MULTIPLE FREQUENCY FLUORESCENCE LIFETIME IMAGING

摘要

The application of a high frequency periodic bias voltage to the photocathode of an image intensifier device results in a repetitive modulation of the gain characteristics of the intensifier. By controlling the bias or DC component of the bias voltage it is possible to control the duration of the 'on' state of the device relative to its 'off' state. The amplitude of the higher harmonic content in the square waveform can thus be controlled. In this mode the image intensifier can be used as a mixing device for the simultaneous homodyne detection at each of the harmonics. Applied in a fluorescence lifetime imaging microscopy (FLIM) application, fluorescence lifetime images can be obtained at multiple frequencies in a harmonic set calculated from a single series of phase dependent images. Both the phase and modulation parameters necessary for spatially resolved heterogeneous lifetime calculations are extracted by Fourier analysis of the series of phase dependent images.