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Conference on 3-dimensional and multidimensional microscopy: image acquisition and processing IX

作者： Knowles, DW Keränen, S Biggin, MD Sudar, D Lawrence Berkeley Lab Dept Cell & Mol Biol Berkeley CA 94720 USA

ISBN: (纸本)0819443603

The development of an animal embryo is orchestrated by a network of genetically determined, temporal and spatial gene expression patterns that determine the animals final form. To understand such networks, we are developing novel quantitative optical imaging techniques to map gene expression levels at cellular and sub-cellular resolution within pregastrula Drosophila. Embryos at different stages of development are labeled for total DNA and specific gene products using different fluorophors and imaged in 3D with confocal microscopy. Innovative steps have been made which allow the DNA-image to be automatically segmented to produce a morphological mask of the individual nuclear boundaries. For each stage of development an "average" morphology is chosen to which images from different embryo are compared. The morphological mask is then used to quantify gene-product on a per nuclei basis. What results is an atlas of the relative amount of the specific gene product expressed within the nucleus of every cell in the embryo at the various stages of development. We are creating a quantitative database of transcription factor and target gene expression patterns in wild-type and factor mutant embryos with single cell resolution. Our goal is to uncover the rules determining how patterns of gene expression are generated.

关键词： confocal imaging image segmentation image registration quantitative fluorescence analysis regulatory gene networks Drosophila

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A state space based approach to localizing single molecules from multi-emitter images 24

A state space based approach to localizing single molecules ...

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

作者： Vahid, Milad R. Chao, Jerry Ward, E. Sally Ober, Raimund J. Texas A&M Univ Dept Biomed Engn College Stn TX 77843 USA Texas A&M Univ Hlth Sci Ctr Dept Mol & Cellular Med College Stn TX 77843 USA Texas A&M Univ Hlth Sci Ctr Dept Microbial Pathogenesis & Immunol College Stn TX 77843 USA

ISBN: (数字)9781510605824

ISBN: (纸本)9781510605817;9781510605824

Single molecule super-resolution microscopy is a powerful tool that enables imaging at sub-diffraction-limit resolution. In this technique, subsets of stochastically photoactivated fluorophores are imaged over a sequence of frames and accurately localized, and the estimated locations are used to construct a high-resolution image of the cellular structures labeled by the fluorophores. Available localization methods typically first determine the regions of the image that contain emitting fluorophores through a process referred to as detection. Then, the locations of the fluorophores are estimated accurately in an estimation step. We propose a novel localization method which combines the detection and estimation steps. The method models the given image as the frequency response of a multi-order system obtained with a balanced state space realization algorithm based on the singular value decomposition of a Hankel matrix, and determines the locations of intensity peaks in the image as the pole locations of the resulting system. The locations of the most significant peaks correspond to the locations of single molecules in the original image. Although the accuracy of the location estimates is reasonably good, we demonstrate that, by using the estimates as the initial conditions for a maximum likelihood estimator, refined estimates can be obtained that have a standard deviation close to the Cram ' er-Rao lower bound-based limit of accuracy. We validate our method using both simulated and experimental multi-emitter images.

关键词： Fluorescence microscopy frequency response multi-emitter localization single molecule localization single molecule microscopy singular value decomposition state space realization super-resolution microscopy

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Multi-beam optical coherence tomography for microvascular imaging of human skin in vivo 24

Multi-beam optical coherence tomography for microvascular im...

