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three-dimensional and multidimensional microscopy: image acquisition and processing xxxI 2024

作者： Saberigarakani, Alireza Zhang, Xinyuan Almasian, Milad Ding, Yichen Department of Bioengineering The University of Texas at Dallas RichardsonTX75080 United States Center for Imaging and Surgical Innovation The University of Texas at Dallas RichardsonTX75080 United States Hamon Center for Regenerative Science and Medicine UT Southwestern Medical Center DallasTX75390 United States

ISBN: (纸本)9781510669550

Paroxysmal arrhythmias caused by medications are challenging to be prospectively identified. Zebrafish have emerged as an ideal model organism for screening small molecule compounds to study cardiac abnormalities, due to their rapid development, optical transparency during early stages, and similarities to the human heart. To overcome the challenges associated with observing cardiac abnormalities in zebrafish, we sought to develop a light-field microscope, a rapid imaging method with high photon efficiency, for the volumetric acquisition in a single snapshot. This method, along with its variations, utilizes a multi-lens array (MLA) to capture angular information. We have customized a light-field system and developed a pipeline to incorporate the MLA into the detection path. A program based on wave optics has also been developed to calculate the point spread function at different depths. The program involves two main steps: calculating the wide-field PSF and applying the MLA effect as a mask. This enables us to simulate the impact of the MLA on the imaging system. The comparison between the simulated and experimental data allows for the determination of MLA position. We aim to capture drug-induced arrhythmias in zebrafish larvae using this method, exploring the contractile dysfunction across the atrium and ventricle during multiple cardiac cycles. This research aims to deepen our understanding of the mechanisms underlying these arrhythmias and their connection to drug effects. © COPYRIGHT SPIE. Downloading of the abstract is permitted for personal use only.

关键词： Optical transfer function

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Design and fabrication of universal resolution targets for microsphere-assisted microscopy 31

Design and fabrication of universal resolution targets for m...

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three-dimensional and multidimensional microscopy: image acquisition and processing xxxI 2024

作者： Abbasian, Vahid Awan, Kashif Masud Darafsheh, Arash Department of Radiation Oncology Washington University School of Medicine in St. Louis St. LouisMO63110 United States Imaging Science Program McKelvey School of Engineering Washington University in St. Louis St. LouisMO63130 United States Institute of Materials Science and Engineering Washington University St. LouisMO63130 United States Department of Mechanical Engineering and Materials Science Washington University in St. Louis St. LouisMO63130 United States

ISBN: (纸本)9781510669550

Determining the achieved spatial resolution in microsphere-assisted microscopy (MAM) has remained an important and challenging task. While fluorescent-based microscopies use fluorescent nano-beads to determine the point spread function (PSF), such a method is not practical in label-free super-resolution techniques. In some previous studies, objects with certain features have been imaged and those feature sizes have been reported as the resolution;however, determining the resolution in that way is not consistent with the definition of resolution based on point sources. Furthermore, the imaging characteristics in MAM may depend on specimen-specific properties, such as materials and substrate. In this study, we propose a robust and objective technique to address these issues. We fabricate arrays of nano-pillars varying in diameter and periodicity, ranging from 50 to 1000 nm. These pillars consist of a 20 nm Ag layer with a 2 nm Cr adhesion layer, deposited on a silicon substrate. The role of specimen-specific properties can be investigated by considering different materials. Fabrication involves the bilayer PMMA resist lift-off process, ensuring accurate deposition of nanometer-scale features to serve as sub-diffraction-limited targets in optical super-resolution microscopy. A deconvolution method that provides a robust platform for resolution measurement was used. By deconvolving the object profile, obtained from scanning electron microscopy, from the image profile, the PSF of the system is determined. Our proposed technique offers a reliable and standardized approach for quantifying resolution, magnification, and field-of-view in MAM or any other label-free microscopy technique, enabling accurate and objective comparison across different novel microscopy techniques. © COPYRIGHT SPIE. Downloading of the abstract is permitted for personal use only.

