High-speed vibrational imaging and spectral analysis of lipid bodies by compound Raman microscopy.

PubMed

Slipchenko, Mikhail N; Le, Thuc T; Chen, Hongtao; Cheng, Ji-Xin

2009-05-28

Cells store excess energy in the form of cytoplasmic lipid droplets. At present, it is unclear how different types of fatty acids contribute to the formation of lipid droplets. We describe a compound Raman microscope capable of both high-speed chemical imaging and quantitative spectral analysis on the same platform. We used a picosecond laser source to perform coherent Raman scattering imaging of a biological sample and confocal Raman spectral analysis at points of interest. The potential of the compound Raman microscope was evaluated on lipid bodies of cultured cells and live animals. Our data indicate that the in vivo fat contains much more unsaturated fatty acids (FAs) than the fat formed via de novo synthesis in 3T3-L1 cells. Furthermore, in vivo analysis of subcutaneous adipocytes and glands revealed a dramatic difference not only in the unsaturation level but also in the thermodynamic state of FAs inside their lipid bodies. Additionally, the compound Raman microscope allows tracking of the cellular uptake of a specific fatty acid and its abundance in nascent cytoplasmic lipid droplets. The high-speed vibrational imaging and spectral analysis capability renders compound Raman microscopy an indispensible analytical tool for the study of lipid-droplet biology.

High-speed imaging using compressed sensing and wavelength-dependent scattering (Conference Presentation)

NASA Astrophysics Data System (ADS)

Shin, Jaewook; Bosworth, Bryan T.; Foster, Mark A.

2017-02-01

The process of multiple scattering has inherent characteristics that are attractive for high-speed imaging with high spatial resolution and a wide field-of-view. A coherent source passing through a multiple-scattering medium naturally generates speckle patterns with diffraction-limited features over an arbitrarily large field-of-view. In addition, the process of multiple scattering is deterministic allowing a given speckle pattern to be reliably reproduced with identical illumination conditions. Here, by exploiting wavelength dependent multiple scattering and compressed sensing, we develop a high-speed 2D time-stretch microscope. Highly chirped pulses from a 90-MHz mode-locked laser are sent through a 2D grating and a ground-glass diffuser to produce 2D speckle patterns that rapidly evolve with the instantaneous frequency of the chirped pulse. To image a scene, we first characterize the high-speed evolution of the generated speckle patterns. Subsequently we project the patterns onto the microscopic region of interest and collect the total light from the scene using a single high-speed photodetector. Thus the wavelength dependent speckle patterns serve as high-speed pseudorandom structured illumination of the scene. An image sequence is then recovered using the time-dependent signal received by the photodetector, the known speckle pattern evolution, and compressed sensing algorithms. Notably, the use of compressed sensing allows for reconstruction of a time-dependent scene using a highly sub-Nyquist number of measurements, which both increases the speed of the imager and reduces the amount of data that must be collected and stored. We will discuss our experimental demonstration of this approach and the theoretical limits on imaging speed.

Automatic analysis for neuron by confocal laser scanning microscope

NASA Astrophysics Data System (ADS)

Satou, Kouhei; Aoki, Yoshimitsu; Mataga, Nobuko; Hensh, Takao K.; Taki, Katuhiko

2005-12-01

The aim of this study is to develop a system that recognizes both the macro- and microscopic configurations of nerve cells and automatically performs the necessary 3-D measurements and functional classification of spines. The acquisition of 3-D images of cranial nerves has been enabled by the use of a confocal laser scanning microscope, although the highly accurate 3-D measurements of the microscopic structures of cranial nerves and their classification based on their configurations have not yet been accomplished. In this study, in order to obtain highly accurate measurements of the microscopic structures of cranial nerves, existing positions of spines were predicted by the 2-D image processing of tomographic images. Next, based on the positions that were predicted on the 2-D images, the positions and configurations of the spines were determined more accurately by 3-D image processing of the volume data. We report the successful construction of an automatic analysis system that uses a coarse-to-fine technique to analyze the microscopic structures of cranial nerves with high speed and accuracy by combining 2-D and 3-D image analyses.

An integrated single- and two-photon non-diffracting light-sheet microscope

NASA Astrophysics Data System (ADS)

Lau, Sze Cheung; Chiu, Hoi Chun; Zhao, Luwei; Zhao, Teng; Loy, M. M. T.; Du, Shengwang

2018-04-01

We describe a fluorescence optical microscope with both single-photon and two-photon non-diffracting light-sheet excitations for large volume imaging. With a special design to accommodate two different wavelength ranges (visible: 400-700 nm and near infrared: 800-1200 nm), we combine the line-Bessel sheet (LBS, for single-photon excitation) and the scanning Bessel beam (SBB, for two-photon excitation) light sheet together in a single microscope setup. For a transparent thin sample where the scattering can be ignored, the LBS single-photon excitation is the optimal imaging solution. When the light scattering becomes significant for a deep-cell or deep-tissue imaging, we use SBB light-sheet two-photon excitation with a longer wavelength. We achieved nearly identical lateral/axial resolution of about 350/270 nm for both imagings. This integrated light-sheet microscope may have a wide application for live-cell and live-tissue three-dimensional high-speed imaging.

Movement measurement of isolated skeletal muscle using imaging microscopy

NASA Astrophysics Data System (ADS)

Elias, David; Zepeda, Hugo; Leija, Lorenzo S.; Sossa, Humberto; de la Rosa, Jose I.

1997-05-01

An imaging-microscopy methodology to measure contraction movement in chemically stimulated crustacean skeletal muscle, whose movement speed is about 0.02 mm/s is presented. For this, a CCD camera coupled to a microscope and a high speed digital image acquisition system, allowing us to capture 960 images per second are used. The images are digitally processed in a PC and displayed in a video monitor. A maximal field of 0.198 X 0.198 mm2 and a spatial resolution of 3.5 micrometers are obtained.

Digital image processing of bone - Problems and potentials

NASA Technical Reports Server (NTRS)

Morey, E. R.; Wronski, T. J.

1980-01-01

The development of a digital image processing system for bone histomorphometry and fluorescent marker monitoring is discussed. The system in question is capable of making measurements of UV or light microscope features on a video screen with either video or computer-generated images, and comprises a microscope, low-light-level video camera, video digitizer and display terminal, color monitor, and PDP 11/34 computer. Capabilities demonstrated in the analysis of an undecalcified rat tibia include the measurement of perimeter and total bone area, and the generation of microscope images, false color images, digitized images and contoured images for further analysis. Software development will be based on an existing software library, specifically the mini-VICAR system developed at JPL. It is noted that the potentials of the system in terms of speed and reliability far exceed any problems associated with hardware and software development.

High-throughput microscopy must re-invent the microscope rather than speed up its functions

PubMed Central

Oheim, M

2007-01-01

Knowledge gained from the revolutions in genomics and proteomics has helped to identify many of the key molecules involved in cellular signalling. Researchers, both in academia and in the pharmaceutical industry, now screen, at a sub-cellular level, where and when these proteins interact. Fluorescence imaging and molecular labelling combine to provide a powerful tool for real-time functional biochemistry with molecular resolution. However, they traditionally have been work-intensive, required trained personnel, and suffered from low through-put due to sample preparation, loading and handling. The need for speeding up microscopy is apparent from the tremendous complexity of cellular signalling pathways, the inherent biological variability, as well as the possibility that the same molecule plays different roles in different sub-cellular compartments. Research institutes and companies have teamed up to develop imaging cytometers of ever-increasing complexity. However, to truly go high-speed, sub-cellular imaging must free itself from the rigid framework of current microscopes. PMID:17603553

Design of a high-speed electrochemical scanning tunneling microscope.

PubMed

Yanson, Y I; Schenkel, F; Rost, M J

2013-02-01

In this paper, we present a bottom-up approach to designing and constructing a high-speed electrochemical scanning tunneling microscope (EC-STM). Using finite element analysis (FEA) calculations of the frequency response of the whole mechanical loop of the STM, we analyzed several geometries to find the most stable one that could facilitate fast scanning. To test the FEA results, we conducted measurements of the vibration amplitudes using a prototype STM setup. Based on the FEA analysis and the measurement results, we identified the potentially most disturbing vibration modes that could impair fast scanning. By modifying the design of some parts of the EC-STM, we reduced the amplitudes as well as increased the resonance frequencies of these modes. Additionally, we designed and constructed an electrochemical flow-cell that allows STM imaging in a flowing electrolyte, and built a bi-potentiostat to achieve electrochemical potential control during the measurements. Finally, we present STM images acquired during high-speed imaging in air as well as in an electrochemical environment using our newly-developed EC-STM.

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.

A high speed multifocal multiphoton fluorescence lifetime imaging microscope for live-cell FRET imaging

PubMed Central

Poland, Simon P.; Krstajić, Nikola; Monypenny, James; Coelho, Simao; Tyndall, David; Walker, Richard J.; Devauges, Viviane; Richardson, Justin; Dutton, Neale; Barber, Paul; Li, David Day-Uei; Suhling, Klaus; Ng, Tony; Henderson, Robert K.; Ameer-Beg, Simon M.

2015-01-01

We demonstrate diffraction limited multiphoton imaging in a massively parallel, fully addressable time-resolved multi-beam multiphoton microscope capable of producing fluorescence lifetime images with sub-50ps temporal resolution. This imaging platform offers a significant improvement in acquisition speed over single-beam laser scanning FLIM by a factor of 64 without compromising in either the temporal or spatial resolutions of the system. We demonstrate FLIM acquisition at 500 ms with live cells expressing green fluorescent protein. The applicability of the technique to imaging protein-protein interactions in live cells is exemplified by observation of time-dependent FRET between the epidermal growth factor receptor (EGFR) and the adapter protein Grb2 following stimulation with the receptor ligand. Furthermore, ligand-dependent association of HER2-HER3 receptor tyrosine kinases was observed on a similar timescale and involved the internalisation and accumulation or receptor heterodimers within endosomes. These data demonstrate the broad applicability of this novel FLIM technique to the spatio-temporal dynamics of protein-protein interaction. PMID:25780724

Medical microscopic image matching based on relativity

NASA Astrophysics Data System (ADS)

Xie, Fengying; Zhu, Liangen; Jiang, Zhiguo

2003-12-01

In this paper, an effective medical micro-optical image matching algorithm based on relativity is described. The algorithm includes the following steps: Firstly, selecting a sub-area that has obvious character in one of the two images as standard image; Secondly, finding the right matching position in the other image; Thirdly, applying coordinate transformation to merge the two images together. As a kind of application of image matching in medical micro-optical image, this method overcomes the shortcoming of microscope whose visual field is little and makes it possible to watch a big object or many objects in one view. Simultaneously it implements adaptive selection of standard image, and has a satisfied matching speed and result.

Programmable Illumination and High-Speed, Multi-Wavelength, Confocal Microscopy Using a Digital Micromirror

PubMed Central

Martial, Franck P.; Hartell, Nicholas A.

