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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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Quantifying the drug response of patient-derived organoid clusters by aggregated morphological indicators with multi-parameters based on optical coherence tomography

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BIOMEDICAL OPTICS EXPRESS 2023年第4期14卷 1703-1717页

作者： Zhang, Linyi Wang, Ling Yang, Shanshan He, Kangxin Bao, Di Xu, Mingen Hangzhou Dianzi Univ Automat Coll Hangzhou Zhejiang Peoples R China Zhejiang Prov Key Lab Med Informat & Biol 3D Print Hangzhou Zhejiang Peoples R China Zhejiang Univ Affiliated Hosp 1 Sch Med Hangzhou Zhejiang Peoples R China

Patient-derived organoids (PDOs) serve as excellent tools for personalized drug screening to predict clinical outcomes of cancer treatment. However, current methods for efficient quantification of drug response are limited. Herein, we develop a method for label-free, continuous tracking imaging and quantitative analysis of drug efficacy using PDOs. A self-developed optical coherence tomography (OCT) system was used to monitor the morphological changes of PDOs within 6 days of drug administration. OCT image acquisition was performed every 24 h. An analytical method for organoid segmentation and morphological quantification was developed based on a deep learning network (EGO-Net) to simultaneously analyze multiple morphological organoid parameters under the drug's effect. Adenosine triphosphate (ATP) testing was conducted on the last day of drug treatment. Finally, a corresponding aggregated morphological indicator (AMI) was established using principal component analysis (PCA) based on the correlation analysis between OCT morphological quantification and ATP testing. Determining the AMI of organoids allowed quantitative evaluation of the PDOs responses to gradient concentrations and combinations of drugs. Results showed that there was a strong correlation (correlation coefficient >90%) between the results using the AMI of organoids and those from ATP testing, which is the standard test used for bioactivity measurement. Compared with single-time-point morphological parameters, the introduction of time-dependent morphological parameters can reflect drug efficacy with improved accuracy. Additionally, the AMI of organoids was found to improve the efficiency of 5-fluorouracil(5FU) against tumor cells by allowing the determination of the optimum concentration, and the discrepancies in response among different PDOs using the same drug combinations could also be measured. Collectively, the AMI established by OCT system combined with PCA could quantify the multidimensional mor

关键词： Analytical techniques image processing Imaging techniques Optical coherence tomography Spectral domain optical coherence tomography three dimensional imaging

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A Targeted Sampling Strategy for Compressive Cryo Focused Ion Beam Scanning Electron microscopy 48

A Targeted Sampling Strategy for Compressive Cryo Focused Io...

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48th IEEE International Conference on Acoustics, Speech and Signal processing, ICASSP 2023

作者： Nicholls, D. Wells, J. Robinson, A.W. Moshtaghpour, A. Kobylynska, M. Fleck, R.A. Kirkland, A.I. Browning, N.D. University of Liverpool Distributed Algorithms Centre for Doctoral Training United Kingdom King's College London Centre for Ultrastructural Imaging London United Kingdom Harwell Science and Innovation Campus Correlated Imaging Group Rosalind Franklin Institute Didcot United Kingdom University of Oxford Department of Materials United Kingdom University of Liverpool Department of Mechanical Materials and Aerospace Engineering United Kingdom Pacific Northwest National Laboratory Physical and Computational Science Richland United States Sivananthan Laboratories 590 Territorial Drive BolingbrookIL United States

ISBN: (纸本)9781728163277

Cryo Focused Ion-Beam Scanning Electron microscopy (cryo FIB-SEM) enables three-dimensional and nanoscale imaging of biological specimens via a slice and view mechanism. The FIB-SEM experiments are, however, limited by a slow (typically, several hours) acquisition process and the high electron doses imposed on the beam sensitive specimen can cause damage. In this work, we present a compressive sensing variant of cryo FIB-SEM capable of reducing the operational electron dose and increasing speed. We propose two Targeted Sampling (TS) strategies that leverage the reconstructed image of the previous sample layer as a prior for designing the next subsampling mask. Our image recovery is based on a blind Bayesian dictionary learning approach, i.e., Beta Process Factor Analysis (BPFA). This method is experimentally viable due to our GPU-based implementation of BPFA. Simulations on artificial compressive FIB-SEM measurements validate the success of proposed methods: the operational electron dose can be reduced by up to 20 times. These methods have large implications for the cryo FIB-SEM community, in which the imaging of beam sensitive biological materials without beam damage is crucial. © 2023 IEEE.

