I have a question regarding calibration of z-step using fluorescent latex
beads. Although the beads are sherical I get an elongated Phi-Z scan. Part of
it is due to the blooming of fluorescence. How can I correct for it. Are there
any other suggestions for calibrating the z-steps? Normally it wont matter much
but I am doing some volumetric calculations an need to know very precisely of
the limitations. Also in these situations what isthe best way to decide the top
and the bottom of the specimen. I have no problem deciding that via a Phi-z scan
on non fluorescent samples.
Shanti