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Conference on three-dimensional and multidimensional microscopy - image acquisition and processing XXVII

作者： Borah, Bhaskar Jyoti Chi, Han-Hsiung Yen, Chen-Tung Sun, Chi-Kuang Natl Taiwan Univ Dept Elect Engn Taipei 10617 Taiwan Natl Taiwan Univ Grad Inst Photon & Optoelect Taipei 10617 Taiwan Natl Taiwan Univ Dept Life Sci Taipei 10617 Taiwan Natl Taiwan Univ Coll Med Taipei 10051 Taiwan Natl Taiwan Univ Mol Imaging Ctr Taipei 10617 Taiwan Natl Taiwan Univ Grad Inst Biomed Elect & Bioinformat Taipei 10617 Taiwan Acad Sinica Res Ctr Appl Sci Taipei 11529 Taiwan

ISBN: (纸本)9781510632547

We developed a nonlinear optical microscopy (NLOM) system with an extended field of view (FOV) of up -to 1.6 mm x 1.6 mm, employing a high numerical aperture (NA) and low magnification objective lens (20x, NA>0.9) maintaining submicron lateral resolution and <2 mu m axial resolution, making the system a suitable candidate for high resolution imaging. An acquisition speed of 95 M samples per second was implemented with synchronized sampling of 1 voxel per optical pulse from a femtosecond laser source with a 95 MHz repetition rate, with the ability of scanning a 1.6 x 1.6 x 1.6 mm(3) volume, with 12000 x 12000 x 2000 (x 4 channels), i.e., 1.152 Tera-voxels in total, capturing similar to 2.1 Terabyte of 16-bit raw data with 14-bit resolution in <53 minutes at 0.8 mu m Z-steps, and maintaining a Nyquist-exceeded voxel-size, a Nyquist-exceeded volume-scanning speed and a Nyquist-exceeded line-scanning speed of <15 attoliter, >1288 mu m(3)/ms and >12 mm/ms, respectively.

关键词： nonlinear optical microscopy field of view high resolution imaging sampling rate Nyquist-exceeded voxel-size

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Hyperspectral three-dimensional Absorption Imaging Using Snapshot Optical Tomography

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Physical Review Applied 2022年第3期18卷 034055-034055页

作者： Cory Juntunen Andrew R. Abramczyk Isabel M. Woller Yongjin Sung College of Engineering and Applied Science University of Wisconsin Milwaukee Wisconsin 53211 USA College of Health Sciences University of Wisconsin Milwaukee Wisconsin 53211 USA

Hyperspectral imaging (HSI) records a series of two-dimensional (2D) images for different wavelengths to provide the chemical fingerprint at each pixel. Combining HSI with a tomographic data-acquisition method, we can obtain the chemical fingerprint of a sample at each point in three-dimensional (3D) space. The so-called 3D HSI typically suffers from low imaging throughput due to the requirement of scanning the wavelength and rotating the beam or sample. In this paper, we present an optical system that captures the entire four-dimensional (4D; i.e., 3D structure and one-dimensional spectrum) data set of a sample with the same throughput of conventional HSI systems. Our system works by combining snapshot projection optical tomography (SPOT), which collects multiple projection images with a single snapshot, and Fourier-transform spectroscopy (FTS), which results in superior spectral resolution by collecting and processing a series of interferogram images. Using this hyperspectral SPOT system, we image the volumetric absorbance of dyed polystyrene microbeads, oxygenated red blood cells (RBCs), and deoxygenated RBCs. The 4D optical system demonstrated in this paper provides a tool for high-throughput chemical imaging of complex microscopic specimens.

关键词： Biomolecular optics Light-matter interaction Imaging & optical processing Optical absorption spectroscopy Optical microscopy Spectroscopy

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Excitation-scan Mirror Array System Advancements to Hyperspectral Imaging Applications 27

Excitation-scan Mirror Array System Advancements to Hyperspe...

