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).
Upon illumination with light of an appropriate color (photon energy), a fluorochrome can absorb that photon and transit from the ground state G to an excited state E. In molecules, these states show a series of sub-states (vibrational states). When excited into one of these vibrational states, the molecule will relax quickly to the lowest of the substates (at low temperatures, e.g. room temperature). From the excited state, the molecule will return to the ground state. The typical time remaining in the excited state depends on the electronic system of the molecule and is called “fluorescence lifetime”. The excited state decays exponentially into the ground state. Under appropriate conditions, the decay is triggered by other photons. This phenomenon is called stimulated emission (see STED super-resolution microscopy). When transiting into the ground state, the energy is released by emission of a photon. In the common fluorescence process (spontaneous emission), the emitted photon’s energy is less than the excitation photon’s energy by the vibrational energy difference. Consequently, there is a Stokes shift from shorter wavelength (excitation) to longer wavelength (emission).
Depending on the molecule, there are also other pathways in which the excited state may be depleted. One of the most prominent non-fluorescence ways is a so-called “inter-system crossing”, ISC. Here the molecule enters a state that is energetically between E and G. For quantum-mechanical reasons this state is called a “triplet state” T, and the decay from here into the ground state is very slow. Consequently, the molecule is “switched off” for the time assuming the triplet state. There are other states, where a fluorochrome is non-fluorescent, especially the most important fluorescent proteins show various kinds of “dark states”, that are not necessarily triplet states. The finding for fluorescence yield reported here will also apply for non-triplet dark states. Irreversible fluorochrome destruction (bleaching) may need a different argumentation.
Triplet states are discussed as one of the main sources for bleaching. Especially excited triplet states tend to correlate with chemical disruption of the molecule. In simulations one can assume for the sake of simplicity that all fluorochromes in triplet states that absorb an additional photon will be irreversibly destroyed (bleached).
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.