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检索条件"任意字段=Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XXIX 2022"

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Normalized level set model for segmentation of low-contrast objects in 2- and 3-dimensional images 31

Normalized level set model for segmentation of low-contrast ...

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

作者： Baig, Mirza M. Junaid Wang, Yao L. Chung, Samuel H. Stepanyants, Armen Department of Physics Northeastern University 360 Huntington Ave BostonMA02115 United States Department of Bioengineering Northeastern University 360 Huntington Ave BostonMA02115 United States

ISBN: (纸本)9781510669550

Analyses of biomedical images often rely on accurate segmentation of structures of interest. Traditional segmentation methods based on thresholding, watershed, fast marching, and level set perform well in high-contrast images containing structures of similar intensities. However, such methods can under-segment or miss entirely low-intensity objects on noisy backgrounds. Machine learning segmentation methods promise superior performance but require large training datasets of labeled images which are difficult to create, particularly in 3D. Here, we propose an algorithm based on the Local Binary Fitting level set method and its application specifically designed to improve the segmentation accuracy for low-contrast structures even with significant noise levels present. The proposed algorithm, the Normalized Local Binary Fitting level set method, shows promise in enhancing the segmentation of low-contrast structures in biomedical images, addressing the limitations of traditional segmentation methods, and offering an alternative to machine learning approaches that require extensive training datasets. © COPYRIGHT SPIE. Downloading of the abstract is permitted for personal use only.

关键词： Machine learning

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High-speed light-sheet microscopy imaging and data post-processing using custom hardware and software solutions 30

High-speed light-sheet microscopy imaging and data post-proc...

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three-dimensional and multidimensional microscopy: image acquisition and processing XXX 2023

作者： Mazzamuto, Giacomo Silvestri, Ludovico Sancataldo, Giuseppe Gavryusev, Vladislav Scardigli, Marina Costantini, Irene Pavone, Francesco S. Sesto Fiorentino Italy Sesto Fiorentino Italy Department of Physics and Astronomy University of Florence Italy Department of Physics and Chemistry University of Palermo Italy Department of Biology University of Florence Italy

ISBN: (纸本)9781510658752

High-speed imaging with Light-Sheet Fluorescence microscopy poses several challenges throughout the whole pipeline, from data acquisition in the lab to image stitching and post-processing. Here we present our custom hardware and software solutions that allow us to map large biological samples at the cellular level, e.g. large portions of human brain cortex. Our custom optical setup — a dual-view, inverted, light-sheet microscope — is capable of simultaneous two-color acquisition at a data rate of 1 GB/s. Our open source tools include the instrument’s data acquisition and control software and also cover volumetric image stitching and post-processing. © 2023 SPIE. All rights reserved.

关键词： Brain

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Validation of Excitation-Scan Hyperspectral Multi-faceted Mirror Array Prototype System Advancements to Hyperspectral Imaging Applications 29

Validation of Excitation-Scan Hyperspectral Multi-faceted Mi...

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

作者： Parker, Marina Mayes, Sam A. Browning, Craig M. Deal, Joshua Gunn-Mayes, Samantha Rich, Thomas C. Leavesley, Silas J. 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)9781510648043;9781510648036

Hyperspectral imaging technologies (HSI) have undergone rapid development since their beginning stages. While original applications were in remote sensing, other uses include agriculture, food safety and medicine. HSI has shown great utility in fluorescence microscopy for detecting signatures from many fluorescent molecules;however, acquisitions speeds have been slow due to light losses associated with spectral filtering. Therefore, we designed a novel light emitting diode (LED)-based rapid excitation scanning hyperspectral imaging platform allowing users to obtain simultaneous measurements of fluorescent labels without compromising acquisition speeds. Previously, we reported our results of the optical ray trace simulations and the geometrical capability of designing a multifaceted mirror imaging system as an initial approach to combine light at many wavelengths. The design utilized LEDs and a multifaceted mirror array to combine light sources into a liquid light guide. The computational model was constructed using Monte Carlo optical ray software (TracePro, Lambda Research Corp.). Recent prototype validation results show that when compared to a commercial emission scanning spectral confocal microscope (Zeiss-LSM-980), the novel LED-based excitation scanning HSI prototype successfully detected and separated six fluorescent labels from a custom 6-label African green monkey kidney epithelial cells. We report on the prototype's ability to overcome limitations of acquisition speeds, sensitivity, and specificity present in conventional systems. Future work will evaluate prototype's light losses to determine latent design modifications needed to demonstrate the system's feasibility as a promising solution for overcoming HSI acquisition speeds. This work was supported by NSF award MRI1725937.

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

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three-dimensional and multidimensional microscopy: image acquisition and processing XXX

Three-Dimensional and Multidimensional Microscopy: Image Acq...

