Natural Science Forum / Physics / Optics / September 2004

I need to count live cells in a microplate with an inverted
microscope.  I know that DAPI, Hoechst and a host of other dyes and
stains can be utilized.  however, I need to find a dye that is not
toxic, won't interfere with the biology of the cells, and won't
interact with the compounds that I will be treating the cells with.

I am interested in finding ways to count the cells using visible
imaging (I have found problems with discerning junk/debris from the
cells) and fluorescent imaging (concerns about dye-cell interactions
and dye-compound interactions).

Solutions that I have thought of
 1.  Use a transient fluorescent dye which degrades faster than it
could interfere with compounds or cells (The time scale of the
compounds' activity is fairly long (hours to days), so a photo
unstable dye might work).

 2.  Complex algorithm for selectively counting cells and not debris.

 3.  Find a relatively inert dye which won't interfere with either
the cells or the compounds (this can be done, but will involve a large
amount of testing to ensure lack of interference).

Does anyone have any advice, suggestions, experience in this?  I
imagine that their will be tradeoffs in any choice, but I am having a
hard time selecting an approach or a dye.

Thanks,

Jason