On Thu, 18 Jul 1996, Natalie James wrote:
>>Hello all,
>>I have a flow chamber to study calcium signalling in endothelial cells under
>>exposure to fluid flow (cell culture medium). The indicator used is Fluo-3.
>>Dye loading of 1.5 um Fluo-3 for 20 min RT is used to give low levels of
>>intracellular dye, in order to minimise buffering of the calcium responses.
>>I have heard that there are potential fixation effects due to the aldehyde
>>degradation products of fluorescein-derived probes which are produced during
>>photobleaching, which occurs during scanning. The original papers on the
>>development of Fluo-3 and its application in living cells (JBC 1989 (264)
>>8171-8 & 8179-84), don't discuss the possibility of this complication (& the
>>illumination was with a xenon lamp rather than a laser).
>>
>>1. Does anyone know whether aldehyde-related fixation is likely at the
>>concentrations of fluo-3 used for calcium measurements?
>>What intracellular concentrations of a fluorescein-derived compounds could
>>yield aldehydes that would give this problem?
>>Does anyone have references discussing this problem?
>>
>>2.In addition this factor was suggested as a possible reason for loss of
>>contractility in experiments involving scanning of smooth muscle cells. Any
>>comments?
On Thu, 18 Jul 1996, Ian Harper wrote:
>I have not personally encountered "fixation" effects in scanned cells
>after loading with Fluo-3. We use 2,5 - 10uM Fluo in cardiac
>myocytes, and we would clearly see if contractility was affected.
>However, I remember Steve Cody showing some examples of contractile
>dysfunction in parts of a cell excessively scanned.........
Thanks Ian,
And I thought you were asleep during that talk :) Whilst working with
David Williams et al., on calcium waves in enzymatically isolated
rat cardiac cells, with Fluo-3 and confocal. We wanted to show that the
fluorescent waves were due to calcium, and not a weird artifact (prior to
any Fluo-3, Ca++ waves in myocyte publications). So we looked at
contracting cells with a calcium INsensitive dye. The dye we choose was
BCECF/AM (a pH sensitive dye). We excited this dye with the 488nm laser
line. However the cells kept "dying" (stopped contracting) as we scanned
them with the laser. If we repeatedly scanned the same line along the
length of the cell (to collect an X-T plot) we found that the line where we
had scanned was "paralysed", while the rest of the cell was still
contracting. This artifact was breifly described in Cody et al. (1993)
Intracellular pH mapping with SNARF-1 and confocal microscopy. I: A
quatitative technique for living tissues and isolated cells. Micron: 24,
573-580.
I shall try and describe the observation so that you can picture it in your
mind. Rat cardiac myocytes are rod shaped cells, aprox. 100 micron long by
25 micron wide. If we continually irradiated the same line along the long
axis of the cell, let's say close to one side of the cell. The cell would
then contract as if a stiff but flexible rod had been inserted along one
side. Instead of simply shortening during contraction, the cell would bend
like a bananna during each contraction! It appeared that the contractile
proteins in irradiated areas had been cross-linked, and so we suspeceted
aldehyde production from the probe. So we started using SNARF-1 instead.
My gut feeling was that it was not just the dye concentration but the
intensity and duration of laser irradiation may have been a major problem.
The good news is, that we did not have this problem with Fluo-3. But it may
be possible given the right circumstances I suppose. Try using more dye and
less laser.
Stephen H. Cody, __ /
Biomedical Confocal Microscopy Research Centre _/ \__/ \
Department of Pharmacology, / \
University of Western Australia, / \
Nedlands WA 6009, \* ____ /
Australia. \_/ \_ _/
email: [log in to unmask] __
Phone: 61 09 346 4569 Fax: 61 09 346 3469 \/
|
