TCSPC for FLIM and FRET in - Boston Electronics Corporation

Cornerstones of Fluorescence Lifetime ImagingTime ResolutionThe lifetimes of highly efficient fluorescence markers are typically in the region of a few ns.However, these dyes are selected for high quantum yield. Lifetimes of less efficient chromophorescan easily be below 100 ps. Quenching effects can reduce the lifetime down to a few 10 ps, and thelifetime of the quenched donor fluorescence in FRET experiments is in the order of 100 to 300 ps.Therefore, a good lifetime system should resolve fluorescence decay functions down to the order of10 ps.The fluorescence decay curves of in biological samples are often multi-exponential. There can beseveral chromophores in the same part of a cell, a single chromophore can be quenched witch nonuniformefficiency, or there can be quenched and unquenched molecules in the same part of the cell.Therefore, the ability to resolve multi-exponential decay functions is an absolute requirement to getquantitative results. Resolving two or even more exponential terms in a decay function requires datawith an excellent signal-to-noise ratio.Signal-to-Noise RatioDue to the short lifetimes, the measurement of fluorescence decay functions requires a detectionbandwidth in the GHz range. The high bandwidth does not only demand for very fast detectors anddetection electronics, it poses also a noise problem. The noise in fast optical measurements is almostessentially shot noise, i.e. the fluctuation of the number of photons detected within the resolvingtime of the measurement system. The best signal-to-noise ratio, SNR, that can be achieved isSNR = n 1/2with n being the number of photons detected within the resolved time interval. Actually the SNRcan be even lower due to background signals originating in the detector or coming from theenvironment, random gain fluctuations in the detector, and inefficient acquisition of the detectedphotons in the subsequent signal processing chain.Acceptable steady state images can be obtained for less than 100 photons detected per pixel of theimage. However, lifetime measurements actually deliver a stack of images for - in case of timedomainmethods - different times after the excitation or - in case of frequency domain methods - fordifferent phases and modulation frequencies. Therefore, the number of photons required to getlifetime information is much larger. Although rough lifetime information for single exponential canbe obtained from only 100 detected photons high accuracy measurements for multi-exponentialdecay analysis can easily require 10.000 or 100.000 photons per pixel [80].Unfortunately the number of photons that can be emitted from the sample in a given time interval islimited by the sample itself.Detection efficiencyThe detection efficiency for the photons emitted be the sample depends on the optical system and onthe detector. The efficiency of a microscope depends on the numerical aperture of the microscopeobjective, NA, and increases with NA 2 . The effective NA can be doubled by using the 4Pi technique[37].It is often claimed that the efficiency for non-descanned detection is considerably higher than fordescanned detection. This is certainly true for deep-tissue imaging when the emission light isscattered in the sample and cannot be fed through a pinhole. However, state-of the art microscopeshave pinholes with adjustable diameter. For imaging single cells there is no noticeable differencebetween descanned and non-descanned detection.13