TCSPC for FLIM and FRET in - Boston Electronics Corporation

TCSPC ImagingTCSPC Imaging is an advanced TCSPC technique used in conjunction with scanning microscopes.The method builds up a three-dimensional histogram of the photon number over the time and thecoordinates of the scan area. The principle is shown in figure 30. The recording electronics consistsof a time measurement channel, a scanning interface, and a large histogram memory.The time measurement channel contains the usual TCSPC building blocks. Two constant fractiondiscriminators, CFD, receive the single photon pulses from the detector and the reference pulsesfrom the laser. The time-to-amplitude converter, TAC, measures the time from the detection of aphoton to the next laser pulse. This ‘reversed start-stop‘ principle is the key to the processing ofhigh laser repetition rates. The analog-digital converter, ADC, converts the TAC output voltage intoan address for the memory.DetectorTimingStartStopfrom LaserTimeMeasurementCFDTAC ADCCFDtTime within decay curveFrame SyncLine SyncPixel ClockfromMicroscopeCounter YScanningInterfaceCounter XyHistogramMemoryxLocation within scanning areaFig. 30: TCSPC ImagingThe scanning interface is a system of counters. It receives the scan control pulses from themicroscope and determines the current position of the laser beam in the scanning area.When a photon is detected the device determines the time, t, within the fluorescence decay curveand the location of the laser spot within the scanning area, x, y. These values are used to address thehistogram memory. Consequently, in the memory the photon distribution versus t, x, and y buildsup. The result can be interpreted as a stack of images for different times after the excitation pulse oras an array of pixels containing a complete fluorescence decay function each.The implementation of TCSPC imaging into Zeiss microscopes is described in [67-69]. Thedescribed instrument consist of a Zeiss LSM-510 NLO Laser scanning microscope, a femtosecondTi:Sa laser, a fast PMT, and a Becker & Hickl SPC-730 TCSPC imaging module [63]. The setup isshown in fig. 31.27