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three-dimensional and multidimensional microscopy: image acquisition and processing XXX 2023

ISBN: (纸本)9781510658752

The proceedings contain 13 papers. The topics discussed include: compressive time-resolved multispectral fluorescence microscopy with single-pixel camera;optical sectioning microscopy with structured illumination and single-pixel detection;high-speed light-sheet microscopy imaging and data post-processing using custom hardware and software solutions;utilizing fluorescent beads to mimic single fluorophores;single-shot capture bright-field and digital holographic microscopy for the analysis of living cells;simulations of imperfect refractive index matching in scanning laser optical tomography and a method for correction;super-resolution in confocal microscopy using generative adversarial networks with paired and unpaired data;estimation of 3D point spread function of a widefield microscope by stochastic least square minimization method;and design and implementation of plenoptic imaging system with reduced aberrations.

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On-the-fly deconvolution of diagonally-scanned Airy lightsheet microscopy 31

On-the-fly deconvolution of diagonally-scanned Airy lightshe...

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

作者： Vettenburg, Tom Valantinas, Laurynas Center for Medical Engineering and Technology School of Science and Engineering University of Dundee Nethergate DD14HN United Kingdom

ISBN: (纸本)9781510669550

Its subcellular resolution and minimal sample exposure make light-sheet microscopy the ideal tool to study biological specimen during their early development. A light-sheet microscope scans the sample with a plane of light and collects fluorescence with an objective orthogonal to the illumination. However, tightly focused Gaussian light-sheets suffer from a shallow depth of focus and are susceptible to scattering-induced aberrations. Light-sheets created by non-diffractive Airy beams can overcome this to yield isotropic sub-cellular resolution over a ten-fold larger field-of-view. Airy beam light-sheets have a characteristically curved structure and a broad transverse structure with side-lobes. Digital deconvolution of the raw data is thus essential to obtain high-fidelity images. Provided that the scan is along the direction of the detection axis, and all recorded data fits within working memory, a simple and efficient Wiener filter can recover accurate 3D images. However, multi-millimeter sized samples must be scanned with a light-sheet that is diagonal to the sample surface. The diagonal movement prevents the use of standard Wiener filtering. Moreover, the associated data sets can become too large to fit within the working memory of a consumer-grade GPU. This demands slow off-line processing, thus breaking a rapid experimental feedback-loop. Here, we investigate the potential of on-the-fly deconvolution of diagonally-scanned Airy light-sheet microscopy. © COPYRIGHT SPIE. Downloading of the abstract is permitted for personal use only.

关键词： Fluorescence

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Quantification of collagen networks in mammary tumors using TPEF and laser-based tomography 29

Quantification of collagen networks in mammary tumors using ...

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

作者： Kamin, Hannes Nolte, Lena Maurer, Jochen Bleilevens, Andreas Stickeler, Elmar Johannsmeier, Sonja Heinemann, Dag Ripken, Tammo Laser Zentrum Hannover eV Ind & Biomed Opt Dept Hollerithallee 8 D-30419 Hannover Germany Univ Hosp Aachen Clin Gynecol & Obstet Pauwelstr 30 D-52074 Aachen Germany Lower Saxony Ctr Biomed Engn Implant Res & Dev NI Stadtfelddamm 34 D-30625 Hannover Germany Leibniz Univ Hannover Inst Hort Prod Syst Dept Phytophoton Herrenhauser Str 2 D-30419 Hannover Germany Leibniz Univ Hannover Hannover Ctr Opt Technol HOT Nienburger Str 17 D-30167 Hannover Germany

ISBN: (纸本)9781510648043;9781510648036

The triple-negative breast cancer (tnbc) is an aggressive subtype linked to a poor outcome of established breast cancer therapy. Increasing evidence points to the role of the tumor's extracellular matrix (ECM) as a determinant of its aggressiveness as well as the effectiveness of chemical therapeutics. three-dimensional imaging techniques can be used to unravel ECM architecture. Label-free contrast mechanisms such as second harmonic generation (SHG) avoid falsification and artifacts introduced by the labeling process. Here, we present the complementary use of two-photon excitation microscopy (TPEF) and Scanning Laser Optical Tomography (SLOT) for the investigation and quantification of tumor ECM. Both methods were used to capture fluorescence from antibody-labeled samples as well as the SHG signal from collagen strands in the ECM. SLOT generally allows for the investigation of larger samples of several mm up to a few cm in size. This work shows the capabilities of the tomographic setup compared to established TPEF, and demonstrates their combined use to maximize the information content of the acquired data. The obtained images served as a basis for ECM quantification. 3D-analysis allowed for determination of length, straightness and orientation of the collagen fibers based on fluorescence imaging as well as SHG imaging. The resulting coordinates might be used for synthetic reconstruction of a patient-specific tumor matrix, serving as a scaffold for pre-clinical therapeutic testing. Collagen imaging and quantification as presented here can therefore be employed for both basic and clinical research, paving the way for patient-specific cancer therapy.

