In Fluorescence Correlation Spectroscopy experiments, we usually calibrate the dimensions of the focal volume from the diffusion coefficients of well-characterized free dyes (like Alexa-488). Assuming a 3D Gaussian detection profile, one can calculate the waist radius (w_xy) and axial extension (w_z) of the focal volume.
When one changes the pinhole size of the confocal microscope, I am assuming that these dimensions also change, however I am not sure how much should they change and if their aspect ratio, S (where S= w_z/w_xy) also changes.
Hi Joseph,
You have to decompose the problem into 2 parts.
1- The excitation light is focused by the objective into a excitation volume. This volume depends on the numerical aperture of your objective and the index of refraction of your immersion medium.
2- The fluorescence light is emitted from this excitation volume. It is collected by the objective and detected through the pinhole. The detection volume depends both on the size of the pinhole and the numerical aperture of the objective.
These volumes can be directly deduced from the excitation PSF and the detection PSF. PSF = Point Spread Function. It is the response of your optical system to a sub-resolution object. That's why you use special dyes to calibrate your system. These dyes are sub resolution and allow to measure your system's PSF.
Basically, the more you increase the size of the pinhole, the larger is the detection volume (in all directions with the same ratio between x,y and z). But it can't theoritically never be greater than the excitation volume (theoritically...).
As a consequence, if you increase the size of the pinhole, it seems the signal is increased whereas the resolution will decrease.
For more info you can have look on this page : http://www.svi.nl/PinholeAndResolution
Hi Joseph,
You have to decompose the problem into 2 parts.
1- The excitation light is focused by the objective into a excitation volume. This volume depends on the numerical aperture of your objective and the index of refraction of your immersion medium.
2- The fluorescence light is emitted from this excitation volume. It is collected by the objective and detected through the pinhole. The detection volume depends both on the size of the pinhole and the numerical aperture of the objective.
These volumes can be directly deduced from the excitation PSF and the detection PSF. PSF = Point Spread Function. It is the response of your optical system to a sub-resolution object. That's why you use special dyes to calibrate your system. These dyes are sub resolution and allow to measure your system's PSF.
Basically, the more you increase the size of the pinhole, the larger is the detection volume (in all directions with the same ratio between x,y and z). But it can't theoritically never be greater than the excitation volume (theoritically...).
As a consequence, if you increase the size of the pinhole, it seems the signal is increased whereas the resolution will decrease.
For more info you can have look on this page : http://www.svi.nl/PinholeAndResolution
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