2012-01-01

Confocal microscopy is routinely used for high-resolution fluorescence imaging of biological specimens. Most standard confocal systems scan a laser across a specimen and collect emitted light passing through a single pinhole to produce an optical section of the sample. Sequential scanning on a point-by-point basis limits the speed of image acquisition and even the fastest commercial instruments struggle to resolve the temporal dynamics of rapid cellular events such as calcium signals. Various approaches have been introduced that increase the speed of confocal imaging. Nipkov disk microscopes, for example, use arrays of pinholes or slits on a spinning disk to achieve parallel scanning which significantly increases the speed of acquisition. Here we report the development of a microscope module that utilises a digital micromirror device as a spatial light modulator to provide programmable confocal optical sectioning with a single camera, at high spatial and axial resolution at speeds limited by the frame rate of the camera. The digital micromirror acts as a solid state Nipkov disk but with the added ability to change the pinholes size and separation and to control the light intensity on a mirror-by-mirror basis. The use of an arrangement of concave and convex mirrors in the emission pathway instead of lenses overcomes the astigmatism inherent with DMD devices, increases light collection efficiency and ensures image collection is achromatic so that images are perfectly aligned at different wavelengths. Combined with non-laser light sources, this allows low cost, high-speed, multi-wavelength image acquisition without the need for complex wavelength-dependent image alignment. The micromirror can also be used for programmable illumination allowing spatially defined photoactivation of fluorescent proteins. We demonstrate the use of this system for high-speed calcium imaging using both a single wavelength calcium indicator and a genetically encoded, ratiometric, calcium sensor. PMID:22937130

Programmable illumination and high-speed, multi-wavelength, confocal microscopy using a digital micromirror.

PubMed

Martial, Franck P; Hartell, Nicholas A

2012-01-01

Confocal microscopy is routinely used for high-resolution fluorescence imaging of biological specimens. Most standard confocal systems scan a laser across a specimen and collect emitted light passing through a single pinhole to produce an optical section of the sample. Sequential scanning on a point-by-point basis limits the speed of image acquisition and even the fastest commercial instruments struggle to resolve the temporal dynamics of rapid cellular events such as calcium signals. Various approaches have been introduced that increase the speed of confocal imaging. Nipkov disk microscopes, for example, use arrays of pinholes or slits on a spinning disk to achieve parallel scanning which significantly increases the speed of acquisition. Here we report the development of a microscope module that utilises a digital micromirror device as a spatial light modulator to provide programmable confocal optical sectioning with a single camera, at high spatial and axial resolution at speeds limited by the frame rate of the camera. The digital micromirror acts as a solid state Nipkov disk but with the added ability to change the pinholes size and separation and to control the light intensity on a mirror-by-mirror basis. The use of an arrangement of concave and convex mirrors in the emission pathway instead of lenses overcomes the astigmatism inherent with DMD devices, increases light collection efficiency and ensures image collection is achromatic so that images are perfectly aligned at different wavelengths. Combined with non-laser light sources, this allows low cost, high-speed, multi-wavelength image acquisition without the need for complex wavelength-dependent image alignment. The micromirror can also be used for programmable illumination allowing spatially defined photoactivation of fluorescent proteins. We demonstrate the use of this system for high-speed calcium imaging using both a single wavelength calcium indicator and a genetically encoded, ratiometric, calcium sensor.

Multiresolution multiscale active mask segmentation of fluorescence microscope images

NASA Astrophysics Data System (ADS)

Srinivasa, Gowri; Fickus, Matthew; Kovačević, Jelena

2009-08-01

We propose an active mask segmentation framework that combines the advantages of statistical modeling, smoothing, speed and flexibility offered by the traditional methods of region-growing, multiscale, multiresolution and active contours respectively. At the crux of this framework is a paradigm shift from evolving contours in the continuous domain to evolving multiple masks in the discrete domain. Thus, the active mask framework is particularly suited to segment digital images. We demonstrate the use of the framework in practice through the segmentation of punctate patterns in fluorescence microscope images. Experiments reveal that statistical modeling helps the multiple masks converge from a random initial configuration to a meaningful one. This obviates the need for an involved initialization procedure germane to most of the traditional methods used to segment fluorescence microscope images. While we provide the mathematical details of the functions used to segment fluorescence microscope images, this is only an instantiation of the active mask framework. We suggest some other instantiations of the framework to segment different types of images.

High-resolution retinal imaging using adaptive optics and Fourier-domain optical coherence tomography

DOEpatents

Olivier, Scot S.; Werner, John S.; Zawadzki, Robert J.; Laut, Sophie P.; Jones, Steven M.

2010-09-07

This invention permits retinal images to be acquired at high speed and with unprecedented resolution in three dimensions (4.times.4.times.6 .mu.m). The instrument achieves high lateral resolution by using adaptive optics to correct optical aberrations of the human eye in real time. High axial resolution and high speed are made possible by the use of Fourier-domain optical coherence tomography. Using this system, we have demonstrated the ability to image microscopic blood vessels and the cone photoreceptor mosaic.

A wide field-of-view microscope based on holographic focus grid

NASA Astrophysics Data System (ADS)

Wu, Jigang; Cui, Xiquan; Zheng, Guoan; Lee, Lap Man; Yang, Changhuei

2010-02-01

We have developed a novel microscope technique that can achieve wide field-of-view (FOV) imaging and yet possess resolution that is comparable to conventional microscope. The principle of wide FOV microscope system breaks the link between resolution and FOV magnitude of traditional microscopes. Furthermore, by eliminating bulky optical elements from its design and utilizing holographic optical elements, the wide FOV microscope system is more cost-effective. In our system, a hologram was made to focus incoming collimated beam into a focus grid. The sample is put in the focal plane and the transmissions of the focuses are detected by an imaging sensor. By scanning the incident angle of the incoming beam, the focus grid will scan across the sample and the time-varying transmission can be detected. We can then reconstruct the transmission image of the sample. The resolution of microscopic image is limited by the size of the focus formed by the hologram. The scanning area of each focus spot is determined by the separation of the focus spots and can be made small for fast imaging speed. We have fabricated a prototype system with a 2.4-mm FOV and 1-μm resolution. The prototype system was used to image onion skin cells for a demonstration. The preliminary experiments prove the feasibility of the wide FOV microscope technique, and the possibility of a wider FOV system with better resolution.

PScan 1.0: flexible software framework for polygon based multiphoton microscopy

NASA Astrophysics Data System (ADS)

Li, Yongxiao; Lee, Woei Ming

2016-12-01

Multiphoton laser scanning microscopes exhibit highly localized nonlinear optical excitation and are powerful instruments for in-vivo deep tissue imaging. Customized multiphoton microscopy has a significantly superior performance for in-vivo imaging because of precise control over the scanning and detection system. To date, there have been several flexible software platforms catered to custom built microscopy systems i.e. ScanImage, HelioScan, MicroManager, that perform at imaging speeds of 30-100fps. In this paper, we describe a flexible software framework for high speed imaging systems capable of operating from 5 fps to 1600 fps. The software is based on the MATLAB image processing toolbox. It has the capability to communicate directly with a high performing imaging card (Matrox Solios eA/XA), thus retaining high speed acquisition. The program is also designed to communicate with LabVIEW and Fiji for instrument control and image processing. Pscan 1.0 can handle high imaging rates and contains sufficient flexibility for users to adapt to their high speed imaging systems.

Vibration compensation for high speed scanning tunneling microscopy

NASA Astrophysics Data System (ADS)

Croft, D.; Devasia, S.

1999-12-01

Low scanning speed is a fundamental limitation of scanning tunneling microscopes (STMs), making real time imaging of surface processes and nanofabrication impractical. The effective scanning bandwidth is currently limited by the smallest resonant vibrational frequency of the piezobased positioning system (i.e., scanner) used in the STM. Due to this limitation, the acquired images are distorted during high speed operations. In practice, the achievable scan rates are much less than 1/10th of the resonant vibrational frequency of the STM scanner. To alleviate the scanning speed limitation, this article describes an inversion-based approach that compensates for the structural vibrations in the scanner and thus, allows STM imaging at high scanning speeds (relative to the smallest resonant vibrational frequency). Experimental results are presented to show the increase in scanning speeds achievable by applying the vibration compensation methods.

A framed, 16-image Kirkpatrick–Baez x-ray microscope

DOE PAGES

Marshall, F. J.; Bahr, R. E.; Goncharov, V. N.; ...

2017-09-08

A 16-image Kirkpatrick–Baez (KB)–type x-ray microscope consisting of compact KB mirrors has been assembled for the first time with mirrors aligned to allow it to be coupled to a high-speed framing camera. The high-speed framing camera has four independently gated strips whose emission sampling interval is ~30 ps. Images are arranged four to a strip with ~60-ps temporal spacing between frames on a strip. By spacing the timing of the strips, a frame spacing of ~15 ps is achieved. A framed resolution of ~6-um is achieved with this combination in a 400-um region of laser–plasma x-ray emission in the 2-more » to 8-keV energy range. A principal use of the microscope is to measure the evolution of the implosion stagnation region of cryogenic DT target implosions on the University of Rochester’s OMEGA Laser System. The unprecedented time and spatial resolution achieved with this framed, multi-image KB microscope have made it possible to accurately determine the cryogenic implosion core emission size and shape at the peak of stagnation. In conclusion, these core size measurements, taken in combination with those of ion temperature, neutron-production temporal width, and neutron yield allow for inference of core pressures, currently exceeding 50 GBar in OMEGA cryogenic target implosions.« less

A framed, 16-image Kirkpatrick–Baez x-ray microscope

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

Marshall, F. J.; Bahr, R. E.; Goncharov, V. N.

A 16-image Kirkpatrick–Baez (KB)–type x-ray microscope consisting of compact KB mirrors has been assembled for the first time with mirrors aligned to allow it to be coupled to a high-speed framing camera. The high-speed framing camera has four independently gated strips whose emission sampling interval is ~30 ps. Images are arranged four to a strip with ~60-ps temporal spacing between frames on a strip. By spacing the timing of the strips, a frame spacing of ~15 ps is achieved. A framed resolution of ~6-um is achieved with this combination in a 400-um region of laser–plasma x-ray emission in the 2-more » to 8-keV energy range. A principal use of the microscope is to measure the evolution of the implosion stagnation region of cryogenic DT target implosions on the University of Rochester’s OMEGA Laser System. The unprecedented time and spatial resolution achieved with this framed, multi-image KB microscope have made it possible to accurately determine the cryogenic implosion core emission size and shape at the peak of stagnation. In conclusion, these core size measurements, taken in combination with those of ion temperature, neutron-production temporal width, and neutron yield allow for inference of core pressures, currently exceeding 50 GBar in OMEGA cryogenic target implosions.« less

Single-channel stereoscopic ophthalmology microscope based on TRD

NASA Astrophysics Data System (ADS)

Radfar, Edalat; Park, Jihoon; Lee, Sangyeob; Ha, Myungjin; Yu, Sungkon; Jang, Seulki; Jung, Byungjo

2016-03-01

A stereoscopic imaging modality was developed for the application of ophthalmology surgical microscopes. A previous study has already introduced a single-channel stereoscopic video imaging modality based on a transparent rotating deflector (SSVIM-TRD), in which two different view angles, image disparity, are generated by imaging through a transparent rotating deflector (TRD) mounted on a stepping motor and is placed in a lens system. In this case, the image disparity is a function of the refractive index and the rotation angle of TRD. Real-time single-channel stereoscopic ophthalmology microscope (SSOM) based on the TRD is improved by real-time controlling and programming, imaging speed, and illumination method. Image quality assessments were performed to investigate images quality and stability during the TRD operation. Results presented little significant difference in image quality in terms of stability of structural similarity (SSIM). A subjective analysis was performed with 15 blinded observers to evaluate the depth perception improvement and presented significant improvement in the depth perception capability. Along with all evaluation results, preliminary results of rabbit eye imaging presented that the SSOM could be utilized as an ophthalmic operating microscopes to overcome some of the limitations of conventional ones.