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Conference on three-dimensional and multidimensional microscopy - image acquisition and processing XXVII

作者： Browning, Craig M. Mayes, Sam A. Deal, Joshua Gunn-Mayes, Samantha Rich, Thomas C. Leavesley, Silas J. Parker, Marina Univ S Alabama Chem & Biomol Engn Mobile AL 36688 USA Univ S Alabama Pharmacol Mobile AL 36688 USA Univ S Alabama Ctr Lung Biol Mobile AL 36688 USA Univ S Alabama Dept Syst Engn Mobile AL 36688 USA

ISBN: (纸本)9781510632547

Hyperspectral imaging (HSI) technology has been applied in a range of fields for target detection and mixture analysis. While its original applications were in remote sensing, modern uses include agriculture, historical document authentications and medicine. HSI has shown great utility in fluorescence microscopy;however, acquisition speeds have been slow due to light losses associated with spectral filtering. We are currently developing a rapid hyperspectral imaging platform for 5-dimensional imaging (RHIP-5D), a confocal imaging system that will allow users to obtain simultaneous measurements of many fluorescent labels. We have previously reported on optical modeling performance of the system. This previous model investigated geometrical capability of designing a multifaceted mirror imaging system as an initial approach to sample light at many wavelengths. The design utilized light-emitting diodes (LEDs) and a multifaceted mirror array to combine light sources into a liquid light guide (LLG). The computational model was constructed using Monte Carlo optical ray software (TracePro, Lambda Research Corp.). Recent results presented here show transmission has increased up to 9% through parametric optimization of each component. Future work will involve system validation using a prototype engineered based on our optimized model. System requirements will be evaluated to determine if potential design changes are needed to improve the system. We will report on-spectral resolution to demonstrate feasibility of the RHIP-5D as a promising solution for overcoming current HSI acquisition speed and sensitivity limitations.

关键词： Systems Design LED Tissue imaging HSI Spectroscopy microscopy Transmission Spectral Fluorescence Molecules Cells

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Cryo-Electron microscopy: The field of 1,000⁺ methods

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JOURNAL OF STRUCTURAL BIOLOGY 2022年第3期214卷 107861页

作者： Sorzano, C. O. S. Carazo, J. M. Univ Autonoma Madrid CSIC Natl Ctr Biotechnol C-Darwin3 Campus Madrid 28049 Spain

Cryo-Electron microscopy (CryoEM) is currently a well-established method to elucidate a biological macro-molecule's three-dimensional (3D) structure. Its success is due to technological and methodological advances in several fronts: sample preparation, electron optics and detection, image acquisition, image processing, and map interpretation. The first methods started in the late 1960s and, since then, new methods on all fronts have continuously been published, maturating the field as we know it *** terms of publications, we can distinguish several periods, witnessing a substantial acceleration of meth-odological publications in recent years, pointing out to an increased interest in the domain. On the other hand, this accelerated increase of methods development may confuse practitioners about which method they should be using (and how) and highlight the importance of paying attention to establishing best practices for methods reporting and *** this paper, we analyze the trends identified in over 1,000 methodological papers. Our focus is primarily on computational image processing methods. However, our list also covers some aspects of sample preparation and image *** interesting ideas stem out from this study: (1) Single Particle Analysis (SPA) has largely accelerated in the last decade and sample preparation methods in the last five years;(2) Electron Tomography is not yet in a rapidly growing phase, but it is foreseeable that it will soon be;(3) the work horses of SPA are 3D classification, 3D reconstruction, and 3D alignment, and there have been many papers on these topics, which are not considered to be solved yet, but ever improving;and (4) since the resolution revolution, atomic modelling has also caught on as a hot topic.

关键词： cryoEM Method development image processing

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Refractive index properties of the retina accessed by multi-wavelength digital holographic microscopy 26

Refractive index properties of the retina accessed by multi-...

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Conference on three-dimensional and multidimensional microscopy - image acquisition and processing XXVI

作者： Barroso, Alvaro Ketelhut, Steffi Heiduschka, Peter Nettels-Hackert, Gerburg Schnekenburger, Juergen Kemper, Bjoern Univ Munster Med Fac Biomed Technol Ctr Mendelstr 17 D-48149 Munster Germany Univ Munster Dept Ophtalmol Med Ctr Domagkstr 15 D-48149 Munster Germany

ISBN: (纸本)9781510624092

The refractive index (RI) of the retina and its dispersion are essential parameters in ophthalmologic imaging. However, the spatial RI distribution in retinal tissue is difficult to access. We explored the capabilities of multispectral quantitative phase imaging (QPI) with digital holographic microscopy (DHM) for label-free refractive index characterization of dissected murine retina. The retrieved tissue refractive indices are in agreement with previously reported values for living cells and dissected tissues. Moreover, the detected spatial refractive index distributions correlate with results from complementary conducted OCT investigations. In summary, multispectral DHM is a promising tool for label-free characterization of optical retina properties.

