Fwd: Live cell imaging with multiphoton confocal

[George McNamara]

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Hi Dave,

Fixed cells, assuming thin and wanting subcellular details and
refractive index 1.515 mounting media (that also suppresses
photobleaching), a new plan apo 63x/1.4 NA lens: point scanning confocal
microscope with galvo-Z or pizeo-Z drive. Possibly in resonant scanner
mode (8000 lines per second, maybe 10 frame averaging per channel).
Sequential scanning (one laser per track). Ideally with GaAsP or hybrid
detector(s) in photon counting mode. Followed by quantitative
deconvolution (hopefully at least Q.D. company has optimized its
algorithm for photon counting confocal and/or multiphoton).

Live cells - depends on cell type and possibly fluorophore(s). Probably
high NA water immersion, glycerol or (if I had one) new silicon oil
immersion lens (speaking of which, I still have not seen papers using
cell culture media R.I. matched to the immersion medium of glycerol or
silicon oil lenses). At one extreme, the following paper gave a clear
win to multiphoton imaging:

Squirrell JM, Wokosin DL, White JG, Bavister BD. Long-term two-photon
fluorescence imaging of mammalian embryos without compromising
viability. Nat Biotechnol. 1999 Aug;17(8):763-7. PubMed PMID: 10429240.

Having just organized a shared instrumentation grant proposal, if I can
change your instrument rules: Applied Precision OMX V4, appropriate lens
for fixed or live cells, "fully loaded" (4 lasers, 4 cameras, with TIRF
... the proposal is for 1 camera but with TIRF).

If someone would buy my core in Miami a Leica STED SMD (single molecule
detection) HyD's instrument (and full set of lenses, and pay for the
service contract, please), I'll get you better data than anyone else on
the list (except those with an identical instrument) for fixed cells. If
the live cells are EGFP-fusion expressing /E coli /cells I'll get you
amazing STED images. If you want to image live hamster embryos, STED is
not going to be tool to use.

Sincerely,

George




-------- Original Message --------
Subject: Live cell imaging with multiphoton confocal
Date: Fri, 22 Apr 2011 13:29:03 -0400
From: David Knecht charter <[hidden email]>
Reply-To: Confocal Microscopy List <[hidden email]>
To: [hidden email]



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I have limited experience with multi-photon confocal microscopes.
I tried imaging live cells with one a while back and was unimpressed with the results as compared to a standard laser scanning instrument.
If you had a new single photon instrument and a new multi-photon instrument sitting side by side in your facility (and I presume some of you do),
which would you choose for imaging  fixed cells or live cells in culture (not trying to go deep into tissue)?
Thanks- Dave

Dr. David Knecht
Department of Molecular and Cell Biology
Co-head Flow Cytometry and Confocal Microscopy Facility
U-3125
91 N. Eagleville Rd.
University of Connecticut
Storrs, CT 06269
860-486-2200
860-486-4331 (fax)