关键词： cancer collagen imaging multimodal imaging extracellular matrix 3D imaging image reconstruction tomography second harmonic generation

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Light-field microscopy to study cardiac arrhythmias in zebrafish larvae 31

Light-field microscopy to study cardiac arrhythmias in zebra...

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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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Single-shot capture bright-field and digital holographic microscopy for the analysis of living cells 30

Single-shot capture bright-field and digital holographic mic...

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three-dimensional and multidimensional microscopy: image acquisition and processing XXX 2023

作者： Picazo-Bueno, José Ángel Barroso, Álvaro Ketelhut, Steffi Schnekenburger, Jürgen Micó, Vicente Kemper, Björn Biomedical Technology Center University of Muenster Mendelstraβe 17 MuensterD-48149 Germany Department of Optics and Optometry and Vision Sciences University of Valencia C/Doctor Moliner 50 Burjassot46100 Spain

ISBN: (纸本)9781510658752

Digital holographic microscopy (DHM) is an emerging label-free modality for quantitative imaging of semi-transparent biological specimens. DHM provides quantitative phase imaging (QPI) of the light transmitted or scattered by an object, which allows to extract information about morphology and dynamics of investigated specimens. DHM can be implemented with other optical microscopy techniques like bright field (BF) or fluorescence, for example, to provide complementary information about cellular processes. Multimodal microscopy is often achieved by sequential acquisition of information from different imaging modalities or simultaneously by capturing images with different acquisition devices. However, such arrangements can be cost-intensive and/or limited in speed for imaging of dynamic processes. Here, we investigate a single-snapshot multimodal approach that enables simultaneous acquisition of DHM-based QPI and BF images and is compatible with common optical microscopes for the analysis of living cells. The technique is based on spatially multiplexed interferometric microscopy (SMIM), where off-axis holograms are generated by spatial multiplexing of the illuminated sample plane utilizing a diffraction grating, which is extended by additional superposition of complementary white-light image information. image separation is achieved by Fourier-transformation-based numerical demultiplexing procedures. The technique is firstly characterized by investigations on microspheres. Then, the application on living adherent and suspended cells is illustrated. © 2023 SPIE. All rights reserved.

关键词： Holograms

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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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Hyperspectral imaging and adaptive thresholding to identify agonist-induced cAMP signals in pulmonary microvascular endothelial cells 29

Hyperspectral imaging and adaptive thresholding to identify ...

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

作者： Annamdevula, N. Johnson, S. Pleshinger, D. J. Castleberry, S. Russell, W. Britain, A. L. Francis, C. M. Rich, T. C. Leavesley, S. J. Univ S Alabama Pharmacol Mobile AL 36688 USA Univ S Alabama Physiol & Cell Biol Mobile AL 36688 USA Univ S Alabama Chem & Biomol Engn Mobile AL 36688 USA Univ West Alabama Livingston AL 35470 USA Auburn Univ Auburn AL 36849 USA

ISBN: (纸本)9781510648043;9781510648036

Cyclic AMP (cAMP) is a second messenger that regulates a wide variety of cellular functions. There is increasing evidence suggesting that signaling specificity is due in part to cAMP compartmentalization. In the last 15 years, development of cAMP-specific Forster resonance energy transfer (FRET) probes have allowed us to visualize spatial distributions of intracellular cAMP signals. The use of FRET-based sensors is not without its limitations, as FRET probes display low signal to noise ratio (SNR). Hyperspectral imaging and analysis approaches have, in part, allowed us to overcome these limitations by improving the SNR of FRET measurements. Here we demonstrate that the combination of hyperspectral imaging approaches, linear unmixing, and adaptive thresholding allow us to visualize regions of elevated cAMP (regions of interest - ROIs) in an unbiased manner. We transfected cDNA encoding the H188 FRET-based cAMP probe into pulmonary microvascular endothelial cells. Application of isoproterenol and prostaglandin E1 (PGE(1)) triggered complex cAMP responses. Spatial and temporal aspects of cAMP responses were quantified using an adaptive thresholding approach and compared between agonist treatment groups. Our data indicate that both the origination sites and spatial/temporal distributions of cAMP signals are agonist dependent in PMVECs. We are currently analyzing the data in order to better quantify the distribution of cAMP signals triggered by different agonists.

关键词： FRET fluorescence microscopy spectral imaging adaptive thresholding second messengers cAMP signals localized signals

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