TCSPC wide-field imaging

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. 

The benefit of TCSPC wide-field imaging is that it can be easily adapted to almost any microscope or other optical system. It may also be a solution for samples that preclude, for whatever reason, scanning by a laser spot of high power density. 

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]. 

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. 

Lifetime imaging can be implemented both in wide field and in scanning microscopes such as confocal microscopes and two-photon excitation microscopes. The most common implementations in time-domain fluorescence lifetime imaging microscopy (FLIM) are based on TCSPC [8, 9] and time-gating (TG) [2, 10],... 

In wide field microscopy, spatial information of the entire image is acquired simultaneously thus providing comparatively short acquisition times compared with scanning microscopy implementations. Combining TCSPC with wide field microscopy is not straightforward. However, a four quadrant anode multichannel plate (MCP) has been used for time- and space-correlated SPC experiments [25, 26]. This detector has excellent timing properties that make it very suitable for FLIM. Unfortunately, it can be operated only at low count-rates ( 105-106 Hz) therefore, it requires comparatively long acquisition times (minutes). 

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. 

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. 

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