Laser scanning confocal microscopy: history, applications, and related optical sectioning techniques.

PubMed

Paddock, Stephen W; Eliceiri, Kevin W

2014-01-01

Confocal microscopy is an established light microscopical technique for imaging fluorescently labeled specimens with significant three-dimensional structure. Applications of confocal microscopy in the biomedical sciences include the imaging of the spatial distribution of macromolecules in either fixed or living cells, the automated collection of 3D data, the imaging of multiple labeled specimens and the measurement of physiological events in living cells. The laser scanning confocal microscope continues to be chosen for most routine work although a number of instruments have been developed for more specific applications. Significant improvements have been made to all areas of the confocal approach, not only to the instruments themselves, but also to the protocols of specimen preparation, to the analysis, the display, the reproduction, sharing and management of confocal images using bioinformatics techniques.

Efficient Parallel Levenberg-Marquardt Model Fitting towards Real-Time Automated Parametric Imaging Microscopy

PubMed Central

Zhu, Xiang; Zhang, Dianwen

2013-01-01

We present a fast, accurate and robust parallel Levenberg-Marquardt minimization optimizer, GPU-LMFit, which is implemented on graphics processing unit for high performance scalable parallel model fitting processing. GPU-LMFit can provide a dramatic speed-up in massive model fitting analyses to enable real-time automated pixel-wise parametric imaging microscopy. We demonstrate the performance of GPU-LMFit for the applications in superresolution localization microscopy and fluorescence lifetime imaging microscopy. PMID:24130785

Recent progress in tissue optical clearing for spectroscopic application

NASA Astrophysics Data System (ADS)

Sdobnov, A. Yu.; Darvin, M. E.; Genina, E. A.; Bashkatov, A. N.; Lademann, J.; Tuchin, V. V.

2018-05-01

This paper aims to review recent progress in optical clearing of the skin and over naturally turbid biological tissues and blood using this technique in vivo and in vitro with multiphoton microscopy, confocal Raman microscopy, confocal microscopy, NIR spectroscopy, optical coherence tomography, and laser speckle contrast imaging. Basic principles of the technique, its safety, advantages and limitations are discussed. The application of optical clearing agent on a tissue allows for controlling the optical properties of tissue. Optical clearing-induced reduction of tissue scattering significantly facilitates the observation of deep-located tissue regions, at the same time improving the resolution and image contrast for a variety of optical imaging methods suitable for clinical applications, such as diagnostics and laser treatment of skin diseases, mucosal tumor imaging, laser disruption of pathological abnormalities, etc. Structural images of different skin layers obtained ex vivo for porcine ear skin samples at application of Omnipaque™ and glycerol solutions during 60 min. Red color corresponds to TPEAF signal channel. Green color corresponds to SHG signal channel.

Biological imaging with coherent Raman scattering microscopy: a tutorial

PubMed Central

Alfonso-García, Alba; Mittal, Richa; Lee, Eun Seong; Potma, Eric O.

2014-01-01

Abstract. Coherent Raman scattering (CRS) microscopy is gaining acceptance as a valuable addition to the imaging toolset of biological researchers. Optimal use of this label-free imaging technique benefits from a basic understanding of the physical principles and technical merits of the CRS microscope. This tutorial offers qualitative explanations of the principles behind CRS microscopy and provides information about the applicability of this nonlinear optical imaging approach for biological research. PMID:24615671

A short feature vector for image matching: The Log-Polar Magnitude feature descriptor

PubMed Central

Hast, Anders; Wählby, Carolina; Sintorn, Ida-Maria

2017-01-01

The choice of an optimal feature detector-descriptor combination for image matching often depends on the application and the image type. In this paper, we propose the Log-Polar Magnitude feature descriptor—a rotation, scale, and illumination invariant descriptor that achieves comparable performance to SIFT on a large variety of image registration problems but with much shorter feature vectors. The descriptor is based on the Log-Polar Transform followed by a Fourier Transform and selection of the magnitude spectrum components. Selecting different frequency components allows optimizing for image patterns specific for a particular application. In addition, by relying only on coordinates of the found features and (optionally) feature sizes our descriptor is completely detector independent. We propose 48- or 56-long feature vectors that potentially can be shortened even further depending on the application. Shorter feature vectors result in better memory usage and faster matching. This combined with the fact that the descriptor does not require a time-consuming feature orientation estimation (the rotation invariance is achieved solely by using the magnitude spectrum of the Log-Polar Transform) makes it particularly attractive to applications with limited hardware capacity. Evaluation is performed on the standard Oxford dataset and two different microscopy datasets; one with fluorescence and one with transmission electron microscopy images. Our method performs better than SURF and comparable to SIFT on the Oxford dataset, and better than SIFT on both microscopy datasets indicating that it is particularly useful in applications with microscopy images. PMID:29190737

Magnetic force microscopy/current contrast imaging: A new technique for internal current probing of ICs

DOE Office of Scientific and Technical Information (OSTI.GOV)

Campbell, A.N.; Cole, E.I. Jr.; Dodd, B.A.

This invited paper describes recently reported work on the application of magnetic force microscopy (MFM) to image currents in IC conductors [1]. A computer model for MFM imaging of IC currents and experimental results demonstrating the ability to determine current direction and magnitude with a resolution of {approximately} 1 mA dc and {approximately} 1 {mu}A ac are presented. The physics of MFM signal generation and applications to current imaging and measurement are described.

Coherent total internal reflection dark-field microscopy: label-free imaging beyond the diffraction limit.

PubMed

von Olshausen, Philipp; Rohrbach, Alexander

2013-10-15

Coherent imaging is barely applicable in life-science microscopy due to multiple interference artifacts. Here, we show how these interferences can be used to improve image resolution and contrast. We present a dark-field microscopy technique with evanescent illumination via total internal reflection that delivers high-contrast images of coherently scattering samples. By incoherent averaging of multiple coherent images illuminated from different directions we can resolve image structures that remain unresolved by conventional (incoherent) fluorescence microscopy. We provide images of 190 nm beads revealing resolution beyond the diffraction limit and slightly increased object distances. An analytical model is introduced that accounts for the observed effects and which is confirmed by numerical simulations. Our approach may be a route to fast, label-free, super-resolution imaging in live-cell microscopy.

[Watching dance of the molecules - CARS microscopy].

PubMed

Korczyński, Jaroslaw; Kubiak, Katarzyna; Węgłowska, Edyta

2017-01-01

CARS (Coherent Anti-Stokes Raman Scattering) microscopy is an imaging method for living cells visualization as well as for food or cosmetics material analysis without the need for staining. The near infrared laser source generates the CARS signal - the characteristic intrinsic vibrational contrast of the molecules in a sample which is no longer caused by staining, but by the molecules themselves. It provides the benefit of a non-toxic, non-destructive and almost noninvasive method for sample imaging. CARS can easily be combined with fluorescence confocal microscopy so it is an excellent complementary imaging method. In this article we showed some of the applications for this technology: imaging of lipid droplets inside human HaCaT cells and analysis of the composition of cosmetic products. Moreover we believe, that soon new fields of application become accessible for this rapidly developing branch of microscopy.

Automated seeding-based nuclei segmentation in nonlinear optical microscopy.

PubMed

Medyukhina, Anna; Meyer, Tobias; Heuke, Sandro; Vogler, Nadine; Dietzek, Benjamin; Popp, Jürgen

2013-10-01

Nonlinear optical (NLO) microscopy based, e.g., on coherent anti-Stokes Raman scattering (CARS) or two-photon-excited fluorescence (TPEF) is a fast label-free imaging technique, with a great potential for biomedical applications. However, NLO microscopy as a diagnostic tool is still in its infancy; there is a lack of robust and durable nuclei segmentation methods capable of accurate image processing in cases of variable image contrast, nuclear density, and type of investigated tissue. Nonetheless, such algorithms specifically adapted to NLO microscopy present one prerequisite for the technology to be routinely used, e.g., in pathology or intraoperatively for surgical guidance. In this paper, we compare the applicability of different seeding and boundary detection methods to NLO microscopic images in order to develop an optimal seeding-based approach capable of accurate segmentation of both TPEF and CARS images. Among different methods, the Laplacian of Gaussian filter showed the best accuracy for the seeding of the image, while a modified seeded watershed segmentation was the most accurate in the task of boundary detection. The resulting combination of these methods followed by the verification of the detected nuclei performs high average sensitivity and specificity when applied to various types of NLO microscopy images.

Applications of microscopy to genetic therapy of cystic fibrosis and other human diseases.

PubMed

Moninger, Thomas O; Nessler, Randy A; Moore, Kenneth C

2006-01-01

Gene therapy has become an extremely important and active field of biomedical research. Microscopy is an integral component of this effort. This chapter presents an overview of imaging techniques used in our facility in support of cystic fibrosis gene therapy research. Instrumentation used in these studies includes light and confocal microscopy, transmission electron microscopy, and scanning electron microscopy. Techniques outlined include negative staining, cryo-electron microscopy, three-dimentional reconstruction, enzyme cytochemistry, immunocytochemistry, and fluorescence imaging.

Technical Review: Microscopy and Image Processing Tools to Analyze Plant Chromatin: Practical Considerations.

PubMed

Baroux, Célia; Schubert, Veit

2018-01-01

In situ nucleus and chromatin analyses rely on microscopy imaging that benefits from versatile, efficient fluorescent probes and proteins for static or live imaging. Yet the broad choice in imaging instruments offered to the user poses orientation problems. Which imaging instrument should be used for which purpose? What are the main caveats and what are the considerations to best exploit each instrument's ability to obtain informative and high-quality images? How to infer quantitative information on chromatin or nuclear organization from microscopy images? In this review, we present an overview of common, fluorescence-based microscopy systems and discuss recently developed super-resolution microscopy systems, which are able to bridge the resolution gap between common fluorescence microscopy and electron microscopy. We briefly present their basic principles and discuss their possible applications in the field, while providing experience-based recommendations to guide the user toward best-possible imaging. In addition to raw data acquisition methods, we discuss commercial and noncommercial processing tools required for optimal image presentation and signal evaluation in two and three dimensions.

Wide-field imaging through scattering media by scattered light fluorescence microscopy

NASA Astrophysics Data System (ADS)

Zhou, Yulan; Li, Xun

2017-08-01

To obtain images through scattering media, scattered light fluorescence (SLF) microscopy that utilizes the optical memory effect has been developed. However, the small field of view (FOV) of SLF microscopy limits its application. In this paper, we have introduced a re-modulation method to achieve wide-field imaging through scattering media by SLF microscopy. In the re-modulation method, to raster scan the focus across the object plane, the incident wavefront is re-modulated via a spatial light modulator (SLM) in the updated phase compensation calculated using the optimized iterative algorithm. Compared with the conventional optical memory effect method, the re-modulation method can greatly increase the FOV of a SLF microscope. With the phase compensation theoretically calculated, the process of updating the phase compensation of a high speed SLM is fast. The re-modulation method does not increase the imaging time. The re-modulation method is, therefore, expected to make SLF microscopy have much wider applications in biology, medicine and physiology.

Intravital Microscopy Imaging Approaches for Image-Guided Drug Delivery Systems

PubMed Central

Kirui, Dickson K.; Ferrari, Mauro

2016-01-01

Rapid technical advances in the field of non-linear microscopy have made intravital microscopy a vital pre-clinical tool for research and development of imaging-guided drug delivery systems. The ability to dynamically monitor the fate of macromolecules in live animals provides invaluable information regarding properties of drug carriers (size, charge, and surface coating), physiological, and pathological processes that exist between point-of-injection and the projected of site of delivery, all of which influence delivery and effectiveness of drug delivery systems. In this Review, we highlight how integrating intravital microscopy imaging with experimental designs (in vitro analyses and mathematical modeling) can provide unique information critical in the design of novel disease-relevant drug delivery platforms with improved diagnostic and therapeutic indexes. The Review will provide the reader an overview of the various applications for which intravital microscopy has been used to monitor the delivery of diagnostic and therapeutic agents and discuss some of their potential clinical applications. PMID:25901526

Evanescent Waves in High Numerical Aperture Aplanatic Solid Immersion Microscopy: Effects of Forbidden Light on Subsurface Imaging (Open Access, Publisher’s Version)

DTIC Science & Technology

2014-03-24

of the aSIL microscopy for semiconductor failure analysis and is applicable to imaging in quantum optics [18], biophotonics [19] and metrology [20...is usually of interest, the model can be adapted to applications in fields such as quantum optics and biophotonics for which the non-resonant

NMR Microscopy - Micron-Level Resolution.

NASA Astrophysics Data System (ADS)

Kwok, Wing-Chi Edmund

1990-01-01

Nuclear Magnetic Resonance Imaging (MRI) has been developed into a powerful and widely used diagnostic tool since the invention of techniques using linear magnetic field gradients in 1973. The variety of imaging contrasts obtainable in MRI, such as spin density, relaxation times and flow rate, gives MRI a significant advantage over other imaging techniques. For common diagnostic applications, image resolutions have been in the order of millimeters with slice thicknesses in centimeters. For many research applications, however, resolutions in the order of tens of microns or smaller are needed. NMR Imaging in these high resolution disciplines is known as NMR microscopy. Compared with conventional microscopy, NMR microscopy has the advantage of being non-invasive and non-destructive. The major obstacles of NMR microscopy are low signal-to-noise ratio and effects due to spin diffusion. To overcome these difficulties, more sensitive RF probes and very high magnetic field gradients have to be used. The most effective way to increase sensitivity is to build smaller probes. Microscope probes of different designs have been built and evaluated. Magnetic field gradient coils that can produce linear field gradients up to 450 Gauss/cm were also assembled. In addition, since microscope probes often employ remote capacitors for RF tuning, the associated signal loss in the transmission line was studied. Imaging experiments have been carried out in a 2.1 Tesla small bore superconducting magnet using the typical two-dimensional spin warp imaging technique. Images have been acquired for both biological and non-biological samples. The highest resolution was obtained in an image of a nerve bundle from the spinal cord of a racoon and has an in-plane resolution of 4 microns. These experiments have demonstrated the potential application of NMR microscopy to pathological research, nervous system study and non -destructive testings of materials. One way to further improve NMR microscopy is to implement a higher static magnetic field which will increase signal strength. In the future, NMR microscopy should prove to be useful in the studies of cell linings, T1 & T2 relaxation mechanisms and NMR contrast agents.

Study of electromechanical and mechanical properties of bacteria using force microscopy

NASA Astrophysics Data System (ADS)

Reukov, Vladimir; Thompson, Gary; Nikiforov, Maxim; Guo, Senli; Ovchinnikov, Oleg; Jesse, Stephen; Kalinin, Sergei; Vertegel, Alexey

2010-03-01

The application of scanning probe microscopy (SPM) to biological systems has evolved over the past decade into a multimodal and spectroscopic instrument that provides multiple information channels at each spatial pixel acquired. Recently, functional recognition imaging based on differing electromechanical properties between Gram negative and Gram positive bacteria was achieved using artificial neural network analysis of band excitation piezoresponse force microscopy (BEPFM) data. The immediate goal of this project was to study mechanical and electromechanical properties of bacterial systems physiologically-relevant solutions using Band-width Excitation Piezoresponce Force Microscopy (BE PFM) in combination with Force Mapping. Electromechanical imaging in physiological environments will improve the versatility of functional recognition imaging and open the way for application of the rapid BEPFM line mode method to other living cell systems.

Research and application on imaging technology of line structure light based on confocal microscopy

NASA Astrophysics Data System (ADS)

Han, Wenfeng; Xiao, Zexin; Wang, Xiaofen

2009-11-01

In 2005, the theory of line structure light confocal microscopy was put forward firstly in China by Xingyu Gao and Zexin Xiao in the Institute of Opt-mechatronics of Guilin University of Electronic Technology. Though the lateral resolution of line confocal microscopy can only reach or approach the level of the traditional dot confocal microscopy. But compared with traditional dot confocal microscopy, it has two advantages: first, by substituting line scanning for dot scanning, plane imaging only performs one-dimensional scanning, with imaging velocity greatly improved and scanning mechanism simplified, second, transfer quantity of light is greatly improved by substituting detection hairline for detection pinhole, and low illumination CCD is used directly to collect images instead of photoelectric intensifier. In order to apply the line confocal microscopy to practical system, based on the further research on the theory of the line confocal microscopy, imaging technology of line structure light is put forward on condition of implementation of confocal microscopy. Its validity and reliability are also verified by experiments.

Spectral mapping tools from the earth sciences applied to spectral microscopy data.

PubMed

Harris, A Thomas

2006-08-01

Spectral imaging, originating from the field of earth remote sensing, is a powerful tool that is being increasingly used in a wide variety of applications for material identification. Several workers have used techniques like linear spectral unmixing (LSU) to discriminate materials in images derived from spectral microscopy. However, many spectral analysis algorithms rely on assumptions that are often violated in microscopy applications. This study explores algorithms originally developed as improvements on early earth imaging techniques that can be easily translated for use with spectral microscopy. To best demonstrate the application of earth remote sensing spectral analysis tools to spectral microscopy data, earth imaging software was used to analyze data acquired with a Leica confocal microscope with mechanical spectral scanning. For this study, spectral training signatures (often referred to as endmembers) were selected with the ENVI (ITT Visual Information Solutions, Boulder, CO) "spectral hourglass" processing flow, a series of tools that use the spectrally over-determined nature of hyperspectral data to find the most spectrally pure (or spectrally unique) pixels within the data set. This set of endmember signatures was then used in the full range of mapping algorithms available in ENVI to determine locations, and in some cases subpixel abundances of endmembers. Mapping and abundance images showed a broad agreement between the spectral analysis algorithms, supported through visual assessment of output classification images and through statistical analysis of the distribution of pixels within each endmember class. The powerful spectral analysis algorithms available in COTS software, the result of decades of research in earth imaging, are easily translated to new sources of spectral data. Although the scale between earth imagery and spectral microscopy is radically different, the problem is the same: mapping material locations and abundances based on unique spectral signatures. (c) 2006 International Society for Analytical Cytology.

Coherent nonlinear optical imaging: beyond fluorescence microscopy.

PubMed

Min, Wei; Freudiger, Christian W; Lu, Sijia; Xie, X Sunney

2011-01-01

The quest for ultrahigh detection sensitivity with spectroscopic contrasts other than fluorescence has led to various novel approaches to optical microscopy of biological systems. Coherent nonlinear optical imaging, especially the recently developed nonlinear dissipation microscopy (including stimulated Raman scattering and two-photon absorption) and pump-probe microscopy (including excited-state absorption, stimulated emission, and ground-state depletion), provides new image contrasts for nonfluorescent species. Thanks to the high-frequency modulation transfer scheme, these imaging techniques exhibit superb detection sensitivity. By directly interrogating vibrational and/or electronic energy levels of molecules, they offer high molecular specificity. Here we review the underlying principles and excitation and detection schemes, as well as exemplary biomedical applications of this emerging class of molecular imaging techniques.

Color calibration and fusion of lens-free and mobile-phone microscopy images for high-resolution and accurate color reproduction.

PubMed

Zhang, Yibo; Wu, Yichen; Zhang, Yun; Ozcan, Aydogan

2016-06-10

Lens-free holographic microscopy can achieve wide-field imaging in a cost-effective and field-portable setup, making it a promising technique for point-of-care and telepathology applications. However, due to relatively narrow-band sources used in holographic microscopy, conventional colorization methods that use images reconstructed at discrete wavelengths, corresponding to e.g., red (R), green (G) and blue (B) channels, are subject to color artifacts. Furthermore, these existing RGB colorization methods do not match the chromatic perception of human vision. Here we present a high-color-fidelity and high-resolution imaging method, termed "digital color fusion microscopy" (DCFM), which fuses a holographic image acquired at a single wavelength with a color-calibrated image taken by a low-magnification lens-based microscope using a wavelet transform-based colorization method. We demonstrate accurate color reproduction of DCFM by imaging stained tissue sections. In particular we show that a lens-free holographic microscope in combination with a cost-effective mobile-phone-based microscope can generate color images of specimens, performing very close to a high numerical-aperture (NA) benchtop microscope that is corrected for color distortions and chromatic aberrations, also matching the chromatic response of human vision. This method can be useful for wide-field imaging needs in telepathology applications and in resource-limited settings, where whole-slide scanning microscopy systems are not available.

Biomedical Applications of Nanodiamonds: An Overview.

PubMed

Passeri, D; Rinaldi, F; Ingallina, C; Carafa, M; Rossi, M; Terranova, M L; Marianecci, C

2015-02-01

Nanodiamonds are a novel class of nanomaterials which have raised much attention for application in biomedical field, as they combine the possibility of being produced on large scale using relatively inexpensive synthetic processes, of being fluorescent as a consequence of the presence of nitrogen vacancies, of having their surfaces functionalized, and of having good biocompatibility. Among other applications, we mainly focus on drug delivery, including cell interaction, targeting, cancer therapy, gene and protein delivery. In addition, nanodiamonds for bone and dental implants and for antibacterial use is discussed. Techniques for detection and imaging of nanodiamonds in biological tissues are also reviewed, including electron microscopy, fluorescence microscopy, Raman mapping, atomic force microscopy, thermal imaging, magnetic resonance imaging, and positron emission tomography, either in vitro, in vivo, or ex vivo. Toxicological aspects related to the use of nanodiamonds are also discussed. Finally, patents, preclinical and clinical trials based on the use of nanodiamonds for biomedical applications are reviewed.

Image improvement and three-dimensional reconstruction using holographic image processing

NASA Technical Reports Server (NTRS)

Stroke, G. W.; Halioua, M.; Thon, F.; Willasch, D. H.

1977-01-01

Holographic computing principles make possible image improvement and synthesis in many cases of current scientific and engineering interest. Examples are given for the improvement of resolution in electron microscopy and 3-D reconstruction in electron microscopy and X-ray crystallography, following an analysis of optical versus digital computing in such applications.

Improved wavefront correction for coherent image restoration.

PubMed

Zelenka, Claudius; Koch, Reinhard

2017-08-07

Coherent imaging has a wide range of applications in, for example, microscopy, astronomy, and radar imaging. Particularly interesting is the field of microscopy, where the optical quality of the lens is the main limiting factor. In this article, novel algorithms for the restoration of blurred images in a system with known optical aberrations are presented. Physically motivated by the scalar diffraction theory, the new algorithms are based on Haugazeau POCS and FISTA, and are faster and more robust than methods presented earlier. With the new approach the level of restoration quality on real images is very high, thereby blurring and ringing caused by defocus can be effectively removed. In classical microscopy, lenses with very low aberration must be used, which puts a practical limit on their size and numerical aperture. A coherent microscope using the novel restoration method overcomes this limitation. In contrast to incoherent microscopy, severe optical aberrations including defocus can be removed, hence the requirements on the quality of the optics are lower. This can be exploited for an essential price reduction of the optical system. It can be also used to achieve higher resolution than in classical microscopy, using lenses with high numerical aperture and high aberration. All this makes the coherent microscopy superior to the traditional incoherent in suited applications.

Automated microscopy for high-content RNAi screening

PubMed Central

2010-01-01

Fluorescence microscopy is one of the most powerful tools to investigate complex cellular processes such as cell division, cell motility, or intracellular trafficking. The availability of RNA interference (RNAi) technology and automated microscopy has opened the possibility to perform cellular imaging in functional genomics and other large-scale applications. Although imaging often dramatically increases the content of a screening assay, it poses new challenges to achieve accurate quantitative annotation and therefore needs to be carefully adjusted to the specific needs of individual screening applications. In this review, we discuss principles of assay design, large-scale RNAi, microscope automation, and computational data analysis. We highlight strategies for imaging-based RNAi screening adapted to different library and assay designs. PMID:20176920

A general system for automatic biomedical image segmentation using intensity neighborhoods.

PubMed

Chen, Cheng; Ozolek, John A; Wang, Wei; Rohde, Gustavo K

2011-01-01

Image segmentation is important with applications to several problems in biology and medicine. While extensively researched, generally, current segmentation methods perform adequately in the applications for which they were designed, but often require extensive modifications or calibrations before being used in a different application. We describe an approach that, with few modifications, can be used in a variety of image segmentation problems. The approach is based on a supervised learning strategy that utilizes intensity neighborhoods to assign each pixel in a test image its correct class based on training data. We describe methods for modeling rotations and variations in scales as well as a subset selection for training the classifiers. We show that the performance of our approach in tissue segmentation tasks in magnetic resonance and histopathology microscopy images, as well as nuclei segmentation from fluorescence microscopy images, is similar to or better than several algorithms specifically designed for each of these applications.

Ophthalmic imaging using multiphoton microscopy

NASA Astrophysics Data System (ADS)

Teng, Shu-Wen; Peng, Ju-Li; Lin, Huei-Hsing; Wu, Hai-Yin; Lo, Wen; Sun, Yen; Lin, Wei-Chou; Lin, Sung-Jan; Jee, Shiou-Hwa; Tan, Hsin-Yuan; Dong, Chen-Yuan

2005-04-01

This purpose of this study is to demonstrate the feasibility of using multiphoton microscopy in ophthalmologic imaging. Without the introduction of extrinsic fluorescence molecules, multiphoton induced autofluorescence and second harmonic generation signals can be used to obtain useful structural information of normal and diseased corneas. Our work can potentially lead to the in vivo application of multiphoton microscopy in investigating corneal physiology and pathologies.

3D correlative light and electron microscopy of cultured cells using serial blockface scanning electron microscopy

PubMed Central

Lerner, Thomas R.; Burden, Jemima J.; Nkwe, David O.; Pelchen-Matthews, Annegret; Domart, Marie-Charlotte; Durgan, Joanne; Weston, Anne; Jones, Martin L.; Peddie, Christopher J.; Carzaniga, Raffaella; Florey, Oliver; Marsh, Mark; Gutierrez, Maximiliano G.

2017-01-01

ABSTRACT The processes of life take place in multiple dimensions, but imaging these processes in even three dimensions is challenging. Here, we describe a workflow for 3D correlative light and electron microscopy (CLEM) of cell monolayers using fluorescence microscopy to identify and follow biological events, combined with serial blockface scanning electron microscopy to analyse the underlying ultrastructure. The workflow encompasses all steps from cell culture to sample processing, imaging strategy, and 3D image processing and analysis. We demonstrate successful application of the workflow to three studies, each aiming to better understand complex and dynamic biological processes, including bacterial and viral infections of cultured cells and formation of entotic cell-in-cell structures commonly observed in tumours. Our workflow revealed new insight into the replicative niche of Mycobacterium tuberculosis in primary human lymphatic endothelial cells, HIV-1 in human monocyte-derived macrophages, and the composition of the entotic vacuole. The broad application of this 3D CLEM technique will make it a useful addition to the correlative imaging toolbox for biomedical research. PMID:27445312

Microscopy techniques in flavivirus research.

PubMed

Chong, Mun Keat; Chua, Anthony Jin Shun; Tan, Terence Tze Tong; Tan, Suat Hoon; Ng, Mah Lee

2014-04-01

The Flavivirus genus is composed of many medically important viruses that cause high morbidity and mortality, which include Dengue and West Nile viruses. Various molecular and biochemical techniques have been developed in the endeavour to study flaviviruses. However, microscopy techniques still have irreplaceable roles in the identification of novel virus pathogens and characterization of morphological changes in virus-infected cells. Fluorescence microscopy contributes greatly in understanding the fundamental viral protein localizations and virus-host protein interactions during infection. Electron microscopy remains the gold standard for visualizing ultra-structural features of virus particles and infected cells. New imaging techniques and combinatory applications are continuously being developed to push the limit of resolution and extract more quantitative data. Currently, correlative live cell imaging and high resolution three-dimensional imaging have already been achieved through the tandem use of optical and electron microscopy in analyzing biological specimens. Microscopy techniques are also used to measure protein binding affinities and determine the mobility pattern of proteins in cells. This chapter will consolidate on the applications of various well-established microscopy techniques in flavivirus research, and discuss how recently developed microscopy techniques can potentially help advance our understanding in these membrane viruses. Copyright © 2013 Elsevier Ltd. All rights reserved.

Hybrid label-free multiphoton and optoacoustic microscopy (MPOM)

NASA Astrophysics Data System (ADS)

Soliman, Dominik; Tserevelakis, George J.; Omar, Murad; Ntziachristos, Vasilis

2015-07-01

Many biological applications require a simultaneous observation of different anatomical features. However, unless potentially harmful staining of the specimens is employed, individual microscopy techniques do generally not provide multi-contrast capabilities. We present a hybrid microscope integrating optoacoustic microscopy and multiphoton microscopy, including second-harmonic generation, into a single device. This combined multiphoton and optoacoustic microscope (MPOM) offers visualization of a broad range of structures by employing different contrast mechanisms and at the same time enables pure label-free imaging of biological systems. We investigate the relative performance of the two microscopy modalities and demonstrate their multi-contrast abilities through the label-free imaging of a zebrafish larva ex vivo, simultaneously visualizing muscles and pigments. This hybrid microscopy application bears great potential for developmental biology studies, enabling more comprehensive information to be obtained from biological specimens without the necessity of staining.

Light Microscopy at Maximal Precision

NASA Astrophysics Data System (ADS)

Bierbaum, Matthew; Leahy, Brian D.; Alemi, Alexander A.; Cohen, Itai; Sethna, James P.

2017-10-01

Microscopy is the workhorse of the physical and life sciences, producing crisp images of everything from atoms to cells well beyond the capabilities of the human eye. However, the analysis of these images is frequently little more accurate than manual marking. Here, we revolutionize the analysis of microscopy images, extracting all the useful information theoretically contained in a complex microscope image. Using a generic, methodological approach, we extract the information by fitting experimental images with a detailed optical model of the microscope, a method we call parameter extraction from reconstructing images (PERI). As a proof of principle, we demonstrate this approach with a confocal image of colloidal spheres, improving measurements of particle positions and radii by 10-100 times over current methods and attaining the maximum possible accuracy. With this unprecedented accuracy, we measure nanometer-scale colloidal interactions in dense suspensions solely with light microscopy, a previously impossible feat. Our approach is generic and applicable to imaging methods from brightfield to electron microscopy, where we expect accuracies of 1 nm and 0.1 pm, respectively.

Quantitative photothermal phase imaging of red blood cells using digital holographic photothermal microscope.

PubMed

Vasudevan, Srivathsan; Chen, George C K; Lin, Zhiping; Ng, Beng Koon

2015-05-10

Photothermal microscopy (PTM), a noninvasive pump-probe high-resolution microscopy, has been applied as a bioimaging tool in many biomedical studies. PTM utilizes a conventional phase contrast microscope to obtain highly resolved photothermal images. However, phase information cannot be extracted from these photothermal images, as they are not quantitative. Moreover, the problem of halos inherent in conventional phase contrast microscopy needs to be tackled. Hence, a digital holographic photothermal microscopy technique is proposed as a solution to obtain quantitative phase images. The proposed technique is demonstrated by extracting phase values of red blood cells from their photothermal images. These phase values can potentially be used to determine the temperature distribution of the photothermal images, which is an important study in live cell monitoring applications.

Optofluidic time-stretch microscopy: recent advances

NASA Astrophysics Data System (ADS)

Lei, Cheng; Nitta, Nao; Ozeki, Yasuyuki; Goda, Keisuke

2018-06-01

Flow cytometry is an indispensable method for valuable applications in numerous fields such as immunology, pathology, pharmacology, molecular biology, and marine biology. Optofluidic time-stretch microscopy is superior to conventional flow cytometry methods for its capability to acquire high-quality images of single cells at a high-throughput exceeding 10,000 cells per second. This makes it possible to extract copious information from cellular images for accurate cell detection and analysis with the assistance of machine learning. Optofluidic time-stretch microscopy has proven its effectivity in various applications, including microalga-based biofuel production, evaluation of thrombotic disorders, as well as drug screening and discovery. In this review, we discuss the principles and recent advances of optofluidic time-stretch microscopy.

Frequency domain phase-shifted confocal microscopy (FDPCM) with array detection

NASA Astrophysics Data System (ADS)

Ge, Baoliang; Huang, Yujia; Fang, Yue; Kuang, Cuifang; Xiu, Peng; Liu, Xu

2017-09-01

We proposed a novel method to reconstruct images taken by array detected confocal microscopy without prior knowledge about its detector distribution. The proposed frequency domain phase-shifted confocal microscopy (FDPCM) shifts the image from each detection channel to its corresponding place by substituting the phase information in Fourier domain. Theoretical analysis shows that our method could approach the resolution nearly twofold of wide-field microscopy. Simulation and experiment results are also shown to verify the applicability and effectiveness of our method. Compared to Airyscan, our method holds the advantage of simplicity and convenience to be applied to array detectors with different structure, which makes FDPCM have great potential in the application of biomedical observation in the future.

Optofluidic time-stretch microscopy: recent advances

NASA Astrophysics Data System (ADS)

Lei, Cheng; Nitta, Nao; Ozeki, Yasuyuki; Goda, Keisuke

2018-04-01

Flow cytometry is an indispensable method for valuable applications in numerous fields such as immunology, pathology, pharmacology, molecular biology, and marine biology. Optofluidic time-stretch microscopy is superior to conventional flow cytometry methods for its capability to acquire high-quality images of single cells at a high-throughput exceeding 10,000 cells per second. This makes it possible to extract copious information from cellular images for accurate cell detection and analysis with the assistance of machine learning. Optofluidic time-stretch microscopy has proven its effectivity in various applications, including microalga-based biofuel production, evaluation of thrombotic disorders, as well as drug screening and discovery. In this review, we discuss the principles and recent advances of optofluidic time-stretch microscopy.

Parallel detecting super-resolution microscopy using correlation based image restoration

NASA Astrophysics Data System (ADS)

Yu, Zhongzhi; Liu, Shaocong; Zhu, Dazhao; Kuang, Cuifang; Liu, Xu

2017-12-01

A novel approach to achieve the image restoration is proposed in which each detector's relative position in the detector array is no longer a necessity. We can identify each detector's relative location by extracting a certain area from one of the detector's image and scanning it on other detectors' images. According to this location, we can generate the point spread functions (PSF) for each detector and perform deconvolution for image restoration. Equipped with this method, the microscope with discretionally designed detector array can be easily constructed without the concern of exact relative locations of detectors. The simulated results and experimental results show the total improvement in resolution with a factor of 1.7 compared to conventional confocal fluorescence microscopy. With the significant enhancement in resolution and easiness for application of this method, this novel method should have potential for a wide range of application in fluorescence microscopy based on parallel detecting.

In vivo multiphoton microscopy beyond 1 mm in the brain

NASA Astrophysics Data System (ADS)

Miller, David R.; Medina, Flor A.; Hassan, Ahmed; Perillo, Evan P.; Hagan, Kristen; Kazmi, S. M. Shams; Zemelman, Boris V.; Dunn, Andrew K.

2017-02-01

We perform high-resolution, non-invasive, in vivo deep-tissue imaging of the mouse neocortex using multiphoton microscopy with a high repetition rate optical parametric amplifier laser source tunable between λ=1,100 and 1,400 nm. We demonstrate an imaging depth of 1,200 μm in vasculature and 1,160 μm in neurons. We also demonstrate deep-tissue imaging using Indocyanine Green (ICG), which is FDA approved and a promising route to translate multiphoton microscopy to human applications.

Automated motion artifact removal for intravital microscopy, without a priori information.

PubMed

Lee, Sungon; Vinegoni, Claudio; Sebas, Matthew; Weissleder, Ralph

2014-03-28

Intravital fluorescence microscopy, through extended penetration depth and imaging resolution, provides the ability to image at cellular and subcellular resolution in live animals, presenting an opportunity for new insights into in vivo biology. Unfortunately, physiological induced motion components due to respiration and cardiac activity are major sources of image artifacts and impose severe limitations on the effective imaging resolution that can be ultimately achieved in vivo. Here we present a novel imaging methodology capable of automatically removing motion artifacts during intravital microscopy imaging of organs and orthotopic tumors. The method is universally applicable to different laser scanning modalities including confocal and multiphoton microscopy, and offers artifact free reconstructions independent of the physiological motion source and imaged organ. The methodology, which is based on raw data acquisition followed by image processing, is here demonstrated for both cardiac and respiratory motion compensation in mice heart, kidney, liver, pancreas and dorsal window chamber.

Automated motion artifact removal for intravital microscopy, without a priori information

PubMed Central

Lee, Sungon; Vinegoni, Claudio; Sebas, Matthew; Weissleder, Ralph

2014-01-01

Intravital fluorescence microscopy, through extended penetration depth and imaging resolution, provides the ability to image at cellular and subcellular resolution in live animals, presenting an opportunity for new insights into in vivo biology. Unfortunately, physiological induced motion components due to respiration and cardiac activity are major sources of image artifacts and impose severe limitations on the effective imaging resolution that can be ultimately achieved in vivo. Here we present a novel imaging methodology capable of automatically removing motion artifacts during intravital microscopy imaging of organs and orthotopic tumors. The method is universally applicable to different laser scanning modalities including confocal and multiphoton microscopy, and offers artifact free reconstructions independent of the physiological motion source and imaged organ. The methodology, which is based on raw data acquisition followed by image processing, is here demonstrated for both cardiac and respiratory motion compensation in mice heart, kidney, liver, pancreas and dorsal window chamber. PMID:24676021

Cardiovascular Imaging Using Two-Photon Microscopy

PubMed Central

Scherschel, John A.; Rubart, Michael

2008-01-01

Two-photon excitation microscopy has become the standard technique for high resolution deep tissue and intravital imaging. It provides intrinsic three-dimensional resolution in combination with increased penetration depth compared to single-photon confocal microscopy. This article will describe the basic physical principles of two-photon excitation and will review its multiple applications to cardiovascular imaging, including second harmonic generation and fluorescence laser scanning microscopy. In particular, the capability and limitations of multiphoton microscopy to assess functional heterogeneity on a cellular scale deep within intact, Langendorff-perfused hearts are demonstrated. It will also discuss the use of two-photon excitation-induced release of caged compounds for the study of intracellular calcium signaling and intercellular dye transfer. PMID:18986603

An overview of state-of-the-art image restoration in electron microscopy.

PubMed

Roels, J; Aelterman, J; Luong, H Q; Lippens, S; Pižurica, A; Saeys, Y; Philips, W

2018-06-08

In Life Science research, electron microscopy (EM) is an essential tool for morphological analysis at the subcellular level as it allows for visualization at nanometer resolution. However, electron micrographs contain image degradations such as noise and blur caused by electromagnetic interference, electron counting errors, magnetic lens imperfections, electron diffraction, etc. These imperfections in raw image quality are inevitable and hamper subsequent image analysis and visualization. In an effort to mitigate these artefacts, many electron microscopy image restoration algorithms have been proposed in the last years. Most of these methods rely on generic assumptions on the image or degradations and are therefore outperformed by advanced methods that are based on more accurate models. Ideally, a method will accurately model the specific degradations that fit the physical acquisition settings. In this overview paper, we discuss different electron microscopy image degradation solutions and demonstrate that dedicated artefact regularisation results in higher quality restoration and is applicable through recently developed probabilistic methods. © 2018 The Authors Journal of Microscopy © 2018 Royal Microscopical Society.

Application and Miniaturization of Linear and Nonlinear Raman Microscopy for Biomedical Imaging

NASA Astrophysics Data System (ADS)

Mittal, Richa

Current diagnostics for several disorders rely on surgical biopsy or evaluation of ex vivo bodily fluids, which have numerous drawbacks. We evaluated the potential for vibrational techniques (both linear and nonlinear Raman) as a reliable and noninvasive diagnostic tool. Raman spectroscopy is an optical technique for molecular analysis that has been used extensively in various biomedical applications. Based on demonstrated capabilities of Raman spectroscopy we evaluated the potential of the technique for providing a noninvasive diagnosis of mucopolysaccharidosis (MPS). These studies show that Raman spectroscopy can detect subtle changes in tissue biochemistry. In applications where sub-micrometer visualization of tissue compositional change is required, a transition from spectroscopy to high quality imaging is necessary. Nonlinear vibrational microscopy is sensitive to the same molecular vibrations as linear Raman, but features fast imaging capabilities. Coherent Raman scattering when combined with other nonlinear optical (NLO) techniques (like two-photon excited fluorescence and second harmonic generation) forms a collection of advanced optical techniques that provide noninvasive chemical contrast at submicron resolution. This capability to examine tissues without external molecular agents is driving the NLO approach towards clinical applications. However, the unique imaging capabilities of NLO microscopy are accompanied by complex instrument requirements. Clinical examination requires portable imaging systems for rapid inspection of tissues. Optical components utilized in NLO microscopy would then need substantial miniaturization and optimization to enable in vivo use. The challenges in designing compact microscope objective lenses and laser beam scanning mechanisms are discussed. The development of multimodal NLO probes for imaging oral cavity tissue is presented. Our prototype has been examined for ex vivo tissue imaging based on intrinsic fluorescence and SHG contrast. These studies show a potential for multiphoton compact probes to be used for real time imaging in the clinic.

Optofluidic time-stretch quantitative phase microscopy.

PubMed

Guo, Baoshan; Lei, Cheng; Wu, Yi; Kobayashi, Hirofumi; Ito, Takuro; Yalikun, Yaxiaer; Lee, Sangwook; Isozaki, Akihiro; Li, Ming; Jiang, Yiyue; Yasumoto, Atsushi; Di Carlo, Dino; Tanaka, Yo; Yatomi, Yutaka; Ozeki, Yasuyuki; Goda, Keisuke

2018-03-01

Innovations in optical microscopy have opened new windows onto scientific research, industrial quality control, and medical practice over the last few decades. One of such innovations is optofluidic time-stretch quantitative phase microscopy - an emerging method for high-throughput quantitative phase imaging that builds on the interference between temporally stretched signal and reference pulses by using dispersive properties of light in both spatial and temporal domains in an interferometric configuration on a microfluidic platform. It achieves the continuous acquisition of both intensity and phase images with a high throughput of more than 10,000 particles or cells per second by overcoming speed limitations that exist in conventional quantitative phase imaging methods. Applications enabled by such capabilities are versatile and include characterization of cancer cells and microalgal cultures. In this paper, we review the principles and applications of optofluidic time-stretch quantitative phase microscopy and discuss its future perspective. Copyright © 2017 Elsevier Inc. All rights reserved.

Tutorial on photoacoustic tomography

NASA Astrophysics Data System (ADS)

Zhou, Yong; Yao, Junjie; Wang, Lihong V.

2016-06-01

Photoacoustic tomography (PAT) has become one of the fastest growing fields in biomedical optics. Unlike pure optical imaging, such as confocal microscopy and two-photon microscopy, PAT employs acoustic detection to image optical absorption contrast with high-resolution deep into scattering tissue. So far, PAT has been widely used for multiscale anatomical, functional, and molecular imaging of biological tissues. We focus on PAT's basic principles, major implementations, imaging contrasts, and recent applications.

Extending Single-Molecule Microscopy Using Optical Fourier Processing

PubMed Central

2015-01-01

This article surveys the recent application of optical Fourier processing to the long-established but still expanding field of single-molecule imaging and microscopy. A variety of single-molecule studies can benefit from the additional image information that can be obtained by modulating the Fourier, or pupil, plane of a widefield microscope. After briefly reviewing several current applications, we present a comprehensive and computationally efficient theoretical model for simulating single-molecule fluorescence as it propagates through an imaging system. Furthermore, we describe how phase/amplitude-modulating optics inserted in the imaging pathway may be modeled, especially at the Fourier plane. Finally, we discuss selected recent applications of Fourier processing methods to measure the orientation, depth, and rotational mobility of single fluorescent molecules. PMID:24745862

Extending single-molecule microscopy using optical Fourier processing.

PubMed

Backer, Adam S; Moerner, W E

2014-07-17

This article surveys the recent application of optical Fourier processing to the long-established but still expanding field of single-molecule imaging and microscopy. A variety of single-molecule studies can benefit from the additional image information that can be obtained by modulating the Fourier, or pupil, plane of a widefield microscope. After briefly reviewing several current applications, we present a comprehensive and computationally efficient theoretical model for simulating single-molecule fluorescence as it propagates through an imaging system. Furthermore, we describe how phase/amplitude-modulating optics inserted in the imaging pathway may be modeled, especially at the Fourier plane. Finally, we discuss selected recent applications of Fourier processing methods to measure the orientation, depth, and rotational mobility of single fluorescent molecules.

Recent Advances in Fiber Lasers for Nonlinear Microscopy

PubMed Central

Xu, C.; Wise, F. W.

2013-01-01

Nonlinear microscopy techniques developed over the past two decades have provided dramatic new capabilities for biological imaging. The initial demonstrations of nonlinear microscopies coincided with the development of solid-state femtosecond lasers, which continue to dominate applications of nonlinear microscopy. Fiber lasers offer attractive features for biological and biomedical imaging, and recent advances are leading to high-performance sources with the potential for robust, inexpensive, integrated instruments. This article discusses recent advances, and identifies challenges and opportunities for fiber lasers in nonlinear bioimaging. PMID:24416074

Direct visualization of lithium via annular bright field scanning transmission electron microscopy: a review.

PubMed

Findlay, Scott David; Huang, Rong; Ishikawa, Ryo; Shibata, Naoya; Ikuhara, Yuichi

2017-02-08

Annular bright field (ABF) scanning transmission electron microscopy has proven able to directly image lithium columns within crystalline environments, offering much insight into the structure and properties of lithium-ion battery materials. We summarize the image formation mechanisms underpinning ABF imaging, review the experimental application of this technique to imaging lithium in materials and overview the conditions that help maximize the visibility of lithium columns. © The Author 2016. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Photocontrollable Fluorescent Proteins for Superresolution Imaging

PubMed Central

Shcherbakova, Daria M.; Sengupta, Prabuddha; Lippincott-Schwartz, Jennifer; Verkhusha, Vladislav V.

2014-01-01

Superresolution fluorescence microscopy permits the study of biological processes at scales small enough to visualize fine subcellular structures that are unresolvable by traditional diffraction-limited light microscopy. Many superresolution techniques, including those applicable to live cell imaging, utilize genetically encoded photocontrollable fluorescent proteins. The fluorescence of these proteins can be controlled by light of specific wavelengths. In this review, we discuss the biochemical and photophysical properties of photocontrollable fluorescent proteins that are relevant to their use in superresolution microscopy. We then describe the recently developed photoactivatable, photoswitchable, and reversibly photoswitchable fluorescent proteins, and we detail their particular usefulness in single-molecule localization–based and nonlinear ensemble–based superresolution techniques. Finally, we discuss recent applications of photocontrollable proteins in superresolution imaging, as well as how these applications help to clarify properties of intracellular structures and processes that are relevant to cell and developmental biology, neuroscience, cancer biology and biomedicine. PMID:24895855

Live-cell stimulated Raman scattering imaging of alkyne-tagged biomolecules.

PubMed

Hong, Senlian; Chen, Tao; Zhu, Yuntao; Li, Ang; Huang, Yanyi; Chen, Xing

2014-06-02

Alkynes can be metabolically incorporated into biomolecules including nucleic acids, proteins, lipids, and glycans. In addition to the clickable chemical reactivity, alkynes possess a unique Raman scattering within the Raman-silent region of a cell. Coupling this spectroscopic signature with Raman microscopy yields a new imaging modality beyond fluorescence and label-free microscopies. The bioorthogonal Raman imaging of various biomolecules tagged with an alkyne by a state-of-the-art Raman imaging technique, stimulated Raman scattering (SRS) microscopy, is reported. This imaging method affords non-invasiveness, high sensitivity, and molecular specificity and therefore should find broad applications in live-cell imaging. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Platinum replica electron microscopy: Imaging the cytoskeleton globally and locally.

PubMed

Svitkina, Tatyana M

2017-05-01

Structural studies reveal how smaller components of a system work together as a whole. However, combining high resolution of details with full coverage of the whole is challenging. In cell biology, light microscopy can image many cells in their entirety, but at a lower resolution, whereas electron microscopy affords very high resolution, but usually at the expense of the sample size and coverage. Structural analyses of the cytoskeleton are especially demanding, because cytoskeletal networks are unresolvable by light microscopy due to their density and intricacy, whereas their proper preservation is a challenge for electron microscopy. Platinum replica electron microscopy can uniquely bridge the gap between the "comfort zones" of light and electron microscopy by allowing high resolution imaging of the cytoskeleton throughout the entire cell and in many cells in the population. This review describes the principles and applications of platinum replica electron microscopy for studies of the cytoskeleton. Copyright © 2017 Elsevier Ltd. All rights reserved.

Platinum Replica Electron Microscopy: Imaging the Cytoskeleton Globally and Locally

PubMed Central

SVITKINA, Tatyana M.

2017-01-01

Structural studies reveal how smaller components of a system work together as a whole. However, combining high resolution of details with full coverage of the whole is challenging. In cell biology, light microscopy can image many cells in their entirety, but at a lower resolution, whereas electron microscopy affords very high resolution, but usually at the expense of the sample size and coverage. Structural analyses of the cytoskeleton are especially demanding, because cytoskeletal networks are unresolvable by light microscopy due to their density and intricacy, whereas their proper preservation is a challenge for electron microscopy. Platinum replica electron microscopy can uniquely bridge the gap between the “comfort zones” of light and electron microscopy by allowing high resolution imaging of the cytoskeleton throughout the entire cell and in many cells in the population. This review describes the principles and applications of platinum replica electron microscopy for studies of the cytoskeleton. PMID:28323208

Optical Coherence Microscopy

NASA Astrophysics Data System (ADS)

Aguirre, Aaron D.; Zhou, Chao; Lee, Hsiang-Chieh; Ahsen, Osman O.; Fujimoto, James G.

Cellular imaging of human tissues remains an important advance for many clinical applications of optical coherence tomography (OCT). Imaging cells with traditional OCT systems has not been possible due to the limited transverse resolution of such techniques. Optical coherence microscopy (OCM) refers to OCT methods that achieve high transverse resolution to visualize cells and subcellular features. This chapter provides a comprehensive discussion of the rationale for cellular imaging in human tissues as well as a review of the key technological advances required to achieve it. Time domain and Fourier domain OCM approaches are described with an emphasis on state of the art system designs, including miniaturized endoscopic imaging probes. Clinical applications are discussed and multiple examples of cellular imaging in human tissues are provided.

Coherent Anti-Stokes Raman Scattering Spectroscopy of Single Molecules in Solution

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sunney Xie, Wei Min, Chris Freudiger, Sijia Lu

2012-01-18

During this funding period, we have developed two breakthrough techniques. The first is stimulated Raman scattering microscopy, providing label-free chemical contrast for chemical and biomedical imaging based on vibrational spectroscopy. Spontaneous Raman microscopy provides specific vibrational signatures of chemical bonds, but is often hindered by low sensitivity. We developed a three-dimensional multiphoton vibrational imaging technique based on stimulated Raman scattering (SRS). The sensitivity of SRS imaging is significantly greater than that of spontaneous Raman microscopy, which is achieved by implementing high-frequency (megahertz) phase-sensitive detection. SRS microscopy has a major advantage over previous coherent Raman techniques in that it offers background-freemore » and readily interpretable chemical contrast. We demonstrated a variety of biomedical applications, such as differentiating distributions of omega-3 fatty acids and saturated lipids in living cells, imaging of brain and skin tissues based on intrinsic lipid contrast, and monitoring drug delivery through the epidermis. This technology offers exciting prospect for medical imaging. The second technology we developed is stimulated emission microscopy. Many chromophores, such as haemoglobin and cytochromes, absorb but have undetectable fluorescence because the spontaneous emission is dominated by their fast non-radiative decay. Yet the detection of their absorption is difficult under a microscope. We use stimulated emission, which competes effectively with the nonradiative decay, to make the chromophores detectable, as a new contrast mechanism for optical microscopy. We demonstrate a variety of applications of stimulated emission microscopy, such as visualizing chromoproteins, non-fluorescent variants of the green fluorescent protein, monitoring lacZ gene expression with a chromogenic reporter, mapping transdermal drug distribu- tions without histological sectioning, and label-free microvascular imaging based on endogenous contrast of haemoglobin. For all these applications, sensitivity is orders of magnitude higher than for spontaneous emission or absorption contrast, permitting nonfluorescent reporters for molecular imaging. Although we did not accomplish the original goal of detecting single-molecule by CARS, our quest for high sensitivity of nonlinear optical microscopy paid off in providing the two brand new enabling technologies. Both techniques were greatly benefited from the use of high frequency modulation for microscopy, which led to orders of magnitude increase in sensitivity. Extensive efforts have been made on optics and electronics to accomplish these breakthroughs.« less

A STED-FLIM microscope applied to imaging the natural killer cell immune synapse

NASA Astrophysics Data System (ADS)

Lenz, M. O.; Brown, A. C. N.; Auksorius, E.; Davis, D. M.; Dunsby, C.; Neil, M. A. A.; French, P. M. W.

2011-03-01

We present a stimulated emission depletion (STED) fluorescence lifetime imaging (FLIM) microscope, excited by a microstructured optical fibre supercontinuum source that is pumped by a femtosecond Ti:Sapphire-laser, which is also used for depletion. Implemented using a piezo-scanning stage on a laser scanning confocal fluorescence microscope system with FLIM realised using time correlated single photon counting (TCSPC), this provides convenient switching between confocal and STED-FLIM with spatial resolution down to below 60 nm. We will present our design considerations to make a robust instrument for biological applications including a comparison between fixed phase plate and spatial light modulator (SLM) approaches to shape the STED beam and the correlation of STED and confocal FLIM microscopy. Following our previous application of FLIM-FRET to study intercellular signalling at the immunological synapse (IS), we are employing STED microscopy to characterize the spatial distribution of cellular molecules with subdiffraction resolution at the IS. In particular, we are imaging cytoskeletal structure at the Natural Killer cell activated immune synapse. We will also present our progress towards multilabel STED microscopy to determine how relative spatial molecular organization, previously undetectable by conventional microscopy techniques, is important for NK cell cytotoxic function. Keywords: STED, Stimulated Emission Depletion Microscopy, Natural Killer (NK) cell, Fluorescence lifetime imaging, FLIM, Super resolution microscopy.

Image scanning fluorescence emission difference microscopy based on a detector array.

PubMed

Li, Y; Liu, S; Liu, D; Sun, S; Kuang, C; Ding, Z; Liu, X

2017-06-01

We propose a novel imaging method that enables the enhancement of three-dimensional resolution of confocal microscopy significantly and achieve experimentally a new fluorescence emission difference method for the first time, based on the parallel detection with a detector array. Following the principles of photon reassignment in image scanning microscopy, images captured by the detector array were arranged. And by selecting appropriate reassign patterns, the imaging result with enhanced resolution can be achieved with the method of fluorescence emission difference. Two specific methods are proposed in this paper, showing that the difference between an image scanning microscopy image and a confocal image will achieve an improvement of transverse resolution by approximately 43% compared with that in confocal microscopy, and the axial resolution can also be enhanced by at least 22% experimentally and 35% theoretically. Moreover, the methods presented in this paper can improve the lateral resolution by around 10% than fluorescence emission difference and 15% than Airyscan. The mechanism of our methods is verified by numerical simulations and experimental results, and it has significant potential in biomedical applications. © 2017 The Authors Journal of Microscopy © 2017 Royal Microscopical Society.

Invited Review Article: Imaging techniques for harmonic and multiphoton absorption fluorescence microscopy

PubMed Central

Carriles, Ramón; Schafer, Dawn N.; Sheetz, Kraig E.; Field, Jeffrey J.; Cisek, Richard; Barzda, Virginijus; Sylvester, Anne W.; Squier, Jeffrey A.

2009-01-01

We review the current state of multiphoton microscopy. In particular, the requirements and limitations associated with high-speed multiphoton imaging are considered. A description of the different scanning technologies such as line scan, multifoci approaches, multidepth microscopy, and novel detection techniques is given. The main nonlinear optical contrast mechanisms employed in microscopy are reviewed, namely, multiphoton excitation fluorescence, second harmonic generation, and third harmonic generation. Techniques for optimizing these nonlinear mechanisms through a careful measurement of the spatial and temporal characteristics of the focal volume are discussed, and a brief summary of photobleaching effects is provided. Finally, we consider three new applications of multiphoton microscopy: nonlinear imaging in microfluidics as applied to chemical analysis and the use of two-photon absorption and self-phase modulation as contrast mechanisms applied to imaging problems in the medical sciences. PMID:19725639

Tutorial on photoacoustic tomography

PubMed Central

Zhou, Yong; Yao, Junjie; Wang, Lihong V.

2016-01-01

Abstract. Photoacoustic tomography (PAT) has become one of the fastest growing fields in biomedical optics. Unlike pure optical imaging, such as confocal microscopy and two-photon microscopy, PAT employs acoustic detection to image optical absorption contrast with high-resolution deep into scattering tissue. So far, PAT has been widely used for multiscale anatomical, functional, and molecular imaging of biological tissues. We focus on PAT’s basic principles, major implementations, imaging contrasts, and recent applications. PMID:27086868

Dielectrophoretic immobilization of proteins: Quantification by atomic force microscopy.

PubMed

Laux, Eva-Maria; Knigge, Xenia; Bier, Frank F; Wenger, Christian; Hölzel, Ralph

2015-09-01

The combination of alternating electric fields with nanometer-sized electrodes allows the permanent immobilization of proteins by dielectrophoretic force. Here, atomic force microscopy is introduced as a quantification method, and results are compared with fluorescence microscopy. Experimental parameters, for example the applied voltage and duration of field application, are varied systematically, and the influence on the amount of immobilized proteins is investigated. A linear correlation to the duration of field application was found by atomic force microscopy, and both microscopical methods yield a square dependence of the amount of immobilized proteins on the applied voltage. While fluorescence microscopy allows real-time imaging, atomic force microscopy reveals immobilized proteins obscured in fluorescence images due to low S/N. Furthermore, the higher spatial resolution of the atomic force microscope enables the visualization of the protein distribution on single nanoelectrodes. The electric field distribution is calculated and compared to experimental results with very good agreement to atomic force microscopy measurements. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Atomic force microscopy as a tool for the investigation of living cells.

PubMed

Morkvėnaitė-Vilkončienė, Inga; Ramanavičienė, Almira; Ramanavičius, Arūnas

2013-01-01

Atomic force microscopy is a valuable and useful tool for the imaging and investigation of living cells in their natural environment at high resolution. Procedures applied to living cell preparation before measurements should be adapted individually for different kinds of cells and for the desired measurement technique. Different ways of cell immobilization, such as chemical fixation on the surface, entrapment in the pores of a membrane, or growing them directly on glass cover slips or on plastic substrates, result in the distortion or appearance of artifacts in atomic force microscopy images. Cell fixation allows the multiple use of samples and storage for a prolonged period; it also increases the resolution of imaging. Different atomic force microscopy modes are used for the imaging and analysis of living cells. The contact mode is the best for cell imaging because of high resolution, but it is usually based on the following: (i) image formation at low interaction force, (ii) low scanning speed, and (iii) usage of "soft," low resolution cantilevers. The tapping mode allows a cell to behave like a very solid material, and destructive shear forces are minimized, but imaging in liquid is difficult. The force spectroscopy mode is used for measuring the mechanical properties of cells; however, obtained results strongly depend on the cell fixation method. In this paper, the application of 3 atomic force microscopy modes including (i) contact, (ii) tapping, and (iii) force spectroscopy for the investigation of cells is described. The possibilities of cell preparation for the measurements, imaging, and determination of mechanical properties of cells are provided. The applicability of atomic force microscopy to diagnostics and other biomedical purposes is discussed.

Imaging cellular structures in super-resolution with SIM, STED and Localisation Microscopy: A practical comparison.

PubMed

Wegel, Eva; Göhler, Antonia; Lagerholm, B Christoffer; Wainman, Alan; Uphoff, Stephan; Kaufmann, Rainer; Dobbie, Ian M

2016-06-06

Many biological questions require fluorescence microscopy with a resolution beyond the diffraction limit of light. Super-resolution methods such as Structured Illumination Microscopy (SIM), STimulated Emission Depletion (STED) microscopy and Single Molecule Localisation Microscopy (SMLM) enable an increase in image resolution beyond the classical diffraction-limit. Here, we compare the individual strengths and weaknesses of each technique by imaging a variety of different subcellular structures in fixed cells. We chose examples ranging from well separated vesicles to densely packed three dimensional filaments. We used quantitative and correlative analyses to assess the performance of SIM, STED and SMLM with the aim of establishing a rough guideline regarding the suitability for typical applications and to highlight pitfalls associated with the different techniques.

Color calibration and fusion of lens-free and mobile-phone microscopy images for high-resolution and accurate color reproduction

NASA Astrophysics Data System (ADS)

Zhang, Yibo; Wu, Yichen; Zhang, Yun; Ozcan, Aydogan

2016-06-01

Lens-free holographic microscopy can achieve wide-field imaging in a cost-effective and field-portable setup, making it a promising technique for point-of-care and telepathology applications. However, due to relatively narrow-band sources used in holographic microscopy, conventional colorization methods that use images reconstructed at discrete wavelengths, corresponding to e.g., red (R), green (G) and blue (B) channels, are subject to color artifacts. Furthermore, these existing RGB colorization methods do not match the chromatic perception of human vision. Here we present a high-color-fidelity and high-resolution imaging method, termed “digital color fusion microscopy” (DCFM), which fuses a holographic image acquired at a single wavelength with a color-calibrated image taken by a low-magnification lens-based microscope using a wavelet transform-based colorization method. We demonstrate accurate color reproduction of DCFM by imaging stained tissue sections. In particular we show that a lens-free holographic microscope in combination with a cost-effective mobile-phone-based microscope can generate color images of specimens, performing very close to a high numerical-aperture (NA) benchtop microscope that is corrected for color distortions and chromatic aberrations, also matching the chromatic response of human vision. This method can be useful for wide-field imaging needs in telepathology applications and in resource-limited settings, where whole-slide scanning microscopy systems are not available.

Color calibration and fusion of lens-free and mobile-phone microscopy images for high-resolution and accurate color reproduction

PubMed Central

Zhang, Yibo; Wu, Yichen; Zhang, Yun; Ozcan, Aydogan

2016-01-01

Lens-free holographic microscopy can achieve wide-field imaging in a cost-effective and field-portable setup, making it a promising technique for point-of-care and telepathology applications. However, due to relatively narrow-band sources used in holographic microscopy, conventional colorization methods that use images reconstructed at discrete wavelengths, corresponding to e.g., red (R), green (G) and blue (B) channels, are subject to color artifacts. Furthermore, these existing RGB colorization methods do not match the chromatic perception of human vision. Here we present a high-color-fidelity and high-resolution imaging method, termed “digital color fusion microscopy” (DCFM), which fuses a holographic image acquired at a single wavelength with a color-calibrated image taken by a low-magnification lens-based microscope using a wavelet transform-based colorization method. We demonstrate accurate color reproduction of DCFM by imaging stained tissue sections. In particular we show that a lens-free holographic microscope in combination with a cost-effective mobile-phone-based microscope can generate color images of specimens, performing very close to a high numerical-aperture (NA) benchtop microscope that is corrected for color distortions and chromatic aberrations, also matching the chromatic response of human vision. This method can be useful for wide-field imaging needs in telepathology applications and in resource-limited settings, where whole-slide scanning microscopy systems are not available. PMID:27283459

Applications of two-photon fluorescence microscopy in deep-tissue imaging

NASA Astrophysics Data System (ADS)

Dong, Chen-Yuan; Yu, Betty; Hsu, Lily L.; Kaplan, Peter D.; Blankschstein, D.; Langer, Robert; So, Peter T. C.

2000-07-01

Based on the non-linear excitation of fluorescence molecules, two-photon fluorescence microscopy has become a significant new tool for biological imaging. The point-like excitation characteristic of this technique enhances image quality by the virtual elimination of off-focal fluorescence. Furthermore, sample photodamage is greatly reduced because fluorescence excitation is limited to the focal region. For deep tissue imaging, two-photon microscopy has the additional benefit in the greatly improved imaging depth penetration. Since the near- infrared laser sources used in two-photon microscopy scatter less than their UV/glue-green counterparts, in-depth imaging of highly scattering specimen can be greatly improved. In this work, we will present data characterizing both the imaging characteristics (point-spread-functions) and tissue samples (skin) images using this novel technology. In particular, we will demonstrate how blind deconvolution can be used further improve two-photon image quality and how this technique can be used to study mechanisms of chemically-enhanced, transdermal drug delivery.

Multiphoton imaging with high peak power VECSELs

NASA Astrophysics Data System (ADS)

Mirkhanov, Shamil; Quarterman, Adrian H.; Swift, Samuel; Praveen, Bavishna B.; Smyth, Conor J. C.; Wilcox, Keith G.

2016-03-01

Multiphoton imaging (MMPI) has become one of thee key non-invasive light microscopy techniques. This technique allows deep tissue imaging with high resolution and less photo-damage than conventional confocal microscopy. MPI is type of laser-scanning microscopy that employs localized nonlinear excitation, so that fluorescence is excited only with is scanned focal volume. For many years, Ti: sapphire femtosecond lasers have been the leading light sources for MPI applications. However, recent developments in laser sources and new types of fluorophores indicate that longer wavelength excitation could be a good alternative for these applications. Mode-locked VECSEELs have the potential to be low cost, compact light sources for MPI systems, with the additional advantage of broad wavelength coverage through use of different semiconductor material systems. Here, we use a femtosecond fibber laser to investigate the effect average power and repetition rate has on MPI image quality, to allow us to optimize our mode-locked VVECSELs for MPI.

Application of a reflective microscope objective for multiphoton microscopy.

PubMed

Kabir, Mohammad M; Choubal, Aakash M; Toussaint, Kimani C

2018-04-20

Reflective objectives (ROs) mitigate chromatic aberration across a broad wavelength range. Yet, a systematic performance characterisation of ROs has not been done. In this paper, we compare the performance of a 0.5 numerical-aperture (NA) reflective objective (RO) with a 0.55 NA standard glass objective (SO), using two-photon fluorescence (TPF) and second-harmonic generation (SHG). For experiments spanning ∼1 octave in the visible and NIR wavelengths, the SO leads to defocusing errors of 25-40% for TPF images of subdiffraction fluorescent beads and 10-12% for SHG images of collagen fibres. The corresponding error for the RO is ∼4% for both imaging modalities. This work emphasises the potential utility of ROs for multimodal multiphoton microscopy applications. © 2018 The Authors Journal of Microscopy © 2018 Royal Microscopical Society.

X-ray microscopy with high-resolution zone plates: recent developments

NASA Astrophysics Data System (ADS)

Schneider, Gerd; Wilhein, Thomas; Niemann, Bastian; Guttman, P.; Schliebe, T.; Lehr, J.; Aschoff, H.; Thieme, Juergen; Rudolph, Dietbert M.; Schmahl, Guenther A.

1995-09-01

In order to expand the applications of x-ray microscopy, developments in the fields of zone plate technology, specimen preparation and imaging techniques have been made. A new cross- linked polymer chain electron beam resist allows us to record zone plate pattern down to 19 nm outermost zone width. High resolution zone plates in germanium with outermost zone widths down to 19 nm have been developed. In addition, phase zone plates in nickel down to 30 nm zone width have been made by electroplating. In order to enhance the image contrast for weak absorbing objects, the phase contrast method for x-ray microscopy was developed and implemented on the Gottingen x-ray microscope at BESSY. The effects of x ray absorption on the structure of biological specimen limits the maximum applicable radiation dose and therefore the achievable signal to noise ratio for an artifact-free x-ray image. To improve the stability especially of biological specimen, a cryogenic object chamber has been developed and tested. It turns out that at the operating temperature T less than or equal to 130 K unfixed biological specimen can be exposed to a radiation dose of 109 - 1010 Gy without any observable structural changes. A multiple-angle viewing stage allows us to take stereoscopic images with the x-ray microscope, giving a 3D-impression of the object. As an example for the applications of x-ray microscopy in biology, erythrocytes infected by malaria parasite have been examined. Studies of the aggregation of hematite by sodium sulfate gives an example for the application of x-ray microscopy in the field of colloid research.

Bioorthogonal Chemical Imaging for Biomedicine

NASA Astrophysics Data System (ADS)

Min, Wei

2017-06-01

Innovations in light microscopy have tremendously revolutionized the way researchers study biological systems with subcellular resolution. Although fluorescence microscopy is currently the method of choice for cellular imaging, it faces fundamental limitations for studying the vast number of small biomolecules. This is because relatively bulky fluorescent labels could introduce considerable perturbation to or even completely alter the native functions of vital small biomolecules. Hence, despite their immense functional importance, these small biomolecules remain largely undetectable by fluorescence microscopy. To address this challenge, we have developed a bioorthogonal chemical imaging platform. By coupling stimulated Raman scattering (SRS) microscopy, an emerging nonlinear Raman microscopy technique, with tiny and Raman-active vibrational probes (e.g., alkynes, nitriles and stable isotopes including 2H and 13C), bioorthogonal chemical imaging exhibits superb sensitivity, specificity, multiplicity and biocompatibility for imaging small biomolecules in live systems including tissues and organisms. Exciting biomedical applications such as imaging fatty acid metabolism related to lipotoxicity, glucose uptake and metabolism, drug trafficking, protein synthesis, DNA replication, protein degradation, RNA synthesis and tumor metabolism will be presented. This bioorthogonal chemical imaging platform is compatible with live-cell biology, thus allowing real-time imaging of small-molecule dynamics. Moreover, further chemical and spectroscopic strategies allow for multicolor bioorthogonal chemical imaging, a valuable technique in the era of "omics". We envision that the coupling of SRS microscopy with vibrational probes would do for small biomolecules what fluorescence microscopy of fluorophores has done for larger molecular species, bringing small molecules under the illumination of modern light microscopy.

Ophthalmic applications of confocal microscopy: diagnostics, refractive surgery, and eye banking

NASA Astrophysics Data System (ADS)

Masters, Barry R.

