to 32 reconstructed images
per second in fixed and living cells.
**
with respect to SCMOS vs EMCCD, I recommend going to supplemental text
page 10: Supplementary Figure 8: Localization precision and effective photon
comparison for a single bead imaged by sCMOS and EMCCD cameras.
Not that everyone should toss your EMCCDs in the recycle bin quite yet -
Chao et al published a paper that depends on the "EM" in EMCCDs (does
not work with either sCMOS or CCD):
Chao J, Ram S, Ward ES, Ober RJ. Ultrahigh accuracy imaging modality for
super-localization microscopy. </pubmed/23455923> Nat Methods. 2013
Apr;10(4):335-8. doi: 10.1038/nmeth.2396. PMID: 23455923.
http://www.nature.com/nmeth/journal/v10/n4/full/nmeth.2396.html Super-localization microscopy encompasses techniques that depend on
the accurate localization of individual molecules from generally
low-light images. The obtainable localization accuracies, however,
are ultimately limited by the image detector's pixelation and noise.
We present the ultrahigh accuracy imaging modality (UAIM), which
allows users to obtain accuracies approaching the accuracy that is
achievable only in the absence of detector pixelation and noise, and
which we found can experimentally provide a >200% accuracy
improvement over conventional low-light imaging.
Enjoy,
George
--
George McNamara, Ph.D.
Single Cells Analyst
L.J.N. Cooper Lab
University of Texas M.D. Anderson Cancer Center
Houston, TX 77054
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