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

作者： Chen, Chaoliang Cheng, Kyle H. Y. Jakubovic, Raphael Jivraj, Jamil Ramjist, Joel Deorajh, Ryan Gao, Wanrong Yang, Victor X. D. Nanjing Univ Sci & Technol Dept Opt Engn Nanjing Jiangsu Peoples R China Ryerson Univ Dept Elect & Comp Engn Biophoton & Bioengn Lab Toronto ON Canada Sunnybrook Hlth Sci Ctr Div Neurosurg Toronto ON Canada Ryerson Univ Dept Biomed Phys Toronto ON Canada Univ Toronto Fac Med Div Neurosurg Toronto ON Canada

ISBN: (数字)9781510605824

ISBN: (纸本)9781510605817;9781510605824

In this paper, a multi-beam optical coherence tomography (OCT) was used to reconstruct the microvascular image of human skin in vivo with phase resolved Doppler OCT (PRDOCT), phase resolved Doppler variance (PRDV) and speckle variance OCT (svOCT), in which the blood flow image was calculated by averaging the four blood flow images obtained by the four beams. In PRDOCT method, it is difficult to detect the blood flow perpendicular to optical axis of the probe beam for single beam OCT, but the multi-beam scanning method can solve this because the input angles of the four probe beams are slightly different from each other. The proposed method can further improve the signal-to-noise ratio (SNR) of the blood flow signals extracted by the three methods mentioned above.

关键词： Optical coherence tomography medical and biological imaging tomographic image processing

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Development of a temporal multiplexed 3D beam-scanning Lissajous trajectory microscope for rapid, multimodal volumetric imaging 13

Development of a temporal multiplexed 3D beam-scanning Lissa...

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

作者： Newman, Justin A. Sullivan, Shane Z. Dinh, Janny Sarkar, Sreya Simpson, Garth J. Purdue Univ Dept Chem 560 Oval Dr W Lafayette IN 47907 USA

ISBN: (纸本)9781628419474

A beam-scanning microscope is described based on a temporally multiplexed Lissajous trajectory for achieving 1 kHz frame rate 3D imaging. The microscope utilizes two fast-scan resonant mirrors to direct the optical beam on a circuitous, Lissajous trajectory through the field of view. acquisition of two simultaneous focal planes is achieved by implementation of an optical delay line, producing a second incident beam at a different focal plane relative to the initial incident beam. High frame rates are achieved by separating the full time-domain data into shorter sub-trajectories resulting in undersampling of the field of view. A model-based image reconstruction (MBIR) 3D in-painting algorithm is utilized for interpolating the missing data to recover full images. The MBIR algorithm uses a maximum a posteriori estimation with a generalized Gaussian Markov random field prior model for image interpolation. Because images are acquired using photomultiplier tubes or photodiodes, parallelization for multi-channel imaging is straightforward. Preliminary results obtained using a Lissajous trajectory beam-scanning approach coupled with temporal multiplexing by the implementation of an optical delay line demonstrate the ability to acquire 2 distinct focal planes simultaneously at frame rates >450 Hz for full 512 x 512 images. The use of multi-channel data acquisition cards allows for simultaneous multimodal image acquisition with perfect image registry between all imaging modalities. Also discussed here is the implementation of Lissajous trajectory beam-scanning on commercially available microscope hardware.

关键词： Lissajous multi-channel beam-scanning temporal multiplexing 3D imaging nonlinear optics image interpolation

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Investigating the performance of reconstruction methods used in structured illumination microscopy as a function of the illumination pattern's modulation frequency 13

Investigating the performance of reconstruction methods used...

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

作者： Shabani, H. Sanchez-Ortiga, E. Preza, C. Univ Memphis Dept Elect & Comp Engn Memphis TN 38152 USA Univ Valencia Dept Opt E-46100 Burjassot Spain