1990-11-01

Confocal microscopy of ocular tissue provides two advantages over traditional imaging techniques: increased range and transverse resolution and increased contrast. The semitransparent cornea and ocular lens in the living eye can be optically sectioned and observed by reflected light confocal microscopy. Within the cornea we observed various cell components nerve fibers nerve cell bodies and fibrous networks. The confocal microscopic images from the in-situ ocular lens show the lens capsule the lens epithelium and the individual lens fibrils. All of the reflected light confocal microscopic images have high contrast and high resolution. Some of the applications of confocal imaging in ophthalmology include: diagnostics of the cornea and the ocular lens examination prior to and after refractive surgery examination of intraocular lenses (IOL) and examination of eye bank material. Other ophthalmic uses of confocal imaging include: studies of wound healing therapeutics and the effects of contact lenses on the cornea. The proposed features of a clinical confocal microscope are reviewed. 2.

4Pi-confocal microscopy of live cells

NASA Astrophysics Data System (ADS)

Bahlmann, Karsten; Jakobs, Stefan; Hell, Stefan W.

2002-06-01

By coherently adding the spherical wavefronts of two opposing lenses, two-photon excitation 4Pi-confocal fluorescence microscopy has achieved three-dimensional imaging with an axial resolution 3-7 times better than confocal microscopy. So far this improvement was possible only in glycerol-mounted, fixed cells. Here we report 4Pi-confocal microscopy of watery objects and its application to the imaging of live cells. Water immersion 4Pi-confocal microscopy of membrane stained live Escherichia coli bacteria attains a 4.3 fold better axial resolution as compared to the best water immersion confocal microscope. The resolution enhancement results into a vastly improved three-dimensional representation of the bacteria. The first images of live biological samples with an all-directional resolution in the 190-280 nm range are presented here, thus establishing a new resolution benchmark in live cell microscopy.

The Open Microscopy Environment: open image informatics for the biological sciences

NASA Astrophysics Data System (ADS)

Blackburn, Colin; Allan, Chris; Besson, Sébastien; Burel, Jean-Marie; Carroll, Mark; Ferguson, Richard K.; Flynn, Helen; Gault, David; Gillen, Kenneth; Leigh, Roger; Leo, Simone; Li, Simon; Lindner, Dominik; Linkert, Melissa; Moore, Josh; Moore, William J.; Ramalingam, Balaji; Rozbicki, Emil; Rustici, Gabriella; Tarkowska, Aleksandra; Walczysko, Petr; Williams, Eleanor; Swedlow, Jason R.

2016-07-01

Despite significant advances in biological imaging and analysis, major informatics challenges remain unsolved: file formats are proprietary, storage and analysis facilities are lacking, as are standards for sharing image data and results. While the open FITS file format is ubiquitous in astronomy, astronomical imaging shares many challenges with biological imaging, including the need to share large image sets using secure, cross-platform APIs, and the need for scalable applications for processing and visualization. The Open Microscopy Environment (OME) is an open-source software framework developed to address these challenges. OME tools include: an open data model for multidimensional imaging (OME Data Model); an open file format (OME-TIFF) and library (Bio-Formats) enabling free access to images (5D+) written in more than 145 formats from many imaging domains, including FITS; and a data management server (OMERO). The Java-based OMERO client-server platform comprises an image metadata store, an image repository, visualization and analysis by remote access, allowing sharing and publishing of image data. OMERO provides a means to manage the data through a multi-platform API. OMERO's model-based architecture has enabled its extension into a range of imaging domains, including light and electron microscopy, high content screening, digital pathology and recently into applications using non-image data from clinical and genomic studies. This is made possible using the Bio-Formats library. The current release includes a single mechanism for accessing image data of all types, regardless of original file format, via Java, C/C++ and Python and a variety of applications and environments (e.g. ImageJ, Matlab and R).

Wide field fluorescence epi-microscopy behind a scattering medium enabled by speckle correlations

NASA Astrophysics Data System (ADS)

Hofer, Matthias; Soeller, Christian; Brasselet, Sophie; Bertolotti, Jacopo

2018-04-01

Fluorescence microscopy is widely used in biological imaging, however scattering from tissues strongly limits its applicability to a shallow depth. In this work we adapt a methodology inspired from stellar speckle interferometry, and exploit the optical memory effect to enable fluorescence microscopy through a turbid layer. We demonstrate efficient reconstruction of micrometer-size fluorescent objects behind a scattering medium in epi-microscopy, and study the specificities of this imaging modality (magnification, field of view, resolution) as compared to traditional microscopy. Using a modified phase retrieval algorithm to reconstruct fluorescent objects from speckle images, we demonstrate robust reconstructions even in relatively low signal to noise conditions. This modality is particularly appropriate for imaging in biological media, which are known to exhibit relatively large optical memory ranges compatible with tens of micrometers size field of views, and large spectral bandwidths compatible with emission fluorescence spectra of tens of nanometers widths.

Quantitative imaging of bilirubin by photoacoustic microscopy

NASA Astrophysics Data System (ADS)

Zhou, Yong; Zhang, Chi; Yao, Da-Kang; Wang, Lihong V.

2013-03-01

Noninvasive detection of both bilirubin concentration and its distribution is important for disease diagnosis. Here we implemented photoacoustic microscopy (PAM) to detect bilirubin distribution. We first demonstrate that our PAM system can measure the absorption spectra of bilirubin and blood. We also image bilirubin distributions in tissuemimicking samples, both without and with blood mixed. Our results show that PAM has the potential to quantitatively image bilirubin in vivo for clinical applications.

Multilayer mounting enables long-term imaging of zebrafish development in a light sheet microscope.

PubMed

Kaufmann, Anna; Mickoleit, Michaela; Weber, Michael; Huisken, Jan

2012-09-01

Light sheet microscopy techniques, such as selective plane illumination microscopy (SPIM), are ideally suited for time-lapse imaging of developmental processes lasting several hours to a few days. The success of this promising technology has mainly been limited by the lack of suitable techniques for mounting fragile samples. Embedding zebrafish embryos in agarose, which is common in conventional confocal microscopy, has resulted in severe growth defects and unreliable results. In this study, we systematically quantified the viability and mobility of zebrafish embryos mounted under more suitable conditions. We found that tubes made of fluorinated ethylene propylene (FEP) filled with low concentrations of agarose or methylcellulose provided an optimal balance between sufficient confinement of the living embryo in a physiological environment over 3 days and optical clarity suitable for fluorescence imaging. We also compared the effect of different concentrations of Tricaine on the development of zebrafish and provide guidelines for its optimal use depending on the application. Our results will make light sheet microscopy techniques applicable to more fields of developmental biology, in particular the multiview long-term imaging of zebrafish embryos and other small organisms. Furthermore, the refinement of sample preparation for in toto and in vivo imaging will promote other emerging optical imaging techniques, such as optical projection tomography (OPT).

Intravital microscopy: a novel tool to study cell biology in living animals.

PubMed

Weigert, Roberto; Sramkova, Monika; Parente, Laura; Amornphimoltham, Panomwat; Masedunskas, Andrius

2010-05-01

Intravital microscopy encompasses various optical microscopy techniques aimed at visualizing biological processes in live animals. In the last decade, the development of non-linear optical microscopy resulted in an enormous increase of in vivo studies, which have addressed key biological questions in fields such as neurobiology, immunology and tumor biology. Recently, few studies have shown that subcellular processes can be imaged dynamically in the live animal at a resolution comparable to that achieved in cell cultures, providing new opportunities to study cell biology under physiological conditions. The overall aim of this review is to give the reader a general idea of the potential applications of intravital microscopy with a particular emphasis on subcellular imaging. An overview of some of the most exciting studies in this field will be presented using resolution as a main organizing criterion. Indeed, first we will focus on those studies in which organs were imaged at the tissue level, then on those focusing on single cells imaging, and finally on those imaging subcellular organelles and structures.

Unwarping confocal microscopy images of bee brains by nonrigid registration to a magnetic resonance microscopy image.

PubMed

Rohlfing, Torsten; Schaupp, Frank; Haddad, Daniel; Brandt, Robert; Haase, Axel; Menzel, Randolf; Maurer, Calvin R

2005-01-01

Confocal microscopy (CM) is a powerful image acquisition technique that is well established in many biological applications. It provides 3-D acquisition with high spatial resolution and can acquire several different channels of complementary image information. Due to the specimen extraction and preparation process, however, the shapes of imaged objects may differ considerably from their in vivo appearance. Magnetic resonance microscopy (MRM) is an evolving variant of magnetic resonance imaging, which achieves microscopic resolutions using a high magnetic field and strong magnetic gradients. Compared to CM imaging, MRM allows for in situ imaging and is virtually free of geometrical distortions. We propose to combine the advantages of both methods by unwarping CM images using a MRM reference image. Our method incorporates a sequence of image processing operators applied to the MRM image, followed by a two-stage intensity-based registration to compute a nonrigid coordinate transformation between the CM images and the MRM image. We present results obtained using CM images from the brains of 20 honey bees and a MRM image of an in situ bee brain. Copyright 2005 Society of Photo-Optical Instrumentation Engineers.

Spectral Demultiplexing in Holographic and Fluorescent On-chip Microscopy

NASA Astrophysics Data System (ADS)

Sencan, Ikbal; Coskun, Ahmet F.; Sikora, Uzair; Ozcan, Aydogan

2014-01-01

Lensfree on-chip imaging and sensing platforms provide compact and cost-effective designs for various telemedicine and lab-on-a-chip applications. In this work, we demonstrate computational solutions for some of the challenges associated with (i) the use of broadband, partially-coherent illumination sources for on-chip holographic imaging, and (ii) multicolor detection for lensfree fluorescent on-chip microscopy. Specifically, we introduce spectral demultiplexing approaches that aim to digitally narrow the spectral content of broadband illumination sources (such as wide-band light emitting diodes or even sunlight) to improve spatial resolution in holographic on-chip microscopy. We also demonstrate the application of such spectral demultiplexing approaches for wide-field imaging of multicolor fluorescent objects on a chip. These computational approaches can be used to replace e.g., thin-film interference filters, gratings or other optical components used for spectral multiplexing/demultiplexing, which can form a desirable solution for cost-effective and compact wide-field microscopy and sensing needs on a chip.

Super-resolution fluorescence microscopy by stepwise optical saturation

PubMed Central

Zhang, Yide; Nallathamby, Prakash D.; Vigil, Genevieve D.; Khan, Aamir A.; Mason, Devon E.; Boerckel, Joel D.; Roeder, Ryan K.; Howard, Scott S.

2018-01-01

Super-resolution fluorescence microscopy is an important tool in biomedical research for its ability to discern features smaller than the diffraction limit. However, due to its difficult implementation and high cost, the super-resolution microscopy is not feasible in many applications. In this paper, we propose and demonstrate a saturation-based super-resolution fluorescence microscopy technique that can be easily implemented and requires neither additional hardware nor complex post-processing. The method is based on the principle of stepwise optical saturation (SOS), where M steps of raw fluorescence images are linearly combined to generate an image with a M-fold increase in resolution compared with conventional diffraction-limited images. For example, linearly combining (scaling and subtracting) two images obtained at regular powers extends the resolution by a factor of 1.4 beyond the diffraction limit. The resolution improvement in SOS microscopy is theoretically infinite but practically is limited by the signal-to-noise ratio. We perform simulations and experimentally demonstrate super-resolution microscopy with both one-photon (confocal) and multiphoton excitation fluorescence. We show that with the multiphoton modality, the SOS microscopy can provide super-resolution imaging deep in scattering samples. PMID:29675306

An open data mining framework for the analysis of medical images: application on obstructive nephropathy microscopy images.

PubMed

Doukas, Charalampos; Goudas, Theodosis; Fischer, Simon; Mierswa, Ingo; Chatziioannou, Aristotle; Maglogiannis, Ilias

2010-01-01

This paper presents an open image-mining framework that provides access to tools and methods for the characterization of medical images. Several image processing and feature extraction operators have been implemented and exposed through Web Services. Rapid-Miner, an open source data mining system has been utilized for applying classification operators and creating the essential processing workflows. The proposed framework has been applied for the detection of salient objects in Obstructive Nephropathy microscopy images. Initial classification results are quite promising demonstrating the feasibility of automated characterization of kidney biopsy images.

Application of environmental scanning electron microscopy to determine biological surface structure.

PubMed

Kirk, S E; Skepper, J N; Donald, A M

2009-02-01

The use of environmental scanning electron microscopy in biology is growing as more becomes understood about the advantages and limitations of the technique. These are discussed and we include new evidence about the effect of environmental scanning electron microscopy imaging on the viability of mammalian cells. We show that although specimen preparation for high-vacuum scanning electron microscopy introduces some artefacts, there are also challenges in the use of environmental scanning electron microscopy, particularly at higher resolutions. This suggests the two technologies are best used in combination. We have used human monocyte-derived macrophages as a test sample, imaging their complicated and delicate membrane ruffles and protrusions. We have also explored the possibility of using environmental scanning electron microscopy for dynamic experiments, finding that mammalian cells cannot be imaged and kept alive in the environmental scanning electron microscopy. The dehydration step in which the cell surface is exposed causes irreversible damage, probably via loss of membrane integrity during liquid removal in the specimen chamber. Therefore, mammalian cells should be imaged after fixation where possible to protect against damage as a result of chamber conditions.

Real-time Image Processing for Microscopy-based Label-free Imaging Flow Cytometry in a Microfluidic Chip.

PubMed

Heo, Young Jin; Lee, Donghyeon; Kang, Junsu; Lee, Keondo; Chung, Wan Kyun

2017-09-14

Imaging flow cytometry (IFC) is an emerging technology that acquires single-cell images at high-throughput for analysis of a cell population. Rich information that comes from high sensitivity and spatial resolution of a single-cell microscopic image is beneficial for single-cell analysis in various biological applications. In this paper, we present a fast image-processing pipeline (R-MOD: Real-time Moving Object Detector) based on deep learning for high-throughput microscopy-based label-free IFC in a microfluidic chip. The R-MOD pipeline acquires all single-cell images of cells in flow, and identifies the acquired images as a real-time process with minimum hardware that consists of a microscope and a high-speed camera. Experiments show that R-MOD has the fast and reliable accuracy (500 fps and 93.3% mAP), and is expected to be used as a powerful tool for biomedical and clinical applications.

Fluorescence confocal microscopy for pathologists.

PubMed

Ragazzi, Moira; Piana, Simonetta; Longo, Caterina; Castagnetti, Fabio; Foroni, Monica; Ferrari, Guglielmo; Gardini, Giorgio; Pellacani, Giovanni

2014-03-01

Confocal microscopy is a non-invasive method of optical imaging that may provide microscopic images of untreated tissue that correspond almost perfectly to hematoxylin- and eosin-stained slides. Nowadays, following two confocal imaging systems are available: (1) reflectance confocal microscopy, based on the natural differences in refractive indices of subcellular structures within the tissues; (2) fluorescence confocal microscopy, based on the use of fluorochromes, such as acridine orange, to increase the contrast epithelium-stroma. In clinical practice to date, confocal microscopy has been used with the goal of obviating the need for excision biopsies, thereby reducing the need for pathological examination. The aim of our study was to test fluorescence confocal microscopy on different types of surgical specimens, specifically breast, lymph node, thyroid, and colon. The confocal images were correlated to the corresponding histological sections in order to provide a morphologic parallel and to highlight current limitations and possible applications of this technology for surgical pathology practice. As a result, neoplastic tissues were easily distinguishable from normal structures and reactive processes such as fibrosis; the use of fluorescence enhanced contrast and image quality in confocal microscopy without compromising final histologic evaluation. Finally, the fluorescence confocal microscopy images of the adipose tissue were as accurate as those of conventional histology and were devoid of the frozen-section-related artefacts that can compromise intraoperative evaluation. Despite some limitations mainly related to black/white images, which require training in imaging interpretation, this study confirms that fluorescence confocal microscopy may represent an alternative to frozen sections in the assessment of margin status in selected settings or when the conservation of the specimen is crucial. This is the first study to employ fluorescent confocal microscopy on surgical specimens other than the skin and to evaluate the diagnostic capability of this technology from pathologists' viewpoint.

Carbon contamination in scanning transmission electron microscopy and its impact on phase-plate applications.

PubMed

Hettler, Simon; Dries, Manuel; Hermann, Peter; Obermair, Martin; Gerthsen, Dagmar; Malac, Marek

2017-05-01

We analyze electron-beam induced carbon contamination in a transmission electron microscope. The study is performed on thin films potentially suitable as phase plates for phase-contrast transmission electron microscopy. Electron energy-loss spectroscopy and phase-plate imaging is utilized to analyze the contamination. The deposited contamination layer is identified as a graphitic carbon layer which is not prone to electrostatic charging whereas a non-conductive underlying substrate charges. Several methods that inhibit contamination are evaluated and the impact of carbon contamination on phase-plate imaging is discussed. The findings are in general interesting for scanning transmission electron microscopy applications. Crown Copyright © 2017. Published by Elsevier Ltd. All rights reserved.

Probing Subdiffraction Limit Separations with Plasmon Coupling Microscopy: Concepts and Applications

PubMed Central

Wu, Linxi

2014-01-01

Due to their advantageous materials properties, noble metal nanoparticles are versatile tools in biosensing and imaging. A characteristic feature of gold and silver nanoparticles is their ability to sustain localized surface plasmons that provide both large optical cross-sections and extraordinary photophysical stability. Plasmon Coupling Microscopy takes advantage of the beneficial optical properties and utilizes electromagnetic near-field coupling between individual noble metal nanoparticle labels to resolve subdiffraction limit separations in an all-optical fashion. This Tutorial provides an introduction into the physical concepts underlying distance dependent plasmon coupling, discusses potential experimental implementations of Plasmon Coupling Microscopy, and reviews applications in the area of biosensing and imaging. PMID:24390574

A low-cost photoacoustic microscopy system with a laser diode excitation

PubMed Central

Wang, Tianheng; Nandy, Sreyankar; Salehi, Hassan S.; Kumavor, Patrick D.; Zhu, Quing

2014-01-01

Photoacoustic microscopy (PAM) is capable of mapping microvasculature networks in biological tissue and has demonstrated great potential for biomedical applications. However, the clinical application of the PAM system is limited due to the use of bulky and expensive pulsed laser sources. In this paper, a low-cost optical-resolution PAM system with a pulsed laser diode excitation has been introduced. The lateral resolution of this PAM system was estimated to be 7 µm by imaging a carbon fiber. The phantoms made of polyethylene tubes filled with blood and a mouse ear were imaged to demonstrate the feasibility of this PAM system for imaging biological tissues. PMID:25401019

Feature evaluation of complex hysteresis smoothing and its practical applications to noisy SEM images.

PubMed

Suzuki, Kazuhiko; Oho, Eisaku

2013-01-01

Quality of a scanning electron microscopy (SEM) image is strongly influenced by noise. This is a fundamental drawback of the SEM instrument. Complex hysteresis smoothing (CHS) has been previously developed for noise removal of SEM images. This noise removal is performed by monitoring and processing properly the amplitude of the SEM signal. As it stands now, CHS may not be so utilized, though it has several advantages for SEM. For example, the resolution of image processed by CHS is basically equal to that of the original image. In order to find wide application of the CHS method in microscopy, the feature of CHS, which has not been so clarified until now is evaluated correctly. As the application of the result obtained by the feature evaluation, cursor width (CW), which is the sole processing parameter of CHS, is determined more properly using standard deviation of noise Nσ. In addition, disadvantage that CHS cannot remove the noise with excessively large amplitude is improved by a certain postprocessing. CHS is successfully applicable to SEM images with various noise amplitudes. © Wiley Periodicals, Inc.

Application of gold nanoparticles as contrast agents in confocal laser scanning microscopy

NASA Astrophysics Data System (ADS)

Lemelle, A.; Veksler, B.; Kozhevnikov, I. S.; Akchurin, G. G.; Piletsky, S. A.; Meglinski, I.

2009-01-01

Confocal laser scanning microscopy (CLSM) is a modern high-resolution optical technique providing detailed image of tissue structure with high (down to microns) spatial resolution. Aiming at a concurrent improvement of imaging depth and image quality the CLSM requires the use of contrast agents. Commonly employed fluorescent contrast agents, such as fluorescent dyes and proteins, suffer from toxicity, photo-bleaching and overlapping with the tissues autofluorescence. Gold nanoparticles are potentially highly attractive to be applied as a contrast agent since they are not subject to photo-bleaching and can target biochemical cells markers associated with the specific diseases. In current report we consider the applicability of gold nano-spheres as a contrast agent to enhance quality of CLSM images of skin tissues in vitro versus the application of optical clearing agent, such as glycerol. The enhancement of CLSM image contrast was observed with an application of gold nano-spheres diffused within the skin tissues. We show that optical clearing agents such as a glycerol provide better CLSM image contrast than gold nano-spheres.

Sparse imaging for fast electron microscopy

NASA Astrophysics Data System (ADS)

Anderson, Hyrum S.; Ilic-Helms, Jovana; Rohrer, Brandon; Wheeler, Jason; Larson, Kurt

2013-02-01

Scanning electron microscopes (SEMs) are used in neuroscience and materials science to image centimeters of sample area at nanometer scales. Since imaging rates are in large part SNR-limited, large collections can lead to weeks of around-the-clock imaging time. To increase data collection speed, we propose and demonstrate on an operational SEM a fast method to sparsely sample and reconstruct smooth images. To accurately localize the electron probe position at fast scan rates, we model the dynamics of the scan coils, and use the model to rapidly and accurately visit a randomly selected subset of pixel locations. Images are reconstructed from the undersampled data by compressed sensing inversion using image smoothness as a prior. We report image fidelity as a function of acquisition speed by comparing traditional raster to sparse imaging modes. Our approach is equally applicable to other domains of nanometer microscopy in which the time to position a probe is a limiting factor (e.g., atomic force microscopy), or in which excessive electron doses might otherwise alter the sample being observed (e.g., scanning transmission electron microscopy).

Microscanners for optical endomicroscopic applications

NASA Astrophysics Data System (ADS)

Hwang, Kyungmin; Seo, Yeong-Hyeon; Jeong, Ki-Hun

2017-12-01

MEMS laser scanning enables the miniaturization of endoscopic catheters for advanced endomicroscopy such as confocal microscopy, multiphoton microscopy, optical coherence tomography, and many other laser scanning microscopy. These advanced biomedical imaging modalities open a great potential for in vivo optical biopsy without surgical excision. They have huge capabilities for detecting on-demand early stage cancer with non-invasiveness. In this article, the scanning arrangement, trajectory, and actuation mechanism of endoscopic microscanners and their endomicroscopic applications will be overviewed.

Lensfree On-Chip Microscopy and Tomography for Bio-Medical Applications

PubMed Central

Isikman, Serhan O.; Bishara, Waheb; Mudanyali, Onur; Sencan, Ikbal; Su, Ting-Wei; Tseng, Derek; Yaglidere, Oguzhan; Sikora, Uzair; Ozcan, Aydogan

2012-01-01

Lensfree on-chip holographic microscopy is an emerging technique that offers imaging of biological specimens over a large field-of-view without using any lenses or bulky optical components. Lending itself to a compact, cost-effective and mechanically robust architecture, lensfree on-chip holographic microscopy can offer an alternative toolset addressing some of the emerging needs of microscopic analysis and diagnostics in low-resource settings, especially for telemedicine applications. In this review, we summarize the latest achievements in lensfree optical microscopy based on partially coherent on-chip holography, including portable telemedicine microscopy, cell-phone based microscopy and field-portable optical tomographic microscopy. We also discuss some of the future directions for telemedicine microscopy and its prospects to help combat various global health challenges. PMID:24478572

The application of laser scanning confocal microscopy to the examination of hairs and textile fibers: an initial investigation.

PubMed

Kirkbride, K Paul; Tridico, Silvana R

2010-02-25

An initial investigation of the application of laser scanning confocal microscopy to the examination of hairs and fibers has been conducted. This technique allows the production of virtual transverse and longitudinal cross-sectional images of a wide range of hairs and fibers. Special mounting techniques are not required; specimens that have been mounted for conventional microscopy require no further treatment. Unlike physical cross-sectioning, in which it is difficult to produce multiple cross-sections from a single hair or fiber and the process is destructive, confocal microscopy allows the examiner to image the cross-section at any point in the field of view along the hair or fiber and it is non-destructive. Confocal microscopy is a fluorescence-based technique. The images described in this article were collected using only the autofluorescence exhibited by the specimen (i.e. fluorescence staining was not necessary). Colorless fibers generally and hairs required excitation at 405 nm in order to stimulate useful autofluorescence; longer wavelength excitation was suitable for dyed fibers. Although confocal microscopy was found to be generally applicable to the generation virtual transverse cross-sections from a wide range of hairs and fibers, on some occasions the autofluorescence signal was attenuated by heavy pigmentation or the presence of an opaque medulla in hairs, and by heavy delustering or the presence of air-filled voids in the case of fibers. In these situations only partial cross-sections were obtained. 2009 Elsevier Ireland Ltd. All rights reserved.

Fourier plane imaging microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dominguez, Daniel, E-mail: daniel.dominguez@ttu.edu; Peralta, Luis Grave de; Nano Tech Center, Texas Tech University, Lubbock, Texas 79409

We show how the image of an unresolved photonic crystal can be reconstructed using a single Fourier plane (FP) image obtained with a second camera that was added to a traditional compound microscope. We discuss how Fourier plane imaging microscopy is an application of a remarkable property of the obtained FP images: they contain more information about the photonic crystals than the images recorded by the camera commonly placed at the real plane of the microscope. We argue that the experimental results support the hypothesis that surface waves, contributing to enhanced resolution abilities, were optically excited in the studied photonicmore » crystals.« less

High-frame-rate imaging of biological samples with optoacoustic micro-tomography

NASA Astrophysics Data System (ADS)

Deán-Ben, X. Luís.; López-Schier, Hernán.; Razansky, Daniel

2018-02-01

Optical microscopy remains a major workhorse in biological discovery despite the fact that light scattering limits its applicability to depths of ˜ 1 mm in scattering tissues. Optoacoustic imaging has been shown to overcome this barrier by resolving optical absorption with microscopic resolution in significantly deeper regions. Yet, the time domain is paramount for the observation of biological dynamics in living systems that exhibit fast motion. Commonly, acquisition of microscopy data involves raster scanning across the imaged volume, which significantly limits temporal resolution in 3D. To overcome these limitations, we have devised a fast optoacoustic micro-tomography (OMT) approach based on simultaneous acquisition of 3D image data with a high-density hemispherical ultrasound array having effective detection bandwidth around 25 MHz. We performed experiments by imaging tissue-mimicking phantoms and zebrafish larvae, demonstrating that OMT can provide nearly cellular resolution and imaging speed of 100 volumetric frames per second. As opposed to other optical microscopy techniques, OMT is a hybrid method that resolves optical absorption contrast acoustically using unfocused light excitation. Thus, no penetration barriers are imposed by light scattering in deep tissues, suggesting it as a powerful approach for multi-scale functional and molecular imaging applications.

Efficient Imaging and Real-Time Display of Scanning Ion Conductance Microscopy Based on Block Compressive Sensing

NASA Astrophysics Data System (ADS)

Li, Gongxin; Li, Peng; Wang, Yuechao; Wang, Wenxue; Xi, Ning; Liu, Lianqing

2014-07-01

Scanning Ion Conductance Microscopy (SICM) is one kind of Scanning Probe Microscopies (SPMs), and it is widely used in imaging soft samples for many distinctive advantages. However, the scanning speed of SICM is much slower than other SPMs. Compressive sensing (CS) could improve scanning speed tremendously by breaking through the Shannon sampling theorem, but it still requires too much time in image reconstruction. Block compressive sensing can be applied to SICM imaging to further reduce the reconstruction time of sparse signals, and it has another unique application that it can achieve the function of image real-time display in SICM imaging. In this article, a new method of dividing blocks and a new matrix arithmetic operation were proposed to build the block compressive sensing model, and several experiments were carried out to verify the superiority of block compressive sensing in reducing imaging time and real-time display in SICM imaging.

Label-Free, High Resolution, Multi-Modal Light Microscopy for Discrimination of Live Stem Cell Differentiation Status.

PubMed

Zhang, Jing; Moradi, Emilia; Somekh, Michael G; Mather, Melissa L

2018-01-15

A label-free microscopy method for assessing the differentiation status of stem cells is presented with potential application for characterization of therapeutic stem cell populations. The microscopy system is capable of characterizing live cells based on the use of evanescent wave microscopy and quantitative phase contrast (QPC) microscopy. The capability of the microscopy system is demonstrated by studying the differentiation of live immortalised neonatal mouse neural stem cells over a 15 day time course. Metrics extracted from microscope images are assessed and images compared with results from endpoint immuno-staining studies to illustrate the system's performance. Results demonstrate the potential of the microscopy system as a valuable tool for cell biologists to readily identify the differentiation status of unlabelled live cells.

Femtosecond laser subsurface scleral treatment in cadaver human sclera and evaluation using two-photon and confocal microscopy

NASA Astrophysics Data System (ADS)

Sun, Hui; Fan, Zhongwei; Yan, Ying; Lian, Fuqiang; Kurtz, Ron; Juhasz, Tibor

2016-03-01

Glaucoma is the second-leading cause of blindness worldwide and is often associated with elevated intraocular pressure (IOP). Partial-thickness drainage channels can be created with femtosecond laser in the translucent sclera for the potential treatment of glaucoma. We demonstrate the creation of partial-thickness subsurface drainage channels with the femtosecond laser in the cadaver human eyeballs and describe the application of two-photon microscopy and confocal microscopy for noninvasive imaging of the femtosecond laser created partial-thickness scleral channels in cadaver human eyes. A femtosecond laser operating at a wavelength of 1700 nm was scanned along a rectangular raster pattern to create the partial thickness subsurface drainage channels in the sclera of cadaver human eyes. Analysis of the dimensions and location of these channels is important in understanding their effects. We describe the application of two-photon microscopy and confocal microscopy for noninvasive imaging of the femtosecond laser created partial-thickness scleral channels in cadaver human eyes. High-resolution images, hundreds of microns deep in the sclera, were obtained to allow determination of the shape and dimension of such partial thickness subsurface scleral channels. Our studies suggest that the confocal and two-photon microscopy can be used to investigate femtosecond-laser created partial-thickness drainage channels in the sclera of cadaver human eyes.

Scanning tunneling spectroscopy under large current flow through the sample.

PubMed

Maldonado, A; Guillamón, I; Suderow, H; Vieira, S

2011-07-01

We describe a method to make scanning tunneling microscopy/spectroscopy imaging at very low temperatures while driving a constant electric current up to some tens of mA through the sample. It gives a new local probe, which we term current driven scanning tunneling microscopy/spectroscopy. We show spectroscopic and topographic measurements under the application of a current in superconducting Al and NbSe(2) at 100 mK. Perspective of applications of this local imaging method includes local vortex motion experiments, and Doppler shift local density of states studies.

Analyzing Protein Clusters on the Plasma Membrane: Application of Spatial Statistical Analysis Methods on Super-Resolution Microscopy Images.

PubMed

Paparelli, Laura; Corthout, Nikky; Pavie, Benjamin; Annaert, Wim; Munck, Sebastian

2016-01-01

The spatial distribution of proteins within the cell affects their capability to interact with other molecules and directly influences cellular processes and signaling. At the plasma membrane, multiple factors drive protein compartmentalization into specialized functional domains, leading to the formation of clusters in which intermolecule interactions are facilitated. Therefore, quantifying protein distributions is a necessity for understanding their regulation and function. The recent advent of super-resolution microscopy has opened up the possibility of imaging protein distributions at the nanometer scale. In parallel, new spatial analysis methods have been developed to quantify distribution patterns in super-resolution images. In this chapter, we provide an overview of super-resolution microscopy and summarize the factors influencing protein arrangements on the plasma membrane. Finally, we highlight methods for analyzing clusterization of plasma membrane proteins, including examples of their applications.

A digital atlas of breast histopathology: an application of web based virtual microscopy

PubMed Central

Lundin, M; Lundin, J; Helin, H; Isola, J

2004-01-01

Aims: To develop an educationally useful atlas of breast histopathology, using advanced web based virtual microscopy technology. Methods: By using a robotic microscope and software adopted and modified from the aerial and satellite imaging industry, a virtual microscopy system was developed that allows fully automated slide scanning and image distribution via the internet. More than 150 slides were scanned at high resolution with an oil immersion ×40 objective (numerical aperture, 1.3) and archived on an image server residing in a high speed university network. Results: A publicly available website was constructed, http://www.webmicroscope.net/breastatlas, which features a comprehensive virtual slide atlas of breast histopathology according to the World Health Organisation 2003 classification. Users can view any part of an entire specimen at any magnification within a standard web browser. The virtual slides are supplemented with concise textual descriptions, but can also be viewed without diagnostic information for self assessment of histopathology skills. Conclusions: Using the technology described here, it is feasible to develop clinically and educationally useful virtual microscopy applications. Web based virtual microscopy will probably become widely used at all levels in pathology teaching. PMID:15563669

Segmentation and learning in the quantitative analysis of microscopy images

NASA Astrophysics Data System (ADS)

Ruggiero, Christy; Ross, Amy; Porter, Reid

2015-02-01

In material science and bio-medical domains the quantity and quality of microscopy images is rapidly increasing and there is a great need to automatically detect, delineate and quantify particles, grains, cells, neurons and other functional "objects" within these images. These are challenging problems for image processing because of the variability in object appearance that inevitably arises in real world image acquisition and analysis. One of the most promising (and practical) ways to address these challenges is interactive image segmentation. These algorithms are designed to incorporate input from a human operator to tailor the segmentation method to the image at hand. Interactive image segmentation is now a key tool in a wide range of applications in microscopy and elsewhere. Historically, interactive image segmentation algorithms have tailored segmentation on an image-by-image basis, and information derived from operator input is not transferred between images. But recently there has been increasing interest to use machine learning in segmentation to provide interactive tools that accumulate and learn from the operator input over longer periods of time. These new learning algorithms reduce the need for operator input over time, and can potentially provide a more dynamic balance between customization and automation for different applications. This paper reviews the state of the art in this area, provides a unified view of these algorithms, and compares the segmentation performance of various design choices.

Thin-film tunable filters for hyperspectral fluorescence microscopy

PubMed Central

Favreau, Peter; Hernandez, Clarissa; Lindsey, Ashley Stringfellow; Alvarez, Diego F.; Rich, Thomas; Prabhat, Prashant

2013-01-01

Abstract. Hyperspectral imaging is a powerful tool that acquires data from many spectral bands, forming a contiguous spectrum. Hyperspectral imaging was originally developed for remote sensing applications; however, hyperspectral techniques have since been applied to biological fluorescence imaging applications, such as fluorescence microscopy and small animal fluorescence imaging. The spectral filtering method largely determines the sensitivity and specificity of any hyperspectral imaging system. There are several types of spectral filtering hardware available for microscopy systems, most commonly acousto-optic tunable filters (AOTFs) and liquid crystal tunable filters (LCTFs). These filtering technologies have advantages and disadvantages. Here, we present a novel tunable filter for hyperspectral imaging—the thin-film tunable filter (TFTF). The TFTF presents several advantages over AOTFs and LCTFs, most notably, a high percentage transmission and a high out-of-band optical density (OD). We present a comparison of a TFTF-based hyperspectral microscopy system and a commercially available AOTF-based system. We have characterized the light transmission, wavelength calibration, and OD of both systems, and have then evaluated the capability of each system for discriminating between green fluorescent protein and highly autofluorescent lung tissue. Our results suggest that TFTFs are an alternative approach for hyperspectral filtering that offers improved transmission and out-of-band blocking. These characteristics make TFTFs well suited for other biomedical imaging devices, such as ophthalmoscopes or endoscopes. PMID:24077519

Modular low-light microscope for imaging cellular bioluminescence and radioluminescence

PubMed Central

Kim, Tae Jin; Türkcan, Silvan; Pratx, Guillem

2017-01-01

Low-light microscopy methods are receiving increased attention as new applications have emerged. One such application is to allow longitudinal imaging of light-sensitive cells with no phototoxicity and no photobleaching of fluorescent biomarkers. Another application is for imaging signals that are inherently dim and undetectable using standard microscopy, such as bioluminescence, chemiluminescence, or radioluminescence. In this protocol, we provide instructions on how to build a modular low-light microscope (1-4 d) by coupling two microscope objective lenses, back-to-back from each other, using standard optomechanical components. We also provide directions on how to image dim signals such as radioluminescence (1-1.5 h), bioluminescence (∼30 min) and low-excitation fluorescence (∼15 min). In particular, radioluminescence microscopy is explained in detail as it is a newly developed technique, which enables the study of small molecule transport (eg. radiolabeled drugs, metabolic precursors, and nuclear medicine contrast agents) by single cells without perturbing endogenous biochemical processes. In this imaging technique, a scintillator crystal (eg. CdWO4) is placed in close proximity to the radiolabeled cells, where it converts the radioactive decays into optical flashes detectable using a sensitive camera. Using the image reconstruction toolkit provided in this protocol, the flashes can be reconstructed to yield high-resolution image of the radiotracer distribution. With appropriate timing, the three aforementioned imaging modalities may be performed altogether on a population of live cells, allowing the user to perform parallel functional studies of cell heterogeneity at the single-cell level. PMID:28426025

Towards low cost photoacoustic Microscopy system for evaluation of skin health

NASA Astrophysics Data System (ADS)

Hariri, Ali; Fatima, Afreen; Mohammadian, Nafiseh; Bely, Nicholas; Nasiriavanaki, Mohammadreza

2016-09-01

Photoacoustic imaging (PAI) involves both optical and ultrasound imaging, owing to this combination the system is capable of generating high resolution images with good penetration depth. With the growing applications of PAI in neurology, vascular biology, dermatology, ophthalmology, tissue engineering, angiogenesis etc., there is a need to make the system more compact, cheap and effective. Therefore we designed an economical and compact version of PAI systems by replacing expensive and sophisticated lasers with a robust pulsed laser diode of 905 nm wavelength. In this study, we determine the feasibility of the Photoacoustic imaging with a very low excitation energy of 0.1uJ in Photoacoustic microscopy. We developed a low cost portable Photoacoustic Imaging including microscopy (both reflection) Phantom study was performed in this configuration and also ex-vivo image was obtained from mouse skin.

Camera array based light field microscopy

PubMed Central

Lin, Xing; Wu, Jiamin; Zheng, Guoan; Dai, Qionghai

2015-01-01

This paper proposes a novel approach for high-resolution light field microscopy imaging by using a camera array. In this approach, we apply a two-stage relay system for expanding the aperture plane of the microscope into the size of an imaging lens array, and utilize a sensor array for acquiring different sub-apertures images formed by corresponding imaging lenses. By combining the rectified and synchronized images from 5 × 5 viewpoints with our prototype system, we successfully recovered color light field videos for various fast-moving microscopic specimens with a spatial resolution of 0.79 megapixels at 30 frames per second, corresponding to an unprecedented data throughput of 562.5 MB/s for light field microscopy. We also demonstrated the use of the reported platform for different applications, including post-capture refocusing, phase reconstruction, 3D imaging, and optical metrology. PMID:26417490

Video-rate volumetric neuronal imaging using 3D targeted illumination.

PubMed

Xiao, Sheng; Tseng, Hua-An; Gritton, Howard; Han, Xue; Mertz, Jerome

2018-05-21

Fast volumetric microscopy is required to monitor large-scale neural ensembles with high spatio-temporal resolution. Widefield fluorescence microscopy can image large 2D fields of view at high resolution and speed while remaining simple and costeffective. A focal sweep add-on can further extend the capacity of widefield microscopy by enabling extended-depth-of-field (EDOF) imaging, but suffers from an inability to reject out-of-focus fluorescence background. Here, by using a digital micromirror device to target only in-focus sample features, we perform EDOF imaging with greatly enhanced contrast and signal-to-noise ratio, while reducing the light dosage delivered to the sample. Image quality is further improved by the application of a robust deconvolution algorithm. We demonstrate the advantages of our technique for in vivo calcium imaging in the mouse brain.

Correlation of live-cell imaging with volume scanning electron microscopy.

PubMed

Lucas, Miriam S; Günthert, Maja; Bittermann, Anne Greet; de Marco, Alex; Wepf, Roger

2017-01-01

Live-cell imaging is one of the most widely applied methods in live science. Here we describe two setups for live-cell imaging, which can easily be combined with volume SEM for correlative studies. The first procedure applies cell culture dishes with a gridded glass support, which can be used for any light microscopy modality. The second approach is a flow-chamber setup based on Ibidi μ-slides. Both live-cell imaging strategies can be followed up with serial blockface- or focused ion beam-scanning electron microscopy. Two types of resin embedding after heavy metal staining and dehydration are presented making best use of the particular advantages of each imaging modality: classical en-bloc embedding and thin-layer plastification. The latter can be used only for focused ion beam-scanning electron microscopy, but is advantageous for studying cell-interactions with specific substrates, or when the substrate cannot be removed. En-bloc embedding has diverse applications and can be applied for both described volume scanning electron microscopy techniques. Finally, strategies for relocating the cell of interest are discussed for both embedding approaches and in respect to the applied light and scanning electron microscopy methods. Copyright © 2017 Elsevier Inc. All rights reserved.

Hybrid Microscopy: Enabling Inexpensive High-Performance Imaging through Combined Physical and Optical Magnifications.

PubMed

Zhang, Yu Shrike; Chang, Jae-Byum; Alvarez, Mario Moisés; Trujillo-de Santiago, Grissel; Aleman, Julio; Batzaya, Byambaa; Krishnadoss, Vaishali; Ramanujam, Aishwarya Aravamudhan; Kazemzadeh-Narbat, Mehdi; Chen, Fei; Tillberg, Paul W; Dokmeci, Mehmet Remzi; Boyden, Edward S; Khademhosseini, Ali

2016-03-15

To date, much effort has been expended on making high-performance microscopes through better instrumentation. Recently, it was discovered that physical magnification of specimens was possible, through a technique called expansion microscopy (ExM), raising the question of whether physical magnification, coupled to inexpensive optics, could together match the performance of high-end optical equipment, at a tiny fraction of the price. Here we show that such "hybrid microscopy" methods--combining physical and optical magnifications--can indeed achieve high performance at low cost. By physically magnifying objects, then imaging them on cheap miniature fluorescence microscopes ("mini-microscopes"), it is possible to image at a resolution comparable to that previously attainable only with benchtop microscopes that present costs orders of magnitude higher. We believe that this unprecedented hybrid technology that combines expansion microscopy, based on physical magnification, and mini-microscopy, relying on conventional optics--a process we refer to as Expansion Mini-Microscopy (ExMM)--is a highly promising alternative method for performing cost-effective, high-resolution imaging of biological samples. With further advancement of the technology, we believe that ExMM will find widespread applications for high-resolution imaging particularly in research and healthcare scenarios in undeveloped countries or remote places.

An update: improvements in imaging perfluorocarbon-mounted plant leaves with implications for studies of plant pathology, physiology, development and cell biology

PubMed Central

Littlejohn, George R.; Mansfield, Jessica C.; Christmas, Jacqueline T.; Witterick, Eleanor; Fricker, Mark D.; Grant, Murray R.; Smirnoff, Nicholas; Everson, Richard M.; Moger, Julian; Love, John

2014-01-01

Plant leaves are optically complex, which makes them difficult to image by light microscopy. Careful sample preparation is therefore required to enable researchers to maximize the information gained from advances in fluorescent protein labeling, cell dyes and innovations in microscope technologies and techniques. We have previously shown that mounting leaves in the non-toxic, non-fluorescent perfluorocarbon (PFC), perfluorodecalin (PFD) enhances the optical properties of the leaf with minimal impact on physiology. Here, we assess the use of the PFCs, PFD, and perfluoroperhydrophenanthrene (PP11) for in vivo plant leaf imaging using four advanced modes of microscopy: laser scanning confocal microscopy (LSCM), two-photon fluorescence microscopy, second harmonic generation microscopy, and stimulated Raman scattering (SRS) microscopy. For every mode of imaging tested, we observed an improved signal when leaves were mounted in PFD or in PP11, compared to mounting the samples in water. Using an image analysis technique based on autocorrelation to quantitatively assess LSCM image deterioration with depth, we show that PP11 outperformed PFD as a mounting medium by enabling the acquisition of clearer images deeper into the tissue. In addition, we show that SRS microscopy can be used to image PFCs directly in the mesophyll and thereby easily delimit the “negative space” within a leaf, which may have important implications for studies of leaf development. Direct comparison of on and off resonance SRS micrographs show that PFCs do not to form intracellular aggregates in live plants. We conclude that the application of PFCs as mounting media substantially increases advanced microscopy image quality of living mesophyll and leaf vascular bundle cells. PMID:24795734

Repurposing a photosynthetic antenna protein as a super-resolution microscopy label.

PubMed

Barnett, Samuel F H; Hitchcock, Andrew; Mandal, Amit K; Vasilev, Cvetelin; Yuen, Jonathan M; Morby, James; Brindley, Amanda A; Niedzwiedzki, Dariusz M; Bryant, Donald A; Cadby, Ashley J; Holten, Dewey; Hunter, C Neil

2017-12-01

Techniques such as Stochastic Optical Reconstruction Microscopy (STORM) and Structured Illumination Microscopy (SIM) have increased the achievable resolution of optical imaging, but few fluorescent proteins are suitable for super-resolution microscopy, particularly in the far-red and near-infrared emission range. Here we demonstrate the applicability of CpcA, a subunit of the photosynthetic antenna complex in cyanobacteria, for STORM and SIM imaging. The periodicity and width of fabricated nanoarrays of CpcA, with a covalently attached phycoerythrobilin (PEB) or phycocyanobilin (PCB) chromophore, matched the lines in reconstructed STORM images. SIM and STORM reconstructions of Escherichia coli cells harbouring CpcA-labelled cytochrome bd 1 ubiquinol oxidase in the cytoplasmic membrane show that CpcA-PEB and CpcA-PCB are suitable for super-resolution imaging in vivo. The stability, ease of production, small size and brightness of CpcA-PEB and CpcA-PCB demonstrate the potential of this largely unexplored protein family as novel probes for super-resolution microscopy.

Methods of Hematoxylin and Erosin Image Information Acquisition and Optimization in Confocal Microscopy

PubMed Central

Yoon, Woong Bae; Kim, Hyunjin; Kim, Kwang Gi; Choi, Yongdoo; Chang, Hee Jin

2016-01-01

Objectives We produced hematoxylin and eosin (H&E) staining-like color images by using confocal laser scanning microscopy (CLSM), which can obtain the same or more information in comparison to conventional tissue staining. Methods We improved images by using several image converting techniques, including morphological methods, color space conversion methods, and segmentation methods. Results An image obtained after image processing showed coloring very similar to that in images produced by H&E staining, and it is advantageous to conduct analysis through fluorescent dye imaging and microscopy rather than analysis based on single microscopic imaging. Conclusions The colors used in CLSM are different from those seen in H&E staining, which is the method most widely used for pathologic diagnosis and is familiar to pathologists. Computer technology can facilitate the conversion of images by CLSM to be very similar to H&E staining images. We believe that the technique used in this study has great potential for application in clinical tissue analysis. PMID:27525165

Image Quality Ranking Method for Microscopy

PubMed Central

Koho, Sami; Fazeli, Elnaz; Eriksson, John E.; Hänninen, Pekka E.

2016-01-01

Automated analysis of microscope images is necessitated by the increased need for high-resolution follow up of events in time. Manually finding the right images to be analyzed, or eliminated from data analysis are common day-to-day problems in microscopy research today, and the constantly growing size of image datasets does not help the matter. We propose a simple method and a software tool for sorting images within a dataset, according to their relative quality. We demonstrate the applicability of our method in finding good quality images in a STED microscope sample preparation optimization image dataset. The results are validated by comparisons to subjective opinion scores, as well as five state-of-the-art blind image quality assessment methods. We also show how our method can be applied to eliminate useless out-of-focus images in a High-Content-Screening experiment. We further evaluate the ability of our image quality ranking method to detect out-of-focus images, by extensive simulations, and by comparing its performance against previously published, well-established microscopy autofocus metrics. PMID:27364703

Methods of Hematoxylin and Erosin Image Information Acquisition and Optimization in Confocal Microscopy.

PubMed

Yoon, Woong Bae; Kim, Hyunjin; Kim, Kwang Gi; Choi, Yongdoo; Chang, Hee Jin; Sohn, Dae Kyung

2016-07-01

We produced hematoxylin and eosin (H&E) staining-like color images by using confocal laser scanning microscopy (CLSM), which can obtain the same or more information in comparison to conventional tissue staining. We improved images by using several image converting techniques, including morphological methods, color space conversion methods, and segmentation methods. An image obtained after image processing showed coloring very similar to that in images produced by H&E staining, and it is advantageous to conduct analysis through fluorescent dye imaging and microscopy rather than analysis based on single microscopic imaging. The colors used in CLSM are different from those seen in H&E staining, which is the method most widely used for pathologic diagnosis and is familiar to pathologists. Computer technology can facilitate the conversion of images by CLSM to be very similar to H&E staining images. We believe that the technique used in this study has great potential for application in clinical tissue analysis.

Brain Slice Staining and Preparation for Three-Dimensional Super-Resolution Microscopy

PubMed Central

German, Christopher L.; Gudheti, Manasa V.; Fleckenstein, Annette E.; Jorgensen, Erik M.

2018-01-01

Localization microscopy techniques – such as photoactivation localization microscopy (PALM), fluorescent PALM (FPALM), ground state depletion (GSD), and stochastic optical reconstruction microscopy (STORM) – provide the highest precision for single molecule localization currently available. However, localization microscopy has been largely limited to cell cultures due to the difficulties that arise in imaging thicker tissue sections. Sample fixation and antibody staining, background fluorescence, fluorophore photoinstability, light scattering in thick sections, and sample movement create significant challenges for imaging intact tissue. We have developed a sample preparation and image acquisition protocol to address these challenges in rat brain slices. The sample preparation combined multiple fixation steps, saponin permeabilization, and tissue clarification. Together, these preserve intracellular structures, promote antibody penetration, reduce background fluorescence and light scattering, and allow acquisition of images deep in a 30 μm thick slice. Image acquisition challenges were resolved by overlaying samples with a permeable agarose pad and custom-built stainless steel imaging adapter, and sealing the imaging chamber. This approach kept slices flat, immobile, bathed in imaging buffer, and prevented buffer oxidation during imaging. Using this protocol, we consistently obtained single molecule localizations of synaptic vesicle and active zone proteins in three-dimensions within individual synaptic terminals of the striatum in rat brain slices. These techniques may be easily adapted to the preparation and imaging of other tissues, substantially broadening the application of super-resolution imaging. PMID:28924666

Recent advancements in structured-illumination microscopy toward live-cell imaging.

PubMed

Hirano, Yasuhiro; Matsuda, Atsushi; Hiraoka, Yasushi

2015-08-01

Fluorescence microscopy allows us to observe fluorescently labeled molecules in diverse biological processes and organelle structures within living cells. However, the diffraction limit restricts its spatial resolution to about half of its wavelength, limiting the capability of biological observation at the molecular level. Structured-illumination microscopy (SIM), a type of super-resolution microscopy, doubles the spatial resolution in all three dimensions by illuminating the sample with a patterned excitation light, followed by computer reconstruction. SIM uses a relatively low illumination power compared with other methods of super-resolution microscopy and is easily available for multicolor imaging. SIM has great potential for meeting the requirements of live-cell imaging. Recent developments in diverse types of SIM have achieved higher spatial (∼50 nm lateral) and temporal (∼100 Hz) resolutions. Here, we review recent advancements in SIM and discuss its application in noninvasive live-cell imaging. © The Author 2015. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Remote focusing for programmable multi-layer differential multiphoton microscopy

PubMed Central

Hoover, Erich E.; Young, Michael D.; Chandler, Eric V.; Luo, Anding; Field, Jeffrey J.; Sheetz, Kraig E.; Sylvester, Anne W.; Squier, Jeff A.

2010-01-01

We present the application of remote focusing to multiphoton laser scanning microscopy and utilize this technology to demonstrate simultaneous, programmable multi-layer imaging. Remote focusing is used to independently control the axial location of multiple focal planes that can be simultaneously imaged with single element detection. This facilitates volumetric multiphoton imaging in scattering specimens and can be practically scaled to a large number of focal planes. Further, it is demonstrated that the remote focusing control can be synchronized with the lateral scan directions, enabling imaging in orthogonal scan planes. PMID:21326641

In vivo confocal microscopy of the cornea: New developments in image acquisition, reconstruction and analysis using the HRT-Rostock Corneal Module

PubMed Central

Petroll, W. Matthew; Robertson, Danielle M.

2015-01-01

The optical sectioning ability of confocal microscopy allows high magnification images to be obtained from different depths within a thick tissue specimen, and is thus ideally suited to the study of intact tissue in living subjects. In vivo confocal microscopy has been used in a variety of corneal research and clinical applications since its development over 25 years ago. In this article we review the latest developments in quantitative corneal imaging with the Heidelberg Retinal Tomograph with Rostock Corneal Module (HRT-RCM). We provide an overview of the unique strengths and weaknesses of the HRT-RCM. We discuss techniques for performing 3-D imaging with the HRT-RCM, including hardware and software modifications that allow full thickness confocal microscopy through focusing (CMTF) of the cornea, which can provide quantitative measurements of corneal sublayer thicknesses, stromal cell and extracellular matrix backscatter, and depth dependent changes in corneal keratocyte density. We also review current approaches for quantitative imaging of the subbasal nerve plexus, which require a combination of advanced image acquisition and analysis procedures, including wide field mapping and 3-D reconstruction of nerve structures. The development of new hardware, software, and acquisition techniques continues to expand the number of applications of the HRT-RCM for quantitative in vivo corneal imaging at the cellular level. Knowledge of these rapidly evolving strategies should benefit corneal clinicians and basic scientists alike. PMID:25998608

Acousto-optical tunable filter for combined wideband, spectral, and optical coherence microscopy.

PubMed

Machikhin, Alexander S; Pozhar, Vitold E; Viskovatykh, Alexander V; Burmak, Ludmila I

2015-09-01

A multimodal technique for inspection of microscopic objects by means of wideband optical microscopy, spectral microscopy, and optical coherence microscopy is described, implemented, and tested. The key feature is the spectral selection of light in the output arm of an interferometer with use of the specialized imaging acousto-optical tunable filter. In this filter, two interfering optical beams are diffracted via the same ultrasound wave without destruction of interference image structure. The basic requirements for the acousto-optical tunable filter are defined, and mathematical formulas for calculation of its parameters are derived. Theoretical estimation of the achievable accuracy of the 3D image reconstruction is presented and experimental proofs are given. It is demonstrated that spectral imaging can also be accompanied by measurement of the quantitative reflectance spectra. Examples of inspection of optically transparent and nontransparent samples demonstrate the applicability of the technique.

High-sensitivity chemical imaging for biomedicine by SRS microscopy (Conference Presentation)

NASA Astrophysics Data System (ADS)

Min, Wei

2017-02-01

Innovations in spectroscopy principles and microscopy technology have significantly impacted modern biology and medicine. While most of the contemporary bio-imaging modalities harness electronic transition, nuclear spin or radioactivity, vibrational spectroscopy has not been widely used yet. Here we will discuss an emerging chemical imaging platform, stimulated Raman scattering (SRS) microscopy, which can enhance the otherwise feeble spontaneous Raman eight orders of magnitude by virtue of stimulated emission. When coupled with stable isotopes (e.g., deuterium and 13C) or bioorthogonal chemical moieties (e.g., alkynes), SRS microscopy is well suited for probing in vivo metabolic dynamics of small bio-molecules which cannot be labeled by bulky fluorophores. Physical principle of the underlying optical spectroscopy and exciting biomedical applications such as imaging lipid metabolism, protein synthesis, DNA replication, protein degradation, RNA synthesis, glucose uptake, drug trafficking and tumor metabolism will be presented.

Nanoscopy for nanoscience: how super-resolution microscopy extends imaging for nanotechnology.

PubMed

Johnson, Sam A

2015-01-01

Imaging methods have presented scientists with powerful means of investigation for centuries. The ability to resolve structures using light microscopes is though limited to around 200 nm. Fluorescence-based super-resolution light microscopy techniques of several principles and methods have emerged in recent years and offer great potential to extend the capabilities of microscopy. This resolution improvement is especially promising for nanoscience where the imaging of nanoscale structures is inherently restricted by the resolution limit of standard forms of light microscopy. Resolution can be improved by several distinct approaches including structured illumination microscopy, stimulated emission depletion, and single-molecule positioning methods such as photoactivated localization microscopy and stochastic optical reconstruction microscopy and several derivative variations of each of these. These methods involve substantial differences in the resolutions achievable in the different axes, speed of acquisition, compatibility with different labels, ease of use, hardware complexity, and compatibility with live biological samples. The field of super-resolution imaging and its application to nanotechnology is relatively new and still rapidly developing. An overview of how these methods may be used with nanomaterials is presented with some examples of pioneering uses of these approaches. © 2014 Wiley Periodicals, Inc.

Fibre optic confocal imaging (FOCI) for subsurface microscopy of the colon in vivo.

PubMed Central

Delaney, P M; King, R G; Lambert, J R; Harris, M R

1994-01-01

Fibre optic confocal imaging (FOCI) is a new type of microscopy which has been recently developed (Delaney et al. 1993). In contrast to conventional light microscopy, FOCI and other confocal techniques allow clear imaging of subsurface structures within translucent objects. However, unlike conventional confocal microscopes which are bulky (because of a need for accurate alignment of large components) FOCI allows the imaging end to be miniaturised and relatively mobile. FOCI is thus particularly suited for clear subsurface imaging of structures within living animals or subjects. The aim of the present study was to assess the suitability of using FOCI for imaging of subsurface structures within the colon, both in vitro (human and rat biopsies) and in vivo (in rats). Images were obtained in fluorescence mode (excitation 488 nm, detection above 515 nm) following topical application of fluorescein. By this technique the glandular structure of the colon was imaged. FOCI is thus suitable for subsurface imaging of the colon in vivo. Images Fig. 2 Fig. 3 PMID:8157487

Towards real-time image deconvolution: application to confocal and STED microscopy

PubMed Central

Zanella, R.; Zanghirati, G.; Cavicchioli, R.; Zanni, L.; Boccacci, P.; Bertero, M.; Vicidomini, G.

2013-01-01

Although deconvolution can improve the quality of any type of microscope, the high computational time required has so far limited its massive spreading. Here we demonstrate the ability of the scaled-gradient-projection (SGP) method to provide accelerated versions of the most used algorithms in microscopy. To achieve further increases in efficiency, we also consider implementations on graphic processing units (GPUs). We test the proposed algorithms both on synthetic and real data of confocal and STED microscopy. Combining the SGP method with the GPU implementation we achieve a speed-up factor from about a factor 25 to 690 (with respect the conventional algorithm). The excellent results obtained on STED microscopy images demonstrate the synergy between super-resolution techniques and image-deconvolution. Further, the real-time processing allows conserving one of the most important property of STED microscopy, i.e the ability to provide fast sub-diffraction resolution recordings. PMID:23982127

Functional imaging for regenerative medicine.

PubMed

Leahy, Martin; Thompson, Kerry; Zafar, Haroon; Alexandrov, Sergey; Foley, Mark; O'Flatharta, Cathal; Dockery, Peter

2016-04-19

In vivo imaging is a platform technology with the power to put function in its natural structural context. With the drive to translate stem cell therapies into pre-clinical and clinical trials, early selection of the right imaging techniques is paramount to success. There are many instances in regenerative medicine where the biological, biochemical, and biomechanical mechanisms behind the proposed function of stem cell therapies can be elucidated by appropriate imaging. Imaging techniques can be divided according to whether labels are used and as to whether the imaging can be done in vivo. In vivo human imaging places additional restrictions on the imaging tools that can be used. Microscopies and nanoscopies, especially those requiring fluorescent markers, have made an extraordinary impact on discovery at the molecular and cellular level, but due to their very limited ability to focus in the scattering tissues encountered for in vivo applications they are largely confined to superficial imaging applications in research laboratories. Nanoscopy, which has tremendous benefits in resolution, is limited to the near-field (e.g. near-field scanning optical microscope (NSNOM)) or to very high light intensity (e.g. stimulated emission depletion (STED)) or to slow stochastic events (photo-activated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM)). In all cases, nanoscopy is limited to very superficial applications. Imaging depth may be increased using multiphoton or coherence gating tricks. Scattering dominates the limitation on imaging depth in most tissues and this can be mitigated by the application of optical clearing techniques that can impose mild (e.g. topical application of glycerol) or severe (e.g. CLARITY) changes to the tissue to be imaged. Progression of therapies through to clinical trials requires some thought as to the imaging and sensing modalities that should be used. Smoother progression is facilitated by the use of comparable imaging modalities throughout the discovery and trial phases, giving label-free techniques an advantage wherever they can be used, although this is seldom considered in the early stages. In this paper, we will explore the techniques that have found success in aiding discovery in stem cell therapies and try to predict the likely technologies best suited to translation and future directions.

Longitudinal in vivo two-photon fluorescence imaging

PubMed Central

Crowe, Sarah E.; Ellis-Davies, Graham C.R.

2014-01-01

Fluorescence microscopy is an essential technique for the basic sciences, especially biomedical research. Since the invention of laser scanning confocal microscopy in 1980s, that enabled imaging both fixed and living biological tissue with three-dimensional precision, high-resolution fluorescence imaging has revolutionized biological research. Confocal microscopy, by its very nature, has one fundamental limitation. Due to the confocal pinhole, deep tissue fluorescence imaging is not practical. In contrast (no pun intended), two-photon fluorescence microscopy allows, in principle, the collection of all emitted photons from fluorophores in the imaged voxel, dramatically extending our ability to see deep into living tissue. Since the development of transgenic mice with genetically encoded fluorescent protein in neocortical cells in 2000, two-photon imaging has enabled the dynamics of individual synapses to be followed for up to two years. Since the initial landmark contributions to this field in 2002, the technique has been used to understand how neuronal structure are changed by experience, learning and memory and various diseases. Here we provide a basic summary of the crucial elements that are required for such studies, and discuss many applications of longitudinal two-photon fluorescence microscopy that have appeared since 2002. PMID:24214350

4D (x-y-z-t) imaging of thick biological samples by means of Two-Photon inverted Selective Plane Illumination Microscopy (2PE-iSPIM)

PubMed Central

Lavagnino, Zeno; Sancataldo, Giuseppe; d’Amora, Marta; Follert, Philipp; De Pietri Tonelli, Davide; Diaspro, Alberto; Cella Zanacchi, Francesca

2016-01-01

In the last decade light sheet fluorescence microscopy techniques, such as selective plane illumination microscopy (SPIM), has become a well established method for developmental biology. However, conventional SPIM architectures hardly permit imaging of certain tissues since the common sample mounting procedure, based on gel embedding, could interfere with the sample morphology. In this work we propose an inverted selective plane microscopy system (iSPIM), based on non-linear excitation, suitable for 3D tissue imaging. First, the iSPIM architecture provides flexibility on the sample mounting, getting rid of the gel-based mounting typical of conventional SPIM, permitting 3D imaging of hippocampal slices from mouse brain. Moreover, all the advantages brought by two photon excitation (2PE) in terms of reduction of scattering effects and contrast improvement are exploited, demonstrating an improved image quality and contrast compared to single photon excitation. The system proposed represents an optimal platform for tissue imaging and it smooths the way to the applicability of light sheet microscopy to a wider range of samples including those that have to be mounted on non-transparent surfaces. PMID:27033347

Three-dimensional nanoscale imaging by plasmonic Brownian microscopy

NASA Astrophysics Data System (ADS)

Labno, Anna; Gladden, Christopher; Kim, Jeongmin; Lu, Dylan; Yin, Xiaobo; Wang, Yuan; Liu, Zhaowei; Zhang, Xiang

2017-12-01

Three-dimensional (3D) imaging at the nanoscale is a key to understanding of nanomaterials and complex systems. While scanning probe microscopy (SPM) has been the workhorse of nanoscale metrology, its slow scanning speed by a single probe tip can limit the application of SPM to wide-field imaging of 3D complex nanostructures. Both electron microscopy and optical tomography allow 3D imaging, but are limited to the use in vacuum environment due to electron scattering and to optical resolution in micron scales, respectively. Here we demonstrate plasmonic Brownian microscopy (PBM) as a way to improve the imaging speed of SPM. Unlike photonic force microscopy where a single trapped particle is used for a serial scanning, PBM utilizes a massive number of plasmonic nanoparticles (NPs) under Brownian diffusion in solution to scan in parallel around the unlabeled sample object. The motion of NPs under an evanescent field is three-dimensionally localized to reconstruct the super-resolution topology of 3D dielectric objects. Our method allows high throughput imaging of complex 3D structures over a large field of view, even with internal structures such as cavities that cannot be accessed by conventional mechanical tips in SPM.

Correlative fluorescence and electron microscopy of quantum dot labeled proteins on whole cells in liquid.

PubMed

Peckys, Diana B; Dukes, Madeline J; de Jonge, Niels

2014-01-01

Correlative fluorescence microscopy and scanning transmission electron microscopy (STEM) of cells fully immersed in liquid is a new methodology with many application areas. Proteins, in live cells immobilized on microchips, are labeled with fluorescent quantum dot (QD) nanoparticles. In this protocol, the epidermal growth factor receptor (EGFR) is labeled. The cells are fixed after a selected labeling time, for example, 5 min as needed to form EGFR dimers. The microchip with cells is then imaged with fluorescence microscopy. Thereafter, the microchip with the labeled cells and one with a spacer are assembled in a special microfluidic device and imaged with STEM.

Automated analysis of high-content microscopy data with deep learning.

PubMed

Kraus, Oren Z; Grys, Ben T; Ba, Jimmy; Chong, Yolanda; Frey, Brendan J; Boone, Charles; Andrews, Brenda J

2017-04-18

Existing computational pipelines for quantitative analysis of high-content microscopy data rely on traditional machine learning approaches that fail to accurately classify more than a single dataset without substantial tuning and training, requiring extensive analysis. Here, we demonstrate that the application of deep learning to biological image data can overcome the pitfalls associated with conventional machine learning classifiers. Using a deep convolutional neural network (DeepLoc) to analyze yeast cell images, we show improved performance over traditional approaches in the automated classification of protein subcellular localization. We also demonstrate the ability of DeepLoc to classify highly divergent image sets, including images of pheromone-arrested cells with abnormal cellular morphology, as well as images generated in different genetic backgrounds and in different laboratories. We offer an open-source implementation that enables updating DeepLoc on new microscopy datasets. This study highlights deep learning as an important tool for the expedited analysis of high-content microscopy data. © 2017 The Authors. Published under the terms of the CC BY 4.0 license.

Click-electron microscopy for imaging metabolically tagged non-protein biomolecules

PubMed Central

Ngo, John T.; Adams, Stephen R.; Deerinck, Thomas J.; Boassa, Daniela; Rodriguez-Rivera, Frances; Palida, Sakina F.; Bertozzi, Carolyn R.; Ellisman, Mark H.; Tsien, Roger Y.

2016-01-01

Electron microscopy (EM) has long been the main technique to image cell structures with nanometer resolution, but has lagged behind light microscopy in the crucial ability to make specific molecules stand out. Here we introduce “Click-EM,” a labeling technique for correlative light microscopy and EM imaging of non-protein biomolecules. In this approach, metabolic labeling substrates containing bioorthogonal functional groups are provided to cells for incorporation into biopolymers by endogenous biosynthetic machinery. The unique chemical functionality of these analogs is exploited for selective attachment of singlet oxygen-generating fluorescent dyes via bioorthogonal “click chemistry” ligations. Illumination of dye-labeled structures generates singlet oxygen to locally catalyze the polymerization of diaminobenzidine into an osmiophilic reaction product that is readily imaged by EM. We describe the application of Click-EM in imaging metabolically tagged DNA, RNA, and lipids in cultured cells and neurons, and highlight its use in tracking peptidoglycan synthesis in the Gram-positive bacterium Listeria monocytogenes. PMID:27110681

Wavelength-multiplexing surface plasmon holographic microscopy.

PubMed

Zhang, Jiwei; Dai, Siqing; Zhong, Jinzhan; Xi, Teli; Ma, Chaojie; Li, Ying; Di, Jianglei; Zhao, Jianlin

2018-05-14

Surface plasmon holographic microscopy (SPHM), which combines surface plasmon microscopy with digital holographic microscopy, can be applied for amplitude- and phase-contrast surface plasmon resonance (SPR) imaging. In this paper, we propose an improved SPHM with the wavelength multiplexing technique based on two laser sources and a common-path hologram recording configuration. Through recording and reconstructing the SPR images at two wavelengths simultaneously employing the improved SPHM, tiny variation of dielectric refractive index in near field is quantitatively monitored with an extended measurement range while maintaining the high sensitivity. Moreover, imaging onion tissues is performed to demonstrate that the detection sensitivities of two wavelengths can compensate for each other in SPR imaging. The proposed wavelength-multiplexing SPHM presents simple structure, high temporal stability and inherent capability of phase curvature compensation, as well as shows great potentials for further applications in monitoring diverse dynamic processes related with refractive index variations and imaging biological tissues with low-contrast refractive index distributions in the near field.

Image recovery from defocused 2D fluorescent images in multimodal digital holographic microscopy.

PubMed

Quan, Xiangyu; Matoba, Osamu; Awatsuji, Yasuhiro

2017-05-01

A technique of three-dimensional (3D) intensity retrieval from defocused, two-dimensional (2D) fluorescent images in the multimodal digital holographic microscopy (DHM) is proposed. In the multimodal DHM, 3D phase and 2D fluorescence distributions are obtained simultaneously by an integrated system of an off-axis DHM and a conventional epifluorescence microscopy, respectively. This gives us more information of the target; however, defocused fluorescent images are observed due to the short depth of field. In this Letter, we propose a method to recover the defocused images based on the phase compensation and backpropagation from the defocused plane to the focused plane using the distance information that is obtained from a 3D phase distribution. By applying Zernike polynomial phase correction, we brought back the fluorescence intensity to the focused imaging planes. The experimental demonstration using fluorescent beads is presented, and the expected applications are suggested.