关键词： digital holographic microscopy quantitative phase imaging multi-wavelength hyperspectral imaging retina refractive index

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Enhanced spectral lightfield fusion microscopy via deep computational optics for whole-slide pathology 26

Enhanced spectral lightfield fusion microscopy via deep comp...

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Conference on three-dimensional and multidimensional microscopy - image acquisition and processing XXVI

作者： Kazemzadeh, Farnoud Li-Chang, Hector Wong, Alexander Univ Waterloo Waterloo ON Canada Waterloo Artificial Intelligence Inst Waterloo ON Canada Royal Victoria Reg Hlth Ctr Barrie ON Canada

ISBN: (纸本)9781510624092

Lensfree on-chip microscopy, which harnesses holography principles to capture interferometric light-field encodings without the need of lenses, is an emerging microscopy modality with widespread interest given the large field-of-view (FOV) compared to lens-based microscopy systems. In particular, there is a growing interest on the development of high-quality lensfree on-chip color microscopy. In this study, we propose a multi-laser spectral lightfield fusion microscopy using deep computational optics for achieving lensfree on-chip color microscopy. We will demonstrate that leveraging deep computational optics can enable imaging resolution beyond the diffraction limit without the use of any complex hardware-based super-resolution techniques, such as aperture scanning. The capabilities of the microscope are examined for whole-slide pathology. The superior imaging resolution of the instrument is demonstrated by imaging of a series of biological specimens demonstrating the true color imaging capability of the instrument while showcasing the large FOV of the instrument.

关键词： on-chip microscopy deep computational optics light-field fusion

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Temporal focusing-based multiphoton excitation fluorescence images with background noise cancellation via Hilbert-Huang transform 26

Temporal focusing-based multiphoton excitation fluorescence ...

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Conference on three-dimensional and multidimensional microscopy - image acquisition and processing XXVI

作者： Hu, Yvonne Yuling Luo, Yuan-Rong Lin, Chun-Yu Chang, Chia-Yuan Chen, Shean-Jen Natl Cheng Kung Univ Dept Photon Tainan Taiwan Natl Cheng Kung Univ Dept Engn Sci Tainan Taiwan Natl Cheng Kung Univ Dept Mech Engn Tainan Taiwan Natl Chiao Tung Univ Inst Imaging & Biomed Photon Coll Photon Tainan Taiwan

ISBN: (纸本)9781510624092

Temporal focusing multiphoton excitation microscopy has wide field-of-view and optical sectioning. By using digital micromirror device, it provides patterned illumination. However, without filling the back aperture of objective lens, the axial confinement is limited to micron-meters, leading the out-of-focus fluorophores excited and image blurred. In this study, Hilbert-Huang transform is proposed to reduce the background noise. The empirical mode decomposition is first applied to disassemble the image into intrinsic mode functions and then reconstruct by Hilbert transform after diminishing background residues. The axial confinement can be enhanced from 2.79 mu m to 0.73 mu m with structure frequency in 1.06 mu m(-1).

关键词： Temporal focusing Digital micromirror device Structured illumination microscopy Hilbert-Huang transform

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Wave-optical calibration of a light-field fluorescence microscopy 26

Wave-optical calibration of a light-field fluorescence micro...

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Conference on three-dimensional and multidimensional microscopy - image acquisition and processing XXVI

作者： Pribosek, J. Steinbrener, J. Baumgart, M. Bereczki, T. Harms, K. Tortschanoff, A. Kenda, A. Carinthian Tech Res Europastr 12 A-9524 Villach Austria Mol Devicess Lagerhausstr 45 A-5071 Wals Austria

ISBN: (纸本)9781510624092

In light-field microscopy, a single point emitter gives rise to a complex diffraction pattern, which varies with the position of the emitter in object space. In order to use deconvolution-based wave-optical reconstruction schemes for light-field imaging systems, established methods rely on theoretical estimation of such diffraction patterns. In this paper we propose a novel method for direct experimental estimation of the light-field point spread function. Our approach relies on a modified reversed micro-Hartmann test to acquire a composite light-field point spread function of several thousand point emitters in the object plane simultaneously. By using fiducial markers and a custom image processing algorithm we separate the contributions of individual point emitters directly in raw light-field images and allow the construction of the forward imaging process without any prior assumption about the optical system required. The constructed forward imaging model can finally be applied in the 3D-deconvolution based wave-optical reconstruction scheme.