关键词： Microspheres

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High-resolution volumetric imaging using hybrid image of Fourier light-field microscopy and wide-field microscopy 29

High-resolution volumetric imaging using hybrid image of Fou...

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

作者： Liu, Wenhao Jia, Shu Georgia Inst Technol Wallace H Coulter Dept Biomed Engn Atlanta GA 30332 USA Emory Univ Atlanta GA 30332 USA

ISBN: (纸本)9781510648043;9781510648036

We have developed wFLFM to further enhance the resolution by taking the advantage of both wide-field image and FLFM. We proposed the principle of wFLFM and verified the approach both numerically using simulated rings, and experimentally using phantom samples on the wFLFM system we constructed as a prototype. The principle of wFLFM requires only additional wild-field image at the aperture plane, making it readily compatible with various extended depth of focus wide-field modules, and the validating results showed a 2-3x enhancement in lateral resolution without cost in axial information, thereby advancing wFLFM as a promising high-resolution 3D imaging method.

关键词： Light-field microscopy Fourier light-field microscopy high resolution volumetric imaging

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Multispectral time-resolved fluorescence microscopy based on compressive acquisitions 29

Multispectral time-resolved fluorescence microscopy based on...

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

作者： Ghezzi, Alberto Farina, Andrea Valentini, Gianluca Bassi, Andrea D'Andrea, Cosimo Politecn Milan Dipartimento Fis Piazza L da Vinci 32 I-20133 Milan Italy CNR IFN Piazza L da Vinci 32 I-20133 Milan Italy Ist Italiano Tecnol Ctr NanoSci & Technol PoliMi Via Pascoli 70-3 I-20133 Milan Italy

ISBN: (纸本)9781510648043;9781510648036

multidimensional fluorescence microscopy techniques produce dataset rich of information (space, emission spectrum and lifetime) to investigate photophysical processes in biological samples. To acquire a 4D dataset, one promising microscope design is based on the single-pixel camera scheme and on compressive sensing acquisitions, thanks to which the measurement time can be reduced. Within this framework, a computational step is required to move from the acquisition space to the pixel space and, subsequently, the analysis can be carried out exploiting the high dimensionality. In this work we present an experimental system and a fast-fit method that can produce a map of fluorophore concentrations in parallel to the measurement routine.

关键词： fluorescence microscopy compressive sensing multispectral imaging time-resolved imaging

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Evaluation of tile artifact correction methods for multiphoton microscopy mosaics of whole-slide tissue sections 29

Evaluation of tile artifact correction methods for multiphot...

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

作者： Knapp, Thomas Lima, Natzem Duan, Suzann Merchant, Juanita L. Sawyer, Travis W. Univ Arizona Dept Biomed Engn Tucson AZ 85721 USA Univ Arizona Wyant Coll Opt Sci Tucson AZ 85721 USA Univ Arizona Coll Med Tucson AZ 85724 USA

ISBN: (纸本)9781510648043;9781510648036

Multi-photon microscopy (MPM) is a useful biomedical imaging tool due, in part, to its capabilities of probing tissue biomarkers at high resolution and with depth-resolved capabilities. Automated MPM tile scanning allows for whole-slide image acquisition but suffers from tile-stitching artifacts that prevent accurate quantitative data analysis. We have investigated a variety of post-processing artifact correction methods using imageJ macros and custom Python/MATLAB code and present a quantitative and qualitative comparison of these methods using whole-slide MPM autofluorescence images of human duodenal tissue. image quality is assessed via evaluation of artifact removal compared to the calculated mean square error (MSE), peak signal-to-noise ratio (PSNR), and structural similarity index (SSIM) of the processed image and its raw counterpart. Consideration of both quantitative and qualitative results suggest a combination of flat-field based correction and frequency filtering processing steps provide improved artifact correction when compared to each method used independently to correct for tiling artifacts of tile-scan MPM images.

关键词： Multiphoton microscopy image processing artifacts whole slide multiwavelength tiling

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Efficient and versatile aberration correction through sensorless adaptive optics 29

Efficient and versatile aberration correction through sensor...