A multimodal imaging platform with integrated simultaneous photoacoustic microscopy, optical coherence tomography, optical Doppler tomography and fluorescence microscopy

NASA Astrophysics Data System (ADS)

Dadkhah, Arash; Zhou, Jun; Yeasmin, Nusrat; Jiao, Shuliang

2018-02-01

Various optical imaging modalities with different optical contrast mechanisms have been developed over the past years. Although most of these imaging techniques are being used in many biomedical applications and researches, integration of these techniques will allow researchers to reach the full potential of these technologies. Nevertheless, combining different imaging techniques is always challenging due to the difference in optical and hardware requirements for different imaging systems. Here, we developed a multimodal optical imaging system with the capability of providing comprehensive structural, functional and molecular information of living tissue in micrometer scale. This imaging system integrates photoacoustic microscopy (PAM), optical coherence tomography (OCT), optical Doppler tomography (ODT) and fluorescence microscopy in one platform. Optical-resolution PAM (OR-PAM) provides absorption-based imaging of biological tissues. Spectral domain OCT is able to provide structural information based on the scattering property of biological sample with no need for exogenous contrast agents. In addition, ODT is a functional extension of OCT with the capability of measurement and visualization of blood flow based on the Doppler effect. Fluorescence microscopy allows to reveal molecular information of biological tissue using autofluoresce or exogenous fluorophores. In-vivo as well as ex-vivo imaging studies demonstrated the capability of our multimodal imaging system to provide comprehensive microscopic information on biological tissues. Integrating all the aforementioned imaging modalities for simultaneous multimodal imaging has promising potential for preclinical research and clinical practice in the near future.

78 FR 46995 - National Institute of Biomedical Imaging and Bioengineering; Notice of Closed Meeting

Federal Register 2010, 2011, 2012, 2013, 2014

2013-08-02

...) and 552b(c)(6), Title 5 U.S.C., as amended. The grant applications and the discussions could disclose... concerning individuals associated with the grant applications, the disclosure of which would constitute a... Imaging and Bioengineering Special Emphasis Panel Computer Integrated Systems for Microscopy...

Biological imaging by soft x-ray diffraction microscopy

DOE PAGES

Shapiro, D.; Thibault, P.; Beetz, T.; ...

2005-10-25

We have used the method of x-ray diffraction microscopy to image the complex-valued exit wave of an intact and unstained yeast cell. The images of the freeze-dried cell, obtained by using 750-eV x-rays from different angular orientations, portray several of the cell's major internal components to 30-nm resolution. The good agreement among the independently recovered structures demonstrates the accuracy of the imaging technique. To obtain the best possible reconstructions, we have implemented procedures for handling noisy and incomplete diffraction data, and we propose a method for determining the reconstructed resolution. This work represents a previously uncharacterized application of x-ray diffractionmore » microscopy to a specimen of this complexity and provides confidence in the feasibility of the ultimate goal of imaging biological specimens at 10-nm resolution in three dimensions.« less

Superresolution Imaging using Single-Molecule Localization

PubMed Central

Patterson, George; Davidson, Michael; Manley, Suliana; Lippincott-Schwartz, Jennifer

2013-01-01

Superresolution imaging is a rapidly emerging new field of microscopy that dramatically improves the spatial resolution of light microscopy by over an order of magnitude (∼10–20-nm resolution), allowing biological processes to be described at the molecular scale. Here, we discuss a form of superresolution microscopy based on the controlled activation and sampling of sparse subsets of photoconvertible fluorescent molecules. In this single-molecule based imaging approach, a wide variety of probes have proved valuable, ranging from genetically encodable photoactivatable fluorescent proteins to photoswitchable cyanine dyes. These have been used in diverse applications of superresolution imaging: from three-dimensional, multicolor molecule localization to tracking of nanometric structures and molecules in living cells. Single-molecule-based superresolution imaging thus offers exciting possibilities for obtaining molecular-scale information on biological events occurring at variable timescales. PMID:20055680

Three-dimensional imaging of sulfides in silicate rocks at submicron resolution with multiphoton microscopy.

PubMed

Bénard, Antoine; Palle, Sabine; Doucet, Luc Serge; Ionov, Dmitri A

2011-12-01

We report the first application of multiphoton microscopy (MPM) to generate three-dimensional (3D) images of natural minerals (micron-sized sulfides) in thick (∼120 μm) rock sections. First, reflection mode (RM) using confocal laser scanning microscopy (CLSM), combined with differential interference contrast (DIC), was tested on polished sections. Second, two-photon fluorescence (TPF) and second harmonic signal (SHG) images were generated using a femtosecond-laser on the same rock section without impregnation by a fluorescent dye. CSLM results show that the silicate matrix is revealed with DIC and RM, while sulfides can be imaged in 3D at low resolution by RM. Sulfides yield strong autofluorescence from 392 to 715 nm with TPF, while SHG is only produced by the embedding medium. Simultaneous recording of TPF and SHG images enables efficient discrimination between different components of silicate rocks. Image stacks obtained with MPM enable complete reconstruction of the 3D structure of a rock slice and of sulfide morphology at submicron resolution, which has not been previously reported for 3D imaging of minerals. Our work suggests that MPM is a highly efficient tool for 3D studies of microstructures and morphologies of minerals in silicate rocks, which may find other applications in geosciences.

Taking a deep look: modern microscopy technologies to optimize the design and functionality of biocompatible scaffolds for tissue engineering in regenerative medicine

PubMed Central

Vielreicher, M.; Schürmann, S.; Detsch, R.; Schmidt, M. A.; Buttgereit, A.; Boccaccini, A.; Friedrich, O.

2013-01-01

This review focuses on modern nonlinear optical microscopy (NLOM) methods that are increasingly being used in the field of tissue engineering (TE) to image tissue non-invasively and without labelling in depths unreached by conventional microscopy techniques. With NLOM techniques, biomaterial matrices, cultured cells and their produced extracellular matrix may be visualized with high resolution. After introducing classical imaging methodologies such as µCT, MRI, optical coherence tomography, electron microscopy and conventional microscopy two-photon fluorescence (2-PF) and second harmonic generation (SHG) imaging are described in detail (principle, power, limitations) together with their most widely used TE applications. Besides our own cell encapsulation, cell printing and collagen scaffolding systems and their NLOM imaging the most current research articles will be reviewed. These cover imaging of autofluorescence and fluorescence-labelled tissue and biomaterial structures, SHG-based quantitative morphometry of collagen I and other proteins, imaging of vascularization and online monitoring techniques in TE. Finally, some insight is given into state-of-the-art three-photon-based imaging methods (e.g. coherent anti-Stokes Raman scattering, third harmonic generation). This review provides an overview of the powerful and constantly evolving field of multiphoton microscopy, which is a powerful and indispensable tool for the development of artificial tissues in regenerative medicine and which is likely to gain importance also as a means for general diagnostic medical imaging. PMID:23864499

Review of quantitative phase-digital holographic microscopy: promising novel imaging technique to resolve neuronal network activity and identify cellular biomarkers of psychiatric disorders

PubMed Central

Marquet, Pierre; Depeursinge, Christian; Magistretti, Pierre J.

2014-01-01

Abstract. Quantitative phase microscopy (QPM) has recently emerged as a new powerful quantitative imaging technique well suited to noninvasively explore a transparent specimen with a nanometric axial sensitivity. In this review, we expose the recent developments of quantitative phase-digital holographic microscopy (QP-DHM). Quantitative phase-digital holographic microscopy (QP-DHM) represents an important and efficient quantitative phase method to explore cell structure and dynamics. In a second part, the most relevant QPM applications in the field of cell biology are summarized. A particular emphasis is placed on the original biological information, which can be derived from the quantitative phase signal. In a third part, recent applications obtained, with QP-DHM in the field of cellular neuroscience, namely the possibility to optically resolve neuronal network activity and spine dynamics, are presented. Furthermore, potential applications of QPM related to psychiatry through the identification of new and original cell biomarkers that, when combined with a range of other biomarkers, could significantly contribute to the determination of high risk developmental trajectories for psychiatric disorders, are discussed. PMID:26157976

Review of quantitative phase-digital holographic microscopy: promising novel imaging technique to resolve neuronal network activity and identify cellular biomarkers of psychiatric disorders.

PubMed

Marquet, Pierre; Depeursinge, Christian; Magistretti, Pierre J

2014-10-01

Quantitative phase microscopy (QPM) has recently emerged as a new powerful quantitative imaging technique well suited to noninvasively explore a transparent specimen with a nanometric axial sensitivity. In this review, we expose the recent developments of quantitative phase-digital holographic microscopy (QP-DHM). Quantitative phase-digital holographic microscopy (QP-DHM) represents an important and efficient quantitative phase method to explore cell structure and dynamics. In a second part, the most relevant QPM applications in the field of cell biology are summarized. A particular emphasis is placed on the original biological information, which can be derived from the quantitative phase signal. In a third part, recent applications obtained, with QP-DHM in the field of cellular neuroscience, namely the possibility to optically resolve neuronal network activity and spine dynamics, are presented. Furthermore, potential applications of QPM related to psychiatry through the identification of new and original cell biomarkers that, when combined with a range of other biomarkers, could significantly contribute to the determination of high risk developmental trajectories for psychiatric disorders, are discussed.

High-Content Microscopy Analysis of Subcellular Structures: Assay Development and Application to Focal Adhesion Quantification.

PubMed

Kroll, Torsten; Schmidt, David; Schwanitz, Georg; Ahmad, Mubashir; Hamann, Jana; Schlosser, Corinne; Lin, Yu-Chieh; Böhm, Konrad J; Tuckermann, Jan; Ploubidou, Aspasia

2016-07-01

High-content analysis (HCA) converts raw light microscopy images to quantitative data through the automated extraction, multiparametric analysis, and classification of the relevant information content. Combined with automated high-throughput image acquisition, HCA applied to the screening of chemicals or RNAi-reagents is termed high-content screening (HCS). Its power in quantifying cell phenotypes makes HCA applicable also to routine microscopy. However, developing effective HCA and bioinformatic analysis pipelines for acquisition of biologically meaningful data in HCS is challenging. Here, the step-by-step development of an HCA assay protocol and an HCS bioinformatics analysis pipeline are described. The protocol's power is demonstrated by application to focal adhesion (FA) detection, quantitative analysis of multiple FA features, and functional annotation of signaling pathways regulating FA size, using primary data of a published RNAi screen. The assay and the underlying strategy are aimed at researchers performing microscopy-based quantitative analysis of subcellular features, on a small scale or in large HCS experiments. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

Real time quantitative phase microscopy based on single-shot transport of intensity equation (ssTIE) method

NASA Astrophysics Data System (ADS)

Yu, Wei; Tian, Xiaolin; He, Xiaoliang; Song, Xiaojun; Xue, Liang; Liu, Cheng; Wang, Shouyu

2016-08-01

Microscopy based on transport of intensity equation provides quantitative phase distributions which opens another perspective for cellular observations. However, it requires multi-focal image capturing while mechanical and electrical scanning limits its real time capacity in sample detections. Here, in order to break through this restriction, real time quantitative phase microscopy based on single-shot transport of the intensity equation method is proposed. A programmed phase mask is designed to realize simultaneous multi-focal image recording without any scanning; thus, phase distributions can be quantitatively retrieved in real time. It is believed the proposed method can be potentially applied in various biological and medical applications, especially for live cell imaging.

Fast globally optimal segmentation of cells in fluorescence microscopy images.

PubMed

Bergeest, Jan-Philip; Rohr, Karl

2011-01-01

Accurate and efficient segmentation of cells in fluorescence microscopy images is of central importance for the quantification of protein expression in high-throughput screening applications. We propose a new approach for segmenting cell nuclei which is based on active contours and convex energy functionals. Compared to previous work, our approach determines the global solution. Thus, the approach does not suffer from local minima and the segmentation result does not depend on the initialization. We also suggest a numeric approach for efficiently computing the solution. The performance of our approach has been evaluated using fluorescence microscopy images of different cell types. We have also performed a quantitative comparison with previous segmentation approaches.

Compact fixed wavelength femtosecond oscillators as an add-on for tunable Ti:sapphire lasers extend the range of applications towards multimodal imaging and optogenetics

NASA Astrophysics Data System (ADS)

Hakulinen, T.; Klein, J.

2016-03-01

Two-photon (2P) microscopy based on tunable Ti:sapphire lasers has become a widespread tool for 3D imaging with sub-cellular resolution in living tissues. In recent years multi-photon microscopy with simpler fixed-wavelength femtosecond oscillators using Yb-doped tungstenates as gain material has raised increasing interest in life-sciences, because these lasers offer one order of magnitude more average power than Ti:sapphire lasers in the wavelength range around 1040 nm: Two-photon (2P) excitation of mainly red or yellow fluorescent dyes and proteins (e.g. YFP, mFruit series) simultaneously has been proven with a single IR laser wavelength. A new approach is to extend the usability of existing tunable Titanium sapphire lasers by adding a fixed IR wavelength with an Yb femtosecond oscillator. By that means a multitude of applications for multimodal imaging and optogenetics can be supported. Furthermore fs Yb-lasers are available with a repetition rate of typically 10 MHz and an average power of typically 5 W resulting in pulse energy of typically 500 nJ, which is comparably high for fs-oscillators. This makes them an ideal tool for two-photon spinning disk laser scanning microscopy and holographic patterning for simultaneous photoactivation of large cell populations. With this work we demonstrate that economical, small-footprint Yb fixed-wavelength lasers can present an interesting add-on to tunable lasers that are commonly used in multiphoton microscopy. The Yb fs-lasers hereby offer higher power for imaging of red fluorescent dyes and proteins, are ideally enhancing existing Ti:sapphire lasers with more power in the IR, and are supporting pulse energy and power hungry applications such as spinning disk microscopy and holographic patterning.

Plasmonics and metamaterials based super-resolution imaging (Conference Presentation)

NASA Astrophysics Data System (ADS)

Liu, Zhaowei

2017-05-01

In recent years, surface imaging of various biological dynamics and biomechanical phenomena has seen a surge of interest. Imaging of processes such as exocytosis and kinesin motion are most effective when depth is limited to a very thin region of interest at the edge of the cell or specimen. However, many objects and processes of interest are of size scales below the diffraction limit for safe, visible wavelength illumination. Super-resolution imaging methods such as structured illumination microscopy and others have offered various compromises between resolution, imaging speed, and bio-compatibility. In this talk, I will present our most recent progress in plasmonic structured illumination microscopy (PSIM) and localized plasmonic structured illumination microscopy (LPSIM), and their applications in bio-imaging. We have achieved wide-field surface imaging with resolution down to 75 nm while maintaining reasonable speed and compatibility with biological specimens. These plasmonic enhanced super resolution techniques offer unique solutions to obtain 50nm spatial resolution and 50 frames per second wide imaging speed at the same time.

3D Filament Network Segmentation with Multiple Active Contours

NASA Astrophysics Data System (ADS)

Xu, Ting; Vavylonis, Dimitrios; Huang, Xiaolei

2014-03-01

Fluorescence microscopy is frequently used to study two and three dimensional network structures formed by cytoskeletal polymer fibers such as actin filaments and microtubules. While these cytoskeletal structures are often dilute enough to allow imaging of individual filaments or bundles of them, quantitative analysis of these images is challenging. To facilitate quantitative, reproducible and objective analysis of the image data, we developed a semi-automated method to extract actin networks and retrieve their topology in 3D. Our method uses multiple Stretching Open Active Contours (SOACs) that are automatically initialized at image intensity ridges and then evolve along the centerlines of filaments in the network. SOACs can merge, stop at junctions, and reconfigure with others to allow smooth crossing at junctions of filaments. The proposed approach is generally applicable to images of curvilinear networks with low SNR. We demonstrate its potential by extracting the centerlines of synthetic meshwork images, actin networks in 2D TIRF Microscopy images, and 3D actin cable meshworks of live fission yeast cells imaged by spinning disk confocal microscopy.

Continuum generation in ultra high numerical aperture fiber with application to multiphoton microscopy

NASA Astrophysics Data System (ADS)

Sayler, Nicholas

Nonlinear microscopy benefits from broadband laser sources, enabling efficient excitation of an array of fluorophores, for example. This work demonstrates broadening of a narrow band input pulse (6 nm to 40 nm) centered at 1040 nm with excellent shot-to-shot stability. In a preliminary demonstration, multiphoton imaging with pulses from the fiber is performed. In particular second harmonic imaging of corn starch is performed.

All-in-one 3D printed microscopy chamber for multidimensional imaging, the UniverSlide.

PubMed

Alessandri, Kevin; Andrique, Laetitia; Feyeux, Maxime; Bikfalvi, Andreas; Nassoy, Pierre; Recher, Gaëlle

2017-02-10

While live 3D high resolution microscopy techniques are developing rapidly, their use for biological applications is partially hampered by practical difficulties such as the lack of a versatile sample chamber. Here, we propose the design of a multi-usage observation chamber adapted for live 3D bio-imaging. We show the usefulness and practicality of this chamber, which we named the UniverSlide, for live imaging of two case examples, namely multicellular systems encapsulated in sub-millimeter hydrogel shells and zebrafish larvae. We also demonstrate its versatility and compatibility with all microscopy devices by using upright or inverted microscope configurations after loading the UniverSlide with fixed or living samples. Further, the device is applicable for medium/high throughput screening and automatized multi-position image acquisition, providing a constraint-free but stable and parallelized immobilization of the samples. The frame of the UniverSlide is fabricated using a stereolithography 3D printer, has the size of a microscopy slide, is autoclavable and sealed with a removable lid, which makes it suitable for use in a controlled culture environment. We describe in details how to build this chamber and we provide all the files necessary to print the different pieces in the lab.

All-in-one 3D printed microscopy chamber for multidimensional imaging, the UniverSlide

PubMed Central

Alessandri, Kevin; Andrique, Laetitia; Feyeux, Maxime; Bikfalvi, Andreas; Nassoy, Pierre; Recher, Gaëlle

2017-01-01

While live 3D high resolution microscopy techniques are developing rapidly, their use for biological applications is partially hampered by practical difficulties such as the lack of a versatile sample chamber. Here, we propose the design of a multi-usage observation chamber adapted for live 3D bio-imaging. We show the usefulness and practicality of this chamber, which we named the UniverSlide, for live imaging of two case examples, namely multicellular systems encapsulated in sub-millimeter hydrogel shells and zebrafish larvae. We also demonstrate its versatility and compatibility with all microscopy devices by using upright or inverted microscope configurations after loading the UniverSlide with fixed or living samples. Further, the device is applicable for medium/high throughput screening and automatized multi-position image acquisition, providing a constraint-free but stable and parallelized immobilization of the samples. The frame of the UniverSlide is fabricated using a stereolithography 3D printer, has the size of a microscopy slide, is autoclavable and sealed with a removable lid, which makes it suitable for use in a controlled culture environment. We describe in details how to build this chamber and we provide all the files necessary to print the different pieces in the lab. PMID:28186188

Correlative atomic force microscopy quantitative imaging-laser scanning confocal microscopy quantifies the impact of stressors on live cells in real-time.

PubMed

Bhat, Supriya V; Sultana, Taranum; Körnig, André; McGrath, Seamus; Shahina, Zinnat; Dahms, Tanya E S

2018-05-29

There is an urgent need to assess the effect of anthropogenic chemicals on model cells prior to their release, helping to predict their potential impact on the environment and human health. Laser scanning confocal microscopy (LSCM) and atomic force microscopy (AFM) have each provided an abundance of information on cell physiology. In addition to determining surface architecture, AFM in quantitative imaging (QI) mode probes surface biochemistry and cellular mechanics using minimal applied force, while LSCM offers a window into the cell for imaging fluorescently tagged macromolecules. Correlative AFM-LSCM produces complimentary information on different cellular characteristics for a comprehensive picture of cellular behaviour. We present a correlative AFM-QI-LSCM assay for the simultaneous real-time imaging of living cells in situ, producing multiplexed data on cell morphology and mechanics, surface adhesion and ultrastructure, and real-time localization of multiple fluorescently tagged macromolecules. To demonstrate the broad applicability of this method for disparate cell types, we show altered surface properties, internal molecular arrangement and oxidative stress in model bacterial, fungal and human cells exposed to 2,4-dichlorophenoxyacetic acid. AFM-QI-LSCM is broadly applicable to a variety of cell types and can be used to assess the impact of any multitude of contaminants, alone or in combination.

All-in-one 3D printed microscopy chamber for multidimensional imaging, the UniverSlide

NASA Astrophysics Data System (ADS)

Alessandri, Kevin; Andrique, Laetitia; Feyeux, Maxime; Bikfalvi, Andreas; Nassoy, Pierre; Recher, Gaëlle

2017-02-01

While live 3D high resolution microscopy techniques are developing rapidly, their use for biological applications is partially hampered by practical difficulties such as the lack of a versatile sample chamber. Here, we propose the design of a multi-usage observation chamber adapted for live 3D bio-imaging. We show the usefulness and practicality of this chamber, which we named the UniverSlide, for live imaging of two case examples, namely multicellular systems encapsulated in sub-millimeter hydrogel shells and zebrafish larvae. We also demonstrate its versatility and compatibility with all microscopy devices by using upright or inverted microscope configurations after loading the UniverSlide with fixed or living samples. Further, the device is applicable for medium/high throughput screening and automatized multi-position image acquisition, providing a constraint-free but stable and parallelized immobilization of the samples. The frame of the UniverSlide is fabricated using a stereolithography 3D printer, has the size of a microscopy slide, is autoclavable and sealed with a removable lid, which makes it suitable for use in a controlled culture environment. We describe in details how to build this chamber and we provide all the files necessary to print the different pieces in the lab.

A simple tool for stereological assessment of digital images: the STEPanizer.

PubMed

Tschanz, S A; Burri, P H; Weibel, E R

2011-07-01

STEPanizer is an easy-to-use computer-based software tool for the stereological assessment of digitally captured images from all kinds of microscopical (LM, TEM, LSM) and macroscopical (radiology, tomography) imaging modalities. The program design focuses on providing the user a defined workflow adapted to most basic stereological tasks. The software is compact, that is user friendly without being bulky. STEPanizer comprises the creation of test systems, the appropriate display of digital images with superimposed test systems, a scaling facility, a counting module and an export function for the transfer of results to spreadsheet programs. Here we describe the major workflow of the tool illustrating the application on two examples from transmission electron microscopy and light microscopy, respectively. © 2011 The Authors Journal of Microscopy © 2011 Royal Microscopical Society.

Imaging genes, chromosomes, and nuclear structures using laser-scanning confocal microscopy

NASA Astrophysics Data System (ADS)

Ballard, Stephen G.

1990-08-01

For 350 years, the optical microscope has had a powerful symbiotic relationship with biology. Until this century, optical microscopy was the only means of examining cellular structure; in return, biologists have contributed greatly to the evolution of microscope design and technique. Recent advances in the detection and processing of optical images, together with methods for labelling specific biological molecules, have brought about a resurgence in the application of optical microscopy to the biological sciences. One of the areas in which optical microscopy is breaking new ground is in elucidating the large scale organization of chromatin in chromosomes and cell nuclei. Nevertheless, imaging the contents of the cell nucleus is a difficult challenge for light microscopy, for two principal reasons. First, the dimensions of all but the largest nuclear structures (nucleoli, vacuoles) are close to or below the resolving power of far field optics. Second, the native optical contrast properties of many important chromatin structures (eg. chromosome domains, centromere regions) are very weak, or essentially zero. As an extreme example, individual genes probably have nothing to distinguish them other than their sequence of DNA bases, which cannot be directly visualized with any current form of microscopy. Similarly, the interphase nucleus shows no direct visible evidence of focal chromatin domains. Thus, imaging of such entities depends heavily on contrast enhancement methods. The most promising of these is labelling DNA in situ using sequence-specific probes that may be visualized using fluorescent dyes. We have applied this method to detecting individual genes in metaphase chromosomes and interphase nuclei, and to imaging a number of DNA-containing structures including chromosome domains, metaphase chromosomes and centromere regions. We have also demonstrated the applicability of in situ fluorescent labelling to detecting numerical and structural abnormalities both in condensed metaphase chromosomes and in interphase nuclei. The ability to image the loci of fluorescent-labelled gene probes hybridized to chromosomes and to interphase nuclei will play a major role in the mapping of the human genome. This presentation is an overview of our laboratory's efforts to use confocal imaging to address fundamental questions about the structure and organization of genes, chromosomes and cell nuclei, and to develop applications useful in clinical diagnosis of inherited diseases.

High resolution imaging and lithography with hard x rays using parabolic compound refractive lenses

NASA Astrophysics Data System (ADS)

Schroer, C. G.; Benner, B.; Günzler, T. F.; Kuhlmann, M.; Zimprich, C.; Lengeler, B.; Rau, C.; Weitkamp, T.; Snigirev, A.; Snigireva, I.; Appenzeller, J.

2002-03-01

Parabolic compound refractive lenses are high quality optical components for hard x rays. They are particularly suited for full field imaging, with applications in microscopy and x-ray lithography. Taking advantage of the large penetration depth of hard x rays, the interior of opaque samples can be imaged with submicrometer resolution. To obtain the three-dimensional structure of a sample, microscopy is combined with tomographic techniques. In a first hard x-ray lithography experiment, parabolic compound refractive lenses have been used to project the reduced image of a lithography mask onto a resist. Future developments are discussed.

A review of cellphone microscopy for disease detection.

PubMed

Dendere, R; Myburg, N; Douglas, T S

2015-12-01

The expansion in global cellphone network coverage coupled with advances in cellphone imaging capabilities present an opportunity for the advancement of cellphone microscopy as a low-cost alternative to conventional microscopy for disease detection in resource-limited regions. The development of cellphone microscopy has also benefitted from the availability of low-cost miniature microscope components such as low-power light-emitting diodes and ball lenses. As a result, researchers are developing hardware and software techniques that would enable such microscopes to produce high-resolution, diagnostic-quality images. This approach may lead to more widespread delivery of diagnostic services in resource-limited areas where there is a shortage of the skilled labour required for conventional microscopy and where prevalence of infectious and other diseases is still high. In this paper, we review current techniques, clinical applications and challenges faced in the field of cellphone microscopy. © 2015 The Authors Journal of Microscopy © 2015 Royal Microscopical Society.

Imaging of surgical margin in pancreatic metastasis using two-photon excited fluorescence microscopy

NASA Astrophysics Data System (ADS)

Chen, Jing; Hong, Zhipeng; Chen, Hong; Chen, Youting; Xu, Yahao; Zhu, Xiaoqin; Zhuo, Shuangmu; Shi, Zheng; Chen, Jianxin

2014-09-01

Two-photon excited fluorescence (TPEF) microscopy, has become a powerful tool for imaging unstained tissue samples at subcellular level in biomedical research. The purpose of this study was to determine whether TPEF imaging of histological sections without H-E staining can be used to identify the boundary between normal pancreas and pancreatic metastasis from renal cell carcinoma (RCC). The typical features such as the significant increase of cancerous nests, the absence of pancreatic ductal, the appearance of cancer cells were observed to present the boundary between normal pancreas and pancreatic metastasis from RCC. These results correlated well with the corresponding histological outcomes. With the advent of clinically miniaturized TPEF microscopy and integrative endoscopy, TPEF microscopy has the potential application on surgical location of pancreatic metastasis from RCC in the near future.

Lensless digital holographic microscopy and its applications in biomedicine and environmental monitoring.

PubMed

Wu, Yichen; Ozcan, Aydogan

2018-03-01

Optical compound microscope has been a major tool in biomedical imaging for centuries. Its performance relies on relatively complicated, bulky and expensive lenses and alignment mechanics. In contrast, the lensless microscope digitally reconstructs microscopic images of specimens without using any lenses, as a result of which it can be made much smaller, lighter and lower-cost. Furthermore, the limited space-bandwidth product of objective lenses in a conventional microscope can be significantly surpassed by a lensless microscope. Such lensless imaging designs have enabled high-resolution and high-throughput imaging of specimens using compact, portable and cost-effective devices to potentially address various point-of-care, global-health and telemedicine related challenges. In this review, we discuss the operation principles and the methods behind lensless digital holographic on-chip microscopy. We also go over various applications that are enabled by cost-effective and compact implementations of lensless microscopy, including some recent work on air quality monitoring, which utilized machine learning for high-throughput and accurate quantification of particulate matter in air. Finally, we conclude with a brief future outlook of this computational imaging technology. Copyright © 2017 Elsevier Inc. All rights reserved.

Identification of nodal tissue in the living heart using rapid scanning fiber-optics confocal microscopy and extracellular fluorophores.

PubMed

Huang, Chao; Kaza, Aditya K; Hitchcock, Robert W; Sachse, Frank B

2013-09-01

Risks associated with pediatric reconstructive heart surgery include injury of the sinoatrial node (SAN) and atrioventricular node (AVN), requiring cardiac rhythm management using implantable pacemakers. These injuries are the result of difficulties in identifying nodal tissues intraoperatively. Here we describe an approach based on confocal microscopy and extracellular fluorophores to quantify tissue microstructure and identify nodal tissue. Using conventional 3-dimensional confocal microscopy we investigated the microstructural arrangement of SAN, AVN, and atrial working myocardium (AWM) in fixed rat heart. AWM exhibited a regular striated arrangement of the extracellular space. In contrast, SAN and AVN had an irregular, reticulated arrangement. AWM, SAN, and AVN tissues were beneath a thin surface layer of tissue that did not obstruct confocal microscopic imaging. Subsequently, we imaged tissues in living rat hearts with real-time fiber-optics confocal microscopy. Fiber-optics confocal microscopy images resembled images acquired with conventional confocal microscopy. We investigated spatial regularity of tissue microstructure from Fourier analysis and second-order image moments. Fourier analysis of fiber-optics confocal microscopy images showed that the spatial regularity of AWM was greater than that of nodal tissues (37.5 ± 5.0% versus 24.3 ± 3.9% for SAN and 23.8 ± 3.7% for AVN; P<0.05). Similar differences of spatial regularities were revealed from second-order image moments (50.0 ± 7.3% for AWM versus 29.3 ± 6.7% for SAN and 27.3 ± 5.5% for AVN; P<0.05). The study demonstrates feasibility of identifying nodal tissue in living heart using extracellular fluorophores and fiber-optics confocal microscopy. Application of the approach in pediatric reconstructive heart surgery may reduce risks of injuring nodal tissues.

Potential of ultraviolet wide-field imaging and multiphoton microscopy for analysis of dehydroergosterol in cellular membranes.

PubMed

Wüstner, Daniel; Brewer, Jonathan R; Bagatolli, Luis; Sage, Daniel

2011-01-01

Dehydroergosterol (DHE) is an intrinsically fluorescent sterol with absorption/emission in the ultraviolet (UV) region and biophysical properties similar to those of cholesterol. We compared the potential of UV-sensitive low-light-level wide-field (UV-WF) imaging with that of multiphoton (MP) excitation microscopy to monitor DHE in living cells. Significantly reduced photobleaching in MP microscopy of DHE enabled us to acquire three-dimensional z-stacks of DHE-stained cells and to obtain high-resolution maps of DHE in surface ruffles, nanotubes, and the apical membrane of epithelial cells. We found that the lateral resolution of MP microscopy is ∼1.5-fold higher than that of UV-WF deconvolution microscopy, allowing for improved spatiotemporal analysis of plasma membrane sterol distribution. Surface intensity patterns of DHE with a diameter of 0.2 μm persisting over several minutes could be resolved by MP time-lapse microscopy. Diffusion coefficients of 0.25-μm-diameter endocytic vesicles containing DHE were determined by MP spatiotemporal image correlation spectroscopy. The requirement of extremely high laser power for visualization of DHE by MP microscopy made this method less potent for multicolor applications with organelle markers like green fluorescent protein-tagged proteins. The signal-to-noise ratio obtainable by UV-WF imaging could be significantly improved by pixelwise bleach rate fitting and calculation of an amplitude image from the decay model and by frame averaging after pixelwise bleaching correction of the image stacks. We conclude that UV-WF imaging and MP microscopy of DHE provide complementary information regarding membrane distribution and intracellular targeting of sterols. © 2010 Wiley-Liss, Inc.

A fast MEMS scanning photoacoustic microscopy system and its application in glioma study

NASA Astrophysics Data System (ADS)

Bi, Renzhe; Balasundaram, Ghayathri; Jeon, Seungwan; Pu, Yang; Tay, Hui Chien; Kim, Chulhong; Olivo, Malini

2018-02-01

We present a water-proof Microelectromechanical systems (MEMS) based scanning optical resolution Photoacoustic Microscopy (OR-PAM) system and its application in glioma tumor mouse model study. The presented OR-PAM system has high optical resolution ( 3 μm) and high scanning speed (up to 50 kHz A-scan rate), which is ideal for cerebral vascular imaging. In this study, the mice with glioma tumor are treated with vascular disrupting agent (VDA). OR-PAM system is utilized to image the cerebral with the whole skull intact before and after the injection of VDA. By image registration, the response of every single blood vessel can be traced. This will provide us deeper understanding of the drug effect.

Correlative 3D superresolution fluorescence and electron microscopy reveal the relationship of mitochondrial nucleoids to membranes

PubMed Central

Kopek, Benjamin G.; Shtengel, Gleb; Xu, C. Shan; Clayton, David A.; Hess, Harald F.

2012-01-01

Microscopic images of specific proteins in their cellular context yield important insights into biological processes and cellular architecture. The advent of superresolution optical microscopy techniques provides the possibility to augment EM with nanometer-resolution fluorescence microscopy to access the precise location of proteins in the context of cellular ultrastructure. Unfortunately, efforts to combine superresolution fluorescence and EM have been stymied by the divergent and incompatible sample preparation protocols of the two methods. Here, we describe a protocol that preserves both the delicate photoactivatable fluorescent protein labels essential for superresolution microscopy and the fine ultrastructural context of EM. This preparation enables direct 3D imaging in 500- to 750-nm sections with interferometric photoactivatable localization microscopy followed by scanning EM images generated by focused ion beam ablation. We use this process to “colorize” detailed EM images of the mitochondrion with the position of labeled proteins. The approach presented here has provided a new level of definition of the in vivo nature of organization of mitochondrial nucleoids, and we expect this straightforward method to be applicable to many other biological questions that can be answered by direct imaging. PMID:22474357

Filling the gap: adding super-resolution to array tomography for correlated ultrastructural and molecular identification of electrical synapses at the C. elegans connectome.

PubMed

Markert, Sebastian Matthias; Britz, Sebastian; Proppert, Sven; Lang, Marietta; Witvliet, Daniel; Mulcahy, Ben; Sauer, Markus; Zhen, Mei; Bessereau, Jean-Louis; Stigloher, Christian

2016-10-01

Correlating molecular labeling at the ultrastructural level with high confidence remains challenging. Array tomography (AT) allows for a combination of fluorescence and electron microscopy (EM) to visualize subcellular protein localization on serial EM sections. Here, we describe an application for AT that combines near-native tissue preservation via high-pressure freezing and freeze substitution with super-resolution light microscopy and high-resolution scanning electron microscopy (SEM) analysis on the same section. We established protocols that combine SEM with structured illumination microscopy (SIM) and direct stochastic optical reconstruction microscopy (dSTORM). We devised a method for easy, precise, and unbiased correlation of EM images and super-resolution imaging data using endogenous cellular landmarks and freely available image processing software. We demonstrate that these methods allow us to identify and label gap junctions in Caenorhabditis elegans with precision and confidence, and imaging of even smaller structures is feasible. With the emergence of connectomics, these methods will allow us to fill in the gap-acquiring the correlated ultrastructural and molecular identity of electrical synapses.

Determination of localization accuracy based on experimentally acquired image sets: applications to single molecule microscopy

PubMed Central

Tahmasbi, Amir; Ward, E. Sally; Ober, Raimund J.

2015-01-01

Fluorescence microscopy is a photon-limited imaging modality that allows the study of subcellular objects and processes with high specificity. The best possible accuracy (standard deviation) with which an object of interest can be localized when imaged using a fluorescence microscope is typically calculated using the Cramér-Rao lower bound, that is, the inverse of the Fisher information. However, the current approach for the calculation of the best possible localization accuracy relies on an analytical expression for the image of the object. This can pose practical challenges since it is often difficult to find appropriate analytical models for the images of general objects. In this study, we instead develop an approach that directly uses an experimentally collected image set to calculate the best possible localization accuracy for a general subcellular object. In this approach, we fit splines, i.e. smoothly connected piecewise polynomials, to the experimentally collected image set to provide a continuous model of the object, which can then be used for the calculation of the best possible localization accuracy. Due to its practical importance, we investigate in detail the application of the proposed approach in single molecule fluorescence microscopy. In this case, the object of interest is a point source and, therefore, the acquired image set pertains to an experimental point spread function. PMID:25837101

Atomic force microscopy of biological samples

DOE Office of Scientific and Technical Information (OSTI.GOV)

Doktycz, Mitchel John

2010-01-01

The ability to evaluate structural-functional relationships in real time has allowed scanning probe microscopy (SPM) to assume a prominent role in post genomic biological research. In this mini-review, we highlight the development of imaging and ancillary techniques that have allowed SPM to permeate many key areas of contemporary research. We begin by examining the invention of the scanning tunneling microscope (STM) by Binnig and Rohrer in 1982 and discuss how it served to team biologists with physicists to integrate high-resolution microscopy into biological science. We point to the problems of imaging nonconductive biological samples with the STM and relate howmore » this led to the evolution of the atomic force microscope (AFM) developed by Binnig, Quate, and Gerber, in 1986. Commercialization in the late 1980s established SPM as a powerful research tool in the biological research community. Contact mode AFM imaging was soon complemented by the development of non-contact imaging modes. These non-contact modes eventually became the primary focus for further new applications including the development of fast scanning methods. The extreme sensitivity of the AFM cantilever was recognized and has been developed into applications for measuring forces required for indenting biological surfaces and breaking bonds between biomolecules. Further functional augmentation to the cantilever tip allowed development of new and emerging techniques including scanning ion-conductance microscopy (SICM), scanning electrochemical microscope (SECM), Kelvin force microscopy (KFM) and scanning near field ultrasonic holography (SNFUH).« less

High-resolution high-sensitivity elemental imaging by secondary ion mass spectrometry: from traditional 2D and 3D imaging to correlative microscopy

NASA Astrophysics Data System (ADS)

Wirtz, T.; Philipp, P.; Audinot, J.-N.; Dowsett, D.; Eswara, S.

2015-10-01

Secondary ion mass spectrometry (SIMS) constitutes an extremely sensitive technique for imaging surfaces in 2D and 3D. Apart from its excellent sensitivity and high lateral resolution (50 nm on state-of-the-art SIMS instruments), advantages of SIMS include high dynamic range and the ability to differentiate between isotopes. This paper first reviews the underlying principles of SIMS as well as the performance and applications of 2D and 3D SIMS elemental imaging. The prospects for further improving the capabilities of SIMS imaging are discussed. The lateral resolution in SIMS imaging when using the microprobe mode is limited by (i) the ion probe size, which is dependent on the brightness of the primary ion source, the quality of the optics of the primary ion column and the electric fields in the near sample region used to extract secondary ions; (ii) the sensitivity of the analysis as a reasonable secondary ion signal, which must be detected from very tiny voxel sizes and thus from a very limited number of sputtered atoms; and (iii) the physical dimensions of the collision cascade determining the origin of the sputtered ions with respect to the impact site of the incident primary ion probe. One interesting prospect is the use of SIMS-based correlative microscopy. In this approach SIMS is combined with various high-resolution microscopy techniques, so that elemental/chemical information at the highest sensitivity can be obtained with SIMS, while excellent spatial resolution is provided by overlaying the SIMS images with high-resolution images obtained by these microscopy techniques. Examples of this approach are given by presenting in situ combinations of SIMS with transmission electron microscopy (TEM), helium ion microscopy (HIM) and scanning probe microscopy (SPM).

Imaging nanoscale lattice variations by machine learning of x-ray diffraction microscopy data

DOE PAGES

Laanait, Nouamane; Zhang, Zhan; Schlepütz, Christian M.

2016-08-09

In this paper, we present a novel methodology based on machine learning to extract lattice variations in crystalline materials, at the nanoscale, from an x-ray Bragg diffraction-based imaging technique. By employing a full-field microscopy setup, we capture real space images of materials, with imaging contrast determined solely by the x-ray diffracted signal. The data sets that emanate from this imaging technique are a hybrid of real space information (image spatial support) and reciprocal lattice space information (image contrast), and are intrinsically multidimensional (5D). By a judicious application of established unsupervised machine learning techniques and multivariate analysis to this multidimensional datamore » cube, we show how to extract features that can be ascribed physical interpretations in terms of common structural distortions, such as lattice tilts and dislocation arrays. Finally, we demonstrate this 'big data' approach to x-ray diffraction microscopy by identifying structural defects present in an epitaxial ferroelectric thin-film of lead zirconate titanate.« less

Imaging nanoscale lattice variations by machine learning of x-ray diffraction microscopy data

DOE Office of Scientific and Technical Information (OSTI.GOV)

Laanait, Nouamane; Zhang, Zhan; Schlepütz, Christian M.

In this paper, we present a novel methodology based on machine learning to extract lattice variations in crystalline materials, at the nanoscale, from an x-ray Bragg diffraction-based imaging technique. By employing a full-field microscopy setup, we capture real space images of materials, with imaging contrast determined solely by the x-ray diffracted signal. The data sets that emanate from this imaging technique are a hybrid of real space information (image spatial support) and reciprocal lattice space information (image contrast), and are intrinsically multidimensional (5D). By a judicious application of established unsupervised machine learning techniques and multivariate analysis to this multidimensional datamore » cube, we show how to extract features that can be ascribed physical interpretations in terms of common structural distortions, such as lattice tilts and dislocation arrays. Finally, we demonstrate this 'big data' approach to x-ray diffraction microscopy by identifying structural defects present in an epitaxial ferroelectric thin-film of lead zirconate titanate.« less

Light microscopy applications in systems biology: opportunities and challenges

PubMed Central

2013-01-01

Biological systems present multiple scales of complexity, ranging from molecules to entire populations. Light microscopy is one of the least invasive techniques used to access information from various biological scales in living cells. The combination of molecular biology and imaging provides a bottom-up tool for direct insight into how molecular processes work on a cellular scale. However, imaging can also be used as a top-down approach to study the behavior of a system without detailed prior knowledge about its underlying molecular mechanisms. In this review, we highlight the recent developments on microscopy-based systems analyses and discuss the complementary opportunities and different challenges with high-content screening and high-throughput imaging. Furthermore, we provide a comprehensive overview of the available platforms that can be used for image analysis, which enable community-driven efforts in the development of image-based systems biology. PMID:23578051

Single particle analysis based on Zernike phase contrast transmission electron microscopy.

PubMed

Danev, Radostin; Nagayama, Kuniaki

2008-02-01

We present the first application of Zernike phase-contrast transmission electron microscopy to single-particle 3D reconstruction of a protein, using GroEL chaperonin as the test specimen. We evaluated the performance of the technique by comparing 3D models derived from Zernike phase contrast imaging, with models from conventional underfocus phase contrast imaging. The same resolution, about 12A, was achieved by both imaging methods. The reconstruction based on Zernike phase contrast data required about 30% fewer particles. The advantages and prospects of each technique are discussed.

Efficient blue conversion from a 1064 nm microchip laser in long photonic crystal fiber tapers for fluorescence microscopy.

PubMed

Kudlinski, A; Lelek, M; Barviau, B; Audry, L; Mussot, A

2010-08-02

Using a low-cost microchip laser and a long photonic crystal fiber taper, we report a supercontinuum source with a very efficient visible conversion, especially in the blue region (around 420 nm). About 30 % of the total average output power is located in the 350-600 nm band, which is of primary importance in a number of biophotonics applications such as flow cytometry or fluorescence imaging microscopy for instance. We successfully demonstrate the use of this visible-enhanced source for a three-color imaging of HeLa cells in wide-field microscopy.

Fusion of lens-free microscopy and mobile-phone microscopy images for high-color-accuracy and high-resolution pathology imaging

NASA Astrophysics Data System (ADS)

Zhang, Yibo; Wu, Yichen; Zhang, Yun; Ozcan, Aydogan

2017-03-01

Digital pathology and telepathology require imaging tools with high-throughput, high-resolution and accurate color reproduction. Lens-free on-chip microscopy based on digital in-line holography is a promising technique towards these needs, as it offers a wide field of view (FOV >20 mm2) and high resolution with a compact, low-cost and portable setup. Color imaging has been previously demonstrated by combining reconstructed images at three discrete wavelengths in the red, green and blue parts of the visible spectrum, i.e., the RGB combination method. However, this RGB combination method is subject to color distortions. To improve the color performance of lens-free microscopy for pathology imaging, here we present a wavelet-based color fusion imaging framework, termed "digital color fusion microscopy" (DCFM), which digitally fuses together a grayscale lens-free microscope image taken at a single wavelength and a low-resolution and low-magnification color-calibrated image taken by a lens-based microscope, which can simply be a mobile phone based cost-effective microscope. We show that the imaging results of an H&E stained breast cancer tissue slide with the DCFM technique come very close to a color-calibrated microscope using a 40x objective lens with 0.75 NA. Quantitative comparison showed 2-fold reduction in the mean color distance using the DCFM method compared to the RGB combination method, while also preserving the high-resolution features of the lens-free microscope. Due to the cost-effective and field-portable nature of both lens-free and mobile-phone microscopy techniques, their combination through the DCFM framework could be useful for digital pathology and telepathology applications, in low-resource and point-of-care settings.

Bessel light sheet structured illumination microscopy

NASA Astrophysics Data System (ADS)

Noshirvani Allahabadi, Golchehr

Biomedical study researchers using animals to model disease and treatment need fast, deep, noninvasive, and inexpensive multi-channel imaging methods. Traditional fluorescence microscopy meets those criteria to an extent. Specifically, two-photon and confocal microscopy, the two most commonly used methods, are limited in penetration depth, cost, resolution, and field of view. In addition, two-photon microscopy has limited ability in multi-channel imaging. Light sheet microscopy, a fast developing 3D fluorescence imaging method, offers attractive advantages over traditional two-photon and confocal microscopy. Light sheet microscopy is much more applicable for in vivo 3D time-lapsed imaging, owing to its selective illumination of tissue layer, superior speed, low light exposure, high penetration depth, and low levels of photobleaching. However, standard light sheet microscopy using Gaussian beam excitation has two main disadvantages: 1) the field of view (FOV) of light sheet microscopy is limited by the depth of focus of the Gaussian beam. 2) Light-sheet images can be degraded by scattering, which limits the penetration of the excitation beam and blurs emission images in deep tissue layers. While two-sided sheet illumination, which doubles the field of view by illuminating the sample from opposite sides, offers a potential solution, the technique adds complexity and cost to the imaging system. We investigate a new technique to address these limitations: Bessel light sheet microscopy in combination with incoherent nonlinear Structured Illumination Microscopy (SIM). Results demonstrate that, at visible wavelengths, Bessel excitation penetrates up to 250 microns deep in the scattering media with single-side illumination. Bessel light sheet microscope achieves confocal level resolution at a lateral resolution of 0.3 micron and an axial resolution of 1 micron. Incoherent nonlinear SIM further reduces the diffused background in Bessel light sheet images, resulting in confocal quality images in thick tissue. The technique was applied to live transgenic zebra fish tg(kdrl:GFP), and the sub-cellular structure of fish vasculature genetically labeled with GFP was captured in 3D. The superior speed of the microscope enables us to acquire signal from 200 layers of a thick sample in 4 minutes. The compact microscope uses exclusively off-the-shelf components and offers a low-cost imaging solution for studying small animal models or tissue samples.

Molecular engineering of two-photon fluorescent probes for bioimaging applications

NASA Astrophysics Data System (ADS)

Liu, Hong-Wen; Liu, Yongchao; Wang, Peng; Zhang, Xiao-Bing

2017-03-01

During the past two decades, two-photon microscopy (TPM), which utilizes two near-infrared photons as the excitation source, has emerged as a novel, attractive imaging tool for biological research. Compared with one-photon microscopy, TPM offers several advantages, such as lowering background fluorescence in living cells and tissues, reducing photodamage to biosamples, and a photobleaching phenomenon, offering better 3D spatial localization, and increasing penetration depth. Small-molecule-based two-photon fluorescent probes have been well developed for the detection and imaging of various analytes in biological systems. In this review, we will give a general introduction of molecular engineering of two-photon fluorescent probes based on different fluorescence response mechanisms for bioimaging applications during the past decade. Inspired by the desired advantages of small-molecule two-photon fluorescent probes in biological imaging applications, we expect that more attention will be devoted to the development of new two-photon fluorophores and applications of TPM in areas of bioanalysis and disease diagnosis.

Terahertz solid immersion microscopy for sub-wavelength-resolution imaging of biological objects and tissues

NASA Astrophysics Data System (ADS)

Chernomyrdin, Nikita V.; Kucheryavenko, Anna S.; Malakhov, Kirill M.; Schadko, Alexander O.; Komandin, Gennady A.; Lebedev, Sergey P.; Dolganova, Irina N.; Kurlov, Vladimir N.; Lavrukhin, Denis V.; Ponomarev, Dmitry S.; Yurchenko, Stanislav O.; Tuchin, Valery V.; Zaytsev, Kirill I.

2018-04-01

We have developed a method of terahertz (THz) solid immersion microscopy for imaging of biological objects and tissues. It relies on the solid immersion lens (SIL) employing the THz beam focusing into the evanescent-field volume and allowing strong reduction in the dimensions of the THz beam caustic. By solving the problems of the sample handling at the focal plane and raster scanning of its surface with the focused THz beam, the THz SIL microscopy has been adapted for imaging of soft tissues. We have assembled an experimental setup based on a backward-wave oscillator, as a continuous-wave source operating at the wavelength of λ = 500 μm, and a Golay cell, as a detector of the THz wave intensity. By imaging of the razor blade, we have demonstrated advanced 0.2λ-resolution of the proposed THz SIL configuration. Using the experimental setup, we have performed THz imaging of a mint leaf revealing its sub-wavelength features. The observed results highlight a potential of the THz SIL microscopy in biomedical applications of THz science and technology.

Development of HiLo Microscope and its use in In-Vivo Applications

NASA Astrophysics Data System (ADS)

Patel, Shreyas J.

The functionality of achieving optical sectioning in biomedical research is invaluable as it allows for visualization of a biological sample at different depths while being free of background scattering. Most current microscopy techniques that offer optical sectioning, unfortunately, require complex instrumentation and thus are generally costly. HiLo microscopy, on the other hand, offers the same functionality and advantage at a relatively low cost. Hence, the work described in this thesis involves the design, build, and application of a HiLo microscope. More specifically, a standalone HiLo microscope was built in addition to implementing HiLo microscopy on a standard fluorescence microscope. In HiLo microscopy, optical sectioning is achieved by acquiring two different types of images per focal plane. One image is acquired under uniform illumination and the other is acquired under speckle illumination. These images are processed using an algorithm that extracts in-focus information and removes features and glare that occur as a result of background fluorescence. To show the benefits of the HiLo microscopy, several imaging experiments on various samples were performed under a HiLo microscope and compared against a traditional fluorescence microscope and a confocal microscope, which is considered the gold standard in optical imaging. In-vitro and ex-vivo imaging was performed on a set of pollen grains, and optically cleared mouse brain and heart slices. Each of these experiments showed great reduction in background scattering at different depths under HiLo microscopy. More importantly, HiLo imaging of optically cleared heart slice demonstrated emergence of different vasculature at different depths. Reduction of out-of-focus light increased the spatial resolution and allowed better visualization of capillary vessels. Furthermore, HiLo imaging was tested in an in-vivo model of a rodent dorsal window chamber model. When imaging the same sample under confocal microscope, the results were comparable between the two modalities. Additionally, a method of achieving blood flow maps at different depth using a combination of HiLo and LSI imaging is also discussed. The significance of this combined technique could help categorize blood flow to particular depths; this can help improve outcomes of medical treatments such pulse dye laser and photodynamic therapy treatments.

Quantitative comparison between full-spectrum and filter-based imaging in hyperspectral fluorescence microscopy

PubMed Central

GAO, L.; HAGEN, N.; TKACZYK, T.S.

2012-01-01

Summary We implement a filterless illumination scheme on a hyperspectral fluorescence microscope to achieve full-range spectral imaging. The microscope employs polarisation filtering, spatial filtering and spectral unmixing filtering to replace the role of traditional filters. Quantitative comparisons between full-spectrum and filter-based microscopy are provided in the context of signal dynamic range and accuracy of measured fluorophores’ emission spectra. To show potential applications, a five-colour cell immunofluorescence imaging experiment is theoretically simulated. Simulation results indicate that the use of proposed full-spectrum imaging technique may result in three times improvement in signal dynamic range compared to that can be achieved in the filter-based imaging. PMID:22356127

Memory-effect based deconvolution microscopy for super-resolution imaging through scattering media

NASA Astrophysics Data System (ADS)

Edrei, Eitan; Scarcelli, Giuliano

2016-09-01

High-resolution imaging through turbid media is a fundamental challenge of optical sciences that has attracted a lot of attention in recent years for its wide range of potential applications. Here, we demonstrate that the resolution of imaging systems looking behind a highly scattering medium can be improved below the diffraction-limit. To achieve this, we demonstrate a novel microscopy technique enabled by the optical memory effect that uses a deconvolution image processing and thus it does not require iterative focusing, scanning or phase retrieval procedures. We show that this newly established ability of direct imaging through turbid media provides fundamental and practical advantages such as three-dimensional refocusing and unambiguous object reconstruction.

Memory-effect based deconvolution microscopy for super-resolution imaging through scattering media.

PubMed

Edrei, Eitan; Scarcelli, Giuliano

2016-09-16

High-resolution imaging through turbid media is a fundamental challenge of optical sciences that has attracted a lot of attention in recent years for its wide range of potential applications. Here, we demonstrate that the resolution of imaging systems looking behind a highly scattering medium can be improved below the diffraction-limit. To achieve this, we demonstrate a novel microscopy technique enabled by the optical memory effect that uses a deconvolution image processing and thus it does not require iterative focusing, scanning or phase retrieval procedures. We show that this newly established ability of direct imaging through turbid media provides fundamental and practical advantages such as three-dimensional refocusing and unambiguous object reconstruction.

Applications of scientific imaging in environmental toxicology

NASA Astrophysics Data System (ADS)

El-Demerdash, Aref M.

The national goals of clean air, clean water, and healthy ecosystems are a few of the primary forces that drive the need for better environmental monitoring. As we approach the end of the 1990s, the environmental questions at regional to global scales are being redefined and refined in the light of developments in environmental understanding and technological capability. Research in the use of scientific imaging data for the study of the environment is urgently needed in order to explore the possibilities of utilizing emerging new technologies. The objective of this research proposal is to demonstrate the usability of a wealth of new technology made available in the last decade to providing a better understanding of environmental problems. Research is focused in two imaging techniques macro and micro imaging. Several examples of applications of scientific imaging in research in the field of environmental toxicology were presented. This was achieved on two scales, micro and macro imaging. On the micro level four specific examples were covered. First, the effect of utilizing scanning electron microscopy as an imaging tool in enhancing taxa identification when studying diatoms was presented. Second, scanning electron microscopy combined with energy dispersive x-ray analyzer were demonstrated as a valuable and effective tool for identifying and analyzing household dust samples. Third, electronic autoradiography combined with FT-IR microscopy were used to study the distribution pattern of [14C]-Malathion in rats as a result of dermal exposure. The results of the autoradiography made on skin sections of the application site revealed the presence of [ 14C]-activity in the first region of the skin. These results were evidenced by FT-IR microscopy. The obtained results suggest that the penetration of Malathion into the skin and other tissues is vehicle and dose dependent. The results also suggest the use of FT-IR microscopy imaging for monitoring the disposition of insecticides in biological tissues. Finally, in the microscale level, the penetration of household insecticides through different types of textiles fabrics. The results obtained from the fluorescence spectra, SFC and SEM showed that cotton-polyester (twill), cotton, wool and cotton thermal underwear were the least penetrable materials for the aerosols. On the other hand, acrylic and artificial silk (rayon) were the most penetrable cloth types. The most protective form of clothing will be more than one layer e.g. cotton/polyester type of clothing. (Abstract shortened by UMI.)

Quantitative Imaging of Single Unstained Magnetotactic Bacteria by Coherent X-ray Diffraction Microscopy.

PubMed

Fan, Jiadong; Sun, Zhibin; Zhang, Jian; Huang, Qingjie; Yao, Shengkun; Zong, Yunbing; Kohmura, Yoshiki; Ishikawa, Tetsuya; Liu, Hong; Jiang, Huaidong

2015-06-16

Novel coherent diffraction microscopy provides a powerful lensless imaging method to obtain a better understanding of the microorganism at the nanoscale. Here we demonstrated quantitative imaging of intact unstained magnetotactic bacteria using coherent X-ray diffraction microscopy combined with an iterative phase retrieval algorithm. Although the signal-to-noise ratio of the X-ray diffraction pattern from single magnetotactic bacterium is weak due to low-scattering ability of biomaterials, an 18.6 nm half-period resolution of reconstructed image was achieved by using a hybrid input-output phase retrieval algorithm. On the basis of the quantitative reconstructed images, the morphology and some intracellular structures, such as nucleoid, polyβ-hydroxybutyrate granules, and magnetosomes, were identified, which were also confirmed by scanning electron microscopy and energy dispersive spectroscopy. With the benefit from the quantifiability of coherent diffraction imaging, for the first time to our knowledge, an average density of magnetotactic bacteria was calculated to be ∼1.19 g/cm(3). This technique has a wide range of applications, especially in quantitative imaging of low-scattering biomaterials and multicomponent materials at nanoscale resolution. Combined with the cryogenic technique or X-ray free electron lasers, the method could image cells in a hydrated condition, which helps to maintain their natural structure.

Advances in imaging secondary ion mass spectrometry for biological samples

DOE PAGES

Boxer, Steven G.; Kraft, Mary L.; Weber, Peter K.

2008-12-16

Imaging mass spectrometry combines the power of mass spectrometry to identify complex molecules based on mass with sample imaging. Recent advances in secondary ion mass spectrometry have improved sensitivity and spatial resolution, so that these methods have the potential to bridge between high-resolution structures obtained by X-ray crystallography and cyro-electron microscopy and ultrastructure visualized by conventional light microscopy. Following background information on the method and instrumentation, we address the key issue of sample preparation. Because mass spectrometry is performed in high vacuum, it is essential to preserve the lateral organization of the sample while removing bulk water, and this hasmore » been a major barrier for applications to biological systems. Furthermore, recent applications of imaging mass spectrometry to cell biology, microbial communities, and biosynthetic pathways are summarized briefly, and studies of biological membrane organization are described in greater depth.« less

Chemical imaging of tissue in vivo with video-rate coherent anti-Stokes Raman scattering microscopy.

PubMed

Evans, Conor L; Potma, Eric O; Puoris'haag, Mehron; Côté, Daniel; Lin, Charles P; Xie, X Sunney

2005-11-15

Imaging living organisms with molecular selectivity typically requires the introduction of specific labels. Many applications in biology and medicine, however, would significantly benefit from a noninvasive imaging technique that circumvents such exogenous probes. In vivo microscopy based on vibrational spectroscopic contrast offers a unique approach for visualizing tissue architecture with molecular specificity. We have developed a sensitive technique for vibrational imaging of tissues by combining coherent anti-Stokes Raman scattering (CARS) with video-rate microscopy. Backscattering of the intense forward-propagating CARS radiation in tissue gives rise to a strong epi-CARS signal that makes in vivo imaging possible. This substantially large signal allows for real-time monitoring of dynamic processes, such as the diffusion of chemical compounds, in tissues. By tuning into the CH(2) stretching vibrational band, we demonstrate CARS imaging and spectroscopy of lipid-rich tissue structures in the skin of a live mouse, including sebaceous glands, corneocytes, and adipocytes, with unprecedented contrast at subcellular resolution.

MULTISCALE TENSOR ANISOTROPIC FILTERING OF FLUORESCENCE MICROSCOPY FOR DENOISING MICROVASCULATURE.

PubMed

Prasath, V B S; Pelapur, R; Glinskii, O V; Glinsky, V V; Huxley, V H; Palaniappan, K

2015-04-01

Fluorescence microscopy images are contaminated by noise and improving image quality without blurring vascular structures by filtering is an important step in automatic image analysis. The application of interest here is to automatically extract the structural components of the microvascular system with accuracy from images acquired by fluorescence microscopy. A robust denoising process is necessary in order to extract accurate vascular morphology information. For this purpose, we propose a multiscale tensor with anisotropic diffusion model which progressively and adaptively updates the amount of smoothing while preserving vessel boundaries accurately. Based on a coherency enhancing flow with planar confidence measure and fused 3D structure information, our method integrates multiple scales for microvasculature preservation and noise removal membrane structures. Experimental results on simulated synthetic images and epifluorescence images show the advantage of our improvement over other related diffusion filters. We further show that the proposed multiscale integration approach improves denoising accuracy of different tensor diffusion methods to obtain better microvasculature segmentation.

4D imaging of transient structures and morphologies in ultrafast electron microscopy.

PubMed

Barwick, Brett; Park, Hyun Soon; Kwon, Oh-Hoon; Baskin, J Spencer; Zewail, Ahmed H

2008-11-21

With advances in spatial resolution reaching the atomic scale, two-dimensional (2D) and 3D imaging in electron microscopy has become an essential methodology in various fields of study. Here, we report 4D imaging, with in situ spatiotemporal resolutions, in ultrafast electron microscopy (UEM). The ability to capture selected-area-image dynamics with pixel resolution and to control the time separation between pulses for temporal cooling of the specimen made possible studies of fleeting structures and morphologies. We demonstrate the potential for applications with two examples, gold and graphite. For gold, after thermally induced stress, we determined the atomic structural expansion, the nonthermal lattice temperature, and the ultrafast transients of warping/bulging. In contrast, in graphite, striking coherent transients of the structure were observed in both image and diffraction, directly measuring, on the nanoscale, the longitudinal resonance period governed by Young's elastic modulus. The success of these studies demonstrates the promise of UEM in real-space imaging of dynamics.

Improving your four-dimensional image: traveling through a decade of light-sheet-based fluorescence microscopy research.

PubMed

Strobl, Frederic; Schmitz, Alexander; Stelzer, Ernst H K

2017-06-01

Light-sheet-based fluorescence microscopy features optical sectioning in the excitation process. This reduces phototoxicity and photobleaching by up to four orders of magnitude compared with that caused by confocal fluorescence microscopy, simplifies segmentation and quantification for three-dimensional cell biology, and supports the transition from on-demand to systematic data acquisition in developmental biology applications.

A smartphone-based chip-scale microscope using ambient illumination.

PubMed

Lee, Seung Ah; Yang, Changhuei

2014-08-21

Portable chip-scale microscopy devices can potentially address various imaging needs in mobile healthcare and environmental monitoring. Here, we demonstrate the adaptation of a smartphone's camera to function as a compact lensless microscope. Unlike other chip-scale microscopy schemes, this method uses ambient illumination as its light source and does not require the incorporation of a dedicated light source. The method is based on the shadow imaging technique where the sample is placed on the surface of the image sensor, which captures direct shadow images under illumination. To improve the image resolution beyond the pixel size, we perform pixel super-resolution reconstruction with multiple images at different angles of illumination, which are captured while the user is manually tilting the device around any ambient light source, such as the sun or a lamp. The lensless imaging scheme allows for sub-micron resolution imaging over an ultra-wide field-of-view (FOV). Image acquisition and reconstruction are performed on the device using a custom-built Android application, constructing a stand-alone imaging device for field applications. We discuss the construction of the device using a commercial smartphone and demonstrate the imaging capabilities of our system.

A smartphone-based chip-scale microscope using ambient illumination

PubMed Central

Lee, Seung Ah; Yang, Changhuei

2014-01-01

Portable chip-scale microscopy devices can potentially address various imaging needs in mobile healthcare and environmental monitoring. Here, we demonstrate the adaptation of a smartphone’s camera to function as a compact lensless microscope. Unlike other chip-scale microscopy schemes, this method uses ambient illumination as its light source and does not require the incorporation of a dedicated light source. The method is based on the shadow imaging technique where the sample is placed on the surface of the image sensor, which captures direct shadow images under illumination. To improve the imaging resolution beyond the pixel size, we perform pixel super-resolution reconstruction with multiple images at different angles of illumination, which are captured while the user is manually tilting the device around any ambient light source, such as the sun or a lamp. The lensless imaging scheme allows for sub-micron resolution imaging over an ultra-wide field-of-view (FOV). Image acquisition and reconstruction is performed on the device using a custom-built android application, constructing a stand-alone imaging device for field applications. We discuss the construction of the device using a commercial smartphone and demonstrate the imaging capabilities of our system. PMID:24964209

Alignment of large image series using cubic B-splines tessellation: application to transmission electron microscopy data.

PubMed

Dauguet, Julien; Bock, Davi; Reid, R Clay; Warfield, Simon K

2007-01-01

3D reconstruction from serial 2D microscopy images depends on non-linear alignment of serial sections. For some structures, such as the neuronal circuitry of the brain, very large images at very high resolution are necessary to permit reconstruction. These very large images prevent the direct use of classical registration methods. We propose in this work a method to deal with the non-linear alignment of arbitrarily large 2D images using the finite support properties of cubic B-splines. After initial affine alignment, each large image is split into a grid of smaller overlapping sub-images, which are individually registered using cubic B-splines transformations. Inside the overlapping regions between neighboring sub-images, the coefficients of the knots controlling the B-splines deformations are blended, to create a virtual large grid of knots for the whole image. The sub-images are resampled individually, using the new coefficients, and assembled together into a final large aligned image. We evaluated the method on a series of large transmission electron microscopy images and our results indicate significant improvements compared to both manual and affine alignment.

Minimal resin embedding of multicellular specimens for targeted FIB-SEM imaging.

PubMed

Schieber, Nicole L; Machado, Pedro; Markert, Sebastian M; Stigloher, Christian; Schwab, Yannick; Steyer, Anna M

2017-01-01

Correlative light and electron microscopy (CLEM) is a powerful tool to perform ultrastructural analysis of targeted tissues or cells. The large field of view of the light microscope (LM) enables quick and efficient surveys of the whole specimen. It is also compatible with live imaging, giving access to functional assays. CLEM protocols take advantage of the features to efficiently retrace the position of targeted sites when switching from one modality to the other. They more often rely on anatomical cues that are visible both by light and electron microscopy. We present here a simple workflow where multicellular specimens are embedded in minimal amounts of resin, exposing their surface topology that can be imaged by scanning electron microscopy (SEM). LM and SEM both benefit from a large field of view that can cover whole model organisms. As a result, targeting specific anatomic locations by focused ion beam-SEM (FIB-SEM) tomography becomes straightforward. We illustrate this application on three different model organisms, used in our laboratory: the zebrafish embryo Danio rerio, the marine worm Platynereis dumerilii, and the dauer larva of the nematode Caenorhabditis elegans. Here we focus on the experimental steps to reduce the amount of resin covering the samples and to image the specimens inside an FIB-SEM. We expect this approach to have widespread applications for volume electron microscopy on multiple model organisms. Copyright © 2017 Elsevier Inc. All rights reserved.

Development and Application of Chemical Probes for Vibrational Imaging by Stimulated Raman Scattering

NASA Astrophysics Data System (ADS)

Hu, Fanghao

During the last decade, Raman microscopy is experiencing rapid development and increasingly applied in biological and medical systems. Especially, stimulated Raman scattering (SRS) microscopy, which significantly improves the sensitivity of Raman scattering through stimulated emission, has allowed direct visualization of many species that are previously challenging with conventional fluorescence imaging. Compared to fluorescence, SRS imaging requires no label or small label on the target molecule, thus with minimal perturbation to the molecule of interest. Moreover, Raman scattering is free from complicated photophysical and photochemical processes such as photobleaching, and has intrinsically narrower linewidth than fluorescence emission. This allows multiplexed Raman imaging with minimal spectral crosstalk and excellent photo-stability. To achieve the full potential of Raman microscopy, vibrational probes have been developed for Raman imaging. Multiple Raman probes with a few atoms in size are applied in Raman imaging with high sensitivity and specificity. An overview of both fluorescence and Raman microscopy and their imaging probes is given in Chapter 1 with a brief discussion on the SRS theory. Built on the current progress of Raman microscopy and vibrational probes, I write on my research in the development of carbon-deuterium, alkyne and nitrile probes for visualizing choline metabolism (Chapter 2), glucose uptake activity (Chapter 3), complex brain metabolism (Chapter 4) and polymeric nanoparticles (Chapter 5) in live cells and tissues, as well as the development of polyyne-based vibrational probes for super-multiplexed imaging, barcoding and analysis (Chapter 6).

SRRF: Universal live-cell super-resolution microscopy.

PubMed

Culley, Siân; Tosheva, Kalina L; Matos Pereira, Pedro; Henriques, Ricardo

2018-08-01

Super-resolution microscopy techniques break the diffraction limit of conventional optical microscopy to achieve resolutions approaching tens of nanometres. The major advantage of such techniques is that they provide resolutions close to those obtainable with electron microscopy while maintaining the benefits of light microscopy such as a wide palette of high specificity molecular labels, straightforward sample preparation and live-cell compatibility. Despite this, the application of super-resolution microscopy to dynamic, living samples has thus far been limited and often requires specialised, complex hardware. Here we demonstrate how a novel analytical approach, Super-Resolution Radial Fluctuations (SRRF), is able to make live-cell super-resolution microscopy accessible to a wider range of researchers. We show its applicability to live samples expressing GFP using commercial confocal as well as laser- and LED-based widefield microscopes, with the latter achieving long-term timelapse imaging with minimal photobleaching. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

Nanoscale live cell optical imaging of the dynamics of intracellular microvesicles in neural cells.

