title: Practical implementation, characterization and applications of a multi-colour time-gated luminescence microscope.
abstract: 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.
authors.affiliation: Advanced Cytometry Labs, ARC Centre of Excellence for Nanoscale BioPhotonics (CNBP), Macquarie University, Sydney, NSW 2109, Australia.
authors.forename: Lixin
authors.initials: L
authors.lastname: Zhang
authors.affiliation: Advanced Cytometry Labs, ARC Centre of Excellence for Nanoscale BioPhotonics (CNBP), Macquarie University, Sydney, NSW 2109, Australia.
authors.forename: Xianlin
authors.initials: X
authors.lastname: Zheng
authors.affiliation: Advanced Cytometry Labs, ARC Centre of Excellence for Nanoscale BioPhotonics (CNBP), Macquarie University, Sydney, NSW 2109, Australia.
authors.forename: Wei
authors.initials: W
authors.lastname: Deng
authors.affiliation: Advanced Cytometry Labs, ARC Centre of Excellence for Nanoscale BioPhotonics (CNBP), Macquarie University, Sydney, NSW 2109, Australia.
authors.forename: Yiqing
authors.initials: Y
authors.lastname: Lu
authors.affiliation: Centre d'Élaboration de Matériaux et d'Etudes Structurales (CERMES - CNRS), Paul Sabatier University, France.
authors.forename: Severine
authors.initials: S
authors.lastname: Lechevallier
authors.affiliation: State Key Laboratory of Fine Chemicals, School of Chemistry, Dalian University of Technology, Dalian 116024, China.
authors.forename: Zhiqiang
authors.initials: Z
authors.lastname: Ye
authors.affiliation: Advanced Cytometry Labs, ARC Centre of Excellence for Nanoscale BioPhotonics (CNBP), Macquarie University, Sydney, NSW 2109, Australia.
authors.forename: Ewa M
authors.initials: EM
authors.lastname: Goldys
authors.affiliation: Advanced Cytometry Labs, ARC Centre of Excellence for Nanoscale BioPhotonics (CNBP), Macquarie University, Sydney, NSW 2109, Australia.
authors.forename: Judith M
authors.initials: JM
authors.lastname: Dawes
authors.affiliation: Advanced Cytometry Labs, ARC Centre of Excellence for Nanoscale BioPhotonics (CNBP), Macquarie University, Sydney, NSW 2109, Australia.
authors.forename: James A
authors.initials: JA
authors.lastname: Piper
authors.affiliation: State Key Laboratory of Fine Chemicals, School of Chemistry, Dalian University of Technology, Dalian 116024, China.
authors.forename: Jingli
authors.initials: J
authors.lastname: Yuan
authors.affiliation: Centre d'Élaboration de Matériaux et d'Etudes Structurales (CERMES - CNRS), Paul Sabatier University, France.
authors.forename: Marc
authors.initials: M
authors.lastname: Verelst
authors.affiliation: Advanced Cytometry Labs, ARC Centre of Excellence for Nanoscale BioPhotonics (CNBP), Macquarie University, Sydney, NSW 2109, Australia.
authors.forename: Dayong
authors.initials: D
authors.lastname: Jin
mesh_terms: D016785:Cryptosporidium parvum / Q000648:ultrastructure*; D005063:Europium / Q000737:chemistry; D008163:Luminescent Measurements; D008853:Microscopy / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods*; D057054:Molecular Imaging
chemical_list: D005063:Europium