title: Time-gated orthogonal scanning automated microscopy (OSAM) for high-speed cell detection and analysis.
abstract: We report a new development of orthogonal scanning automated microscopy (OSAM) incorporating time-gated detection to locate rare-event organisms regardless of autofluorescent background. The necessity of using long-lifetime (hundreds of microseconds) luminescent biolabels for time-gated detection implies long integration (dwell) time, resulting in slow scan speed. However, here we achieve high scan speed using a new 2-step orthogonal scanning strategy to realise on-the-fly time-gated detection and precise location of 1-μm lanthanide-doped microspheres with signal-to-background ratio of 8.9. This enables analysis of a 15 mm × 15 mm slide area in only 3.3 minutes. We demonstrate that detection of only a few hundred photoelectrons within 100 μs is sufficient to distinguish a target event in a prototype system using ultraviolet LED excitation. Cytometric analysis of lanthanide labelled Giardia cysts achieved a signal-to-background ratio of two orders of magnitude. Results suggest that time-gated OSAM represents a new opportunity for high-throughput background-free biosensing applications.
authors.affiliation: Advanced Cytometry Laboratories, MQ Biofocus Research Centre, Macquarie University, Sydney, NSW 2109, Australia. yiqing.lu@mq.edu.au
authors.forename: Yiqing
authors.initials: Y
authors.lastname: Lu
authors.forename: Peng
authors.initials: P
authors.lastname: Xi
authors.forename: James A
authors.initials: JA
authors.lastname: Piper
authors.forename: Yujing
authors.initials: Y
authors.lastname: Huo
authors.forename: Dayong
authors.initials: D
authors.lastname: Jin
mesh_terms: D001331:Automation; D015374:Biosensing Techniques; D005872:Giardia / Q000166:cytology*; D028581:Lanthanoid Series Elements / Q000737:chemistry; D008163:Luminescent Measurements; D008853:Microscopy / Q000295:instrumentation / Q000379:methods*; D008863:Microspheres; D013997:Time Factors
chemical_list: D028581:Lanthanoid Series Elements