PubMed

Lee, Sohee; Heo, Chaejeong; Suh, Minah; Lee, Young Hee

2013-11-01

Recent advances in biotechnology and imaging technology have provided great opportunities to investigate cellular dynamics. Conventional imaging methods such as transmission electron microscopy, scanning electron microscopy, and atomic force microscopy are powerful techniques for cellular imaging, even at the nanoscale level. However, these techniques have limitations applications in live cell imaging because of the experimental preparation required, namely cell fixation, and the innately small field of view. In this study, we developed a nanoscale optical imaging (NOI) system that combines a conventional optical microscope with a high resolution dark-field condenser (Cytoviva, Inc.) and halogen illuminator. The NOI system's maximum resolution for live cell imaging is around 100 nm. We utilized NOI to investigate the dynamics of intracellular microvesicles of neural cells without immunocytological analysis. In particular, we studied direct, active random, and moderate random dynamic motions of intracellular microvesicles and visualized lysosomal vesicle changes after treatment of cells with a lysosomal inhibitor (NH4Cl). Our results indicate that the NOI system is a feasible, high-resolution optical imaging system for live small organelles that does not require complicated optics or immunocytological staining processes.

Evaluation of three methods for retrospective correction of vignetting on medical microscopy images utilizing two open source software tools.

PubMed

Babaloukas, Georgios; Tentolouris, Nicholas; Liatis, Stavros; Sklavounou, Alexandra; Perrea, Despoina

2011-12-01

Correction of vignetting on images obtained by a digital camera mounted on a microscope is essential before applying image analysis. The aim of this study is to evaluate three methods for retrospective correction of vignetting on medical microscopy images and compare them with a prospective correction method. One digital image from four different tissues was used and a vignetting effect was applied on each of these images. The resulted vignetted image was replicated four times and in each replica a different method for vignetting correction was applied with fiji and gimp software tools. The highest peak signal-to-noise ratio from the comparison of each method to the original image was obtained from the prospective method in all tissues. The morphological filtering method provided the highest peak signal-to-noise ratio value amongst the retrospective methods. The prospective method is suggested as the method of choice for correction of vignetting and if it is not applicable, then the morphological filtering may be suggested as the retrospective alternative method. © 2011 The Authors Journal of Microscopy © 2011 Royal Microscopical Society.

Sample holder for axial rotation of specimens in 3D microscopy.

PubMed

Bruns, T; Schickinger, S; Schneckenburger, H

2015-10-01

In common light microscopy, observation of samples is only possible from one perspective. However, especially for larger three-dimensional specimens observation from different views is desirable. Therefore, we are presenting a sample holder permitting rotation of the specimen around an axis perpendicular to the light path of the microscope. Thus, images can be put into a defined multidimensional context, enabling reliable three-dimensional reconstructions. The device can be easily adapted to a great variety of common light microscopes and is suitable for various applications in science, education and industry, where the observation of three-dimensional specimens is essential. Fluorescence z-projection images of copepods and ixodidae ticks at different rotation angles obtained by confocal laser scanning microscopy and light sheet fluorescence microscopy are reported as representative results. © 2015 The Authors Journal of Microscopy © 2015 Royal Microscopical Society.

Superresolution Microscopy of the Nuclear Envelope and Associated Proteins.

PubMed

Xie, Wei; Horn, Henning F; Wright, Graham D

2016-01-01

Superresolution microscopy is undoubtedly one of the most exciting technologies since the invention of the optical microscope. Capable of nanometer-scale resolution to surpass the diffraction limit and coupled with the versatile labeling techniques available, it is revolutionizing the study of cell biology. Our understanding of the nucleus, the genetic and architectural center of the cell, has gained great advancements through the application of various superresolution microscopy techniques. This chapter describes detailed procedures of multichannel superresolution imaging of the mammalian nucleus, using structured illumination microscopy and single-molecule localization microscopy.

Bioconjugation of luminescent silicon quantum dots to gadolinium ions for bioimaging applications

NASA Astrophysics Data System (ADS)

Erogbogbo, Folarin; Chang, Ching-Wen; May, Jasmine L.; Liu, Liwei; Kumar, Rajiv; Law, Wing-Cheung; Ding, Hong; Yong, Ken Tye; Roy, Indrajit; Sheshadri, Mukund; Swihart, Mark T.; Prasad, Paras N.

2012-08-01

Luminescent imaging agents and MRI contrast agents are desirable components in the rational design of multifunctional nanoconstructs for biological imaging applications. Luminescent biocompatible silicon quantum dots (SiQDs) and gadolinium chelates can be applied for fluorescence microscopy and MRI, respectively. Here, we report the first synthesis of a nanocomplex incorporating SiQDs and gadolinium ions (Gd3+) for biological applications. The nanoconstruct is composed of a PEGylated micelle, with hydrophobic SiQDs in its core, covalently bound to DOTA-chelated Gd3+. Dynamic light scattering reveals a radius of 85 nm for these nanoconstructs, which is consistent with the electron microscopy results depicting radii ranging from 25 to 60 nm. Cellular uptake of the probes verified that they maintain their optical properties within the intracellular environment. The magnetic resonance relaxivity of the nanoconstruct was 2.4 mM-1 s-1 (in terms of Gd3+ concentration), calculated to be around 6000 mM-1 s-1 per nanoconstruct. These desirable optical and relaxivity properties of the newly developed probe open the door for use of SiQDs in future multimodal applications such as tumour imaging.Luminescent imaging agents and MRI contrast agents are desirable components in the rational design of multifunctional nanoconstructs for biological imaging applications. Luminescent biocompatible silicon quantum dots (SiQDs) and gadolinium chelates can be applied for fluorescence microscopy and MRI, respectively. Here, we report the first synthesis of a nanocomplex incorporating SiQDs and gadolinium ions (Gd3+) for biological applications. The nanoconstruct is composed of a PEGylated micelle, with hydrophobic SiQDs in its core, covalently bound to DOTA-chelated Gd3+. Dynamic light scattering reveals a radius of 85 nm for these nanoconstructs, which is consistent with the electron microscopy results depicting radii ranging from 25 to 60 nm. Cellular uptake of the probes verified that they maintain their optical properties within the intracellular environment. The magnetic resonance relaxivity of the nanoconstruct was 2.4 mM-1 s-1 (in terms of Gd3+ concentration), calculated to be around 6000 mM-1 s-1 per nanoconstruct. These desirable optical and relaxivity properties of the newly developed probe open the door for use of SiQDs in future multimodal applications such as tumour imaging. Electronic supplementary information (ESI) available: SEM images of MSiQD-Gd3+, DLS plot of MSiQD-NH2, images of Gd3+-functionalized Si QDs micelles in water, plot of percentages of Gd3+ leaked from original sample, and determination of free Gd3+ in solutions of Gd chelates. See DOI: 10.1039/c2nr31002c

A portable microscopy system for fluorescence, polarized, and brightfield imaging

NASA Astrophysics Data System (ADS)

Gordon, Paul; Wattinger, Rolla; Lewis, Cody; Venancio, Vinicius Paula; Mertens-Talcott, Susanne U.; Coté, Gerard

2018-02-01

The use of mobile phones to conduct diagnostic microscopy at the point-of-care presents intriguing possibilities for the advancement of high-quality medical care in remote settings. However, it is challenging to create a single device that can adapt to the ever-varying camera technologies in phones or that can image with the customization that multiple modalities require for applications such as malaria diagnosis. A portable multi-modal microscope system is presented that utilizes a Raspberry Pi to collect and transmit data wirelessly to a myriad of electronic devices for image analysis. The microscopy system is capable of providing to the user correlated brightfield, polarized, and fluorescent images of samples fixed on traditional microscopy slides. The multimodal diagnostic capabilities of the microscope were assessed by measuring parasitemia of Plasmodium falciparum-infected thin blood smears. The device is capable of detecting fluorescently-labeled DNA using FITC excitation (490 nm) and emission (525 nm), the birefringent P. falciparum byproduct hemozoin, and detecting brightfield absorption with a resolution of 0.78 micrometers (element 9-3 of a 1951 Air Force Target). This microscopy system is a novel portable imaging tool that may be a viable candidate for field implementation if challenges of system durability, cost considerations, and full automation can be overcome.

Hybrid Microscopy: Enabling Inexpensive High-Performance Imaging through Combined Physical and Optical Magnifications

NASA Astrophysics Data System (ADS)

Zhang, Yu Shrike; Chang, Jae-Byum; Alvarez, Mario Moisés; Trujillo-de Santiago, Grissel; Aleman, Julio; Batzaya, Byambaa; Krishnadoss, Vaishali; Ramanujam, Aishwarya Aravamudhan; Kazemzadeh-Narbat, Mehdi; Chen, Fei; Tillberg, Paul W.; Dokmeci, Mehmet Remzi; Boyden, Edward S.; Khademhosseini, Ali

2016-03-01

To date, much effort has been expended on making high-performance microscopes through better instrumentation. Recently, it was discovered that physical magnification of specimens was possible, through a technique called expansion microscopy (ExM), raising the question of whether physical magnification, coupled to inexpensive optics, could together match the performance of high-end optical equipment, at a tiny fraction of the price. Here we show that such “hybrid microscopy” methods—combining physical and optical magnifications—can indeed achieve high performance at low cost. By physically magnifying objects, then imaging them on cheap miniature fluorescence microscopes (“mini-microscopes”), it is possible to image at a resolution comparable to that previously attainable only with benchtop microscopes that present costs orders of magnitude higher. We believe that this unprecedented hybrid technology that combines expansion microscopy, based on physical magnification, and mini-microscopy, relying on conventional optics—a process we refer to as Expansion Mini-Microscopy (ExMM)—is a highly promising alternative method for performing cost-effective, high-resolution imaging of biological samples. With further advancement of the technology, we believe that ExMM will find widespread applications for high-resolution imaging particularly in research and healthcare scenarios in undeveloped countries or remote places.

Focus measure method based on the modulus of the gradient of the color planes for digital microscopy

NASA Astrophysics Data System (ADS)

Hurtado-Pérez, Román; Toxqui-Quitl, Carina; Padilla-Vivanco, Alfonso; Aguilar-Valdez, J. Félix; Ortega-Mendoza, Gabriel

2018-02-01

The modulus of the gradient of the color planes (MGC) is implemented to transform multichannel information to a grayscale image. This digital technique is used in two applications: (a) focus measurements during autofocusing (AF) process and (b) extending the depth of field (EDoF) by means of multifocus image fusion. In the first case, the MGC procedure is based on an edge detection technique and is implemented in over 15 focus metrics that are typically handled in digital microscopy. The MGC approach is tested on color images of histological sections for the selection of in-focus images. An appealing attribute of all the AF metrics working in the MGC space is their monotonic behavior even up to a magnification of 100×. An advantage of the MGC method is its computational simplicity and inherent parallelism. In the second application, a multifocus image fusion algorithm based on the MGC approach has been implemented on graphics processing units (GPUs). The resulting fused images are evaluated using a nonreference image quality metric. The proposed fusion method reveals a high-quality image independently of faulty illumination during the image acquisition. Finally, the three-dimensional visualization of the in-focus image is shown.

Neural network control of focal position during time-lapse microscopy of cells.

PubMed

Wei, Ling; Roberts, Elijah

2018-05-09

Live-cell microscopy is quickly becoming an indispensable technique for studying the dynamics of cellular processes. Maintaining the specimen in focus during image acquisition is crucial for high-throughput applications, especially for long experiments or when a large sample is being continuously scanned. Automated focus control methods are often expensive, imperfect, or ill-adapted to a specific application and are a bottleneck for widespread adoption of high-throughput, live-cell imaging. Here, we demonstrate a neural network approach for automatically maintaining focus during bright-field microscopy. Z-stacks of yeast cells growing in a microfluidic device were collected and used to train a convolutional neural network to classify images according to their z-position. We studied the effect on prediction accuracy of the various hyperparameters of the neural network, including downsampling, batch size, and z-bin resolution. The network was able to predict the z-position of an image with ±1 μm accuracy, outperforming human annotators. Finally, we used our neural network to control microscope focus in real-time during a 24 hour growth experiment. The method robustly maintained the correct focal position compensating for 40 μm of focal drift and was insensitive to changes in the field of view. About ~100 annotated z-stacks were required to train the network making our method quite practical for custom autofocus applications.

Development of an add-on kit for scanning confocal microscopy (Conference Presentation)

NASA Astrophysics Data System (ADS)

Guo, Kaikai; Zheng, Guoan

2017-03-01

Scanning confocal microscopy is a standard choice for many fluorescence imaging applications in basic biomedical research. It is able to produce optically sectioned images and provide acquisition versatility to address many samples and application demands. However, scanning a focused point across the specimen limits the speed of image acquisition. As a result, scanning confocal microscope only works well with stationary samples. Researchers have performed parallel confocal scanning using digital-micromirror-device (DMD), which was used to project a scanning multi-point pattern across the sample. The DMD based parallel confocal systems increase the imaging speed while maintaining the optical sectioning ability. In this paper, we report the development of an add-on kit for high-speed and low-cost confocal microscopy. By adapting this add-on kit to an existing regular microscope, one can convert it into a confocal microscope without significant hardware modifications. Compared with current DMD-based implementations, the reported approach is able to recover multiple layers along the z axis simultaneously. It may find applications in wafer inspection and 3D metrology of semiconductor circuit. The dissemination of the proposed add-on kit under $1000 budget could also lead to new types of experimental designs for biological research labs, e.g., cytology analysis in cell culture experiments, genetic studies on multicellular organisms, pharmaceutical drug profiling, RNA interference studies, investigation of microbial communities in environmental systems, and etc.

New Application of Hyperspectral Imaging for Bacterial Cell Classification

USDA-ARS?s Scientific Manuscript database

Hyperspectral microscopy has shown potential as a method for rapid detection of foodborne pathogenic bacteria with spectral characteristics from bacterial cells. Hyperspectral microscope images (HMIs) are collected from broiler chicken isolates of Salmonella serotypes Enteritidis, Typhimurium, Infa...

Sensorless adaptive optics for isoSTED nanoscopy

NASA Astrophysics Data System (ADS)

Antonello, Jacopo; Hao, Xiang; Allgeyer, Edward S.; Bewersdorf, Joerg; Rittscher, Jens; Booth, Martin J.

2018-02-01

The presence of aberrations is a major concern when using fluorescence microscopy to image deep inside tissue. Aberrations due to refractive index mismatch and heterogeneity of the specimen under investigation cause severe reduction in the amount of fluorescence emission that is collected by the microscope. Furthermore, aberrations adversely affect the resolution, leading to loss of fine detail in the acquired images. These phenomena are particularly troublesome for super-resolution microscopy techniques such as isotropic stimulated-emission-depletion microscopy (isoSTED), which relies on accurate control of the shape and co-alignment of multiple excitation and depletion foci to operate as expected and to achieve the super-resolution effect. Aberrations can be suppressed by implementing sensorless adaptive optics techniques, whereby aberration correction is achieved by maximising a certain image quality metric. In confocal microscopy for example, one can employ the total image brightness as an image quality metric. Aberration correction is subsequently achieved by iteratively changing the settings of a wavefront corrector device until the metric is maximised. This simplistic approach has limited applicability to isoSTED microscopy where, due to the complex interplay between the excitation and depletion foci, maximising the total image brightness can lead to introducing aberrations in the depletion foci. In this work we first consider the effects that different aberration modes have on isoSTED microscopes. We then propose an iterative, wavelet-based aberration correction algorithm and evaluate its benefits.

Video image processing greatly enhances contrast, quality, and speed in polarization-based microscopy

PubMed Central

1981-01-01

Video cameras with contrast and black level controls can yield polarized light and differential interference contrast microscope images with unprecedented image quality, resolution, and recording speed. The theoretical basis and practical aspects of video polarization and differential interference contrast microscopy are discussed and several applications in cell biology are illustrated. These include: birefringence of cortical structures and beating cilia in Stentor, birefringence of rotating flagella on a single bacterium, growth and morphogenesis of echinoderm skeletal spicules in culture, ciliary and electrical activity in a balancing organ of a nudibranch snail, and acrosomal reaction in activated sperm. PMID:6788777

Imaging without lenses: achievements and remaining challenges of wide-field on-chip microscopy

PubMed Central

Greenbaum, Alon; Luo, Wei; Su, Ting-Wei; Göröcs, Zoltán; Xue, Liang; Isikman, Serhan O; Coskun, Ahmet F; Mudanyali, Onur; Ozcan, Aydogan

2012-01-01

We discuss unique features of lens-free computational imaging tools and report some of their emerging results for wide-field on-chip microscopy, such as the achievement of a numerical aperture (NA) of ~0.8–0.9 across a field of view (FOV) of more than 20 mm2 or an NA of ~0.1 across a FOV of ~18 cm2, which corresponds to an image with more than 1.5 gigapixels. We also discuss the current challenges that these computational on-chip microscopes face, shedding light on their future directions and applications. PMID:22936170

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2011-08-08

... Number: 11-032. Applicant: Southern Illinois University, Integrated Microscopy and Graphic Expertise (IMAGE) Center, 750 Communications Drive--Mailcode 4402, Carbondale, IL 62901. Instrument: Quanta 450...

Understanding Super-Resolution Nanoscopy and Its Biological Applications in Cell Imaging

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hu, Dehong; Zhao, Baoming; Xie, Yumei

2013-01-01

Optical microscopy has been an ideal tool to study phenomena in live cells because visible light at reasonable intensity does not perturb much of the normal biological functions. However, optical resolution using visible light is significantly limited by the wavelength. Overcoming this diffraction-limit barrier will reveal biological mechanisms, cellular structures, and physiological processes at nanometer scale, orders of magnitude lower than current optical microscopy. Although this appears to be a daunting task, recently developed photoswitchable probes enable reconstruction of individual images into a super-resolution image, thus the emergence of nanoscopy. Harnessing the resolution power of nanoscopy, we report here nano-resolutionmore » fluorescence imaging of microtubules and their network structures in biological cells. The super-resolution nanoscopy successfully resolved nanostructures of microtubule network—a daunting task that cannot be completed using conventional wide-field microscopy.« less

Simultaneous multicolor imaging of wide-field epi-fluorescence microscopy with four-bucket detection

PubMed Central

Park, Kwan Seob; Kim, Dong Uk; Lee, Jooran; Kim, Geon Hee; Chang, Ki Soo

2016-01-01

We demonstrate simultaneous imaging of multiple fluorophores using wide-field epi-fluorescence microscopy with a monochrome camera. The intensities of the three lasers are modulated by a sinusoidal waveform in order to excite each fluorophore with the same modulation frequency and a different time-delay. Then, the modulated fluorescence emissions are simultaneously detected by a camera operating at four times the excitation frequency. We show that two different fluorescence beads having crosstalk can be clearly separated using digital processing based on the phase information. In addition, multiple organelles within multi-stained single cells are shown with the phase mapping method, demonstrating an improved dynamic range and contrast compared to the conventional fluorescence image. These findings suggest that wide-field epi-fluorescence microscopy with four-bucket detection could be utilized for high-contrast multicolor imaging applications such as drug delivery and fluorescence in situ hybridization. PMID:27375944

Nanoscale deformation analysis with high-resolution transmission electron microscopy and digital image correlation

DOE PAGES

Wang, Xueju; Pan, Zhipeng; Fan, Feifei; ...

2015-09-10

We present an application of the digital image correlation (DIC) method to high-resolution transmission electron microscopy (HRTEM) images for nanoscale deformation analysis. The combination of DIC and HRTEM offers both the ultrahigh spatial resolution and high displacement detection sensitivity that are not possible with other microscope-based DIC techniques. We demonstrate the accuracy and utility of the HRTEM-DIC technique through displacement and strain analysis on amorphous silicon. Two types of error sources resulting from the transmission electron microscopy (TEM) image noise and electromagnetic-lens distortions are quantitatively investigated via rigid-body translation experiments. The local and global DIC approaches are applied for themore » analysis of diffusion- and reaction-induced deformation fields in electrochemically lithiated amorphous silicon. As a result, the DIC technique coupled with HRTEM provides a new avenue for the deformation analysis of materials at the nanometer length scales.« less

Evaluation of human sclera after femtosecond laser ablation using two photon and confocal microscopy

NASA Astrophysics Data System (ADS)

Sun, Hui; Kurtz, Ronald; Juhasz, Tibor

2012-08-01

Glaucoma is the second-leading cause of blindness worldwide and is often associated with elevated intraocular pressure (IOP). Partial thickness intrascleral channels can be created with a femtosecond laser operating at a wavelength of 1700 nm. Such channels have the potential to increase outflow facility and reduce elevated IOP. Analysis of the dimensions and location of these channels is important in understanding their effects. We describe the application of two-photon microscopy and confocal microscopy for noninvasive imaging of the femtosecond laser created partial-thickness scleral channels in human cadaver eyes. High-resolution images, hundreds of microns deep in the sclera, were obtained to allow determination of the shape and dimension of such channels. This demonstrates that concept of integrating femtosecond laser surgery, and two-photon and confocal imaging has the future potential for image-guided high-precision surgery in transparent and translucent tissue.

Fluorescence lifetime FRET imaging of receptor-ligand complexes in tumor cells in vitro and in vivo

NASA Astrophysics Data System (ADS)

Rudkouskaya, Alena; Sinsuebphon, Nattawut; Intes, Xavier; Mazurkiewicz, Joseph E.; Barroso, Margarida

2017-02-01

To guide the development of targeted therapies with improved efficacy and accelerated clinical acceptance, novel imaging methodologies need to be established. Toward this goal, fluorescence lifetime Förster resonance energy transfer (FLIM-FRET) imaging assays capitalize on the ability of antibodies or protein ligands to bind dimerized membrane bound receptors to measure their target engagement levels in cancer cells. Conventional FLIM FRET microscopy has been widely applied at visible wavelengths to detect protein-protein interactions in vitro. However, operation at these wavelengths restricts imaging quality and ability to quantitate lifetime changes in in vivo small animal optical imaging due to high auto-fluorescence and light scattering. Here, we have analyzed the uptake of iron-bound transferrin (Tf) probes into human breast cancer cells using FLIM-FRET microscopy in the visible and near-infrared (NIR) range. The development of NIR FLIM FRET microscopy allows for the use of quantitative lifetime-based molecular assays to measure drug-target engagement levels at multiple scales: from in vitro microscopy to in vivo small animal optical imaging (macroscopy). This novel approach can be extended to other receptors, currently targeted in oncology. Hence, lifetime-based molecular imaging can find numerous applications in drug delivery and targeted therapy assessment and optimization.

Multimodal Nonlinear Optical Microscopy

PubMed Central

Yue, Shuhua; Slipchenko, Mikhail N.; Cheng, Ji-Xin

2013-01-01

Because each nonlinear optical (NLO) imaging modality is sensitive to specific molecules or structures, multimodal NLO imaging capitalizes the potential of NLO microscopy for studies of complex biological tissues. The coupling of multiphoton fluorescence, second harmonic generation, and coherent anti-Stokes Raman scattering (CARS) has allowed investigation of a broad range of biological questions concerning lipid metabolism, cancer development, cardiovascular disease, and skin biology. Moreover, recent research shows the great potential of using CARS microscope as a platform to develop more advanced NLO modalities such as electronic-resonance-enhanced four-wave mixing, stimulated Raman scattering, and pump-probe microscopy. This article reviews the various approaches developed for realization of multimodal NLO imaging as well as developments of new NLO modalities on a CARS microscope. Applications to various aspects of biological and biomedical research are discussed. PMID:24353747

Double-clad photonic crystal fiber coupler for compact nonlinear optical microscopy imaging.

PubMed

Fu, Ling; Gu, Min

2006-05-15

A 1 x 2 double-clad photonic crystal fiber coupler is fabricated by the fused tapered method, showing a low excess loss of 1.1 dB and a splitting ratio of 97/3 over the entire visible and near-infrared wavelength range. In addition to the property of splitting the laser power, the double-clad feature of the coupler facilitates the separation of a near-infrared single-mode beam from a visible multimode beam, which is ideal for nonlinear optical microscopy imaging. In conjunction with a gradient-index lens, this coupler is used to construct a miniaturized microscope based on two-photon fluorescence and second-harmonic generation. Three-dimensional nonlinear optical images demonstrate potential applications of the coupler to compact all-fiber and nonlinear optical microscopy and endoscopy.

Comparative Performance Analysis of Intel Xeon Phi, GPU, and CPU: A Case Study from Microscopy Image Analysis

PubMed Central

Teodoro, George; Kurc, Tahsin; Kong, Jun; Cooper, Lee; Saltz, Joel

2014-01-01

We study and characterize the performance of operations in an important class of applications on GPUs and Many Integrated Core (MIC) architectures. Our work is motivated by applications that analyze low-dimensional spatial datasets captured by high resolution sensors, such as image datasets obtained from whole slide tissue specimens using microscopy scanners. Common operations in these applications involve the detection and extraction of objects (object segmentation), the computation of features of each extracted object (feature computation), and characterization of objects based on these features (object classification). In this work, we have identify the data access and computation patterns of operations in the object segmentation and feature computation categories. We systematically implement and evaluate the performance of these operations on modern CPUs, GPUs, and MIC systems for a microscopy image analysis application. Our results show that the performance on a MIC of operations that perform regular data access is comparable or sometimes better than that on a GPU. On the other hand, GPUs are significantly more efficient than MICs for operations that access data irregularly. This is a result of the low performance of MICs when it comes to random data access. We also have examined the coordinated use of MICs and CPUs. Our experiments show that using a performance aware task strategy for scheduling application operations improves performance about 1.29× over a first-come-first-served strategy. This allows applications to obtain high performance efficiency on CPU-MIC systems - the example application attained an efficiency of 84% on 192 nodes (3072 CPU cores and 192 MICs). PMID:25419088

Three-dimensional imaging of porous media using confocal laser scanning microscopy.

PubMed

Shah, S M; Crawshaw, J P; Boek, E S

2017-02-01

In the last decade, imaging techniques capable of reconstructing three-dimensional (3-D) pore-scale model have played a pivotal role in the study of fluid flow through complex porous media. In this study, we present advances in the application of confocal laser scanning microscopy (CLSM) to image, reconstruct and characterize complex porous geological materials with hydrocarbon reservoir and CO 2 storage potential. CLSM has a unique capability of producing 3-D thin optical sections of a material, with a wide field of view and submicron resolution in the lateral and axial planes. However, CLSM is limited in the depth (z-dimension) that can be imaged in porous materials. In this study, we introduce a 'grind and slice' technique to overcome this limitation. We discuss the practical and technical aspects of the confocal imaging technique with application to complex rock samples including Mt. Gambier and Ketton carbonates. We then describe the complete workflow of image processing to filtering and segmenting the raw 3-D confocal volumetric data into pores and grains. Finally, we use the resulting 3-D pore-scale binarized confocal data obtained to quantitatively determine petrophysical pore-scale properties such as total porosity, macro- and microporosity and single-phase permeability using lattice Boltzmann (LB) simulations, validated by experiments. © 2016 The Authors Journal of Microscopy © 2016 Royal Microscopical Society.

Digital Imaging

NASA Technical Reports Server (NTRS)

1986-01-01

Digital Imaging is the computer processed numerical representation of physical images. Enhancement of images results in easier interpretation. Quantitative digital image analysis by Perceptive Scientific Instruments, locates objects within an image and measures them to extract quantitative information. Applications are CAT scanners, radiography, microscopy in medicine as well as various industrial and manufacturing uses. The PSICOM 327 performs all digital image analysis functions. It is based on Jet Propulsion Laboratory technology, is accurate and cost efficient.

Application of optical coherence tomography based microangiography for cerebral imaging

NASA Astrophysics Data System (ADS)

Baran, Utku; Wang, Ruikang K.

2016-03-01

Requirements of in vivo rodent brain imaging are hard to satisfy using traditional technologies such as magnetic resonance imaging and two-photon microscopy. Optical coherence tomography (OCT) is an emerging tool that can easily reach at high speeds and provide high resolution volumetric images with a relatively large field of view for rodent brain imaging. Here, we provide the overview of recent developments of functional OCT based imaging techniques for neuroscience applications on rodents. Moreover, a summary of OCT-based microangiography (OMAG) studies for stroke and traumatic brain injury cases on rodents are provided.

Integrated one- and two-photon scanned oblique plane illumination (SOPi) microscopy for rapid volumetric imaging

NASA Astrophysics Data System (ADS)

Kumar, Manish; Kishore, Sandeep; Nasenbeny, Jordan; McLean, David L.; Kozorovitskiy, Yevgenia

2018-05-01

Versatile, sterically accessible imaging systems capable of in vivo rapid volumetric functional and structural imaging deep in the brain continue to be a limiting factor in neuroscience research. Towards overcoming this obstacle, we present integrated one- and two-photon scanned oblique plane illumination (SOPi) microscopy which uses a single front-facing microscope objective to provide light-sheet scanning based rapid volumetric imaging capability at subcellular resolution. Our planar scan-mirror based optimized light-sheet architecture allows for non-distorted scanning of volume samples, simplifying accurate reconstruction of the imaged volume. Integration of both one-photon (1P) and two-photon (2P) light-sheet microscopy in the same system allows for easy selection between rapid volumetric imaging and higher resolution imaging in scattering media. Using SOPi, we demonstrate deep, large volume imaging capability inside scattering mouse brain sections and rapid imaging speeds up to 10 volumes per second in zebrafish larvae expressing genetically encoded fluorescent proteins GFP or GCaMP6s. SOPi flexibility and steric access makes it adaptable for numerous imaging applications and broadly compatible with orthogonal techniques for actuating or interrogating neuronal structure and activity.

Integrated one- and two-photon scanned oblique plane illumination (SOPi) microscopy for rapid volumetric imaging.

PubMed

Kumar, Manish; Kishore, Sandeep; Nasenbeny, Jordan; McLean, David L; Kozorovitskiy, Yevgenia

2018-05-14

Versatile, sterically accessible imaging systems capable of in vivo rapid volumetric functional and structural imaging deep in the brain continue to be a limiting factor in neuroscience research. Towards overcoming this obstacle, we present integrated one- and two-photon scanned oblique plane illumination (SOPi, /sōpī/) microscopy which uses a single front-facing microscope objective to provide light-sheet scanning based rapid volumetric imaging capability at subcellular resolution. Our planar scan-mirror based optimized light-sheet architecture allows for non-distorted scanning of volume samples, simplifying accurate reconstruction of the imaged volume. Integration of both one-photon (1P) and two-photon (2P) light-sheet microscopy in the same system allows for easy selection between rapid volumetric imaging and higher resolution imaging in scattering media. Using SOPi, we demonstrate deep, large volume imaging capability inside scattering mouse brain sections and rapid imaging speeds up to 10 volumes per second in zebrafish larvae expressing genetically encoded fluorescent proteins GFP or GCaMP6s. SOPi's flexibility and steric access makes it adaptable for numerous imaging applications and broadly compatible with orthogonal techniques for actuating or interrogating neuronal structure and activity.

Optical coherent tomography and fluorescent microscopy for the study of meningeal lymphatic systems

NASA Astrophysics Data System (ADS)

Semyachkina-Glushkovskaya, O.; Abdurashitov, A.; Namykin, A.; Fedosov, I.; Pavlov, A.; Karavaev, A.; Sindeeva, O.; Shirokov, A.; Ulanova, M.; Shushunova, N.; Khorovodov, A.; Agranovich, I.; Bodrova, A.; Sagatova, M.; Shareef, Ali Esmat; Saranceva, E.; Dvoryatkina, M.; Tuchin, V.

2018-04-01

The development of novel technologies for the imaging of meningeal lymphatic vessels is one of the amazing trends of biophotonics thanks to discovery of brain lymphatics over several years ago. However, there is the limited technologies exist for the study of lymphatics in vivo because lymphatic vessels are transparent with a low speed flow of lymph. Here we demonstrate the successful application of fluorescent microscopy for the imaging of lymphatic system in the mouse brain in vivo.

Quantitative comparison of 3D third harmonic generation and fluorescence microscopy images.

PubMed

Zhang, Zhiqing; Kuzmin, Nikolay V; Groot, Marie Louise; de Munck, Jan C

2018-01-01

Third harmonic generation (THG) microscopy is a label-free imaging technique that shows great potential for rapid pathology of brain tissue during brain tumor surgery. However, the interpretation of THG brain images should be quantitatively linked to images of more standard imaging techniques, which so far has been done qualitatively only. We establish here such a quantitative link between THG images of mouse brain tissue and all-nuclei-highlighted fluorescence images, acquired simultaneously from the same tissue area. For quantitative comparison of a substantial pair of images, we present here a segmentation workflow that is applicable for both THG and fluorescence images, with a precision of 91.3 % and 95.8 % achieved respectively. We find that the correspondence between the main features of the two imaging modalities amounts to 88.9 %, providing quantitative evidence of the interpretation of dark holes as brain cells. Moreover, 80 % bright objects in THG images overlap with nuclei highlighted in the fluorescence images, and they are 2 times smaller than the dark holes, showing that cells of different morphologies can be recognized in THG images. We expect that the described quantitative comparison is applicable to other types of brain tissue and with more specific staining experiments for cell type identification. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Optofluidic bioimaging platform for quantitative phase imaging of lab on a chip devices using digital holographic microscopy.

PubMed

Pandiyan, Vimal Prabhu; John, Renu

2016-01-20

We propose a versatile 3D phase-imaging microscope platform for real-time imaging of optomicrofluidic devices based on the principle of digital holographic microscopy (DHM). Lab-on-chip microfluidic devices fabricated on transparent polydimethylsiloxane (PDMS) and glass substrates have attained wide popularity in biological sensing applications. However, monitoring, visualization, and characterization of microfluidic devices, microfluidic flows, and the biochemical kinetics happening in these devices is difficult due to the lack of proper techniques for real-time imaging and analysis. The traditional bright-field microscopic techniques fail in imaging applications, as the microfluidic channels and the fluids carrying biological samples are transparent and not visible in bright light. Phase-based microscopy techniques that can image the phase of the microfluidic channel and changes in refractive indices due to the fluids and biological samples present in the channel are ideal for imaging the fluid flow dynamics in a microfluidic channel at high resolutions. This paper demonstrates three-dimensional imaging of a microfluidic device with nanometric depth precisions and high SNR. We demonstrate imaging of microelectrodes of nanometric thickness patterned on glass substrate and the microfluidic channel. Three-dimensional imaging of a transparent PDMS optomicrofluidic channel, fluid flow, and live yeast cell flow in this channel has been demonstrated using DHM. We also quantify the average velocity of fluid flow through the channel. In comparison to any conventional bright-field microscope, the 3D depth information in the images illustrated in this work carry much information about the biological system under observation. The results demonstrated in this paper prove the high potential of DHM in imaging optofluidic devices; detection of pathogens, cells, and bioanalytes on lab-on-chip devices; and in studying microfluidic dynamics in real time based on phase changes.

Optical magnetic imaging of living cells

PubMed Central

Le Sage, D.; Arai, K.; Glenn, D. R.; DeVience, S. J.; Pham, L. M.; Rahn-Lee, L.; Lukin, M. D.; Yacoby, A.; Komeili, A.; Walsworth, R. L.

2013-01-01

Magnetic imaging is a powerful tool for probing biological and physical systems. However, existing techniques either have poor spatial resolution compared to optical microscopy and are hence not generally applicable to imaging of sub-cellular structure (e.g., magnetic resonance imaging [MRI]1), or entail operating conditions that preclude application to living biological samples while providing sub-micron resolution (e.g., scanning superconducting quantum interference device [SQUID] microscopy2, electron holography3, and magnetic resonance force microscopy [MRFM]4). Here we demonstrate magnetic imaging of living cells (magnetotactic bacteria) under ambient laboratory conditions and with sub-cellular spatial resolution (400 nm), using an optically-detected magnetic field imaging array consisting of a nanoscale layer of nitrogen-vacancy (NV) colour centres implanted at the surface of a diamond chip. With the bacteria placed on the diamond surface, we optically probe the NV quantum spin states and rapidly reconstruct images of the vector components of the magnetic field created by chains of magnetic nanoparticles (magnetosomes) produced in the bacteria, and spatially correlate these magnetic field maps with optical images acquired in the same apparatus. Wide-field sCMOS acquisition allows parallel optical and magnetic imaging of multiple cells in a population with sub-micron resolution and >100 micron field-of-view. Scanning electron microscope (SEM) images of the bacteria confirm that the correlated optical and magnetic images can be used to locate and characterize the magnetosomes in each bacterium. The results provide a new capability for imaging bio-magnetic structures in living cells under ambient conditions with high spatial resolution, and will enable the mapping of a wide range of magnetic signals within cells and cellular networks5, 6. PMID:23619694

Color normalization for robust evaluation of microscopy images

NASA Astrophysics Data System (ADS)

Švihlík, Jan; Kybic, Jan; Habart, David

2015-09-01

This paper deals with color normalization of microscopy images of Langerhans islets in order to increase robustness of the islet segmentation to illumination changes. The main application is automatic quantitative evaluation of the islet parameters, useful for determining the feasibility of islet transplantation in diabetes. First, background illumination inhomogeneity is compensated and a preliminary foreground/background segmentation is performed. The color normalization itself is done in either lαβ or logarithmic RGB color spaces, by comparison with a reference image. The color-normalized images are segmented using color-based features and pixel-wise logistic regression, trained on manually labeled images. Finally, relevant statistics such as the total islet area are evaluated in order to determine the success likelihood of the transplantation.

Smooth 2D manifold extraction from 3D image stack

PubMed Central

Shihavuddin, Asm; Basu, Sreetama; Rexhepaj, Elton; Delestro, Felipe; Menezes, Nikita; Sigoillot, Séverine M; Del Nery, Elaine; Selimi, Fekrije; Spassky, Nathalie; Genovesio, Auguste

2017-01-01

Three-dimensional fluorescence microscopy followed by image processing is routinely used to study biological objects at various scales such as cells and tissue. However, maximum intensity projection, the most broadly used rendering tool, extracts a discontinuous layer of voxels, obliviously creating important artifacts and possibly misleading interpretation. Here we propose smooth manifold extraction, an algorithm that produces a continuous focused 2D extraction from a 3D volume, hence preserving local spatial relationships. We demonstrate the usefulness of our approach by applying it to various biological applications using confocal and wide-field microscopy 3D image stacks. We provide a parameter-free ImageJ/Fiji plugin that allows 2D visualization and interpretation of 3D image stacks with maximum accuracy. PMID:28561033

Quantitative analysis of phosphoinositide 3-kinase (PI3K) signaling using live-cell total internal reflection fluorescence (TIRF) microscopy.

PubMed

Johnson, Heath E; Haugh, Jason M

2013-12-02

This unit focuses on the use of total internal reflection fluorescence (TIRF) microscopy and image analysis methods to study the dynamics of signal transduction mediated by class I phosphoinositide 3-kinases (PI3Ks) in mammalian cells. The first four protocols cover live-cell imaging experiments, image acquisition parameters, and basic image processing and segmentation. These methods are generally applicable to live-cell TIRF experiments. The remaining protocols outline more advanced image analysis methods, which were developed in our laboratory for the purpose of characterizing the spatiotemporal dynamics of PI3K signaling. These methods may be extended to analyze other cellular processes monitored using fluorescent biosensors. Copyright © 2013 John Wiley & Sons, Inc.

On-Chip Biomedical Imaging

PubMed Central

Göröcs, Zoltán; Ozcan, Aydogan

2012-01-01

Lab-on-a-chip systems have been rapidly emerging to pave the way toward ultra-compact, efficient, mass producible and cost-effective biomedical research and diagnostic tools. Although such microfluidic and micro electromechanical systems achieved high levels of integration, and are capable of performing various important tasks on the same chip, such as cell culturing, sorting and staining, they still rely on conventional microscopes for their imaging needs. Recently several alternative on-chip optical imaging techniques have been introduced, which have the potential to substitute conventional microscopes for various lab-on-a-chip applications. Here we present a critical review of these recently emerging on-chip biomedical imaging modalities, including contact shadow imaging, lensfree holographic microscopy, fluorescent on-chip microscopy and lensfree optical tomography. PMID:23558399

Gamete competence assessment by polarizing optics in assisted reproduction.

PubMed

Montag, Markus; Köster, Maria; van der Ven, Katrin; van der Ven, Hans

2011-01-01

The purpose of this study was first to give an overview of the historical development of polarization microscopy, second to describe the various applications of this technique in assisted reproduction techniques (ART) and third to discuss the potential benefit of polarization microscopy as a predictor for IVF success. The history of polarization microscopy was undertaken by performing a backward search in the scientific literature using Google and internet sites of several Societies for Microscopy and Cell Biology. Studies of polarization microscopy in ART were identified by using a systematic literature search in PubMed and Scopus. A total of 62 articles were identified by the direct search and further relevant articles were found by screening the cited literature in these articles. The topics relevant for assisted reproduction were spindle and zona imaging in combination with IVF success, meiotic cell cycle progression, pharmaceutical studies and cryopreservation. A separate topic was the use of sperm birefringence in ART. The majority of studies are observational studies and were not performed in a randomized manner and there is no direct comparison of techniques using other gamete selection markers. Despite this, most studies show that polarization microscopy may help us to further increase our knowledge on gametes and meiosis. Whether certain applications such as spindle or zona imaging may lead to an increase in IVF success is unclear at present. Publications on the use of polarization microscopy on sperm are still very limited.

Backscattered helium spectroscopy in the helium ion microscope: Principles, resolution and applications

NASA Astrophysics Data System (ADS)

van Gastel, R.; Hlawacek, G.; Dutta, S.; Poelsema, B.

2015-02-01

We demonstrate the possibilities and limitations for microstructure characterization using backscattered particles from a sharply focused helium ion beam. The interaction of helium ions with matter enables the imaging, spectroscopic characterization, as well as the nanometer scale modification of samples. The contrast that is seen in helium ion microscopy (HIM) images differs from that in scanning electron microscopy (SEM) and is generally a result of the higher surface sensitivity of the method. It allows, for instance, a much better visualization of low-Z materials as a result of the small secondary electron escape depth. However, the same differences in beam interaction that give HIM an edge over other imaging techniques, also impose limitations for spectroscopic applications using backscattered particles. Here we quantify those limitations and discuss opportunities to further improve the technique.

Atomic force-multi-optical imaging integrated microscope for monitoring molecular dynamics in live cells.

PubMed

Trache, Andreea; Meininger, Gerald A

2005-01-01

A novel hybrid imaging system is constructed integrating atomic force microscopy (AFM) with a combination of optical imaging techniques that offer high spatial resolution. The main application of this instrument (the NanoFluor microscope) is the study of mechanotransduction with an emphasis on extracellular matrix-integrin-cytoskeletal interactions and their role in the cellular responses to changes in external chemical and mechanical factors. The AFM allows the quantitative assessment of cytoskeletal changes, binding probability, adhesion forces, and micromechanical properties of the cells, while the optical imaging applications allow thin sectioning of the cell body at the coverslip-cell interface, permitting the study of focal adhesions using total internal reflection fluorescence (TIRF) and internal reflection microscopy (IRM). Combined AFM-optical imaging experiments show that mechanical stimulation at the apical surface of cells induces a force-generating cytoskeletal response, resulting in focal contact reorganization on the basal surface that can be monitored in real time. The NanoFluor system is also equipped with a novel mechanically aligned dual camera acquisition system for synthesized Forster resonance energy transfer (FRET). The integrated NanoFluor microscope system is described, including its characteristics, applications, and limitations.

Differential Multiphoton Laser Scanning Microscopy

PubMed Central

Field, Jeffrey J.; Sheetz, Kraig E.; Chandler, Eric V.; Hoover, Erich E.; Young, Michael D.; Ding, Shi-you; Sylvester, Anne W.; Kleinfeld, David; Squier, Jeff A.

2016-01-01

Multifocal multiphoton microscopy (MMM) in the biological and medical sciences has become an important tool for obtaining high resolution images at video rates. While current implementations of MMM achieve very high frame rates, they are limited in their applicability to essentially those biological samples that exhibit little or no scattering. In this paper, we report on a method for MMM in which imaging detection is not necessary (single element point detection is implemented), and is therefore fully compatible for use in imaging through scattering media. Further, we demonstrate that this method leads to a new type of MMM wherein it is possible to simultaneously obtain multiple images and view differences in excitation parameters in a single shot. PMID:27390511

Seeing cilia: imaging modalities for ciliary motion and clinical connections.

PubMed

Peabody, Jacelyn E; Shei, Ren-Jay; Bermingham, Brent M; Phillips, Scott E; Turner, Brett; Rowe, Steven M; Solomon, George M

2018-06-01

The respiratory tract is lined with multiciliated epithelial cells that function to move mucus and trapped particles via the mucociliary transport apparatus. Genetic and acquired ciliopathies result in diminished mucociliary clearance, contributing to disease pathogenesis. Recent innovations in imaging technology have advanced our understanding of ciliary motion in health and disease states. Application of imaging modalities including transmission electron microscopy, high-speed video microscopy, and micron-optical coherence tomography could improve diagnostics and be applied for precision medicine. In this review, we provide an overview of ciliary motion, imaging modalities, and ciliopathic diseases of the respiratory system including primary ciliary dyskinesia, cystic fibrosis, chronic obstructive pulmonary disease, and idiopathic pulmonary fibrosis.

Nano-scale measurement of biomolecules by optical microscopy and semiconductor nanoparticles

PubMed Central

Ichimura, Taro; Jin, Takashi; Fujita, Hideaki; Higuchi, Hideo; Watanabe, Tomonobu M.

2014-01-01

Over the past decade, great developments in optical microscopy have made this technology increasingly compatible with biological studies. Fluorescence microscopy has especially contributed to investigating the dynamic behaviors of live specimens and can now resolve objects with nanometer precision and resolution due to super-resolution imaging. Additionally, single particle tracking provides information on the dynamics of individual proteins at the nanometer scale both in vitro and in cells. Complementing advances in microscopy technologies has been the development of fluorescent probes. The quantum dot, a semi-conductor fluorescent nanoparticle, is particularly suitable for single particle tracking and super-resolution imaging. This article overviews the principles of single particle tracking and super resolution along with describing their application to the nanometer measurement/observation of biological systems when combined with quantum dot technologies. PMID:25120488

Shedding new light on lipid functions with CARS and SRS microscopy

PubMed Central

Yu, Yong; Ramachandran, Prasanna V.; Wang, Meng C.

2014-01-01

Modern optical microscopy has granted biomedical scientists unprecedented access to the inner workings of a cell, and revolutionized our understanding of the molecular mechanisms underlying physiological and disease states. In spite of these advances, however, visualization of certain classes of molecules (e.g. lipids) at the sub-cellular level has remained elusive. Recently developed chemical imaging modalities – Coherent Anti-Stokes Raman Scattering (CARS) microscopy and Stimulated Raman Scattering (SRS) microscopy – have helped bridge this gap. By selectively imaging the vibration of a specific chemical group, these non-invasive techniques allow high-resolution imaging of individual molecules in vivo, and circumvent the need for potentially perturbative extrinsic labels. These tools have already been applied to the study of fat metabolism, helping uncover novel regulators of lipid storage. Here we review the underlying principle of CARS and SRS microscopy, and discuss the advantages and caveats of each technique. We also review recent applications of these tools in the study of lipids as well as other biomolecules, and conclude with a brief guide for interested researchers to build and use CARS/SRS systems for their own research. PMID:24576891

Correlative fluorescence and scanning transmission electron microscopy of quantum dot-labeled proteins on whole cells in liquid.

PubMed

Peckys, Diana B; Bandmann, Vera; de Jonge, Niels

2014-01-01

Correlative fluorescence microscopy combined with scanning transmission electron microscopy (STEM) of cells fully immersed in liquid is a new methodology with many application areas. Proteins, in live cells immobilized on microchips, are labeled with fluorescent quantum dot nanoparticles. In this protocol, the epidermal growth factor receptor (EGFR) is labeled. The cells are fixed after a selected labeling time, for example, 5 min as needed to form EGFR dimers. The microchip with cells is then imaged with fluorescence microscopy. Thereafter, STEM can be accomplished in two ways. The microchip with the labeled cells and one microchip with a spacer are assembled into a special microfluidic device and imaged with dedicated high-voltage STEM. Alternatively, thin edges of cells can be studied with environmental scanning electron microscopy with a STEM detector, by placing a microchip with cells in a cooled wet environment. © 2014 Elsevier Inc. All rights reserved.

Probing Membrane Order and Topography in Supported Lipid Bilayers by Combined Polarized Total Internal Reflection Fluorescence-Atomic Force Microscopy

PubMed Central

Oreopoulos, John; Yip, Christopher M.

2009-01-01

Determining the local structure, dynamics, and conformational requirements for protein-protein and protein-lipid interactions in membranes is critical to understanding biological processes ranging from signaling to the translocating and membranolytic action of antimicrobial peptides. We report here the application of a combined polarized total internal reflection fluorescence microscopy-in situ atomic force microscopy platform. This platform's ability to image membrane orientational order was demonstrated on DOPC/DSPC/cholesterol model membranes containing the fluorescent membrane probe, DiI-C20 or BODIPY-PC. Spatially resolved order parameters and fluorophore tilt angles extracted from the polarized total internal reflection fluorescence microscopy images were in good agreement with the topographical details resolved by in situ atomic force microscopy, portending use of this technique for high-resolution characterization of membrane domain structures and peptide-membrane interactions. PMID:19254557

High-speed X-ray microscopy by use of high-resolution zone plates and synchrotron radiation.

PubMed

Hou, Qiyue; Wang, Zhili; Gao, Kun; Pan, Zhiyun; Wang, Dajiang; Ge, Xin; Zhang, Kai; Hong, Youli; Zhu, Peiping; Wu, Ziyu

2012-09-01

X-ray microscopy based on synchrotron radiation has become a fundamental tool in biology and life sciences to visualize the morphology of a specimen. These studies have particular requirements in terms of radiation damage and the image exposure time, which directly determines the total acquisition speed. To monitor and improve these key parameters, we present a novel X-ray microscopy method using a high-resolution zone plate as the objective and the matching condenser. Numerical simulations based on the scalar wave field theory validate the feasibility of the method and also indicate the performance of X-ray microscopy is optimized most with sub-10-nm-resolution zone plates. The proposed method is compatible with conventional X-ray microscopy techniques, such as computed tomography, and will find wide applications in time-resolved and/or dose-sensitive studies such as living cell imaging.

Qualitative and quantitative interpretation of SEM image using digital image processing.

PubMed

Saladra, Dawid; Kopernik, Magdalena

2016-10-01

The aim of the this study is improvement of qualitative and quantitative analysis of scanning electron microscope micrographs by development of computer program, which enables automatic crack analysis of scanning electron microscopy (SEM) micrographs. Micromechanical tests of pneumatic ventricular assist devices result in a large number of micrographs. Therefore, the analysis must be automatic. Tests for athrombogenic titanium nitride/gold coatings deposited on polymeric substrates (Bionate II) are performed. These tests include microshear, microtension and fatigue analysis. Anisotropic surface defects observed in the SEM micrographs require support for qualitative and quantitative interpretation. Improvement of qualitative analysis of scanning electron microscope images was achieved by a set of computational tools that includes binarization, simplified expanding, expanding, simple image statistic thresholding, the filters Laplacian 1, and Laplacian 2, Otsu and reverse binarization. Several modifications of the known image processing techniques and combinations of the selected image processing techniques were applied. The introduced quantitative analysis of digital scanning electron microscope images enables computation of stereological parameters such as area, crack angle, crack length, and total crack length per unit area. This study also compares the functionality of the developed computer program of digital image processing with existing applications. The described pre- and postprocessing may be helpful in scanning electron microscopy and transmission electron microscopy surface investigations. © 2016 The Authors Journal of Microscopy © 2016 Royal Microscopical Society.

Investigation of Local Ordering in Amorphous Materials.

NASA Astrophysics Data System (ADS)

Fan, Gary Guoyou

The intent of the research described in this dissertation, as indicated by the title, is to provide a better understanding of the structure of amorphous material. The possibility of using electron microscopy to study the amorphous structure is investigated. Chapter 1 gives a brief introduction to the understanding and modeling of the amorphous structure, electron microscopy and the image analysis in general. The difficulty of using 2-D images to infer 3-D structures information is illustrated in Chapter 2, where it is shown that some high resolution images are not qualitatively different from images of white -noises weak-phase objects or those of random atomic arrangements. The means of obtaining statistical information from these images is given in Chapters 3 and 5, where the quantitative differences between experimental images and simulated white-noise or simulated images corresponding to random arrangements are revealed. The use of image processing techniques in electron microscopy and the possible artifacts are presented in Chapter 4. The pattern recognition technique outlined in Chapter 6 demonstrates a feasible mode of scanning transition electron microscope operation. Statistical analysis can be effectively performed on a large number of nano-diffraction patterns from, for example, locally ordered samples. Some recent developments in physics as well as in electron microscopy are briefly reviewed, and their possible applications in the study of amorphous structures are discussed in Chapter 7.

(Machine-)Learning to analyze in vivo microscopy: Support vector machines.

PubMed

Wang, Michael F Z; Fernandez-Gonzalez, Rodrigo

2017-11-01

The development of new microscopy techniques for super-resolved, long-term monitoring of cellular and subcellular dynamics in living organisms is revealing new fundamental aspects of tissue development and repair. However, new microscopy approaches present several challenges. In addition to unprecedented requirements for data storage, the analysis of high resolution, time-lapse images is too complex to be done manually. Machine learning techniques are ideally suited for the (semi-)automated analysis of multidimensional image data. In particular, support vector machines (SVMs), have emerged as an efficient method to analyze microscopy images obtained from animals. Here, we discuss the use of SVMs to analyze in vivo microscopy data. We introduce the mathematical framework behind SVMs, and we describe the metrics used by SVMs and other machine learning approaches to classify image data. We discuss the influence of different SVM parameters in the context of an algorithm for cell segmentation and tracking. Finally, we describe how the application of SVMs has been critical to study protein localization in yeast screens, for lineage tracing in C. elegans, or to determine the developmental stage of Drosophila embryos to investigate gene expression dynamics. We propose that SVMs will become central tools in the analysis of the complex image data that novel microscopy modalities have made possible. This article is part of a Special Issue entitled: Biophysics in Canada, edited by Lewis Kay, John Baenziger, Albert Berghuis and Peter Tieleman. Copyright © 2017 Elsevier B.V. All rights reserved.

Analytical Model of the Nonlinear Dynamics of Cantilever Tip-Sample Surface Interactions for Various Acoustic-Atomic Force Microscopies

NASA Technical Reports Server (NTRS)

Cantrell, John H., Jr.; Cantrell, Sean A.

2008-01-01

A comprehensive analytical model of the interaction of the cantilever tip of the atomic force microscope (AFM) with the sample surface is developed that accounts for the nonlinearity of the tip-surface interaction force. The interaction is modeled as a nonlinear spring coupled at opposite ends to linear springs representing cantilever and sample surface oscillators. The model leads to a pair of coupled nonlinear differential equations that are solved analytically using a standard iteration procedure. Solutions are obtained for the phase and amplitude signals generated by various acoustic-atomic force microscope (A-AFM) techniques including force modulation microscopy, atomic force acoustic microscopy, ultrasonic force microscopy, heterodyne force microscopy, resonant difference-frequency atomic force ultrasonic microscopy (RDF-AFUM), and the commonly used intermittent contact mode (TappingMode) generally available on AFMs. The solutions are used to obtain a quantitative measure of image contrast resulting from variations in the Young modulus of the sample for the amplitude and phase images generated by the A-AFM techniques. Application of the model to RDF-AFUM and intermittent soft contact phase images of LaRC-cp2 polyimide polymer is discussed. The model predicts variations in the Young modulus of the material of 24 percent from the RDF-AFUM image and 18 percent from the intermittent soft contact image. Both predictions are in good agreement with the literature value of 21 percent obtained from independent, macroscopic measurements of sheet polymer material.

Out-of-focal plane imaging by leakage radiation microscopy

NASA Astrophysics Data System (ADS)

Zhu, Liangfu; Zhang, Douguo; Wang, Ruxue; Wen, Xiaolei; Wang, Pei; Ming, Hai; Badugu, Ramachandram; Lakowicz, Joseph R.

2017-09-01

Leakage radiation microscopy (LRM) is used to investigate the optical properties of surfaces. The front-focal plane (FFP) image with LRM reveals the structural features on the surfaces. A back-focal plane (BFP) image with LRM reveals the angular distribution of the radiation. Herein, we experimentally demonstrate that the out-of-focal plane (OFP) images present a link between the FFP and BFP images and provide optical information that cannot be resolved by either FFP or BFP images. The OFP image provides a link between the spatial location of the emission and the angular distribution from the same location, and thus information about the film’s discontinuity, nonuniformity or variable thickness can be uncovered. The use of OFP imaging will extend the scope and applications of the LRM and coupled emission imaging, which are powerful tools in nanophotonics and high throughput fluorescence screening.

Quantitative Image Restoration in Bright Field Optical Microscopy.

PubMed

Gutiérrez-Medina, Braulio; Sánchez Miranda, Manuel de Jesús

2017-11-07

Bright field (BF) optical microscopy is regarded as a poor method to observe unstained biological samples due to intrinsic low image contrast. We introduce quantitative image restoration in bright field (QRBF), a digital image processing method that restores out-of-focus BF images of unstained cells. Our procedure is based on deconvolution, using a point spread function modeled from theory. By comparing with reference images of bacteria observed in fluorescence, we show that QRBF faithfully recovers shape and enables quantify size of individual cells, even from a single input image. We applied QRBF in a high-throughput image cytometer to assess shape changes in Escherichia coli during hyperosmotic shock, finding size heterogeneity. We demonstrate that QRBF is also applicable to eukaryotic cells (yeast). Altogether, digital restoration emerges as a straightforward alternative to methods designed to generate contrast in BF imaging for quantitative analysis. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

A microwave-assisted solution combustion synthesis to produce europium-doped calcium phosphate nanowhiskers for bioimaging applications.

PubMed

Wagner, Darcy E; Eisenmann, Kathryn M; Nestor-Kalinoski, Andrea L; Bhaduri, Sarit B

2013-09-01

Biocompatible nanoparticles possessing fluorescent properties offer attractive possibilities for multifunctional bioimaging and/or drug and gene delivery applications. Many of the limitations with current imaging systems center on the properties of the optical probes in relation to equipment technical capabilities. Here we introduce a novel high aspect ratio and highly crystalline europium-doped calcium phosphate nanowhisker produced using a simple microwave-assisted solution combustion synthesis method for use as a multifunctional bioimaging probe. X-ray diffraction confirmed the material phase as europium-doped hydroxyapatite. Fluorescence emission and excitation spectra and their corresponding peaks were identified using spectrofluorimetry and validated with fluorescence, confocal and multiphoton microscopy. The nanowhiskers were found to exhibit red and far red wavelength fluorescence under ultraviolet excitation with an optimal peak emission of 696 nm achieved with a 350 nm excitation. Relatively narrow emission bands were observed, which may permit their use in multicolor imaging applications. Confocal and multiphoton microscopy confirmed that the nanoparticles provide sufficient intensity to be utilized in imaging applications. Copyright © 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Direct manipulation of metallic nanosheets by shear force microscopy.

PubMed

Bi, Z; Cai, W; Wang, Y; Shang, G

2018-05-15

Micro/nanomanipulation is a rapidly growing technology and holds promising applications in various fields, including photonic/electronic devices, chemical/biosensors etc. In this work, we present that shear force microscopy (ShFM) can be exploited to manipulate metallic nanosheets besides imaging. The manipulation is realized via controlling the shear force sensor probe position and shear force magnitude based on our homemade ShFM system under an optical microscopy for in situ observation. The main feature of the ShFM system is usage of a piezoelectric bimorph sensor, which has the ability of self-excitation and detection. Moreover, the shear force magnitude as a function of the spring constant of the sensor and setpoint is obtained, which indicates that operation modes can be switched between imaging and manipulation through designing the spring constant before experiment and changing the setpoint during manipulation process, respectively. We believe that this alternative manipulation technique could be used to assemble other nanostructures with different shapes, sizes and compositions for new properties and wider applications. © 2018 The Authors Journal of Microscopy © 2018 Royal Microscopical Society.

3D image restoration for confocal microscopy: toward a wavelet deconvolution for the study of complex biological structures

NASA Astrophysics Data System (ADS)

Boutet de Monvel, Jacques; Le Calvez, Sophie; Ulfendahl, Mats

2000-05-01

Image restoration algorithms provide efficient tools for recovering part of the information lost in the imaging process of a microscope. We describe recent progress in the application of deconvolution to confocal microscopy. The point spread function of a Biorad-MRC1024 confocal microscope was measured under various imaging conditions, and used to process 3D-confocal images acquired in an intact preparation of the inner ear developed at Karolinska Institutet. Using these experiments we investigate the application of denoising methods based on wavelet analysis as a natural regularization of the deconvolution process. Within the Bayesian approach to image restoration, we compare wavelet denoising with the use of a maximum entropy constraint as another natural regularization method. Numerical experiments performed with test images show a clear advantage of the wavelet denoising approach, allowing to `cool down' the image with respect to the signal, while suppressing much of the fine-scale artifacts appearing during deconvolution due to the presence of noise, incomplete knowledge of the point spread function, or undersampling problems. We further describe a natural development of this approach, which consists of performing the Bayesian inference directly in the wavelet domain.

Non-interferometric quantitative phase imaging of yeast cells

NASA Astrophysics Data System (ADS)

Poola, Praveen K.; Pandiyan, Vimal Prabhu; John, Renu

2015-12-01

Real-time imaging of live cells is quite difficult without the addition of external contrast agents. Various methods for quantitative phase imaging of living cells have been proposed like digital holographic microscopy and diffraction phase microscopy. In this paper, we report theoretical and experimental results of quantitative phase imaging of live yeast cells with nanometric precision using transport of intensity equations (TIE). We demonstrate nanometric depth sensitivity in imaging live yeast cells using this technique. This technique being noninterferometric, does not need any coherent light sources and images can be captured through a regular bright-field microscope. This real-time imaging technique would deliver the depth or 3-D volume information of cells and is highly promising in real-time digital pathology applications, screening of pathogens and staging of diseases like malaria as it does not need any preprocessing of samples.

Scanning tunneling microscopy and atomic force microscopy: application to biology and technology.

PubMed

Hansma, P K; Elings, V B; Marti, O; Bracker, C E

1988-10-14

The scanning tunneling microscope (STM) and the atomic force microscope (AFM) are scanning probe microscopes capable of resolving surface detail down to the atomic level. The potential of these microscopes for revealing subtle details of structure is illustrated by atomic resolution images including graphite, an organic conductor, an insulating layered compound, and individual adsorbed oxygen atoms on a semiconductor. Application of the STM for imaging biological materials directly has been hampered by the poor electron conductivity of most biological samples. The use of thin conductive metal coatings and replicas has made it possible to image some biological samples, as indicated by recently obtained images of a recA-DNA complex, a phospholipid bilayer, and an enzyme crystal. The potential of the AFM, which does not require a conductive sample, is shown with molecular resolution images of a nonconducting organic monolayer and an amino acid crystal that reveals individual methyl groups on the ends of the amino acids. Applications of these new microscopes to technology are demonstrated with images of an optical disk stamper, a diffraction grating, a thin-film magnetic recording head, and a diamond cutting tool. The STM has even been used to improve the quality of diffraction gratings and magnetic recording heads.

Re-scan confocal microscopy: scanning twice for better resolution.

PubMed

De Luca, Giulia M R; Breedijk, Ronald M P; Brandt, Rick A J; Zeelenberg, Christiaan H C; de Jong, Babette E; Timmermans, Wendy; Azar, Leila Nahidi; Hoebe, Ron A; Stallinga, Sjoerd; Manders, Erik M M

2013-01-01

We present a new super-resolution technique, Re-scan Confocal Microscopy (RCM), based on standard confocal microscopy extended with an optical (re-scanning) unit that projects the image directly on a CCD-camera. This new microscope has improved lateral resolution and strongly improved sensitivity while maintaining the sectioning capability of a standard confocal microscope. This simple technology is typically useful for biological applications where the combination high-resolution and high-sensitivity is required.

Light sheet microscopy.

PubMed

Weber, Michael; Mickoleit, Michaela; Huisken, Jan

2014-01-01

This chapter introduces the concept of light sheet microscopy along with practical advice on how to design and build such an instrument. Selective plane illumination microscopy is presented as an alternative to confocal microscopy due to several superior features such as high-speed full-frame acquisition, minimal phototoxicity, and multiview sample rotation. Based on our experience over the last 10 years, we summarize the key concepts in light sheet microscopy, typical implementations, and successful applications. In particular, sample mounting for long time-lapse imaging and the resulting challenges in data processing are discussed in detail. © 2014 Elsevier Inc. All rights reserved.

High-resolution photoluminescence electro-modulation microscopy by scanning lock-in

NASA Astrophysics Data System (ADS)

Koopman, W.; Muccini, M.; Toffanin, S.

2018-04-01

Morphological inhomogeneities and structural defects in organic semiconductors crucially determine the charge accumulation and lateral transport in organic thin-film transistors. Photoluminescence Electro-Modulation (PLEM) microscopy is a laser-scanning microscopy technique that relies on the modulation of the thin-film fluorescence in the presence of charge-carriers to image the spatial distribution of charges within the active organic semiconductor. Here, we present a lock-in scheme based on a scanning beam approach for increasing the PLEM microscopy resolution and contrast. The charge density in the device is modulated by a sinusoidal electrical signal, phase-locked to the scanning beam of the excitation laser. The lock-in detection scheme is achieved by acquiring a series of images with different phases between the beam scan and the electrical modulation. Application of high resolution PLEM to an organic transistor in accumulation mode demonstrates its potential to image local variations in the charge accumulation. A diffraction-limited precision of sub-300 nm and a signal to noise ratio of 21.4 dB could be achieved.

Under the Microscope: Single-Domain Antibodies for Live-Cell Imaging and Super-Resolution Microscopy.

PubMed

Traenkle, Bjoern; Rothbauer, Ulrich

2017-01-01

Single-domain antibodies (sdAbs) have substantially expanded the possibilities of advanced cellular imaging such as live-cell or super-resolution microscopy to visualize cellular antigens and their dynamics. In addition to their unique properties including small size, high stability, and solubility in many environments, sdAbs can be efficiently functionalized according to the needs of the respective imaging approach. Genetically encoded intrabodies fused to fluorescent proteins (chromobodies) have become versatile tools to study dynamics of endogenous proteins in living cells. Additionally, sdAbs conjugated to organic dyes were shown to label cellular structures with high density and minimal fluorophore displacement making them highly attractive probes for super-resolution microscopy. Here, we review recent advances of the chromobody technology to visualize localization and dynamics of cellular targets and the application of chromobody-based cell models for compound screening. Acknowledging the emerging importance of super-resolution microscopy in cell biology, we further discuss advantages and challenges of sdAbs for this technology.

Intravital imaging by simultaneous label-free autofluorescence-multiharmonic microscopy.

PubMed

You, Sixian; Tu, Haohua; Chaney, Eric J; Sun, Yi; Zhao, Youbo; Bower, Andrew J; Liu, Yuan-Zhi; Marjanovic, Marina; Sinha, Saurabh; Pu, Yang; Boppart, Stephen A

2018-05-29

Intravital microscopy (IVM) emerged and matured as a powerful tool for elucidating pathways in biological processes. Although label-free multiphoton IVM is attractive for its non-perturbative nature, its wide application has been hindered, mostly due to the limited contrast of each imaging modality and the challenge to integrate them. Here we introduce simultaneous label-free autofluorescence-multiharmonic (SLAM) microscopy, a single-excitation source nonlinear imaging platform that uses a custom-designed excitation window at 1110 nm and shaped ultrafast pulses at 10 MHz to enable fast (2-orders-of-magnitude improvement), simultaneous, and efficient acquisition of autofluorescence (FAD and NADH) and second/third harmonic generation from a wide array of cellular and extracellular components (e.g., tumor cells, immune cells, vesicles, and vessels) in living tissue using only 14 mW for extended time-lapse investigations. Our work demonstrates the versatility and efficiency of SLAM microscopy for tracking cellular events in vivo, and is a major enabling advance in label-free IVM.

Neurite density from magnetic resonance diffusion measurements at ultrahigh field: Comparison with light microscopy and electron microscopy

PubMed Central

Jespersen, Sune N.; Bjarkam, Carsten R.; Nyengaard, Jens R.; Chakravarty, M. Mallar; Hansen, Brian; Vosegaard, Thomas; Østergaard, Leif; Yablonskiy, Dmitriy; Nielsen, Niels Chr.; Vestergaard-Poulsen, Peter

2010-01-01

Due to its unique sensitivity to tissue microstructure, diffusion-weighted magnetic resonance imaging (MRI) has found many applications in clinical and fundamental science. With few exceptions, a more precise correspondence between physiological or biophysical properties and the obtained diffusion parameters remain uncertain due to lack of specificity. In this work, we address this problem by comparing diffusion parameters of a recently introduced model for water diffusion in brain matter to light microscopy and quantitative electron microscopy. Specifically, we compare diffusion model predictions of neurite density in rats to optical myelin staining intensity and stereological estimation of neurite volume fraction using electron microscopy. We find that the diffusion model describes data better and that its parameters show stronger correlation with optical and electron microscopy, and thus reflect myelinated neurite density better than the more frequently used diffusion tensor imaging (DTI) and cumulant expansion methods. Furthermore, the estimated neurite orientations capture dendritic architecture more faithfully than DTI diffusion ellipsoids. PMID:19732836

Bleed-through correction for rendering and correlation analysis in multi-colour localization microscopy

PubMed Central

Kim, Dahan; Curthoys, Nikki M.; Parent, Matthew T.; Hess, Samuel T.