Miniaturized integration of a fluorescence microscope

PubMed Central

Ghosh, Kunal K.; Burns, Laurie D.; Cocker, Eric D.; Nimmerjahn, Axel; Ziv, Yaniv; Gamal, Abbas El; Schnitzer, Mark J.

2013-01-01

The light microscope is traditionally an instrument of substantial size and expense. Its miniaturized integration would enable many new applications based on mass-producible, tiny microscopes. Key prospective usages include brain imaging in behaving animals towards relating cellular dynamics to animal behavior. Here we introduce a miniature (1.9 g) integrated fluorescence microscope made from mass-producible parts, including semiconductor light source and sensor. This device enables high-speed cellular-level imaging across ∼0.5 mm2 areas in active mice. This capability allowed concurrent tracking of Ca2+ spiking in >200 Purkinje neurons across nine cerebellar microzones. During mouse locomotion, individual microzones exhibited large-scale, synchronized Ca2+ spiking. This is a mesoscopic neural dynamic missed by prior techniques for studying the brain at other length scales. Overall, the integrated microscope is a potentially transformative technology that permits distribution to many animals and enables diverse usages, such as portable diagnostics or microscope arrays for large-scale screens. PMID:21909102

Miniaturized integration of a fluorescence microscope.

PubMed

Ghosh, Kunal K; Burns, Laurie D; Cocker, Eric D; Nimmerjahn, Axel; Ziv, Yaniv; Gamal, Abbas El; Schnitzer, Mark J

2011-09-11

The light microscope is traditionally an instrument of substantial size and expense. Its miniaturized integration would enable many new applications based on mass-producible, tiny microscopes. Key prospective usages include brain imaging in behaving animals for relating cellular dynamics to animal behavior. Here we introduce a miniature (1.9 g) integrated fluorescence microscope made from mass-producible parts, including a semiconductor light source and sensor. This device enables high-speed cellular imaging across ∼0.5 mm2 areas in active mice. This capability allowed concurrent tracking of Ca2+ spiking in >200 Purkinje neurons across nine cerebellar microzones. During mouse locomotion, individual microzones exhibited large-scale, synchronized Ca2+ spiking. This is a mesoscopic neural dynamic missed by prior techniques for studying the brain at other length scales. Overall, the integrated microscope is a potentially transformative technology that permits distribution to many animals and enables diverse usages, such as portable diagnostics or microscope arrays for large-scale screens.

Fluorescence Microscopy Gets Faster and Clearer: Roles of Photochemistry and Selective Illumination

PubMed Central

Wolenski, Joseph S.; Julich, Doerthe

2014-01-01

Significant advances in fluorescence microscopy tend be a balance between two competing qualities wherein improvements in resolution and low light detection are typically accompanied by losses in acquisition rate and signal-to-noise, respectively. These trade-offs are becoming less of a barrier to biomedical research as recent advances in optoelectronic microscopy and developments in fluorophore chemistry have enabled scientists to see beyond the diffraction barrier, image deeper into live specimens, and acquire images at unprecedented speed. Selective plane illumination microscopy has provided significant gains in the spatial and temporal acquisition of fluorescence specimens several mm in thickness. With commercial systems now available, this method promises to expand on recent advances in 2-photon deep-tissue imaging with improved speed and reduced photobleaching compared to laser scanning confocal microscopy. Superresolution microscopes are also available in several modalities and can be coupled with selective plane illumination techniques. The combination of methods to increase resolution, acquisition speed, and depth of collection are now being married to common microscope systems, enabling scientists to make significant advances in live cell and in situ imaging in real time. We show that light sheet microscopy provides significant advantages for imaging live zebrafish embryos compared to laser scanning confocal microscopy. PMID:24600334

High speed quantitative digital microscopy

NASA Technical Reports Server (NTRS)

Castleman, K. R.; Price, K. H.; Eskenazi, R.; Ovadya, M. M.; Navon, M. A.

1984-01-01

Modern digital image processing hardware makes possible quantitative analysis of microscope images at high speed. This paper describes an application to automatic screening for cervical cancer. The system uses twelve MC6809 microprocessors arranged in a pipeline multiprocessor configuration. Each processor executes one part of the algorithm on each cell image as it passes through the pipeline. Each processor communicates with its upstream and downstream neighbors via shared two-port memory. Thus no time is devoted to input-output operations as such. This configuration is expected to be at least ten times faster than previous systems.

Visualization of Electrical Field of Electrode Using Voltage-Controlled Fluorescence Release

PubMed Central

Jia, Wenyan; Wu, Jiamin; Gao, Di; Wang, Hao; Sun, Mingui

2016-01-01

In this study we propose an approach to directly visualize electrical current distribution at the electrode-electrolyte interface of a biopotential electrode. High-speed fluorescent microscopic images are acquired when an electric potential is applied across the interface to trigger the release of fluorescent material from the surface of the electrode. These images are analyzed computationally to obtain the distribution of the electric field from the fluorescent intensity of each pixel. Our approach allows direct observation of microscopic electrical current distribution around the electrode. Experiments are conducted to validate the feasibility of the fluorescent imaging method. PMID:27253615

Compact multi-band fluorescent microscope with an electrically tunable lens for autofocusing

PubMed Central

Wang, Zhaojun; Lei, Ming; Yao, Baoli; Cai, Yanan; Liang, Yansheng; Yang, Yanlong; Yang, Xibin; Li, Hui; Xiong, Daxi

2015-01-01

Autofocusing is a routine technique in redressing focus drift that occurs in time-lapse microscopic image acquisition. To date, most automatic microscopes are designed on the distance detection scheme to fulfill the autofocusing operation, which may suffer from the low contrast of the reflected signal due to the refractive index mismatch at the water/glass interface. To achieve high autofocusing speed with minimal motion artifacts, we developed a compact multi-band fluorescent microscope with an electrically tunable lens (ETL) device for autofocusing. A modified searching algorithm based on equidistant scanning and curve fitting is proposed, which no longer requires a single-peak focus curve and then efficiently restrains the impact of external disturbance. This technique enables us to achieve an autofocusing time of down to 170 ms and the reproductivity of over 97%. The imaging head of the microscope has dimensions of 12 cm × 12 cm × 6 cm. This portable instrument can easily fit inside standard incubators for real-time imaging of living specimens. PMID:26601001

Nanoscale characterization of local structures and defects in photonic crystals using synchrotron-based transmission soft X-ray microscopy

PubMed Central

Nho, Hyun Woo; Kalegowda, Yogesh; Shin, Hyun-Joon; Yoon, Tae Hyun

2016-01-01

For the structural characterization of the polystyrene (PS)-based photonic crystals (PCs), fast and direct imaging capabilities of full field transmission X-ray microscopy (TXM) were demonstrated at soft X-ray energy. PS-based PCs were prepared on an O2-plasma treated Si3N4 window and their local structures and defects were investigated using this label-free TXM technique with an image acquisition speed of ~10 sec/frame and marginal radiation damage. Micro-domains of face-centered cubic (FCC (111)) and hexagonal close-packed (HCP (0001)) structures were dominantly found in PS-based PCs, while point and line defects, FCC (100), and 12-fold symmetry structures were also identified as minor components. Additionally, in situ observation capability for hydrated samples and 3D tomographic reconstruction of TXM images were also demonstrated. This soft X-ray full field TXM technique with faster image acquisition speed, in situ observation, and 3D tomography capability can be complementally used with the other X-ray microscopic techniques (i.e., scanning transmission X-ray microscopy, STXM) as well as conventional characterization methods (e.g., electron microscopic and optical/fluorescence microscopic techniques) for clearer structure identification of self-assembled PCs and better understanding of the relationship between their structures and resultant optical properties. PMID:27087141

Performance comparison between the high-speed Yokogawa spinning disc confocal system and single-point scanning confocal systems.

PubMed

Wang, E; Babbey, C M; Dunn, K W

2005-05-01

Fluorescence microscopy of the dynamics of living cells presents a special challenge to a microscope imaging system, simultaneously requiring both high spatial resolution and high temporal resolution, but with illumination levels low enough to prevent fluorophore damage and cytotoxicity. We have compared the high-speed Yokogawa CSU10 spinning disc confocal system with several conventional single-point scanning confocal (SPSC) microscopes, using the relationship between image signal-to-noise ratio and fluorophore photobleaching as an index of system efficiency. These studies demonstrate that the efficiency of the CSU10 consistently exceeds that of the SPSC systems. The high efficiency of the CSU10 means that quality images can be collected with much lower levels of illumination; the CSU10 was capable of achieving the maximum signal-to-noise of an SPSC system at illumination levels that incur only at 1/15th of the rate of the photobleaching of the SPSC system. Although some of the relative efficiency of the CSU10 system may be attributed to the use of a CCD rather than a photomultiplier detector system, our analyses indicate that high-speed imaging with the SPSC system is limited by fluorescence saturation at the high levels of illumination frequently needed to collect images at high frame rates. The high speed, high efficiency and freedom from fluorescence saturation combine to make the CSU10 effective for extended imaging of living cells at rates capable of capturing the three-dimensional motion of endosomes moving up to several micrometres per second.

Isometric multimodal photoacoustic microscopy based on optically transparent micro-ring ultrasonic detection.