关键词： Beta-process factor analysis Compressive sensing Focused ion beam Scanning electron microscopy

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DOF adjustment and 3D free viewpoint viewing of Z stack data of fluorescent microscope using image processing 28

DOF adjustment and 3D free viewpoint viewing of Z stack data...

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three-dimensional and multidimensional microscopy: image acquisition and processing XXVIII 2021

作者： Hagiwara, Tsuneyuki 3rd Research Section Optical Research Laboratory Research and Development Division Nikon Corporation Japan

ISBN: (数字)9781510641341

ISBN: (纸本)9781510641334

In recent years, confocal fluorescence microscopes have become widespread and many Z-stack data have been collected. However, it is the current situation that there is hardly a method for three-dimensional observation of huge amounts of data with sufficient resolution and without loss of information. In light of this situation, we have developed an image processing algorithm, referred to as arbitrary blur algorithm (ABA), which enables post-adjustment of DOF and 3D image observation from a free viewpoint, with high resolution and without loss of information. In this report, we first explain the principle of image processing that enables depth of field (DOF) adjustment and 3D image observation from a free viewpoint using Z-stack data. Next, the results of the simulation are introduced. In the simulation, we show the comparison result of the point image by MIP (maximum intensity projection) method and the point image of the proposed method and clarify the superiority of the proposed method. Next, we introduce the results of the experimental verification. For experimental verification, we show the results using wide field fluorescence microscope Z-stack data and confocal fluorescence microscope Z-stack data. As confocal data, observation examples of mouse hippocampal neuron synapses are shown in comparison with the results displayed by existing software. © 2021 Copyright SPIE.

关键词： Microscopes

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Robust gaussian fitting for super-resolution microscopy with fast parameters initial estimation 28

Robust gaussian fitting for super-resolution microscopy with...

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three-dimensional and multidimensional microscopy: image acquisition and processing XXVIII 2021

作者： Qin, Shun Chan, Wai Kin Tsinghua Shenzhen International Graduate School Shenzhen China

ISBN: (数字)9781510641341

ISBN: (纸本)9781510641334

Efficient accurate Gaussian fitting is an important topic in many applications, take localization based super-resolution microscopy and image scanning microscopy for example, which requires localizing plenty of Gaussian patterns for accurately reconstructing a super-resolution image. Existing Gaussian fitting methods usually require inputting a good initial value for all parameters for efficient and robust fitting, which apparently is not suitable for the task of large scale Gaussian fitting with a computer. It would be even more challenge to estimate an appropriate initial value for all parameters and guarantee the robustness of the fitting algorithm for low signal-to-noise ratio measured data with strong background. In this paper, we propose a two-step fitting algorithm for robust and accurate 2D Gaussian fitting without inputting any initial parameter value. Our simulation shows that the performance of the fitting algorithm can be improved significantly with the proposed parameter initial value estimation algorithm. © 2021 Copyright SPIE.

关键词： Optical resolving power

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Sparsity-based approach for 3D super-resolution microscopy from correlation information of high emitter-density frames 28

Sparsity-based approach for 3D super-resolution microscopy f...

