5 March 2015 2D simultaneous spatial and temporal focusing multiphoton microscopy for fast volume imaging with improved sectioning ability
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Abstract
Simultaneous spatial and temporal focusing (SSTF) multiphoton microscopy offers us widefield imaging with sectioning ability. As extending the idea to 2D SSTF, people can utilize a 2D spectral disperser. In this study, we use a 2D spectral disperser via a virtually-imaged phased-array (VIPA) and a diffraction grating to fulfill the back aperture of objective lens with a spectrum matrix. This offers us an axial resolution enhanced by a factor of ~1.7 compared with conventional SSTF microscopy. Furthermore, the small free spectral range (FSR) of VIPA will reduce the temporal self-imaging effect around out-of-focus region and thus will reduce the out-of-focus multiphoton excited fluorescence (MPEF) signal of 2D SSTF microscopy. We experimentally show that inside a sample with dense MPEF, the contrast of the sectioning image is increased in our 2D SSTF microscope compared with SSTF microscope. In our microscope, we use a 1 kHz chirped amplification laser, a piezo stage and a sCMOS camera integrated with 2D SSTF to realize high speed volume imaging at a speed of 50 volumes per second as well as improved sectioning ability. Volume imaging of Brownian motions of fluorescent beads as small as 1μm has been demonstrated. Not only the lateral motion but also the axial motion could be traced.
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Qiyuan Song, Keisuke Isobe, Kenichi Hirosawa, Katsumi Midorikawa, and Fumihiko Kannari "2D simultaneous spatial and temporal focusing multiphoton microscopy for fast volume imaging with improved sectioning ability", Proc. SPIE 9329, Multiphoton Microscopy in the Biomedical Sciences XV, 93292N (5 March 2015); doi: 10.1117/12.2078093; https://doi.org/10.1117/12.2078093
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