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Measurement of the chirp dependent fluorescence response: a window to excited state population dynamics

Proceedings of SPIE - The International Society for Optical Engineering 1999年 3605卷 40-47页

作者： Brakenhoff, G.J. Buist, Arjan H. Mueller, Michiel Squier, Jeff A. Bardeen, Chris J. Yakovlev, Vladislav V. Wilson, Kent R. Univ of Amsterdam Amsterdam Netherlands

ISBN: (纸本)0819430757;9780819430755

High intensity chirped pulses can be used for probing microscopic chemical environments through the use of a particular choice of dye, for instance SNAFL2. The basis for this technique is that the excited state populations can be manipulated through control over the temporal order of the excitation frequencies in the excitation pulse - i.e. chirp - with the outcoming fluorescence as the reporting parameter. A chirp dependent fluorescence response can also be observed in larger molecular systems with more degrees of freedom like for instance green fluorescent proteins. In preparation for application of the technique to microscopy we use a facility permitting observation of this phenomenon in various dyes with high sensitivity. High power, 30 fs pulses from an OPA, tunable from 400 nm to 1.5 micron are used. These pulses with a repetition rate of 1 kHz are sufficiently intense that a relatively large sample region can be excited to saturation from which then a sub-region with uniform excitation conditions can be selected for signal collection.

关键词： Optical microscopy

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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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Imaging modalities in confocal correlation microscopy

Imaging modalities in confocal correlation microscopy

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Proceedings of the 1999 three-dimensional and multidimensional microscopy: image acquisition and processing VI

作者： Juskaitis, Rimas Neil, Mark A.A. Wilson, Tony Oxford Univ Oxford United Kingdom

ISBN: (纸本)0819430757

We present a novel light efficient technique for obtaining optically sectioned images in wide-field microscopy. The technique is a further development of correlation microscopy and is based on using complementary structured light patterns to illuminate the specimen together with uncomplicated post-processing of the captured images.

关键词： Optical 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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Feature extraction and 3D reconstruction of Nomarski DIC microscope images using local energy and neural net techniques

Feature extraction and 3D reconstruction of Nomarski DIC mic...

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Conference on 3-dimensional and multidimensional microscopy - image acquisition and processing VI

作者： Kyan, M Guan, L Arison, M Cogswell, C Univ Sydney Dept Elect Engn Sydney NSW 2006 Australia

ISBN: (纸本)0819430757

The Nomarski differential interference contrast (DIC) mode is commonly used for imaging translucent biological specimens and it exhibits several major advantages over other phase contrast techniques, including a boost in high spatial frequencies in the region of focus. However, DIC images (unlike confocal) are limited by the presence of low spatial frequency blur and also by a differential shading gradient at feature boundaries, which make normal confocal visualisation techniques unsuitable for feature extraction or for 3D volume rendering of focus-series datasets. To remedy this problem, we employ a neural network technique based on competitive learning, known as Kohonen's self-organising feature map (SOFM), to perform segmentation, using a collection of statistics (know as features) defining the image. Our past investigation showed that standard features such as the localised mean and variance of pixel intensities provided reasonable extraction of objects such asmitotic chromosomes, but surface detail was only moderately resolved. In this current work, local energy is investigated as an alternative image statistic based on phase congruency in the image. This, along with different combinations of other image statistics, is applied in a SOFM, producing 3D images exhibiting vast improvement in the level of detail and clearly isolating the chromosomes from the background.

关键词： differential interference contrast DIC feature extraction feature space image segmentation self organising feature map SOFM phase congruency local energy Morlet wavelet

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Tissomics:: two- and threedimensional distribution of nuclei in brain tissue using Laser Scanning Cytometry (LSC)

Tissomics:: two- and threedimensional distribution of nuclei...