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three-dimensional and multidimensional microscopy: image acquisition and processing XXVIII 2021

作者： Dardikman-Yoffe, Gili Eldar, Yonina C. Faculty of Mathematics and Computer Science Weizmann Institute of Science Israel

ISBN: (数字)9781510641341

ISBN: (纸本)9781510641334

Breakthroughs in the field of chemistry have enabled surpassing the classical optical diffraction limit by utilizing photo-activated fluorescent molecules. In the single-molecule localization microscopy (SMLM) approach, a sequence of diffraction-limited images, produced by a sparse set of emitting fluorophores with minimally overlapping point-spread functions is acquired, allowing the emitters to be localized with high precision by simple post-processing. However, the low emitter density concept requires lengthy imaging times to achieve full coverage of the imaged specimen on the one hand, and minimal overlap on the other. Thus, this concept in its classical form has low temporal resolution, limiting its application to slow-changing specimens. In recent years, a variety of approaches have been suggested to reduce imaging times by allowing the use of higher emitter densities. One of these methods is the sparsity-based approach for super-resolution microscopy from correlation information of high emitter-density frames, dubbed SPARCOM, which utilizes sparsity in the correlation domain while assuming that the blinking emitters are uncorrelated over time and space, yielding both high temporal and spatial resolution. However, SPARCOM has only been formulated for the two-dimensional setting, where the sample is assumed to be an infinitely thin single-layer, and thus is unsuitable to most biological specimens. In this work, we present an extension of SPARCOM to the more challenging three-dimensional scenario, where we recover a volume from a set of recorded frames, rather than an image. © 2021 Copyright SPIE.

关键词： Optical transfer function

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Time-resolved multi-spectral wide-field fluorescence lifetime imaging microscopy with a SPAD array detector 28

Time-resolved multi-spectral wide-field fluorescence lifetim...

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three-dimensional and multidimensional microscopy: image acquisition and processing XXVIII 2021

作者： Ghezzi, A. Farina, A. Bassi, A. Valentini, G. Labanca, I. Acconcia, G. Rech, I. D'Andrea, C. Politecnico di Milano Dipartimento di Fisica Piazza L. da Vinci 32 Milan20133 Italy Consiglio Nazionale delle Ricerche IFN Piazza L. da Vinci 32 Milan20133 Italy Politecnico di Milano Dipartimento di Elettronica Informazione e Bioingegneria via Golgi 40 Milan20133 Italy Center for NanoScience and Technology @PoliMi Istituto Italiano di Tecnologia via Pascoli 70/3 Milan20133 Italy

ISBN: (数字)9781510641341

ISBN: (纸本)9781510641334

Multispectral Fluorescence Lifetime Imaging microscopy (FLIM) is a fundamental tool to study multifold processes in biology and material science. The growing demand for acquisition time reduction requires the parallel acquisition of a multi-dimensional dataset and the exploitation of compressive sensing techniques. In this work we present a multispectral FLIM set-up based on wide-field structured illumination coupled with a spectrometer and a novel time-resolved parallel 18x1 SPAD array detector, working in a single pixel camera scheme. We show the system characterization and its imaging properties varying the compression ratio. © 2021 Copyright SPIE.

关键词： Compressed sensing

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Evaluation of different illumination designs for tunable 3D structured illumination microscopy through model-based restoration 28

Evaluation of different illumination designs for tunable 3D ...

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three-dimensional and multidimensional microscopy: image acquisition and processing XXVIII 2021

作者： Mohammed, Jules Van, Cong T. S. Preza, Chrysanthe Computational Imaging Research Laboratory Department of Electrical and Computer Engineering University of Memphis MemphisTN38152 United States