ISBN: (纸本)9781628419474

Surpassing the resolution of optical microscopy defined by the Abbe diffraction limit, while simultaneously achieving optical sectioning, is a challenging problem particularly for live cell imaging of thick samples. Among a few developing techniques, structured illumination microscopy (SIM) addresses this challenge by imposing higher frequency information into the observable frequency band confined by the optical transfer function (OTF) of a conventional microscope either doubling the spatial resolution or filling the missing cone based on the spatial frequency of the pattern when the patterned illumination is two-dimensional. Standard reconstruction methods for SIM decompose the low and high frequency components from the recorded low-resolution images and then combine them to reach a high-resolution image. In contrast, model-based approaches rely on iterative optimization approaches to minimize the error between estimated and forward images. In this paper, we study the performance of both groups of methods by simulating fluorescence microscopy images from different type of objects ( ranging from simulated two-point sources to extended objects). These simulations are used to investigate the methods' effectiveness on restoring objects with various types of power spectrum when modulation frequency of the patterned illumination is changing from zero to the incoherent cut-off frequency of the imaging system. Our results show that increasing the amount of imposed information by using a higher modulation frequency of the illumination pattern does not always yield a better restoration performance, which was found to be depended on the underlying object. Results from model-based restoration show performance improvement, quantified by an up to 62% drop in the mean square error compared to standard reconstruction, with increasing modulation frequency. However, we found cases for which results obtained with standard reconstruction methods do not always follow the same trend.

关键词： structured illumination modulation frequency 3D microscopy optical sectioning super-resolution

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In-vivo Third-Harmonic Generation microscopy at 1550nm three-dimensional long-term time-lapse studies in living C. elegans embryos

In-vivo Third-Harmonic Generation Microscopy at 1550nm three...

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

作者： Aviles-Espinosa, Rodrigo Santos, Susana I. C. O. Brodschelm, Andreas Kaenders, Wilhelm G. Alonso-Ortega, Cesar Artigas, David Loza-Alvarez, Pablo ICFO Inst Photon Sci Mediterranean Techn PkAv Canal Olimp S-N Castelldefels 08860 Barcelona Spain TOPTICA Photon AG D-82166 Graefelfing Germany Univ Politecn Cataluna Dept Signal Theory & Commun E-08034 Barcelona Spain

ISBN: (纸本)9780819484413

In-vivo microscopic long term time-lapse studies require controlled imaging conditions to preserve sample viability. Therefore it is crucial to meet specific exposure conditions as these may limit the applicability of established techniques. In this work we demonstrate the use of third harmonic generation (THG) microscopy for long term time-lapse three-dimensional studies (4D) in living Caenorhabditis elegans embryos employing a 1550 nm femtosecond fiber laser. We take advantage of the fact that THG only requires the existence of interfaces to generate signal or a change in the refractive index or in the chi(3) nonlinear coefficient, therefore no markers are required. In addition, by using this wavelength the emitted THG signal is generated at visible wavelengths (516 nm) enabling the use of standard collection optics and detectors operating near their maximum efficiency. This enables the reduction of the incident light intensity at the sample plane allowing to image the sample for several hours. THG signal is obtained through all embryo development stages, providing different tissue/structure information. By means of control samples, we demonstrate that the expected water absorption at this wavelength does not severely compromise sample viability. Certainly, this technique reduces the complexity of sample preparation (i.e. genetic modification) required by established linear and nonlinear fluorescence based techniques. We demonstrate the non-invasiveness, reduced specimen interference, and strong potential of this particular wavelength to be used to perform long-term 4D recordings.

关键词： Multiphoton processes Nonlinear microscopy Fiber Laser Biological in-vivo imaging three dimensional Time-lapse imaging

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High Brightness LED in Confocal microscopy 22

High Brightness LED in Confocal Microscopy

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

作者： Vakili, Ali Xiong, Daxi Rajadhyaksha, Milind DiMarzio, Charles A. Northeastern Univ Boston MA 02115 USA Chinese Acad Sci Suzhou Inst Biomed Engn & Technol Jiangsu Key Lab Med Opt Suzhou Peoples R China Mem Sloan Kettering Canc Ctr New York NY USA

