title: High-Precision Pinpointing of Luminescent Targets in Encoder-Assisted Scanning Microscopy Allowing High-Speed Quantitative Analysis.
abstract: Compared with routine microscopy imaging of a few analytes at a time, rapid scanning through the whole sample area of a microscope slide to locate every single target object offers many advantages in terms of simplicity, speed, throughput, and potential for robust quantitative analysis. Existing techniques that accommodate solid-phase samples incorporating individual micrometer-sized targets generally rely on digital microscopy and image analysis, with intrinsically low throughput and reliability. Here, we report an advanced on-the-fly stage scanning method to achieve high-precision target location across the whole slide. By integrating X- and Y-axis linear encoders to a motorized stage as the virtual "grids" that provide real-time positional references, we demonstrate an orthogonal scanning automated microscopy (OSAM) technique which can search a coverslip area of 50 × 24 mm(2) in just 5.3 min and locate individual 15 μm lanthanide luminescent microspheres with standard deviations of 1.38 and 1.75 μm in X and Y directions. Alongside implementation of an autofocus unit that compensates the tilt of a slide in the Z-axis in real time, we increase the luminescence detection efficiency by 35% with an improved coefficient of variation. We demonstrate the capability of advanced OSAM for robust quantification of luminescence intensities and lifetimes for a variety of micrometer-scale luminescent targets, specifically single down-shifting and upconversion microspheres, crystalline microplates, and color-barcoded microrods, as well as quantitative suspension array assays of biotinylated-DNA functionalized upconversion nanoparticles.
authors.affiliation: Advanced Cytometry Laboratories, ARC Centre of Excellence for Nanoscale BioPhotonics (CNBP), Macquarie University , Sydney, New South Wales 2109, Australia.
authors.forename: Xianlin
authors.initials: X
authors.lastname: Zheng
authors.affiliation: Advanced Cytometry Laboratories, ARC Centre of Excellence for Nanoscale BioPhotonics (CNBP), Macquarie University , Sydney, New South Wales 2109, Australia.
authors.forename: Yiqing
authors.initials: Y
authors.lastname: Lu
authors.affiliation: Advanced Cytometry Laboratories, ARC Centre of Excellence for Nanoscale BioPhotonics (CNBP), Macquarie University , Sydney, New South Wales 2109, Australia.
authors.forename: Jiangbo
authors.initials: J
authors.lastname: Zhao
authors.affiliation: Department of Chemistry, National University of Singapore , 3 Science Drive 3, Singapore 117543, Singapore.
authors.forename: Yuhai
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Institute for Biomedical Materials and Devices, Faculty of Science, University of Technology Sydney , Sydney, New South Wales 2007, Australia.
authors.forename: Wei
authors.initials: W
authors.lastname: Ren
authors.affiliation: Advanced Cytometry Laboratories, ARC Centre of Excellence for Nanoscale BioPhotonics (CNBP), Macquarie University , Sydney, New South Wales 2109, Australia.
authors.forename: Deming
authors.initials: D
authors.lastname: Liu
authors.affiliation: Advanced Cytometry Laboratories, ARC Centre of Excellence for Nanoscale BioPhotonics (CNBP), Macquarie University , Sydney, New South Wales 2109, Australia.
authors.forename: Jie
authors.initials: J
authors.lastname: Lu
authors.affiliation: Advanced Cytometry Laboratories, ARC Centre of Excellence for Nanoscale BioPhotonics (CNBP), Macquarie University , Sydney, New South Wales 2109, Australia.
authors.forename: James A
authors.initials: JA
authors.lastname: Piper
authors.affiliation: Newport Instruments , 3345 Hopi Place, San Diego, California 92117-3516, United States.
authors.forename: Robert C
authors.initials: RC
authors.lastname: Leif
authors.affiliation: Department of Chemistry, National University of Singapore , 3 Science Drive 3, Singapore 117543, Singapore.|Institute of Materials Research and Engineering, A*STAR (Agency for Science, Technology and Research), 3 Research Link, Singapore 117602, Singapore.
authors.forename: Xiaogang
authors.initials: X
authors.lastname: Liu
authors.affiliation: Advanced Cytometry Laboratories, ARC Centre of Excellence for Nanoscale BioPhotonics (CNBP), Macquarie University , Sydney, New South Wales 2109, Australia.|Institute for Biomedical Materials and Devices, Faculty of Science, University of Technology Sydney , Sydney, New South Wales 2007, Australia.
authors.forename: Dayong
authors.initials: D
authors.lastname: Jin