PubMed

Dong, Biqin; Li, Hao; Zhang, Zhen; Zhang, Kevin; Chen, Siyu; Sun, Cheng; Zhang, Hao F

2015-01-01

Photoacoustic microscopy (PAM) is an attractive imaging tool complementary to established optical microscopic modalities by providing additional molecular specificities through imaging optical absorption contrast. While the development of optical resolution photoacoustic microscopy (ORPAM) offers high lateral resolution, the acoustically-determined axial resolution is limited due to the constraint in ultrasonic detection bandwidth. ORPAM with isometric spatial resolution along both axial and lateral direction is yet to be developed. Although recently developed sophisticated optical illumination and reconstruction methods offer improved axial resolution in ORPAM, the image acquisition procedures are rather complicated, limiting their capabilities for high-speed imaging and being easily integrated with established optical microscopic modalities. Here we report an isometric ORPAM based on an optically transparent micro-ring resonator ultrasonic detector and a commercial inverted microscope platform. Owing to the superior spatial resolution and the ease of integrating our ORPAM with established microscopic modalities, single cell imaging with extrinsic fluorescence staining, intrinsic autofluorescence, and optical absorption can be achieved simultaneously. This technique holds promise to greatly improve the accessibility of PAM to the broader biomedical researchers.

Single-photon counting multicolor multiphoton fluorescence microscope.

PubMed

Buehler, Christof; Kim, Ki H; Greuter, Urs; Schlumpf, Nick; So, Peter T C

2005-01-01

We present a multicolor multiphoton fluorescence microscope with single-photon counting sensitivity. The system integrates a standard multiphoton fluorescence microscope, an optical grating spectrograph operating in the UV-Vis wavelength region, and a 16-anode photomultiplier tube (PMT). The major technical innovation is in the development of a multichannel photon counting card (mC-PhCC) for direct signal collection from multi-anode PMTs. The electronic design of the mC-PhCC employs a high-throughput, fully-parallel, single-photon counting scheme along with a high-speed electrical or fiber-optical link interface to the data acquisition computer. There is no electronic crosstalk among the detection channels of the mC-PhCC. The collected signal remains linear up to an incident photon rate of 10(8) counts per second. The high-speed data interface offers ample bandwidth for real-time readout: 2 MByte lambda-stacks composed of 16 spectral channels, 256 x 256 pixel image with 12-bit dynamic range can be transferred at 30 frames per second. The modular design of the mC-PhCC can be readily extended to accommodate PMTs of more anodes. Data acquisition from a 64-anode PMT has been verified. As a demonstration of system performance, spectrally resolved images of fluorescent latex spheres and ex-vivo human skin are reported. The multicolor multiphoton microscope is suitable for highly sensitive, real-time, spectrally-resolved three-dimensional imaging in biomedical applications.

Calcium imaging of neural circuits with extended depth-of-field light-sheet microscopy

PubMed Central

Quirin, Sean; Vladimirov, Nikita; Yang, Chao-Tsung; Peterka, Darcy S.; Yuste, Rafael; Ahrens, Misha B.

2016-01-01

Increasing the volumetric imaging speed of light-sheet microscopy will improve its ability to detect fast changes in neural activity. Here, a system is introduced for brain-wide imaging of neural activity in the larval zebrafish by coupling structured illumination with cubic phase extended depth-of-field (EDoF) pupil encoding. This microscope enables faster light-sheet imaging and facilitates arbitrary plane scanning—removing constraints on acquisition speed, alignment tolerances, and physical motion near the sample. The usefulness of this method is demonstrated by performing multi-plane calcium imaging in the fish brain with a 416 × 832 × 160 µm field of view at 33 Hz. The optomotor response behavior of the zebrafish is monitored at high speeds, and time-locked correlations of neuronal activity are resolved across its brain. PMID:26974063

Proper alignment of the microscope.

PubMed

Rottenfusser, Rudi

2013-01-01

The light microscope is merely the first element of an imaging system in a research facility. Such a system may include high-speed and/or high-resolution image acquisition capabilities, confocal technologies, and super-resolution methods of various types. Yet more than ever, the proverb "garbage in-garbage out" remains a fact. Image manipulations may be used to conceal a suboptimal microscope setup, but an artifact-free image can only be obtained when the microscope is optimally aligned, both mechanically and optically. Something else is often overlooked in the quest to get the best image out of the microscope: Proper sample preparation! The microscope optics can only do its job when its design criteria are matched to the specimen or vice versa. The specimen itself, the mounting medium, the cover slip, and the type of immersion medium (if applicable) are all part of the total optical makeup. To get the best results out of a microscope, understanding the functions of all of its variable components is important. Only then one knows how to optimize these components for the intended application. Different approaches might be chosen to discuss all of the microscope's components. We decided to follow the light path which starts with the light source and ends at the camera or the eyepieces. To add more transparency to this sequence, the section up to the microscope stage was called the "Illuminating Section", to be followed by the "Imaging Section" which starts with the microscope objective. After understanding the various components, we can start "working with the microscope." To get the best resolution and contrast from the microscope, the practice of "Koehler Illumination" should be understood and followed by every serious microscopist. Step-by-step instructions as well as illustrations of the beam path in an upright and inverted microscope are included in this chapter. A few practical considerations are listed in Section 3. Copyright © 2013 Elsevier Inc. All rights reserved.

Brandaris 128 ultra-high-speed imaging facility: 10 years of operation, updates, and enhanced features

NASA Astrophysics Data System (ADS)

Gelderblom, Erik C.; Vos, Hendrik J.; Mastik, Frits; Faez, Telli; Luan, Ying; Kokhuis, Tom J. A.; van der Steen, Antonius F. W.; Lohse, Detlef; de Jong, Nico; Versluis, Michel

2012-10-01

The Brandaris 128 ultra-high-speed imaging facility has been updated over the last 10 years through modifications made to the camera's hardware and software. At its introduction the camera was able to record 6 sequences of 128 images (500 × 292 pixels) at a maximum frame rate of 25 Mfps. The segmented mode of the camera was revised to allow for subdivision of the 128 image sensors into arbitrary segments (1-128) with an inter-segment time of 17 μs. Furthermore, a region of interest can be selected to increase the number of recordings within a single run of the camera from 6 up to 125. By extending the imaging system with a laser-induced fluorescence setup, time-resolved ultra-high-speed fluorescence imaging of microscopic objects has been enabled. Minor updates to the system are also reported here.

A compact CCD-monitored atomic force microscope with optical vision and improved performances.

PubMed

Mingyue, Liu; Haijun, Zhang; Dongxian, Zhang

2013-09-01

A novel CCD-monitored atomic force microscope (AFM) with optical vision and improved performances has been developed. Compact optical paths are specifically devised for both tip-sample microscopic monitoring and cantilever's deflection detecting with minimized volume and optimal light-amplifying ratio. The ingeniously designed AFM probe with such optical paths enables quick and safe tip-sample approaching, convenient and effective tip-sample positioning, and high quality image scanning. An image stitching method is also developed to build a wider-range AFM image under monitoring. Experiments show that this AFM system can offer real-time optical vision for tip-sample monitoring with wide visual field and/or high lateral optical resolution by simply switching the objective; meanwhile, it has the elegant performances of nanometer resolution, high stability, and high scan speed. Furthermore, it is capable of conducting wider-range image measurement while keeping nanometer resolution. Copyright © 2013 Wiley Periodicals, Inc.

Ultra-large field-of-view two-photon microscopy.

PubMed

Tsai, Philbert S; Mateo, Celine; Field, Jeffrey J; Schaffer, Chris B; Anderson, Matthew E; Kleinfeld, David

2015-06-01

We present a two-photon microscope that images the full extent of murine cortex with an objective-limited spatial resolution across an 8 mm by 10 mm field. The lateral resolution is approximately 1 µm and the maximum scan speed is 5 mm/ms. The scan pathway employs large diameter compound lenses to minimize aberrations and performs near theoretical limits. We demonstrate the special utility of the microscope by recording resting-state vasomotion across both hemispheres of the murine brain through a transcranial window and by imaging histological sections without the need to stitch.

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

Large scale infrared imaging of tissue micro arrays (TMAs) using a tunable Quantum Cascade Laser (QCL) based microscope.

PubMed

Bassan, Paul; Weida, Miles J; Rowlette, Jeremy; Gardner, Peter

2014-08-21

Chemical imaging in the field of vibrational spectroscopy is developing into a promising tool to complement digital histopathology. Applications include screening of biopsy tissue via automated recognition of tissue/cell type and disease state based on the chemical information from the spectrum. For integration into clinical practice, data acquisition needs to be speeded up to implement a rack based system where specimens are rapidly imaged to compete with current visible scanners where 100's of slides can be scanned overnight. Current Fourier transform infrared (FTIR) imaging with focal plane array (FPA) detectors are currently the state-of-the-art instrumentation for infrared absorption chemical imaging, however recent development in broadly tunable lasers in the mid-IR range is considered the most promising potential candidate for next generation microscopes. In this paper we test a prototype quantum cascade laser (QCL) based spectral imaging microscope with a focus on discrete frequency chemical imaging. We demonstrate how a protein chemical image of the amide I band (1655 cm(-1)) of a 2 × 2.4 cm(2) breast tissue microarray (TMA) containing over 200 cores can be measured in 9 min. This result indicates that applications requiring chemical images from a few key wavelengths would be ideally served by laser-based microscopes.

A high sensitivity 20Mfps CMOS image sensor with readout speed of 1Tpixel/sec for visualization of ultra-high speed phenomena

NASA Astrophysics Data System (ADS)

Kuroda, R.; Sugawa, S.

2017-02-01

Ultra-high speed (UHS) CMOS image sensors with on-chop analog memories placed on the periphery of pixel array for the visualization of UHS phenomena are overviewed in this paper. The developed UHS CMOS image sensors consist of 400H×256V pixels and 128 memories/pixel, and the readout speed of 1Tpixel/sec is obtained, leading to 10 Mfps full resolution video capturing with consecutive 128 frames, and 20 Mfps half resolution video capturing with consecutive 256 frames. The first development model has been employed in the high speed video camera and put in practical use in 2012. By the development of dedicated process technologies, photosensitivity improvement and power consumption reduction were simultaneously achieved, and the performance improved version has been utilized in the commercialized high-speed video camera since 2015 that offers 10 Mfps with ISO16,000 photosensitivity. Due to the improved photosensitivity, clear images can be captured and analyzed even under low light condition, such as under a microscope as well as capturing of UHS light emission phenomena.

Error mapping of high-speed AFM systems

NASA Astrophysics Data System (ADS)

Klapetek, Petr; Picco, Loren; Payton, Oliver; Yacoot, Andrew; Miles, Mervyn

2013-02-01

In recent years, there have been several advances in the development of high-speed atomic force microscopes (HSAFMs) to obtain images with nanometre vertical and lateral resolution at frame rates in excess of 1 fps. To date, these instruments are lacking in metrology for their lateral scan axes; however, by imaging a series of two-dimensional lateral calibration standards, it has been possible to obtain information about the errors associated with these HSAFM scan axes. Results from initial measurements are presented in this paper and show that the scan speed needs to be taken into account when performing a calibration as it can lead to positioning errors of up to 3%.

