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Guy,

Did you have any problems with autofluorescence in the mitochondria?
I've got a user coming in who is having that issue, and we're looking
for the simplest way to quench the autofluorescence. This is on
muscle formalin fixed after cryosectioning, and stained with
fluorochromes attached to mtDNA probes.
The thoughts so far are NaBH4, eliminating the formamide from the
steps making the DNA probes, and getting rid of the formalin fixation
step.
Any thoughts?
Thanks.

Phil

>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
>We did something similar with neurons - comparing viability
>between confocal and multiphoton for mitochondrial membrane
>potential imaging.
>
>Dedov, B. Roufogalis & G.C. Cox, 2001.  Visualisation of mitochondria in
>living neurons with single and two-photon fluorescence laser microscopy.
>Micron 32, 653-660
>
>                                                 Guy
>
>
>>Dear list
>>
>>I’m trying to design an experiment to see if confocal imaging is harmful to
>>chondrocytes in culture. Has anyone done this sort of thing with other cell
>>types before? any suggestions?
>>the only related article i have found imaged cultured cells in combination
>>with stains for extended periods of time (1hr). the maximum laser power used
>>here was only 30uW using a x20/0.75NA
>>
>>Knight, M.M., et al., Live cell imaging using confocal microscopy induces
>>intracellular calcium transients and cell death. Am J Physiol Cell Physiol,
>>2003. 284(4): p. C1083-C1089.
>>
>>
>>I was thinking of translating the culture slide in the Y direction while
>>only scanning the X thus irradiating a larger area so biochemical changes
>>will be easier to detect.
>>
>>Is there a practical/mathematical way of determining the focused spot
>>diameter to obtain the laser power density (mW/mm2) using NA of a lens,
>>which I imagine would be the critical factor for cell damage/death?
>>
>>Does total energy irradiated (J/mm2) onto the cells have any bearing on
>>cytotoxicity? i.e. repeated scans of the same area (is time between scans a
>>factor?) or a slower raster scan at the same power density?
>>
>>Thank you in advance
>>Dan
>>
>>
>>Daniel Smolinski
>>School of Mechanical Engineering
>>University of Western Australia
>>MDBP MO50
>>35 Stirling Highway
>>CRAWLEY WA 6009
>>Australia
>>
>>
>>T:   +61 08 9380 3609
>>F:   +61 08 9380 1024
>>E:   [log in to unmask]
>
>
>Assoc. Prof. Guy Cox,                 ooOOOOOOoo
>E.M. Unit, F09            #       oOOOO  |  |  OOOOo       #
>University of Sydney     ###    OOO|  |  |  |  |  |OOO    ###
>NSW 2006, Australia      ###  OOO  |  |  |  |  |  |  OOO  ###
>Ph:  02 9351 3176        ### OO |  |  |  |  |  |  |  | OO ###
>Fax: 02 9351 7682       #####   |  |  |  |  |  |  |  |   #####
>                       ==#####============================#####==
>http://www.guycox.net   #####                            #####
>http://www.guycox.com ~~#####~~~~~~~~~~~~~~~~~~~~~~~~~~~~#####~~

--
Philip Oshel
Supervisor, BBPIC microscopy facility
Department of Animal Sciences
University of Wisconsin
1675 Observatory Drive
Madison,  WI  53706 - 1284
voice: (608) 263-4162
fax: (608) 262-5157 (dept. fax)