TCSPC position-sensitive

To make multispectral time-resolved fluorescence measurements into a diagnostic tool, the technique must be combined with imaging. Direct imaging techniques by position-sensitive TCSPC are optically easy. They can be used with wide-field illumination and are easily integrated into endoscopes. However, it is difficult to obtain simultaneous spectral and temporal resolution. Several detectors must be used or several images of different wavelength be projected on one detector. True multispectral resolution cannot be obtained this way. 

A few comments should be made about the differences between the TCSPC scanning technique and TCSPC wide-field imaging. The obvious difference is that wide-field imaging by position-sensitive TCSPC imaging does not yield any depth resolution or out-of-focus suppression. Moreover, two-photon excitation cannot be used. Wide-field TCSPC therefore lacks the contrast of the TCSPC scanning technique and is not useful for deep tissue imaging. 

Fig. 3.12 TCSPC system for position-sensitive detection by detectors based on charge division...

Wide-field TCSPC [162, 262] achieves high efficiency and high time resolution. A position-sensitive detector delivers the position and the time of the photons, from which the lifetime image is built up see Sect. 3.5, page 39. For reasons described below, wide-field (or camera) systems are not fully compatible with the scanning microscope. 

TCSPC with two-dimensional position-sensitive detection can be used to acquire time-resolved images with wide-field illumination. The complete sample is illuminated by the laser and a fluorescence image of the sample is projected on the detector. For each photon, the coordinates in the image area and the time in the laser pulse sequence are determined. These values are used to build up the photon distribution over the image coordinates and the time (see Fig. 3.12, page 40). The technique dates back to the 70s [312] and is described in detail in [262]. Lifetime imaging with a TCSPC wide-field system and its application to GFP-DsRed FRET is described in [162]. A spatially one-dimensional lifetime system based on a delay-line MCP is described in [509]. 

To obtain position sensitivity, the single anode can be replaced with an array of individual anode elements [297, 298] see Fig. 6.4. The position of the corresponding photon on the photocathode can be determined by individually detecting the pulses from the anode elements. Multianode PMTs are particularly interesting in conjunction with the multidetector capability of advanced TCSPC techniques. 

An additional push can be expected from new technical developments in TCSPC itself. The largest potential is probably in the development of new detectors. The introduction of direct (wide-field) imaging techniques is clearly hampered by the limited availability of position-sensitive detectors. In addition the selection of multianode PMTs is still very limited, especially for NIR-sensitive versions. Large-area detectors with 64 or more channels may result in considerable improvements in DOT techniques. Single photon APDs with improved timing stability are urgently required for single-molecule spectroscopy and time-resolved microscopy. 

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