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Area of Science:

  • Biomedical Imaging
  • Microscopy techniques
  • Analytical Chemistry

Background:

  • Autofluorescence in biological samples hinders high-contrast imaging.
  • Long-lifetime molecular probes offer a solution by enabling time-gated luminescence microscopy.
  • Simultaneous visualization of multiple microbial species requires advanced imaging strategies.

Purpose of the Study:

  • To develop and demonstrate a novel time-gated imaging strategy for simultaneous visualization of multiple microorganism species.
  • To enhance signal-to-background ratio and overall sensitivity in low-background imaging.
  • To assess the compatibility of the developed time-gating unit with standard microscopy equipment.

Main Methods:

  • Utilized time-gated luminescence microscopy with long-lived europium and terbium probes.
  • Imaged pathogenic organisms (Giardia lamblia and Cryptosporidium parvum) at UV wavelengths (320-400 nm).
  • Synchronized a high-repetition rate (1 kHz) flash lamp with a time-gating detection unit.

Main Results:

  • Achieved simultaneous visualization of Giardia lamblia (red probe) and Cryptosporidium parvum (green probe).
  • Demonstrated a four-fold enhancement in signal-to-background ratio compared to non-time-gated imaging.
  • Observed a six-fold increase in average signal intensity compared to UV LED excitation.
  • Successfully imaged single europium-doped Y₂O₂S nanocrystals, confirming high sensitivity.

Conclusions:

  • The developed time-gating imaging strategy enables high-contrast, simultaneous visualization of multiple microbial species.
  • This approach significantly improves signal-to-background ratio and sensitivity under low-background conditions.
  • The time-gating detection unit is compatible with commercial microscopes, offering a valuable tool for biological research.