Principle of Time-resolved Luminescence Microscopy

Autofiuorescence occurs in a short span time (typieally in the ns range), so that replacement of the fluorescent probes by phosphoreseent stains considerably attenuates this problem if time-resolved (or time-gated) deteetion (TRD) is applied. The microscope is then operated in time domain and detects only the longer time-scale events. The principle of this powerfiil technique is outlined in Fig. 4.2. 

The latter time is too long for microscopy experiments, but integration times of this order of magnimde are common for time-resolved immunoassays, considerably boosting their sensitivity. 

Both delay and acquisition times have to be chosen carefully in order to maximise SNR. For instance, a too long delay time cuts out the most intense emission from the phosphorescent probes and a too long acquisition time leads to noise integration at the end of the probe decay. If the decay is a single exponential function, the phosphorescence signal 

Technically, early time-resolved luminescence microscopes employed two chopper wheels [29] to isolate the excitation and detection phases in a TRLM cycle as demonstrated in Fig. 4.3. The excitation chopper can however be advantageously replaced with a pulsed light source. 

Another point stemming out of Table 4.1 is that, ideally, the delay and acquisition times should be adapted to the decay time of the luminescent probe. This is not quite easy with chopper-fitted microscopes since the delay and acquisition times not only depend on the rotation speed of the chopper but, also, on the number of blades. Optimum working conditions would therefore require changing the chopper wheel, depending on the luminescent stain, which is not very practical. Microscopes are therefore usually fitted with chopper wheels adequate for one class of LLBs (e.g. lanthanide polyaminocarboxylates, with lifetimes around 600-700 ps). There are other ways of continuously modifying the time delay, but they also have their drawbacks (see below). 

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