A simple way to higher speed atomic force microscopy by retrofitting with a novel high-speed flexure-guided scanner

NASA Astrophysics Data System (ADS)

Ouma Alunda, Bernard; Lee, Yong Joong; Park, Soyeun

2018-06-01

A typical line-scan rate for a commercial atomic force microscope (AFM) is about 1 Hz. At such a rate, more than four minutes of scanning time is required to obtain an image of 256 × 256 pixels. Despite control electronics of most commercial AFMs permit faster scan rates, default piezoelectric X–Y scanners limit the overall speed of the system. This is a direct consequence of manufacturers choosing a large scan range over the maximum operating speed for a X–Y scanner. Although some AFM manufacturers offer reduced-scan area scanners as an option, the speed improvement is not significant because such scanners do not have large enough reduction in the scan range and are mainly targeted to reducing the overall cost of the AFM systems. In this article, we present a simple parallel-kinematic substitute scanner for a commercial atomic force microscope to afford a higher scanning speed with no other hardware or software upgrade to the original system. Although the scan area reduction is unavoidable, our modified commercial XE-70 AFM from Park Systems has achieved a line scan rate of over 50 Hz, more than 10 times faster than the original, unmodified system. Our flexure-guided X–Y scanner can be a simple drop-in replacement option for enhancing the speed of various aging atomic force microscopes.

Scanning electron microscope image signal-to-noise ratio monitoring for micro-nanomanipulation.

PubMed

Marturi, Naresh; Dembélé, Sounkalo; Piat, Nadine

2014-01-01

As an imaging system, scanning electron microscope (SEM) performs an important role in autonomous micro-nanomanipulation applications. When it comes to the sub micrometer range and at high scanning speeds, the images produced by the SEM are noisy and need to be evaluated or corrected beforehand. In this article, the quality of images produced by a tungsten gun SEM has been evaluated by quantifying the level of image signal-to-noise ratio (SNR). In order to determine the SNR, an efficient and online monitoring method is developed based on the nonlinear filtering using a single image. Using this method, the quality of images produced by a tungsten gun SEM is monitored at different experimental conditions. The derived results demonstrate the developed method's efficiency in SNR quantification and illustrate the imaging quality evolution in SEM. © 2014 Wiley Periodicals, Inc.

Free and open-source automated 3-D microscope.

PubMed

Wijnen, Bas; Petersen, Emily E; Hunt, Emily J; Pearce, Joshua M

2016-11-01

Open-source technology not only has facilitated the expansion of the greater research community, but by lowering costs it has encouraged innovation and customizable design. The field of automated microscopy has continued to be a challenge in accessibility due the expense and inflexible, noninterchangeable stages. This paper presents a low-cost, open-source microscope 3-D stage. A RepRap 3-D printer was converted to an optical microscope equipped with a customized, 3-D printed holder for a USB microscope. Precision measurements were determined to have an average error of 10 μm at the maximum speed and 27 μm at the minimum recorded speed. Accuracy tests yielded an error of 0.15%. The machine is a true 3-D stage and thus able to operate with USB microscopes or conventional desktop microscopes. It is larger than all commercial alternatives, and is thus capable of high-depth images over unprecedented areas and complex geometries. The repeatability is below 2-D microscope stages, but testing shows that it is adequate for the majority of scientific applications. The open-source microscope stage costs less than 3-9% of the closest proprietary commercial stages. This extreme affordability vastly improves accessibility for 3-D microscopy throughout the world. © 2016 The Authors Journal of Microscopy © 2016 Royal Microscopical Society.

Parallel detection experiment of fluorescence confocal microscopy using DMD.

PubMed

Wang, Qingqing; Zheng, Jihong; Wang, Kangni; Gui, Kun; Guo, Hanming; Zhuang, Songlin

2016-05-01

Parallel detection of fluorescence confocal microscopy (PDFCM) system based on Digital Micromirror Device (DMD) is reported in this paper in order to realize simultaneous multi-channel imaging and improve detection speed. DMD is added into PDFCM system, working to take replace of the single traditional pinhole in the confocal system, which divides the laser source into multiple excitation beams. The PDFCM imaging system based on DMD is experimentally set up. The multi-channel image of fluorescence signal of potato cells sample is detected by parallel lateral scanning in order to verify the feasibility of introducing the DMD into fluorescence confocal microscope. In addition, for the purpose of characterizing the microscope, the depth response curve is also acquired. The experimental result shows that in contrast to conventional microscopy, the DMD-based PDFCM system has higher axial resolution and faster detection speed, which may bring some potential benefits in the biology and medicine analysis. SCANNING 38:234-239, 2016. © 2015 Wiley Periodicals, Inc. © Wiley Periodicals, Inc.

Detecting Phase Boundaries in Hard-Sphere Suspensions

NASA Technical Reports Server (NTRS)

McDowell, Mark; Rogers, Richard B.; Gray, Elizabeth

2009-01-01

A special image-data-processing technique has been developed for use in experiments that involve observation, via optical microscopes equipped with electronic cameras, of moving boundaries between the colloidal-solid and colloidal-liquid phases of colloidal suspensions of monodisperse hard spheres. During an experiment, it is necessary to adjust the position of a microscope to keep the phase boundary within view. A boundary typically moves at a speed of the order of microns per hour. Because an experiment can last days or even weeks, it is impractical to require human intervention to keep the phase boundary in view. The present image-data-processing technique yields results within a computation time short enough to enable generation of automated-microscope-positioning commands to track the moving phase boundary

Pushing the physical limits of spectroscopic imaging for new biology and better medicine (Conference Presentation)

NASA Astrophysics Data System (ADS)

Cheng, Ji-Xin

2017-02-01

In vivo molecular spectroscopic imaging is not a simple addition of a spectrometer to a microscope. Innovations are needed to break the physical limits in sensitivity, depth, speed and resolution perspectives. I will present our most recent advances in modality development, biological application, and clinical translation. My talk will focus on the development of mid-infrared photothermal microscope for depth-resolved vibrational imaging of living cells (Science Advances, in press), the discovery of a metabolic signature in cancer stem cells by hyperspectral stimulated Raman scattering imaging (Cell Stem Cell, in press), and the development of an intravascular vibrational photoacoustic catheter for label-free sensing of lipid laden plaques (Scientific Report 2016, 6:25236).

Chronic monitoring of cortical hemodynamics in behaving, freely-moving rats using a miniaturized head-mounted optical microscope

NASA Astrophysics Data System (ADS)

Sigal, Iliya; Gad, Raanan; Koletar, Margaret; Ringuette, Dene; Stefanovic, Bojana; Levi, Ofer

2016-03-01

Growing interest within the neurophysiology community in assessing healthy and pathological brain activity in animals that are awake and freely-behaving has triggered the need for optical systems that are suitable for such longitudinal studies. In this work we report label-free multi-modal imaging of cortical hemodynamics in the somatosensory cortex of awake, freely-behaving rats, using a novel head-mounted miniature optical microscope. The microscope employs vertical cavity surface emitting lasers (VCSELs) at three distinct wavelengths (680 nm, 795 nm, and 850 nm) to provide measurements of four hemodynamic markers: blood flow speeds, HbO, HbR, and total Hb concentration, across a > 2 mm field of view. Blood flow speeds are extracted using Laser Speckle Contrast Imaging (LSCI), while oxygenation measurements are performed using Intrinsic Optical Signal Imaging (IOSI). Longitudinal measurements on the same animal are made possible over the course of > 6 weeks using a chronic window that is surgically implanted into the skull. We use the device to examine changes in blood flow and blood oxygenation in superficial cortical blood vessels and tissue in response to drug-induced absence-like seizures, correlating motor behavior with changes in blood flow and blood oxygenation in the brain.

Ultra-large field-of-view two-photon microscopy

PubMed Central

Tsai, Philbert S.; Mateo, Celine; Field, Jeffrey J.; Schaffer, Chris B.; Anderson, Matthew E.; Kleinfeld, David

2015-01-01

We present a two-photon microscope that images the full extent of murine cortex with an objective-limited spatial resolution across an 8 mm by 10 mm field. The lateral resolution is approximately 1 µm and the maximum scan speed is 5 mm/ms. The scan pathway employs large diameter compound lenses to minimize aberrations and performs near theoretical limits. We demonstrate the special utility of the microscope by recording resting-state vasomotion across both hemispheres of the murine brain through a transcranial window and by imaging histological sections without the need to stitch. PMID:26072755

High-speed quantitative phase imaging using time-stretch spectral shearing contrast (Conference Presentation)

NASA Astrophysics Data System (ADS)

Bosworth, Bryan; Foster, Mark A.

2017-02-01

Photonic time-stretch microscopy (TSM) provides an ideal platform for high-throughput imaging flow cytometry, affording extremely high shutter speeds and frame rates with high sensitivity. In order to resolve weakly scattering cells in biofluid and solve the issue of signal-to-noise in cell labeling specificity of biomarkers in imaging flow cytometry, several quantitative phase (QP) techniques have recently been adapted to TSM. However, these techniques have relied primarily on sensitive free-space optical configurations to generate full electric field measurements. The present work draws from the field of ultrashort pulse characterization to leverage the coherence of the ultrashort optical pulses integral to all TSM systems in order to do self-referenced single-shot quantitative phase imaging in a TSM system. Self-referencing is achieved via spectral shearing interferometry in an exceptionally stable and straightforward Sagnac loop incorporating an electro-optic phase modulator and polarization-maintaining fiber that produce sheared and unsheared copies of the pulse train with an inter-pulse delay determined by polarization mode dispersion. The spectral interferogram then yields a squared amplitude and a phase derivative image that can be integrated for conventional phase. We apply this spectral shearing contrast microscope to acquire QP images on a high-speed flow microscope at 90-MHz line rates with <400 pixels per line. We also consider the extension of this technique to compressed sensing (CS) acquisition by intensity modulating the interference spectra with pseudorandom binary waveforms to reconstruct the images from a highly sub-Nyquist number of random inner products, providing a path to even higher operating rates and reduced data storage requirements.

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.

Confocal reflectance quantitative phase microscope system for cellular membranes dynamics study (Conference Presentation)

NASA Astrophysics Data System (ADS)

Singh, Vijay Raj; Yaqoob, Zahid; So, Peter T. C.

2017-02-01

Quantitative phase microscopy (QPM) techniques developed so far primarily belongs to high speed transmitted light based systems that has enough sensitivity to resolve membrane fluctuations and dynamics, but has no depth resolution. Therefore, most biomechanics studies using QPM today is confined to simple cells, such as RBCs, without internal organelles. An important instrument that will greatly extend the biomedical applications of QPM is to develop next generation microscope with 3D capability and sufficient temporal resolution to study biomechanics of complex eukaryotic cells including the mechanics of their internal compartments. For eukaryotic cells, the depth sectioning capability is critical and should be sufficient to distinguish nucleic membrane fluctuations from plasma membrane fluctuations. Further, this microscope must provide high temporal resolution since typical eukaryotes membranes are substantially stiffer than RBCs. A confocal reflectance quantitative phase microscope is presented based on multi-pinhole scanning, with the capabilities of higher temporal resolution and sensitivity for nucleic and plasma membranes of eukaryotic cells. System hardware is developed based on an array of confocal pinhole generated by using the `ON' state of subset of micro-mirrors of digital micro-mirror device (DMD, from Texas Instruments) and high-speed raster scanning provides 14ms imaging speed in wide-field mode. A common path interferometer is integrated at the imaging arm for detection of specimens' quantitative phase information. Theoretical investigation of quantitative phase reconstructed from system is investigated and application of system is presented for dimensional fluctuations measurements of both cellular plasma and nucleic membranes of embryonic stem cells.

