The point spread function of a confocal fluorescence microscope defines its performance capability, describing how an infinitely small point object is imaged onto the detector. Diffraction inherently spreads the image of such a point (see Sec. A.3). Additional aberrations from the microscope objective or the alignment of the microscope will induce further spreading, and hence decrease the attainable resolution of the instrument. Vice versa, measurement of the PSF of a confocal microscope provides information about its performance. Such a measurement should be done regularly to ensure that the instrument maintains optimal performance over the course of time.
The PSF of a confocal fluorescence microscope can be measured by imaging a very small fluorescing point object, which mimics an infinitely small point source. To this end, fluorescing beads of various sizes are commercially available. Only when the beads are significantly smaller than the PSF can the influence of the finite bead size be ignored and the recorded image directly represents the microscope PSF. When the bead is of similar size to or larger than the instrument's PSF, the image is a convolution of the PSF with the bead and deconvolution (see Sec. 5.2) is required to obtain the PSF. On the other hand, the smaller the bead, the less signal will be detected, decreasing the SNR of the measurement.
What SNR is required? Suppose one wants to measure the PSF of a confocal fluorescence microscope with sufficient SNR to visualize the first Airy disk of the lateral distribution. From theory, we know that the intensity of this first sidelobe of the distribution is 0.09% of the main peak. Thus, with a dark count of, say, 300, a signal level of 106 counts is required. That implies a 20-bit (220) detector. Most confocal microscopes only have a 12-bit (and often only 8-bit) detector. Thus the signal must be averaged over multiple scans of the same bead, or over the signal measured from a number of different beads.
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