2015-01-01

Multi-colour localization microscopy has enabled sub-diffraction studies of colocalization between multiple biological species and quantification of their correlation at length scales previously inaccessible with conventional fluorescence microscopy. However, bleed-through, or misidentification of probe species, creates false colocalization and artificially increases certain types of correlation between two imaged species, affecting the reliability of information provided by colocalization and quantified correlation. Despite the potential risk of these artefacts of bleed-through, neither the effect of bleed-through on correlation nor methods of its correction in correlation analyses has been systematically studied at typical rates of bleed-through reported to affect multi-colour imaging. Here, we present a reliable method of bleed-through correction applicable to image rendering and correlation analysis of multi-colour localization microscopy. Application of our bleed-through correction shows our method accurately corrects the artificial increase in both types of correlations studied (Pearson coefficient and pair correlation), at all rates of bleed-through tested, in all types of correlations examined. In particular, anti-correlation could not be quantified without our bleed-through correction, even at rates of bleed-through as low as 2%. Demonstrated with dichroic-based multi-colour FPALM here, our presented method of bleed-through correction can be applied to all types of localization microscopy (PALM, STORM, dSTORM, GSDIM, etc.), including both simultaneous and sequential multi-colour modalities, provided the rate of bleed-through can be reliably determined. PMID:26185614

Bleed-through correction for rendering and correlation analysis in multi-colour localization microscopy.

PubMed

Kim, Dahan; Curthoys, Nikki M; Parent, Matthew T; Hess, Samuel T

2013-09-01

Multi-colour localization microscopy has enabled sub-diffraction studies of colocalization between multiple biological species and quantification of their correlation at length scales previously inaccessible with conventional fluorescence microscopy. However, bleed-through, or misidentification of probe species, creates false colocalization and artificially increases certain types of correlation between two imaged species, affecting the reliability of information provided by colocalization and quantified correlation. Despite the potential risk of these artefacts of bleed-through, neither the effect of bleed-through on correlation nor methods of its correction in correlation analyses has been systematically studied at typical rates of bleed-through reported to affect multi-colour imaging. Here, we present a reliable method of bleed-through correction applicable to image rendering and correlation analysis of multi-colour localization microscopy. Application of our bleed-through correction shows our method accurately corrects the artificial increase in both types of correlations studied (Pearson coefficient and pair correlation), at all rates of bleed-through tested, in all types of correlations examined. In particular, anti-correlation could not be quantified without our bleed-through correction, even at rates of bleed-through as low as 2%. Demonstrated with dichroic-based multi-colour FPALM here, our presented method of bleed-through correction can be applied to all types of localization microscopy (PALM, STORM, dSTORM, GSDIM, etc.), including both simultaneous and sequential multi-colour modalities, provided the rate of bleed-through can be reliably determined.

Sensitivity and Specificity of Cardiac Tissue Discrimination Using Fiber-Optics Confocal Microscopy.

PubMed

Huang, Chao; Sachse, Frank B; Hitchcock, Robert W; Kaza, Aditya K

2016-01-01

Disturbances of the cardiac conduction system constitute a major risk after surgical repair of complex cases of congenital heart disease. Intraoperative identification of the conduction system may reduce the incidence of these disturbances. We previously developed an approach to identify cardiac tissue types using fiber-optics confocal microscopy and extracellular fluorophores. Here, we applied this approach to investigate sensitivity and specificity of human and automated classification in discriminating images of atrial working myocardium and specialized tissue of the conduction system. Two-dimensional image sequences from atrial working myocardium and nodal tissue of isolated perfused rodent hearts were acquired using a fiber-optics confocal microscope (Leica FCM1000). We compared two methods for local application of extracellular fluorophores: topical via pipette and with a dye carrier. Eight blinded examiners evaluated 162 randomly selected images of atrial working myocardium (n = 81) and nodal tissue (n = 81). In addition, we evaluated the images using automated classification. Blinded examiners achieved a sensitivity and specificity of 99.2 ± 0.3% and 98.0 ± 0.7%, respectively, with the dye carrier method of dye application. Sensitivity and specificity was similar for dye application via a pipette (99.2 ± 0.3% and 94.0 ± 2.4%, respectively). Sensitivity and specificity for automated methods of tissue discrimination were similarly high. Human and automated classification achieved high sensitivity and specificity in discriminating atrial working myocardium and nodal tissue. We suggest that our findings facilitate clinical translation of fiber-optics confocal microscopy as an intraoperative imaging modality to reduce the incidence of conduction disturbances during surgical correction of congenital heart disease.

Influence of sample preparation and reliability of automated numerical refocusing in stain-free analysis of dissected tissues with quantitative phase digital holographic microscopy

NASA Astrophysics Data System (ADS)

Kemper, Björn; Lenz, Philipp; Bettenworth, Dominik; Krausewitz, Philipp; Domagk, Dirk; Ketelhut, Steffi

2015-05-01

Digital holographic microscopy (DHM) has been demonstrated to be a versatile tool for high resolution non-destructive quantitative phase imaging of surfaces and multi-modal minimally-invasive monitoring of living cell cultures in-vitro. DHM provides quantitative monitoring of physiological processes through functional imaging and structural analysis which, for example, gives new insight into signalling of cellular water permeability and cell morphology changes due to toxins and infections. Also the analysis of dissected tissues quantitative DHM phase contrast prospects application fields by stain-free imaging and the quantification of tissue density changes. We show that DHM allows imaging of different tissue layers with high contrast in unstained tissue sections. As the investigation of fixed samples represents a very important application field in pathology, we also analyzed the influence of the sample preparation. The retrieved data demonstrate that the quality of quantitative DHM phase images of dissected tissues depends strongly on the fixing method and common staining agents. As in DHM the reconstruction is performed numerically, multi-focus imaging is achieved from a single digital hologram. Thus, we evaluated the automated refocussing feature of DHM for application on different types of dissected tissues and revealed that on moderately stained samples highly reproducible holographic autofocussing can be achieved. Finally, it is demonstrated that alterations of the spatial refractive index distribution in murine and human tissue samples represent a reliable absolute parameter that is related of different degrees of inflammation in experimental colitis and Crohn's disease. This paves the way towards the usage of DHM in digital pathology for automated histological examinations and further studies to elucidate the translational potential of quantitative phase microscopy for the clinical management of patients, e.g., with inflammatory bowel disease.

Atomic force microscopic imaging of Acanthamoeba castellanii and Balamuthia mandrillaris trophozoites and cysts.

PubMed

Aqeel, Yousuf; Siddiqui, Ruqaiyyah; Ateeq, Muhammad; Raza Shah, Muhammad; Kulsoom, Huma; Khan, Naveed Ahmed

2015-01-01

Light microscopy and electron microscopy have been successfully used in the study of microbes, as well as free-living protists. Unlike light microscopy, which enables us to observe living organisms or the electron microscope which provides a two-dimensional image, atomic force microscopy provides a three-dimensional surface profile. Here, we observed two free-living amoebae, Acanthamoeba castellanii and Balamuthia mandrillaris under the phase contrast inverted microscope, transmission electron microscope and atomic force microscope. Although light microscopy was of lower magnification, it revealed functional biology of live amoebae such as motility and osmoregulation using contractile vacuoles of the trophozoite stage, but it is of limited value in defining the cyst stage. In contrast, transmission electron microscopy showed significantly greater magnification and resolution to reveal the ultra-structural features of trophozoites and cysts including intracellular organelles and cyst wall characteristics but it only produced a snapshot in time of a dead amoeba cell. Atomic force microscopy produced three-dimensional images providing detailed topographic description of shape and surface, phase imaging measuring boundary stiffness, and amplitude measurements including width, height and length of A. castellanii and B. mandrillaris trophozoites and cysts. These results demonstrate the importance of the application of various microscopic methods in the biological and structural characterization of the whole cell, ultra-structural features, as well as surface components and cytoskeleton of protist pathogens. © 2014 The Author(s) Journal of Eukaryotic Microbiology © 2014 International Society of Protistologists.

Atomic force microscopy of atomic-scale ledges and etch pits formed during dissolution of quartz

NASA Technical Reports Server (NTRS)

Gratz, A. J.; Manne, S.; Hansma, P. K.

1991-01-01

The processes involved in the dissolution and growth of crystals are closely related. Atomic force microscopy (AFM) of faceted pits (called negative crystals) formed during quartz dissolution reveals subtle details of these underlying physical mechanisms for silicates. In imaging these surfaces, the AFM detected ledges less than 1 nm high that were spaced 10 to 90 nm apart. A dislocation pit, invisible to optical and scanning electron microscopy measurements and serving as a ledge source, was also imaged. These observations confirm the applicability of ledge-motion models to dissolution and growth of silicates; coupled with measurements of dissolution rate on facets, these methods provide a powerful tool for probing mineral surface kinetics.

Flipping interferometry and its application for quantitative phase microscopy in a micro-channel.

PubMed

Roitshtain, Darina; Turko, Nir A; Javidi, Bahram; Shaked, Natan T

2016-05-15

We present a portable, off-axis interferometric module for quantitative phase microscopy of live cells, positioned at the exit port of a coherently illuminated inverted microscope. The module creates on the digital camera an interference pattern between the image of the sample and its flipped version. The proposed simplified module is based on a retro-reflector modification in an external Michelson interferometer. The module does not contain any lenses, pinholes, or gratings and its alignment is straightforward. Still, it allows full control of the off-axis angle and does not suffer from ghost images. As experimentally demonstrated, the module is useful for quantitative phase microscopy of live cells rapidly flowing in a micro-channel.

Active voltage contrast imaging of cross-sectional surface of multilayer ceramic capacitor using helium ion microscopy

NASA Astrophysics Data System (ADS)

Sakai, C.; Ishida, N.; Masuda, H.; Nagano, S.; Kitahara, M.; Ogata, Y.; Fujita, D.

2016-08-01

We studied active voltage contrast (AVC) imaging using helium ion microscopy (HIM). We observed secondary electron (SE) images of the cross-sectional surface of multilayer ceramic capacitors (MLCCs) with and without a voltage applied to the internal electrodes. When no voltage was applied, we obtained an image reflecting the material contrast between the Ni internal electrode region and the BaTiO3 dielectric region of the cross-sectional surface of the MLCC. When a voltage was applied, the electrical potential difference between the grounded and the positively biased internal electrodes affected the contrast (voltage contrast). Moreover, attenuation of the SE intensity from the grounded to the positively biased internal electrodes was observed in the dielectric region. Kelvin probe force microscopy (KPFM) measurements of the contact potential difference (CPD) were performed on the same sample. By using the AVC image from the HIM observation and the CPD image from the KPFM measurement, we could quantitatively evaluate the electrical potential. We think that the results of this study will lead to an expansion in the number of applications of HIM.

Toward giga-pixel nanoscopy on a chip: a computational wide-field look at the nano-scale without the use of lenses

PubMed Central

McLeod, Euan; Luo, Wei; Mudanyali, Onur; Greenbaum, Alon

2013-01-01

The development of lensfree on-chip microscopy in the past decade has opened up various new possibilities for biomedical imaging across ultra-large fields of view using compact, portable, and cost-effective devices. However, until recently, its ability to resolve fine features and detect ultra-small particles has not rivalled the capabilities of the more expensive and bulky laboratory-grade optical microscopes. In this Frontier Review, we highlight the developments over the last two years that have enabled computational lensfree holographic on-chip microscopy to compete with and, in some cases, surpass conventional bright-field microscopy in its ability to image nano-scale objects across large fields of view, yielding giga-pixel phase and amplitude images. Lensfree microscopy has now achieved a numerical aperture as high as 0.92, with a spatial resolution as small as 225 nm across a large field of view e.g., >20 mm2. Furthermore, the combination of lensfree microscopy with self-assembled nanolenses, forming nano-catenoid minimal surfaces around individual nanoparticles has boosted the image contrast to levels high enough to permit bright-field imaging of individual particles smaller than 100 nm. These capabilities support a number of new applications, including, for example, the detection and sizing of individual virus particles using field-portable computational on-chip microscopes. PMID:23592185

Inhomogeneity Based Characterization of Distribution Patterns on the Plasma Membrane

PubMed Central

Paparelli, Laura; Corthout, Nikky; Wakefield, Devin L.; Sannerud, Ragna; Jovanovic-Talisman, Tijana; Annaert, Wim; Munck, Sebastian

2016-01-01

Cell surface protein and lipid molecules are organized in various patterns: randomly, along gradients, or clustered when segregated into discrete micro- and nano-domains. Their distribution is tightly coupled to events such as polarization, endocytosis, and intracellular signaling, but challenging to quantify using traditional techniques. Here we present a novel approach to quantify the distribution of plasma membrane proteins and lipids. This approach describes spatial patterns in degrees of inhomogeneity and incorporates an intensity-based correction to analyze images with a wide range of resolutions; we have termed it Quantitative Analysis of the Spatial distributions in Images using Mosaic segmentation and Dual parameter Optimization in Histograms (QuASIMoDOH). We tested its applicability using simulated microscopy images and images acquired by widefield microscopy, total internal reflection microscopy, structured illumination microscopy, and photoactivated localization microscopy. We validated QuASIMoDOH, successfully quantifying the distribution of protein and lipid molecules detected with several labeling techniques, in different cell model systems. We also used this method to characterize the reorganization of cell surface lipids in response to disrupted endosomal trafficking and to detect dynamic changes in the global and local organization of epidermal growth factor receptors across the cell surface. Our findings demonstrate that QuASIMoDOH can be used to assess protein and lipid patterns, quantifying distribution changes and spatial reorganization at the cell surface. An ImageJ/Fiji plugin of this analysis tool is provided. PMID:27603951

Toward giga-pixel nanoscopy on a chip: a computational wide-field look at the nano-scale without the use of lenses.

PubMed

McLeod, Euan; Luo, Wei; Mudanyali, Onur; Greenbaum, Alon; Ozcan, Aydogan

2013-06-07

The development of lensfree on-chip microscopy in the past decade has opened up various new possibilities for biomedical imaging across ultra-large fields of view using compact, portable, and cost-effective devices. However, until recently, its ability to resolve fine features and detect ultra-small particles has not rivalled the capabilities of the more expensive and bulky laboratory-grade optical microscopes. In this Frontier Review, we highlight the developments over the last two years that have enabled computational lensfree holographic on-chip microscopy to compete with and, in some cases, surpass conventional bright-field microscopy in its ability to image nano-scale objects across large fields of view, yielding giga-pixel phase and amplitude images. Lensfree microscopy has now achieved a numerical aperture as high as 0.92, with a spatial resolution as small as 225 nm across a large field of view e.g., >20 mm(2). Furthermore, the combination of lensfree microscopy with self-assembled nanolenses, forming nano-catenoid minimal surfaces around individual nanoparticles has boosted the image contrast to levels high enough to permit bright-field imaging of individual particles smaller than 100 nm. These capabilities support a number of new applications, including, for example, the detection and sizing of individual virus particles using field-portable computational on-chip microscopes.

Confocal Microscopy and Molecular-Specific Optical Contrast Agents for the Detection of Oral Neoplasia

PubMed Central

Carlson, Alicia L.; Gillenwater, Ann M.; Williams, Michelle D.; El-Naggar, Adel K.; Richards-Kortum, R. R.

2009-01-01

Using current clinical diagnostic techniques, it is difficult to visualize tumor morphology and architecture at the cellular level, which is necessary for diagnostic localization of pathologic lesions. Optical imaging techniques have the potential to address this clinical need by providing real-time, sub-cellular resolution images. This paper describes the use of dual mode confocal microscopy and optical molecular-specific contrast agents to image tissue architecture, cellular morphology, and sub-cellular molecular features of normal and neoplastic oral tissues. Fresh tissue slices were prepared from 33 biopsies of clinically normal and abnormal oral mucosa obtained from 14 patients. Reflectance confocal images were acquired after the application of 6% acetic acid, and fluorescence confocal images were acquired after the application of a fluorescence contrast agent targeting the epidermal growth factor receptor (EGFR). The dual imaging modes provided images similar to light microscopy of hematoxylin and eosin and immunohistochemistry staining, but from thick fresh tissue slices. Reflectance images provided information on the architecture of the tissue and the cellular morphology. The nuclear-to-cytoplasmic (N/C) ratio from the reflectance images was at least 7.5 times greater for the carcinoma than the corresponding normal samples, except for one case of highly keratinized carcinoma. Separation of carcinoma from normal and mild dysplasia was achieved using this ratio (p<0.01). Fluorescence images of EGFR expression yielded a mean fluorescence labeling intensity (FLI) that was at least 2.7 times higher for severe dysplasia and carcinoma samples than for the corresponding normal sample, and could be used to distinguish carcinoma from normal and mild dysplasia (p<0.01). Analyzed together, the N/C ratio and the mean FLI may improve the ability to distinguish carcinoma from normal squamous epithelium. PMID:17877424

Improved localization of cellular membrane receptors using combined fluorescence microscopy and simultaneous topography and recognition imaging

NASA Astrophysics Data System (ADS)

Duman, M.; Pfleger, M.; Zhu, R.; Rankl, C.; Chtcheglova, L. A.; Neundlinger, I.; Bozna, B. L.; Mayer, B.; Salio, M.; Shepherd, D.; Polzella, P.; Moertelmaier, M.; Kada, G.; Ebner, A.; Dieudonne, M.; Schütz, G. J.; Cerundolo, V.; Kienberger, F.; Hinterdorfer, P.

2010-03-01

The combination of fluorescence microscopy and atomic force microscopy has a great potential in single-molecule-detection applications, overcoming many of the limitations coming from each individual technique. Here we present a new platform of combined fluorescence and simultaneous topography and recognition imaging (TREC) for improved localization of cellular receptors. Green fluorescent protein (GFP) labeled human sodium-glucose cotransporter (hSGLT1) expressed Chinese Hamster Ovary (CHO) cells and endothelial cells (MyEnd) from mouse myocardium stained with phalloidin-rhodamine were used as cell systems to study AFM topography and fluorescence microscopy on the same surface area. Topographical AFM images revealed membrane features such as lamellipodia, cytoskeleton fibers, F-actin filaments and small globular structures with heights ranging from 20 to 30 nm. Combined fluorescence and TREC imaging was applied to detect density, distribution and localization of YFP-labeled CD1d molecules on α-galactosylceramide (αGalCer)-loaded THP1 cells. While the expression level, distribution and localization of CD1d molecules on THP1 cells were detected with fluorescence microscopy, the nanoscale distribution of binding sites was investigated with molecular recognition imaging by using a chemically modified AFM tip. Using TREC on the inverted light microscope, the recognition sites of cell receptors were detected in recognition images with domain sizes ranging from ~ 25 to ~ 160 nm, with the smaller domains corresponding to a single CD1d molecule.

Improved localization of cellular membrane receptors using combined fluorescence microscopy and simultaneous topography and recognition imaging.

PubMed

Duman, M; Pfleger, M; Zhu, R; Rankl, C; Chtcheglova, L A; Neundlinger, I; Bozna, B L; Mayer, B; Salio, M; Shepherd, D; Polzella, P; Moertelmaier, M; Kada, G; Ebner, A; Dieudonne, M; Schütz, G J; Cerundolo, V; Kienberger, F; Hinterdorfer, P

2010-03-19

The combination of fluorescence microscopy and atomic force microscopy has a great potential in single-molecule-detection applications, overcoming many of the limitations coming from each individual technique. Here we present a new platform of combined fluorescence and simultaneous topography and recognition imaging (TREC) for improved localization of cellular receptors. Green fluorescent protein (GFP) labeled human sodium-glucose cotransporter (hSGLT1) expressed Chinese Hamster Ovary (CHO) cells and endothelial cells (MyEnd) from mouse myocardium stained with phalloidin-rhodamine were used as cell systems to study AFM topography and fluorescence microscopy on the same surface area. Topographical AFM images revealed membrane features such as lamellipodia, cytoskeleton fibers, F-actin filaments and small globular structures with heights ranging from 20 to 30 nm. Combined fluorescence and TREC imaging was applied to detect density, distribution and localization of YFP-labeled CD1d molecules on alpha-galactosylceramide (alphaGalCer)-loaded THP1 cells. While the expression level, distribution and localization of CD1d molecules on THP1 cells were detected with fluorescence microscopy, the nanoscale distribution of binding sites was investigated with molecular recognition imaging by using a chemically modified AFM tip. Using TREC on the inverted light microscope, the recognition sites of cell receptors were detected in recognition images with domain sizes ranging from approximately 25 to approximately 160 nm, with the smaller domains corresponding to a single CD1d molecule.

PREFACE: Ultrafast biophotonics Ultrafast biophotonics

NASA Astrophysics Data System (ADS)

Gu, Min; Reid, Derryck; Ben-Yakar, Adela

2010-08-01

The use of light to explore biology can be traced to the first observations of tissue made with early microscopes in the mid-seventeenth century, and has today evolved into the discipline which we now know as biophotonics. This field encompasses a diverse range of activities, each of which shares the common theme of exploiting the interaction of light with biological material. With the rapid advancement of ultrafast optical technologies over the last few decades, ultrafast lasers have increasingly found applications in biophotonics, to the extent that the distinctive new field of ultrafast biophotonics has now emerged, where robust turnkey ultrafast laser systems are facilitating cutting-edge studies in the life sciences to take place in everyday laboratories. The broad spectral bandwidths, precision timing resolution, low coherence and high peak powers of ultrafast optical pulses provide unique opportunities for imaging and manipulating biological systems. Time-resolved studies of bio-molecular dynamics exploit the short pulse durations from such lasers, while other applications such as optical coherence tomography benefit from the broad optical bandwidths possible by using super-continuum generation and additionally allowing for high speed imaging with speeds as high as 47 000 scans per second. Continuing progress in laser-system technology is accelerating the adoption of ultrafast techniques across the life sciences, both in research laboratories and in clinical applications, such as laser-assisted in situ keratomileusis (LASIK) eye surgery. Revolutionizing the field of optical microscopy, two-photon excitation fluorescence (TPEF) microscopy has enabled higher spatial resolution with improved depth penetration into biological specimens. Advantages of this nonlinear optical process include: reduced photo-interactions, allowing for extensive imaging time periods; simultaneously exciting multiple fluorescent molecules with only one excitation wavelength; and reduced chromatic aberration effects. These extensive advantages have led to further exploration of nonlinear processes including second-harmonic generation (SHG) microscopy and third-harmonic generation (THG) microscopy. Second-harmonic generation has provided biologists with an extremely powerful tool for generating contrast in biological imaging, with the additional benefit of non-invasive three-dimensional imaging. The recent popularity of THG microscopy is largely due to the fact that three-dimensional imaging is achievable without the need for any labels, but rather relying on the intrinsic properties of the biological specimen itself. This optical nonlinear technique has attracted much attention recently from the biological community due to its non-invasive capabilities. Users of ultrafast lasers in the biological and medical fields are becoming a fast-growing community, employing pulse-shaping microscopy, resolution-enhancing microscopy techniques, linear and nonlinear micro-spectroscopy, functional deep-tissue imaging, optical coherence tomography, nonlinear fluorescence microscopy, molecular imaging and control, harmonic microscopy and femtosecond lifetime imaging, for cutting-edge research concerning the interaction of light with biological dynamics. The adaptability of ultrafast lasers to interact with a large array of materials through nonlinear excitation has enabled precise control of laser fluence allowing for highly localized material interactions, permitting micro-structured fabricated surfaces. The resultant multi-dimensional fabricated micro-structures are capable of replicating and/or manipulating microenvironments for controlled cell biology. In this special issue of Journal of Optics readers have a chance to view a collection of new contributions to the growing research field of ultrafast biophotonics. They are presented with recent advances in ultrafast technology applied to biological and medical investigations, where topics include advances in the visualization and identification of photo-reaction dynamics of biological functions under relevant physiological conditions, theoretically proposed imaging designs for obtaining super-resolved optical sectioned images in single exposures and fabricated micro-structured surfaces for biological micro-environments. We hope the collection will stimulate innovative new research in this growing field by showcasing new techniques for the visualization and manipulation of complex biological systems using linear and and nonlinear optical processes. Professor Min Gu would like to acknowledge Dr Betty Kouskousis for her contribution and support towards this editorial.

DotLens smartphone microscopy for biological and biomedical applications (Conference Presentation)

NASA Astrophysics Data System (ADS)

Sung, Yu-Lung; Zhao, Fusheng; Shih, Wei-Chuan

2017-02-01

Recent advances in inkjet-printed optics have created a new class of lens fabrication technique. Lenses with a tunable geometry, magnification, and focal length can be fabricated by dispensing controlled amounts of liquid polymer onto a heated surface. This fabrication technique is highly cost-effective, and can achieve optically smooth surface finish. Dubbed DotLens, a single of which weighs less than 50 mg and occupies a volume less than 50 μL. DotLens can be attached onto any smartphone camera akin to a contact lens, and enable smartphones to obtain image resolution as fine as 1 µm. The surface curvature modifies the optical path of light to the image sensor, and enables the camera to focus as close as 2 mm. This enables microscopic imaging on a smartphone without any additional attachments, and has shown great potential in mobile point-of-care diagnostic systems, particularly for histology of tissue sections and cytology of blood cells. DotLens Smartphone Microscopy represents an innovative approach fundamentally different from other smartphone microscopes. In this paper, we describe the application and performance of DotLens smartphone microscopy in biological and biomedical research. In particular, we show recent results from images collected from pathology tissue slides with cancer features. In addition, we show performance in cytological analysis of blood smear. This tool has empowered Citizen Science investigators to collect microscopic images from various interesting objects.

A Microfluidic Platform for Correlative Live-Cell and Super-Resolution Microscopy

PubMed Central

Tam, Johnny; Cordier, Guillaume Alan; Bálint, Štefan; Sandoval Álvarez, Ángel; Borbely, Joseph Steven; Lakadamyali, Melike

2014-01-01

Recently, super-resolution microscopy methods such as stochastic optical reconstruction microscopy (STORM) have enabled visualization of subcellular structures below the optical resolution limit. Due to the poor temporal resolution, however, these methods have mostly been used to image fixed cells or dynamic processes that evolve on slow time-scales. In particular, fast dynamic processes and their relationship to the underlying ultrastructure or nanoscale protein organization cannot be discerned. To overcome this limitation, we have recently developed a correlative and sequential imaging method that combines live-cell and super-resolution microscopy. This approach adds dynamic background to ultrastructural images providing a new dimension to the interpretation of super-resolution data. However, currently, it suffers from the need to carry out tedious steps of sample preparation manually. To alleviate this problem, we implemented a simple and versatile microfluidic platform that streamlines the sample preparation steps in between live-cell and super-resolution imaging. The platform is based on a microfluidic chip with parallel, miniaturized imaging chambers and an automated fluid-injection device, which delivers a precise amount of a specified reagent to the selected imaging chamber at a specific time within the experiment. We demonstrate that this system can be used for live-cell imaging, automated fixation, and immunostaining of adherent mammalian cells in situ followed by STORM imaging. We further demonstrate an application by correlating mitochondrial dynamics, morphology, and nanoscale mitochondrial protein distribution in live and super-resolution images. PMID:25545548

In vivo imaging of neural activity

PubMed Central

Yang, Weijian; Yuste, Rafael

2017-01-01

Since the introduction of calcium imaging to monitor neuronal activity with single-cell resolution, optical imaging methods have revolutionized neuroscience by enabling systematic recordings of neuronal circuits in living animals. The plethora of methods for functional neural imaging can be daunting to the nonexpert to navigate. Here we review advanced microscopy techniques for in vivo functional imaging and offer guidelines for which technologies are best suited for particular applications. PMID:28362436

Laser interference microscopy: a novel approach to the visualization of structural changes in myelin during the propagation of nerve impulses

NASA Astrophysics Data System (ADS)

Yusipovich, A. I.; Cherkashin, A. A.; Verdiyan, E. E.; Sogomonyan, I. A.; Maksimov, G. V.

2016-08-01

We used 3D phase images obtained by laser interference microscopy (LIM) for ex vivo evaluation of changes in the structure of myelin during repetitive stimulation. In this work we propose a simple model of myelinated nerve fiber (mNF), which describes phase images as a result of different geometry and membrane-to-cytoplasm ratio in various regions, particularly, the internode and paranodal-nodal-paranodal region, including the node of Ranvier. Application of this model provides clear interpretation of the phase images and also demonstrates that repetitive action potentials are accompanied by structural changes in myelin in the internode and cytoplasmic modification in the node of Ranvier. The first 20 min of stimulation did not induce significant changes in the measured parameters, but then the optical path difference at the periphery of mNF and at the node of Ranvier declined reversibly. We believe that our model is also applicable to other modifications of interference and non-interference imaging.

Nonlinear plasmonic imaging techniques and their biological applications

NASA Astrophysics Data System (ADS)

Deka, Gitanjal; Sun, Chi-Kuang; Fujita, Katsumasa; Chu, Shi-Wei

2017-01-01

Nonlinear optics, when combined with microscopy, is known to provide advantages including novel contrast, deep tissue observation, and minimal invasiveness. In addition, special nonlinearities, such as switch on/off and saturation, can enhance the spatial resolution below the diffraction limit, revolutionizing the field of optical microscopy. These nonlinear imaging techniques are extremely useful for biological studies on various scales from molecules to cells to tissues. Nevertheless, in most cases, nonlinear optical interaction requires strong illumination, typically at least gigawatts per square centimeter intensity. Such strong illumination can cause significant phototoxicity or even photodamage to fragile biological samples. Therefore, it is highly desirable to find mechanisms that allow the reduction of illumination intensity. Surface plasmon, which is the collective oscillation of electrons in metal under light excitation, is capable of significantly enhancing the local field around the metal nanostructures and thus boosting up the efficiency of nonlinear optical interactions of the surrounding materials or of the metal itself. In this mini-review, we discuss the recent progress of plasmonics in nonlinear optical microscopy with a special focus on biological applications. The advancement of nonlinear imaging modalities (including incoherent/coherent Raman scattering, two/three-photon luminescence, and second/third harmonic generations that have been amalgamated with plasmonics), as well as the novel subdiffraction limit imaging techniques based on nonlinear behaviors of plasmonic scattering, is addressed.

Diverse protocols for correlative super-resolution fluorescence imaging and electron microscopy of chemically fixed samples

PubMed Central

Kopek, Benjamin G.; Paez-Segala, Maria G.; Shtengel, Gleb; Sochacki, Kem A.; Sun, Mei G.; Wang, Yalin; Xu, C. Shan; van Engelenburg, Schuyler B.; Taraska, Justin W.; Looger, Loren L.; Hess, Harald F.

2017-01-01

Our groups have recently developed related approaches for sample preparation for super-resolution imaging within endogenous cellular environments using correlative light and electron microscopy (CLEM). Four distinct techniques for preparing and acquiring super-resolution CLEM datasets on aldehyde-fixed specimens are provided, including Tokuyasu cryosectioning, whole-cell mount, cell unroofing and platinum replication, and resin embedding and sectioning. Choice of the best protocol for a given application depends on a number of criteria that are discussed in detail. Tokuyasu cryosectioning is relatively rapid but is limited to small, delicate specimens. Whole-cell mount has the simplest sample preparation but is restricted to surface structures. Cell unroofing and platinum replica creates high-contrast, 3-dimensional images of the cytoplasmic surface of the plasma membrane, but is more challenging than whole-cell mount. Resin embedding permits serial sectioning of large samples, but is limited to osmium-resistant probes, and is technically difficult. Expected results from these protocols include super-resolution localization (~10–50 nm) of fluorescent targets within the context of electron microscopy ultrastructure, which can help address cell biological questions. These protocols can be completed in 2–7 days, are compatible with a number of super-resolution imaging protocols, and are broadly applicable across biology. PMID:28384138

Spatial Resolution in Scanning Electron Microscopy and Scanning Transmission Electron Microscopy Without a Specimen Vacuum Chamber.

PubMed

Nguyen, Kayla X; Holtz, Megan E; Richmond-Decker, Justin; Muller, David A

2016-08-01

A long-standing goal of electron microscopy has been the high-resolution characterization of specimens in their native environment. However, electron optics require high vacuum to maintain an unscattered and focused probe, a challenge for specimens requiring atmospheric or liquid environments. Here, we use an electron-transparent window at the base of a scanning electron microscope's objective lens to separate column vacuum from the specimen, enabling imaging under ambient conditions, without a specimen vacuum chamber. We demonstrate in-air imaging of specimens at nanoscale resolution using backscattered scanning electron microscopy (airSEM) and scanning transmission electron microscopy. We explore resolution and contrast using Monte Carlo simulations and analytical models. We find that nanometer-scale resolution can be obtained at gas path lengths up to 400 μm, although contrast drops with increasing gas path length. As the electron-transparent window scatters considerably more than gas at our operating conditions, we observe that the densities and thicknesses of the electron-transparent window are the dominant limiting factors for image contrast at lower operating voltages. By enabling a variety of detector configurations, the airSEM is applicable to a wide range of environmental experiments including the imaging of hydrated biological specimens and in situ chemical and electrochemical processes.

Spatial Resolution in Scanning Electron Microscopy and Scanning Transmission Electron Microscopy Without a Specimen Vacuum Chamber

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nguyen, Kayla X.; Holtz, Megan E.; Richmond-Decker, Justin

2016-07-25

Abstract A long-standing goal of electron microscopy has been the high-resolution characterization of specimens in their native environment. However, electron optics require high vacuum to maintain an unscattered and focused probe, a challenge for specimens requiring atmospheric or liquid environments. Here, we use an electron-transparent window at the base of a scanning electron microscope’s objective lens to separate column vacuum from the specimen, enabling imaging under ambient conditions, without a specimen vacuum chamber. We demonstrate in-air imaging of specimens at nanoscale resolution using backscattered scanning electron microscopy (airSEM) and scanning transmission electron microscopy. We explore resolution and contrast using Montemore » Carlo simulations and analytical models. We find that nanometer-scale resolution can be obtained at gas path lengths up to 400μm, although contrast drops with increasing gas path length. As the electron-transparent window scatters considerably more than gas at our operating conditions, we observe that the densities and thicknesses of the electron-transparent window are the dominant limiting factors for image contrast at lower operating voltages. By enabling a variety of detector configurations, the airSEM is applicable to a wide range of environmental experiments including the imaging of hydrated biological specimens andin situchemical and electrochemical processes.« less

Multi-pass transmission electron microscopy

DOE PAGES

Juffmann, Thomas; Koppell, Stewart A.; Klopfer, Brannon B.; ...

2017-05-10

Feynman once asked physicists to build better electron microscopes to be able to watch biology at work. While electron microscopes can now provide atomic resolution, electron beam induced specimen damage precludes high resolution imaging of sensitive materials, such as single proteins or polymers. Here, we use simulations to show that an electron microscope based on a multi-pass measurement protocol enables imaging of single proteins, without averaging structures over multiple images. While we demonstrate the method for particular imaging targets, the approach is broadly applicable and is expected to improve resolution and sensitivity for a range of electron microscopy imaging modalities,more » including, for example, scanning and spectroscopic techniques. The approach implements a quantum mechanically optimal strategy which under idealized conditions can be considered interaction-free.« less

Opportunities of using Stimulated Emission from Biological Tissue

NASA Astrophysics Data System (ADS)

Yun, S. H. Andy

Fluorescence or spontaneous emission has been a powerful tool in biomedical applications ranging from biochemical assays and cytometry to microscopy and medical imaging. Here I present the opportunities in the generation and applications of coherent stimulated emission within biological samples.

Ex-vivo multiphoton analysis of rabbit corneal wound healing following photorefractive keratectomy

NASA Astrophysics Data System (ADS)

Wang, Tsung-Jen; Lo, Wen; Dong, Chen-Yuan; Hu, Fung-Rong

2008-02-01

The aim of this study is to assess the application of multiphoton autofluorescence and second harmonic generation (SHG) microscopy for investigating corneal wound healing after high myopic (-10.0D) photorefractive keratectomy (PRK) procedures on the rabbit eyes. The effect of PRK on the morphology and distribution of keratocytes were investigated using multiphoton excited autofluorescence imaging, while the effect of PRK on the arrangement of collagen fibers was monitored by second-harmonic generation imaging. Without histological processing, multiphoton microscopy is able to characterize corneal damage and wound healing from PRK. Our results show that this technique has potential application in the clinical evaluation of corneal damage due to refractive surgery, and may be used to study the unwanted side effects of these procedures.

Fluorescence lifetime imaging and reflectance confocal microscopy for multiscale imaging of oral precancer

NASA Astrophysics Data System (ADS)

Jabbour, Joey M.; Cheng, Shuna; Malik, Bilal H.; Cuenca, Rodrigo; Jo, Javier A.; Wright, John; Cheng, Yi-Shing Lisa; Maitland, Kristen C.

2013-04-01

Optical imaging techniques using a variety of contrast mechanisms are under evaluation for early detection of epithelial precancer; however, tradeoffs in field of view (FOV) and resolution may limit their application. Therefore, we present a multiscale multimodal optical imaging system combining macroscopic biochemical imaging of fluorescence lifetime imaging (FLIM) with subcellular morphologic imaging of reflectance confocal microscopy (RCM). The FLIM module images a 16×16 mm2 tissue area with 62.5 μm lateral and 320 ps temporal resolution to guide cellular imaging of suspicious regions. Subsequently, coregistered RCM images are acquired at 7 Hz with 400 μm diameter FOV, <1 μm lateral and 3.5 μm axial resolution. FLIM-RCM imaging was performed on a tissue phantom, normal porcine buccal mucosa, and a hamster cheek pouch model of oral carcinogenesis. While FLIM is sensitive to biochemical and macroscopic architectural changes in tissue, RCM provides images of cell nuclear morphology, all key indicators of precancer progression.

Fabrication and characterization of novel microsphere-embedded optical devices for enhancing microscopy resolution

NASA Astrophysics Data System (ADS)

Darafsheh, Arash

2018-02-01

Microsphere-assisted imaging can be incorporated onto conventional light microscopes allowing wide-field and flourescence imaging with enhanced resolution. We demonstrated that imaging of specimens containing subdiffraction-limited features is achievable through high-index microspheres embedded in a transparent thin film placed over the specimen. We fabricated novel microsphere-embedded microscope slides composed of barium titanate glass microspheres (with diameter 10-100 μm and refractive index 1.9-2.2) embedded in a transparent polydimethylsiloxane (PDMS) elastomer layer with controllable thickness. We characterized the imaging performance of such microsphere-embedded devices in white-light microscopies, by measuring the imaging resolution, field-of-view, and magnification as a function of microsphere size. Our results inform on the design of novel optical devices, such as microsphere-embedded microscope slides for imaging applications.

Quantitative single-molecule imaging by confocal laser scanning microscopy.

PubMed

Vukojevic, Vladana; Heidkamp, Marcus; Ming, Yu; Johansson, Björn; Terenius, Lars; Rigler, Rudolf

2008-11-25

A new approach to quantitative single-molecule imaging by confocal laser scanning microscopy (CLSM) is presented. It relies on fluorescence intensity distribution to analyze the molecular occurrence statistics captured by digital imaging and enables direct determination of the number of fluorescent molecules and their diffusion rates without resorting to temporal or spatial autocorrelation analyses. Digital images of fluorescent molecules were recorded by using fast scanning and avalanche photodiode detectors. In this way the signal-to-background ratio was significantly improved, enabling direct quantitative imaging by CLSM. The potential of the proposed approach is demonstrated by using standard solutions of fluorescent dyes, fluorescently labeled DNA molecules, quantum dots, and the Enhanced Green Fluorescent Protein in solution and in live cells. The method was verified by using fluorescence correlation spectroscopy. The relevance for biological applications, in particular, for live cell imaging, is discussed.

A correlative optical microscopy and scanning electron microscopy approach to locating nanoparticles in brain tumors.

PubMed

Kempen, Paul J; Kircher, Moritz F; de la Zerda, Adam; Zavaleta, Cristina L; Jokerst, Jesse V; Mellinghoff, Ingo K; Gambhir, Sanjiv S; Sinclair, Robert

2015-01-01

The growing use of nanoparticles in biomedical applications, including cancer diagnosis and treatment, demands the capability to exactly locate them within complex biological systems. In this work a correlative optical and scanning electron microscopy technique was developed to locate and observe multi-modal gold core nanoparticle accumulation in brain tumor models. Entire brain sections from mice containing orthotopic brain tumors injected intravenously with nanoparticles were imaged using both optical microscopy to identify the brain tumor, and scanning electron microscopy to identify the individual nanoparticles. Gold-based nanoparticles were readily identified in the scanning electron microscope using backscattered electron imaging as bright spots against a darker background. This information was then correlated to determine the exact location of the nanoparticles within the brain tissue. The nanoparticles were located only in areas that contained tumor cells, and not in the surrounding healthy brain tissue. This correlative technique provides a powerful method to relate the macro- and micro-scale features visible in light microscopy with the nanoscale features resolvable in scanning electron microscopy. Copyright © 2014 Elsevier Ltd. All rights reserved.

Functional Scanning Probe Imaging of Nanostructured Solar Energy Materials.

PubMed

Giridharagopal, Rajiv; Cox, Phillip A; Ginger, David S

2016-09-20

From hybrid perovskites to semiconducting polymer/fullerene blends for organic photovoltaics, many new materials being explored for energy harvesting and storage exhibit performance characteristics that depend sensitively on their nanoscale morphology. At the same time, rapid advances in the capability and accessibility of scanning probe microscopy methods over the past decade have made it possible to study processing/structure/function relationships ranging from photocurrent collection to photocarrier lifetimes with resolutions on the scale of tens of nanometers or better. Importantly, such scanning probe methods offer the potential to combine measurements of local structure with local function, and they can be implemented to study materials in situ or devices in operando to better understand how materials evolve in time in response to an external stimulus or environmental perturbation. This Account highlights recent advances in the development and application of scanning probe microscopy methods that can help address such questions while filling key gaps between the capabilities of conventional electron microscopy and newer super-resolution optical methods. Focusing on semiconductor materials for solar energy applications, we highlight a range of electrical and optoelectronic scanning probe microscopy methods that exploit the local dynamics of an atomic force microscope tip to probe key properties of the solar cell material or device structure. We discuss how it is possible to extract relevant device properties using noncontact scanning probe methods as well as how these properties guide materials development. Specifically, we discuss intensity-modulated scanning Kelvin probe microscopy (IM-SKPM), time-resolved electrostatic force microscopy (trEFM), frequency-modulated electrostatic force microscopy (FM-EFM), and cantilever ringdown imaging. We explain these developments in the context of classic atomic force microscopy (AFM) methods that exploit the physics of cantilever motion and photocarrier generation to provide robust, nanoscale measurements of materials physics that are correlated with device operation. We predict that the multidimensional data sets made possible by these types of methods will become increasingly important as advances in data science expand capabilities and opportunities for image correlation and discovery.

In vivo label-free photoacoustic microscopy of the anterior segment of the mouse eye

NASA Astrophysics Data System (ADS)

Rao, Bin; Hu, Song; Li, Li; Maslov, Konstantin; Wang, Lihong V.

2010-02-01

Both iris fluorescein angiography (IFA) and indocyanine green angiography (ICGA) provide ophthalmologists imaging tools in studying the microvasculature structure and hemodynamics of the anterior segment of the eye in normal and diseased status. However, a non-invasive, endogenous imaging modality is preferable for the monitoring of hemodynamics of the iris microvasculature. We investigated the in vivo, label-free ocular anterior segment imaging with photo-acoustic microscopy (PAM) in mouse eyes. We demonstrated the unique advantage of endogenous contrast that is not available in both IFA and ICGA. The laser radiation was maintained within the ANSI laser safety limit. The in vivo, label-free nature of our imaging technology has the potential for ophthalmic applications.

Advancement of X-Ray Microscopy Technology and its Application to Metal Solidification Studies

NASA Technical Reports Server (NTRS)

Kaukler, William F.; Curreri, Peter A.

1996-01-01

The technique of x-ray projection microscopy is being used to view, in real time, the structures and dynamics of the solid-liquid interface during solidification. By employing a hard x-ray source with sub-micron dimensions, resolutions of 2 micrometers can be obtained with magnifications of over 800 X. Specimen growth conditions need to be optimized and the best imaging technologies applied to maintain x-ray image resolution, contrast and sensitivity. It turns out that no single imaging technology offers the best solution and traditional methods like radiographic film cannot be used due to specimen motion (solidification). In addition, a special furnace design is required to permit controlled growth conditions and still offer maximum resolution and image contrast.

Time-Lapse Video Microscopy for Assessment of EYFP-Parkin Aggregation as a Marker for Cellular Mitophagy

PubMed Central

Di Sante, Gabriele; Casimiro, Mathew C.; Pestell, Timothy G.; Pestell, Richard G.

2016-01-01

Time-lapse video microscopy can be defined as the real time imaging of living cells. This technique relies on the collection of images at different time points. Time intervals can be set through a computer interface that controls the microscope-integrated camera. This kind of microscopy requires both the ability to acquire very rapid events and the signal generated by the observed cellular structure during these events. After the images have been collected, a movie of the entire experiment is assembled to show the dynamic of the molecular events of interest. Time-lapse video microscopy has a broad range of applications in the biomedical research field and is a powerful and unique tool for following the dynamics of the cellular events in real time. Through this technique, we can assess cellular events such as migration, division, signal transduction, growth, and death. Moreover, using fluorescent molecular probes we are able to mark specific molecules, such as DNA, RNA or proteins and follow them through their molecular pathways and functions. Time-lapse video microscopy has multiple advantages, the major one being the ability to collect data at the single-cell level, that make it a unique technology for investigation in the field of cell biology. However, time-lapse video microscopy has limitations that can interfere with the acquisition of high quality images. Images can be compromised by both external factors; temperature fluctuations, vibrations, humidity and internal factors; pH, cell motility. Herein, we describe a protocol for the dynamic acquisition of a specific protein, Parkin, fused with the enhanced yellow fluorescent protein (EYFP) in order to track the selective removal of damaged mitochondria, using a time-lapse video microscopy approach. PMID:27168174

Time-Lapse Video Microscopy for Assessment of EYFP-Parkin Aggregation as a Marker for Cellular Mitophagy.

PubMed

Di Sante, Gabriele; Casimiro, Mathew C; Pestell, Timothy G; Pestell, Richard G

2016-05-04

Time-lapse video microscopy can be defined as the real time imaging of living cells. This technique relies on the collection of images at different time points. Time intervals can be set through a computer interface that controls the microscope-integrated camera. This kind of microscopy requires both the ability to acquire very rapid events and the signal generated by the observed cellular structure during these events. After the images have been collected, a movie of the entire experiment is assembled to show the dynamic of the molecular events of interest. Time-lapse video microscopy has a broad range of applications in the biomedical research field and is a powerful and unique tool for following the dynamics of the cellular events in real time. Through this technique, we can assess cellular events such as migration, division, signal transduction, growth, and death. Moreover, using fluorescent molecular probes we are able to mark specific molecules, such as DNA, RNA or proteins and follow them through their molecular pathways and functions. Time-lapse video microscopy has multiple advantages, the major one being the ability to collect data at the single-cell level, that make it a unique technology for investigation in the field of cell biology. However, time-lapse video microscopy has limitations that can interfere with the acquisition of high quality images. Images can be compromised by both external factors; temperature fluctuations, vibrations, humidity and internal factors; pH, cell motility. Herein, we describe a protocol for the dynamic acquisition of a specific protein, Parkin, fused with the enhanced yellow fluorescent protein (EYFP) in order to track the selective removal of damaged mitochondria, using a time-lapse video microscopy approach.

Re-scan confocal microscopy: scanning twice for better resolution

PubMed Central

De Luca, Giulia M.R.; Breedijk, Ronald M.P.; Brandt, Rick A.J.; Zeelenberg, Christiaan H.C.; de Jong, Babette E.; Timmermans, Wendy; Azar, Leila Nahidi; Hoebe, Ron A.; Stallinga, Sjoerd; Manders, Erik M.M.

2013-01-01

We present a new super-resolution technique, Re-scan Confocal Microscopy (RCM), based on standard confocal microscopy extended with an optical (re-scanning) unit that projects the image directly on a CCD-camera. This new microscope has improved lateral resolution and strongly improved sensitivity while maintaining the sectioning capability of a standard confocal microscope. This simple technology is typically useful for biological applications where the combination high-resolution and high-sensitivity is required. PMID:24298422

Diamond-Based Magnetic Imaging with Fourier Optical Processing

NASA Astrophysics Data System (ADS)

Backlund, Mikael P.; Kehayias, Pauli; Walsworth, Ronald L.

2017-11-01

Diamond-based magnetic field sensors have attracted great interest in recent years. In particular, wide-field magnetic imaging using nitrogen-vacancy (NV) centers in diamond has been previously demonstrated in condensed matter, biological, and paleomagnetic applications. Vector magnetic imaging with NV ensembles typically requires a significant applied field (>10 G ) to resolve the contributions from four crystallographic orientations, hindering studies of magnetic samples that require measurement in low or independently specified bias fields. Here we model and measure the complex amplitude distribution of NV emission at the microscope's Fourier plane and show that by modulating this collected light at the Fourier plane, one can decompose the NV ensemble magnetic resonance spectrum into its constituent orientations by purely optical means. This decomposition effectively extends the dynamic range at a given bias field and enables wide-field vector magnetic imaging at arbitrarily low bias fields, thus broadening potential applications of NV imaging and sensing. Our results demonstrate that NV-based microscopy stands to benefit greatly from Fourier optical approaches, which have already found widespread utility in other branches of microscopy.

Depth-resolved mid-infrared photothermal imaging of living cells and organisms with submicrometer spatial resolution

PubMed Central

Zhang, Delong; Li, Chen; Zhang, Chi; Slipchenko, Mikhail N.; Eakins, Gregory; Cheng, Ji-Xin

2016-01-01

Chemical contrast has long been sought for label-free visualization of biomolecules and materials in complex living systems. Although infrared spectroscopic imaging has come a long way in this direction, it is thus far only applicable to dried tissues because of the strong infrared absorption by water. It also suffers from low spatial resolution due to long wavelengths and lacks optical sectioning capabilities. We overcome these limitations through sensing vibrational absorption–induced photothermal effect by a visible laser beam. Our mid-infrared photothermal (MIP) approach reached 10 μM detection sensitivity and submicrometer lateral spatial resolution. This performance has exceeded the diffraction limit of infrared microscopy and allowed label-free three-dimensional chemical imaging of live cells and organisms. Distributions of endogenous lipid and exogenous drug inside single cells were visualized. We further demonstrated in vivo MIP imaging of lipids and proteins in Caenorhabditis elegans. The reported MIP imaging technology promises broad applications from monitoring metabolic activities to high-resolution mapping of drug molecules in living systems, which are beyond the reach of current infrared microscopy. PMID:27704043

Depth-resolved mid-infrared photothermal imaging of living cells and organisms with submicrometer spatial resolution.

PubMed

Zhang, Delong; Li, Chen; Zhang, Chi; Slipchenko, Mikhail N; Eakins, Gregory; Cheng, Ji-Xin

2016-09-01

Chemical contrast has long been sought for label-free visualization of biomolecules and materials in complex living systems. Although infrared spectroscopic imaging has come a long way in this direction, it is thus far only applicable to dried tissues because of the strong infrared absorption by water. It also suffers from low spatial resolution due to long wavelengths and lacks optical sectioning capabilities. We overcome these limitations through sensing vibrational absorption-induced photothermal effect by a visible laser beam. Our mid-infrared photothermal (MIP) approach reached 10 μM detection sensitivity and submicrometer lateral spatial resolution. This performance has exceeded the diffraction limit of infrared microscopy and allowed label-free three-dimensional chemical imaging of live cells and organisms. Distributions of endogenous lipid and exogenous drug inside single cells were visualized. We further demonstrated in vivo MIP imaging of lipids and proteins in Caenorhabditis elegans . The reported MIP imaging technology promises broad applications from monitoring metabolic activities to high-resolution mapping of drug molecules in living systems, which are beyond the reach of current infrared microscopy.

Automated Cell Detection and Morphometry on Growth Plate Images of Mouse Bone

PubMed Central

Ascenzi, Maria-Grazia; Du, Xia; Harding, James I; Beylerian, Emily N; de Silva, Brian M; Gross, Ben J; Kastein, Hannah K; Wang, Weiguang; Lyons, Karen M; Schaeffer, Hayden

2014-01-01

Microscopy imaging of mouse growth plates is extensively used in biology to understand the effect of specific molecules on various stages of normal bone development and on bone disease. Until now, such image analysis has been conducted by manual detection. In fact, when existing automated detection techniques were applied, morphological variations across the growth plate and heterogeneity of image background color, including the faint presence of cells (chondrocytes) located deeper in tissue away from the image’s plane of focus, and lack of cell-specific features, interfered with identification of cell. We propose the first method of automated detection and morphometry applicable to images of cells in the growth plate of long bone. Through ad hoc sequential application of the Retinex method, anisotropic diffusion and thresholding, our new cell detection algorithm (CDA) addresses these challenges on bright-field microscopy images of mouse growth plates. Five parameters, chosen by the user in respect of image characteristics, regulate our CDA. Our results demonstrate effectiveness of the proposed numerical method relative to manual methods. Our CDA confirms previously established results regarding chondrocytes’ number, area, orientation, height and shape of normal growth plates. Our CDA also confirms differences previously found between the genetic mutated mouse Smad1/5CKO and its control mouse on fluorescence images. The CDA aims to aid biomedical research by increasing efficiency and consistency of data collection regarding arrangement and characteristics of chondrocytes. Our results suggest that automated extraction of data from microscopy imaging of growth plates can assist in unlocking information on normal and pathological development, key to the underlying biological mechanisms of bone growth. PMID:25525552

Computer-assisted image processing to detect spores from the fungus Pandora neoaphidis.

PubMed

Korsnes, Reinert; Westrum, Karin; Fløistad, Erling; Klingen, Ingeborg

2016-01-01

This contribution demonstrates an example of experimental automatic image analysis to detect spores prepared on microscope slides derived from trapping. The application is to monitor aerial spore counts of the entomopathogenic fungus Pandora neoaphidis which may serve as a biological control agent for aphids. Automatic detection of such spores can therefore play a role in plant protection. The present approach for such detection is a modification of traditional manual microscopy of prepared slides, where autonomous image recording precedes computerised image analysis. The purpose of the present image analysis is to support human visual inspection of imagery data - not to replace it. The workflow has three components:•Preparation of slides for microscopy.•Image recording.•Computerised image processing where the initial part is, as usual, segmentation depending on the actual data product. Then comes identification of blobs, calculation of principal axes of blobs, symmetry operations and projection on a three parameter egg shape space.

Designing Image Analysis Pipelines in Light Microscopy: A Rational Approach.

PubMed

Arganda-Carreras, Ignacio; Andrey, Philippe

2017-01-01

With the progress of microscopy techniques and the rapidly growing amounts of acquired imaging data, there is an increased need for automated image processing and analysis solutions in biological studies. Each new application requires the design of a specific image analysis pipeline, by assembling a series of image processing operations. Many commercial or free bioimage analysis software are now available and several textbooks and reviews have presented the mathematical and computational fundamentals of image processing and analysis. Tens, if not hundreds, of algorithms and methods have been developed and integrated into image analysis software, resulting in a combinatorial explosion of possible image processing sequences. This paper presents a general guideline methodology to rationally address the design of image processing and analysis pipelines. The originality of the proposed approach is to follow an iterative, backwards procedure from the target objectives of analysis. The proposed goal-oriented strategy should help biologists to better apprehend image analysis in the context of their research and should allow them to efficiently interact with image processing specialists.

A streaming multi-GPU implementation of image simulation algorithms for scanning transmission electron microscopy

DOE PAGES

Pryor, Alan; Ophus, Colin; Miao, Jianwei

2017-10-25

Simulation of atomic-resolution image formation in scanning transmission electron microscopy can require significant computation times using traditional methods. A recently developed method, termed plane-wave reciprocal-space interpolated scattering matrix (PRISM), demonstrates potential for significant acceleration of such simulations with negligible loss of accuracy. In this paper, we present a software package called Prismatic for parallelized simulation of image formation in scanning transmission electron microscopy (STEM) using both the PRISM and multislice methods. By distributing the workload between multiple CUDA-enabled GPUs and multicore processors, accelerations as high as 1000 × for PRISM and 15 × for multislice are achieved relative to traditionalmore » multislice implementations using a single 4-GPU machine. We demonstrate a potentially important application of Prismatic, using it to compute images for atomic electron tomography at sufficient speeds to include in the reconstruction pipeline. Prismatic is freely available both as an open-source CUDA/C++ package with a graphical user interface and as a Python package, PyPrismatic.« less

A streaming multi-GPU implementation of image simulation algorithms for scanning transmission electron microscopy.

PubMed

Pryor, Alan; Ophus, Colin; Miao, Jianwei

2017-01-01

Simulation of atomic-resolution image formation in scanning transmission electron microscopy can require significant computation times using traditional methods. A recently developed method, termed plane-wave reciprocal-space interpolated scattering matrix (PRISM), demonstrates potential for significant acceleration of such simulations with negligible loss of accuracy. Here, we present a software package called Prismatic for parallelized simulation of image formation in scanning transmission electron microscopy (STEM) using both the PRISM and multislice methods. By distributing the workload between multiple CUDA-enabled GPUs and multicore processors, accelerations as high as 1000 × for PRISM and 15 × for multislice are achieved relative to traditional multislice implementations using a single 4-GPU machine. We demonstrate a potentially important application of Prismatic , using it to compute images for atomic electron tomography at sufficient speeds to include in the reconstruction pipeline. Prismatic is freely available both as an open-source CUDA/C++ package with a graphical user interface and as a Python package, PyPrismatic .

3D imaging of cement-based materials at submicron resolution by combining laser scanning confocal microscopy with serial sectioning.

PubMed

Yio, M H N; Mac, M J; Wong, H S; Buenfeld, N R

2015-05-01

In this paper, we present a new method to reconstruct large volumes of nontransparent porous materials at submicron resolution. The proposed method combines fluorescence laser scanning confocal microscopy with serial sectioning to produce a series of overlapping confocal z-stacks, which are then aligned and stitched based on phase correlation. The method can be extended in the XY plane to further increase the overall image volume. Resolution of the reconstructed image volume does not degrade with increase in sample size. We have used the method to image cementitious materials, hardened cement paste and concrete and the results obtained show that the method is reliable. Possible applications of the method such as three-dimensional characterization of the pores and microcracks in hardened concrete, three-dimensional particle shape characterization of cementitious materials and three-dimensional characterization of other porous materials such as rocks and bioceramics are discussed. © 2015 The Authors Journal of Microscopy © 2015 Royal Microscopical Society.

4D multiple-cathode ultrafast electron microscopy

PubMed Central

Baskin, John Spencer; Liu, Haihua; Zewail, Ahmed H.

2014-01-01

Four-dimensional multiple-cathode ultrafast electron microscopy is developed to enable the capture of multiple images at ultrashort time intervals for a single microscopic dynamic process. The dynamic process is initiated in the specimen by one femtosecond light pulse and probed by multiple packets of electrons generated by one UV laser pulse impinging on multiple, spatially distinct, cathode surfaces. Each packet is distinctly recorded, with timing and detector location controlled by the cathode configuration. In the first demonstration, two packets of electrons on each image frame (of the CCD) probe different times, separated by 19 picoseconds, in the evolution of the diffraction of a gold film following femtosecond heating. Future elaborations of this concept to extend its capabilities and expand the range of applications of 4D ultrafast electron microscopy are discussed. The proof-of-principle demonstration reported here provides a path toward the imaging of irreversible ultrafast phenomena of materials, and opens the door to studies involving the single-frame capture of ultrafast dynamics using single-pump/multiple-probe, embedded stroboscopic imaging. PMID:25006261

4D multiple-cathode ultrafast electron microscopy.

PubMed

Baskin, John Spencer; Liu, Haihua; Zewail, Ahmed H

2014-07-22

Four-dimensional multiple-cathode ultrafast electron microscopy is developed to enable the capture of multiple images at ultrashort time intervals for a single microscopic dynamic process. The dynamic process is initiated in the specimen by one femtosecond light pulse and probed by multiple packets of electrons generated by one UV laser pulse impinging on multiple, spatially distinct, cathode surfaces. Each packet is distinctly recorded, with timing and detector location controlled by the cathode configuration. In the first demonstration, two packets of electrons on each image frame (of the CCD) probe different times, separated by 19 picoseconds, in the evolution of the diffraction of a gold film following femtosecond heating. Future elaborations of this concept to extend its capabilities and expand the range of applications of 4D ultrafast electron microscopy are discussed. The proof-of-principle demonstration reported here provides a path toward the imaging of irreversible ultrafast phenomena of materials, and opens the door to studies involving the single-frame capture of ultrafast dynamics using single-pump/multiple-probe, embedded stroboscopic imaging.

A streaming multi-GPU implementation of image simulation algorithms for scanning transmission electron microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pryor, Alan; Ophus, Colin; Miao, Jianwei

Simulation of atomic-resolution image formation in scanning transmission electron microscopy can require significant computation times using traditional methods. A recently developed method, termed plane-wave reciprocal-space interpolated scattering matrix (PRISM), demonstrates potential for significant acceleration of such simulations with negligible loss of accuracy. In this paper, we present a software package called Prismatic for parallelized simulation of image formation in scanning transmission electron microscopy (STEM) using both the PRISM and multislice methods. By distributing the workload between multiple CUDA-enabled GPUs and multicore processors, accelerations as high as 1000 × for PRISM and 15 × for multislice are achieved relative to traditionalmore » multislice implementations using a single 4-GPU machine. We demonstrate a potentially important application of Prismatic, using it to compute images for atomic electron tomography at sufficient speeds to include in the reconstruction pipeline. Prismatic is freely available both as an open-source CUDA/C++ package with a graphical user interface and as a Python package, PyPrismatic.« less

Measuring single-cell gene expression dynamics in bacteria using fluorescence time-lapse microscopy

PubMed Central

Young, Jonathan W; Locke, James C W; Altinok, Alphan; Rosenfeld, Nitzan; Bacarian, Tigran; Swain, Peter S; Mjolsness, Eric; Elowitz, Michael B

2014-01-01

Quantitative single-cell time-lapse microscopy is a powerful method for analyzing gene circuit dynamics and heterogeneous cell behavior. We describe the application of this method to imaging bacteria by using an automated microscopy system. This protocol has been used to analyze sporulation and competence differentiation in Bacillus subtilis, and to quantify gene regulation and its fluctuations in individual Escherichia coli cells. The protocol involves seeding and growing bacteria on small agarose pads and imaging the resulting microcolonies. Images are then reviewed and analyzed using our laboratory's custom MATLAB analysis code, which segments and tracks cells in a frame-to-frame method. This process yields quantitative expression data on cell lineages, which can illustrate dynamic expression profiles and facilitate mathematical models of gene circuits. With fast-growing bacteria, such as E. coli or B. subtilis, image acquisition can be completed in 1 d, with an additional 1–2 d for progressing through the analysis procedure. PMID:22179594

Characterization and improvement of highly inclined optical sheet microscopy

NASA Astrophysics Data System (ADS)

Vignolini, T.; Curcio, V.; Gardini, L.; Capitanio, M.; Pavone, F. S.

2018-02-01

Highly Inclined and Laminated Optical sheet (HILO) microscopy is an optical technique that employs a highly inclined laser beam to illuminate the sample with a thin sheet of light that can be scanned through the sample volume1 . HILO is an efficient illumination technique when applied to fluorescence imaging of thick samples owing to the confined illumination volume that allows high contrast imaging while retaining deep scanning capability in a wide-field configuration. The restricted illumination volume is crucial to limit background fluorescence originating from portions of the sample far from the focal plane, especially in applications such as single molecule localization and super-resolution imaging2-4. Despite its widespread use, current literature lacks comprehensive reports of the actual advantages of HILO in these kinds of microscopies. Here, we thoroughly characterize the propagation of a highly inclined beam through fluorescently labeled samples and implement appropriate beam shaping for optimal application to single molecule and super-resolution imaging. We demonstrate that, by reducing the beam size along the refracted axis only, the excitation volume is consequently reduced while maintaining a field of view suitable for single cell imaging. We quantify the enhancement in signal-tobackground ratio with respect to the standard HILO technique and apply our illumination method to dSTORM superresolution imaging of the actin and vimentin cytoskeleton. We define the conditions to achieve localization precisions comparable to state-of-the-art reports, obtain a significant improvement in the image contrast, and enhanced plane selectivity within the sample volume due to the further confinement of the inclined beam.

Atomic species identification at the (101) anatase surface by simultaneous scanning tunnelling and atomic force microscopy

PubMed Central

Stetsovych, Oleksandr; Todorović, Milica; Shimizu, Tomoko K.; Moreno, César; Ryan, James William; León, Carmen Pérez; Sagisaka, Keisuke; Palomares, Emilio; Matolín, Vladimír; Fujita, Daisuke; Perez, Ruben; Custance, Oscar

2015-01-01

Anatase is a pivotal material in devices for energy-harvesting applications and catalysis. Methods for the accurate characterization of this reducible oxide at the atomic scale are critical in the exploration of outstanding properties for technological developments. Here we combine atomic force microscopy (AFM) and scanning tunnelling microscopy (STM), supported by first-principles calculations, for the simultaneous imaging and unambiguous identification of atomic species at the (101) anatase surface. We demonstrate that dynamic AFM-STM operation allows atomic resolution imaging within the material's band gap. Based on key distinguishing features extracted from calculations and experiments, we identify candidates for the most common surface defects. Our results pave the way for the understanding of surface processes, like adsorption of metal dopants and photoactive molecules, that are fundamental for the catalytic and photovoltaic applications of anatase, and demonstrate the potential of dynamic AFM-STM for the characterization of wide band gap materials. PMID:26118408

Optimizing single-nanoparticle two-photon microscopy by in situ adaptive control of femtosecond pulses

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Donghai; Deng, Yongkai; Chu, Saisai

2016-07-11

Single-nanoparticle two-photon microscopy shows great application potential in super-resolution cell imaging. Here, we report in situ adaptive optimization of single-nanoparticle two-photon luminescence signals by phase and polarization modulations of broadband laser pulses. For polarization-independent quantum dots, phase-only optimization was carried out to compensate the phase dispersion at the focus of the objective. Enhancement of the two-photon excitation fluorescence intensity under dispersion-compensated femtosecond pulses was achieved. For polarization-dependent single gold nanorod, in situ polarization optimization resulted in further enhancement of two-photon photoluminescence intensity than phase-only optimization. The application of in situ adaptive control of femtosecond pulse provides a way for object-orientedmore » optimization of single-nanoparticle two-photon microscopy for its future applications.« less

Automated method for the rapid and precise estimation of adherent cell culture characteristics from phase contrast microscopy images.

PubMed

Jaccard, Nicolas; Griffin, Lewis D; Keser, Ana; Macown, Rhys J; Super, Alexandre; Veraitch, Farlan S; Szita, Nicolas

2014-03-01

The quantitative determination of key adherent cell culture characteristics such as confluency, morphology, and cell density is necessary for the evaluation of experimental outcomes and to provide a suitable basis for the establishment of robust cell culture protocols. Automated processing of images acquired using phase contrast microscopy (PCM), an imaging modality widely used for the visual inspection of adherent cell cultures, could enable the non-invasive determination of these characteristics. We present an image-processing approach that accurately detects cellular objects in PCM images through a combination of local contrast thresholding and post hoc correction of halo artifacts. The method was thoroughly validated using a variety of cell lines, microscope models and imaging conditions, demonstrating consistently high segmentation performance in all cases and very short processing times (<1 s per 1,208 × 960 pixels image). Based on the high segmentation performance, it was possible to precisely determine culture confluency, cell density, and the morphology of cellular objects, demonstrating the wide applicability of our algorithm for typical microscopy image processing pipelines. Furthermore, PCM image segmentation was used to facilitate the interpretation and analysis of fluorescence microscopy data, enabling the determination of temporal and spatial expression patterns of a fluorescent reporter. We created a software toolbox (PHANTAST) that bundles all the algorithms and provides an easy to use graphical user interface. Source-code for MATLAB and ImageJ is freely available under a permissive open-source license. © 2013 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

Automated Method for the Rapid and Precise Estimation of Adherent Cell Culture Characteristics from Phase Contrast Microscopy Images

PubMed Central

Jaccard, Nicolas; Griffin, Lewis D; Keser, Ana; Macown, Rhys J; Super, Alexandre; Veraitch, Farlan S; Szita, Nicolas

2014-01-01

The quantitative determination of key adherent cell culture characteristics such as confluency, morphology, and cell density is necessary for the evaluation of experimental outcomes and to provide a suitable basis for the establishment of robust cell culture protocols. Automated processing of images acquired using phase contrast microscopy (PCM), an imaging modality widely used for the visual inspection of adherent cell cultures, could enable the non-invasive determination of these characteristics. We present an image-processing approach that accurately detects cellular objects in PCM images through a combination of local contrast thresholding and post hoc correction of halo artifacts. The method was thoroughly validated using a variety of cell lines, microscope models and imaging conditions, demonstrating consistently high segmentation performance in all cases and very short processing times (<1 s per 1,208 × 960 pixels image). Based on the high segmentation performance, it was possible to precisely determine culture confluency, cell density, and the morphology of cellular objects, demonstrating the wide applicability of our algorithm for typical microscopy image processing pipelines. Furthermore, PCM image segmentation was used to facilitate the interpretation and analysis of fluorescence microscopy data, enabling the determination of temporal and spatial expression patterns of a fluorescent reporter. We created a software toolbox (PHANTAST) that bundles all the algorithms and provides an easy to use graphical user interface. Source-code for MATLAB and ImageJ is freely available under a permissive open-source license. Biotechnol. Bioeng. 2014;111: 504–517. © 2013 Wiley Periodicals, Inc. PMID:24037521

Moxifloxacin: Clinically compatible contrast agent for multiphoton imaging

NASA Astrophysics Data System (ADS)

Wang, Taejun; Jang, Won Hyuk; Lee, Seunghun; Yoon, Calvin J.; Lee, Jun Ho; Kim, Bumju; Hwang, Sekyu; Hong, Chun-Pyo; Yoon, Yeoreum; Lee, Gilgu; Le, Viet-Hoan; Bok, Seoyeon; Ahn, G.-One; Lee, Jaewook; Gho, Yong Song; Chung, Euiheon; Kim, Sungjee; Jang, Myoung Ho; Myung, Seung-Jae; Kim, Myoung Joon; So, Peter T. C.; Kim, Ki Hean

2016-06-01

Multiphoton microscopy (MPM) is a nonlinear fluorescence microscopic technique widely used for cellular imaging of thick tissues and live animals in biological studies. However, MPM application to human tissues is limited by weak endogenous fluorescence in tissue and cytotoxicity of exogenous probes. Herein, we describe the applications of moxifloxacin, an FDA-approved antibiotic, as a cell-labeling agent for MPM. Moxifloxacin has bright intrinsic multiphoton fluorescence, good tissue penetration and high intracellular concentration. MPM with moxifloxacin was demonstrated in various cell lines, and animal tissues of cornea, skin, small intestine and bladder. Clinical application is promising since imaging based on moxifloxacin labeling could be 10 times faster than imaging based on endogenous fluorescence.