Harnessing the damping properties of materials for high-speed atomic force microscopy.

PubMed

Adams, Jonathan D; Erickson, Blake W; Grossenbacher, Jonas; Brugger, Juergen; Nievergelt, Adrian; Fantner, Georg E

2016-02-01

The success of high-speed atomic force microscopy in imaging molecular motors, enzymes and microbes in liquid environments suggests that the technique could be of significant value in a variety of areas of nanotechnology. However, the majority of atomic force microscopy experiments are performed in air, and the tapping-mode detection speed of current high-speed cantilevers is an order of magnitude lower in air than in liquids. Traditional approaches to increasing the imaging rate of atomic force microscopy have involved reducing the size of the cantilever, but further reductions in size will require a fundamental change in the detection method of the microscope. Here, we show that high-speed imaging in air can instead be achieved by changing the cantilever material. We use cantilevers fabricated from polymers, which can mimic the high damping environment of liquids. With this approach, SU-8 polymer cantilevers are developed that have an imaging-in-air detection bandwidth that is 19 times faster than those of conventional cantilevers of similar size, resonance frequency and spring constant.

Micromirror structured illumination microscope for high-speed in vivo drosophila brain imaging.

PubMed

Masson, A; Pedrazzani, M; Benrezzak, S; Tchenio, P; Preat, T; Nutarelli, D

2014-01-27

Genetic tools and especially genetically encoded fluorescent reporters have given a special place to optical microscopy in drosophila neurobiology research. In order to monitor neural networks activity, high speed and sensitive techniques, with high spatial resolution are required. Structured illumination microscopies are wide-field approaches with optical sectioning ability. Despite the large progress made with the introduction of the HiLo principle, they did not meet the criteria of speed and/or spatial resolution for drosophila brain imaging. We report on a new implementation that took advantage of micromirror matrix technology to structure the illumination. Thus, we showed that the developed instrument exhibits a spatial resolution close to that of confocal microscopy but it can record physiological responses with a speed improved by more than an order a magnitude.

Resolution and throughput optimized intraoperative spectrally encoded coherence tomography and reflectometry (iSECTR) for multimodal imaging during ophthalmic microsurgery

NASA Astrophysics Data System (ADS)

Malone, Joseph D.; El-Haddad, Mohamed T.; Leeburg, Kelsey C.; Terrones, Benjamin D.; Tao, Yuankai K.

2018-02-01

Limited visualization of semi-transparent structures in the eye remains a critical barrier to improving clinical outcomes and developing novel surgical techniques. While increases in imaging speed has enabled intraoperative optical coherence tomography (iOCT) imaging of surgical dynamics, several critical barriers to clinical adoption remain. Specifically, these include (1) static field-of-views (FOVs) requiring manual instrument-tracking; (2) high frame-rates require sparse sampling, which limits FOV; and (3) small iOCT FOV also limits the ability to co-register data with surgical microscopy. We previously addressed these limitations in image-guided ophthalmic microsurgery by developing microscope-integrated multimodal intraoperative swept-source spectrally encoded scanning laser ophthalmoscopy and optical coherence tomography. Complementary en face images enabled orientation and coregistration with the widefield surgical microscope view while OCT imaging enabled depth-resolved visualization of surgical instrument positions relative to anatomic structures-of-interest. In addition, we demonstrated novel integrated segmentation overlays for augmented-reality surgical guidance. Unfortunately, our previous system lacked the resolution and optical throughput for in vivo retinal imaging and necessitated removal of cornea and lens. These limitations were predominately a result of optical aberrations from imaging through a shared surgical microscope objective lens, which was modeled as a paraxial surface. Here, we present an optimized intraoperative spectrally encoded coherence tomography and reflectometry (iSECTR) system. We use a novel lens characterization method to develop an accurate model of surgical microscope objective performance and balance out inherent aberrations using iSECTR relay optics. Using this system, we demonstrate in vivo multimodal ophthalmic imaging through a surgical microscope

OCT in the field of laryngology: further perspectives

NASA Astrophysics Data System (ADS)

Just, T.; Pau, H. W.; Lankenau, E.; Hüttmann, G.

2011-03-01

Early detection of cancerous lesions of the larynx may be the best method of improving patient quality of life and survival rates. New in-vivo technologies may be of great clinical relevance in improving the accuracy of sampling during microlaryngeal surgery. Optical coherence tomography (OCT) is an optical imaging technique that clearly identifies basement membrane violation caused by laryngeal cancer. With a microscope-based spectral domain OCT (SD-OCT) we reached in vivo a fairly accurate assessment of benign and dysplastic laryngeal lesions. Recent improvements in OCT technology have led to the development of high-speed OCT systems displaying millions of pixels per second. These systems allow non-contact real-time imaging of large sections of laryngeal tissue. Polarization contrast OCT (PS-OCT) may provide additional information about the lamina propria of the true vocal cord because of the birefringence of connective tissue. We present microscope-based high-speed SD-OCT images with and without polarization contrast and 3D volumes of selected laryngeal pathologies in order to demonstrate our current concepts for the intended intraoperative application. High-speed SD-OCT and polarization contrast can also be complemented by our recently developed rigid confocal endoscopic system to obtain cellular and sub-cellular information about the tissue. Further perspectives will be presented.

New deconvolution method for microscopic images based on the continuous Gaussian radial basis function interpolation model.

PubMed

Chen, Zhaoxue; Chen, Hao

2014-01-01

A deconvolution method based on the Gaussian radial basis function (GRBF) interpolation is proposed. Both the original image and Gaussian point spread function are expressed as the same continuous GRBF model, thus image degradation is simplified as convolution of two continuous Gaussian functions, and image deconvolution is converted to calculate the weighted coefficients of two-dimensional control points. Compared with Wiener filter and Lucy-Richardson algorithm, the GRBF method has an obvious advantage in the quality of restored images. In order to overcome such a defect of long-time computing, the method of graphic processing unit multithreading or increasing space interval of control points is adopted, respectively, to speed up the implementation of GRBF method. The experiments show that based on the continuous GRBF model, the image deconvolution can be efficiently implemented by the method, which also has a considerable reference value for the study of three-dimensional microscopic image deconvolution.

Measurement of flow speed in the channels of novel threadlike structures on the surfaces of mammalian organs

NASA Astrophysics Data System (ADS)

Sung, Baeckkyoung; Kim, Min Su; Lee, Byung-Cheon; Yoo, Jung Sun; Lee, Sang-Hee; Kim, Youn-Joong; Kim, Ki-Woo; Soh, Kwang-Sup

2008-02-01

There have been several reports on novel threadlike structures (NTSs) on the surfaces of the internal organs of rats and rabbits since their first observation by Bonghan Kim in 1963. To confirm this novel circulatory function, it is necessary to observe the flow of liquid through the NTS as well as the structurally corroborating channels in the NTS. In this article, we report on the measurement of the flow speed of Alcian blue solution in the NTSs on the organ surfaces of rabbits, and we present electron microscopic images depicting the cribrous cross-section with channels. The speed was measured as 0.3 ± 0.1 mm/s, and the flow distance was up to 12 cm. The flow was unidirectional, and the phase contrast microscopic images showed that the NTSs were strongly stained with Alcian blue. The ultrastructure of the NTSs revealed by cryo-scanning electron microscopy and high-voltage electron microscopy showed that (1) there were cell-like bodies and globular clumps of matter inside the sinus of the channel with thin strands of segregated zones which is a microscopic evidence of the liquid flow, (2) the sinuses have wall structures surrounded with extracellular matrices of collagenous fibers, and (3) there exists a cribriform structure of sinuses. To understand the mechanism for the circulation, a quantitative analysis of the flow speed has been undertaken applying a simplified windkessel model. In this analysis, it was shown that the liquid flow through the NTSs could be due to peristaltic motion of the NTS itself.

Enhancing the performance of the light field microscope using wavefront coding.

PubMed

Cohen, Noy; Yang, Samuel; Andalman, Aaron; Broxton, Michael; Grosenick, Logan; Deisseroth, Karl; Horowitz, Mark; Levoy, Marc

2014-10-06

Light field microscopy has been proposed as a new high-speed volumetric computational imaging method that enables reconstruction of 3-D volumes from captured projections of the 4-D light field. Recently, a detailed physical optics model of the light field microscope has been derived, which led to the development of a deconvolution algorithm that reconstructs 3-D volumes with high spatial resolution. However, the spatial resolution of the reconstructions has been shown to be non-uniform across depth, with some z planes showing high resolution and others, particularly at the center of the imaged volume, showing very low resolution. In this paper, we enhance the performance of the light field microscope using wavefront coding techniques. By including phase masks in the optical path of the microscope we are able to address this non-uniform resolution limitation. We have also found that superior control over the performance of the light field microscope can be achieved by using two phase masks rather than one, placed at the objective's back focal plane and at the microscope's native image plane. We present an extended optical model for our wavefront coded light field microscope and develop a performance metric based on Fisher information, which we use to choose adequate phase masks parameters. We validate our approach using both simulated data and experimental resolution measurements of a USAF 1951 resolution target; and demonstrate the utility for biological applications with in vivo volumetric calcium imaging of larval zebrafish brain.

Spectroscopic imaging of biomaterials and biological systems with FTIR microscopy or with quantum cascade lasers.

PubMed

Kimber, James A; Kazarian, Sergei G

2017-10-01

Spectroscopic imaging of biomaterials and biological systems has received increased interest within the last decade because of its potential to aid in the detection of disease using biomaterials/biopsy samples and to probe the states of live cells in a label-free manner. The factors behind this increased attention include the availability of improved infrared microscopes and systems that do not require the use of a synchrotron as a light source, as well as the decreasing costs of these systems. This article highlights the current technical challenges and future directions of mid-infrared spectroscopic imaging within this field. Specifically, these are improvements in spatial resolution and spectral quality through the use of novel added lenses and computational algorithms, as well as quantum cascade laser imaging systems, which offer advantages over traditional Fourier transform infrared systems with respect to the speed of acquisition and field of view. Overcoming these challenges will push forward spectroscopic imaging as a viable tool for disease diagnostics and medical research. Graphical abstract Absorbance images of a biopsy obtained using an FTIR imaging microscope with and without an added lens, and also using a QCL microscope with high-NA objective.

Electron microscopy of whole cells in liquid with nanometer resolution

PubMed Central

de Jonge, N.; Peckys, D. B.; Kremers, G. J.; Piston, D. W.

