With the concepts of pulsed illumination in a scanned image and the fluorescence process that involves a triplet state (which is most common for standard fluorochromes), we can now understand the findings with fast true confocal scanning systems (compared to lower speed scanners) that suggested a better fluorescence signal and less bleaching when employing resonant scanning systems.
If no triplets were present, the amount of fluorescence photons would depend on the illumination intensity and the quantum yield of the fluorophore. When the illumination is switched on, the emission would increase (the speed of increase depending on fluorescence lifetime) and stay at a constant level. As soon as we allow some excited molecules to move away into the triplet state, they are no longer available for generating fluorescence photons. Consequently, the emission would decrease. We could even assume a situation where all molecules are in the triplet state, and only very occasionally, when a triplet state relaxes to the ground state, the fluorescence machinery could work for a short moment, emitting some photons and stopping as soon as the molecule undergoes intersystem transition and stays dark in the triplet state. This extreme situation is the basis of a modern super-resolution technique: ground state depletion (GSD). But even under less extreme conditions, the triplet state reduces the amount of fluorochromes ready to emit. And the relaxation from T to G is slow (depending on the molecules’ quantum parameters).
If we stop illumination, fluorescence will decrease (showing the actual fluorescence decay time). And gradually, at a much slower rate, the triplet-state molecules will also return to the ground state, when they are ready to generate fluorescence again. Obviously, it matters whether we try to get the emission in one single, long illumination (triplet states reduce the intensity), or in a couple of smaller doses with some time to relax in between (still assuming the total illumination dose is constant!) If the breaks in the latter case are in the range of the triplet relaxation (R) or longer, we will collect more photons at the end. This is identical with brighter imaging at equivalent illumination doses.
Avoiding the triplet state has an additional benefit: the sample will bleach less. As pointed out, a great part of bleaching is due to excitation of triplet states. If we can avoid accumulation of those states, the molecules are at less risk of photochemical disintegration.
- Low line frequencies increase the recovery time between illumination pulses but also increase the illumination pulse time, as the spot moves more slowly over the sample. Usually, this slower motion increases the triplet concentration. Only very low intensities can avoid this effect.
- High line frequencies shorten the time between illumination pulses, which increases triplet accumulation. On the other hand, the illumination time per pulse is shorter and consequently fewer triplets are accumulated.
- If the same dose of light is applied in small doses by multiplying the frequency n times and accumulating or averaging n times, the total fluorescence will increase if the decay time is not significantly longer than the break between the pulses. Here, the resonant scanner is most beneficial.
- If the triplet decay is slower than the line frequency, interlaced oversampling will improve the signal. Here, subsequent frames collect information from otherwise overlapping lines. E.g. first scan every 5th line, 2nd scan every 5th + 1 line … and so forth. The allowed time for triplet recovery between illumination pulses in this case is ruled by the frame frequency fF.
- In any case, it is advisable to use short illumination pulses, i.e. fast line frequencies. A resonant scanning system (currently, galvanometric optical scanner devices provide up to 20 kHz) is therefore a general option for improving signal-to-noise and reducing photobleaching and phototoxicity.