Confocal Microscopy Principle
—— RA note to self
“Confocal” refers to both illumination and detection are focused to the same spot. The foci coincide. To achieve this, confocal microscopy have a structure called “pinhole”. There are 2 pinholes: a pinhole between light source and sample, generating a tiny light spot to avoid unnecessary emission. Another pinhole (the detection pinhole) is located between sample and detector which blocks any out-of-focus light from reaching the detector (shown in the figure below).
Therefore, the detection pinhole can also be called “spatial filter” or “optical knife”.
How big or small should a pinhole be?
The inner part of the diffraction limited feature of the pinhole is called “Airy disc”. Generally, emission light transmit through Airy disc so the size of the pinhole is determined by it. The diameter in this case is referred to as “Airy Unit, AU”. Airy Unit depends on the wavelength (color), the objective lens (numerical aperture NA and magnification) and the internal magnification of the optical elements in the microscope. Usually, 1 AU is recommended. You can also choose smaller pinhole to increase resolution, sharpen the image or bigger pinhole to increase the intensity(but the noise might increase as well, not recommended).
By this mean, the sample can be scanned by the tiny light spot one-by-one and then the tiny spot will be combined to form a high-resolution image.