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Conference on 3-dimensional and multidimensional microscopy - image acquisition and processing XII

作者： Lenz, D Mittag, A Mosch, B Bocsi, J Arendt, T Tárnok, A Univ Leipzig Heart Ctr Leipzig Leipzig Germany

ISBN: (纸本)0819456756

Automated quantitative (i.e. stochiometric) analysis of tissues is of eminent importance in the understanding of all interactions between cells in their natural environment. In tissue cytometry a solid trigger is necessary in order to unequivocally differentiate between cellular and non-cellular events. This can be best performed by nuclear staining. Aim of this study was to analyze a brain tissue section by laser scanning cytometry (LSC) in order to depict the threedimensional distribution of nuclei in the tissue. To this end the section was measured in several foci and different nuclei detected in several depths of the tissue were assigned to the respective layer. Frozen sections of formalin-fixed rat or human brain tissue (120 mu m thickness) were incubated with propidiumiodide (PI) (50 mu g/ml) and covered on slides. For analysis by the LSC propidiumiodide was used as trigger. After a first analysis focussed on the top of the tissue, the focus was adjusted in 30 mu m steps deeper into the tissue. Per analysis data of at least 50,000 cells were acquired. After finishing measurements from all depths of the field were merged, i.e. data were combined into a composite data file. With the special features of the LSC it was possible to develop a method depicting the threedimensional distribution of the nuclei in solid tissue sections. LSC can be useful tool for this relatively new field of solid tissue cytometry termed tissomics. After evaluation of methods like this, so far not available data can be analysed for diagnostic purposes. By these studies we intend to demonstrate the power of the LSC for the routine pathological use. This should add up to the bright versatility of applications for the LSC as a cytometric instrument suitable for high throughput and high content analysis.

关键词： Alzheimer's disease cell cycle DNA-polyploidy laser scanning cytometry cytomics

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Tunable thin-film optical filters for hyperspectral microscopy

Tunable thin-film optical filters for hyperspectral microsco...

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

作者： Favreau, Peter F. Rich, Thomas C. Prabhat, Prashant Leavesley, Silas J. Univ S Alabama Biomol Chem engn Mobile AL 36688 USA Univ S Alabama Pharmacol Mobile AL 36688 USA Univ S Alabama Ctr Lung Biol Mobile AL 36688 USA Semrock Inc A Unit Index Mobile AL 36688 USA

ISBN: (纸本)9780819493583

Hyperspectral imaging was originally developed for use in remote sensing applications. More recently, it has been applied to biological imaging systems, such as fluorescence microscopes. The ability to distinguish molecules based on spectral differences has been especially advantageous for identifying fluorophores in highly autofluorescent tissues. A key component of hyperspectral imaging systems is wavelength filtering. Each filtering technology used for hyperspectral imaging has corresponding advantages and disadvantages. Recently, a new optical filtering technology has been developed that uses multi-layered thin-film optical filters that can be rotated, with respect to incident light, to control the center wavelength of the pass-band. Compared to the majority of tunable filter technologies, these filters have superior optical performance including greater than 90% transmission, steep spectral edges and high out-of-band blocking. Hence, tunable thin-film optical filters present optical characteristics that may make them well-suited for many biological spectral imaging applications. An array of tunable thin-film filters was implemented on an inverted fluorescence microscope (TE 2000, Nikon Instruments) to cover the full visible wavelength range. images of a previously published model, GFP-expressing endothelial cells in the lung, were acquired using a charge-coupled device camera (Rolera EM-C2, Q-Imaging). This model sample presents fluorescently-labeled cells in a highly autofluorescent environment. Linear unmixing of hyperspectral images indicates that thin-film tunable filters provide equivalent spectral discrimination to our previous acousto-optic tunable filter-based approach, with increased signal-to-noise characteristics. Hence, tunable multi-layered thin film optical filters may provide greatly improved spectral filtering characteristics and therefore enable wider acceptance of hyperspectral widefield microscopy.

关键词： Hyperspectral imaging spectral imaging microscopy AOTF acousto-optic tunable filter TFTF thin-film tunable filter VersaChrome

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3D+time acquisitions of 3D cell culture by means of lens-free tomographic microscopy 25

3D+time acquisitions of 3D cell culture by means of lens-fre...