ISBN: (纸本)9781628414202

We have introduced a novel illumination system for line scanning confocal microscopy. Confocal microscopy is a popular imaging tool in many applications specifically in medical imaging. Line scanning confocal microscopes have been proven to provide images with resolution comparable to point scanning microscopes. In the point scanning microscopes, the light is focused onto a diffraction limited spot. A pinhole is placed conjugate to the diffraction limited spot, in front of the detector to reject the light coming from out-of-focus planes. Therefore, confocal microscopy can provide optical sectioning. The size of the pinhole determines the amount of light that reaches the detector. A large pinhole results in a blurry image since more of the out-of-focus light contribute to the image. On the other hand, a smaller pinhole rejects more of the light, leading to a lower signal-to-noise ratio. Ideally it is desired to deliver a larger amount of optical power to the diffraction limited spot to increase the signal-to-noise ratio and have a smaller pinhole to reject more of the out-of-focus light. This is the property of the illumination system. In order to get a good signal-to noise ratio in the image, the light source has to provide sufficient radiance. We have introduced a new illumination system utilizing a high brightness LED in the line scanning confocal microscope. High brightness LEDs provide more optical power compared to ordinary LEDs from a smaller area;they have higher radiance. Preliminary results from our line scanning confocal microscope show that the high brightness LED is able to provide enough radiance to obtain an image with resolution comparable with the same microscope utilizing the laser diode. However, in high frame-rate application higher radiance or lower-noise detection system is required.

关键词： High Brightness LED Confocal Line-scan Radiance

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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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Point spread function engineering with multiphoton SPIFI 13

Point spread function engineering with multiphoton SPIFI

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

作者： Wernsing, Keith A. Field, Jeffrey J. Domingue, Scott R. Allende-Motz, Alyssa M. DeLuca, Keith F. Levi, Dean H. DeLuca, Jennifer G. Young, Michael D. Squier, Jeff A. Bartels, Randy A. Colorado State Univ Dept Elect & Comp Engn Ft Collins CO 80523 USA Colorado State Univ Dept Biochem & Mol Biol Ft Collins CO 80523 USA Colorado State Univ Microscopy Core Facil Ft Collins CO 80523 USA Colorado State Univ Inst Genome Architecture & Funct Ft Collins CO 80523 USA Colorado Sch Mines Dept Phys Golden CO 80401 USA Natl Renewable Energy Lab Golden CO USA Colorado State Univ Sch Biomed Engn Ft Collins CO 80523 USA

ISBN: (纸本)9781628419474

MultiPhoton SPatIal Frequency modulated Imaging (MP-SPIFI) has recently demonstrated the ability to simultaneously obtain super-resolved images in both coherent and incoherent scattering processes - namely, second harmonic generation and two-photon fluorescence, respectively. 1 In our previous analysis, we considered image formation produced by the zero and first diffracted orders from the SPIFI modulator. However, the modulator is a binary amplitude mask, and therefore produces multiple diffracted orders. In this work, we extend our analysis to image formation in the presence of higher diffracted orders. We find that tuning the mask duty cycle offers a measure of control over the shape of super-resolved point spread functions in an MP-SPIFI microscope.

关键词： multiphoton microscopy super-resolution SPIFI

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Multi-dimensional Raman spectroscopy: from the UV to the near-IR

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Proceedings of SPIE - The International Society for Optical Engineering 1999年 3605卷 308-316页

作者： Barnett, Steven M. Bormett, Richard W. Whitley, Andrew Renishaw Inc Schaumburg United States

ISBN: (纸本)0819430757;9780819430755

The structural elucidation of complex systems may be simplified with multi-dimensional spectroscopic techniques with some combination of spatial and spectral resolution. Raman spectroscopy permits the addition of another variable to this scenario -excitation wavelength. Data obtained using excitation wavelengths from the UV (244 nm) to near-IR (785 nm) regions will be presented showing the qualitative and quantitative study of diamond-like carbon (DLC), silicon, and other systems of an industrial or biomedical nature. The choice of appropriate wavelength provides an additional advantage over other spectroscopic techniques for elucidating specific structural information from these systems. The advantages of UV-Raman for materials science and thin film studies will be considered. The design of instruments and probes for the application of Raman spectroscopy to industrial process control and the development of Raman spectroscopic libraries for contaminant analysis will be discussed.

关键词： Raman spectroscopy

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