TCSPC scanning

Fig. 5.45 Phantom (left) and images obtained from earliest 10% of the photons centre) and all photons (right) of a TCSPC scan. The glass sphere shows up dimly in the image of the early photons. From [34]...

A laser scanning ophthalmoscope can relatively easily be combined with the TCSPC scanning technique (see Sect. 3.4, page 37). The fluorescence light from the retina is split off by a dichroic mirror and detected by a second PMT. The detection wavelength of the PMTs is selected by filters, FI and F2. The photon pulses from the fluorescence channel PMT are fed into the start input of the TCSPC module. The stop pulses come from the diode laser. 

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 metal-channel design of the R5600 and R7400 results in slight periodical variations of the efficiency and the IRF over the active area, see Fig. 6.41. Consequently, either the entire active area should be illuminated or the position of the illuminated spot should be kept stable. Variations in the position can be a pitfall in TCSPC scanning applications (see Fig. 5.93, page 158). 

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],... 

At present dedicated TCSPC FLIM boards are commercially available. They are compatible with most LSMS and are easily synchronized with the scanning microscope and pulsed laser. These boards, often plug-in cards for PCs, have a lower deadtime than do the conventional TCSPC electronics intended for use in spectroscopy and the memory bottle neck of the histogram-ming memory has been removed [21, 22], Consequently, these dedicated boards provide higher acquisition speeds. 

Dedicated TCSPC electronics is used in all practical TCSPC-FLIM implementations [21, 22]. There are several issues that should be noted. First of all, the lifetime acquisition has to be synchronized with the scanning of the confocal or multiphoton microscope. To this end, the pixel clock and often the line and frame synchronization signals of the scanning microscope are used. 

Both TCSPC and TG benefit from operation in SPC mode. SPC results in little or no noise and a high photon-economy [10]. Therefore, TCSPC and TG are ideal for high spatial and lifetime resolution imaging [24], Both techniques offer high image contrast also on dim samples. However, the dead-time of the detectors and the point scanning character limit the throughput of these systems. 

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). 

Compared with the very limited capacity of the more or less discrete control electronics of classic TCSPC systems, the use of FPGAs led to a breakthrough in functionality. Advanced TCSPC devices use a multidimensional histogramming process. They record the photon density not only as a function of the time in the signal period, but also of other parameters, such as wavelength, spatial coordinates, location within a scanning area, the time from the start of the experiment, or other externally measured variables. It is actually the multidimensionality and flexibility in the data acquisition process that makes new TCSPC techniques really advanced . 

The scan rate in the Scan-Syne-In mode is determined essentially by the sean-ner. Therefore, the scan rate, zoom, or region of interest seleeted in a seanning microscope automatically acts on the TCSPC reeording. Seanning ean simply be started und continued until a suffieient number of photons has been eolleeted. 

With a dead time of 100 ns per TCSPC channel, total useful count rates of the order of 20 MHz can be achieved. All four channels can be used for multidetector operation. The high count rate and the high number of channels make multimodule TCSPC systems exceptionally useful for diffuse optical tomography [34], and high count rate applications in laser scanning microscopy [39]. Details are described under Sect. 5.5, page 97 and Sect. 5.7, page 129. 

Advanced TCSPC devices usually have spectrum-scan modes that reeord several spectra in different time windows simultaneously. The prineiple is shown in Fig. 5.20. The wavelength is scanned, and for each wavelength a fluorescence decay curve is recorded. The counts in the time channels of the deeay eurve are averaged within selectable time intervals. The averaged counts are stored as functions of the wavelength. Several independent time windows can be used simultaneously. Therefore the efficiency is better than for a system that uses a single window discriminator. 

Applications of multiwavelength TCSPC to laser scanning microscopy have been demonstrated in [35, 60]. Spectrally resolved detection in diffuse optical tomography is described in [23]. A multianode MCP PMT and an SPC-330 TCSPC module were used to resolve the luminescence of alkali halides under N, Ar, Kr, and Xe ion irradiation [266]. 

Fig. 536 Continuous-Flow mixer with TCSPC detection. Left Single-point detection with scanning. Right Multipoint detection with a multianode PMT...

The principle of a typical TCSPC-based scanning mammograph is shown in Fig. 5.44. 

In both cases the data acquisition in the TCSPC channels is synchronised with the scanning by clock pulses from the scan controller. It must, however, be taken into account that the length of the lines of the scan varies since the return points of the scan are controlled by the detector overload signals. Therefore, the scan software must store the positions of the return points and the number of pixels between. These positions are used later to adjust the lines horizontally. 

Dual-wavelength TCSPC detection in two-photon laser scanning microscopes is relatively simple [37]. Multispectral TCSPC detection in a two-photon laser scanning microscope requires a suitable relay optics between the objective lens and the polychromator [35, 60]. Details are described under TCSPC Laser Scanning Microscopy . 

To build up lifetime images, the TCSPC module needs scan synchronisation pulses from the scanner. These can be either obtained directly from the scanner or separated from the video signal of the reflection channel. 

The application of an ophthalmic scanning TCSPC system to autofluorescence imaging at the fundus is described in [450, 451, 452, 453, 454]. A typical result is shown in Fig. 5.69. A double-exponential deconvolution was run on the complete pixel array. It delivered a fast lifetime component of the order of T = 400 ps and a... 

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. 

Conventional TCSPC in combination with slow-scan systems has been used in [74, 76]. However, high count rates and high scan rates cannot be achieved with this technique. 

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