Many optical visualization techniques applied to the study of living tissues have to work at photon-limiting levels of signal. This is particularly the case for in vivo fluorescence studies. The requirment to work at ultra-low light levels has spurred the development of advanced imaging detectors (such as cooled, charge-coupled devices) with high photon detection efficiencies and microscopes with low-loss optics. In order to make optimum use of such low-light level microscopes, advanced digital electronics is needed together with computer algorithms that can enhance photon-limited images.

Charge coupled detectors (CCDs) are two-dimensional arrays of silicon photodiodes. CCDs are currently the detectors with the highest quantum efficienies (QE). Typically a CCD array has a QE of around 40% but back-thinning can increase the QE to around 80% at 600nm. CCD arrays are imaging devices, not single detectors as are used in laser scanning microscopes. CCDs are extensively used for wide-field, in vivo fluorescence microscopy because of their high-sensitivity and relative simplicity. Computer deconvolution can be used to remove out-of-focus interference and thereby achieve optical sectioning. Such systems have the advantage that no scanning systems are needed and simple mercury arc lamps can be used as light sources instead of lasers. Unfortunately, scientific CCDs arrays are not suitable as a point detector for laser scanning microscopy due to the presence of readout noise. Although a CCD array can accumulate charge generated by photoelectrons almost noiselessly, noise is produced in the act of commutating the charge out to a charge detector. Read-out noise also tends to increase with increasing readout speeds; typically, the best CCD camera systems currently available give around 5 electrons read-out noise per pixel. Recently, a CCD device specifically designed for use as a point detector in a laser-scanning microscope has been described (Pawley et al. 1996 Proceedings of SPIE, 2655: 125) that has lower levels of read-out noise.

Avalanche photodiodes (APDs) are a promising technology for detectors for laser scanning microscopy. They can have QEs approaching that of back-thinned CCDs yet do not suffer form readout noise. Avalanche photodiodes are reverse-biased to near their break-down voltage. Photons cause the generation of electron-hole pairs in the depletion layer which get accelerated by the strong electric field in this region causing the generation of secondary electrons by impact ionization. These in turn become accelerated producing more ionizations giving rise to an avalanche multiplication of electrons. Usually, APDs are run with a reverse bias less than the breakdown value. In this mode they have a gain of 200-300 which is too low for photon counting. However, APDs run in the Geiger mode (with reverse a bias slightly greater than the breakdown value) can give similar gains to a photomultiplier (PMT), around 106, and can be used for photon counting of low level signals. One of the problems associated with APDs used in the Geiger mode is that the critical reverse bias voltage value has to be set accurately, and is also a function of temperature. The chip heats up at high counting rates and accurately thermally tracking the reverse bias voltage is still problematic. Another problem is that detectors with a high photon counting rate capability are rather small (1<mm). Larger area detectors tend to be slower and have lower QEs. The development of APDs as high-performance photodetectors is currently an active field of research. Ultimately Geiger-mode APD detectors may become the detectors of choice for laser scanning microscopy.

Photomultipliers (PMTs) are used almost universally as the detector in laser scanning microscopes. They consist of a vacuum photodiode coupled to an integral electron multiplier chain. PMTs provide high internal gains and can be used for photon counting. Very high counting rates are possible for PMTs optimized for this mode of operation (>100MHz). PMTs do not suffer from readout noise and provide QEs of around 25% at the peak of the spectral response of the photocathode. This value is lower than is obtained by CCDs or APDs but can be somewhat enhanced by optical enhancement.