The presence of a pinhole helps in rejecting out of plane fluorescence and thus helps in collecting information voxel by voxel which gives us the much needed 3-D information for which a confocal microscope is famous for. It is this aspect which makes a confocal expensive. Without this feature the confocal is as good as an ordinary fluorescence microscope, which comes at a much lower cost. Why do you want to a do a job which a less expensive microscope can do with an expensive setup like a confocal.

It is the true that removing the pinhole leads to increases in S/N but this comes at a cost. The cost being the loss of 3-D resolution.

Hi Ravi,

Thanks for responding. I am doing this to increase the photon count rate as well as S/N ratio. We are trying to measure a average protein folding transition path time, where I cannot get the desired count rate if the pinhole is present. So I am trying to remove the pinhole and place a detector with a smaller diameter (the the beam) as a spatial filter.

Now the question is not about the cost of the set up as I already have the home-build FCS in the lab and that system is well functional. I can remove the pinhole and re align the whole setup to its max count. Before doing that, I am trying to find someone who could give some insight about the this.

If I remove the pinhole will it really that help the count rate or the effect will be something in reverse?

Do you know anybody who did it?

If spatial resolution is not important to you then you can go ahead with your plan. It will not have any effect.

But your answer reveals that even spatial resolution is important to you.

Now your idea of removing the pin-hole and replacing it with a small diameter detector is a good one. But my personal feeling is that you are not going to gain much. Both S/N and photon throughput, both will decrease. How much will it decrease and is it better than the previous arrangement with the pinhole will depend on the diameter of the active area of the detector and alignment issues.