Construction of a high-NFOM multiphoton microscope with large-angle resonant raster scanning

Borah, Bhaskar Jyoti; Sun, Chi‐Kuang

doi:10.1016/j.xpro.2022.101330

Cited by 2 publications

(6 citation statements)

References 19 publications

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“…1a . An objective lens (OBJ) focuses an excitation beam (EXC, 70 MHz, 1070 nm) steered with a large-angle optical raster scanning (LAORS) system 37 , 49 over a specimen placed on a motorized 3D stage. A dichroic beam splitter D1 reflects the emerging signal to a multi-channel detection unit comprising multiple relay systems with focusing lenses L0-2.…”

Section: Resultsmentioning

confidence: 99%

“…It is noted that CUDA (v11.4)-accelerated OpenCV (C++) v4.5.2 was utilized for streamlined data/image processing powered by a Quadro RTX 8000 (NVIDIA Corporation, USA) graphics card. A C++-based software LASERaster + (v4.1.0.4) 49 was employed for data acquisition and processing. For a detailed illustration of the idea and construction of a high-NFOM imaging system, see our previously published papers 37 , 49 .…”

Section: Methodsmentioning

confidence: 99%

“…A C++-based software LASERaster + (v4.1.0.4) 49 was employed for data acquisition and processing. For a detailed illustration of the idea and construction of a high-NFOM imaging system, see our previously published papers 37 , 49 .…”

Section: Methodsmentioning

confidence: 99%

“…Resonant-galvo large-angle raster scanning system. The mNLOG platform reported in this study is powered by a Large Angle Optical Raster Scanning (LAORS) system 37,49 . For nonlinear optical excitation, we employed a 70 MHz pulsed laser source (Fidelity-2 Yb-fiber laser, Coherent, Inc., USA) with a central wavelength of 1070 nm.…”

Section: Methodsmentioning

confidence: 99%

“…A free version of the compiled standalone control and data acquisition software 49 ( LASERaster + ) in the form of object codes with enabled stable features would be available from the corresponding author upon reasonable request, for non-commercial use only, subject to regulations of National Taiwan University, Taiwan.…”

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confidence: 99%

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Rapid digital pathology of H&E-stained fresh human brain specimens as an alternative to frozen biopsy

et al. 2023

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Background Hematoxylin and Eosin (H&E)-based frozen section (FS) pathology is presently the global standard for intraoperative tumor assessment (ITA). Preparation of frozen section is labor intensive, which might consume up-to 30 minutes, and is susceptible to freezing artifacts. An FS-alternative technique is thus necessary, which is sectioning-free, artifact-free, fast, accurate, and reliably deployable without machine learning and/or additional interpretation training. Methods We develop a training-free true-H&E Rapid Fresh digital-Pathology (the-RFP) technique which is 4 times faster than the conventional preparation of frozen sections. The-RFP is assisted by a mesoscale Nonlinear Optical Gigascope (mNLOG) platform with a streamlined rapid artifact-compensated 2D large-field mosaic-stitching (rac2D-LMS) approach. A sub-6-minute True-H&E Rapid whole-mount-Soft-Tissue Staining (the-RSTS) protocol is introduced for soft/frangible fresh brain specimens. The mNLOG platform utilizes third harmonic generation (THG) and two-photon excitation fluorescence (TPEF) signals from H and E dyes, respectively, to yield the-RFP images. Results We demonstrate the-RFP technique on fresh excised human brain specimens. The-RFP enables optically-sectioned high-resolution 2D scanning and digital display of a 1 cm2 area in <120 seconds with 3.6 Gigapixels at a sustained effective throughput of >700 M bits/sec, with zero post-acquisition data/image processing. Training-free blind tests considering 50 normal and tumor-specific brain specimens obtained from 8 participants reveal 100% match to the respective formalin-fixed paraffin-embedded (FFPE)-biopsy outcomes. Conclusions We provide a digital ITA solution: the-RFP, which is potentially a fast and reliable alternative to FS-pathology. With H&E-compatibility, the-RFP eliminates color- and morphology-specific additional interpretation training for a pathologist, and the-RFP-assessed specimen can reliably undergo FFPE-biopsy confirmation.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Rapid digital pathology of H&E-stained fresh human brain specimens as an alternative to frozen biopsy

et al. 2023

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Suppression of Subpixel Jitter in Resonant Scanning Systems With Phase-locked Sampling

Ching-Roa,

Huang,

Giacomelli

2024

IEEE Trans. Med. Imaging

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Resonant scanning is critical to high speed and in vivo imaging in many applications of laser scanning microscopy. However, resonant scanning suffers from well-known image artifacts due to scanner jitter, limiting adoption of high-speed imaging technologies. Here, we introduce a real-time, inexpensive and all electrical method to suppress jitter more than an order of magnitude below the diffraction limit that can be applied to most existing microscope systems with no software changes. By phase-locking imaging to the resonant scanner period, we demonstrate an 86% reduction in pixel jitter, a 15% improvement in point spread function with resonant scanning and show that this approach enables two widely used models of resonant scanners to achieve comparable accuracy to galvanometer scanners running two orders of magnitude slower. Finally, we demonstrate the versatility of this method by retrofitting a commercial two photon microscope and show that this approach enables significant quantitative and qualitative improvements in biological imaging.

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Construction of a high-NFOM multiphoton microscope with large-angle resonant raster scanning

Cited by 2 publications

References 19 publications

Rapid digital pathology of H&E-stained fresh human brain specimens as an alternative to frozen biopsy

Rapid digital pathology of H&E-stained fresh human brain specimens as an alternative to frozen biopsy

Suppression of Subpixel Jitter in Resonant Scanning Systems With Phase-locked Sampling

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