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High contrast imaging and flexible photomanipulation for quantitative in vivo multiphoton imaging with polygon scanning microscope |
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| Authors: | Yongxiao Li Samantha J Montague Anne Brüstle Xuefei He Cathy Gillespie Katharina Gaus Elizabeth E Gardiner Woei Ming Lee |
| Institution: | 1. Research School of Engineering, College of Engineering and Computer Science, The Australian National University, Canberra, ACT, Australia;2. ACRF Department of Cancer Biology and Therapeutics, The John Curtin School of Medical Research, The Australian National University, Canberra, ACT, Australia;3. Department of Immunology and Infectious Disease, The John Curtin School of Medical Research, The Australian National University, Canberra, ACT, Australia;4. Imaging and Cytometry Facility, The John Curtin School of Medical Research, The Australian National University, Canberra, ACT, Australia;5. EMBL Australia Node in Single Molecule Science, School of Medical Sciences, University of New South Wales, Sydney, NSW, Australia;6. Australia Research Council Centre of Excellence in Advanced Molecular Imaging, University of New South Wales, Sydney, NSW, Australia;7. ARC Centre of Excellence in Advanced Molecular Imaging, The Australian National University, Canberra, ACT, Australia |
| Abstract: | In this study, we introduce two key improvements that overcome limitations of existing polygon scanning microscopes while maintaining high spatial and temporal imaging resolution over large field of view (FOV). First, we proposed a simple and straightforward means to control the scanning angle of the polygon mirror to carry out photomanipulation without resorting to high speed optical modulators. Second, we devised a flexible data sampling method directly leading to higher image contrast by over 2‐fold and digital images with 100 megapixels (10 240 × 10 240) per frame at 0.25 Hz. This generates sub‐diffraction limited pixels (60 nm per pixels over the FOV of 512 μm) which increases the degrees of freedom to extract signals computationally. The unique combined optical and digital control recorded fine fluorescence recovery after localized photobleaching (r ~10 μm) within fluorescent giant unilamellar vesicles and micro‐vascular dynamics after laser‐induced injury during thrombus formation in vivo. These new improvements expand the quantitative biological‐imaging capacity of any polygon scanning microscope system.  |
| Keywords: | multiphoton microscopy optical design and fabrication scanning microscopy |