2009-01-01

Single gold-tagged epidermal growth factor (EGF) molecules bound to cellular EGF receptors of fixed fibroblast cells were imaged in liquid with a scanning transmission electron microscope (STEM). The cells were placed in buffer solution in a microfluidic device with electron transparent windows inside the vacuum of the electron microscope. A spatial resolution of 4 nm and a pixel dwell time of 20 μs were obtained. The liquid layer was sufficiently thick to contain the cells with a thickness of 7 ± 1 μm. The experimental findings are consistent with a theoretical calculation. Liquid STEM is a unique approach for imaging single molecules in whole cells with significantly improved resolution and imaging speed over existing methods. PMID:19164524

A sub-sampled approach to extremely low-dose STEM

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

Stevens, A.; Luzi, L.; Yang, H.

The inpainting of randomly sub-sampled images acquired by scanning transmission electron microscopy (STEM) is an attractive method for imaging under low-dose conditions (≤ 1 e -Å 2) without changing either the operation of the microscope or the physics of the imaging process. We show that 1) adaptive sub-sampling increases acquisition speed, resolution, and sensitivity; and 2) random (non-adaptive) sub-sampling is equivalent, but faster than, traditional low-dose techniques. Adaptive sub-sampling opens numerous possibilities for the analysis of beam sensitive materials and in-situ dynamic processes at the resolution limit of the aberration corrected microscope and is demonstrated here for the analysis ofmore » the node distribution in metal-organic frameworks (MOFs).« less

High-speed transport-of-intensity phase microscopy with an electrically tunable lens.

PubMed

Zuo, Chao; Chen, Qian; Qu, Weijuan; Asundi, Anand

2013-10-07

We present a high-speed transport-of-intensity equation (TIE) quantitative phase microscopy technique, named TL-TIE, by combining an electrically tunable lens with a conventional transmission microscope. This permits the specimen at different focus position to be imaged in rapid succession, with constant magnification and no physically moving parts. The simplified image stack collection significantly reduces the acquisition time, allows for the diffraction-limited through-focus intensity stack collection at 15 frames per second, making dynamic TIE phase imaging possible. The technique is demonstrated by profiling of microlens array using optimal frequency selection scheme, and time-lapse imaging of live breast cancer cells by inversion the defocused phase optical transfer function to correct the phase blurring in traditional TIE. Experimental results illustrate its outstanding capability of the technique for quantitative phase imaging, through a simple, non-interferometric, high-speed, high-resolution, and unwrapping-free approach with prosperous applications in micro-optics, life sciences and bio-photonics.

High-speed visualization of fuel spray impingement in the near-wall region using a DISI injector

NASA Astrophysics Data System (ADS)

Kawahara, N.; Kintaka, K.; Tomita, E.

2017-02-01

We used a multi-hole injector to spray isooctane under atmospheric conditions and observed droplet impingement behaviors. It is generally known that droplet impact regimes such as splashing, deposition, or bouncing are governed by the Weber number. However, owing to its complexity, little has been reported on microscopic visualization of poly-dispersed spray. During the spray impingement process, a large number of droplets approach, hit, then interact with the wall. It is therefore difficult to focus on a single droplet and observe the impingement process. We solved this difficulty using high-speed microscopic visualization. The spray/wall interaction processes were recorded by a high-speed camera (Shimadzu HPV-X2) with a long-distance microscope. We captured several impinging microscopic droplets. After optimizing the magnification and frame rate, the atomization behaviors, splashing and deposition, were recorded. Then, we processed the images obtained to determine droplet parameters such as the diameter, velocity, and impingement angle. Based on this information, the critical threshold between splashing and deposition was investigated in terms of the normal and parallel components of the Weber number with respect to the wall. The results suggested that, on a dry wall, we should set the normal critical Weber number to 300.

Combined multi-plane phase retrieval and super-resolution optical fluctuation imaging for 4D cell microscopy

NASA Astrophysics Data System (ADS)

Descloux, A.; Grußmayer, K. S.; Bostan, E.; Lukes, T.; Bouwens, A.; Sharipov, A.; Geissbuehler, S.; Mahul-Mellier, A.-L.; Lashuel, H. A.; Leutenegger, M.; Lasser, T.

2018-03-01

Super-resolution fluorescence microscopy provides unprecedented insight into cellular and subcellular structures. However, going `beyond the diffraction barrier' comes at a price, since most far-field super-resolution imaging techniques trade temporal for spatial super-resolution. We propose the combination of a novel label-free white light quantitative phase imaging with fluorescence to provide high-speed imaging and spatial super-resolution. The non-iterative phase retrieval relies on the acquisition of single images at each z-location and thus enables straightforward 3D phase imaging using a classical microscope. We realized multi-plane imaging using a customized prism for the simultaneous acquisition of eight planes. This allowed us to not only image live cells in 3D at up to 200 Hz, but also to integrate fluorescence super-resolution optical fluctuation imaging within the same optical instrument. The 4D microscope platform unifies the sensitivity and high temporal resolution of phase imaging with the specificity and high spatial resolution of fluorescence microscopy.

AOTF hyperspectral microscope imaging for foodborne bacteria detection

USDA-ARS?s Scientific Manuscript database

Food safety is an important public health issue worldwide. Researchers have developed many different methods for detecting foodborne pathogens; however, most technologies currently being used have limitations, in terms of speed, sensitivity and selectivity, for practical use in the food industry. Ac...

Recent advances in high-throughput QCL-based infrared microspectral imaging (Conference Presentation)

NASA Astrophysics Data System (ADS)

Rowlette, Jeremy A.; Fotheringham, Edeline; Nichols, David; Weida, Miles J.; Kane, Justin; Priest, Allen; Arnone, David B.; Bird, Benjamin; Chapman, William B.; Caffey, David B.; Larson, Paul; Day, Timothy

2017-02-01

The field of infrared spectral imaging and microscopy is advancing rapidly due in large measure to the recent commercialization of the first high-throughput, high-spatial-definition quantum cascade laser (QCL) microscope. Having speed, resolution and noise performance advantages while also eliminating the need for cryogenic cooling, its introduction has established a clear path to translating the well-established diagnostic capability of infrared spectroscopy into clinical and pre-clinical histology, cytology and hematology workflows. Demand for even higher throughput while maintaining high-spectral fidelity and low-noise performance continues to drive innovation in QCL-based spectral imaging instrumentation. In this talk, we will present for the first time, recent technological advances in tunable QCL photonics which have led to an additional 10X enhancement in spectral image data collection speed while preserving the high spectral fidelity and SNR exhibited by the first generation of QCL microscopes. This new approach continues to leverage the benefits of uncooled microbolometer focal plane array cameras, which we find to be essential for ensuring both reproducibility of data across instruments and achieving the high-reliability needed in clinical applications. We will discuss the physics underlying these technological advancements as well as the new biomedical applications these advancements are enabling, including automated whole-slide infrared chemical imaging on clinically relevant timescales.

High-speed broadband nanomechanical property quantification and imaging of life science materials using atomic force microscope

NASA Astrophysics Data System (ADS)

Ren, Juan

Nanoscale morphological characterization and mechanical properties quantification of soft and biological materials play an important role in areas ranging from nano-composite material synthesis and characterization, cellular mechanics to drug design. Frontier studies in these areas demand the coordination between nanoscale morphological evolution and mechanical behavior variations through simultaneous measurement of these two aspects of properties. Atomic force microscope (AFM) is very promising in achieving such simultaneous measurements at high-speed and broadband owing to its unique capability in applying force stimuli and then, measuring the response at specific locations in a physiologically friendly environment with pico-newton force and nanometer spatial resolution. Challenges, however, arise as current AFM systems are unable to account for the complex and coupled dynamics of the measurement system and probe-sample interaction during high-speed imaging and broadband measurements. In this dissertation, the creation of a set of dynamics and control tools to probe-based high-speed imaging and rapid broadband nanomechanical spectroscopy of soft and biological materials are presented. Firstly, advanced control-based approaches are presented to improve the imaging performance of AFM imaging both in air and in liquid. An adaptive contact mode (ACM) imaging scheme is proposed to replace the traditional contact mode (CM) imaging by addressing the major concerns in both the speed and the force exerted to the sample. In this work, the image distortion caused by the topography tracking error is accounted for in the topography quantification and the quantified sample topography is utilized in a gradient-based optimization method to adjust the cantilever deflection set-point for each scanline closely around the minimal level needed for maintaining a stable probe-sample contact, and a data-driven iterative feedforward control that utilizes a prediction of the next-line tracking is implemented to enhance the sample topography tracking. An adaptive multi-loop mode (AMLM) imaging approach is proposed to substantially increase the imaging speed of tapping mode (TM) while preserving the advantages of TM over CM by integrating an inner-outer feedback control loop to regulate the TM-deflection on top of the conventional TM-amplitude feedback control to improve the sample topography tracking. Experiments demonstrated that the proposed ACM and AMLM are capable of increasing the imaging speed by at least 20 times for conventional contact and tapping mode imaging, respectively, with no loss of imaging quality and well controlled tip-sample interaction force. In addition, an adaptive mode imaging for in-liquid topography quantification on live cells is presented. The experiment results demonstrated that instead of keeping constant scanning speed, the proposed speed optimization scheme is able to increase the imaging speed on live human prostate cancer cells by at least eight-fold with no loss of imaging quality. Secondly, control based approaches to accurate nanomechanical quantification on soft materials for both broadband and in-liquid force-curve measurements are proposed to address the adverse effects caused by the system coupling dynamics and the cantilever acceleration, which were not compensated for by the conventional AFM measurement approach. The proposed nanomechanical measurement approaches are demonstrated through experiments to measure the viscoelastic properties of different polymer samples in air and live human cells in liquid to study the variation of rate-dependent elastic modulus of cervix cancer cell during the epithelial-mesenchymal transition process.

Imaging inflammation in mouse colon using a rapid stage-scanning confocal fluorescence microscope

NASA Astrophysics Data System (ADS)

Saldua, Meagan A.; Olsovsky, Cory A.; Callaway, Evelyn S.; Chapkin, Robert S.; Maitland, Kristen C.

2012-01-01

Large area confocal microscopy may provide fast, high-resolution image acquisition for evaluation of tissue in pre-clinical studies with reduced tissue processing in comparison to histology. We present a rapid beam and stage-scanning confocal fluorescence microscope to image cellular and tissue features along the length of the entire excised mouse colon. The beam is scanned at 8,333 lines/sec by a polygon scanning mirror while the specimen is scanned in the orthogonal axis by a motorized translation stage with a maximum speed of 7 mm/sec. A single 1×60 mm2 field of view image spanning the length of the mouse colon is acquired in 10 s. Z-projection images generated from axial image stacks allow high resolution imaging of the surface of non-flat specimens. In contrast to the uniform size, shape, and distribution of colon crypts in confocal images of normal colon, confocal images of chronic bowel inflammation exhibit heterogeneous tissue structure with localized severe crypt distortion.