Portable optical-resolution photoacoustic microscopy for volumetric imaging of multiscale organisms.

PubMed

Jin, Tian; Guo, Heng; Yao, Lei; Xie, Huikai; Jiang, Huabei; Xi, Lei

2018-04-01

Photoacoustic microscopy (PAM) provides a fundamentally new tool for a broad range of studies of biological structures and functions. However, the use of PAM has been largely limited to small vertebrates due to the large size/weight and the inconvenience of the equipment. Here, we describe a portable optical-resolution photoacoustic microscopy (pORPAM) system for 3-dimensional (3D) imaging of small-to-large rodents and humans with a high spatiotemporal resolution and a large field of view. We show extensive applications of pORPAM to multiscale animals including mice and rabbits. In addition, we image the 3D vascular networks of human lips, and demonstrate the feasibility of pORPAM to observe the recovery process of oral ulcer and cancer-associated capillary loops in human oral cavities. This technology is promising for broad biomedical studies from fundamental biology to clinical diseases. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Advances in imaging and quantification of electrical properties at the nanoscale using Scanning Microwave Impedance Microscopy (sMIM)

NASA Astrophysics Data System (ADS)

Friedman, Stuart; Yang, Yongliang; Amster, Oskar

2015-03-01

Scanning Microwave Impedance Microscopy (sMIM) is a mode for Atomic Force Microscopy (AFM) enabling imaging of unique contrast mechanisms and measurement of local permittivity and conductivity at the 10's of nm length scale. Recent results will be presented illustrating high-resolution electrical features such as sub 15 nm Moire' patterns in Graphene, carbon nanotubes of various electrical states and ferro-electrics. In addition to imaging, the technique is suited to a variety of metrology applications where specific physical properties are determined quantitatively. We will present research activities on quantitative measurements using multiple techniques to determine dielectric constant (permittivity) and conductivity (e.g. dopant concentration) for a range of materials. Examples include bulk dielectrics, low-k dielectric thin films, capacitance standards and doped semiconductors. Funded in part by DOE SBIR DE-SC0009586.

Efficient globally optimal segmentation of cells in fluorescence microscopy images using level sets and convex energy functionals.

PubMed

Bergeest, Jan-Philip; Rohr, Karl

2012-10-01

In high-throughput applications, accurate and efficient segmentation of cells in fluorescence microscopy images is of central importance for the quantification of protein expression and the understanding of cell function. We propose an approach for segmenting cell nuclei which is based on active contours using level sets and convex energy functionals. Compared to previous work, our approach determines the global solution. Thus, the approach does not suffer from local minima and the segmentation result does not depend on the initialization. We consider three different well-known energy functionals for active contour-based segmentation and introduce convex formulations of these functionals. We also suggest a numeric approach for efficiently computing the solution. The performance of our approach has been evaluated using fluorescence microscopy images from different experiments comprising different cell types. We have also performed a quantitative comparison with previous segmentation approaches. Copyright © 2012 Elsevier B.V. All rights reserved.

Detecting overlapping instances in microscopy images using extremal region trees.

PubMed

Arteta, Carlos; Lempitsky, Victor; Noble, J Alison; Zisserman, Andrew

2016-01-01

In many microscopy applications the images may contain both regions of low and high cell densities corresponding to different tissues or colonies at different stages of growth. This poses a challenge to most previously developed automated cell detection and counting methods, which are designed to handle either the low-density scenario (through cell detection) or the high-density scenario (through density estimation or texture analysis). The objective of this work is to detect all the instances of an object of interest in microscopy images. The instances may be partially overlapping and clustered. To this end we introduce a tree-structured discrete graphical model that is used to select and label a set of non-overlapping regions in the image by a global optimization of a classification score. Each region is labeled with the number of instances it contains - for example regions can be selected that contain two or three object instances, by defining separate classes for tuples of objects in the detection process. We show that this formulation can be learned within the structured output SVM framework and that the inference in such a model can be accomplished using dynamic programming on a tree structured region graph. Furthermore, the learning only requires weak annotations - a dot on each instance. The candidate regions for the selection are obtained as extremal region of a surface computed from the microscopy image, and we show that the performance of the model can be improved by considering a proxy problem for learning the surface that allows better selection of the extremal regions. Furthermore, we consider a number of variations for the loss function used in the structured output learning. The model is applied and evaluated over six quite disparate data sets of images covering: fluorescence microscopy, weak-fluorescence molecular images, phase contrast microscopy and histopathology images, and is shown to exceed the state of the art in performance. Copyright © 2015 Elsevier B.V. All rights reserved.

LASER BIOLOGY: Peculiarities of studying an isolated neuron by the method of laser interference microscopy

NASA Astrophysics Data System (ADS)

Yusipovich, Alexander I.; Novikov, Sergey M.; Kazakova, Tatiana A.; Erokhova, Liudmila A.; Brazhe, Nadezda A.; Lazarev, Grigory L.; Maksimov, Georgy V.

2006-09-01

Actual aspects of using a new method of laser interference microscopy (LIM) for studying nerve cells are discussed. The peculiarities of the LIM display of neurons are demonstrated by the example of isolated neurons of a pond snail Lymnaea stagnalis. A comparative analysis of the images of the cell and subcellular structures of a neuron obtained by the methods of interference microscopy, optical transmission microscopy, and confocal microscopy is performed. Various aspects of the application of LIM for studying the lateral dimensions and internal structure of the cytoplasm and organelles of a neuron in cytology and cell physiology are discussed.

High-speed atomic force microscopy imaging of live mammalian cells

PubMed Central

Shibata, Mikihiro; Watanabe, Hiroki; Uchihashi, Takayuki; Ando, Toshio; Yasuda, Ryohei

2017-01-01

Direct imaging of morphological dynamics of live mammalian cells with nanometer resolution under physiological conditions is highly expected, but yet challenging. High-speed atomic force microscopy (HS-AFM) is a unique technique for capturing biomolecules at work under near physiological conditions. However, application of HS-AFM for imaging of live mammalian cells was hard to be accomplished because of collision between a huge mammalian cell and a cantilever during AFM scanning. Here, we review our recent improvements of HS-AFM for imaging of activities of live mammalian cells without significant damage to the cell. The improvement of an extremely long (~3 μm) AFM tip attached to a cantilever enables us to reduce severe damage to soft mammalian cells. In addition, a combination of HS-AFM with simple fluorescence microscopy allows us to quickly locate the cell in the AFM scanning area. After these improvements, we demonstrate that developed HS-AFM for live mammalian cells is possible to image morphogenesis of filopodia, membrane ruffles, pits open-close formations, and endocytosis in COS-7, HeLa cells as well as hippocampal neurons. PMID:28900590

Multiple excitation nano-spot generation and confocal detection for far-field microscopy.

PubMed

Mondal, Partha Pratim

2010-03-01

An imaging technique is developed for the controlled generation of multiple excitation nano-spots for far-field microscopy. The system point spread function (PSF) is obtained by interfering two counter-propagating extended depth-of-focus PSF (DoF-PSF), resulting in highly localized multiple excitation spots along the optical axis. The technique permits (1) simultaneous excitation of multiple planes in the specimen; (2) control of the number of spots by confocal detection; and (3) overcoming the point-by-point based excitation. Fluorescence detection from the excitation spots can be efficiently achieved by Z-scanning the detector/pinhole assembly. The technique complements most of the bioimaging techniques and may find potential application in high resolution fluorescence microscopy and nanoscale imaging.

Multiple excitation nano-spot generation and confocal detection for far-field microscopy

NASA Astrophysics Data System (ADS)

Mondal, Partha Pratim

2010-03-01

An imaging technique is developed for the controlled generation of multiple excitation nano-spots for far-field microscopy. The system point spread function (PSF) is obtained by interfering two counter-propagating extended depth-of-focus PSF (DoF-PSF), resulting in highly localized multiple excitation spots along the optical axis. The technique permits (1) simultaneous excitation of multiple planes in the specimen; (2) control of the number of spots by confocal detection; and (3) overcoming the point-by-point based excitation. Fluorescence detection from the excitation spots can be efficiently achieved by Z-scanning the detector/pinhole assembly. The technique complements most of the bioimaging techniques and may find potential application in high resolution fluorescence microscopy and nanoscale imaging.

Lensfree Computational Microscopy Tools and their Biomedical Applications

NASA Astrophysics Data System (ADS)

Sencan, Ikbal

Conventional microscopy has been a revolutionary tool for biomedical applications since its invention several centuries ago. Ability to non-destructively observe very fine details of biological objects in real time enabled to answer many important questions about their structures and functions. Unfortunately, most of these advance microscopes are complex, bulky, expensive, and/or hard to operate, so they could not reach beyond the walls of well-equipped laboratories. Recent improvements in optoelectronic components and computational methods allow creating imaging systems that better fulfill the specific needs of clinics or research related biomedical applications. In this respect, lensfree computational microscopy aims to replace bulky and expensive optical components with compact and cost-effective alternatives through the use of computation, which can be particularly useful for lab-on-a-chip platforms as well as imaging applications in low-resource settings. Several high-throughput on-chip platforms are built with this approach for applications including, but not limited to, cytometry, micro-array imaging, rare cell analysis, telemedicine, and water quality screening. The lack of optical complexity in these lensfree on-chip imaging platforms is compensated by using computational techniques. These computational methods are utilized for various purposes in coherent, incoherent and fluorescent on-chip imaging platforms e.g. improving the spatial resolution, to undo the light diffraction without using lenses, localization of objects in a large volume and retrieval of the phase or the color/spectral content of the objects. For instance, pixel super resolution approaches based on source shifting are used in lensfree imaging platforms to prevent under sampling, Bayer pattern, and aliasing artifacts. Another method, iterative phase retrieval, is utilized to compensate the lack of lenses by undoing the diffraction and removing the twin image noise of in-line holograms. This technique enables recovering the complex optical field from its intensity measurement(s) by using additional constraints in iterations, such as spatial boundaries and other known properties of objects. Another computational tool employed in lensfree imaging is compressive sensing (or decoding), which is a novel method taking advantage of the fact that natural signals/objects are mostly sparse or compressible in known bases. This inherent property of objects enables better signal recovery when the number of measurement is low, even below the Nyquist rate, and increases the additive noise immunity of the system.

Application of Image Intensifier Technology to the Military, Scientific, Industrial, Educational, and Medical Communities.

DTIC Science & Technology

1980-05-30

afflicted with Retinitis Pigmentosa , commonly called night blindness. People who suffer from this are virtually blind in absence of normal room light...image intensification 5. Low light ophthalmological surgery 6. Retinitis Pigmentosa patients 7. Mine rescue and first aid 8. TV microscopy 9

Molecular and Cellular Quantitative Microscopy: theoretical investigations, technological developments and applications to neurobiology

NASA Astrophysics Data System (ADS)

Esposito, Alessandro

2006-05-01

This PhD project aims at the development and evaluation of microscopy techniques for the quantitative detection of molecular interactions and cellular features. The primarily investigated techniques are Fαrster Resonance Energy Transfer imaging and Fluorescence Lifetime Imaging Microscopy. These techniques have the capability to quantitatively probe the biochemical environment of fluorophores. An automated microscope capable of unsupervised operation has been developed that enables the investigation of molecular and cellular properties at high throughput levels and the analysis of cellular heterogeneity. State-of-the-art Förster Resonance Energy Transfer imaging, Fluorescence Lifetime Imaging Microscopy, Confocal Laser Scanning Microscopy and the newly developed tools have been combined with cellular and molecular biology techniques for the investigation of protein-protein interactions, oligomerization and post-translational modifications of α-Synuclein and Tau, two proteins involved in Parkinson’s and Alzheimer’s disease, respectively. The high inter-disciplinarity of this project required the merging of the expertise of both the Molecular Biophysics Group at the Debye Institute - Utrecht University and the Cell Biophysics Group at the European Neuroscience Institute - Gαttingen University. This project was conducted also with the support and the collaboration of the Center for the Molecular Physiology of the Brain (Göttingen), particularly with the groups associated with the Molecular Quantitative Microscopy and Parkinson’s Disease and Aggregopathies areas. This work demonstrates that molecular and cellular quantitative microscopy can be used in combination with high-throughput screening as a powerful tool for the investigation of the molecular mechanisms of complex biological phenomena like those occurring in neurodegenerative diseases.

Atomic resolution elemental mapping using energy-filtered imaging scanning transmission electron microscopy with chromatic aberration correction.

PubMed

Krause, F F; Rosenauer, A; Barthel, J; Mayer, J; Urban, K; Dunin-Borkowski, R E; Brown, H G; Forbes, B D; Allen, L J

2017-10-01

This paper addresses a novel approach to atomic resolution elemental mapping, demonstrating a method that produces elemental maps with a similar resolution to the established method of electron energy-loss spectroscopy in scanning transmission electron microscopy. Dubbed energy-filtered imaging scanning transmission electron microscopy (EFISTEM) this mode of imaging is, by the quantum mechanical principle of reciprocity, equivalent to tilting the probe in energy-filtered transmission electron microscopy (EFTEM) through a cone and incoherently averaging the results. In this paper we present a proof-of-principle EFISTEM experimental study on strontium titanate. The present approach, made possible by chromatic aberration correction, has the advantage that it provides elemental maps which are immune to spatial incoherence in the electron source, coherent aberrations in the probe-forming lens and probe jitter. The veracity of the experiment is supported by quantum mechanical image simulations, which provide an insight into the image-forming process. Elemental maps obtained in EFTEM suffer from the effect known as preservation of elastic contrast, which, for example, can lead to a given atomic species appearing to be in atomic columns where it is not to be found. EFISTEM very substantially reduces the preservation of elastic contrast and yields images which show stability of contrast with changing thickness. The experimental application is demonstrated in a proof-of-principle study on strontium titanate. Copyright © 2017 Elsevier B.V. All rights reserved.

Measurement of absorption spectrum of deuterium oxide (D{sub 2}O) and its application to signal enhancement in multiphoton microscopy at the 1700-nm window

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Yuxin; Wen, Wenhui; Wang, Kai

2016-01-11

1700-nm window has been demonstrated to be a promising excitation window for deep-tissue multiphoton microscopy (MPM). Long working-distance water immersion objective lenses are typically used for deep-tissue imaging. However, absorption due to immersion water at 1700 nm is still high and leads to dramatic decrease in signals. In this paper, we demonstrate measurement of absorption spectrum of deuterium oxide (D{sub 2}O) from 1200 nm to 2600 nm, covering the three low water-absorption windows potentially applicable for deep-tissue imaging (1300 nm, 1700 nm, and 2200 nm). We apply this measured result to signal enhancement in MPM at the 1700-nm window. Compared with water immersion, D{sub 2}O immersionmore » enhances signal levels in second-harmonic generation imaging, 3-photon fluorescence imaging, and third-harmonic generation imaging by 8.1, 24.8, and 24.7 times with 1662-nm excitation, in good agreement with theoretical calculation based on our absorption measurement. This suggests D{sub 2}O a promising immersion medium for deep-tissue imaging.« less

Optical resolution photoacoustic microscopy using novel high-repetition-rate passively Q-switched microchip and fiber lasers.

PubMed

Shi, Wei; Kerr, Shaun; Utkin, Ilya; Ranasinghesagara, Janaka; Pan, Lei; Godwal, Yogesh; Zemp, Roger J; Fedosejevs, Robert

2010-01-01

Optical-resolution photoacoustic microscopy (OR-PAM) is a novel imaging technology for visualizing optically absorbing superficial structures in vivo with lateral spatial resolution determined by optical focusing rather than acoustic detection. Since scanning of the illumination spot is required, OR-PAM imaging speed is limited by both scanning speed and laser pulse repetition rate. Unfortunately, lasers with high repetition rates and suitable pulse durations and energies are not widely available and can be cost-prohibitive and bulky. We are developing compact, passively Q-switched fiber and microchip laser sources for this application. The properties of these lasers are discussed, and pulse repetition rates up to 100 kHz are demonstrated. OR-PAM imaging was conducted using a previously developed photoacoustic probe, which enabled flexible scanning of the focused output of the lasers. Phantom studies demonstrate the ability to image with lateral spatial resolution of 7±2 μm with the microchip laser system and 15±5 μm with the fiber laser system. We believe that the high pulse repetition rates and the potentially compact and fiber-coupled nature of these lasers will prove important for clinical imaging applications where real-time imaging performance is essential.

Imaging live cells at high spatiotemporal resolution for lab-on-a-chip applications.

PubMed

Chin, Lip Ket; Lee, Chau-Hwang; Chen, Bi-Chang

2016-05-24

Conventional optical imaging techniques are limited by the diffraction limit and difficult-to-image biomolecular and sub-cellular processes in living specimens. Novel optical imaging techniques are constantly evolving with the desire to innovate an imaging tool that is capable of seeing sub-cellular processes in a biological system, especially in three dimensions (3D) over time, i.e. 4D imaging. For fluorescence imaging on live cells, the trade-offs among imaging depth, spatial resolution, temporal resolution and photo-damage are constrained based on the limited photons of the emitters. The fundamental solution to solve this dilemma is to enlarge the photon bank such as the development of photostable and bright fluorophores, leading to the innovation in optical imaging techniques such as super-resolution microscopy and light sheet microscopy. With the synergy of microfluidic technology that is capable of manipulating biological cells and controlling their microenvironments to mimic in vivo physiological environments, studies of sub-cellular processes in various biological systems can be simplified and investigated systematically. In this review, we provide an overview of current state-of-the-art super-resolution and 3D live cell imaging techniques and their lab-on-a-chip applications, and finally discuss future research trends in new and breakthrough research areas of live specimen 4D imaging in controlled 3D microenvironments.

Distinguishing human normal or cancerous esophagus tissue ex vivo using multiphoton microscopy

NASA Astrophysics Data System (ADS)

Liu, N. R.; Chen, G. N.; Wu, S. S.; Chen, R.

2014-02-01

Application of multiphoton microscopy (MPM) to clinical cancer research has greatly developed over the last few years. In this paper, we mainly focus on two-photon excitation fluorescence (TPEF) and second harmonic generation (SHG) for investigating esophageal cancer. We chiefly discuss the SHG/TPEF image and spectral characteristics of normal and cancerous esophagus submucosa with the combined multi-channel imaging mode and Lambda mode of a multiphoton microscope (LSM 510 META). Great differences can be detected, such as collagen content and morphology, glandular-shaped cancer cells, TPEF/SHG intensity ratio, and so on, which demonstrate that the multiphoton imaging technique has the potential ability for minimally-invasive early cancer diagnosis.

A study approach on ferroelectric domains in BaTiO{sub 3}

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rocha, L.S.R.; Cavalcanti, C.S.

Atomic Force Acoustic Microscopy (AFAM) and Piezoresponse Force Microscopy (PFM) were used to study local elastic and electromechanical response in BaTiO{sub 3} ceramics. A commercial multi-mode Scanning Probe Microscopy (SPM) and AFAM mode to image contact stiffness were employed to accomplish the aforementioned purposes. Stiffness parameters along with Young's moduli and piezo coefficients were quantitatively determined. PFM studies were based on electrostatic and electromechanical response from localized tip-surface contact. Comparison was made regarding the Young's moduli obtained by AFAM and PFM. In addition, phase and amplitude images were analyzed based on poling behavior, obtained via the application of − 10more » V to + 10 V local voltage. - Highlights: •Nanoscale behavior of piezo domains in BaTiO{sub 3} ferroelectric materials •Use of Atomic Force Acoustic Microscopy (AFAM) and Piezo Force Microscopy (PFM) •Local elastic and electromechanical response in BaTiO{sub 3} ceramics •The young's moduli obtained from AFAM and PFM.« less

Application Performance Analysis and Efficient Execution on Systems with multi-core CPUs, GPUs and MICs: A Case Study with Microscopy Image Analysis

PubMed Central

Teodoro, George; Kurc, Tahsin; Andrade, Guilherme; Kong, Jun; Ferreira, Renato; Saltz, Joel

2015-01-01

We carry out a comparative performance study of multi-core CPUs, GPUs and Intel Xeon Phi (Many Integrated Core-MIC) with a microscopy image analysis application. We experimentally evaluate the performance of computing devices on core operations of the application. We correlate the observed performance with the characteristics of computing devices and data access patterns, computation complexities, and parallelization forms of the operations. The results show a significant variability in the performance of operations with respect to the device used. The performances of operations with regular data access are comparable or sometimes better on a MIC than that on a GPU. GPUs are more efficient than MICs for operations that access data irregularly, because of the lower bandwidth of the MIC for random data accesses. We propose new performance-aware scheduling strategies that consider variabilities in operation speedups. Our scheduling strategies significantly improve application performance compared to classic strategies in hybrid configurations. PMID:28239253

Graphene-enhanced infrared near-field microscopy.

PubMed

Li, Peining; Wang, Tao; Böckmann, Hannes; Taubner, Thomas

2014-08-13

Graphene is a promising two-dimensional platform for widespread nanophotonic applications. Recent theories have predicted that graphene can also enhance evanescent fields for subdiffraction-limited imaging. Here, for the first time we experimentally demonstrate that monolayer graphene offers a 7-fold enhancement of evanescent information, improving conventional infrared near-field microscopy to resolve buried structures at a 500 nm depth with λ/11-resolution.

Label-free quantitative cell division monitoring of endothelial cells by digital holographic microscopy

NASA Astrophysics Data System (ADS)

Kemper, Björn; Bauwens, Andreas; Vollmer, Angelika; Ketelhut, Steffi; Langehanenberg, Patrik; Müthing, Johannes; Karch, Helge; von Bally, Gert

2010-05-01

Digital holographic microscopy (DHM) enables quantitative multifocus phase contrast imaging for nondestructive technical inspection and live cell analysis. Time-lapse investigations on human brain microvascular endothelial cells demonstrate the use of DHM for label-free dynamic quantitative monitoring of cell division of mother cells into daughter cells. Cytokinetic DHM analysis provides future applications in toxicology and cancer research.

Imaging modes of atomic force microscopy for application in molecular and cell biology.

PubMed

Dufrêne, Yves F; Ando, Toshio; Garcia, Ricardo; Alsteens, David; Martinez-Martin, David; Engel, Andreas; Gerber, Christoph; Müller, Daniel J

2017-04-06

Atomic force microscopy (AFM) is a powerful, multifunctional imaging platform that allows biological samples, from single molecules to living cells, to be visualized and manipulated. Soon after the instrument was invented, it was recognized that in order to maximize the opportunities of AFM imaging in biology, various technological developments would be required to address certain limitations of the method. This has led to the creation of a range of new imaging modes, which continue to push the capabilities of the technique today. Here, we review the basic principles, advantages and limitations of the most common AFM bioimaging modes, including the popular contact and dynamic modes, as well as recently developed modes such as multiparametric, molecular recognition, multifrequency and high-speed imaging. For each of these modes, we discuss recent experiments that highlight their unique capabilities.

Atomic-resolution transmission electron microscopy of electron beam–sensitive crystalline materials

NASA Astrophysics Data System (ADS)

Zhang, Daliang; Zhu, Yihan; Liu, Lingmei; Ying, Xiangrong; Hsiung, Chia-En; Sougrat, Rachid; Li, Kun; Han, Yu

2018-02-01

High-resolution imaging of electron beam–sensitive materials is one of the most difficult applications of transmission electron microscopy (TEM). The challenges are manifold, including the acquisition of images with extremely low beam doses, the time-constrained search for crystal zone axes, the precise image alignment, and the accurate determination of the defocus value. We develop a suite of methods to fulfill these requirements and acquire atomic-resolution TEM images of several metal organic frameworks that are generally recognized as highly sensitive to electron beams. The high image resolution allows us to identify individual metal atomic columns, various types of surface termination, and benzene rings in the organic linkers. We also apply our methods to other electron beam–sensitive materials, including the organic-inorganic hybrid perovskite CH3NH3PbBr3.

High-resolution electron microscopy and its applications.

PubMed

Li, F H

1987-12-01

A review of research on high-resolution electron microscopy (HREM) carried out at the Institute of Physics, the Chinese Academy of Sciences, is presented. Apart from the direct observation of crystal and quasicrystal defects for some alloys, oxides, minerals, etc., and the structure determination for some minute crystals, an approximate image-contrast theory named pseudo-weak-phase object approximation (PWPOA), which shows the image contrast change with crystal thickness, is described. Within the framework of PWPOA, the image contrast of lithium ions in the crystal of R-Li2Ti3O7 has been observed. The usefulness of diffraction analysis techniques such as the direct method and Patterson method in HREM is discussed. Image deconvolution and resolution enhancement for weak-phase objects by use of the direct method are illustrated. In addition, preliminary results of image restoration for thick crystals are given.

Confocal multispot microscope for fast and deep imaging in semicleared tissues

NASA Astrophysics Data System (ADS)

Adam, Marie-Pierre; Müllenbroich, Marie Caroline; Di Giovanna, Antonino Paolo; Alfieri, Domenico; Silvestri, Ludovico; Sacconi, Leonardo; Pavone, Francesco Saverio

2018-02-01

Although perfectly transparent specimens are imaged faster with light-sheet microscopy, less transparent samples are often imaged with two-photon microscopy leveraging its robustness to scattering; however, at the price of increased acquisition times. Clearing methods that are capable of rendering strongly scattering samples such as brain tissue perfectly transparent specimens are often complex, costly, and time intensive, even though for many applications a slightly lower level of tissue transparency is sufficient and easily achieved with simpler and faster methods. Here, we present a microscope type that has been geared toward the imaging of semicleared tissue by combining multispot two-photon excitation with rolling shutter wide-field detection to image deep and fast inside semicleared mouse brain. We present a theoretical and experimental evaluation of the point spread function and contrast as a function of shutter size. Finally, we demonstrate microscope performance in fixed brain slices by imaging dendritic spines up to 400-μm deep.

Extending Whole Slide Imaging: Color Darkfield Internal Reflection Illumination (DIRI) for Biological Applications

PubMed Central

Namiki, Kana; Miyawaki, Atsushi; Ishikawa, Takuji

2017-01-01

Whole slide imaging (WSI) is a useful tool for multi-modal imaging, and in our work, we have often combined WSI with darkfield microscopy. However, traditional darkfield microscopy cannot use a single condenser to support high- and low-numerical-aperture objectives, which limits the modality of WSI. To overcome this limitation, we previously developed a darkfield internal reflection illumination (DIRI) microscope using white light-emitting diodes (LEDs). Although the developed DIRI is useful for biological applications, substantial problems remain to be resolved. In this study, we propose a novel illumination technique called color DIRI. The use of three-color LEDs dramatically improves the capability of the system, such that color DIRI (1) enables optimization of the illumination color; (2) can be combined with an oil objective lens; (3) can produce fluorescence excitation illumination; (4) can adjust the wavelength of light to avoid cell damage or reactions; and (5) can be used as a photostimulator. These results clearly illustrate that the proposed color DIRI can significantly extend WSI modalities for biological applications. PMID:28085892

Advanced microscopy of star-shaped gold nanoparticles and their adsorption-uptake by macrophages

PubMed Central

Plascencia-Villa, Germán; Bahena, Daniel; Rodríguez, Annette R.; Ponce, Arturo; José-Yacamán, Miguel

2013-01-01

Metallic nanoparticles have diverse applications in biomedicine, as diagnostics, image contrast agents, nanosensors and drug delivery systems. Anisotropic metallic nanoparticles possess potential applications in cell imaging and therapy+diagnostics (theranostics), but controlled synthesis and growth of these anisotropic or branched nanostructures has been challenging and usually require use of high concentrations of surfactants. Star-shaped gold nanoparticles were synthesized in high yield through a seed mediated route using HEPES as a precise shape-directing capping agent. Characterization was performed using advanced electron microscopy techniques including atomic resolution TEM, obtaining a detailed characterization of nanostructure and atomic arrangement. Spectroscopy techniques showed that particles have narrow size distribution, monodispersity and high colloidal stability, with absorbance into NIR region and high efficiency for SERS applications. Gold nanostars showed to be biocompatible and efficiently adsorbed and internalized by macrophages, as revealed by advanced FE-SEM and backscattered electron imaging techniques of complete unstained uncoated cells. Additionally, low voltage STEM and X-ray microanalysis revealed the ultra-structural location and confirmed stability of nanoparticles after endocytosis with high spatial resolution. PMID:23443314

The 2015 super-resolution microscopy roadmap

NASA Astrophysics Data System (ADS)

Hell, Stefan W.; Sahl, Steffen J.; Bates, Mark; Zhuang, Xiaowei; Heintzmann, Rainer; Booth, Martin J.; Bewersdorf, Joerg; Shtengel, Gleb; Hess, Harald; Tinnefeld, Philip; Honigmann, Alf; Jakobs, Stefan; Testa, Ilaria; Cognet, Laurent; Lounis, Brahim; Ewers, Helge; Davis, Simon J.; Eggeling, Christian; Klenerman, David; Willig, Katrin I.; Vicidomini, Giuseppe; Castello, Marco; Diaspro, Alberto; Cordes, Thorben

2015-11-01

Far-field optical microscopy using focused light is an important tool in a number of scientific disciplines including chemical, (bio)physical and biomedical research, particularly with respect to the study of living cells and organisms. Unfortunately, the applicability of the optical microscope is limited, since the diffraction of light imposes limitations on the spatial resolution of the image. Consequently the details of, for example, cellular protein distributions, can be visualized only to a certain extent. Fortunately, recent years have witnessed the development of ‘super-resolution’ far-field optical microscopy (nanoscopy) techniques such as stimulated emission depletion (STED), ground state depletion (GSD), reversible saturated optical (fluorescence) transitions (RESOLFT), photoactivation localization microscopy (PALM), stochastic optical reconstruction microscopy (STORM), structured illumination microscopy (SIM) or saturated structured illumination microscopy (SSIM), all in one way or another addressing the problem of the limited spatial resolution of far-field optical microscopy. While SIM achieves a two-fold improvement in spatial resolution compared to conventional optical microscopy, STED, RESOLFT, PALM/STORM, or SSIM have all gone beyond, pushing the limits of optical image resolution to the nanometer scale. Consequently, all super-resolution techniques open new avenues of biomedical research. Because the field is so young, the potential capabilities of different super-resolution microscopy approaches have yet to be fully explored, and uncertainties remain when considering the best choice of methodology. Thus, even for experts, the road to the future is sometimes shrouded in mist. The super-resolution optical microscopy roadmap of Journal of Physics D: Applied Physics addresses this need for clarity. It provides guidance to the outstanding questions through a collection of short review articles from experts in the field, giving a thorough discussion on the concepts underlying super-resolution optical microscopy, the potential of different approaches, the importance of label optimization (such as reversible photoswitchable proteins) and applications in which these methods will have a significant impact. Mark Bates, Christian Eggeling

Emulation and design of terahertz reflection-mode confocal scanning microscopy based on virtual pinhole

NASA Astrophysics Data System (ADS)

Yang, Yong-fa; Li, Qi

2014-12-01

In the practical application of terahertz reflection-mode confocal scanning microscopy, the size of detector pinhole is an important factor that determines the performance of spatial resolution characteristic of the microscopic system. However, the use of physical pinhole brings some inconvenience to the experiment and the adjustment error has a great influence on the experiment result. Through reasonably selecting the parameter of matrix detector virtual pinhole (VPH), it can efficiently approximate the physical pinhole. By using this approach, the difficulty of experimental calibration is reduced significantly. In this article, an imaging scheme of terahertz reflection-mode confocal scanning microscopy that is based on the matrix detector VPH is put forward. The influence of detector pinhole size on the axial resolution of confocal scanning microscopy is emulated and analyzed. Then, the parameter of VPH is emulated when the best axial imaging performance is reached.

Single-exposure super-resolved interferometric microscopy by RGB multiplexing in lensless configuration

NASA Astrophysics Data System (ADS)

Granero, Luis; Ferreira, Carlos; Zalevsky, Zeev; García, Javier; Micó, Vicente

2016-07-01

Single-Exposure Super-Resolved Interferometric Microscopy (SESRIM) reports on a way to achieve one-dimensional (1-D) superresolved imaging in digital holographic microscopy (DHM) by a single illumination shot and digital recording. SESRIM provides color-coded angular multiplexing of the accessible sample's range of spatial frequencies and it allows their recording in a single CCD (color or monochrome) snapshot by adding 3 RGB coherent reference beams at the output plane. In this manuscript, we extend the applicability of SESRIM to the field of digital in-line holographic microscopy (DIHM), that is, working without lenses. As consequence of the in-line configuration, an additional restriction concerning the object field of view (FOV) must be imposed to the technique. Experimental results are reported for both a synthetic object (USAF resolution test target) and a biological sample (swine sperm sample) validating this new kind of superresolution imaging method named as lensless SESRIM (L-SESRIM).

Multifunctional scanning ion conductance microscopy

PubMed Central

Page, Ashley; Unwin, Patrick R.

2017-01-01

Scanning ion conductance microscopy (SICM) is a nanopipette-based technique that has traditionally been used to image topography or to deliver species to an interface, particularly in a biological setting. This article highlights the recent blossoming of SICM into a technique with a much greater diversity of applications and capability that can be used either standalone, with advanced control (potential–time) functions, or in tandem with other methods. SICM can be used to elucidate functional information about interfaces, such as surface charge density or electrochemical activity (ion fluxes). Using a multi-barrel probe format, SICM-related techniques can be employed to deposit nanoscale three-dimensional structures and further functionality is realized when SICM is combined with scanning electrochemical microscopy (SECM), with simultaneous measurements from a single probe opening up considerable prospects for multifunctional imaging. SICM studies are greatly enhanced by finite-element method modelling for quantitative treatment of issues such as resolution, surface charge and (tip) geometry effects. SICM is particularly applicable to the study of living systems, notably single cells, although applications extend to materials characterization and to new methods of printing and nanofabrication. A more thorough understanding of the electrochemical principles and properties of SICM provides a foundation for significant applications of SICM in electrochemistry and interfacial science. PMID:28484332

To boldly glow ... applications of laser scanning confocal microscopy in developmental biology.

PubMed

Paddock, S W

1994-05-01

The laser scanning confocal microscope (LSCM) is now established as an invaluable tool in developmental biology for improved light microscope imaging of fluorescently labelled eggs, embryos and developing tissues. The universal application of the LSCM in biomedical research has stimulated improvements to the microscopes themselves and the synthesis of novel probes for imaging biological structures and physiological processes. Moreover the ability of the LSCM to produce an optical series in perfect register has made computer 3-D reconstruction and analysis of light microscope images a practical option.

New Algorithm to Enable Construction and Display of 3D Structures from Scanning Probe Microscopy Images Acquired Layer-by-Layer.

PubMed

Deng, William Nanqiao; Wang, Shuo; Ventrici de Souza, Joao; Kuhl, Tonya L; Liu, Gang-Yu

2018-06-25

Scanning probe microscopy (SPM), such as atomic force microscopy (AFM), is widely known for high-resolution imaging of surface structures and nanolithography in two dimensions (2D), providing important physical insights into surface science and material science. This work reports a new algorithm to enable construction and display of layer-by-layer 3D structures from SPM images. The algorithm enables alignment of SPM images acquired during layer-by-layer deposition and removal of redundant features and faithfully constructs the deposited 3D structures. The display uses a "see-through" strategy to enable the structure of each layer to be visible. The results demonstrate high spatial accuracy as well as algorithm versatility; users can set parameters for reconstruction and display as per image quality and research needs. To the best of our knowledge, this method represents the first report to enable SPM technology for 3D imaging construction and display. The detailed algorithm is provided to facilitate usage of the same approach in any SPM software. These new capabilities support wide applications of SPM that require 3D image reconstruction and display, such as 3D nanoprinting and 3D additive and subtractive manufacturing and imaging.

microMS: A Python Platform for Image-Guided Mass Spectrometry Profiling

NASA Astrophysics Data System (ADS)

Comi, Troy J.; Neumann, Elizabeth K.; Do, Thanh D.; Sweedler, Jonathan V.

2017-09-01

Image-guided mass spectrometry (MS) profiling provides a facile framework for analyzing samples ranging from single cells to tissue sections. The fundamental workflow utilizes a whole-slide microscopy image to select targets of interest, determine their spatial locations, and subsequently perform MS analysis at those locations. Improving upon prior reported methodology, a software package was developed for working with microscopy images. microMS, for microscopy-guided mass spectrometry, allows the user to select and profile diverse samples using a variety of target patterns and mass analyzers. Written in Python, the program provides an intuitive graphical user interface to simplify image-guided MS for novice users. The class hierarchy of instrument interactions permits integration of new MS systems while retaining the feature-rich image analysis framework. microMS is a versatile platform for performing targeted profiling experiments using a series of mass spectrometers. The flexibility in mass analyzers greatly simplifies serial analyses of the same targets by different instruments. The current capabilities of microMS are presented, and its application for off-line analysis of single cells on three distinct instruments is demonstrated. The software has been made freely available for research purposes. [Figure not available: see fulltext.

microMS: A Python Platform for Image-Guided Mass Spectrometry Profiling.

PubMed

Comi, Troy J; Neumann, Elizabeth K; Do, Thanh D; Sweedler, Jonathan V

2017-09-01

Image-guided mass spectrometry (MS) profiling provides a facile framework for analyzing samples ranging from single cells to tissue sections. The fundamental workflow utilizes a whole-slide microscopy image to select targets of interest, determine their spatial locations, and subsequently perform MS analysis at those locations. Improving upon prior reported methodology, a software package was developed for working with microscopy images. microMS, for microscopy-guided mass spectrometry, allows the user to select and profile diverse samples using a variety of target patterns and mass analyzers. Written in Python, the program provides an intuitive graphical user interface to simplify image-guided MS for novice users. The class hierarchy of instrument interactions permits integration of new MS systems while retaining the feature-rich image analysis framework. microMS is a versatile platform for performing targeted profiling experiments using a series of mass spectrometers. The flexibility in mass analyzers greatly simplifies serial analyses of the same targets by different instruments. The current capabilities of microMS are presented, and its application for off-line analysis of single cells on three distinct instruments is demonstrated. The software has been made freely available for research purposes. Graphical Abstract ᅟ.

Active voltage contrast imaging of cross-sectional surface of multilayer ceramic capacitor using helium ion microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sakai, C., E-mail: SAKAI.Chikako@nims.go.jp; Ishida, N.; Masuda, H.

2016-08-01

We studied active voltage contrast (AVC) imaging using helium ion microscopy (HIM). We observed secondary electron (SE) images of the cross-sectional surface of multilayer ceramic capacitors (MLCCs) with and without a voltage applied to the internal electrodes. When no voltage was applied, we obtained an image reflecting the material contrast between the Ni internal electrode region and the BaTiO{sub 3} dielectric region of the cross-sectional surface of the MLCC. When a voltage was applied, the electrical potential difference between the grounded and the positively biased internal electrodes affected the contrast (voltage contrast). Moreover, attenuation of the SE intensity from themore » grounded to the positively biased internal electrodes was observed in the dielectric region. Kelvin probe force microscopy (KPFM) measurements of the contact potential difference (CPD) were performed on the same sample. By using the AVC image from the HIM observation and the CPD image from the KPFM measurement, we could quantitatively evaluate the electrical potential. We think that the results of this study will lead to an expansion in the number of applications of HIM.« less

Orientational imaging of a single plasmonic nanoparticle using dark-field hyperspectral imaging

NASA Astrophysics Data System (ADS)

Mehta, Nishir; Mahigir, Amirreza; Veronis, Georgios; Gartia, Manas Ranjan

2017-08-01

Orientation of plasmonic nanostructures is an important feature in many nanoscale applications such as catalyst, biosensors DNA interactions, protein detections, hotspot of surface enhanced Raman spectroscopy (SERS), and fluorescence resonant energy transfer (FRET) experiments. However, due to diffraction limit, it is challenging to obtain the exact orientation of the nanostructure using standard optical microscope. Hyperspectral Imaging Microscopy is a state-of-the-art visualization technology that combines modern optics with hyperspectral imaging and computer system to provide the identification and quantitative spectral analysis of nano- and microscale structures. In this work, initially we use transmitted dark field imaging technique to locate single nanoparticle on a glass substrate. Then we employ hyperspectral imaging technique at the same spot to investigate orientation of single nanoparticle. No special tagging or staining of nanoparticle has been done, as more likely required in traditional microscopy techniques. Different orientations have been identified by carefully understanding and calibrating shift in spectral response from each different orientations of similar sized nanoparticles. Wavelengths recorded are between 300 nm to 900 nm. The orientations measured by hyperspectral microscopy was validated using finite difference time domain (FDTD) electrodynamics calculations and scanning electron microscopy (SEM) analysis. The combination of high resolution nanometer-scale imaging techniques and the modern numerical modeling capacities thus enables a meaningful advance in our knowledge of manipulating and fabricating shaped nanostructures. This work will advance our understanding of the behavior of small nanoparticle clusters useful for sensing, nanomedicine, and surface sciences.

[application of the analytical transmission electron microscopy techniques for detection, identification and visualization of localization of nanoparticles of titanium and cerium oxides in mammalian cells].

PubMed

Shebanova, A S; Bogdanov, A G; Ismagulova, T T; Feofanov, A V; Semenyuk, P I; Muronets, V I; Erokhina, M V; Onishchenko, G E; Kirpichnikov, M P; Shaitan, K V

2014-01-01

This work represents the results of the study on applicability of the modern methods of analytical transmission electron microscopy for detection, identification and visualization of localization of nanoparticles of titanium and cerium oxides in A549 cell, human lung adenocarcinoma cell line. A comparative analysis of images of the nanoparticles in the cells obtained in the bright field mode of transmission electron microscopy, under dark-field scanning transmission electron microscopy and high-angle annular dark field scanning transmission electron was performed. For identification of nanoparticles in the cells the analytical techniques, energy-dispersive X-ray spectroscopy and electron energy loss spectroscopy, were compared when used in the mode of obtaining energy spectrum from different particles and element mapping. It was shown that the method for electron tomography is applicable to confirm that nanoparticles are localized in the sample but not coated by contamination. The possibilities and fields of utilizing different techniques for analytical transmission electron microscopy for detection, visualization and identification of nanoparticles in the biological samples are discussed.

Optimizing pulse compressibility in completely all-fibered Ytterbium chirped pulse amplifiers for in vivo two photon laser scanning microscopy

PubMed Central

Fernández, A.; Grüner-Nielsen, L.; Andreana, M.; Stadler, M.; Kirchberger, S.; Sturtzel, C.; Distel, M.; Zhu, L.; Kautek, W.; Leitgeb, R.; Baltuska, A.; Jespersen, K.; Verhoef, A.

2017-01-01

A simple and completely all-fiber Yb chirped pulse amplifier that uses a dispersion matched fiber stretcher and a spliced-on hollow core photonic bandgap fiber compressor is applied in nonlinear optical microscopy. This stretching-compression approach improves compressibility and helps to maximize the fluorescence signal in two-photon laser scanning microscopy as compared with approaches that use standard single mode fibers as stretcher. We also show that in femtosecond all-fiber systems, compensation of higher order dispersion terms is relevant even for pulses with relatively narrow bandwidths for applications relying on nonlinear optical effects. The completely all-fiber system was applied to image green fluorescent beads, a stained lily-of-the-valley root and rat-tail tendon. We also demonstrated in vivo imaging in zebrafish larvae, where we simultaneously measure second harmonic and fluorescence from two-photon excited red-fluorescent protein. Since the pulses are compressed in a fiber, this source is especially suited for upgrading existing laser scanning (confocal) microscopes with multiphoton imaging capabilities in space restricted settings or for incorporation in endoscope-based microscopy. PMID:28856032

Low-cost mobile phone microscopy with a reversed mobile phone camera lens.

PubMed

Switz, Neil A; D'Ambrosio, Michael V; Fletcher, Daniel A

2014-01-01

The increasing capabilities and ubiquity of mobile phones and their associated digital cameras offer the possibility of extending low-cost, portable diagnostic microscopy to underserved and low-resource areas. However, mobile phone microscopes created by adding magnifying optics to the phone's camera module have been unable to make use of the full image sensor due to the specialized design of the embedded camera lens, exacerbating the tradeoff between resolution and field of view inherent to optical systems. This tradeoff is acutely felt for diagnostic applications, where the speed and cost of image-based diagnosis is related to the area of the sample that can be viewed at sufficient resolution. Here we present a simple and low-cost approach to mobile phone microscopy that uses a reversed mobile phone camera lens added to an intact mobile phone to enable high quality imaging over a significantly larger field of view than standard microscopy. We demonstrate use of the reversed lens mobile phone microscope to identify red and white blood cells in blood smears and soil-transmitted helminth eggs in stool samples.

Review of combined isotopic and optical nanoscopy

PubMed Central

Richter, Katharina N.; Rizzoli, Silvio O.; Jähne, Sebastian; Vogts, Angela; Lovric, Jelena

2017-01-01

Abstract. Investigating the detailed substructure of the cell is beyond the ability of conventional optical microscopy. Electron microscopy, therefore, has been the only option for such studies for several decades. The recent implementation of several super-resolution optical microscopy techniques has rendered the investigation of cellular substructure easier and more efficient. Nevertheless, optical microscopy only provides an image of the present structure of the cell, without any information on its long-temporal changes. These can be investigated by combining super-resolution optics with a nonoptical imaging technique, nanoscale secondary ion mass spectrometry, which investigates the isotopic composition of the samples. The resulting technique, combined isotopic and optical nanoscopy, enables the investigation of both the structure and the “history” of the cellular elements. The age and the turnover of cellular organelles can be read by isotopic imaging, while the structure can be analyzed by optical (fluorescence) approaches. We present these technologies, and we discuss their implementation for the study of biological samples. We conclude that, albeit complex, this type of technology is reliable enough for mass application to cell biology. PMID:28466025

Low-Cost Mobile Phone Microscopy with a Reversed Mobile Phone Camera Lens

PubMed Central

Fletcher, Daniel A.

2014-01-01

The increasing capabilities and ubiquity of mobile phones and their associated digital cameras offer the possibility of extending low-cost, portable diagnostic microscopy to underserved and low-resource areas. However, mobile phone microscopes created by adding magnifying optics to the phone's camera module have been unable to make use of the full image sensor due to the specialized design of the embedded camera lens, exacerbating the tradeoff between resolution and field of view inherent to optical systems. This tradeoff is acutely felt for diagnostic applications, where the speed and cost of image-based diagnosis is related to the area of the sample that can be viewed at sufficient resolution. Here we present a simple and low-cost approach to mobile phone microscopy that uses a reversed mobile phone camera lens added to an intact mobile phone to enable high quality imaging over a significantly larger field of view than standard microscopy. We demonstrate use of the reversed lens mobile phone microscope to identify red and white blood cells in blood smears and soil-transmitted helminth eggs in stool samples. PMID:24854188

Quantitative Aspects of Single Molecule Microscopy

PubMed Central

Ober, Raimund J.; Tahmasbi, Amir; Ram, Sripad; Lin, Zhiping; Ward, E. Sally

2015-01-01

Single molecule microscopy is a relatively new optical microscopy technique that allows the detection of individual molecules such as proteins in a cellular context. This technique has generated significant interest among biologists, biophysicists and biochemists, as it holds the promise to provide novel insights into subcellular processes and structures that otherwise cannot be gained through traditional experimental approaches. Single molecule experiments place stringent demands on experimental and algorithmic tools due to the low signal levels and the presence of significant extraneous noise sources. Consequently, this has necessitated the use of advanced statistical signal and image processing techniques for the design and analysis of single molecule experiments. In this tutorial paper, we provide an overview of single molecule microscopy from early works to current applications and challenges. Specific emphasis will be on the quantitative aspects of this imaging modality, in particular single molecule localization and resolvability, which will be discussed from an information theoretic perspective. We review the stochastic framework for image formation, different types of estimation techniques and expressions for the Fisher information matrix. We also discuss several open problems in the field that demand highly non-trivial signal processing algorithms. PMID:26167102

Optimizing pulse compressibility in completely all-fibered Ytterbium chirped pulse amplifiers for in vivo two photon laser scanning microscopy.

PubMed

Fernández, A; Grüner-Nielsen, L; Andreana, M; Stadler, M; Kirchberger, S; Sturtzel, C; Distel, M; Zhu, L; Kautek, W; Leitgeb, R; Baltuska, A; Jespersen, K; Verhoef, A

2017-08-01

A simple and completely all-fiber Yb chirped pulse amplifier that uses a dispersion matched fiber stretcher and a spliced-on hollow core photonic bandgap fiber compressor is applied in nonlinear optical microscopy. This stretching-compression approach improves compressibility and helps to maximize the fluorescence signal in two-photon laser scanning microscopy as compared with approaches that use standard single mode fibers as stretcher. We also show that in femtosecond all-fiber systems, compensation of higher order dispersion terms is relevant even for pulses with relatively narrow bandwidths for applications relying on nonlinear optical effects. The completely all-fiber system was applied to image green fluorescent beads, a stained lily-of-the-valley root and rat-tail tendon. We also demonstrated in vivo imaging in zebrafish larvae, where we simultaneously measure second harmonic and fluorescence from two-photon excited red-fluorescent protein. Since the pulses are compressed in a fiber, this source is especially suited for upgrading existing laser scanning (confocal) microscopes with multiphoton imaging capabilities in space restricted settings or for incorporation in endoscope-based microscopy.

Femtosecond few- to single-electron point-projection microscopy for nanoscale dynamic imaging

PubMed Central

Bainbridge, A. R.; Barlow Myers, C. W.; Bryan, W. A.

2016-01-01

Femtosecond electron microscopy produces real-space images of matter in a series of ultrafast snapshots. Pulses of electrons self-disperse under space-charge broadening, so without compression, the ideal operation mode is a single electron per pulse. Here, we demonstrate femtosecond single-electron point projection microscopy (fs-ePPM) in a laser-pump fs-e-probe configuration. The electrons have an energy of only 150 eV and take tens of picoseconds to propagate to the object under study. Nonetheless, we achieve a temporal resolution with a standard deviation of 114 fs (equivalent to a full-width at half-maximum of 269 ± 40 fs) combined with a spatial resolution of 100 nm, applied to a localized region of charge at the apex of a nanoscale metal tip induced by 30 fs 800 nm laser pulses at 50 kHz. These observations demonstrate real-space imaging of reversible processes, such as tracking charge distributions, is feasible whilst maintaining femtosecond resolution. Our findings could find application as a characterization method, which, depending on geometry, could resolve tens of femtoseconds and tens of nanometres. Dynamically imaging electric and magnetic fields and charge distributions on sub-micron length scales opens new avenues of ultrafast dynamics. Furthermore, through the use of active compression, such pulses are an ideal seed for few-femtosecond to attosecond imaging applications which will access sub-optical cycle processes in nanoplasmonics. PMID:27158637

Accurate Construction of Photoactivated Localization Microscopy (PALM) Images for Quantitative Measurements

PubMed Central

Coltharp, Carla; Kessler, Rene P.; Xiao, Jie

2012-01-01

Localization-based superresolution microscopy techniques such as Photoactivated Localization Microscopy (PALM) and Stochastic Optical Reconstruction Microscopy (STORM) have allowed investigations of cellular structures with unprecedented optical resolutions. One major obstacle to interpreting superresolution images, however, is the overcounting of molecule numbers caused by fluorophore photoblinking. Using both experimental and simulated images, we determined the effects of photoblinking on the accurate reconstruction of superresolution images and on quantitative measurements of structural dimension and molecule density made from those images. We found that structural dimension and relative density measurements can be made reliably from images that contain photoblinking-related overcounting, but accurate absolute density measurements, and consequently faithful representations of molecule counts and positions in cellular structures, require the application of a clustering algorithm to group localizations that originate from the same molecule. We analyzed how applying a simple algorithm with different clustering thresholds (tThresh and dThresh) affects the accuracy of reconstructed images, and developed an easy method to select optimal thresholds. We also identified an empirical criterion to evaluate whether an imaging condition is appropriate for accurate superresolution image reconstruction with the clustering algorithm. Both the threshold selection method and imaging condition criterion are easy to implement within existing PALM clustering algorithms and experimental conditions. The main advantage of our method is that it generates a superresolution image and molecule position list that faithfully represents molecule counts and positions within a cellular structure, rather than only summarizing structural properties into ensemble parameters. This feature makes it particularly useful for cellular structures of heterogeneous densities and irregular geometries, and allows a variety of quantitative measurements tailored to specific needs of different biological systems. PMID:23251611

Self-phase modulation and two-photon absorption imaging of cells and active neurons

NASA Astrophysics Data System (ADS)

Fischer, Martin C.; Liu, Henry; Piletic, Ivan R.; Ye, Tong; Yasuda, Ryohei; Warren, Warren S.

2007-02-01

Even though multi-photon fluorescence microscopy offers higher resolution and better penetration depth than traditional fluorescence microscopy, its use is restricted to the detection of molecules that fluoresce. Two-photon absorption (TPA) imaging can provide contrast in non-fluorescent molecules while retaining the high resolution and sectioning capabilities of nonlinear imaging modalities. In the long-wavelength water window, tissue TPA is dominated by the endogenous molecules melanin and hemoglobin with an almost complete absence of endogenous two-photon fluorescence. A complementary nonlinear contrast mechanism is self-phase modulation (SPM), which can provide intrinsic signatures that can depend on local tissue anisotropy, chemical environment, or other structural properties. We have developed a spectral hole refilling measurement technique for TPA and SPM measurements using shaped ultrafast laser pulses. Here we report on a microscopy setup to simultaneously acquire 3D, high-resolution TPA and SPM images. We have acquired data in mounted B16 melanoma cells with very modest laser power levels. We will also discuss the possible application of this measurement technique to neuronal imaging. Since SPM is sensitive to material structure we can expect SPM properties of neurons to change during neuronal firing. Using our hole-refilling technique we have now demonstrated strong novel intrinsic nonlinear signatures of neuronal activation in a hippocampal brain slice. The observed changes in nonlinear signal upon collective activation were up to factors of two, unlike other intrinsic optical signal changes on the percent level. These results show that TPA and SPM imaging can provide important novel functional contrast in tissue using very modest power levels suitable for in vivo applications.

High-quality imaging in environmental scanning electron microscopy--optimizing the pressure limiting system and the secondary electron detection of a commercially available ESEM.

PubMed

Fitzek, H; Schroettner, H; Wagner, J; Hofer, F; Rattenberger, J

2016-04-01

In environmental scanning electron microscopy applications in the kPa regime are of increasing interest for the investigation of wet and biological samples, because neither sample preparation nor extensive cooling are necessary. Unfortunately, the applications are limited by poor image quality. In this work the image quality at high pressures of a FEI Quanta 600 (field emission gun) and a FEI Quanta 200 (thermionic gun) is greatly improved by optimizing the pressure limiting system and the secondary electron (SE) detection system. The scattering of the primary electron beam strongly increases with pressure and thus the image quality vanishes. The key to high-image quality at high pressures is to reduce scattering as far as possible while maintaining ideal operation conditions for the SE-detector. The amount of scattering is reduced by reducing both the additional stagnation gas thickness (aSGT) and the environmental distance (ED). A new aperture holder is presented that significantly reduces the aSGT while maintaining the same field-of-view (FOV) as the original design. With this aperture holder it is also possible to make the aSGT even smaller at the expense of a smaller FOV. A new blade-shaped SE-detector is presented yielding better image quality than usual flat SE-detectors. The electrode of the new SE detector is positioned on the sample table, which allows the SE-detector to operate at ideal conditions regardless of pressure and ED. © 2015 The Authors Journal of Microscopy © 2015 Royal Microscopical Society.

High-performance serial block-face SEM of nonconductive biological samples enabled by focal gas injection-based charge compensation.

PubMed

Deerinck, T J; Shone, T M; Bushong, E A; Ramachandra, R; Peltier, S T; Ellisman, M H

2018-05-01

A longstanding limitation of imaging with serial block-face scanning electron microscopy is specimen surface charging. This charging is largely due to the difficulties in making biological specimens and the resins in which they are embedded sufficiently conductive. Local accumulation of charge on the specimen surface can result in poor image quality and distortions. Even minor charging can lead to misalignments between sequential images of the block-face due to image jitter. Typically, variable-pressure SEM is used to reduce specimen charging, but this results in a significant reduction to spatial resolution, signal-to-noise ratio and overall image quality. Here we show the development and application of a simple system that effectively mitigates specimen charging by using focal gas injection of nitrogen over the sample block-face during imaging. A standard gas injection valve is paired with a precisely positioned but retractable application nozzle, which is mechanically coupled to the reciprocating action of the serial block-face ultramicrotome. This system enables the application of nitrogen gas precisely over the block-face during imaging while allowing the specimen chamber to be maintained under high vacuum to maximise achievable SEM image resolution. The action of the ultramicrotome drives the nozzle retraction, automatically moving it away from the specimen area during the cutting cycle of the knife. The device described was added to a Gatan 3View system with minimal modifications, allowing high-resolution block-face imaging of even the most charge prone of epoxy-embedded biological samples. © 2017 The Authors Journal of Microscopy © 2017 Royal Microscopical Society.

Adaptive platform for fluorescence microscopy-based high-content screening

NASA Astrophysics Data System (ADS)

Geisbauer, Matthias; Röder, Thorsten; Chen, Yang; Knoll, Alois; Uhl, Rainer

2010-04-01

Fluorescence microscopy has become a widely used tool for the study of medically relevant intra- and intercellular processes. Extracting meaningful information out of a bulk of acquired images is usually performed during a separate post-processing task. Thus capturing raw data results in an unnecessary huge number of images, whereas usually only a few images really show the particular information that is searched for. Here we propose a novel automated high-content microscope system, which enables experiments to be carried out with only a minimum of human interaction. It facilitates a huge speed-increase for cell biology research and its applications compared to the widely performed workflows. Our fluorescence microscopy system can automatically execute application-dependent data processing algorithms during the actual experiment. They are used for image contrast enhancement, cell segmentation and/or cell property evaluation. On-the-fly retrieved information is used to reduce data and concomitantly control the experiment process in real-time. Resulting in a closed loop of perception and action the system can greatly decrease the amount of stored data on one hand and increases the relative valuable data content on the other hand. We demonstrate our approach by addressing the problem of automatically finding cells with a particular combination of labeled receptors and then selectively stimulate them with antagonists or agonists. The results are then compared against the results of traditional, static systems.

Confocal fluorescence microscopy for rapid evaluation of invasive tumor cellularity of inflammatory breast carcinoma core needle biopsies.

PubMed

Dobbs, Jessica; Krishnamurthy, Savitri; Kyrish, Matthew; Benveniste, Ana Paula; Yang, Wei; Richards-Kortum, Rebecca

2015-01-01

Tissue sampling is a problematic issue for inflammatory breast carcinoma, and immediate evaluation following core needle biopsy is needed to evaluate specimen adequacy. We sought to determine if confocal fluorescence microscopy provides sufficient resolution to evaluate specimen adequacy by comparing invasive tumor cellularity estimated from standard histologic images to invasive tumor cellularity estimated from confocal images of breast core needle biopsy specimens. Grayscale confocal fluorescence images of breast core needle biopsy specimens were acquired following proflavine application. A breast-dedicated pathologist evaluated invasive tumor cellularity in histologic images with hematoxylin and eosin staining and in grayscale and false-colored confocal images of cores. Agreement between cellularity estimates was quantified using a kappa coefficient. 23 cores from 23 patients with suspected inflammatory breast carcinoma were imaged. Confocal images were acquired in an average of less than 2 min per core. Invasive tumor cellularity estimated from histologic and grayscale confocal images showed moderate agreement by kappa coefficient: κ = 0.48 ± 0.09 (p < 0.001). Grayscale confocal images require less than 2 min for acquisition and allow for evaluation of invasive tumor cellularity in breast core needle biopsy specimens with moderate agreement to histologic images. We show that confocal fluorescence microscopy can be performed immediately following specimen acquisition and could indicate the need for additional biopsies at the initial visit.

Whole-organ atlas imaged by label-free high-resolution photoacoustic microscopy assisted by a microtome

NASA Astrophysics Data System (ADS)

Wong, Terence T. W.; Zhang, Ruiying; Hsu, Hsun-Chia; Maslov, Konstantin I.; Shi, Junhui; Chen, Ruimin; Shung, K. Kirk; Zhou, Qifa; Wang, Lihong V.

2018-02-01

In biomedical imaging, all optical techniques face a fundamental trade-off between spatial resolution and tissue penetration. Therefore, obtaining an organelle-level resolution image of a whole organ has remained a challenging and yet appealing scientific pursuit. Over the past decade, optical microscopy assisted by mechanical sectioning or chemical clearing of tissue has been demonstrated as a powerful technique to overcome this dilemma, one of particular use in imaging the neural network. However, this type of techniques needs lengthy special preparation of the tissue specimen, which hinders broad application in life sciences. Here, we propose a new label-free three-dimensional imaging technique, named microtomy-assisted photoacoustic microscopy (mPAM), for potentially imaging all biomolecules with 100% endogenous natural staining in whole organs with high fidelity. We demonstrate the first label-free mPAM, using UV light for label-free histology-like imaging, in whole organs (e.g., mouse brains), most of them formalin-fixed and paraffin- or agarose-embedded for minimal morphological deformation. Furthermore, mPAM with dual wavelength illuminations is also employed to image a mouse brain slice, demonstrating the potential for imaging of multiple biomolecules without staining. With visible light illumination, mPAM also shows its deep tissue imaging capability, which enables less slicing and hence reduces sectioning artifacts. mPAM could potentially provide a new insight for understanding complex biological organs.

3D Actin Network Centerline Extraction with Multiple Active Contours

PubMed Central

Xu, Ting; Vavylonis, Dimitrios; Huang, Xiaolei

2013-01-01

Fluorescence microscopy is frequently used to study two and three dimensional network structures formed by cytoskeletal polymer fibers such as actin filaments and actin cables. While these cytoskeletal structures are often dilute enough to allow imaging of individual filaments or bundles of them, quantitative analysis of these images is challenging. To facilitate quantitative, reproducible and objective analysis of the image data, we propose a semi-automated method to extract actin networks and retrieve their topology in 3D. Our method uses multiple Stretching Open Active Contours (SOACs) that are automatically initialized at image intensity ridges and then evolve along the centerlines of filaments in the network. SOACs can merge, stop at junctions, and reconfigure with others to allow smooth crossing at junctions of filaments. The proposed approach is generally applicable to images of curvilinear networks with low SNR. We demonstrate its potential by extracting the centerlines of synthetic meshwork images, actin networks in 2D Total Internal Reflection Fluorescence Microscopy images, and 3D actin cable meshworks of live fission yeast cells imaged by spinning disk confocal microscopy. Quantitative evaluation of the method using synthetic images shows that for images with SNR above 5.0, the average vertex error measured by the distance between our result and ground truth is 1 voxel, and the average Hausdorff distance is below 10 voxels. PMID:24316442

Two-photon excitation fluorescence bioassays.

PubMed

Hänninen, Pekka; Soukka, Jori; Soini, Juhani T

2008-01-01

Application of two-photon excitation of fluorescence in microscopy is one of the major discoveries of the "renaissance" of light microscopy that started in the 1980s. The technique derives its advantages from the biologically "smooth" wavelength of the excitation light and the confinement of the excitation. Difficult, and seemingly nontransparent, samples may be imaged with the technique with good resolution. Although the bioresearch has been concentrating mostly on the positive properties of the technique for imaging, the same properties may be applied successfully to nonimaging bioassays. This article focuses on the development path of two-photon excitation-based assay system.

Nomarski differential interference contrast microscopy for surface slope measurements: an examination of techniques

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hartman, J.S.; Gordon, R.L.; Lessor, D.L.

1981-08-01

Alternate measurement and data analysis procedures are discussed and compared for the application of reflective Nomarski differential interference contrast microscopy for the determination of surface slopes. The discussion includes the interpretation of a previously reported iterative procedure using the results of a detailed optical model and the presentation of a new procedure based on measured image intensity extrema. Surface slope determinations from these procedures are presented and compared with results from a previously reported curve fit analysis of image intensity data. The accuracy and advantages of the different procedures are discussed.

Noninvasive characterization of the fission yeast cell cycle by monitoring dry mass with digital holographic microscopy.

PubMed

Rappaz, Benjamin; Cano, Elena; Colomb, Tristan; Kühn, Jonas; Depeursinge, Christian; Simanis, Viesturs; Magistretti, Pierre J; Marquet, Pierre

2009-01-01

Digital holography microscopy (DHM) is an optical technique which provides phase images yielding quantitative information about cell structure and cellular dynamics. Furthermore, the quantitative phase images allow the derivation of other parameters, including dry mass production, density, and spatial distribution. We have applied DHM to study the dry mass production rate and the dry mass surface density in wild-type and mutant fission yeast cells. Our study demonstrates the applicability of DHM as a tool for label-free quantitative analysis of the cell cycle and opens the possibility for its use in high-throughput screening.

The role of gas in determining image quality and resolution during in situ scanning transmission electron microscopy experiments

DOE PAGES

Zhu, Yuanyuan; Browning, Nigel D.

2017-05-24

As gas-solid heterogeneous catalytic reactions are molecular in nature, a full mechanistic understanding of the process requires atomic scale characterization under realistic operating conditions. While atomic resolution imaging has become a routine in modern high-vacuum (scanning) transmission electron microscopy ((S)TEM), both image quality and resolution nominally degrade when reaction gases are introduced. In this work, we systematically assess the effects of different gases at various pressures on the quality and resolution of images obtained at room temperature in the annular dark field STEM imaging mode using a differentially pumped (DP) gas cell. This imaging mode is largely free from inelasticmore » scattering effects induced by the presence of gases and retains good imaging properties over a wide range of gas mass/pressures. Furthermore, we demonstrate the application of the ESTEM with atomic resolution images of a complex oxide alkane oxidation catalyst MoVNbTeOx (M1) immersed in light and heavy gas environments.« less

EnLightenment: High resolution smartphone microscopy as an educational and public engagement platform.

PubMed

Wicks, Laura C; Cairns, Gemma S; Melnyk, Jacob; Bryce, Scott; Duncan, Rory R; Dalgarno, Paul A

2017-01-01

We developed a simple, cost-effective smartphone microscopy platform for use in educational and public engagement programs. We demonstrated its effectiveness, and potential for citizen science through a national imaging initiative, EnLightenment . The cost effectiveness of the instrument allowed for the program to deliver over 500 microscopes to more than 100 secondary schools throughout Scotland, targeting 1000's of 12-14 year olds. Through careful, quantified, selection of a high power, low-cost objective lens, our smartphone microscope has an imaging resolution of microns, with a working distance of 3 mm. It is therefore capable of imaging single cells and sub-cellular features, and retains usability for young children. The microscopes were designed in kit form and provided an interdisciplinary educational tool. By providing full lesson plans and support material, we developed a framework to explore optical design, microscope performance, engineering challenges on construction and real-world applications in life sciences, biological imaging, marine biology, art, and technology. A national online imaging competition framed EnLightenment ; with over 500 high quality images submitted of diverse content, spanning multiple disciplines. With examples of cellular and sub-cellular features clearly identifiable in some submissions, we show how young public can use these instruments for research-level imaging applications, and the potential of the instrument for citizen science programs.

Application of two-dimensional crystallography and image processing to atomic resolution Z-contrast images.

PubMed

Morgan, David G; Ramasse, Quentin M; Browning, Nigel D

2009-06-01

Zone axis images recorded using high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM or Z-contrast imaging) reveal the atomic structure with a resolution that is defined by the probe size of the microscope. In most cases, the full images contain many sub-images of the crystal unit cell and/or interface structure. Thanks to the repetitive nature of these images, it is possible to apply standard image processing techniques that have been developed for the electron crystallography of biological macromolecules and have been used widely in other fields of electron microscopy for both organic and inorganic materials. These methods can be used to enhance the signal-to-noise present in the original images, to remove distortions in the images that arise from either the instrumentation or the specimen itself and to quantify properties of the material in ways that are difficult without such data processing. In this paper, we describe briefly the theory behind these image processing techniques and demonstrate them for aberration-corrected, high-resolution HAADF-STEM images of Si(46) clathrates developed for hydrogen storage.

Digital micromirror devices: principles and applications in imaging.

PubMed

Bansal, Vivek; Saggau, Peter

2013-05-01

A digital micromirror device (DMD) is an array of individually switchable mirrors that can be used in many advanced optical systems as a rapid spatial light modulator. With a DMD, several implementations of confocal microscopy, hyperspectral imaging, and fluorescence lifetime imaging can be realized. The DMD can also be used as a real-time optical processor for applications such as the programmable array microscope and compressive sensing. Advantages and disadvantages of the DMD for these applications as well as methods to overcome some of the limitations will be discussed in this article. Practical considerations when designing with the DMD and sample optical layouts of a completely DMD-based imaging system and one in which acousto-optic deflectors (AODs) are used in the illumination pathway are also provided.

Development of multifunctional nanoparticles towards applications in non-invasive magnetic resonance imaging and axonal tracing.