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three-dimensional and multidimensional microscopy: image acquisition and processing XXV 2018

作者： Berdeu, Anthony Laperrousaz, Bastien Bordy, Thomas Morales, S. Gidrol, Xavier Picollet-D'Hahan, Nathalie Allier, Cédric Univ. Grenoble Alpes CEA LETI DTBS LISA GrenobleF-38000 France Université Grenoble-Alpes CEA BIG INSERM GrenobleF-38054 France

ISBN: (数字)9781510614840

ISBN: (纸本)9781510614833

We propose a three-dimensional (3D) imaging platform based on lens-free microscopy to perform multi-angle acquisitions on 3D cell cultures embedded in extracellular matrix (ECM). We developed algorithms based on the Fourier diffraction theorem to perform fully 3D reconstructions of biological samples and we adapted the lens-free microscope to incubator conditions. Here we demonstrate for the first time, 3D+time lens-free acquisitions of 3D cell culture over 8 days directly into the incubator. The 3D reconstructed volume is as large as ∼5 mm3 and provides a unique way to observe in the same 3D cell culture experiment multiple cell migration strategies. Namely, in a 3D cell culture of prostate epithelial cells embedded within a Matrigel® matrix, we are able to distinguish single cell 'leaders', migration of cell clusters, migration of large aggregates of cells, and also close-gap and large-scale branching. In addition, we observe long-scale 3D deformations of the ECM that modify the geometry of the 3D cell culture. Interestingly, we also observed the opposite, i.e. we found that large aggregates of cells may deform the ECM by generating traction forces over very long distances. In sum we put forward a novel 3D lens-free microscopy tomographic technique to study the single and collective cell migrations, the cell-to-cell interactions and the cell-to-matrix interactions. © COPYRIGHT SPIE. Downloading of the abstract is permitted for personal use only.

关键词： Cells

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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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Hyperspectral imaging fluorescence excitation scanning (HIFEX) microscopy for live cell imaging 26

Hyperspectral imaging fluorescence excitation scanning (HIFE...

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

作者： Leavesley, Silas J. Griswold, John Robert Deal, Joshua McAlister, Kathleen Mayes, Sam Browning, Craig Parker, Marina Mayes, Samantha Gunn Rich, Thomas C. Univ S Alabama Dept Chem & Biomol Engn Mobile AL 36688 USA Univ S Alabama Dept Pharmacol Mobile AL 36688 USA Univ S Alabama Ctr Lung Biol Mobile AL 36688 USA Univ S Alabama Dept Biomed Sci Mobile AL 36688 USA Univ S Alabama Syst Engn Mobile AL 36688 USA

ISBN: (纸本)9781510624092

In the past two decades, spectral imaging technologies have expanded the capacity of fluorescence microscopy for accurate detection of multiple labels, separation of labels from cellular and tissue autofluorescence, and analysis of autofluorescence signatures. These technologies have been implemented using a range of optical techniques, such as tunable filters, diffraction gratings, prisms, interferometry, and custom Bayer filters. Each of these techniques has associated strengths and weaknesses with regard to spectral resolution, spatial resolution, temporal resolution, and signal-to-noise characteristics. We have previously shown that spectral scanning of the fluorescence excitation spectrum can provide greatly increased signal strength compared to traditional emission-scanning approaches. Here, we present results from utilizing a Hyperspectral Imaging Fluorescence Excitation Scanning (HIFEX) microscope system for live cell imaging. Live cell signaling studies were performed using HEK 293 and rat pulmonary microvascular endothelial cells (PMVECs), transfected with either a cAMP FRET reporter or a Ca2+ reporter. Cells were further labeled to visualize subcellular structures (nuclei, membrane, mitochondria, etc.). Spectral images were acquired using a custom inverted microscope (TE2000, Nikon Instruments) equipped with a 300W Xe arc lamp and tunable excitation filter (VF-5, Sutter Instrument Co., equipped with VersaChrome filters, Semrock), and run through MicroManager. Timelapse spectral images were acquired from 350-550 nm, in 5 nm increments. Spectral image data were linearly unmixed using custom MATLAB scripts. Results indicate that the HIFEX microscope system can acquire live cell image data at acquisition speeds of 8 ms/wavelength band with minimal photobleaching, sufficient for studying moderate speed cAMP and Ca2+ events.

关键词： Spectral Spectroscopy Signature Fingerprint Optical Biopsy Classification EEM

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