Pinhole size?? - Confocal Microscopy (Jun/23/2005 )
I have a question...
I am actually looking for the colocalization of two proteins in the cytoplasm using confocal microscopy. Therefore, I am using two different antibodies with two different excitation and emmission wavelengths. The question i have is : Do I have to keep the pinhole size of the two differnt channels the same?? Will this ensure that I am looking at the same region within the cell.
The way I understand it the Pinhole is like the condenser in a camera it allows extra light in or shuts it off. By alterring the pin hole you do not change the field of view at all. What you do have to be careful of however, is that what you are seeing with each filter is infact real and not artifactual by increasing the light through the pin hole.
To do this what you should do is find the optimal Pin hole setting for viewing in the red spectrum and then check using the green filter for spill over. The same should then be completed with optimal Pin hole setting for the green spectrum and checking for spill over in the red.
Hope this helps, I haven't explained it very well.