On Wed, 5 Aug 1992, Shanti J. Aggarwal wrote:
 
> 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
 
Elongation occurs, even on a perfectly accurate z-stepper because the x-y
resolution of the confocal exceeds the z resolution. In our hands, the
z-1/2 is maximally about 200nm and the x- or y-1/2 is about 100-150 nm
with a 1.4 n.a. objective (MRC-600, Nikon 60x, 1.4 n.a.). If your samples
are very bright, it is possible to use the gain/offset of the PMT to
increase the resolution, but you sacrifice sensitivity. Otherwise, you can
calculate your x-, y-, and z-1/2's and correct you data set for the
difference. If you do that, be sure to measure the resolutions and more
than one depth under the coverslip and you should attempt to match the
refractive index of the sample you are going to be doing your volumetric
measurements in.
 
Paul Goodwin
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