PubMed

Du, Yan; Qin, Yubo; Li, Zizhen; Yang, Xiuying; Zhang, Jingchang; Westwick, Harrison; Tsai, Eve; Cao, Xudong

2017-12-01

A multifunctional nanobiomaterial has been developed by deliberately combining functions of superparamagnetism, fluorescence, and axonal tracing into one material. Superparamagnetic iron oxide nanoparticles were first synthesized and coated with a silica layer to prevent emission quenching through core-dye interactions; a fluorescent molecule, fluorescein isothiocyanate, was doped inside second layer of silica shell to improve photo-stability and to enable further thiol functionalization. Subsequently, biotinylated dextran amine, a sensitive axonal tracing reagent, was immobilized on the thiol-functionalized nanoparticle surfaces. The resulting nanoparticles were characterized by transmission electron microscopy, dynamic light scattering, X-ray diffraction, X-ray photoelectron spectroscopy, UV-Vis spectroscopy, magnetic resonance imaging and fluorescence confocal microscopy. In vitro cell experiments using both undifferentiated and differentiated Neuro-2a cells showed that the cells were able to take up the nanoparticles intracellularly and that the nanoparticles showed good biocompatibilities. In summary, this new material demonstrated promising performances for both optical and magnetic resonance imaging modalities, suggesting its promising potentials in applications such as in non-invasive imaging, particularly in neuronal tracing.

The application of atomic force microscopy in mineral flotation.

PubMed

Xing, Yaowen; Xu, Mengdi; Gui, Xiahui; Cao, Yijun; Babel, Bent; Rudolph, Martin; Weber, Stefan; Kappl, Michael; Butt, Hans-Jürgen

2018-06-01

During the past years, atomic force microscopy (AFM) has matured to an indispensable tool to characterize nanomaterials in colloid and interface science. For imaging, a sharp probe mounted near to the end of a cantilever scans over the sample surface providing a high resolution three-dimensional topographic image. In addition, the AFM tip can be used as a force sensor to detect local properties like adhesion, stiffness, charge etc. After the invention of the colloidal probe technique it has also become a major method to measure surface forces. In this review, we highlight the advances in the application of AFM in the field of mineral flotation, such as mineral morphology imaging, water at mineral surface, reagent adsorption, inter-particle force, and bubble-particle interaction. In the coming years, the complementary characterization of chemical composition such as using infrared spectroscopy and Raman spectroscopy for AFM topography imaging and the synchronous measurement of the force and distance involving deformable bubble as a force sensor will further assist the fundamental understanding of flotation mechanism. Copyright © 2018 Elsevier B.V. All rights reserved.

Peculiarities of studying an isolated neuron by the method of laser interference microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yusipovich, Alexander I; Kazakova, Tatiana A; Erokhova, Liudmila A

2006-09-30

Actual aspects of using a new method of laser interference microscopy (LIM) for studying nerve cells are discussed. The peculiarities of the LIM display of neurons are demonstrated by the example of isolated neurons of a pond snail Lymnaea stagnalis. A comparative analysis of the images of the cell and subcellular structures of a neuron obtained by the methods of interference microscopy, optical transmission microscopy, and confocal microscopy is performed. Various aspects of the application of LIM for studying the lateral dimensions and internal structure of the cytoplasm and organelles of a neuron in cytology and cell physiology are discussed.more » (laser biology)« less

3D Image Analysis of Geomaterials using Confocal Microscopy

NASA Astrophysics Data System (ADS)

Mulukutla, G.; Proussevitch, A.; Sahagian, D.

2009-05-01

Confocal microscopy is one of the most significant advances in optical microscopy of the last century. It is widely used in biological sciences but its application to geomaterials lingers due to a number of technical problems. Potentially the technique can perform non-invasive testing on a laser illuminated sample that fluoresces using a unique optical sectioning capability that rejects out-of-focus light reaching the confocal aperture. Fluorescence in geomaterials is commonly induced using epoxy doped with a fluorochrome that is impregnated into the sample to enable discrimination of various features such as void space or material boundaries. However, for many geomaterials, this method cannot be used because they do not naturally fluoresce and because epoxy cannot be impregnated into inaccessible parts of the sample due to lack of permeability. As a result, the confocal images of most geomaterials that have not been pre-processed with extensive sample preparation techniques are of poor quality and lack the necessary image and edge contrast necessary to apply any commonly used segmentation techniques to conduct any quantitative study of its features such as vesicularity, internal structure, etc. In our present work, we are developing a methodology to conduct a quantitative 3D analysis of images of geomaterials collected using a confocal microscope with minimal amount of prior sample preparation and no addition of fluorescence. Two sample geomaterials, a volcanic melt sample and a crystal chip containing fluid inclusions are used to assess the feasibility of the method. A step-by-step process of image analysis includes application of image filtration to enhance the edges or material interfaces and is based on two segmentation techniques: geodesic active contours and region competition. Both techniques have been applied extensively to the analysis of medical MRI images to segment anatomical structures. Preliminary analysis suggests that there is distortion in the shapes of the segmented vesicles, vapor bubbles, and void spaces due to the optical measurements, so corrective actions are being explored. This will establish a practical and reliable framework for an adaptive 3D image processing technique for the analysis of geomaterials using confocal microscopy.

Video-rate in vivo fluorescence imaging with a line-scanned dual-axis confocal microscope.

PubMed

Chen, Ye; Wang, Danni; Khan, Altaz; Wang, Yu; Borwege, Sabine; Sanai, Nader; Liu, Jonathan T C

2015-10-01

Video-rate optical-sectioning microscopy of living organisms would allow for the investigation of dynamic biological processes and would also reduce motion artifacts, especially for in vivo imaging applications. Previous feasibility studies, with a slow stage-scanned line-scanned dual-axis confocal (LS-DAC) microscope, have demonstrated that LS-DAC microscopy is capable of imaging tissues with subcellular resolution and high contrast at moderate depths of up to several hundred microns. However, the sensitivity and performance of a video-rate LS-DAC imaging system, with low-numerical aperture optics, have yet to be demonstrated. Here, we report on the construction and validation of a video-rate LS-DAC system that possesses sufficient sensitivity to visualize fluorescent contrast agents that are topically applied or systemically delivered in animal and human tissues. We present images of murine oral mucosa that are topically stained with methylene blue, and images of protoporphyrin IX-expressing brain tumor from glioma patients that have been administered 5-aminolevulinic acid prior to surgery. In addition, we demonstrate in vivo fluorescence imaging of red blood cells trafficking within the capillaries of a mouse ear, at frame rates of up to 30 fps. These results can serve as a benchmark for miniature in vivo microscopy devices under development.

Video-rate in vivo fluorescence imaging with a line-scanned dual-axis confocal microscope

NASA Astrophysics Data System (ADS)

Chen, Ye; Wang, Danni; Khan, Altaz; Wang, Yu; Borwege, Sabine; Sanai, Nader; Liu, Jonathan T. C.

2015-10-01

Video-rate optical-sectioning microscopy of living organisms would allow for the investigation of dynamic biological processes and would also reduce motion artifacts, especially for in vivo imaging applications. Previous feasibility studies, with a slow stage-scanned line-scanned dual-axis confocal (LS-DAC) microscope, have demonstrated that LS-DAC microscopy is capable of imaging tissues with subcellular resolution and high contrast at moderate depths of up to several hundred microns. However, the sensitivity and performance of a video-rate LS-DAC imaging system, with low-numerical aperture optics, have yet to be demonstrated. Here, we report on the construction and validation of a video-rate LS-DAC system that possesses sufficient sensitivity to visualize fluorescent contrast agents that are topically applied or systemically delivered in animal and human tissues. We present images of murine oral mucosa that are topically stained with methylene blue, and images of protoporphyrin IX-expressing brain tumor from glioma patients that have been administered 5-aminolevulinic acid prior to surgery. In addition, we demonstrate in vivo fluorescence imaging of red blood cells trafficking within the capillaries of a mouse ear, at frame rates of up to 30 fps. These results can serve as a benchmark for miniature in vivo microscopy devices under development.

Global gray-level thresholding based on object size.

PubMed

Ranefall, Petter; Wählby, Carolina

2016-04-01

In this article, we propose a fast and robust global gray-level thresholding method based on object size, where the selection of threshold level is based on recall and maximum precision with regard to objects within a given size interval. The method relies on the component tree representation, which can be computed in quasi-linear time. Feature-based segmentation is especially suitable for biomedical microscopy applications where objects often vary in number, but have limited variation in size. We show that for real images of cell nuclei and synthetic data sets mimicking fluorescent spots the proposed method is more robust than all standard global thresholding methods available for microscopy applications in ImageJ and CellProfiler. The proposed method, provided as ImageJ and CellProfiler plugins, is simple to use and the only required input is an interval of the expected object sizes. © 2016 International Society for Advancement of Cytometry. © 2016 International Society for Advancement of Cytometry.

Nucleocytoplasmic shuttling: the ins and outs of quantitative imaging.

PubMed

Molenaar, Chris; Weeks, Kate L

2018-05-17

Nucleocytoplasmic protein shuttling is integral to the transmission of signals between the nucleus and the cytoplasm. The nuclear/cytoplasmic distribution of proteins of interest can be determined via fluorescence microscopy, following labelling of the target protein with fluorophore-conjugated antibodies (immunofluorescence) or by tagging the target protein with an autofluorescent protein, such as green fluorescent protein (GFP). The latter enables live cell imaging, a powerful approach that precludes many of the artefacts associated with indirect immunofluorescence in fixed cells. In this review, we discuss important considerations for the design and implementation of fluorescence microscopy experiments to quantify the nuclear/cytoplasmic distribution of a protein of interest. We summarise the pros and cons of detecting endogenous proteins in fixed cells by immunofluorescence and ectopically-expressed fluorescent fusion proteins in living cells. We discuss the suitability of widefield fluorescence microscopy and of 2D, 3D and 4D imaging by confocal microscopy for different applications, and describe two different methods for quantifying the nuclear/cytoplasmic distribution of a protein of interest from the fluorescent signal. Finally, we discuss the importance of eliminating sources of bias and subjectivity during image acquisition and post-imaging analyses. This is critical for the accurate and reliable quantification of nucleocytoplasmic shuttling. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Classification of cryo electron microscopy images, noisy tomographic images recorded with unknown projection directions, by simultaneously estimating reconstructions and application to an assembly mutant of Cowpea Chlorotic Mottle Virus and portals of the bacteriophage P22

NASA Astrophysics Data System (ADS)

Lee, Junghoon; Zheng, Yili; Yin, Zhye; Doerschuk, Peter C.; Johnson, John E.

2010-08-01

Cryo electron microscopy is frequently used on biological specimens that show a mixture of different types of object. Because the electron beam rapidly destroys the specimen, the beam current is minimized which leads to noisy images (SNR substantially less than 1) and only one projection image per object (with an unknown projection direction) is collected. For situations where the objects can reasonably be described as coming from a finite set of classes, an approach based on joint maximum likelihood estimation of the reconstruction of each class and then use of the reconstructions to label the class of each image is described and demonstrated on two challenging problems: an assembly mutant of Cowpea Chlorotic Mottle Virus and portals of the bacteriophage P22.

Knowledge Extraction from Atomically Resolved Images.

PubMed

Vlcek, Lukas; Maksov, Artem; Pan, Minghu; Vasudevan, Rama K; Kalinin, Sergei V

2017-10-24

Tremendous strides in experimental capabilities of scanning transmission electron microscopy and scanning tunneling microscopy (STM) over the past 30 years made atomically resolved imaging routine. However, consistent integration and use of atomically resolved data with generative models is unavailable, so information on local thermodynamics and other microscopic driving forces encoded in the observed atomic configurations remains hidden. Here, we present a framework based on statistical distance minimization to consistently utilize the information available from atomic configurations obtained from an atomically resolved image and extract meaningful physical interaction parameters. We illustrate the applicability of the framework on an STM image of a FeSe x Te 1-x superconductor, with the segregation of the chalcogen atoms investigated using a nonideal interacting solid solution model. This universal method makes full use of the microscopic degrees of freedom sampled in an atomically resolved image and can be extended via Bayesian inference toward unbiased model selection with uncertainty quantification.

AutoMicromanager: A microscopy scripting toolkit for LABVIEW and other programming environments

NASA Astrophysics Data System (ADS)

Ashcroft, Brian Alan; Oosterkamp, Tjerk

2010-11-01

We present a scripting toolkit for the acquisition and analysis of a wide variety of imaging data by integrating the ease of use of various programming environments such as LABVIEW, IGOR PRO, MATLAB, SCILAB, and others. This toolkit is designed to allow the user to quickly program a variety of standard microscopy components for custom microscopy applications allowing much more flexibility than other packages. Included are both programming tools as well as graphical user interface classes allowing a standard, consistent, and easy to maintain scripting environment. This programming toolkit allows easy access to most commonly used cameras, stages, and shutters through the Micromanager project so the scripter can focus on their custom application instead of boilerplate code generation.

AutoMicromanager: a microscopy scripting toolkit for LABVIEW and other programming environments.

PubMed

Ashcroft, Brian Alan; Oosterkamp, Tjerk

2010-11-01

We present a scripting toolkit for the acquisition and analysis of a wide variety of imaging data by integrating the ease of use of various programming environments such as LABVIEW, IGOR PRO, MATLAB, SCILAB, and others. This toolkit is designed to allow the user to quickly program a variety of standard microscopy components for custom microscopy applications allowing much more flexibility than other packages. Included are both programming tools as well as graphical user interface classes allowing a standard, consistent, and easy to maintain scripting environment. This programming toolkit allows easy access to most commonly used cameras, stages, and shutters through the Micromanager project so the scripter can focus on their custom application instead of boilerplate code generation.

EFM data mapped into 2D images of tip-sample contact potential difference and capacitance second derivative.

PubMed

Lilliu, S; Maragliano, C; Hampton, M; Elliott, M; Stefancich, M; Chiesa, M; Dahlem, M S; Macdonald, J E

2013-11-27

We report a simple technique for mapping Electrostatic Force Microscopy (EFM) bias sweep data into 2D images. The method allows simultaneous probing, in the same scanning area, of the contact potential difference and the second derivative of the capacitance between tip and sample, along with the height information. The only required equipment consists of a microscope with lift-mode EFM capable of phase shift detection. We designate this approach as Scanning Probe Potential Electrostatic Force Microscopy (SPP-EFM). An open-source MATLAB Graphical User Interface (GUI) for images acquisition, processing and analysis has been developed. The technique is tested with Indium Tin Oxide (ITO) and with poly(3-hexylthiophene) (P3HT) nanowires for organic transistor applications.

New approaches for the analysis of confluent cell layers with quantitative phase digital holographic microscopy

NASA Astrophysics Data System (ADS)

Pohl, L.; Kaiser, M.; Ketelhut, S.; Pereira, S.; Goycoolea, F.; Kemper, Björn

2016-03-01

Digital holographic microscopy (DHM) enables high resolution non-destructive inspection of technical surfaces and minimally-invasive label-free live cell imaging. However, the analysis of confluent cell layers represents a challenge as quantitative DHM phase images in this case do not provide sufficient information for image segmentation, determination of the cellular dry mass or calculation of the cell thickness. We present novel strategies for the analysis of confluent cell layers with quantitative DHM phase contrast utilizing a histogram based-evaluation procedure. The applicability of our approach is illustrated by quantification of drug induced cell morphology changes and it is shown that the method is capable to quantify reliable global morphology changes of confluent cell layers.

Characterization of a reflective objective with multiphoton microscopy

NASA Astrophysics Data System (ADS)

Kabir, Mohammad M.; Choubal, Aakash M.; Sivaguru, Mayandi; Toussaint, Kimani C.

2018-02-01

Reflective objectives (ROs) can reduce chromatic aberration across a wide wavelength range in multiphoton microscopy (MPM). However, a systematic characterization of the performance of ROs has not been carried out. In this paper, we analyze the performance of a 0.5 numerical-aperture (NA) RO and compare it with a 0.55 NA standard glass objective (SO), using two-photon fluorescence (TPF) and second-harmonic generation (SHG). For experiments extending 1 octave in visible and NIR wavelengths, the SO introduces defocusing errors of 25% for TPF images of sub-diffraction fluorescent beads and 10% for SHG images of collagen fibers. For both imaging systems, the RO provides a corresponding error of 4%. This work highlights the potential usefulness of ROs for multimodal MPM applications.

Visible continuum pulses based on enhanced dispersive wave generation for endogenous fluorescence imaging.

PubMed

Cui, Quan; Chen, Zhongyun; Liu, Qian; Zhang, Zhihong; Luo, Qingming; Fu, Ling

2017-09-01

In this study, we demonstrate endogenous fluorescence imaging using visible continuum pulses based on 100-fs Ti:sapphire oscillator and a nonlinear photonic crystal fiber. Broadband (500-700 nm) and high-power (150 mW) continuum pulses are generated through enhanced dispersive wave generation by pumping femtosecond pulses at the anomalous dispersion region near zero-dispersion wavelength of high-nonlinear photonic crystal fibers. We also minimize the continuum pulse width by determining the proper fiber length. The visible-wavelength two-photon microscopy produces NADH and tryptophan images of mice tissues simultaneously. Our 500-700 nm continuum pulses support extending nonlinear microscopy to visible wavelength range that is inaccessible to 100-fs Ti:sapphire oscillators and other applications requiring visible laser pulses.

Multi-modal Registration for Correlative Microscopy using Image Analogies

PubMed Central

Cao, Tian; Zach, Christopher; Modla, Shannon; Powell, Debbie; Czymmek, Kirk; Niethammer, Marc

2014-01-01

Correlative microscopy is a methodology combining the functionality of light microscopy with the high resolution of electron microscopy and other microscopy technologies for the same biological specimen. In this paper, we propose an image registration method for correlative microscopy, which is challenging due to the distinct appearance of biological structures when imaged with different modalities. Our method is based on image analogies and allows to transform images of a given modality into the appearance-space of another modality. Hence, the registration between two different types of microscopy images can be transformed to a mono-modality image registration. We use a sparse representation model to obtain image analogies. The method makes use of corresponding image training patches of two different imaging modalities to learn a dictionary capturing appearance relations. We test our approach on backscattered electron (BSE) scanning electron microscopy (SEM)/confocal and transmission electron microscopy (TEM)/confocal images. We perform rigid, affine, and deformable registration via B-splines and show improvements over direct registration using both mutual information and sum of squared differences similarity measures to account for differences in image appearance. PMID:24387943

The multi-modal Australian ScienceS Imaging and Visualization Environment (MASSIVE) high performance computing infrastructure: applications in neuroscience and neuroinformatics research

PubMed Central

Goscinski, Wojtek J.; McIntosh, Paul; Felzmann, Ulrich; Maksimenko, Anton; Hall, Christopher J.; Gureyev, Timur; Thompson, Darren; Janke, Andrew; Galloway, Graham; Killeen, Neil E. B.; Raniga, Parnesh; Kaluza, Owen; Ng, Amanda; Poudel, Govinda; Barnes, David G.; Nguyen, Toan; Bonnington, Paul; Egan, Gary F.

2014-01-01

The Multi-modal Australian ScienceS Imaging and Visualization Environment (MASSIVE) is a national imaging and visualization facility established by Monash University, the Australian Synchrotron, the Commonwealth Scientific Industrial Research Organization (CSIRO), and the Victorian Partnership for Advanced Computing (VPAC), with funding from the National Computational Infrastructure and the Victorian Government. The MASSIVE facility provides hardware, software, and expertise to drive research in the biomedical sciences, particularly advanced brain imaging research using synchrotron x-ray and infrared imaging, functional and structural magnetic resonance imaging (MRI), x-ray computer tomography (CT), electron microscopy and optical microscopy. The development of MASSIVE has been based on best practice in system integration methodologies, frameworks, and architectures. The facility has: (i) integrated multiple different neuroimaging analysis software components, (ii) enabled cross-platform and cross-modality integration of neuroinformatics tools, and (iii) brought together neuroimaging databases and analysis workflows. MASSIVE is now operational as a nationally distributed and integrated facility for neuroinfomatics and brain imaging research. PMID:24734019

Fisher information theory for parameter estimation in single molecule microscopy: tutorial

PubMed Central

Chao, Jerry; Ward, E. Sally; Ober, Raimund J.

2016-01-01

Estimation of a parameter of interest from image data represents a task that is commonly carried out in single molecule microscopy data analysis. The determination of the positional coordinates of a molecule from its image, for example, forms the basis of standard applications such as single molecule tracking and localization-based superresolution image reconstruction. Assuming that the estimator used recovers, on average, the true value of the parameter, its accuracy, or standard deviation, is then at best equal to the square root of the Cramér-Rao lower bound. The Cramér-Rao lower bound can therefore be used as a benchmark in the evaluation of the accuracy of an estimator. Additionally, as its value can be computed and assessed for different experimental settings, it is useful as an experimental design tool. This tutorial demonstrates a mathematical framework that has been specifically developed to calculate the Cramér-Rao lower bound for estimation problems in single molecule microscopy and, more broadly, fluorescence microscopy. The material includes a presentation of the photon detection process that underlies all image data, various image data models that describe images acquired with different detector types, and Fisher information expressions that are necessary for the calculation of the lower bound. Throughout the tutorial, examples involving concrete estimation problems are used to illustrate the effects of various factors on the accuracy of parameter estimation, and more generally, to demonstrate the flexibility of the mathematical framework. PMID:27409706

Multimodal hyperspectral optical microscopy

DOE PAGES

Novikova, Irina V.; Smallwood, Chuck R.; Gong, Yu; ...

2017-09-02

We describe a unique and convenient approach to multimodal hyperspectral optical microscopy, herein achieved by coupling a portable and transferable hyperspectral imager to various optical microscopes. The experimental and data analysis schemes involved in recording spectrally and spatially resolved fluorescence, dark field, and optical absorption micrographs are illustrated through prototypical measurements targeting selected model systems. Namely, hyperspectral fluorescence micrographs of isolated fluorescent beads are employed to ensure spectral calibration of our detector and to gauge the attainable spatial resolution of our measurements; the recorded images are diffraction-limited. Moreover, spatially over-sampled absorption spectroscopy of a single lipid (18:1 Liss Rhod PE)more » layer reveals that optical densities on the order of 10-3 may be resolved by spatially averaging the recorded optical signatures. We also briefly illustrate two applications of our setup in the general areas of plasmonics and cell biology. Most notably, we deploy hyperspectral optical absorption microscopy to identify and image algal pigments within a single live Tisochrysis lutea cell. Overall, this work paves the way for multimodal multidimensional spectral imaging measurements spanning the realms of several scientific disciples.« less

Multifocus microscopy with precise color multi-phase diffractive optics applied in functional neuronal imaging.

PubMed

Abrahamsson, Sara; Ilic, Rob; Wisniewski, Jan; Mehl, Brian; Yu, Liya; Chen, Lei; Davanco, Marcelo; Oudjedi, Laura; Fiche, Jean-Bernard; Hajj, Bassam; Jin, Xin; Pulupa, Joan; Cho, Christine; Mir, Mustafa; El Beheiry, Mohamed; Darzacq, Xavier; Nollmann, Marcelo; Dahan, Maxime; Wu, Carl; Lionnet, Timothée; Liddle, J Alexander; Bargmann, Cornelia I

2016-03-01

Multifocus microscopy (MFM) allows high-resolution instantaneous three-dimensional (3D) imaging and has been applied to study biological specimens ranging from single molecules inside cells nuclei to entire embryos. We here describe pattern designs and nanofabrication methods for diffractive optics that optimize the light-efficiency of the central optical component of MFM: the diffractive multifocus grating (MFG). We also implement a "precise color" MFM layout with MFGs tailored to individual fluorophores in separate optical arms. The reported advancements enable faster and brighter volumetric time-lapse imaging of biological samples. In live microscopy applications, photon budget is a critical parameter and light-efficiency must be optimized to obtain the fastest possible frame rate while minimizing photodamage. We provide comprehensive descriptions and code for designing diffractive optical devices, and a detailed methods description for nanofabrication of devices. Theoretical efficiencies of reported designs is ≈90% and we have obtained efficiencies of > 80% in MFGs of our own manufacture. We demonstrate the performance of a multi-phase MFG in 3D functional neuronal imaging in living C. elegans.

Simultaneous fluorescence and quantitative phase microscopy with single-pixel detectors

NASA Astrophysics Data System (ADS)

Liu, Yang; Suo, Jinli; Zhang, Yuanlong; Dai, Qionghai

2018-02-01

Multimodal microscopy offers high flexibilities for biomedical observation and diagnosis. Conventional multimodal approaches either use multiple cameras or a single camera spatially multiplexing different modes. The former needs expertise demanding alignment and the latter suffers from limited spatial resolution. Here, we report an alignment-free full-resolution simultaneous fluorescence and quantitative phase imaging approach using single-pixel detectors. By combining reference-free interferometry with single-pixel detection, we encode the phase and fluorescence of the sample in two detection arms at the same time. Then we employ structured illumination and the correlated measurements between the sample and the illuminations for reconstruction. The recovered fluorescence and phase images are inherently aligned thanks to single-pixel detection. To validate the proposed method, we built a proof-of-concept setup for first imaging the phase of etched glass with the depth of a few hundred nanometers and then imaging the fluorescence and phase of the quantum dot drop. This method holds great potential for multispectral fluorescence microscopy with additional single-pixel detectors or a spectrometer. Besides, this cost-efficient multimodal system might find broad applications in biomedical science and neuroscience.

The application of Fresnel zone plate based projection in optofluidic microscopy.

PubMed

Wu, Jigang; Cui, Xiquan; Lee, Lap Man; Yang, Changhuei

2008-09-29

Optofluidic microscopy (OFM) is a novel technique for low-cost, high-resolution on-chip microscopy imaging. In this paper we report the use of the Fresnel zone plate (FZP) based projection in OFM as a cost-effective and compact means for projecting the transmission through an OFM's aperture array onto a sensor grid. We demonstrate this approach by employing a FZP (diameter = 255 microm, focal length = 800 microm) that has been patterned onto a glass slide to project the transmission from an array of apertures (diameter = 1 microm, separation = 10 microm) onto a CMOS sensor. We are able to resolve the contributions from 44 apertures on the sensor under the illumination from a HeNe laser (wavelength = 633 nm). The imaging quality of the FZP determines the effective field-of-view (related to the number of resolvable transmissions from apertures) but not the image resolution of such an OFM system--a key distinction from conventional microscope systems. We demonstrate the capability of the integrated system by flowing the protist Euglena gracilis across the aperture array microfluidically and performing OFM imaging of the samples.

Multimodal hyperspectral optical microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Novikova, Irina V.; Smallwood, Chuck R.; Gong, Yu

We describe a unique and convenient approach to multimodal hyperspectral optical microscopy, herein achieved by coupling a portable and transferable hyperspectral imager to various optical microscopes. The experimental and data analysis schemes involved in recording spectrally and spatially resolved fluorescence, dark field, and optical absorption micrographs are illustrated through prototypical measurements targeting selected model systems. Namely, hyperspectral fluorescence micrographs of isolated fluorescent beads are employed to ensure spectral calibration of our detector and to gauge the attainable spatial resolution of our measurements; the recorded images are diffraction-limited. Moreover, spatially over-sampled absorption spectroscopy of a single lipid (18:1 Liss Rhod PE)more » layer reveals that optical densities on the order of 10-3 may be resolved by spatially averaging the recorded optical signatures. We also briefly illustrate two applications of our setup in the general areas of plasmonics and cell biology. Most notably, we deploy hyperspectral optical absorption microscopy to identify and image algal pigments within a single live Tisochrysis lutea cell. Overall, this work paves the way for multimodal multidimensional spectral imaging measurements spanning the realms of several scientific disciples.« less

Development of carbon electrodes for electrochemistry, solid-state electronics and multimodal atomic force microscopy imaging

NASA Astrophysics Data System (ADS)

Morton, Kirstin Claire

Carbon is one of the most remarkable elements due to its wide abundance on Earth and its many allotropes, which include diamond and graphite. Many carbon allotropes are conductive and in recent decades scientists have discovered and synthesized many new forms of carbon, including graphene and carbon nanotubes. The work in this thesis specifically focuses on the fabrication and characterization of pyrolyzed parylene C (PPC), a conductive pyrocarbon, as an electrode material for diodes, as a conductive coating for atomic force microscopy (AFM) probes and as an ultramicroelectrode (UME) for the electrochemical interrogation of cellular systems in vitro. Herein, planar and three-dimensional (3D) PPC electrodes were microscopically, spectroscopically and electrochemically characterized. First, planar PPC films and PPC-coated nanopipettes were utilized to detect a model redox species, Ru(NH3) 6Cl3. Then, free-standing PPC thin films were chemically doped, with hydrazine and concentrated nitric acid, to yield p- and n-type carbon films. Doped PPC thin films were positioned in conjunction with doped silicon to create Schottky and p-n junction diodes for use in an alternating current half-wave rectifier circuit. Pyrolyzed parylene C has found particular merit as a 3D electrode coating of AFM probes. Current sensing-atomic force microscopy imaging in air of nanoscale metallic features was undertaken to demonstrate the electronic imaging applicability of PPC AFM probes. Upon further insulation with parylene C and modification with a focused ion beam, a PPC UME was microfabricated near the AFM probe apex and utilized for electrochemical imaging. Subsequently, scanning electrochemical microscopy-atomic force microscopy imaging was undertaken to electrochemically quantify and image the spatial location of dopamine exocytotic release, elicited mechanically via the AFM probe itself, from differentiated pheochromocytoma 12 cells in vitro.

A highly sensitive protocol for microscopy of alkyne lipids and fluorescently tagged or immunostained proteins[S

PubMed Central

Gaebler, Anne; Penno, Anke; Kuerschner, Lars; Thiele, Christoph

2016-01-01

The demand to study the cellular localization of specific lipids has led to recent advances in lipid probes and microscopy. Alkyne lipids bear a small, noninterfering tag and can be detected upon click reaction with an azide-coupled reporter. Fluorescent alkyne lipid imaging crucially depends on appropriate azide reporters and labeling protocols that allow for an efficient click reaction and therefore a sensitive detection. We synthesized several azide reporters with different spacer components and tested their suitability for alkyne lipid imaging in fixed cells. The implementation of a copper-chelating picolyl moiety into fluorescent or biotin-based azide reagents strongly increased the sensitivity of the imaging routine. We demonstrate the applicability and evaluate the performance of this approach using different lipid classes and experimental setups. As azide picolyl reporters allow for reduced copper catalyst concentrations, they also enable coimaging of alkyne lipids with multiple fluorescent proteins including enhanced green fluorescent protein. Alternatively, and as we also show, microscopy of alkyne lipids can be combined with protein detection by immunocytochemistry. In summary, we present a robust, sensitive, and highly versatile protocol for the labeling of alkyne lipids with azide-coupled reporters for fluorescence microscopy that can be combined with different protein detection and imaging techniques. PMID:27565170

Perspective: Differential dynamic microscopy extracts multi-scale activity in complex fluids and biological systems

NASA Astrophysics Data System (ADS)

Cerbino, Roberto; Cicuta, Pietro

2017-09-01

Differential dynamic microscopy (DDM) is a technique that exploits optical microscopy to obtain local, multi-scale quantitative information about dynamic samples, in most cases without user intervention. It is proving extremely useful in understanding dynamics in liquid suspensions, soft materials, cells, and tissues. In DDM, image sequences are analyzed via a combination of image differences and spatial Fourier transforms to obtain information equivalent to that obtained by means of light scattering techniques. Compared to light scattering, DDM offers obvious advantages, principally (a) simplicity of the setup; (b) possibility of removing static contributions along the optical path; (c) power of simultaneous different microscopy contrast mechanisms; and (d) flexibility of choosing an analysis region, analogous to a scattering volume. For many questions, DDM has also advantages compared to segmentation/tracking approaches and to correlation techniques like particle image velocimetry. The very straightforward DDM approach, originally demonstrated with bright field microscopy of aqueous colloids, has lately been used to probe a variety of other complex fluids and biological systems with many different imaging methods, including dark-field, differential interference contrast, wide-field, light-sheet, and confocal microscopy. The number of adopting groups is rapidly increasing and so are the applications. Here, we briefly recall the working principles of DDM, we highlight its advantages and limitations, we outline recent experimental breakthroughs, and we provide a perspective on future challenges and directions. DDM can become a standard primary tool in every laboratory equipped with a microscope, at the very least as a first bias-free automated evaluation of the dynamics in a system.

Large-scale Scanning Transmission Electron Microscopy (Nanotomy) of Healthy and Injured Zebrafish Brain.

PubMed

Kuipers, Jeroen; Kalicharan, Ruby D; Wolters, Anouk H G; van Ham, Tjakko J; Giepmans, Ben N G

2016-05-25

Large-scale 2D electron microscopy (EM), or nanotomy, is the tissue-wide application of nanoscale resolution electron microscopy. Others and we previously applied large scale EM to human skin pancreatic islets, tissue culture and whole zebrafish larvae(1-7). Here we describe a universally applicable method for tissue-scale scanning EM for unbiased detection of sub-cellular and molecular features. Nanotomy was applied to investigate the healthy and a neurodegenerative zebrafish brain. Our method is based on standardized EM sample preparation protocols: Fixation with glutaraldehyde and osmium, followed by epoxy-resin embedding, ultrathin sectioning and mounting of ultrathin-sections on one-hole grids, followed by post staining with uranyl and lead. Large-scale 2D EM mosaic images are acquired using a scanning EM connected to an external large area scan generator using scanning transmission EM (STEM). Large scale EM images are typically ~ 5 - 50 G pixels in size, and best viewed using zoomable HTML files, which can be opened in any web browser, similar to online geographical HTML maps. This method can be applied to (human) tissue, cross sections of whole animals as well as tissue culture(1-5). Here, zebrafish brains were analyzed in a non-invasive neuronal ablation model. We visualize within a single dataset tissue, cellular and subcellular changes which can be quantified in various cell types including neurons and microglia, the brain's macrophages. In addition, nanotomy facilitates the correlation of EM with light microscopy (CLEM)(8) on the same tissue, as large surface areas previously imaged using fluorescent microscopy, can subsequently be subjected to large area EM, resulting in the nano-anatomy (nanotomy) of tissues. In all, nanotomy allows unbiased detection of features at EM level in a tissue-wide quantifiable manner.

Large-scale Scanning Transmission Electron Microscopy (Nanotomy) of Healthy and Injured Zebrafish Brain

PubMed Central

Kuipers, Jeroen; Kalicharan, Ruby D.; Wolters, Anouk H. G.

2016-01-01

Large-scale 2D electron microscopy (EM), or nanotomy, is the tissue-wide application of nanoscale resolution electron microscopy. Others and we previously applied large scale EM to human skin pancreatic islets, tissue culture and whole zebrafish larvae1-7. Here we describe a universally applicable method for tissue-scale scanning EM for unbiased detection of sub-cellular and molecular features. Nanotomy was applied to investigate the healthy and a neurodegenerative zebrafish brain. Our method is based on standardized EM sample preparation protocols: Fixation with glutaraldehyde and osmium, followed by epoxy-resin embedding, ultrathin sectioning and mounting of ultrathin-sections on one-hole grids, followed by post staining with uranyl and lead. Large-scale 2D EM mosaic images are acquired using a scanning EM connected to an external large area scan generator using scanning transmission EM (STEM). Large scale EM images are typically ~ 5 - 50 G pixels in size, and best viewed using zoomable HTML files, which can be opened in any web browser, similar to online geographical HTML maps. This method can be applied to (human) tissue, cross sections of whole animals as well as tissue culture1-5. Here, zebrafish brains were analyzed in a non-invasive neuronal ablation model. We visualize within a single dataset tissue, cellular and subcellular changes which can be quantified in various cell types including neurons and microglia, the brain's macrophages. In addition, nanotomy facilitates the correlation of EM with light microscopy (CLEM)8 on the same tissue, as large surface areas previously imaged using fluorescent microscopy, can subsequently be subjected to large area EM, resulting in the nano-anatomy (nanotomy) of tissues. In all, nanotomy allows unbiased detection of features at EM level in a tissue-wide quantifiable manner. PMID:27285162

Three-Dimensional Reconstruction of Three-Way FRET Microscopy Improves Imaging of Multiple Protein-Protein Interactions.

PubMed

Scott, Brandon L; Hoppe, Adam D

2016-01-01

Fluorescence resonance energy transfer (FRET) microscopy is a powerful tool for imaging the interactions between fluorescently tagged proteins in two-dimensions. For FRET microscopy to reach its full potential, it must be able to image more than one pair of interacting molecules and image degradation from out-of-focus light must be reduced. Here we extend our previous work on the application of maximum likelihood methods to the 3-dimensional reconstruction of 3-way FRET interactions within cells. We validated the new method (3D-3Way FRET) by simulation and fluorescent protein test constructs expressed in cells. In addition, we improved the computational methods to create a 2-log reduction in computation time over our previous method (3DFSR). We applied 3D-3Way FRET to image the 3D subcellular distributions of HIV Gag assembly. Gag fused to three different FPs (CFP, YFP, and RFP), assembled into viral-like particles and created punctate FRET signals that become visible on the cell surface when 3D-3Way FRET was applied to the data. Control experiments in which YFP-Gag, RFP-Gag and free CFP were expressed, demonstrated localized FRET between YFP and RFP at sites of viral assembly that were not associated with CFP. 3D-3Way FRET provides the first approach for quantifying multiple FRET interactions while improving the 3D resolution of FRET microscopy data without introducing bias into the reconstructed estimates. This method should allow improvement of widefield, confocal and superresolution FRET microscopy data.

Functional Scanning Probe Imaging of Nanostructured Solar Energy Materials

DOE Office of Scientific and Technical Information (OSTI.GOV)

Giridharagopal, Rajiv; Cox, Phillip A.; Ginger, David S.

From hybrid perovskites to semiconducting polymer/fullerene blends for organic photovoltaics, many new materials being explored for energy harvesting and storage exhibit performance characteristics that depend sensitively on their nanoscale morphology. At the same time, rapid advances in the capability and accessibility of scanning probe microscopy methods over the past decade have made it possible to study processing/structure/function relationships ranging from photocurrent collection to photocarrier lifetimes with resolutions on the scale of tens of nanometers or better. Importantly, such scanning probe methods offer the potential to combine measurements of local structure with local function, and they can be implemented to studymore » materials in situ or devices in operando to better understand how materials evolve in time in response to an external stimulus or environmental perturbation. This Account highlights recent advances in the development and application of scanning probe microscopy methods that can help address such questions while filling key gaps between the capabilities of conventional electron microscopy and newer super-resolution optical methods. Focusing on semiconductor materials for solar energy applications, we highlight a range of electrical and optoelectronic scanning probe microscopy methods that exploit the local dynamics of an atomic force microscope tip to probe key properties of the solar cell material or device structure. We discuss how it is possible to extract relevant device properties using noncontact scanning probe methods as well as how these properties guide materials development. Specifically, we discuss intensity-modulated scanning Kelvin probe microscopy (IM-SKPM), time-resolved electrostatic force microscopy (trEFM), frequency-modulated electrostatic force microscopy (FM-EFM), and cantilever ringdown imaging. We explain these developments in the context of classic atomic force microscopy (AFM) methods that exploit the physics of cantilever motion and photocarrier generation to provide robust, nanoscale measurements of materials physics that are correlated with device operation. We predict that the multidimensional data sets made possible by these types of methods will become increasingly important as advances in data science expand capabilities and opportunities for image correlation and discovery.« less

Functional Scanning Probe Imaging of Nanostructured Solar Energy Materials

DOE PAGES

Giridharagopal, Rajiv; Cox, Phillip A.; Ginger, David S.

2016-08-30

From hybrid perovskites to semiconducting polymer/fullerene blends for organic photovoltaics, many new materials being explored for energy harvesting and storage exhibit performance characteristics that depend sensitively on their nanoscale morphology. At the same time, rapid advances in the capability and accessibility of scanning probe microscopy methods over the past decade have made it possible to study processing/structure/function relationships ranging from photocurrent collection to photocarrier lifetimes with resolutions on the scale of tens of nanometers or better. Importantly, such scanning probe methods offer the potential to combine measurements of local structure with local function, and they can be implemented to studymore » materials in situ or devices in operando to better understand how materials evolve in time in response to an external stimulus or environmental perturbation. This Account highlights recent advances in the development and application of scanning probe microscopy methods that can help address such questions while filling key gaps between the capabilities of conventional electron microscopy and newer super-resolution optical methods. Focusing on semiconductor materials for solar energy applications, we highlight a range of electrical and optoelectronic scanning probe microscopy methods that exploit the local dynamics of an atomic force microscope tip to probe key properties of the solar cell material or device structure. We discuss how it is possible to extract relevant device properties using noncontact scanning probe methods as well as how these properties guide materials development. Specifically, we discuss intensity-modulated scanning Kelvin probe microscopy (IM-SKPM), time-resolved electrostatic force microscopy (trEFM), frequency-modulated electrostatic force microscopy (FM-EFM), and cantilever ringdown imaging. We explain these developments in the context of classic atomic force microscopy (AFM) methods that exploit the physics of cantilever motion and photocarrier generation to provide robust, nanoscale measurements of materials physics that are correlated with device operation. We predict that the multidimensional data sets made possible by these types of methods will become increasingly important as advances in data science expand capabilities and opportunities for image correlation and discovery.« less

High-speed particle tracking in microscopy using SPAD image sensors

NASA Astrophysics Data System (ADS)

Gyongy, Istvan; Davies, Amy; Miguelez Crespo, Allende; Green, Andrew; Dutton, Neale A. W.; Duncan, Rory R.; Rickman, Colin; Henderson, Robert K.; Dalgarno, Paul A.

2018-02-01

Single photon avalanche diodes (SPADs) are used in a wide range of applications, from fluorescence lifetime imaging microscopy (FLIM) to time-of-flight (ToF) 3D imaging. SPAD arrays are becoming increasingly established, combining the unique properties of SPADs with widefield camera configurations. Traditionally, the photosensitive area (fill factor) of SPAD arrays has been limited by the in-pixel digital electronics. However, recent designs have demonstrated that by replacing the complex digital pixel logic with simple binary pixels and external frame summation, the fill factor can be increased considerably. A significant advantage of such binary SPAD arrays is the high frame rates offered by the sensors (>100kFPS), which opens up new possibilities for capturing ultra-fast temporal dynamics in, for example, life science cellular imaging. In this work we consider the use of novel binary SPAD arrays in high-speed particle tracking in microscopy. We demonstrate the tracking of fluorescent microspheres undergoing Brownian motion, and in intra-cellular vesicle dynamics, at high frame rates. We thereby show how binary SPAD arrays can offer an important advance in live cell imaging in such fields as intercellular communication, cell trafficking and cell signaling.

Automated 100-Position Specimen Loader and Image Acquisition System for Transmission Electron Microscopy

PubMed Central

Lefman, Jonathan; Morrison, Robert; Subramaniam, Sriram

2007-01-01

We report the development of a novel, multi-specimen imaging system for high-throughput transmission electron microscopy. Our cartridge-based loading system, called the “Gatling”, permits the sequential examination of as many as 100 specimens in the microscope for room temperature electron microscopy using mechanisms for rapid and automated specimen exchange. The software for the operation of the Gatling and automated data acquisition has been implemented in an updated version of our in-house program AutoEM. In the current implementation of the system, the time required to deliver 95 specimens into the microscope and collect overview images from each is about 13 hours. Regions of interest are identified from a low magnification atlas generation from each specimen and an unlimited number of higher magnifications images can be subsequently acquired from these regions using fully automated data acquisition procedures that can be controlled from a remote interface. We anticipate that the availability of the Gatling will greatly accelerate the speed of data acquisition for a variety of applications in biology, materials science and nanotechnology that require rapid screening and image analysis of multiple specimens. PMID:17240161

New techniques for motion-artifact-free in vivo cardiac microscopy

PubMed Central

Vinegoni, Claudio; Lee, Sungon; Aguirre, Aaron D.; Weissleder, Ralph

2015-01-01

Intravital imaging microscopy (i.e., imaging in live animals at microscopic resolution) has become an indispensable tool for studying the cellular micro-dynamics in cancer, immunology and neurobiology. High spatial and temporal resolution, combined with large penetration depth and multi-reporter visualization capability make fluorescence intravital microscopy compelling for heart imaging. However, tissue motion caused by cardiac contraction and respiration critically limits its use. As a result, in vitro cell preparations or non-contracting explanted heart models are more commonly employed. Unfortunately, these approaches fall short of understanding the more complex host physiology that may be dynamic and occur over longer periods of time. In this review, we report on novel technologies, which have been recently developed by our group and others, aimed at overcoming motion-induced artifacts and capable of providing in vivo subcellular resolution imaging in the beating mouse heart. The methods are based on mechanical stabilization, image processing algorithms, gated/triggered acquisition schemes or a combination of both. We expect that in the immediate future all these methodologies will have considerable applications in expanding our understanding of the cardiac biology, elucidating cardiomyocyte function and interactions within the organism in vivo, and ultimately improving the treatment of cardiac diseases. PMID:26029116

Gold nanoparticle flow sensors designed for dynamic X-ray imaging in biofluids.

PubMed

Ahn, Sungsook; Jung, Sung Yong; Lee, Jin Pyung; Kim, Hae Koo; Lee, Sang Joon

2010-07-27

X-ray-based imaging is one of the most powerful and convenient methods in terms of versatility in applicable energy and high performance in use. Different from conventional nuclear medicine imaging, contrast agents are required in X-ray imaging especially for effectively targeted and molecularly specific functions. Here, in contrast to much reported static accumulation of the contrast agents in targeted organs, dynamic visualization in a living organism is successfully accomplished by the particle-traced X-ray imaging for the first time. Flow phenomena across perforated end walls of xylem vessels in rice are monitored by a gold nanoparticle (AuNP) (approximately 20 nm in diameter) as a flow tracing sensor working in nontransparent biofluids. AuNPs are surface-modified to control the hydrodynamic properties such as hydrodynamic size (DH), zeta-potential, and surface plasmonic properties in aqueous conditions. Transmission electron microscopy (TEM), scanning electron microscopy (SEM), X-ray nanoscopy (XN), and X-ray microscopy (XM) are used to correlate the interparticle interactions with X-ray absorption ability. Cluster formation and X-ray contrast ability of the AuNPs are successfully modulated by controlling the interparticle interactions evaluated as flow-tracing sensors.

Nonlinear optical microscopy: use of second harmonic generation and two-photon microscopy for automated quantitative liver fibrosis studies.

PubMed

Sun, Wanxin; Chang, Shi; Tai, Dean C S; Tan, Nancy; Xiao, Guangfa; Tang, Huihuan; Yu, Hanry

2008-01-01

Liver fibrosis is associated with an abnormal increase in an extracellular matrix in chronic liver diseases. Quantitative characterization of fibrillar collagen in intact tissue is essential for both fibrosis studies and clinical applications. Commonly used methods, histological staining followed by either semiquantitative or computerized image analysis, have limited sensitivity, accuracy, and operator-dependent variations. The fibrillar collagen in sinusoids of normal livers could be observed through second-harmonic generation (SHG) microscopy. The two-photon excited fluorescence (TPEF) images, recorded simultaneously with SHG, clearly revealed the hepatocyte morphology. We have systematically optimized the parameters for the quantitative SHG/TPEF imaging of liver tissue and developed fully automated image analysis algorithms to extract the information of collagen changes and cell necrosis. Subtle changes in the distribution and amount of collagen and cell morphology are quantitatively characterized in SHG/TPEF images. By comparing to traditional staining, such as Masson's trichrome and Sirius red, SHG/TPEF is a sensitive quantitative tool for automated collagen characterization in liver tissue. Our system allows for enhanced detection and quantification of sinusoidal collagen fibers in fibrosis research and clinical diagnostics.

Halo-free phase contrast microscopy (Conference Presentation)

NASA Astrophysics Data System (ADS)

Nguyen, Tan H.; Kandel, Mikhail E.; Shakir, Haadi M.; Best, Catherine; Do, Minh N.; Popescu, Gabriel

2017-02-01

The phase contrast (PC) method is one of the most impactful developments in the four-century long history of microscopy. It allows for intrinsic, nondestructive contrast of transparent specimens, such as live cells. However, PC is plagued by the halo artifact, a result of insufficient spatial coherence in the illumination field, which limits its applicability. We present a new approach for retrieving halo-free phase contrast microscopy (hfPC) images by upgrading the conventional PC microscope with an external interferometric module, which generates sufficient data for reversing the halo artifact. Measuring four independent intensity images, our approach first measures haloed phase maps of the sample. We solve for the halo-free sample transmission function by using a physical model of the image formation under partial spatial coherence. Using this halo-free sample transmission, we can numerically generate artifact-free PC images. Furthermore, this transmission can be further used to obtain quantitative information about the sample, e.g., the thickness with known refractive indices, dry mass of live cells during their cycles. We tested our hfPC method on various control samples, e.g., beads, pillars and validated its potential for biological investigation by imaging live HeLa cells, red blood cells, and neurons.

Design and demonstration of multimodal optical scanning microscopy for confocal and two-photon imaging

NASA Astrophysics Data System (ADS)

Chun, Wanhee; Do, Dukho; Gweon, Dae-Gab

2013-01-01

We developed a multimodal microscopy based on an optical scanning system in order to obtain diverse optical information of the same area of a sample. Multimodal imaging researches have mostly depended on a commercial microscope platform, easy to use but restrictive to extend imaging modalities. In this work, the beam scanning optics, especially including a relay lens, was customized to transfer broadband (400-1000 nm) lights to a sample without any optical error or loss. The customized scanning optics guarantees the best performances of imaging techniques utilizing the lights within the design wavelength. Confocal reflection, confocal fluorescence, and two-photon excitation fluorescence images were obtained, through respective implemented imaging channels, to demonstrate imaging feasibility for near-UV, visible, near-IR continuous light, and pulsed light in the scanning optics. The imaging performances for spatial resolution and image contrast were verified experimentally; the results were satisfactory in comparison with theoretical results. The advantages of customization, containing low cost, outstanding combining ability and diverse applications, will contribute to vitalize multimodal imaging researches.

Augmented microscopy: real-time overlay of bright-field and near-infrared fluorescence images.

PubMed

Watson, Jeffrey R; Gainer, Christian F; Martirosyan, Nikolay; Skoch, Jesse; Lemole, G Michael; Anton, Rein; Romanowski, Marek

2015-10-01

Intraoperative applications of near-infrared (NIR) fluorescent contrast agents can be aided by instrumentation capable of merging the view of surgical field with that of NIR fluorescence. We demonstrate augmented microscopy, an intraoperative imaging technique in which bright-field (real) and electronically processed NIR fluorescence (synthetic) images are merged within the optical path of a stereomicroscope. Under luminance of 100,000 lx, representing typical illumination of the surgical field, the augmented microscope detects 189 nM concentration of indocyanine green and produces a composite of the real and synthetic images within the eyepiece of the microscope at 20 fps. Augmentation described here can be implemented as an add-on module to visualize NIR contrast agents, laser beams, or various types of electronic data within the surgical microscopes commonly used in neurosurgical, cerebrovascular, otolaryngological, and ophthalmic procedures.

Augmented microscopy: real-time overlay of bright-field and near-infrared fluorescence images

NASA Astrophysics Data System (ADS)

Watson, Jeffrey R.; Gainer, Christian F.; Martirosyan, Nikolay; Skoch, Jesse; Lemole, G. Michael, Jr.; Anton, Rein; Romanowski, Marek

2015-10-01

Intraoperative applications of near-infrared (NIR) fluorescent contrast agents can be aided by instrumentation capable of merging the view of surgical field with that of NIR fluorescence. We demonstrate augmented microscopy, an intraoperative imaging technique in which bright-field (real) and electronically processed NIR fluorescence (synthetic) images are merged within the optical path of a stereomicroscope. Under luminance of 100,000 lx, representing typical illumination of the surgical field, the augmented microscope detects 189 nM concentration of indocyanine green and produces a composite of the real and synthetic images within the eyepiece of the microscope at 20 fps. Augmentation described here can be implemented as an add-on module to visualize NIR contrast agents, laser beams, or various types of electronic data within the surgical microscopes commonly used in neurosurgical, cerebrovascular, otolaryngological, and ophthalmic procedures.

Two-photon excitation based photochemistry and neural imaging

NASA Astrophysics Data System (ADS)

Hatch, Kevin Andrew

Two-photon microscopy is a fluorescence imaging technique which provides distinct advantages in three-dimensional cellular and molecular imaging. The benefits of this technology may extend beyond imaging capabilities through exploitation of the quantum processes responsible for fluorescent events. This study utilized a two-photon microscope to investigate a synthetic photoreactive collagen peptidomimetic, which may serve as a potential material for tissue engineering using the techniques of two-photon photolysis and two-photon polymerization. The combination of these techniques could potentially be used to produce a scaffold for the vascularization of engineered three-dimensional tissues in vitro to address the current limitations of tissue engineering. Additionally, two-photon microscopy was used to observe the effects of the application of the neurotransmitter dopamine to the mushroom body neural structures of Drosophila melanogaster to investigate dopamine's connection to cognitive degeneration.

Partially coherent lensfree tomographic microscopy⋄

PubMed Central

Isikman, Serhan O.; Bishara, Waheb; Ozcan, Aydogan

2012-01-01

Optical sectioning of biological specimens provides detailed volumetric information regarding their internal structure. To provide a complementary approach to existing three-dimensional (3D) microscopy modalities, we have recently demonstrated lensfree optical tomography that offers high-throughput imaging within a compact and simple platform. In this approach, in-line holograms of objects at different angles of partially coherent illumination are recorded using a digital sensor-array, which enables computing pixel super-resolved tomographic images of the specimen. This imaging modality, which forms the focus of this review, offers micrometer-scale 3D resolution over large imaging volumes of, for example, 10–15 mm3, and can be assembled in light weight and compact architectures. Therefore, lensfree optical tomography might be particularly useful for lab-on-a-chip applications as well as for microscopy needs in resource-limited settings. PMID:22193016

Imaging TiO2 nanoparticles on GaN nanowires with electrostatic force microscopy

NASA Astrophysics Data System (ADS)

Xie, Ting; Wen, Baomei; Liu, Guannan; Guo, Shiqi; Motayed, Abhishek; Murphy, Thomas; Gomez, R. D.

Gallium nitride (GaN) nanowires that are functionalized with metal-oxides nanoparticles have been explored extensively for gas sensing applications in the past few years. These sensors have several advantages over conventional schemes, including miniature size, low-power consumption and fast response and recovery times. The morphology of the oxide functionalization layer is critical to achieve faster response and recovery times, with the optimal size distribution of nanoparticles being in the range of 10 to 30 nm. However, it is challenging to characterize these nanoparticles on GaN nanowires using common techniques such as scanning electron microscopy, transmission electron microscopy, and x-ray diffraction. Here, we demonstrate electrostatic force microscopy in combination with atomic force microscopy as a non-destructive technique for morphological characterization of the dispersed TiO2 nanoparticles on GaN nanowires. We also discuss the applicability of this method to other material systems with a proposed tip-surface capacitor model. This project was sponsored through N5 Sensors and the Maryland Industrial Partnerships (MIPS, #5418).

Application of Nomarski DIC and cathodoluminescence (CL) microscopy to building materials

DOE Office of Scientific and Technical Information (OSTI.GOV)

Goetze, J., E-mail: goetze@mineral.tu-freiberg.de

2009-07-15

The present study discusses the potential of an integrated application of Nomarski differential interference contrast and cathodoluminescence microscopy for the investigation of building materials such as natural stone, cement, mortar and concrete. Nomarski differential interference contrast microscopy is a modern technique applied in materials sciences to visualize different phases and/or to image the surface relief on the scale of 50 nm. It is based on the principle of beam splitting by a double-crystal prism split, resulting in the superposition of laterally shifted wave fronts. In cathodoluminescence microscopy, the luminescence signal is excited by an electron beam and is generated bymore » different point defects within the material. Therefore, cathodoluminescence is a powerful method to characterize the defect structure of solid materials, to distinguish different phases and to reveal detailed information about their chemical composition. By combining Nomarski differential interference contrast and cathodoluminescence microscopy, textural, crystallographic and chemical information can be obtained from the same sample area in a polished thin section.« less

Evaluation of a Fluorochlorozirconate Glass-Ceramic Storage Phosphor Plate for Gamma-Ray Computed Radiography

DOE PAGES

Leonard, Russell L.; Gray, Sharon K.; Alvarez, Carlos J.; ...

2015-05-21

In this paper, a fluorochlorozirconate (FCZ) glass-ceramic containing orthorhombic barium chloride crystals doped with divalent europium was evaluated for use as a storage phosphor in gamma-ray imaging. X-ray diffraction and phosphorimetry of the glass-ceramic sample showed the presence of a significant amount of orthorhombic barium chloride crystals in the glass matrix. Transmission electron microscopy and scanning electron microscopy were used to identify crystal size, structure, and morphology. The size of the orthorhombic barium chloride crystals in the FCZ glass matrix was very large, ~0.5–0.7 μm, which can limit image resolution. The FCZ glass-ceramic sample was exposed to 1 MeV gammamore » rays to determine its photostimulated emission characteristics at high energies, which were found to be suitable for imaging applications. Test images were made at 2 MeV energies using gap and step wedge phantoms. Gaps as small as 101.6 μm in a 440 stainless steel phantom were imaged using the sample imaging plate. Analysis of an image created using a depleted uranium step wedge phantom showed that emission is proportional to incident energy at the sample and the estimated absorbed dose. Finally, the results showed that the sample imaging plate has potential for gamma-ray-computed radiography and dosimetry applications.« less

Region Templates: Data Representation and Management for High-Throughput Image Analysis

PubMed Central

Pan, Tony; Kurc, Tahsin; Kong, Jun; Cooper, Lee; Klasky, Scott; Saltz, Joel

2015-01-01

We introduce a region template abstraction and framework for the efficient storage, management and processing of common data types in analysis of large datasets of high resolution images on clusters of hybrid computing nodes. The region template abstraction provides a generic container template for common data structures, such as points, arrays, regions, and object sets, within a spatial and temporal bounding box. It allows for different data management strategies and I/O implementations, while providing a homogeneous, unified interface to applications for data storage and retrieval. A region template application is represented as a hierarchical dataflow in which each computing stage may be represented as another dataflow of finer-grain tasks. The execution of the application is coordinated by a runtime system that implements optimizations for hybrid machines, including performance-aware scheduling for maximizing the utilization of computing devices and techniques to reduce the impact of data transfers between CPUs and GPUs. An experimental evaluation on a state-of-the-art hybrid cluster using a microscopy imaging application shows that the abstraction adds negligible overhead (about 3%) and achieves good scalability and high data transfer rates. Optimizations in a high speed disk based storage implementation of the abstraction to support asynchronous data transfers and computation result in an application performance gain of about 1.13×. Finally, a processing rate of 11,730 4K×4K tiles per minute was achieved for the microscopy imaging application on a cluster with 100 nodes (300 GPUs and 1,200 CPU cores). This computation rate enables studies with very large datasets. PMID:26139953

Aberration control in 4Pi nanoscopy: definitions, properties, and applications (Conference Presentation)

NASA Astrophysics Data System (ADS)

Hao, Xiang; Allgeyer, Edward S.; Velasco, Mary Grace M.; Booth, Martin J.; Bewersdorf, Joerg

2016-03-01

The development of fluorescence microscopy, which allows live-cell imaging with high labeling specificity, has made the visualization of cellular architecture routine. However, for centuries, the spatial resolution of optical microscopy was fundamentally limited by diffraction. The past two decades have seen a revolution in far-field optical nanoscopy (or "super-resolution" microscopy). The best 3D resolution is achieved by optical nanoscopes like the isoSTED or the iPALM/4Pi-SMS, which utilize two opposing objective lenses in a coherent manner. These system are, however, also more complex and the required interference conditions demand precise aberration control. Our research involves developing novel adaptive optics techniques that enable high spatial and temporal resolution imaging for biological applications. In this talk, we will discuss how adaptive optics can enhance dual-objective lens nanoscopes. We will demonstrate how adaptive optics devices provide unprecedented freedom to manipulate the light field in isoSTED nanoscopy, allow to realize automatic beam alignment, suppress the inherent side-lobes of the point-spread function, and dynamically compensate for sample-induced aberrations. We will present both the theoretical groundwork and the experimental confirmations.

Super-Resolution Microscopy Techniques and Their Potential for Applications in Radiation Biophysics.

PubMed

Eberle, Jan Philipp; Rapp, Alexander; Krufczik, Matthias; Eryilmaz, Marion; Gunkel, Manuel; Erfle, Holger; Hausmann, Michael

2017-01-01

Fluorescence microscopy is an essential tool for imaging tagged biological structures. Due to the wave nature of light, the resolution of a conventional fluorescence microscope is limited laterally to about 200 nm and axially to about 600 nm, which is often referred to as the Abbe limit. This hampers the observation of important biological structures and dynamics in the nano-scaled range ~10 nm to ~100 nm. Consequentially, various methods have been developed circumventing this limit of resolution. Super-resolution microscopy comprises several of those methods employing physical and/or chemical properties, such as optical/instrumental modifications and specific labeling of samples. In this article, we will give a brief insight into a variety of selected optical microscopy methods reaching super-resolution beyond the Abbe limit. We will survey three different concepts in connection to biological applications in radiation research without making a claim to be complete.

Digital cytology: current state of the art and prospects for the future.

PubMed

Wilbur, David C

2011-01-01

The growth of digital methods in pathology is accelerating. Digital images can be used for a variety of applications in cytology, including rapid interpretations, primary diagnosis and second opinions, continuing education and proficiency testing. All of these functions can be performed using small static digital images, real-time dynamic digital microscopy, or whole-slide images. This review will discuss the general principles of digital pathology, its methods and applications to cytologic specimens. As cytologic specimens have unique features compared to histopathology specimens, the key differences will be discussed. Technical and administrative issues in digital pathology applications and the outlook for the future of the field will be presented. Copyright © 2011 S. Karger AG, Basel.

Optical imaging beyond the diffraction limit by SNEM: effects of AFM tip modifications with thiol monolayers on imaging quality.

PubMed

Cumurcu, Aysegul; Diaz, Jordi; Lindsay, Ian D; de Beer, Sissi; Duvigneau, Joost; Schön, Peter; Julius Vancso, G

2015-03-01

Tip-enhanced nanoscale optical imaging techniques such as apertureless scanning near-field optical microscopy (a-SNOM) and scanning near-field ellipsometric microscopy (SNEM) applications can suffer from a steady degradation in performance due to adhesion of atmospheric contaminants to the metal coated tip. Here, we demonstrate that a self-assembled monolayer (SAM) of ethanethiol (EtSH) is an effective means of protecting gold-coated atomic force microscopy (AFM) probe tips from accumulation of surface contaminants during prolonged exposure to ambient air. The period over which they yield consistent and reproducible results for scanning near-field ellipsometric microscopy (SNEM) imaging is thus extended. SNEM optical images of a microphase separated polystyrene-block-poly (methylmethacrylate) (PS-b-PMMA) diblock copolymer film, which were captured with bare and SAM-protected gold-coated AFM probes, both immediately after coating and following five days of storage in ambient air, were compared. During this period the intensity of the optical signals from the untreated gold tip fell by 66%, while those from the SAM protected tip fell by 14%. Additionally, gold coated AFM probe tips were modified with various lengths of alkanethiols to measure the change in intensity variation in the optical images with SAM layer thickness. The experimental results were compared to point dipole model calculations. While a SAM of 1-dodecanethiol (DoSH) was found to strongly suppress field enhancement we find that it can be locally removed from the tip apex by deforming the molecules under load, restoring SNEM image contrast. Copyright © 2014 Elsevier B.V. All rights reserved.

Strategies for rare-event detection: an approach for automated fetal cell detection in maternal blood.

PubMed Central

Oosterwijk, J C; Knepflé, C F; Mesker, W E; Vrolijk, H; Sloos, W C; Pattenier, H; Ravkin, I; van Ommen, G J; Kanhai, H H; Tanke, H J

1998-01-01

This article explores the feasibility of the use of automated microscopy and image analysis to detect the presence of rare fetal nucleated red blood cells (NRBCs) circulating in maternal blood. The rationales for enrichment and for automated image analysis for "rare-event" detection are reviewed. We also describe the application of automated image analysis to 42 maternal blood samples, using a protocol consisting of one-step enrichment followed by immunocytochemical staining for fetal hemoglobin (HbF) and FISH for X- and Y-chromosomal sequences. Automated image analysis consisted of multimode microscopy and subsequent visual evaluation of image memories containing the selected objects. The FISH results were compared with the results of conventional karyotyping of the chorionic villi. By use of manual screening, 43% of the slides were found to be positive (>=1 NRBC), with a mean number of 11 NRBCs (range 1-40). By automated microscopy, 52% were positive, with on average 17 NRBCs (range 1-111). There was a good correlation between both manual and automated screening, but the NRBC yield from automated image analysis was found to be superior to that from manual screening (P=.0443), particularly when the NRBC count was >15. Seven (64%) of 11 XY fetuses were correctly diagnosed by FISH analysis of automatically detected cells, and all discrepancies were restricted to the lower cell-count range. We believe that automated microscopy and image analysis reduce the screening workload, are more sensitive than manual evaluation, and can be used to detect rare HbF-containing NRBCs in maternal blood. PMID:9837832

Quantitative 3D comparison of biofilm imaged by X-ray micro-tomography and two-photon laser scanning microscopy.

PubMed

Larue, A E; Swider, P; Duru, P; Daviaud, D; Quintard, M; Davit, Y

2018-06-21

Optical imaging techniques for biofilm observation, like laser scanning microscopy, are not applicable when investigating biofilm formation in opaque porous media. X-ray micro-tomography (X-ray CMT) might be an alternative but it finds limitations in similarity of X-ray absorption coefficients for the biofilm and aqueous phases. To overcome this difficulty, barium sulphate was used in Davit et al. (2011) to enable high-resolution 3D imaging of biofilm via X-ray CMT. However, this approach lacks comparison with well-established imaging methods, which are known to capture the fine structures of biofilms, as well as uncertainty quantification. Here, we compare two-photon laser scanning microscopy (TPLSM) images of Pseudomonas Aeruginosa biofilm grown in glass capillaries against X-ray CMT using an improved protocol where barium sulphate is combined with low-gelling temperature agarose to avoid sedimentation. Calibrated phantoms consisting of mono-dispersed fluorescent and X-ray absorbent beads were used to evaluate the uncertainty associated with our protocol along with three different segmentation techniques, namely hysteresis, watershed and region growing, to determine the bias relative to image binarization. Metrics such as volume, 3D surface area and thickness were measured and comparison of both imaging modalities shows that X-ray CMT of biofilm using our protocol yields an accuracy that is comparable and even better in certain respects than TPLSM, even in a nonporous system that is largely favourable to TPLSM. © 2018 The Authors Journal of Microscopy © 2018 Royal Microscopical Society.

Visualization of collagen regeneration in mouse dorsal skin using second harmonic generation microscopy

NASA Astrophysics Data System (ADS)

Luo, T.; Chen, J. X.; Zhuo, S. M.; Lu, K. C.; Jiang, X. S.; Liu, Q. G.

2009-03-01

The purpose of this study is to highlight a clearer understanding of the process of collagen regeneration during wound healing. By means of second harmonic generation (SHG) microscopy, the changes of collagen arrangement at the wound margin were analyzed at 0, 3, 5, 7, 11 and 13 days post injury. The degree of collagen disorders associated with the healing process was quantitatively obtained using the aspect ratio of polar plot image of collagen azimuthal angles and the healing status of collagen could be estimated by arithmetical mean deviation ( Ra) of the collagen SHG images. Our results suggest that SHG microscopy has potential advances in the collagen studies during wound healing and the arrangement of collagen fibers gradually transformed from disorder to order so as to contract the wound. It is capable of promoting clinical application of the noninvasive imaging tool and the analysis methods of collagen disorder as an effective scar management for prevention and treatment about aberrant healing.

Nanoparticle-assisted-multiphoton microscopy for in vivo brain imaging of mice

NASA Astrophysics Data System (ADS)

Qian, Jun

2015-03-01

Neuro/brain study has attracted much attention during past few years, and many optical methods have been utilized in order to obtain accurate and complete neural information inside the brain. Relying on simultaneous absorption of two or more near-infrared photons by a fluorophore, multiphoton microscopy can achieve deep tissue penetration and efficient light detection noninvasively, which makes it very suitable for thick-tissue and in vivo bioimaging. Nanoparticles possess many unique optical and chemical properties, such as anti-photobleaching, large multiphoton absorption cross-section, and high stability in biological environment, which facilitates their applications in long-term multiphoton microscopy as contrast agents. In this paper, we will introduce several typical nanoparticles (e.g. organic dye doped polymer nanoparticles and gold nanorods) with high multiphoton fluorescence efficiency. We further applied them in two- and three-photon in vivo functional brain imaging of mice, such as brain-microglia imaging, 3D architecture reconstruction of brain blood vessel, and blood velocity measurement.

High-resolution light-sheet microscopy: a simulation of an optical illumination system for oil immersion

NASA Astrophysics Data System (ADS)

Lu, Xiang; Heintzmann, Rainer; Leischner, Ulrich

2015-09-01

Light sheet microscopy is a microscopy technique characterized by an illumination from the side, perpendicular to the direction of observation. While this is often used and easy to implement for imaging samples with water-immersion, the application in combination with oil-immersion is less often used and requires a specific optimization. In this paper we present our design of a light-sheet illumination optical system with a ~1μm illumination thickness, a long working distance through the immersion oil, and including a focusing system allowing for moving the focus-spot of the lightsheet laterally through the field of view. This optical design allows for the acquisition of fluorescence images in 3D with isotropic resolution of below 1 micrometer of whole-mount samples with a size of ~1mm diameter. This technique enables high-resolution insights in the 3D structure of biological samples, e.g. for research of insect anatomy or for imaging of biopsies in medical diagnostics.

Superresolution Imaging with Standard Fluorescent Probes

PubMed Central

Burnette, Dylan T.; Lippincott-Schwartz, Jennifer; Kachar, Bechara

2013-01-01

For more than 100 years, the ultimate resolution of a light microscope (~200 nm) has been constrained by the fundamental physical phenomenon of diffraction, as described by Ernst Abbe in 1873. While this limitation is just as applicable to today’s light microscopes, it is the combination of high-end optics, clever methods of sample illumination, and computational techniques that has enabled researchers to access high-resolution information an order of magnitude greater than once thought possible. This combination, broadly termed superresolution microscopy, has been increasingly practical for many labs to implement from both a hardware and software standpoint, but as with many cutting-edge techniques, it also comes with limitations. One of the current drawbacks to superresolution microscopy is the limited number of probes and conditions that have been suitable for imaging. Here, a technique termed bleaching/blinking assisted localization microscopy (BaLM) makes use of almost all fluorophore’s inherent blinking and bleaching properties as a means to generate superresolution images. PMID:24510788

High spatial resolution soft-x-ray microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Meyer-Ilse, W.; Medecki, H.; Brown, J.T.

