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      Temporal focusing multiphoton microscopy with optimized parallel multiline scanning for fast biotissue imaging


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          Significance: Line scanning-based temporal focusing multiphoton microscopy (TFMPM) has superior axial excitation confinement (AEC) compared to conventional widefield TFMPM, but the frame rate is limited due to the limitation of the single line-to-line scanning mechanism. The development of the multiline scanning-based TFMPM requires only eight multiline patterns for full-field uniform multiphoton excitation and it still maintains superior AEC.

          Aim: The optimized parallel multiline scanning TFMPM is developed, and the performance is verified with theoretical simulation. The system provides a sharp AEC equivalent to the line scanning-based TFMPM, but fewer scans are required.

          Approach: A digital micromirror device is integrated in the TFMPM system and generates the multiline pattern for excitation. Based on the result of single-line pattern with sharp AEC, we can further model the multiline pattern to find the best structure that has the highest duty cycle together with the best AEC performance.

          Results: The AEC is experimentally improved to 1.7    μ m from the 3.5    μ m of conventional TFMPM. The adopted multiline pattern is akin to a pulse-width-modulation pattern with a spatial period of four times the diffraction-limited line width. In other words, ideally only four π / 2 spatial phase-shift scans are required to form a full two-dimensional image with superior AEC instead of image-size-dependent line-to-line scanning.

          Conclusions: We have demonstrated the developed parallel multiline scanning-based TFMPM has the multiline pattern for sharp AEC and the least scans required for full-field uniform excitation. In the experimental results, the temporal focusing-based multiphoton images of disordered biotissue of mouse skin with improved axial resolution due to the near-theoretical limit AEC are shown to clearly reduce background scattering.

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          Most cited references67

          • Record: found
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          Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution.

          Contrary to the well known diffraction limit, the fluorescence microscope is in principle capable of unlimited resolution. The necessary elements are spatially structured illumination light and a nonlinear dependence of the fluorescence emission rate on the illumination intensity. As an example of this concept, this article experimentally demonstrates saturated structured-illumination microscopy, a recently proposed method in which the nonlinearity arises from saturation of the excited state. This method can be used in a simple, wide-field (nonscanning) microscope, uses only a single, inexpensive laser, and requires no unusual photophysical properties of the fluorophore. The practical resolving power is determined by the signal-to-noise ratio, which in turn is limited by photobleaching. Experimental results show that a 2D point resolution of <50 nm is possible on sufficiently bright and photostable samples.
            • Record: found
            • Abstract: found
            • Article: not found

            Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation.

            Multicolor nonlinear microscopy of living tissue using two- and three-photon-excited intrinsic fluorescence combined with second harmonic generation by supermolecular structures produces images with the resolution and detail of standard histology without the use of exogenous stains. Imaging of intrinsic indicators within tissue, such as nicotinamide adenine dinucleotide, retinol, indoleamines, and collagen provides crucial information for physiology and pathology. The efficient application of multiphoton microscopy to intrinsic imaging requires knowledge of the nonlinear optical properties of specific cell and tissue components. Here we compile and demonstrate applications involving a range of intrinsic molecules and molecular assemblies that enable direct visualization of tissue morphology, cell metabolism, and disease states such as Alzheimer's disease and cancer.
              • Record: found
              • Abstract: found
              • Article: not found

              Deep tissue two-photon microscopy.

              With few exceptions biological tissues strongly scatter light, making high-resolution deep imaging impossible for traditional-including confocal-fluorescence microscopy. Nonlinear optical microscopy, in particular two photon-excited fluorescence microscopy, has overcome this limitation, providing large depth penetration mainly because even multiply scattered signal photons can be assigned to their origin as the result of localized nonlinear signal generation. Two-photon microscopy thus allows cellular imaging several hundred microns deep in various organs of living animals. Here we review fundamental concepts of nonlinear microscopy and discuss conditions relevant for achieving large imaging depths in intact tissue.

                Author and article information

                J Biomed Opt
                J Biomed Opt
                Journal of Biomedical Optics
                Society of Photo-Optical Instrumentation Engineers
                1 January 2021
                January 2021
                1 January 2021
                : 26
                : 1
                : 016501
                [a ]National Cheng Kung University , Department of Mechanical Engineering, Tainan, Taiwan
                [b ]National Chiao Tung University , College of Photonics, Tainan, Taiwan
                [c ]National Cheng Kung University , Department of Photonics, Tainan, Taiwan
                [d ]National Cheng Kung University , Department of Cell Biology and Anatomy, Tainan, Taiwan
                Author notes
                [* ]Address all correspondence to Shean-Jen Chen, sheanjen@ 123456nctu.edu.tw
                Author information
                JBO-200171RR 200171RR
                © 2021 The Authors

                Published by SPIE under a Creative Commons Attribution 4.0 Unported License. Distribution or reproduction of this work in whole or in part requires full attribution of the original publication, including its DOI.

                : 16 June 2020
                : 9 December 2020
                Page count
                Figures: 9, Tables: 0, References: 67, Pages: 16
                Funded by: Ministry of Science and Technology of Taiwan
                Award ID: 109-2811-E-009-503
                Award ID: 109-2636-E-006-018
                Award ID: 107-2221-E-009-168-MY2
                Custom metadata
                Chang et al.: Temporal focusing multiphoton microscopy with optimized parallel multiline scanning…

                Biomedical engineering
                medical and biological imaging,fluorescence microscopy,nonlinear microscopy,three-dimensional microscopy


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