Abstract:
A method of measuring fluorescence from a location, the method comprising applying to the location a first fluorescence excitation signal having a first duty cycle, accumulating as a first result fluorescence that emanates from the location in response to the first excitation signal, applying to the location a second fluorescence excitation signal having a second duty cycle, accumulating as a second result fluorescence that emanates from the location in response to the second excitation signal, and comparing the first and second results to provide a comparison result for the location. The invention also relates to apparatus for performing the method.

Description:
FIELD 
       [0001]    The invention relates to the field of assessing sample material based on the fluorescence lifetime of fluorescent material in the sample material. 
       BACKGROUND 
       [0002]    Fluorescence lifetime imaging (FLIM) is a well known microscopy technique. There are two main types of FLIM. These are time domain FLIM and frequency domain FLIM. 
         [0003]    In time domain FLIM, it is typically the case that an impulse of laser energy is used to excite fluorescence in a microscopy sample. A high sample rate detector is then used to sample the resulting fluorescence and the lifetime is extracted from the exponential decay trend that should be manifest in the captured sample sequence. The sample rate of the detector must typically be in the 10 9  Hertz range, and such components with such performance are relatively costly. 
         [0004]    In frequency domain FLIM, it is typically the case that a sinusoidally modulated light beam is used to excite fluorescence in a microscopy sample. As in time domain FLIM, a relatively fast detector is required to sample the fluorescence, which should exhibit a sinusoidal modulation offset in phase relative to, but of frequency equal to, the modulation applied to the stimulating laser. Furthermore, relatively high clock rate electronics is needed to synchronise the modulation of the stimulating laser with the waveform of the detected fluorescence. 
       BRIEF DESCRIPTION OF THE INVENTION 
       [0005]    The invention is defined by the appended claims, to which reference should now be made. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0006]    By way of example only, certain embodiments of the invention will be described with reference to the accompanying drawings in which: 
           [0007]      FIG. 1  is a block diagram schematically illustrating a fluorescence lifetime imaging microscope (FLIM); and 
           [0008]      FIG. 2  is a chart plotting variation in a parameter calculated from results produced by the microscope of  FIG. 1 . 
       
    
    
     DETAILED DESCRIPTION 
       [0009]      FIG. 1  shows an optical system  10  comprising a high energy, pulsed laser  12 , an input optical system  14 , an output optical system  16 , a fluorescence detector  18  and a computer  20 . As shown, a sample  22  is installed in the system  10 . In the present example, the sample is a slide on which is fixed a group of cells that have been stained with fluorophores in the form of fluorescent nanocrystals (quantum dots) that have been introduced to the sample  22 . The input optical system  14  serves to channel light from the laser  12  into the sample  22  where it stimulates the fluorophores. Fluorescence emitted by the fluorophores is then collected by the output optical system  16  and registered by the detector  18 . In this example, the detector  18  is a charge coupled device (CCD) camera. The digital signals produced by the detector are supplied to the computer  20  for processing. 
         [0010]    The laser  12  emits pulses of radiation to excite the fluorophores. The duty cycle of the radiation emitted by the laser  12  is characterised by a pulse of picosecond scale duration at a repetition rate that can be varied up to hundreds of MHz. 
         [0011]    In this example, the laser  12  illuminates an area of the slide that is broad in comparison with the cells under examination and the detector  18  captures images of the fluorescence from the illuminated area. Of course, in other embodiments, the input optical system  14  provides point-like illumination of the sample  22  and includes a scanning arrangement to allow the illumination point to be moved over the sample and in such cases the detector  18  typically employs a relatively simple photodetector rather than a more complicated CCD camera. 
         [0012]    The computer  20  processes the output of each CCD to produce a corresponding pixel for an image of the illuminated area of the sample  22 . As a precursor to describing that processing, the physics of the fluorescence excitation and decay of the fluorophores will now be briefly discussed. 
         [0013]    When a fluorophore absorbs light from a laser pulse, it moves from a ground state to an excited state and, some time later, decays back to the ground state emitting fluorescence in the process. Therefore, after excitation by a laser pulse, the fluorescence emitted by the sample  22  will decay and can be described using an exponential function characterised by a fluorescence lifetime of τ. That is to say, at time t after an excitation pulse, the intensity of the fluorescence will be proportional to 
         [0000]    
       
         
           
             
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         [0014]    Assume now that the pulses of the laser  12  have a repetition frequency f such that the duration between the starts of two consecutive pulses is T. If it is the case that T is less than τ, then the majority of the fluorophores do not have time to decay from the excited state to the ground state with the result that there is a permanent subpopulation of fluorophores in the excited state. In this situation, there will be saturation of the overall absorption of the pulsed laser radiation by the fluorophores, leading to reduced efficiency in the excitation of the fluorophores and a reduced fluorescence integrated over the duty cycle of T of the laser. 
         [0015]    Mathematically, E, the energy of the fluorescence light that is incident upon a CCD of the detector  18  over the course of one duty cycle of the laser  12 , is: 
         [0000]    
       
         
           
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         [0000]    where κ is the Boltzmann constant and αP is related to the number of excitation events per cycle. The output value from a CCD of the camera will be proportional to the accumulation of (or in other words proportional to the integral of) E over the duration of the sampling time of the camera. 
         [0016]      FIG. 2  demonstrates how E varies with T and plots E versus 1/T (i.e. against f) when the fluorophores are excited by the laser  12 . The solid line  24  represents the result where the fluorophore lifetime is τ 1  and the dashed line  26  represents the result where the flurophore lifetime is τ 2 , where τ 1 &gt;τ 2 . It will be apparent that, for both τ 1  and τ 2 , E is steady at low f and then falls off as f increases, the fall off occurring sooner (i.e. at lower f) in the τ 1  case. In each case, the departure from the plateau commences when T becomes less than approximately twice the fluorophore lifetime. 
         [0017]    The computer  20  captures first and second images of the sample  22  at respective laser pulse frequencies f 1  and f 2 . For the j th  CCD within the camera, its output value for the first image (i.e. when the laser pulse frequency is f 1 ) is S 1,j  and its output value for second image (i.e. when the laser pulse frequency is f 2 ) is S 2,j . The computer  20  calculates a ratio R for the j th  CCD which is defined as: 
         [0000]    
       
         
           
             
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         [0018]    This is a ratio of the values S 1,j  and S 2,j  after normalisation to account for the difference in their excitation pulse frequencies f 1  and f 2 . If this scaling were not performed, the ratio would be biased by the fact that S 2,j  is a measurement that is an integral over f 2 /f 1  more excitation cycles than S 1,j . The frequencies f 1  and f 2  are chosen such that E for the fluorophore being imaged is markedly different at f 1  and f 2  so that a contrast picture can be created. Clearly, contrast would be largely unobtainable if both f 1  and f 2  where within the plateau of the E function illustrated in  FIG. 2 . Typically then, 1/f 1  is set greater than twice the fluorophore lifetime and 1/f 2  is set to be less than the fluorophore lifetime. 
         [0019]    The computer  20  calculates the value R for each CCD of the camera of the detector  18 . This set of R values is then plotted as an array of pixels making up an image of the sample. 
         [0020]    Thus, a contrast image of the sample can be obtained using a CCD camera which has a slow response (relative, that is, to the electronics required in time domain FLIM and frequency domain FLIM), with each CCD of the camera generating an output value which is in effect an integral of the received fluorescence light over many duty cycles of the laser  12 . 
         [0021]    In an alternative embodiment, a pulsed LED is used in place of the laser  12 .