Frequency-domain lifetime imaging methods at unilever research

Fluorescence lifetime imaging methodology has been successfully implemented at Unilever Research in a frequency-domain manner. The experimental rig constructed comprises a wide-band-width electrooptic modulator operating on a CW argon-ion laser. The modulated excitation with a typical upper modulation frequency limit of 200 MHz falls on macroscopic samples and the resultant scattered light or fluorescence emission is then imaged onto a custom gain-modulatable image intensifier and slow-scan CCD camera combination. Phase adjustment of the image intensifier relative to the laser modulator is achieved by the RF function generator driving the intensifier. Both homodyne and heterodyne (500-Hz) strobing modes are employed to generate a double image stack (scattered light reference and fluorescence emission) comprising an image sequence as a function of instrumental phase difference. These image stacks are analyzed by Fourier least-squares methods to yield lifetime images by both phase delay and normalized demodulation. Correct operation of the apparatus is deduced from the direct imaging of a quencher-induced lifetime variation of BODIPY disulfonate over a range of concentrations. A typical industrially relevant sample, comprising an investigation of the lifetime aspects of human dental enamel autofluorescence at 50-MHz modulation frequency, is given. This shows that there are real emission lifetime decreases of about 0.5 nsec in white-spot lesion areas compared to the surrounding sound enamel. © 1997 Plenum Publishing Corporation.