ISBN: (数字)9781510641341

ISBN: (纸本)9781510641334

In a tunable 3D structured illumination microscopy (3D-SIM) based on an illumination system comprised by a multi-slit array and a Fresnel biprism, the 3D structured illumination (SI) pattern depends on the design of the slit array. Previous work studied the impact of the illumination pattern design on the achieved extension of the compact support of the tunable 3D-SIM optical transfer function. In this contribution, we use simulations with different illumination designs that utilize a different number of slits, to evaluate system performance by investigating the lateral and axial resolution achieved in the restored images computed with a regularized iterative 3D model-based algorithm, which minimizes the mean square error between the model and data. three illumination designs are investigated in which the number of slits is N = 3, 9 and 11 while the corresponding distance between the slits is x0 = 488 μm, 122 μm and 100 μm, respectively. A 3D star-like object, commonly used in resolution analysis of imaging systems, is used in the simulations. Results are quantified using the mean square error and the structured similarity index as well as intensity profiles that show the achieved resolution and robustness to noise in each case. The lateral and axial 3D-SIM theoretical resolution limits are achieved in the presence of 20-dB Poisson noise while small differences are evident in the results from 15-dB simulated data obtained using the 3 investigated designs. © 2021 Copyright SPIE.

关键词： Fluorescence microscopy

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Deconvolution optical-resolution photoacoustic microscope for high-resolution imaging of cerebral vasculature 28

Deconvolution optical-resolution photoacoustic microscope fo...

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three-dimensional and multidimensional microscopy: image acquisition and processing XXVIII 2021

作者： Song, Xianlin Song, Lingfang Chen, Anqing Wei, Jianshuang Zhao, Aojie Teng, Ao School of Information Engineering Nanchang University Nanchang330031 China Nanchang Normal University Nanchang330031 China Britton Chance Center for Biomedical Photonics Wuhan National Laboratory for Optoelectronics Huazhong University of Science and Technology Wuhan430074 China Moe Key Laboratory of Biomedical Photonics of Ministry of Education Department of Biomedical Engineering Huazhong University of Science and Technology Wuhan430074 China

ISBN: (数字)9781510641341

ISBN: (纸本)9781510641334

Photoacoustic imaging is becoming a very promising tool for the research of living organisms. It combines the high contrast of optical imaging and the high resolution of acoustic imaging to realize the imaging of absorption clusters in biological tissues. Since the scattering of ultrasound signals in biological tissues is 2-3 orders of magnitude weaker than the scattering of light in biological tissues, the endogenous absorption difference of tissues is directly used in the imaging process, so photoacoustic imaging has the advantages of deep imaging depth and non-destructive. As an important branch of photoacoustic imaging, photoacoustic microscopy can provide micron-level or even sub-micron-level imaging resolution, which is of great significance for biological research such as blood vessel detection. Since the lateral resolution of the photoacoustic microscopy imaging system depends on the focus of the laser, a higher resolution can be obtained by increasing the numerical aperture of the condenser objective. However, a large numerical aperture usually means a shorter working distance and makes the entire imaging system very sensitive to small optical defects. Therefore, the improvement of resolution through this method will be limited in practical applications. This paper implements a method of using iterative deconvolution to obtain a high-resolution photoacoustic image of the brain. The focal spot of the photoacoustic microscopy is measured to obtain the lateral PSF (point spread function) of the system. Making the measured PSF as the initial system PSF to perform Lucy-Richardson (LR) deconvolution. The image resolution of cerebral vasculature obtained by this method is higher. The full width at half maximum (FWHM) of width of the same cerebral capillaries before and after deconvolution are 7 μm and 3.6 μm, respectively, and the image definition is increased by about 1.9 times. Experiments show that this method can further improve the clarity of photoacoustic im

关键词： Photoacoustic microscopy

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Multi-immersion open-top light-sheet microscopy 27

Multi-immersion open-top light-sheet microscopy

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

作者： Glaser, Adam K. Liu, Jonathan T. C. Univ Washington Dept Mech Engn Seattle WA 98195 USA

We present an easy-to-use multi-immersion open-top light-sheet microscope designed specifically for high-throughput imaging of a diverse set of tissues prepared with a variety of clearing protocols.

ISBN: (纸本)9781510632547

We present an easy-to-use multi-immersion open-top light-sheet microscope designed specifically for high-throughput imaging of a diverse set of tissues prepared with a variety of clearing protocols.

关键词： light-sheet microscopy selective-plane illumination microscopy fluorescence microscopy

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