Characterization of fluorescence emissions from cells often leads to conclusive results in the early detection of cellular abnormalities. Cellular abnormalities can be characterized by their difference in the fluorescence lifetime, which may be less than nanoseconds. A sensitive frequency domain technique, also called a phase-resolved fluorescence imaging technique, is proposed in which fluorescence emissions at the same wavelengths can more effectively be separated with subnanosecond resolution in their lifetime difference. The system configuration is optimized by incorporating even-step phase shifting in the homodyne-assisted signal-processing concept along with the phase-resolved fluorescence technique to eliminate the dc offsets of emission. Experiments are carried out with simulated samples composed of two fluorescence emissions of the same wavelength but with different lifetime values. Suppression of either of the fluorescence emissions by selective imaging of the other validates the superiority of the proposed technique. Hence, this technique can potentially be applied in the early detection of cellular abnormalities.
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