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Hi,

I have a couple of examples where cellular function was (as far as we
could tell) unaffected by photobleaching of a fluorophore.

We were interested in the lumenal continuity of mitochondria. The
specific question was "is the perinuclear aggregation of mitochondria
lumenally continuous?". Cells expressing mitochondrially targetted DsRed
  were photobleached in the perinuclear region. A small (5-10 um) are
was bleached and we waited for fluorescence recovery. There was very
little after 20 minutes, and only a little after 60 min, i.e. the
mitohchondria are not lumenally continuous. We then added the
potentiometric dye TMRE to see if the mitochondria in the photobleached
region were depolorised. The mitochondria took up the TMRE. Here's the
figure from the article (should be freely available):
http://jeb.biologists.org/cgi/content/full/206/12/1993/FIG2

We also tried to use photodamage to knock out GFP-tagged IP3-receptors.
GFP-IP3R cells were loaded with Fura2 and put on a confocal scope that
had a video imaging system bolted on another port. The cell was treated
with histamine to determine the basal calcium response.
Then, one half of a cell expressing GFP-IP3R was then phhotobleached
with the confocal in an attmept to knock out the IP3R (CALI?). The cell
was then immediately treated with a low dose of histamine and the
calcium signal was monitored with Fura2. Unfortunalty the cells response
to histamine was unaffected by the photobleaching of the IP3R!

While we didn't exhaustively investigate the health of the cells, in
these two cases the cells seemed to tolerate the photobleaching
protocol. However, these were HeLa cells and I think you could probably
stamp on them and they wouldn't mind...

Tony




Kevin Braeckmans wrote:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> Dear all,
>
> in the discussion on FRAP last week, James Pawley rightly expressed his
> concern, both on and off list, about damage to live cells during the
> bleach phase in FRAP experiments. As we don't have any practical
> experience as yet in our lab on FRAP on live cells, is there anyone else
> who can comment on that from experience?
>
>
> JP also asked to give some numbers about actual light doses in the
> bleach phase of a FRAP experiment. I'll give it a try:
>
> If P is the power (W) of your laser beam at the sample, and r the radius
> (m) of the diffraction limited spot in the focal plane, the local
> (average) irradiance is H = P/(pi*r^2), with units W/m^2 or J/(s*m^2).
> Hence, the amount of energy per unit of area is H*t, where t is the time
> of illumination.
>
> The bleach time in a FRAP experiment is usually about 1% of the
> characteritic lateral diffusion time: tau = R^2/(4*D), where R is the
> radius of the bleached area and D the diffusion coefficient.
>
> Now, there are roughly speaking two types of bleach methods: 1) with a
> stationary laser beam (spot photobleaching) or 2) with a scanning laser
> beam on a confocal microscope. An example:
>
> 1) spot photobleaching: the radius of the spot is approx. 1 micron, so
> assuming a typical diffusion coefficient of 10 mum^2/s,  the
> characteristic recovery time is tau = 25 ms and hence the bleach time
> approx. 0.25 ms. For a typical bleach power of 1mW the total energy
> recieved per unit area is therefore 1mW/(pi*1mum^2)*0.25ms = 80 kJ/m^2.
>
> 2) scanning laser beam: To calculate the total amount of light received
> in this case, the calculation is a little more complicated because it
> now depends on the scanning speed and the distance between the adjacent
> scanning lines. So in stead of the formula H*t, one now needs to use
> H/(v*dy), where v is the scanning speed of the laser beam and dy the
> distance between the adjacent scanning lines (see our article on FRAP
> for details). v and dy depend on the zoom setting of course, but will be
> typically in the order of: v = 0.1 m/s and dy = 0.2 mum. Hence, the
> total amount of energy per unit area is: 1mW/(pi*1mum^2)/(0.1m/s*0.2mum)
> = 6 J/m^2.
>
> As this little calculation shows, it is much more likely to inflict
> damage to your live cell when doing spot photobleaching, compared to
> scanning photobleaching. Luckily, hardly anyone is still doing spot
> photobleaching experiments I guess.
>
> As a final note, those numbers don't tell anything about the actual
> damage to a live cell of course, they're just quantities of energy.  To
> relate them to actual damage to a particular structure in the cell, one
> would have to take the absorption cross section (wavelength dependent)
> into account. Does anyone have any data on that?
>
> Best regards,
>
> Kevin
>
>
> Kevin Braeckmans, Ph.D.
> Lab. General Biochemistry and Physical Pharmacy
> Ghent University
> Belgium

--
Tony Collins, Ph.D.
Facility Manager
Wright Cell Imaging Facility
Toronto Western Research Institute
13-407 McLaughlin Pavilion
399 Bathurst Street
Toronto, ON. M5T 2S8
tel. (416) 603 5367 fax: (416) 603 5745
http://www.uhnresearch.ca/wcif