1997-04-01

A new soft x-ray microscope (XM-1) with high spatial resolution has been constructed by the Center for X-ray Optics. It uses bending magnet radiation from beamline 6.1 at the Advanced Light Source, and is used in a variety of projects and applications in the life and physical sciences. Most of these projects are ongoing. The instrument uses zone plate lenses and achieves a resolution of 43 nm, measured over 10% to 90% intensity with a knife edge test sample. X-ray microscopy permits the imaging of relatively thick samples, up to 10 {mu}m thick, in water. XM-1 has an easy tomore » use interface, that utilizes visible light microscopy to precisely position and focus the specimen. The authors describe applications of this device in the biological sciences, as well as in studying industrial applications including structured polymer samples.« less

Performance of a three-dimensional-printed microscanner in a laser scanning microscopy application

NASA Astrophysics Data System (ADS)

Oyman, Hilmi Artun; Gokdel, Yigit Daghan; Ferhanoglu, Onur; Yalcinkaya, Arda Deniz

2018-04-01

A magnetically actuated microscanner is used in a laser scanning microscopy application. Stress distribution along the circular-profiled flexure is compared with a rectangular counterpart in finite-element environment. Magnetic actuation mechanism of the scanning unit is explained in detail. Moreover, reliability of the scanner is tested for 3×106 cycle. The scanning device is designed to meet a confocal microscopy application providing 100 μm×100 μm field of view and <3-μm lateral resolution. The resonance frequencies of the device were analytically modeled, where we obtained 130- and 268-Hz resonance values for the out-of-plane and torsion modes, respectively. The scanning device provided an optical scan angle about 2.5 deg for 170-mA drive current, enabling the desired field of view for our custom built confocal microscope setup. Finally, imaging experiments were conducted on a resolution target, showcasing the desired scan area and resolution.

Nonlinear excitation fluorescence microscopy: source considerations for biological applications

NASA Astrophysics Data System (ADS)

Wokosin, David L.

2008-02-01

Ultra-short-pulse solid-state laser sources have improved contrast within fluorescence imaging and also opened new windows of investigation in biological imaging applications. Additionally, the pulsed illumination enables harmonic scattering microscopy which yields intrinsic structure, symmetry and contrast from viable embryos, cells and tissues. Numerous human diseases are being investigated by the combination of (more) intact dynamic tissue imaging of cellular function with gene-targeted specificity and electrophysiology context. The major limitation to more widespread use of multi-photon microscopy has been the complete system cost and added complexity above and beyond commercial camera and confocal systems. The current status of all-solid-state ultrafast lasers as excitation sources will be reviewed since these lasers offer tremendous potential for affordable, reliable, "turnkey" multiphoton imaging systems. This effort highlights the single box laser systems currently commercially available, with defined suggestions for the ranges for individual laser parameters as derived from a biological and fluorophore limited perspective. The standard two-photon dose is defined by 800nm, 10mW, 200fs, and 80Mhz - at the sample plane for tissue culture cells, i.e. after the full scanning microscope system. Selected application-derived excitation wavelengths are well represented by 700nm, 780nm, ~830nm, ~960nm, 1050nm, and 1250nm. Many of the one-box lasers have fixed or very limited excitation wavelengths available, so the lasers will be lumped near 780nm, 800nm, 900nm, 1050nm, and 1250nm. The following laser parameter ranges are discussed: average power from 200mW to 2W, pulse duration from 70fs to 700fs, pulse repetition rate from 20MHz to 200MHz, with the laser output linearly polarized with an extinction ratio at least 100:1.

Quantitative pathology in virtual microscopy: history, applications, perspectives.

PubMed

Kayser, Gian; Kayser, Klaus

2013-07-01

With the emerging success of commercially available personal computers and the rapid progress in the development of information technologies, morphometric analyses of static histological images have been introduced to improve our understanding of the biology of diseases such as cancer. First applications have been quantifications of immunohistochemical expression patterns. In addition to object counting and feature extraction, laws of thermodynamics have been applied in morphometric calculations termed syntactic structure analysis. Here, one has to consider that the information of an image can be calculated for separate hierarchical layers such as single pixels, cluster of pixels, segmented small objects, clusters of small objects, objects of higher order composed of several small objects. Using syntactic structure analysis in histological images, functional states can be extracted and efficiency of labor in tissues can be quantified. Image standardization procedures, such as shading correction and color normalization, can overcome artifacts blurring clear thresholds. Morphometric techniques are not only useful to learn more about biological features of growth patterns, they can also be helpful in routine diagnostic pathology. In such cases, entropy calculations are applied in analogy to theoretical considerations concerning information content. Thus, regions with high information content can automatically be highlighted. Analysis of the "regions of high diagnostic value" can deliver in the context of clinical information, site of involvement and patient data (e.g. age, sex), support in histopathological differential diagnoses. It can be expected that quantitative virtual microscopy will open new possibilities for automated histological support. Automated integrated quantification of histological slides also serves for quality assurance. The development and theoretical background of morphometric analyses in histopathology are reviewed, as well as their application and potential future implementation in virtual microscopy. Copyright © 2012 Elsevier GmbH. All rights reserved.

A fast image registration approach of neural activities in light-sheet fluorescence microscopy images

NASA Astrophysics Data System (ADS)

Meng, Hui; Hui, Hui; Hu, Chaoen; Yang, Xin; Tian, Jie

2017-03-01

The ability of fast and single-neuron resolution imaging of neural activities enables light-sheet fluorescence microscopy (LSFM) as a powerful imaging technique in functional neural connection applications. The state-of-art LSFM imaging system can record the neuronal activities of entire brain for small animal, such as zebrafish or C. elegans at single-neuron resolution. However, the stimulated and spontaneous movements in animal brain result in inconsistent neuron positions during recording process. It is time consuming to register the acquired large-scale images with conventional method. In this work, we address the problem of fast registration of neural positions in stacks of LSFM images. This is necessary to register brain structures and activities. To achieve fast registration of neural activities, we present a rigid registration architecture by implementation of Graphics Processing Unit (GPU). In this approach, the image stacks were preprocessed on GPU by mean stretching to reduce the computation effort. The present image was registered to the previous image stack that considered as reference. A fast Fourier transform (FFT) algorithm was used for calculating the shift of the image stack. The calculations for image registration were performed in different threads while the preparation functionality was refactored and called only once by the master thread. We implemented our registration algorithm on NVIDIA Quadro K4200 GPU under Compute Unified Device Architecture (CUDA) programming environment. The experimental results showed that the registration computation can speed-up to 550ms for a full high-resolution brain image. Our approach also has potential to be used for other dynamic image registrations in biomedical applications.

Orbital angular momentum light in microscopy

PubMed Central

2017-01-01

Light with a helical phase has had an impact on optical imaging, pushing the limits of resolution or sensitivity. Here, special emphasis will be given to classical light microscopy of phase samples and to Fourier filtering techniques with a helical phase profile, such as the spiral phase contrast technique in its many variants and areas of application. This article is part of the themed issue ‘Optical orbital angular momentum’. PMID:28069768

Raman Microscopy and Imaging: Applications to Skin Pharmacology and Wound Healing

NASA Astrophysics Data System (ADS)

Flach, Carol R.; Zhang, Guojin; Mendelsohn, Richard

The utility of confocal Raman microscopy to study biological events in skin is demonstrated with three examples. (i) monitoring the spatial and structural differences between native and cultured skin, (ii) tracking the permeation and biochemical transformation in skin of a Vitamin E derivative and (iii) tracking the spatial distribution of three major skin proteins (keratin, collagen, and elastin) during wound healing in an explant skin model.

In vivo assessment of cytological changes by means of reflectance confocal microscopy - demonstration of the effect of topical vitamin E on skin irritation caused by sodium lauryl sulfate.

PubMed

Casari, Alice; Farnetani, Francesca; De Pace, Barbara; Losi, Amanda; Pittet, Jean-Christophe; Pellacani, Giovanni; Longo, Caterina

2017-03-01

Irritant contact dermatitis is caused by skin barrier damage. Vitamin E is an antioxidant that is commonly used in cosmetics to prevent photo-damage. To show the usefulness of reflectance confocal microscopy in the assessment of irritant skin damage caused by sodium lauryl sulfate (SLS) and of the protective action of vitamin E applied prior to skin irritation. Ten healthy volunteers were enrolled. Irritation was induced by the application of a patch test containing SLS 5% aq. for 24 h. Three sites were compared: one site on which a product with vitamin E was applied before SLS treatment, one site on which the same product was applied after SLS treatment, and one control site (SLS only). Each site was evaluated with reflectance confocal microscopy, providing in vivo tissue images at nearly histological resolution. We also performed a computerized analysis of the VivaStack® images. Reflectance confocal microscopy is able to identify signs of skin irritation and the preventive effect of vitamin E application. Reflectance confocal microscopy is useful in the objective assessment of irritative skin damage. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Challenges of microtome‐based serial block‐face scanning electron microscopy in neuroscience

PubMed Central

WANNER, A. A.; KIRSCHMANN, M. A.

2015-01-01

Summary Serial block‐face scanning electron microscopy (SBEM) is becoming increasingly popular for a wide range of applications in many disciplines from biology to material sciences. This review focuses on applications for circuit reconstruction in neuroscience, which is one of the major driving forces advancing SBEM. Neuronal circuit reconstruction poses exceptional challenges to volume EM in terms of resolution, field of view, acquisition time and sample preparation. Mapping the connections between neurons in the brain is crucial for understanding information flow and information processing in the brain. However, information on the connectivity between hundreds or even thousands of neurons densely packed in neuronal microcircuits is still largely missing. Volume EM techniques such as serial section TEM, automated tape‐collecting ultramicrotome, focused ion‐beam scanning electron microscopy and SBEM (microtome serial block‐face scanning electron microscopy) are the techniques that provide sufficient resolution to resolve ultrastructural details such as synapses and provides sufficient field of view for dense reconstruction of neuronal circuits. While volume EM techniques are advancing, they are generating large data sets on the terabyte scale that require new image processing workflows and analysis tools. In this review, we present the recent advances in SBEM for circuit reconstruction in neuroscience and an overview of existing image processing and analysis pipelines. PMID:25907464

Atmospheric scanning electron microscope observes cells and tissues in open medium through silicon nitride film.

PubMed

Nishiyama, Hidetoshi; Suga, Mitsuo; Ogura, Toshihiko; Maruyama, Yuusuke; Koizumi, Mitsuru; Mio, Kazuhiro; Kitamura, Shinichi; Sato, Chikara

2010-03-01

Direct observation of subcellular structures and their characterization is essential for understanding their physiological functions. To observe them in open environment, we have developed an inverted scanning electron microscope with a detachable, open-culture dish, capable of 8 nm resolution, and combined with a fluorescence microscope quasi-simultaneously observing the same area from the top. For scanning electron microscopy from the bottom, a silicon nitride film window in the base of the dish maintains a vacuum between electron gun and open sample dish while allowing electrons to pass through. Electrons are backscattered from the sample and captured by a detector under the dish. Cells cultured on the open dish can be externally manipulated under optical microscopy, fixed, and observed using scanning electron microscopy. Once fine structures have been revealed by scanning electron microscopy, their component proteins may be identified by comparison with separately prepared fluorescence-labeled optical microscopic images of the candidate proteins, with their heavy-metal-labeled or stained ASEM images. Furthermore, cell nuclei in a tissue block stained with platinum-blue were successfully observed without thin-sectioning, which suggests the applicability of this inverted scanning electron microscope to cancer diagnosis. This microscope visualizes mesoscopic-scale structures, and is also applicable to non-bioscience fields including polymer chemistry. (c) 2010 Elsevier Inc. All rights reserved.

The Empirical Foundations of Telepathology: Evidence of Feasibility and Intermediate Effects

PubMed Central

Krupinski, Elizabeth A.; Weinstein, Ronald S.; Dunn, Matthew R.; Bashshur, Noura

2017-01-01

Abstract Introduction: Telepathology evolved from video microscopy (i.e., “television microscopy”) research in the early 1950s to video microscopy used in basic research in the biological sciences to a basic diagnostic tool in telemedicine clinical applications. Its genesis can be traced to pioneering feasibility studies regarding the importance of color and other image-based parameters for rendering diagnoses and a series of studies assessing concordance of virtual slide and light microscopy diagnoses. This article documents the empirical foundations of telepathology. Methods: A selective review of the research literature during the past decade (2005–2016) was conducted using robust research design and adequate sample size as criteria for inclusion. Conclusions: The evidence regarding feasibility/acceptance of telepathology and related information technology applications has been well documented for several decades. The majority of evidentiary studies focused on intermediate outcomes, as indicated by comparability between telepathology and conventional light microscopy. A consistent trend of concordance between the two modalities was observed in terms of diagnostic accuracy and reliability. Additional benefits include use of telepathology and whole slide imaging for teaching, research, and outreach to resource-limited countries. Challenges still exist, however, in terms of use of telepathology as an effective diagnostic modality in clinical practice. PMID:28170313

Multiphoton microscopy and image guided light activated therapy using nanomaterials (Conference Presentation)

NASA Astrophysics Data System (ADS)

Prasad, Paras N.

2017-02-01

This talk will focus on design and applications of nanomaterials exhibiting strong multiphoton upconversion for multiphoton microscopy as well as for image-guided and light activated therapy .1-3 Such processes can occur by truly nonlinear optical interactions proceeding through virtual intermediate states or by stepwise coupled linear excitations through real intermediate states. Multiphoton processes in biocompatible multifunctional nanoparticles allow for 3D deep tissue imaging. In addition, they can produce in-situ photon conversion of deep tissue penetrating near IR light into a needed shorter wavelength light for photo-activated therapy at a targeted site, thus overcoming the limited penetration of UV or visible light into biological media. We are using near IR emitters such as silicon quantum dots which also exhibit strong multiphoton excitation for multiphoton microscopy. Another approach involves nonlinear nanocrystals such as ZnO which can produce four wave mixing, sum frequency generation as well as second harmonic generation to convert a deep tissue penetrating Near IR light at the targeted biological site to a desired shorter wavelength light suitable for bio imaging or activation of a therapy. We have utilized this approach to activate a photosensitizer for photodynamic therapy. Yet another type of upconversion materials is rare-earth ion doped optical nanotransformers which transform a Near IR (NIR) light from an external source by sequential single photon absorption, in situ and on demand, to a needed wavelength. Applications of these nanotransformers in multiphoton photoacoustic imaging will also be presented. An exciting direction pursued by us using these multiphoton nanoparticles, is functional imaging of brain. Simultaneously, they can effect optogenetics for regioselective stimulation of neurons for providing an effective intervention/augmentation strategy to enhance the cognitive state and lead to a foundation for futuristic vision of super human capabilities. Challenges and opportunities will be discussed.

In vivo subsurface morphological and functional cellular and subcellular imaging of the gastrointestinal tract with confocal mini-microscopy

PubMed Central

Goetz, Martin; Memadathil, Beena; Biesterfeld, Stefan; Schneider, Constantin; Gregor, Sebastian; Galle, Peter R; Neurath, Markus F; Kiesslich, Ralf

2007-01-01

AIM: To evaluate a newly developed hand-held confocal probe for in vivo microscopic imaging of the complete gastrointestinal tract in rodents. METHODS: A novel rigid confocal probe (diameter 7 mm) was designed with optical features similar to the flexible endomicroscopy system for use in humans using a 488 nm single line laser for fluorophore excitation. Light emission was detected at 505 to 750 nm. The field of view was 475 μm × 475 μm. Optical slice thickness was 7 μm with a lateral resolution of 0.7 μm. Subsurface serial images at different depths (surface to 250 μm) were generated in real time at 1024 × 1024 pixels (0.8 frames/s) by placing the probe onto the tissue in gentle, stable contact. Tissue specimens were sampled for histopathological correlation. RESULTS: The esophagus, stomach, small and large intestine and meso, liver, pancreas and gall bladder were visualised in vivo at high resolution in n = 48 mice. Real time microscopic imaging with the confocal mini-microscopy probe was easy to achieve. The different staining protocols (fluorescein, acriflavine, FITC-labelled dextran and L. esculentum lectin) each highlighted specific aspects of the tissue, and in vivo imaging correlated excellently with conventional histology. In vivo blood flow monitoring added a functional quality to morphologic imaging. CONCLUSION: Confocal microscopy is feasible in vivo allowing the visualisation of the complete GI tract at high resolution even of subsurface tissue structures. The new confocal probe design evaluated in this study is compatible with laparoscopy and significantly expands the field of possible applications to intra-abdominal organs. It allows immediate testing of new in vivo staining and application options and therefore permits rapid transfer from animal studies to clinical use in patients. PMID:17465494

Fully integrated reflection-mode photoacoustic, two-photon, and second harmonic generation microscopy in vivo

NASA Astrophysics Data System (ADS)

Song, Wei; Xu, Qiang; Zhang, Yang; Zhan, Yang; Zheng, Wei; Song, Liang

2016-08-01

The ability to obtain comprehensive structural and functional information from intact biological tissue in vivo is highly desirable for many important biomedical applications, including cancer and brain studies. Here, we developed a fully integrated multimodal microscopy that can provide photoacoustic (optical absorption), two-photon (fluorescence), and second harmonic generation (SHG) information from tissue in vivo, with intrinsically co-registered images. Moreover, using a delicately designed optical-acoustic coupling configuration, a high-frequency miniature ultrasonic transducer was integrated into a water-immersion optical objective, thus allowing all three imaging modalities to provide a high lateral resolution of ~290 nm with reflection-mode imaging capability, which is essential for studying intricate anatomy, such as that of the brain. Taking advantage of the complementary and comprehensive contrasts of the system, we demonstrated high-resolution imaging of various tissues in living mice, including microvasculature (by photoacoustics), epidermis cells, cortical neurons (by two-photon fluorescence), and extracellular collagen fibers (by SHG). The intrinsic image co-registration of the three modalities conveniently provided improved visualization and understanding of the tissue microarchitecture. The reported results suggest that, by revealing complementary tissue microstructures in vivo, this multimodal microscopy can potentially facilitate a broad range of biomedical studies, such as imaging of the tumor microenvironment and neurovascular coupling.

Improving spinning disk confocal microscopy by preventing pinhole cross-talk for intravital imaging

PubMed Central

Shimozawa, Togo; Yamagata, Kazuo; Kondo, Takefumi; Hayashi, Shigeo; Shitamukai, Atsunori; Konno, Daijiro; Matsuzaki, Fumio; Takayama, Jun; Onami, Shuichi; Nakayama, Hiroshi; Kosugi, Yasuhito; Watanabe, Tomonobu M.; Fujita, Katsumasa; Mimori-Kiyosue, Yuko

2013-01-01

A recent key requirement in life sciences is the observation of biological processes in their natural in vivo context. However, imaging techniques that allow fast imaging with higher resolution in 3D thick specimens are still limited. Spinning disk confocal microscopy using a Yokogawa Confocal Scanner Unit, which offers high-speed multipoint confocal live imaging, has been found to have wide utility among cell biologists. A conventional Confocal Scanner Unit configuration, however, is not optimized for thick specimens, for which the background noise attributed to “pinhole cross-talk,” which is unintended pinhole transmission of out-of-focus light, limits overall performance in focal discrimination and reduces confocal capability. Here, we improve spinning disk confocal microscopy by eliminating pinhole cross-talk. First, the amount of pinhole cross-talk is reduced by increasing the interpinhole distance. Second, the generation of out-of-focus light is prevented by two-photon excitation that achieves selective-plane illumination. We evaluate the effect of these modifications and test the applicability to the live imaging of green fluorescent protein-expressing model animals. As demonstrated by visualizing the fine details of the 3D cell shape and submicron-size cytoskeletal structures inside animals, these strategies dramatically improve higher-resolution intravital imaging. PMID:23401517

Improving spinning disk confocal microscopy by preventing pinhole cross-talk for intravital imaging.

PubMed

Shimozawa, Togo; Yamagata, Kazuo; Kondo, Takefumi; Hayashi, Shigeo; Shitamukai, Atsunori; Konno, Daijiro; Matsuzaki, Fumio; Takayama, Jun; Onami, Shuichi; Nakayama, Hiroshi; Kosugi, Yasuhito; Watanabe, Tomonobu M; Fujita, Katsumasa; Mimori-Kiyosue, Yuko

2013-02-26

A recent key requirement in life sciences is the observation of biological processes in their natural in vivo context. However, imaging techniques that allow fast imaging with higher resolution in 3D thick specimens are still limited. Spinning disk confocal microscopy using a Yokogawa Confocal Scanner Unit, which offers high-speed multipoint confocal live imaging, has been found to have wide utility among cell biologists. A conventional Confocal Scanner Unit configuration, however, is not optimized for thick specimens, for which the background noise attributed to "pinhole cross-talk," which is unintended pinhole transmission of out-of-focus light, limits overall performance in focal discrimination and reduces confocal capability. Here, we improve spinning disk confocal microscopy by eliminating pinhole cross-talk. First, the amount of pinhole cross-talk is reduced by increasing the interpinhole distance. Second, the generation of out-of-focus light is prevented by two-photon excitation that achieves selective-plane illumination. We evaluate the effect of these modifications and test the applicability to the live imaging of green fluorescent protein-expressing model animals. As demonstrated by visualizing the fine details of the 3D cell shape and submicron-size cytoskeletal structures inside animals, these strategies dramatically improve higher-resolution intravital imaging.

2D hybrid analysis: Approach for building three-dimensional atomic model by electron microscopy image matching.

PubMed

Matsumoto, Atsushi; Miyazaki, Naoyuki; Takagi, Junichi; Iwasaki, Kenji

2017-03-23

In this study, we develop an approach termed "2D hybrid analysis" for building atomic models by image matching from electron microscopy (EM) images of biological molecules. The key advantage is that it is applicable to flexible molecules, which are difficult to analyze by 3DEM approach. In the proposed approach, first, a lot of atomic models with different conformations are built by computer simulation. Then, simulated EM images are built from each atomic model. Finally, they are compared with the experimental EM image. Two kinds of models are used as simulated EM images: the negative stain model and the simple projection model. Although the former is more realistic, the latter is adopted to perform faster computations. The use of the negative stain model enables decomposition of the averaged EM images into multiple projection images, each of which originated from a different conformation or orientation. We apply this approach to the EM images of integrin to obtain the distribution of the conformations, from which the pathway of the conformational change of the protein is deduced.

Recent applications of hyperspectral imaging in microbiology.

PubMed

Gowen, Aoife A; Feng, Yaoze; Gaston, Edurne; Valdramidis, Vasilis

2015-05-01

Hyperspectral chemical imaging (HSI) is a broad term encompassing spatially resolved spectral data obtained through a variety of modalities (e.g. Raman scattering, Fourier transform infrared microscopy, fluorescence and near-infrared chemical imaging). It goes beyond the capabilities of conventional imaging and spectroscopy by obtaining spatially resolved spectra from objects at spatial resolutions varying from the level of single cells up to macroscopic objects (e.g. foods). In tandem with recent developments in instrumentation and sampling protocols, applications of HSI in microbiology have increased rapidly. This article gives a brief overview of the fundamentals of HSI and a comprehensive review of applications of HSI in microbiology over the past 10 years. Technical challenges and future perspectives for these techniques are also discussed. Copyright © 2015 Elsevier B.V. All rights reserved.

Ultra-high resolution coded wavefront sensor.

PubMed

Wang, Congli; Dun, Xiong; Fu, Qiang; Heidrich, Wolfgang

2017-06-12

Wavefront sensors and more general phase retrieval methods have recently attracted a lot of attention in a host of application domains, ranging from astronomy to scientific imaging and microscopy. In this paper, we introduce a new class of sensor, the Coded Wavefront Sensor, which provides high spatio-temporal resolution using a simple masked sensor under white light illumination. Specifically, we demonstrate megapixel spatial resolution and phase accuracy better than 0.1 wavelengths at reconstruction rates of 50 Hz or more, thus opening up many new applications from high-resolution adaptive optics to real-time phase retrieval in microscopy.

Overview of Single-Molecule Speckle (SiMS) Microscopy and Its Electroporation-Based Version with Efficient Labeling and Improved Spatiotemporal Resolution.

PubMed

Yamashiro, Sawako; Watanabe, Naoki

2017-07-06

Live-cell single-molecule imaging was introduced more than a decade ago, and has provided critical information on remodeling of the actin cytoskeleton, the motion of plasma membrane proteins, and dynamics of molecular motor proteins. Actin remodeling has been the best target for this approach because actin and its associated proteins stop diffusing when assembled, allowing visualization of single-molecules of fluorescently-labeled proteins in a state specific manner. The approach based on this simple principle is called Single-Molecule Speckle (SiMS) microscopy. For instance, spatiotemporal regulation of actin polymerization and lifetime distribution of actin filaments can be monitored directly by tracking actin SiMS. In combination with fluorescently labeled probes of various actin regulators, SiMS microscopy has contributed to clarifying the processes underlying recycling, motion and remodeling of the live-cell actin network. Recently, we introduced an electroporation-based method called eSiMS microscopy, with high efficiency, easiness and improved spatiotemporal precision. In this review, we describe the application of live-cell single-molecule imaging to cellular actin dynamics and discuss the advantages of eSiMS microscopy over previous SiMS microscopy.

Particle image diffusometry: Resolving diffusion coefficient field from microscopy movie data without particle tracking

NASA Astrophysics Data System (ADS)

Hanasaki, Itsuo; Ooi, Yuto

2018-06-01

We propose a technique to evaluate the field of diffusion coefficient for particle dispersion where the Brownian motion is heterogeneous in space and single particle tracking (SPT) analysis is hindered by high concentration of the particles and/or their small size. We realize this "particle image diffusometry" by the principle of the differential dynamic microscopy (DDM). We extend the DDM by introducing the automated objective decision of the scaling regime itself. Label-free evaluation of spatially non-uniform diffusion coefficients without SPT is useful in the diverse applications including crystal nucleation and glass transition where non-invasive observation is desired.

Multiplane and Spectrally-Resolved Single Molecule Localization Microscopy with Industrial Grade CMOS cameras.

PubMed

Babcock, Hazen P

2018-01-29

This work explores the use of industrial grade CMOS cameras for single molecule localization microscopy (SMLM). We show that industrial grade CMOS cameras approach the performance of scientific grade CMOS cameras at a fraction of the cost. This makes it more economically feasible to construct high-performance imaging systems with multiple cameras that are capable of a diversity of applications. In particular we demonstrate the use of industrial CMOS cameras for biplane, multiplane and spectrally resolved SMLM. We also provide open-source software for simultaneous control of multiple CMOS cameras and for the reduction of the movies that are acquired to super-resolution images.

Observation of the immune response of cells and tissue through multimodal label-free microscopy

NASA Astrophysics Data System (ADS)

Pavillon, Nicolas; Smith, Nicholas I.

2017-02-01

We present applications of a label-free approach to assess the immune response based on the combination of interferometric microscopy and Raman spectroscopy, which makes it possible to simultaneously acquire morphological and molecular information of live cells. We employ this approach to derive statistical models for predicting the activation state of macrophage cells based both on morphological parameters extracted from the high-throughput full-field quantitative phase imaging, and on the molecular content information acquired through Raman spectroscopy. We also employ a system for 3D imaging based on coherence gating, enabling specific targeting of the Raman channel to structures of interest within tissue.

High-speed bioimaging with frequency-division-multiplexed fluorescence confocal microscopy

NASA Astrophysics Data System (ADS)

Mikami, Hideharu; Harmon, Jeffrey; Ozeki, Yasuyuki; Goda, Keisuke

2017-04-01

We present methods of fluorescence confocal microscopy that enable unprecedentedly high frame rate of > 10,000 fps. The methods are based on a frequency-division multiplexing technique, which was originally developed in the field of communication engineering. Specifically, we achieved a broad bandwidth ( 400 MHz) of detection signals using a dual- AOD method and overcame limitations in frame rate, due to a scanning device, by using a multi-line focusing method, resulting in a significant increase in frame rate. The methods have potential biomedical applications such as observation of sub-millisecond dynamics in biological tissues, in-vivo three-dimensional imaging, and fluorescence imaging flow cytometry.

Core-shell designs of photoluminescent nanodiamonds with porous silica coatings for bioimaging and drug delivery II: application.

PubMed

Prabhakar, Neeraj; Näreoja, Tuomas; von Haartman, Eva; Karaman, Didem Şen; Jiang, Hua; Koho, Sami; Dolenko, Tatiana A; Hänninen, Pekka E; Vlasov, Denis I; Ralchenko, Victor G; Hosomi, Satoru; Vlasov, Igor I; Sahlgren, Cecilia; Rosenholm, Jessica M

2013-05-07

Recent advances within materials science and its interdisciplinary applications in biomedicine have emphasized the potential of using a single multifunctional composite material for concurrent drug delivery and biomedical imaging. Here we present a novel composite material consisting of a photoluminescent nanodiamond (ND) core with a porous silica (SiO2) shell. This novel multifunctional probe serves as an alternative nanomaterial to address the existing problems with delivery and subsequent tracing of the particles. Whereas the unique optical properties of ND allows for long-term live cell imaging and tracking of cellular processes, mesoporous silica nanoparticles (MSNs) have proven to be efficient drug carriers. The advantages of both ND and MSNs were hereby integrated in the new composite material, ND@MSN. The optical properties provided by the ND core rendered the nanocomposite suitable for microscopy imaging in fluorescence and reflectance mode, as well as super-resolution microscopy as a STED label; whereas the porous silica coating provided efficient intracellular delivery capacity, especially in surface-functionalized form. This study serves as a demonstration how this novel nanomaterial can be exploited for both bioimaging and drug delivery for future theranostic applications.

Tomographic phase microscopy: principles and applications in bioimaging [Invited

PubMed Central

Jin, Di; Zhou, Renjie; Yaqoob, Zahid; So, Peter T. C.

2017-01-01

Tomographic phase microscopy (TPM) is an emerging optical microscopic technique for bioimaging. TPM uses digital holographic measurements of complex scattered fields to reconstruct three-dimensional refractive index (RI) maps of cells with diffraction-limited resolution by solving inverse scattering problems. In this paper, we review the developments of TPM from the fundamental physics to its applications in bioimaging. We first provide a comprehensive description of the tomographic reconstruction physical models used in TPM. The RI map reconstruction algorithms and various regularization methods are discussed. Selected TPM applications for cellular imaging, particularly in hematology, are reviewed. Finally, we examine the limitations of current TPM systems, propose future solutions, and envision promising directions in biomedical research. PMID:29386746

Chemically Resolved Imaging of Biological Cells and Thin Films by Infrared Scanning Near-Field Optical Microscopy

PubMed Central

Cricenti, Antonio; Generosi, Renato; Luce, Marco; Perfetti, Paolo; Margaritondo, Giorgio; Talley, David; Sanghera, Jas S.; Aggarwal, Ishwar D.; Tolk, Norman H.; Congiu-Castellano, Agostina; Rizzo, Mark A.; Piston, David W.

2003-01-01

The infrared (IR) absorption of a biological system can potentially report on fundamentally important microchemical properties. For example, molecular IR profiles are known to change during increases in metabolic flux, protein phosphorylation, or proteolytic cleavage. However, practical implementation of intracellular IR imaging has been problematic because the diffraction limit of conventional infrared microscopy results in low spatial resolution. We have overcome this limitation by using an IR spectroscopic version of scanning near-field optical microscopy (SNOM), in conjunction with a tunable free-electron laser source. The results presented here clearly reveal different chemical constituents in thin films and biological cells. The space distribution of specific chemical species was obtained by taking SNOM images at IR wavelengths (λ) corresponding to stretch absorption bands of common biochemical bonds, such as the amide bond. In our SNOM implementation, this chemical sensitivity is combined with a lateral resolution of 0.1 μm (≈λ/70), well below the diffraction limit of standard infrared microscopy. The potential applications of this approach touch virtually every aspect of the life sciences and medical research, as well as problems in materials science, chemistry, physics, and environmental research. PMID:14507733

Emerging optical nanoscopy techniques

PubMed Central

Montgomery, Paul C; Leong-Hoi, Audrey

2015-01-01

To face the challenges of modern health care, new imaging techniques with subcellular resolution or detection over wide fields are required. Far field optical nanoscopy presents many new solutions, providing high resolution or detection at high speed. We present a new classification scheme to help appreciate the growing number of optical nanoscopy techniques. We underline an important distinction between superresolution techniques that provide improved resolving power and nanodetection techniques for characterizing unresolved nanostructures. Some of the emerging techniques within these two categories are highlighted with applications in biophysics and medicine. Recent techniques employing wider angle imaging by digital holography and scattering lens microscopy allow superresolution to be achieved for subcellular and even in vivo, imaging without labeling. Nanodetection techniques are divided into four subcategories using contrast, phase, deconvolution, and nanomarkers. Contrast enhancement is illustrated by means of a polarized light-based technique and with strobed phase-contrast microscopy to reveal nanostructures. Very high sensitivity phase measurement using interference microscopy is shown to provide nanometric surface roughness measurement or to reveal internal nanometric structures. Finally, the use of nanomarkers is illustrated with stochastic fluorescence microscopy for mapping intracellular structures. We also present some of the future perspectives of optical nanoscopy. PMID:26491270

Sub-diffraction-limit localization imaging of a plasmonic nanoparticle pair with wavelength-resolved dark-field microscopy.

PubMed

Wei, Lin; Ma, Yanhong; Zhu, Xupeng; Xu, Jianghong; Wang, Yaxin; Duan, Huigao; Xiao, Lehui

2017-06-29

In this work, with wavelength-resolved dark-field microscopy, the center-of-mass localization information from nanoparticle pairs (i.e., spherical (45 nm in diameter) and rod (45 × 70 nm) shaped gold nanoparticle pairs with different gap distances and orientations) was explored and compared with the results determined by scanning electron microscopy (SEM) measurements. When the gap distance was less than 20 nm, the scattering spectrum of the nanoparticle pair was seriously modulated by the plasmonic coupling effect. The measured coordinate information determined by the optical method (Gaussian fitting) was not consistent with the true results determined by SEM measurement. A good correlation between the optical and SEM measurements was achieved when the gap distance was further increased (e.g., 20, 40 and 60 nm). Under these conditions, well-defined scattering peaks assigned to the corresponding individual nanoparticles could be distinguished from the obtained scattering spectrum. These results would afford valuable information for the studies on single plasmonic nanoparticle imaging applications with the optical microscopy method such as super-localization imaging, high precision single particle tracking in a crowding environment and so on.

A New Class of Stable Heptamethine Cyanine Fluorophores and Biomedical Applications Thereof | NCI Technology Transfer Center | TTC

Cancer.gov

Researchers at the National Cancer Institute (NCI) have developed an improved class of heptamethine cyanine fluorophore dyes useful for imaging applications in the near-IR range (750-850 nm). A new chemical reaction has been developed that provides easy access to novel molecules with improved properties. Specifically, the dyes display greater resistance to thiol nucleophiles, and are more robust while maintaining excellent optical properties. The dyes have been successfully employed in various in vivo imaging applications and in vitro labeling and microscopy applications. The NCI seek co-development or licensees to develop them as targetable agents for optical-guided surgical interventions.

Automated quantitative cytological analysis using portable microfluidic microscopy.

PubMed

Jagannadh, Veerendra Kalyan; Murthy, Rashmi Sreeramachandra; Srinivasan, Rajesh; Gorthi, Sai Siva

2016-06-01

In this article, a portable microfluidic microscopy based approach for automated cytological investigations is presented. Inexpensive optical and electronic components have been used to construct a simple microfluidic microscopy system. In contrast to the conventional slide-based methods, the presented method employs microfluidics to enable automated sample handling and image acquisition. The approach involves the use of simple in-suspension staining and automated image acquisition to enable quantitative cytological analysis of samples. The applicability of the presented approach to research in cellular biology is shown by performing an automated cell viability assessment on a given population of yeast cells. Further, the relevance of the presented approach to clinical diagnosis and prognosis has been demonstrated by performing detection and differential assessment of malaria infection in a given sample. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Mosaicing of single plane illumination microscopy images using groupwise registration and fast content-based image fusion

NASA Astrophysics Data System (ADS)

Preibisch, Stephan; Rohlfing, Torsten; Hasak, Michael P.; Tomancak, Pavel

2008-03-01

Single Plane Illumination Microscopy (SPIM; Huisken et al., Nature 305(5686):1007-1009, 2004) is an emerging microscopic technique that enables live imaging of large biological specimens in their entirety. By imaging the living biological sample from multiple angles SPIM has the potential to achieve isotropic resolution throughout even relatively large biological specimens. For every angle, however, only a relatively shallow section of the specimen is imaged with high resolution, whereas deeper regions appear increasingly blurred. In order to produce a single, uniformly high resolution image, we propose here an image mosaicing algorithm that combines state of the art groupwise image registration for alignment with content-based image fusion to prevent degrading of the fused image due to regional blurring of the input images. For the registration stage, we introduce an application-specific groupwise transformation model that incorporates per-image as well as groupwise transformation parameters. We also propose a new fusion algorithm based on Gaussian filters, which is substantially faster than fusion based on local image entropy. We demonstrate the performance of our mosaicing method on data acquired from living embryos of the fruit fly, Drosophila, using four and eight angle acquisitions.

Gold Nanoparticle Quantitation by Whole Cell Tomography.

PubMed

Sanders, Aric W; Jeerage, Kavita M; Schwartz, Cindi L; Curtin, Alexandra E; Chiaramonti, Ann N

2015-12-22

Many proposed biomedical applications for engineered gold nanoparticles require their incorporation by mammalian cells in specific numbers and locations. Here, the number of gold nanoparticles inside of individual mammalian stem cells was characterized using fast focused ion beam-scanning electron microscopy based tomography. Enhanced optical microscopy was used to provide a multiscale map of the in vitro sample, which allows cells of interest to be identified within their local environment. Cells were then serially sectioned using a gallium ion beam and imaged using a scanning electron beam. To confirm the accuracy of single cross sections, nanoparticles in similar cross sections were imaged using transmission electron microscopy and scanning helium ion microscopy. Complete tomographic series were then used to count the nanoparticles inside of each cell and measure their spatial distribution. We investigated the influence of slice thickness on counting single particles and clusters as well as nanoparticle packing within clusters. For 60 nm citrate stabilized particles, the nanoparticle cluster packing volume is 2.15 ± 0.20 times the volume of the bare gold nanoparticles.

EnLightenment: High resolution smartphone microscopy as an educational and public engagement platform

PubMed Central

Wicks, Laura C.; Cairns, Gemma S.; Melnyk, Jacob; Bryce, Scott; Duncan, Rory R.; Dalgarno, Paul A.

2018-01-01

We developed a simple, cost-effective smartphone microscopy platform for use in educational and public engagement programs. We demonstrated its effectiveness, and potential for citizen science through a national imaging initiative, EnLightenment. The cost effectiveness of the instrument allowed for the program to deliver over 500 microscopes to more than 100 secondary schools throughout Scotland, targeting 1000’s of 12-14 year olds. Through careful, quantified, selection of a high power, low-cost objective lens, our smartphone microscope has an imaging resolution of microns, with a working distance of 3 mm. It is therefore capable of imaging single cells and sub-cellular features, and retains usability for young children. The microscopes were designed in kit form and provided an interdisciplinary educational tool. By providing full lesson plans and support material, we developed a framework to explore optical design, microscope performance, engineering challenges on construction and real-world applications in life sciences, biological imaging, marine biology, art, and technology. A national online imaging competition framed EnLightenment ; with over 500 high quality images submitted of diverse content, spanning multiple disciplines. With examples of cellular and sub-cellular features clearly identifiable in some submissions, we show how young public can use these instruments for research-level imaging applications, and the potential of the instrument for citizen science programs. PMID:29623296

Three-dimensional Imaging and Scanning: Current and Future Applications for Pathology

PubMed Central

Farahani, Navid; Braun, Alex; Jutt, Dylan; Huffman, Todd; Reder, Nick; Liu, Zheng; Yagi, Yukako; Pantanowitz, Liron

2017-01-01

Imaging is vital for the assessment of physiologic and phenotypic details. In the past, biomedical imaging was heavily reliant on analog, low-throughput methods, which would produce two-dimensional images. However, newer, digital, and high-throughput three-dimensional (3D) imaging methods, which rely on computer vision and computer graphics, are transforming the way biomedical professionals practice. 3D imaging has been useful in diagnostic, prognostic, and therapeutic decision-making for the medical and biomedical professions. Herein, we summarize current imaging methods that enable optimal 3D histopathologic reconstruction: Scanning, 3D scanning, and whole slide imaging. Briefly mentioned are emerging platforms, which combine robotics, sectioning, and imaging in their pursuit to digitize and automate the entire microscopy workflow. Finally, both current and emerging 3D imaging methods are discussed in relation to current and future applications within the context of pathology. PMID:28966836

Magnetic nanoparticles as contrast agents for molecular imaging in medicine

NASA Astrophysics Data System (ADS)

O'Donnell, Matthew

2018-05-01

For over twenty years, superparamagnetic nanoparticles have been developed for a number of medical applications ranging from bioseparations, magnetic drug targeting, hyperthermia and imaging. Recent studies have shown that they can be functionalized for in vivo biological targeting, potentially enabling nanoagents for molecular imaging and site-localized drug delivery. Here we review several imaging technologies developed using functionalized superparamagnetic iron oxide nanoparticles (SPIONs) as targeted molecular agents. Several imaging modalities have exploited the large induced magnetic moment of SPIONs to create local mechanical force. Magnetic force microscopy can probe nanoparticle uptake in single cells. For in vivo applications, magnetomotive modulation of primary images in ultrasound (US), photoacoustics (PA), and optical coherence tomography (OCT) can help identify very small concentrations of nanoagents while simultaneously suppressing intrinsic background signals from tissue.

Quantitative X-ray Differential Interference Contrast Microscopy

NASA Astrophysics Data System (ADS)

Nakamura, Takashi

Full-field soft x-ray microscopes are widely used in many fields of sciences. Advances in nanofabrication technology enabled short wavelength focusing elements with significantly improved spatial resolution. In the soft x-ray spectral region, samples as small as 12 nm can be resolved using micro zone-plates as the objective lens. In addition to conventional x-ray microscopy in which x-ray absorption difference provides the image contrast, phase contrast mechanisms such as differential phase contrast (DIC) and Zernike phase contrast have also been demonstrated These phase contrast imaging mechanisms are especially attractive at the x-ray wavelengths where phase contrast of most materials is typically 10 times stronger than the absorption contrast. With recent progresses in plasma-based x- ray sources and increasing accessibility to synchrotron user facilities, x-ray microscopes are quickly becoming standard measurement equipment in the laboratory. To further the usefulness of x-ray DIC microscopy this thesis explicitly addresses three known issues with this imaging modality by introducing new techniques and devices First, as opposed to its visible-light counterpart, no quantitative phase imaging technique exists for x-ray DIC microscopy. To address this issue, two nanoscale x-ray quantitative phase imaging techniques, using exclusive OR (XOR) patterns and zone-plate doublets, respectively, are proposed. Unlike existing x-ray quantitative phase imaging techniques such as Talbot interferometry and ptychography, no dedicated experimental setups or stringent illumination coherence are needed for quantitative phase retrieval. Second, to the best of our knowledge, no quantitative performance characterization of DIC microscopy exists to date. Therefore the imaging system's response to sample's spatial frequency is not known In order to gain in-depth understanding of this imaging modality, performance of x-ray DIC microscopy is quantified using modulation transfer function. A new illumination apparatus required for the transfer function analysis under partially coherent illumination is also proposed. Such a characterization is essential for a proper selection of DIC optics for various transparent samples under study. Finally, optical elements used for x-ray DIC microscopy are highly absorptive and high brilliance x-ray sources such as synchrotrons are generally needed for image contrast. To extend the use of x-ray DIC microscopy to a wider variety of applications, a high efficiency large numerical aperture optical element consisting of high reflective Bragg reflectors is proposed. Using Bragg reflectors, which have 70% ˜99% reflectivity at extreme ultraviolet and soft x-rays for all angles of glancing incidence, the first order focusing efficiency is expected to increase by ˜ 8 times compared to that of a typical Fresnel zone-plate. This thesis contributes to current nanoscale x-ray phase contrast imaging research and provides new insights for biological, material, and magnetic sciences

Multiscale nonlinear microscopy and widefield white light imaging enables rapid histological imaging of surgical specimen margins

PubMed Central

Giacomelli, Michael G.; Yoshitake, Tadayuki; Cahill, Lucas C.; Vardeh, Hilde; Quintana, Liza M.; Faulkner-Jones, Beverly E.; Brooker, Jeff; Connolly, James L.; Fujimoto, James G.

2018-01-01

The ability to histologically assess surgical specimens in real-time is a long-standing challenge in cancer surgery, including applications such as breast conserving therapy (BCT). Up to 40% of women treated with BCT for breast cancer require a repeat surgery due to postoperative histological findings of close or positive surgical margins using conventional formalin fixed paraffin embedded histology. Imaging technologies such as nonlinear microscopy (NLM), combined with exogenous fluorophores can rapidly provide virtual H&E imaging of surgical specimens without requiring microtome sectioning, facilitating intraoperative assessment of margin status. However, the large volume of typical surgical excisions combined with the need for rapid assessment, make comprehensive cellular resolution margin assessment during surgery challenging. To address this limitation, we developed a multiscale, real-time microscope with variable magnification NLM and real-time, co-registered position display using a widefield white light imaging system. Margin assessment can be performed rapidly under operator guidance to image specific regions of interest located using widefield imaging. Using simulated surgical margins dissected from human breast excisions, we demonstrate that multi-centimeter margins can be comprehensively imaged at cellular resolution, enabling intraoperative margin assessment. These methods are consistent with pathology assessment performed using frozen section analysis (FSA), however NLM enables faster and more comprehensive assessment of surgical specimens because imaging can be performed without freezing and cryo-sectioning. Therefore, NLM methods have the potential to be applied to a wide range of intra-operative applications. PMID:29761001

Automated sample area definition for high-throughput microscopy.

PubMed

Zeder, M; Ellrott, A; Amann, R

2011-04-01

High-throughput screening platforms based on epifluorescence microscopy are powerful tools in a variety of scientific fields. Although some applications are based on imaging geometrically defined samples such as microtiter plates, multiwell slides, or spotted gene arrays, others need to cope with inhomogeneously located samples on glass slides. The analysis of microbial communities in aquatic systems by sample filtration on membrane filters followed by multiple fluorescent staining, or the investigation of tissue sections are examples. Therefore, we developed a strategy for flexible and fast definition of sample locations by the acquisition of whole slide overview images and automated sample recognition by image analysis. Our approach was tested on different microscopes and the computer programs are freely available (http://www.technobiology.ch). Copyright © 2011 International Society for Advancement of Cytometry.

Murine fetal echocardiography.

PubMed

Kim, Gene H

2013-02-15

Transgenic mice displaying abnormalities in cardiac development and function represent a powerful tool for the understanding the molecular mechanisms underlying both normal cardiovascular function and the pathophysiological basis of human cardiovascular disease. Fetal and perinatal death is a common feature when studying genetic alterations affecting cardiac development. In order to study the role of genetic or pharmacologic alterations in the early development of cardiac function, ultrasound imaging of the live fetus has become an important tool for early recognition of abnormalities and longitudinal follow-up. Noninvasive ultrasound imaging is an ideal method for detecting and studying congenital malformations and the impact on cardiac function prior to death. It allows early recognition of abnormalities in the living fetus and the progression of disease can be followed in utero with longitudinal studies. Until recently, imaging of fetal mouse hearts frequently involved invasive methods. The fetus had to be sacrificed to perform magnetic resonance microscopy and electron microscopy or surgically delivered for transillumination microscopy. An application of high-frequency probes with conventional 2-D and pulsed-wave Doppler imaging has been shown to provide measurements of cardiac contraction and heart rates during embryonic development with databases of normal developmental changes now available. M-mode imaging further provides important functional data, although, the proper imaging planes are often difficult to obtain. High-frequency ultrasound imaging of the fetus has improved 2-D resolution and can provide excellent information on the early development of cardiac structures.

Identification of Metal Oxide Nanoparticles in Histological Samples by Enhanced Darkfield Microscopy and Hyperspectral Mapping.

PubMed

Roth, Gary A; Sosa Peña, Maria del Pilar; Neu-Baker, Nicole M; Tahiliani, Sahil; Brenner, Sara A

2015-12-08

Nanomaterials are increasingly prevalent throughout industry, manufacturing, and biomedical research. The need for tools and techniques that aid in the identification, localization, and characterization of nanoscale materials in biological samples is on the rise. Currently available methods, such as electron microscopy, tend to be resource-intensive, making their use prohibitive for much of the research community. Enhanced darkfield microscopy complemented with a hyperspectral imaging system may provide a solution to this bottleneck by enabling rapid and less expensive characterization of nanoparticles in histological samples. This method allows for high-contrast nanoscale imaging as well as nanomaterial identification. For this technique, histological tissue samples are prepared as they would be for light-based microscopy. First, positive control samples are analyzed to generate the reference spectra that will enable the detection of a material of interest in the sample. Negative controls without the material of interest are also analyzed in order to improve specificity (reduce false positives). Samples can then be imaged and analyzed using methods and software for hyperspectral microscopy or matched against these reference spectra in order to provide maps of the location of materials of interest in a sample. The technique is particularly well-suited for materials with highly unique reflectance spectra, such as noble metals, but is also applicable to other materials, such as semi-metallic oxides. This technique provides information that is difficult to acquire from histological samples without the use of electron microscopy techniques, which may provide higher sensitivity and resolution, but are vastly more resource-intensive and time-consuming than light microscopy.

Real-time digital signal processing in multiphoton and time-resolved microscopy

NASA Astrophysics Data System (ADS)

Wilson, Jesse W.; Warren, Warren S.; Fischer, Martin C.

2016-03-01

The use of multiphoton interactions in biological tissue for imaging contrast requires highly sensitive optical measurements. These often involve signal processing and filtering steps between the photodetector and the data acquisition device, such as photon counting and lock-in amplification. These steps can be implemented as real-time digital signal processing (DSP) elements on field-programmable gate array (FPGA) devices, an approach that affords much greater flexibility than commercial photon counting or lock-in devices. We will present progress toward developing two new FPGA-based DSP devices for multiphoton and time-resolved microscopy applications. The first is a high-speed multiharmonic lock-in amplifier for transient absorption microscopy, which is being developed for real-time analysis of the intensity-dependence of melanin, with applications in vivo and ex vivo (noninvasive histopathology of melanoma and pigmented lesions). The second device is a kHz lock-in amplifier running on a low cost (50-200) development platform. It is our hope that these FPGA-based DSP devices will enable new, high-speed, low-cost applications in multiphoton and time-resolved microscopy.

Imaging of surface spin textures on bulk crystals by scanning electron microscopy

NASA Astrophysics Data System (ADS)

Akamine, Hiroshi; Okumura, So; Farjami, Sahar; Murakami, Yasukazu; Nishida, Minoru

2016-11-01

Direct observation of magnetic microstructures is vital for advancing spintronics and other technologies. Here we report a method for imaging surface domain structures on bulk samples by scanning electron microscopy (SEM). Complex magnetic domains, referred to as the maze state in CoPt/FePt alloys, were observed at a spatial resolution of less than 100 nm by using an in-lens annular detector. The method allows for imaging almost all the domain walls in the mazy structure, whereas the visualisation of the domain walls with the classical SEM method was limited. Our method provides a simple way to analyse surface domain structures in the bulk state that can be used in combination with SEM functions such as orientation or composition analysis. Thus, the method extends applications of SEM-based magnetic imaging, and is promising for resolving various problems at the forefront of fields including physics, magnetics, materials science, engineering, and chemistry.

Augmented microscopy: real-time overlay of bright-field and near-infrared fluorescence images

PubMed Central

Watson, Jeffrey R.; Gainer, Christian F.; Martirosyan, Nikolay; Skoch, Jesse; Lemole, G. Michael; Anton, Rein; Romanowski, Marek

2015-01-01

Abstract. Intraoperative applications of near-infrared (NIR) fluorescent contrast agents can be aided by instrumentation capable of merging the view of surgical field with that of NIR fluorescence. We demonstrate augmented microscopy, an intraoperative imaging technique in which bright-field (real) and electronically processed NIR fluorescence (synthetic) images are merged within the optical path of a stereomicroscope. Under luminance of 100,000 lx, representing typical illumination of the surgical field, the augmented microscope detects 189 nM concentration of indocyanine green and produces a composite of the real and synthetic images within the eyepiece of the microscope at 20 fps. Augmentation described here can be implemented as an add-on module to visualize NIR contrast agents, laser beams, or various types of electronic data within the surgical microscopes commonly used in neurosurgical, cerebrovascular, otolaryngological, and ophthalmic procedures. PMID:26440760

Signal and noise modeling in confocal laser scanning fluorescence microscopy.

PubMed

Herberich, Gerlind; Windoffer, Reinhard; Leube, Rudolf E; Aach, Til

2012-01-01

Fluorescence confocal laser scanning microscopy (CLSM) has revolutionized imaging of subcellular structures in biomedical research by enabling the acquisition of 3D time-series of fluorescently-tagged proteins in living cells, hence forming the basis for an automated quantification of their morphological and dynamic characteristics. Due to the inherently weak fluorescence, CLSM images exhibit a low SNR. We present a novel model for the transfer of signal and noise in CLSM that is both theoretically sound as well as corroborated by a rigorous analysis of the pixel intensity statistics via measurement of the 3D noise power spectra, signal-dependence and distribution. Our model provides a better fit to the data than previously proposed models. Further, it forms the basis for (i) the simulation of the CLSM imaging process indispensable for the quantitative evaluation of CLSM image analysis algorithms, (ii) the application of Poisson denoising algorithms and (iii) the reconstruction of the fluorescence signal.

Calcium neuroimaging in behaving zebrafish larvae using a turn-key light field camera

NASA Astrophysics Data System (ADS)

Cruz Perez, Carlos; Lauri, Antonella; Symvoulidis, Panagiotis; Cappetta, Michele; Erdmann, Arne; Westmeyer, Gil Gregor

2015-09-01

Reconstructing a three-dimensional scene from multiple simultaneously acquired perspectives (the light field) is an elegant scanless imaging concept that can exceed the temporal resolution of currently available scanning-based imaging methods for capturing fast cellular processes. We tested the performance of commercially available light field cameras on a fluorescent microscopy setup for monitoring calcium activity in the brain of awake and behaving reporter zebrafish larvae. The plenoptic imaging system could volumetrically resolve diverse neuronal response profiles throughout the zebrafish brain upon stimulation with an aversive odorant. Behavioral responses of the reporter fish could be captured simultaneously together with depth-resolved neuronal activity. Overall, our assessment showed that with some optimizations for fluorescence microscopy applications, commercial light field cameras have the potential of becoming an attractive alternative to custom-built systems to accelerate molecular imaging research on cellular dynamics.

Calcium neuroimaging in behaving zebrafish larvae using a turn-key light field camera.

PubMed

Perez, Carlos Cruz; Lauri, Antonella; Symvoulidis, Panagiotis; Cappetta, Michele; Erdmann, Arne; Westmeyer, Gil Gregor

2015-09-01

Reconstructing a three-dimensional scene from multiple simultaneously acquired perspectives (the light field) is an elegant scanless imaging concept that can exceed the temporal resolution of currently available scanning-based imaging methods for capturing fast cellular processes. We tested the performance of commercially available light field cameras on a fluorescent microscopy setup for monitoring calcium activity in the brain of awake and behaving reporter zebrafish larvae. The plenoptic imaging system could volumetrically resolve diverse neuronal response profiles throughout the zebrafish brain upon stimulation with an aversive odorant. Behavioral responses of the reporter fish could be captured simultaneously together with depth-resolved neuronal activity. Overall, our assessment showed that with some optimizations for fluorescence microscopy applications, commercial light field cameras have the potential of becoming an attractive alternative to custom-built systems to accelerate molecular imaging research on cellular dynamics.

Automated classification of multiphoton microscopy images of ovarian tissue using deep learning.

PubMed

Huttunen, Mikko J; Hassan, Abdurahman; McCloskey, Curtis W; Fasih, Sijyl; Upham, Jeremy; Vanderhyden, Barbara C; Boyd, Robert W; Murugkar, Sangeeta

2018-06-01

Histopathological image analysis of stained tissue slides is routinely used in tumor detection and classification. However, diagnosis requires a highly trained pathologist and can thus be time-consuming, labor-intensive, and potentially risk bias. Here, we demonstrate a potential complementary approach for diagnosis. We show that multiphoton microscopy images from unstained, reproductive tissues can be robustly classified using deep learning techniques. We fine-train four pretrained convolutional neural networks using over 200 murine tissue images based on combined second-harmonic generation and two-photon excitation fluorescence contrast, to classify the tissues either as healthy or associated with high-grade serous carcinoma with over 95% sensitivity and 97% specificity. Our approach shows promise for applications involving automated disease diagnosis. It could also be readily applied to other tissues, diseases, and related classification problems. (2018) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE).

Ultrafast Method for the Analysis of Fluorescence Lifetime Imaging Microscopy Data Based on the Laguerre Expansion Technique

PubMed Central

Jo, Javier A.; Fang, Qiyin; Marcu, Laura

2007-01-01

We report a new deconvolution method for fluorescence lifetime imaging microscopy (FLIM) based on the Laguerre expansion technique. The performance of this method was tested on synthetic and real FLIM images. The following interesting properties of this technique were demonstrated. 1) The fluorescence intensity decay can be estimated simultaneously for all pixels, without a priori assumption of the decay functional form. 2) The computation speed is extremely fast, performing at least two orders of magnitude faster than current algorithms. 3) The estimated maps of Laguerre expansion coefficients provide a new domain for representing FLIM information. 4) The number of images required for the analysis is relatively small, allowing reduction of the acquisition time. These findings indicate that the developed Laguerre expansion technique for FLIM analysis represents a robust and extremely fast deconvolution method that enables practical applications of FLIM in medicine, biology, biochemistry, and chemistry. PMID:19444338

Application of the Indirect Immunoperoxidase Stain Technique to the Flagella of Azospirillum brasilense

PubMed Central

Hall, Patrick G.; Krieg, Noel R.

1984-01-01

An indirect immunoperoxidase stain was used to demonstrate by electron microscopy that an antigenic difference exists between the polar flagellum and the lateral flagella of Azospirillum brasilense ATCC 29145. Images PMID:16346482

Quantitative chemical imaging with background-free multiplex coherent anti-Stokes Raman scattering by dual-soliton Stokes pulses

PubMed Central

Chen, Kun; Wu, Tao; Wei, Haoyun; Zhou, Tian; Li, Yan

2016-01-01

Coherent anti-Stokes Raman microscopy (CARS) is a quantitative, chemically specific, and label-free optical imaging technique for studying inhomogeneous systems. However, the complicating influence of the nonresonant response on the CARS signal severely limits its sensitivity and specificity and especially limits the extent to which CARS microscopy has been used as a fully quantitative imaging technique. On the basis of spectral focusing mechanism, we establish a dual-soliton Stokes based CARS microspectroscopy and microscopy scheme capable of quantifying the spatial information of densities and chemical composition within inhomogeneous samples, using a single fiber laser. Dual-soliton Stokes scheme not only removes the nonresonant background but also allows robust acquisition of multiple characteristic vibrational frequencies. This all-fiber based laser source can cover the entire fingerprint (800-2200 cm−1) region with a spectral resolution of 15 cm−1. We demonstrate that quantitative degree determination of lipid-chain unsaturation in the fatty acids mixture can be achieved by the characterization of C = C stretching and CH2 deformation vibrations. For microscopy purposes, we show that the spatially inhomogeneous distribution of lipid droplets can be further quantitatively visualized using this quantified degree of lipid unsaturation in the acyl chain for contrast in the hyperspectral CARS images. The combination of compact excitation source and background-free capability to facilitate extraction of quantitative composition information with multiplex spectral peaks will enable wider applications of quantitative chemical imaging in studying biological and material systems. PMID:27867704

Backscattered electron SEM imaging of resin sections from plant specimens: observation of histological to subcellular structure and CLEM.

PubMed

Rizzo, N W; Duncan, K E; Bourett, T M; Howard, R J

2016-08-01

We have refined methods for biological specimen preparation and low-voltage backscattered electron imaging in the scanning electron microscope that allow for observation at continuous magnifications of ca. 130-70 000 X, and documentation of tissue and subcellular ultrastructure detail. The technique, based upon early work by Ogura & Hasegawa (1980), affords use of significantly larger sections from fixed and resin-embedded specimens than is possible with transmission electron microscopy while providing similar data. After microtomy, the sections, typically ca. 750 nm thick, were dried onto the surface of glass or silicon wafer and stained with heavy metals-the use of grids avoided. The glass/wafer support was then mounted onto standard scanning electron microscopy sample stubs, carbon-coated and imaged directly at an accelerating voltage of 5 kV, using either a yttrium aluminum garnet or ExB backscattered electron detector. Alternatively, the sections could be viewed first by light microscopy, for example to document signal from a fluorescent protein, and then by scanning electron microscopy to provide correlative light/electron microscope (CLEM) data. These methods provide unobstructed access to ultrastructure in the spatial context of a section ca. 7 × 10 mm in size, significantly larger than the typical 0.2 × 0.3 mm section used for conventional transmission electron microscopy imaging. Application of this approach was especially useful when the biology of interest was rare or difficult to find, e.g. a particular cell type, developmental stage, large organ, the interface between cells of interacting organisms, when contextual information within a large tissue was obligatory, or combinations of these factors. In addition, the methods were easily adapted for immunolocalizations. © 2015 The Author. Journal of Microscopy published by John Wiley & Sons, Ltd on behalf of the Royal Microscopical Society.

A small molecular pH-dependent fluorescent probe for cancer cell imaging in living cell.

PubMed

Ma, Junbao; Li, Wenqi; Li, Juanjuan; Shi, Rongguang; Yin, Gui; Wang, Ruiyong

2018-05-15

A novel pH-dependent two-photon fluorescent molecular probe ABMP has been prepared based on the fluorophore of 2, 4, 6-trisubstituted pyridine. The probe has an absorption wavelength at 354 nm and corresponding emission wavelength at 475 nm with the working pH range from 2.20 to 7.00, especially owning a good liner response from pH = 2.40 to pH = 4.00. ABMP also has excellent reversibility, photostability and selectivity which promotes its ability in analytical application. The probe can be excited with a two-photon fluorescence microscopy and the fluorescence cell imaging indicated that the probe can distinguish Hela cancer cells out of normal cells with a two-photon fluorescence microscopy which suggested its potential application in tumor cell detection. Copyright © 2018 Elsevier B.V. All rights reserved.

Vapor-condensation-assisted optical microscopy for ultralong carbon nanotubes and other nanostructures.

PubMed

Wang, Jiangtao; Li, Tianyi; Xia, Bingyu; Jin, Xiang; Wei, Haoming; Wu, Wenyun; Wei, Yang; Wang, Jiaping; Liu, Peng; Zhang, Lina; Li, Qunqing; Fan, Shoushan; Jiang, Kaili

2014-06-11

Here we present a simple yet powerful approach for the imaging of nanostructures under an optical microscope with the help of vapor condensation on their surfaces. Supersaturated water vapor will first form a nanometer-sized water droplet on the condensation nuclei on the surface of nanostructures, and then the water droplet will grow bigger and scatter more light to make the outline of the nanostructure be visible under dark-field optical microscope. This vapor-condensation-assisted (VCA) optical microscopy is applicable to a variety of nanostructures from ultralong carbon nanotubes to functional groups, generating images with contrast coming from the difference in density of the condensation sites, and does not induce any impurities to the specimens. Moreover, this low-cost and efficient technique can be conveniently integrated with other facilities, such as Raman spectroscope and so forth, which will pave the way for widespread applications.

Local delivery of fluorescent dye for fiber-optics confocal microscopy of the living heart.

PubMed

Huang, Chao; Kaza, Aditya K; Hitchcock, Robert W; Sachse, Frank B

2014-01-01

Fiber-optics confocal microscopy (FCM) is an emerging imaging technology with various applications in basic research and clinical diagnosis. FCM allows for real-time in situ microscopy of tissue at sub-cellular scale. Recently FCM has been investigated for cardiac imaging, in particular, for discrimination of cardiac tissue during pediatric open-heart surgery. FCM relies on fluorescent dyes. The current clinical approach of dye delivery is based on systemic injection, which is associated with high dye consumption, and adverse clinical events. In this study, we investigated approaches for local dye delivery during FCM imaging based on dye carriers attached to the imaging probe. Using three-dimensional confocal microscopy, automated bench tests, and FCM imaging we quantitatively characterized dye release of carriers composed of open-pore foam only and foam loaded with agarose hydrogel. In addition, we compared local dye delivery with a model of systemic dye delivery in the isolated perfused rodent heart. We measured the signal-to-noise ratio (SNR) of images acquired in various regions of the heart. Our evaluations showed that foam-agarose dye carriers exhibited a prolonged dye release vs. foam-only carriers. Foam-agarose dye carriers allowed reliable imaging of 5-9 lines, which is comparable to 4-8 min of continuous dye release. Our study in the living heart revealed that the SNR of FCM images using local and systemic dye delivery is not different. However, we observed differences in the imaged tissue microstructure with the two approaches. Structural features characteristic of microvasculature were solely observed for systemic dye delivery. Our findings suggest that local dye delivery approach for FCM imaging constitutes an important alternative to systemic dye delivery. We suggest that the approach for local dye delivery will facilitate clinical translation of FCM, for instance, for FCM imaging during pediatric heart surgery.

Hybrid Imaging for Extended Depth of Field Microscopy

NASA Astrophysics Data System (ADS)

Zahreddine, Ramzi Nicholas

An inverse relationship exists in optical systems between the depth of field (DOF) and the minimum resolvable feature size. This trade-off is especially detrimental in high numerical aperture microscopy systems where resolution is pushed to the diffraction limit resulting in a DOF on the order of 500 nm. Many biological structures and processes of interest span over micron scales resulting in significant blurring during imaging. This thesis explores a two-step computational imaging technique known as hybrid imaging to create extended DOF (EDF) microscopy systems with minimal sacrifice in resolution. In the first step a mask is inserted at the pupil plane of the microscope to create a focus invariant system over 10 times the traditional DOF, albeit with reduced contrast. In the second step the contrast is restored via deconvolution. Several EDF pupil masks from the literature are quantitatively compared in the context of biological microscopy. From this analysis a new mask is proposed, the incoherently partitioned pupil with binary phase modulation (IPP-BPM), that combines the most advantageous properties from the literature. Total variation regularized deconvolution models are derived for the various noise conditions and detectors commonly used in biological microscopy. State of the art algorithms for efficiently solving the deconvolution problem are analyzed for speed, accuracy, and ease of use. The IPP-BPM mask is compared with the literature and shown to have the highest signal-to-noise ratio and lowest mean square error post-processing. A prototype of the IPP-BPM mask is fabricated using a combination of 3D femtosecond glass etching and standard lithography techniques. The mask is compared against theory and demonstrated in biological imaging applications.

High Frequency Electromechanical Imaging of Ferroelectrics in a Liquid Environment

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jesse, Stephen; Chu, Ying-Hao; Kalinin, Sergei V

The coupling between electrical and mechanical phenomena is a ubiquitous feature of many information and energy storage materials and devices. In addition to involvement in performance and degradation mechanisms, electromechanical effects underpin a broad spectrum of nanoscale imaging and spectroscopies including piezoresponse force and electrochemical strain microscopies. Traditionally, these studies are conducted under ambient conditions. However, applications related to imaging energy storage and electrophysiological phenomena require operation in a liquid phase and therefore the development of electromechanical probing techniques suitable to liquid environments. Due to the relative high conductivity of most liquids and liquid decomposition at low voltages, the transfermore » of characterization techniques from ambient to liquid is not straightforward. Here we present a detailed study of ferroelectric domain imaging and manipulation in thin film BiFeO{sub 3} using piezoresponse force microscopy in liquid environments as model systems for electromechanical phenomena in general. We explore the use of contact resonance enhancement and the application of multifrequency excitation and detection principles to overcome the experimental problems introduced by a liquid environment. Understanding electromechanical sample characterization in liquid is a key aspect not only for ferroelectric oxides but also for biological and electrochemical sample systems.« less

A uniplanar three-axis gradient set for in vivo magnetic resonance microscopy.

PubMed

Demyanenko, Andrey V; Zhao, Lin; Kee, Yun; Nie, Shuyi; Fraser, Scott E; Tyszka, J Michael

2009-09-01

We present an optimized uniplanar magnetic resonance gradient design specifically tailored for MR imaging applications in developmental biology and histology. Uniplanar gradient designs sacrifice gradient uniformity for high gradient efficiency and slew rate, and are attractive for surface imaging applications where open access from one side of the sample is required. However, decreasing the size of the uniplanar gradient set presents several unique engineering challenges, particularly for heat dissipation and thermal insulation of the sample from gradient heating. We demonstrate a new three-axis, target-field optimized uniplanar gradient coil design that combines efficient cooling and insulation to significantly reduce sample heating at sample-gradient distances of less than 5mm. The instrument is designed for microscopy in horizontal bore magnets. Empirical gradient current efficiencies in the prototype coils lie between 3.75G/cm/A and 4.5G/cm/A with current and heating-limited maximum gradient strengths between 235G/cm and 450G/cm at a 2% duty cycle. The uniplanar gradient prototype is demonstrated with non-linearity corrections for both high-resolution structural imaging of tissue slices and for long time-course imaging of live, developing amphibian embryos in a horizontal bore 7T magnet.