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

作者： Hu, Qi Hailstone, Martin Booth, Martin Univ Oxford Oxford England

ISBN: (纸本)9781510648043;9781510648036

Wavefront-sensorless adaptive optics methods are often used to correct phase aberrations in optical systems and thus to improve imaging quality. However, sensorless methods have an intrinsic disadvantage of requiring multiple images that can lead to non-desirable photo-bleaching. We have proposed a machine learning assisted aberration correction method which could correct aberrations consisting of not fewer than five Zernike modes with as few as two images. We showed that our method could be used in microscopes to provide instant aberration predictions when imaging biological samples of non-specific structures. We showed that compared to conventional function fitting sensorless adaptive optics methods, the new method corrected much faster with observable advantages. This novel method has a great potential to be used in any adaptive optics equipped microscopy for efficient sensorless aberration correction for biomedical microscopy.

关键词： Aberrations adaptive optics microscopy

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Enabling hyperspectral acquisition for Scanning Laser Optical Tomography 29

Enabling hyperspectral acquisition for Scanning Laser Optica...

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

作者： Benecke, Hannes Johannsmeier, Sonja May, Tobias Ripken, Tammo Laser Zentrum Hannover eV Ind & Biomed Opt Dept Hollerithallee 8 D-30419 Hannover Germany InSCREENeX GmbH Inhoffenstr 7 D-38124 Braunschweig Germany

ISBN: (纸本)9781510648043;9781510648036

Scanning Laser Optical Tomography (SLOT) is a three-dimensional imaging technique usable on a micro- to mesoscale. The technique is equivalent to computer tomography, using a laser instead of x-rays. With this technology, a set of two-dimensional images is acquired at different angles and subsequently processed into volume information by reconstruction algorithms. Different contrast mechanisms can be used, depending on the application. Up until now transmission, scattering, fluorescence and second harmonic generation have been established for SLOT imaging. All of these contrast mechanisms are coupled to a specific narrow bandwidth, which is determined before the acquisition starts and is dependent on the setup and application. In order to collect true hyperspectral information, a spectrometer and a broadband light source have been integrated. This way, the amount of information is increased with each measurable wavelength, leading to various improvements of the SLOT technology. The entire transmission and absorption spectra of three-dimensional samples can now be measured and reconstructed. Here, we present the current state of development of the hyperspectral SLOT. This includes the technical construction of the hardware setup and the development of the software integration. Many challenges need to be overcome when implementing spectroscopy in a tomographic setup. Solutions to specific problems, such as decreased resolution and focal shift, will be presented. Finally, we will show the first results of hyperspectral SLOT imaging.

关键词： Scanning Laser Optical Tomography 3D microscopy Tomographic imaging Hyperspectral imaging fluorescence spectroscopy

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A compact open-top light-sheet microscope for Optofluidic imaging 29

A compact open-top light-sheet microscope for Optofluidic im...

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

作者： Son, Jeonghwan Mandracchia, Biagio Jia, Shu Georgia Inst Technol Wallace H Coulter Dept Biomed Engn Atlanta GA 30332 USA Emory Univ Atlanta GA 30332 USA

ISBN: (纸本)9781510648043;9781510648036

The fluorescence Optofluidic imaging have recently transformed conventional optical investigations of biological systems by exploiting the synergic combination of recent microscopy and integrated chips technologies. The implementation of light-sheet microscopy on Optofluidic imaging has further pushed the systemic capabilities towards fast and 3D-volumetric imaging. Here, we propose a new compact open-top light-sheet microscope to extend the study of 3D-fluorescence Optofluidic imaging. The proposed portable architecture of the system and customized reconstruction algorithms enable providing high throughput and volumetric reconstruction capability with an isometric single-cell resolution. The experimental results demonstrate the applicability of open-top light-sheet configuration to single-cell imaging in a flow. We anticipate the system to offer promising applications in clinical settings where the portability and 3D-volumetric analysis are required for fast diagnostics.

