High contrast imaging and flexible photomanipulation for quantitative in vivo multiphoton imaging with polygon scanning microscope

In this paper, we introduce two key improvements that overcome limitations of existing polygon scanning microscopes whilst maintaining high spatial and temporal imaging resolution over large fields 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 folds and digital images with 100 megapixels (10240 x 10240) 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 (GUVs) 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.

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