Brighter Fluorescence by Resonant Scanning

True confocal imaging requires illumination and observation at a single spot at a time. In order to create a two- dimensional image, this spot needs to be scanned over the area that is to be imaged. Scanning is typically performed by two mirrors that can point the spot in x- and y-directions. Like in other scan systems (e.g. raster electron microscopes or 20th century TV tubes), the spot is scanned in lines from left to right (x-direction) and frames from top to bottom (y-direction).

A certain position in the sample (e.g. the position of a fluorochrome), experiences a light pulse each time the beam moves over that position. The spot is ideally Airy-shaped. The duration of illumination, i.e. the time τp taken to pass the position, depends on wavelength and NA, on the actual scan speed in the sample and on the height at which the diffraction pattern crosses the position. Diffraction patterns are usually much larger than fluorochromes (150 … 1,000 nm vs. 2 … 20 nm).

For any image scanned, a fluorochrome will experience a pattern of illumination pulses as shown in Figure 1. The time between the illuminations due to oversampling is 1/fL, where fL indicates the line frequency. Typical line frequencies in true confocal microscopy lie around 1 kHz, but may range from 10 Hz to 2 kHz.

For each image recorded, the flourochrome will experience an illumination pulse pattern as described above. The time between the pulse patterns is governed by the image repetition time 1/fF, either limited by the scan speed (then usually referred to as “frames per second” fps) or intentionally extended, which is typical for time-lapse experiments in physiology. fF indicates the “frame frequency”.

(Comment: For simulations, one can assume rectangular pulses that cross only once per frame. The latter assumption is in contradiction with the requirement of oversampling (Nyquist-Shannon), but does not principally interfere with the effects of triplet accumulations).

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