关键词： light-field microscopy light-field point spread function wave-optical calibration fluorescence imaging

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three-dimensional Fluorescence Imaging with an Automated Light-field Microscope 26

Three-dimensional Fluorescence Imaging with an Automated Lig...

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Conference on three-dimensional and multidimensional microscopy - image acquisition and processing XXVI

作者： Steinbrener, J. Pribosek, J. Baumgart, M. Bereczki, T. Harms, K. Ripper, D. Tortschanoff, A. Kenda, A. CTR Carinthian Tech Res AG Europastr 12 A-9524 Villach Austria IMC Fachhsch Krems GmbH Piaristengasse 1 A-3500 Krems Austria Mol Devices Austria GmbH Lagerhausstr 45 A-5071 Salzburg Austria

ISBN: (纸本)9781510624092

In addition to the two-dimensional intensity distribution in the image plane, light field microscopes capture information about the angle of the incident radiation. This information can be used to extract depth information about the object, calculate all-in-focus images and perform three-dimensional reconstructions from a single exposure. In combination with automated microscopy setups, this makes the technique a promising tool for high-throughput, three-dimensional cell assay evaluation which could substantially improve drug development and screening. To this end, we have developed a novel generalized calibration and three-dimensional reconstruction scheme for a light-field fluorescence microscope setup. The scheme can handle Keplerian and Galilean light field camera configurations added to infinity corrected microscopes configured to be telecentric as well as non-telecentric or hypercentric. The latter provides a significant advantage over the state of the art as it allows for an application specific optimization of lateral and axial resolution, field-of-view, and depth-of-focus. The reconstruction itself is performed iteratively using an expectation maximization algorithm. Super-resolved reconstructions can be achieved by including experimentally measured point-spread-functions. To reduce the required computational power, sparsity and periodicity of the system matrix relating object space to light field space is exploited. This is particularly challenging for the non-telecentric cases, where the voxel size of the reconstructed object space depends on the axial coordinate. We provide details on the experimental setup and the reconstruction algorithm, and present results on the experimental verification of theoretical performance parameters as well as successful reconstructions of fluorescent beads and three-dimensional cell spheroids.

关键词： Light-field microscopy 3D fluorescence imaging Reconstruction algorithms

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Projection multiplexing for enhanced acquisition speed in holographic tomography 26

Projection multiplexing for enhanced acquisition speed in ho...

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Conference on three-dimensional and multidimensional microscopy - image acquisition and processing XXVI

作者： Kus, Arkadiusz Baczewska, Maria Ziemczonok, Michal Kujawinska, Malgorzata Warsaw Univ Technol Inst Micromech & Photon Sw Boboli 8 St PL-02525 Warsaw Poland

ISBN: (纸本)9781510624092

In holographic tomography (HT), the 3D refractive index distribution within weakly-scattering, phase-only biological object is retrieved. This key property of the technique is one of its most significant strengths compared to labelling-based methods of cell analysis such as fluorescence microscopy. As a consequence, however, it is required to acquire a set of holograms at several viewing directions, which hinders the measurement speed. In this paper we explore the prospect of multiplexing projections in order to decrease the number of scanning positions required for the full measurements. The presented analysis is based on experimental data acquired in a limited-angle holographic tomography system and emulates the performance of a spatial-light-modulator-based system in which multiple projections may be acquired simultaneously by generating a distribution of multiple point sources in the Fourier plane of the condenser lens. For this reason, the increase in acquisition speed strictly depends on the number of multiplexed holograms and results in decreased reconstruction quality. The performance of the system is demonstrated and analyzed with biological objects - human keratinocyte cells.

关键词： Holographic tomography optical diffraction tomography tomographic phase microscopy quantitative phase imaging 3D refractive index hologram multiplexing

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