Hi Adrian,
Fluo-3, Fluo-4, Fura-2 ... the latter needing 340/380 nm excitation (see
Gary Brooker's papers for alternative excitation wavelength pair, and 360
nm is valuable as isobestic point), which you are not going to get on
your confocal. Xu and Webb have published Fura-2 multiphoton absorption
cross-sections, check with your vendors on how - and how much money! - to
get fast two wavelength MP output.
Mark David, Stanford U, at least through 2003 (latest T-cell Calcium ion
paper from his lab that I had methods details for) was still using
Fura-2. If you have access to a ratiometric Fura-2 rig, I encourage
benchmarking your confocal/MP responses against the Fura-2 rig.
I have not read all of the paper below, yet, but the series of
Nano-Cameleon's look great. Ratiometric dynamic range over 10x (vs older
generations ~2x for CaMeleons, GCamp-# etc):
Spontaneous
network activity visualized by ultrasensitive Ca(2+) indicators, yellow
Cameleon-Nano.
Horikawa K, Yamada Y, Matsuda T, Kobayashi K, Hashimoto M, Matsu-ura T,
Miyawaki A, Michikawa T, Mikoshiba K, Nagai T.
Nat Methods. 2010 Sep;7(9):729-32. Epub 2010 Aug 8.PMID:
20693999
If your SP5 has an Argon ion laser with 458 and 514 nm, "clone by
email" the series of Nano-CaMeleons (if you also want a high FRET
control for any reason, ask also for CY11.5). Please note that the 458 nm
laser line may change power unpredictably - potentially resulting in
oscillations due to laser power changes instead of biology. You can to
some extent monitor laser power by turning on the transmitted light
channel. Some apparent power changes could be due to polarization changes
(re: fiber coupling), which might or might not impact your data.
Best wishes,
George
p.s. if you use Fura-2, or any other -AM ester loading dye, be sure to
avoid overloading the cells. This can lead to failure to cleave all the
molecules - Fura-2/AM is bright but not Calcium sensitive, and the AM
cleaving reaction produces formaldehyde in the cytoplasm of your cell.
Very bright Fura-2 cells reminds me of something Harvey Florman told me:
"physiology is the study of dead and dying cells".
At 04:15 AM 9/10/2010, you wrote:
Hi all,
We are just getting started with setting up some calcium studies in mouse
T cells. We've got some dyes to try but there seems to be a very wide
range currently available.
Any recommendations/tips/references would be greatly
appreciated.
Ultimately we want to image on both a confocal (SP5 with 405, Arg, 561
and 633 lasers) and multiphoton.
Regards,
Adrian Smith
Centenary Institute, Sydney, Australia
George McNamara, Ph.D.
Image Core Manager
Analytical Imaging Core Facility
University of Miami, Miller School of Medicine
Miami, FL 33136
305-243-8436 office
http://www.sylvester.org/AICF (Analytical Imaging Core Facility)
http://www.sylvester.org/AICF/pubspectra.zip (the entire 2000+
spectra .xlsx file is in the zip file)
http://home.earthlink.net/~geomcnamara
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