Practical implementation, characterization and applications of a multi-colour time-gated luminescence microscope

Time-gated luminescence microscopy using long-lifetime molecular probes can effectively eliminate autofluorescence to enable high contrast imaging. Here we investigate a new strategy of time-gated imaging for simultaneous visualisation of multiple species of microorganisms stained with long-lived complexes under low-background conditions. This is realized by imaging two pathogenic organisms (Giardia lamblia stained with a red europium probe and Cryptosporidium parvum with a green terbium probe) at UV wavelengths (320-400 nm) through synchronization of a flash lamp with high repetition rate (1 kHz) to a robust time-gating detection unit. This approach provides four times enhancement in signal-to-background ratio over non-time-gated imaging, while the average signal intensity also increases six-fold compared with that under UV LED excitation. The high sensitivity is further confirmed by imaging the single europium-doped Y₂O₂S nanocrystals (150 nm). We report technical details regarding the time-gating detection unit and demonstrate its compatibility with commercial epi-fluorescence microscopes, providing a valuable and convenient addition to standard laboratory equipment.