TCSPC for FLIM and FRET in - Boston Electronics Corporation

Other TCSPC TechniquesApplication of TCSPC to diffusion in cellsDiffusion constants in cells are usually determined byfluorescence correlation (FCS) techniques. Recordingan FCS function requires to detect the fluorescenceintensity in a fixed spot of the sample and to calculatethe autocorrelation function of the intensity or of thephoton detection times [14,15,16]. Although FCS isnot an imaging technique it is often used incombination with imaging to define the location in acell where an FCS measurement has to be run.Generally, the TCSPC technique is not only able torecord time-resolved images in laser scanningmicroscopes, but also to recorded FCS data. An FCSmeasurement can even be combined with a singlepoint lifetime measurement. The multi-detectortechnique described above for TCSPC imaging is alsoavailable for FCS measurements. With theintroduction of the SPC-830 it is not longer necessaryto use independent modules for imaging and FCS. TheSPC-830 works for imaging and FCS recording aswell.FCS data recording does not build up histograms asthe TCSPC imaging techniques do. Instead, it recordsthe full information about each photon. Each entrycontains the time of the photon in the laser pulseps time from TAC / ADCmicro timeresolution 25 pssequence, the time from the start of the experiment, and the detector channel. The data structure isshown in Fig. 40. For each detector an individual correlation spectrum and a fluorescence decaycurve can be calculated. An instrument like this was used to detect and identify single molecules ona substrate. By using four wavelength channels and a piezo scanning stage different molecules couldbe identified in a time of the order of 1 ms [17]. Moreover, data from different detectors can becross-correlated. By detecting different chromophores in different wavelength intervals crosscorrelationcan show whether the molecules of both chromophores and the associated proteinstructures are linked or diffuse independently.LaserFIFOBufferPhotonHistogram ofmicro timemicro timemicro time...micro timemicro timepicosecondsLaserFluorescencedecaycurvesDetectorChannelDet. NoDet. NoDet. NoDet. NoReadoutHard disktime from start of experimentStartofexperiment Photonsmacro timeresolution 50 nsmacro timemacro time...macro timemacro timeAutocorrelation ofmacro timens to secondsFluorescencecorrelationspectraFig. 40: Combined liftime / FCS data acquisition byTCSPCTCSPC Wide Field ImagingTCSPC can be used for wide field imaging if a special detector is used. The detector consists of theusual photocathode, a microchannel plate and a delay line system or a four-element anode system atthe output. By measuring the delay of the output pulses at the ends of the delay lines or the chargedistribution at the four anode sectors the location of a photon can be determined, see fig. 41.35