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-bandwidth 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 50MHz 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.