Posted by
samjlord on
URL: http://confocal-microscopy-list.275.s1.nabble.com/widefield-getting-better-images-than-spinning-disk-tp7585177p7585213.html
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I realize that for fast calcium imaging, my recommendation for a longer exposure
time may not actually help because the fluorescent bursts are so short. And Jeff
Reece's original response was a very good point: Unless your sample is thicker
than 20 um, there should be little change in the detection side using 40 um
pinholes and 10x/0.45NA objective.
Therefore, I suspect the major culprit is the significant reduction in excitation
intensity that you have not been able to compensate for by simply turning up the
illumination power. With a 40 um pinhole disk, I think the fill-factor of 4.2% means
that only 4.2% of the excitation light hits the sample (compared to wide field).
Unless you have very high-power lasers, or you were using them on very low
power for your wide field images, your excitation irradiance will be significantly
lower with the disk in, so your images will appear noisier. The up-side is that your
sample should bleach more slowly. :)
On Sun, 22 May 2016 05:55:14 +0000, Guy Cox <
[hidden email]>
wrote:
>Repeat 100 times:
>
>CONFOCAL ONLY THROWS OUT
>OUT OF FOCUS LIGHT!
My statement that "confocal is designed to throw out light in order to improve
optical slicing" is correct technically and in a practically sense. The pinholes both
block a significant portion of the excitation light and also reject out-of-focus
fluorescence. Both these facts mean that fewer photons hit the camera, giving the
low S/N result that the original poster observed.
I understand that a pinhole does not reject in-focus light, but that doesn't change
the fact that the excitation light is dimmer. And for thicker features in a sample,
rejecting the out-of-focus light actually makes each feature dimmer, because you
are throwing out fluorescence that originated from a different part of the same
feature.
Anyway, I was just trying to assist someone who has never used a spinning disk
before by offering some practical pointers.
-Sam