Posted by
Gavin Rumbaugh on
URL: http://confocal-microscopy-list.275.s1.nabble.com/DsRed-vs-2P-tp5830859p5846805.html
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There is a very nice paper showing that the DsRED and variants are excited by two-photon energy from 660-780nm (depending on the variant;
http://pubs.acs.org/doi/abs/10.1021/jp8087379 ). IN fact, some of these variants are reported to be excited more efficiently in this range versus the >1000nm peak (
http://f1000.com/1147971). The paper was highlighted in Faculty of 1000, and people have have commented that this excitation peak exists when DsRed variants are expressed in neurons in vivo. Personally, i have excited mCHerry in brain slices at 760 and signal to noise was as good as single photon excitation at 560. We are presently experimenting with TdTomato in neurons from brain slice. Will report our observations in the future if people are interested.
Best, Gavin
Gavin Rumbaugh, PhD
Assistant Professor
Department of Neuroscience, #3C2
The Scripps Research Institute
Jupiter, FL 33458
205-306-7096
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From: Confocal Microscopy List [
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Sent: Monday, December 13, 2010 9:04 AM
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Subject: DsRed vs 2P
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Dear list members,
Has anyone any experience with visualizing DsRed using Ti:Saph (2 photon)?
I have problems getting any decent fluorescence signal through between 700 and 1000nm excitation and there is significant bleedthrough
with EGFP. In the end I gave up since the "regular" 1 photon excitation way gave a much better S/N ratio with minimal bleed through.
Any tips? We have an upright Zeiss LSM system and mostly use 40X 1.0 NA water dipping objectives.
Best
Staffan Nystrom
PhD student
MBB / Oncology & Pathology
Karolinska Institutet
Stockholm, Sweden