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

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Automated chondrocyte viability analysis of articular cartilage based on deep learning segmentation and classification of two-photon microscopic images 29

Automated chondrocyte viability analysis of articular cartil...

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

作者： Fan, Hongming Xu, Pei Le, Michael Hsu, Jennifer Chen, Xun Li, Yang Zhang, Zhao Gao, Bruce Woolf, Shane Ye, Tong Clemson Univ Dept Bioengn Clemson SC 29634 USA Clemson Univ Sch Comp Clemson SC USA Med Univ South Carolina Dept Orthopaed & Phys Med Charleston SC 29425 USA Med Univ South Carolina Dept Regenerat Med & Cell Biol Charleston SC 29425 USA

ISBN: (纸本)9781510648043;9781510648036

Chondrocyte viability is an important measure to consider when assessing cartilage health. Dye-based cell viability assays are not suitable for in vivo or long-term studies. We have introduced a non-labeling viability assay based on the assessment of high-resolution images of cells and collagen structure using two-photon stimulated autofluorescence and second harmonic generation microscopy. By either the visual or quantitative assessment, we were able to differentiate living from dead chondrocytes in those images. However, both techniques require human participation and have limited throughputs. Throughput can be increased by using methods for automated cell-based image processing. Due to the poor image contrast, traditional image processing methods are ineffective on autofluorescence images produced by nonlinear microscopes. In this work, we examined chondrocyte segmentation and classification using Mask R-CNN, a deep learning approach to implement automated viability analysis. It has been demonstrated an 85% accuracy in chondrocyte viability assessment with proper training. This study demonstrates that automated and highly accurate image analysis is achievable with the use of deep learning methods. This image processing approach can be helpful to other imaging applications in clinical medicine and biological research.

关键词： nonlinear optical microscopy autofluorescence second harmonic generation deep learning viability

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Low dosage 3D volume fluorescence microscopy imaging using compressive sensing 29

Low dosage 3D volume fluorescence microscopy imaging using c...

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

作者： Mannam, Varun Brandt, Jacob Smith, Cody J. Howard, Scott Univ Notre Dame Dept Elect Engn Notre Dame IN 46556 USA Univ Notre Dame Dept Biol Sci Notre Dame IN 46556 USA

ISBN: (纸本)9781510648043;9781510648036

Fluorescence microscopy has been a significant tool to observe long-term imaging of embryos (in vivo) growth over time. However, cumulative exposure is phototoxic to such sensitive live samples. While techniques like light-sheet fluorescence microscopy (LSFM) allows for reduced exposure, it is not well suited for deep imaging models. Other computational techniques are computationally expensive and often lack restoration quality. To address this challenge, one can use various low-dosage imaging techniques that are developed to achieve the 3D volume reconstruction using a few slices in the axial direction (z-axis);however, they often lack restoration quality. Also, acquiring dense images (with small steps) in the axial direction is computationally expensive. To address this challenge, we present a compressive sensing (CS) based approach to fully reconstruct 3D volumes with the same signal-to-noise ratio (SNR) with less than half of the excitation dosage. We present the theory and experimentally validate the approach. To demonstrate our technique, we capture a 3D volume of the RFP labeled neurons in the zebrafish embryo spinal cord (30 mu m thickness) with the axial sampling of 0.1 mu m using a confocal microscope. From the results, we observe the CS-based approach achieves accurate 3D volume reconstruction from less than 20% of the entire stack optical sections. The developed CS-based methodology in this work can be easily applied to other deep imaging modalities such as two-photon and light-sheet microscopy, where reducing sample photo-toxicity is a critical challenge.

关键词： Low-dosage imaging 3D volume reconstruction fluorescence microscopy compressive sensing (CS) discrete cosine transform (DCT) deep imaging in vivo imaging

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

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

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three-dimensional and multidimensional microscopy: image acquisition and processing XXVI 2019

ISBN: (纸本)9781510624085

The proceedings contain 20 papers. The topics discussed include: fluorescence recovery after photobleaching (FRAP) with simultaneous fluorescence lifetime and time-resolved fluorescence anisotropy imaging (FLIM & tr-FAIM);evaluation of a high-speed multispectral light source for stroboscopic differential imaging for endocardial examination of daphnia magna;fast stimulated Raman projection tomography with iterative reconstruction from sparse projections;Monte Carlo model for speckle contrast imaging;model-based iterative reconstruction for spectral-domain optical coherence tomography;and feasibility study of limited-angle reconstruction based in vivo optical projection tomography.

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