关键词： Light-sheet microscopy Open-top Light-sheet microscopy Portable microscopy Optofluidic Optofluidic imaging 3D-Volumetric imaging Volumetric imaging High-throughput imaging

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An integrated platform for high-throughput nanoscopy

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NATURE BIOTECHNOLOGY 2023年第11期41卷 1549-+页

作者： Barentine, Andrew E. S. Lin, Yu Courvan, Edward M. Kidd, Phylicia Liu, Miao Balduf, Leonhard Phan, Timy Rivera-Molina, Felix Grace, Michael R. Marin, Zach Lessard, Mark Chen, Juliana Rios Wang, Siyuan Neugebauer, Karla M. Bewersdorf, Joerg Baddeley, David Yale Sch Med Dept Cell Biol New Haven CT 06510 USA Yale Univ Dept Biomed Engn New Haven CT 06520 USA Yale Sch Med Dept Mol Biophys & Biochem New Haven CT USA Yale Sch Med Dept Genet New Haven CT USA Univ Appl Sci Dept Comp Sci & Math Munich Germany Univ Auckland Auckland Bioengn Inst Auckland New Zealand Yale Univ Dept Phys New Haven CT 06520 USA Yale Univ Nanobiol Inst West Haven CT 06520 USA

Single-molecule localization microscopy enables three-dimensional fluorescence imaging at tens-of-nanometer resolution, but requires many camera frames to reconstruct a super-resolved image. This limits the typical throughput to tens of cells per day. While frame rates can now be increased by over an order of magnitude, the large data volumes become limiting in existing workflows. Here we present an integrated acquisition and analysis platform leveraging microscopy-specific data compression, distributed storage and distributed analysis to enable an acquisition and analysis throughput of 10,000 cells per day. The platform facilitates graphically reconfigurable analyses to be automatically initiated from the microscope during acquisition and remotely executed, and can even feed back and queue new acquisition tasks on the microscope. We demonstrate the utility of this framework by imaging hundreds of cells per well in multi-well sample formats. Our platform, implemented within the PYthon-microscopy Environment (PYME), is easily configurable to control custom microscopes, and includes a plugin framework for user-defined extensions. A fast data processing platform enables super-resolution microscopy with increased throughput.

关键词： Biotechnology Fluorescence imaging Imaging Super-resolution microscopy

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Flexible conjugate adaptive optics with a refractive wavefront modulator 29

Flexible conjugate adaptive optics with a refractive wavefro...

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

作者： Dorn, Alex Banerjee, Kaustubh Rajaeipour, Pouya Zappe, Hans Ataman, Caglar Univ Freiburg Dept Microsyst Engn Gisela & Erwin Sick Lab Microopt Freiburg Germany Phaseform GmbH Freiburg Germany

ISBN: (纸本)9781510648043;9781510648036

Diffraction-limited imaging in microscopy is only possible if different layers within the objective's working distance all have an uniform refractive index. However, in many practical imaging problems the samples are inhomogeneous in optical density, and refraction through them introduces field-dependent wavefront aberrations. On the image plane these are manifested in varying degrees of resolution and contrast degradation across the field-of-view. In pupil adaptive optics (AO), where a wavefront modulator is accommodated at the objective's pupil-plane, a single correction profile is applied for all fields, and the correctable field-of-view (FoV) is limited by the isoplanatic patch. In the alternative configuration of conjugate adaptive optics, where the corrective element and the dominant aberrating layer within the sample sit at optically conjugate planes, effective correction across the entire FoV is possible in principle. This configuration, however, is relatively difficult to implement with deformable mirrors for wavefront modulation, since several folded optical paths have to be constructed. In this work, we present the design and evaluation of a completely in-line conjugate-AO system based on a refractive wavefront modulator (DELTA7 from Phaseform GmbH, Germany). We evaluate the performance of the system on a Zeiss Axiovert inverted microscope by imaging fluorescent beads through custom-fabricated phase plates.

关键词： conjugate adaptive optics refractive adaptive optics microscopy

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