Enhancing the performance of the light field microscope using wavefront coding

PubMed Central

Cohen, Noy; Yang, Samuel; Andalman, Aaron; Broxton, Michael; Grosenick, Logan; Deisseroth, Karl; Horowitz, Mark; Levoy, Marc

2014-01-01

Light field microscopy has been proposed as a new high-speed volumetric computational imaging method that enables reconstruction of 3-D volumes from captured projections of the 4-D light field. Recently, a detailed physical optics model of the light field microscope has been derived, which led to the development of a deconvolution algorithm that reconstructs 3-D volumes with high spatial resolution. However, the spatial resolution of the reconstructions has been shown to be non-uniform across depth, with some z planes showing high resolution and others, particularly at the center of the imaged volume, showing very low resolution. In this paper, we enhance the performance of the light field microscope using wavefront coding techniques. By including phase masks in the optical path of the microscope we are able to address this non-uniform resolution limitation. We have also found that superior control over the performance of the light field microscope can be achieved by using two phase masks rather than one, placed at the objective’s back focal plane and at the microscope’s native image plane. We present an extended optical model for our wavefront coded light field microscope and develop a performance metric based on Fisher information, which we use to choose adequate phase masks parameters. We validate our approach using both simulated data and experimental resolution measurements of a USAF 1951 resolution target; and demonstrate the utility for biological applications with in vivo volumetric calcium imaging of larval zebrafish brain. PMID:25322056

Microscopic image analysis for reticulocyte based on watershed algorithm

NASA Astrophysics Data System (ADS)

Wang, J. Q.; Liu, G. F.; Liu, J. G.; Wang, G.

2007-12-01

We present a watershed-based algorithm in the analysis of light microscopic image for reticulocyte (RET), which will be used in an automated recognition system for RET in peripheral blood. The original images, obtained by micrography, are segmented by modified watershed algorithm and are recognized in term of gray entropy and area of connective area. In the process of watershed algorithm, judgment conditions are controlled according to character of the image, besides, the segmentation is performed by morphological subtraction. The algorithm was simulated with MATLAB software. It is similar for automated and manual scoring and there is good correlation(r=0.956) between the methods, which is resulted from 50 pieces of RET images. The result indicates that the algorithm for peripheral blood RETs is comparable to conventional manual scoring, and it is superior in objectivity. This algorithm avoids time-consuming calculation such as ultra-erosion and region-growth, which will speed up the computation consequentially.

High contrast imaging and flexible photomanipulation for quantitative in vivo multiphoton imaging with polygon scanning microscope.

PubMed

Li, Yongxiao; Montague, Samantha J; Brüstle, Anne; He, Xuefei; Gillespie, Cathy; Gaus, Katharina; Gardiner, Elizabeth E; Lee, Woei Ming

2018-02-28

In this study, we introduce two key improvements that overcome limitations of existing polygon scanning microscopes while maintaining high spatial and temporal imaging resolution over large field of view (FOV). First, we proposed a simple and straightforward means to control the scanning angle of the polygon mirror to carry out photomanipulation without resorting to high speed optical modulators. Second, we devised a flexible data sampling method directly leading to higher image contrast by over 2-fold and digital images with 100 megapixels (10 240 × 10 240) per frame at 0.25 Hz. This generates sub-diffraction limited pixels (60 nm per pixels over the FOV of 512 μm) which increases the degrees of freedom to extract signals computationally. The unique combined optical and digital control recorded fine fluorescence recovery after localized photobleaching (r ~10 μm) within fluorescent giant unilamellar vesicles and micro-vascular dynamics after laser-induced injury during thrombus formation in vivo. These new improvements expand the quantitative biological-imaging capacity of any polygon scanning microscope system. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Parallel-multiplexed excitation light-sheet microscopy (Conference Presentation)

NASA Astrophysics Data System (ADS)

Xu, Dongli; Zhou, Weibin; Peng, Leilei

2017-02-01

Laser scanning light-sheet imaging allows fast 3D image of live samples with minimal bleach and photo-toxicity. Existing light-sheet techniques have very limited capability in multi-label imaging. Hyper-spectral imaging is needed to unmix commonly used fluorescent proteins with large spectral overlaps. However, the challenge is how to perform hyper-spectral imaging without sacrificing the image speed, so that dynamic and complex events can be captured live. We report wavelength-encoded structured illumination light sheet imaging (λ-SIM light-sheet), a novel light-sheet technique that is capable of parallel multiplexing in multiple excitation-emission spectral channels. λ-SIM light-sheet captures images of all possible excitation-emission channels in true parallel. It does not require compromising the imaging speed and is capable of distinguish labels by both excitation and emission spectral properties, which facilitates unmixing fluorescent labels with overlapping spectral peaks and will allow more labels being used together. We build a hyper-spectral light-sheet microscope that combined λ-SIM with an extended field of view through Bessel beam illumination. The system has a 250-micron-wide field of view and confocal level resolution. The microscope, equipped with multiple laser lines and an unlimited number of spectral channels, can potentially image up to 6 commonly used fluorescent proteins from blue to red. Results from in vivo imaging of live zebrafish embryos expressing various genetic markers and sensors will be shown. Hyper-spectral images from λ-SIM light-sheet will allow multiplexed and dynamic functional imaging in live tissue and animals.

Direct microscopic image and measurement of the atomization process of a port fuel injector

NASA Astrophysics Data System (ADS)

Esmail, Mohamed; Kawahara, Nobuyuki; Tomita, Eiji; Sumida, Mamoru

2010-07-01

The main objective of this study is to observe and investigate the phenomena of atomization, i.e. the fuel break-up process very close to the nozzle exit of a practical port fuel injector (PFI). In order to achieve this objective, direct microscopic images of the atomization process were obtained using an ultra-high-speed video camera that could record 102 frames at rates of up to 1 Mfps, coupled with a long-distance microscope and Barlow lens. The experiments were carried out using a PFI in a closed chamber at atmospheric pressure. Time-series images of the spray behaviour were obtained with a high temporal resolution using backlighting. The direct microscopic images of a liquid column break-up were compared with experimental results from laser-induced exciplex fluorescence (LIEF), and the wavelength obtained from the experimental results compared with that predicated from the Kelvin-Helmholtz break-up model. The droplet size diameters from a ligament break-up were compared with results predicated from Weber's analysis. Furthermore, experimental results of the mean droplet diameter from a direct microscopic image were compared with the results obtained from phase Doppler anemometry (PDA) experimental results. Three conclusions were obtained from this study. The atomization processes and detailed characterizations of the break-up of a liquid column were identified; the direct microscopic image results were in good agreement with the results obtained from LIEF, experimental results of the wavelength were in good agreement with those from the Kelvin-Helmholtz break-up model. The break-up process of liquid ligaments into droplets was investigated, and Weber's analysis of the predicated droplet diameter from ligament break-up was found to be applicable only at larger wavelengths. Finally, the direct microscopic image method and PDA method give qualitatively similar trends for droplet size distribution and quantitatively similar values of Sauter mean diameter.

Fast scanning mode and its realization in a scanning acoustic microscope

NASA Astrophysics Data System (ADS)

Ju, Bing-Feng; Bai, Xiaolong; Chen, Jian

2012-03-01

The scanning speed of the two-dimensional stage dominates the efficiency of mechanical scanning measurement systems. This paper focused on a detailed scanning time analysis of conventional raster and spiral scan modes and then proposed two fast alternative scanning modes. Performed on a self-developed scanning acoustic microscope (SAM), the measured images obtained by using the conventional scan mode and fast scan modes are compared. The total scanning time is reduced by 29% of the two proposed fast scan modes. It will offer a better solution for high speed scanning without sacrificing the system stability, and will not introduce additional difficulties to the configuration of scanning measurement systems. They can be easily applied to the mechanical scanning measuring systems with different driving actuators such as piezoelectric, linear motor, dc motor, and so on. The proposed fast raster and square spiral scan modes are realized in SAM, but not specially designed for it. Therefore, they have universal adaptability and can be applied to other scanning measurement systems with two-dimensional mechanical scanning stages, such as atomic force microscope or scanning tunneling microscope.

Intraoperative video-rate hemodynamic response assessment in human cortex using snapshot hyperspectral optical imaging

PubMed Central

Pichette, Julien; Laurence, Audrey; Angulo, Leticia; Lesage, Frederic; Bouthillier, Alain; Nguyen, Dang Khoa; Leblond, Frederic

2016-01-01

Abstract. Using light, we are able to visualize the hemodynamic behavior of the brain to better understand neurovascular coupling and cerebral metabolism. In vivo optical imaging of tissue using endogenous chromophores necessitates spectroscopic detection to ensure molecular specificity as well as sufficiently high imaging speed and signal-to-noise ratio, to allow dynamic physiological changes to be captured, isolated, and used as surrogate of pathophysiological processes. An optical imaging system is introduced using a 16-bands on-chip hyperspectral camera. Using this system, we show that up to three dyes can be imaged and quantified in a tissue phantom at video-rate through the optics of a surgical microscope. In vivo human patient data are presented demonstrating brain hemodynamic response can be measured intraoperatively with molecular specificity at high speed. PMID:27752519

Ultrawidefield microscope for high-speed fluorescence imaging and targeted optogenetic stimulation.

PubMed

Werley, Christopher A; Chien, Miao-Ping; Cohen, Adam E

2017-12-01

The rapid increase in the number and quality of fluorescent reporters and optogenetic actuators has yielded a powerful set of tools for recording and controlling cellular state and function. To achieve the full benefit of these tools requires improved optical systems with high light collection efficiency, high spatial and temporal resolution, and patterned optical stimulation, in a wide field of view (FOV). Here we describe our 'Firefly' microscope, which achieves these goals in a Ø6 mm FOV. The Firefly optical system is optimized for simultaneous photostimulation and fluorescence imaging in cultured cells. All but one of the optical elements are commercially available, yet the microscope achieves 10-fold higher light collection efficiency at its design magnification than the comparable commercially available microscope using the same objective. The Firefly microscope enables all-optical electrophysiology ('Optopatch') in cultured neurons with a throughput and information content unmatched by other neuronal phenotyping systems. This capability opens possibilities in disease modeling and phenotypic drug screening. We also demonstrate applications of the system to voltage and calcium recordings in human induced pluripotent stem cell derived cardiomyocytes.

Ultrawidefield microscope for high-speed fluorescence imaging and targeted optogenetic stimulation

PubMed Central

Werley, Christopher A.; Chien, Miao-Ping; Cohen, Adam E.

2017-01-01

The rapid increase in the number and quality of fluorescent reporters and optogenetic actuators has yielded a powerful set of tools for recording and controlling cellular state and function. To achieve the full benefit of these tools requires improved optical systems with high light collection efficiency, high spatial and temporal resolution, and patterned optical stimulation, in a wide field of view (FOV). Here we describe our ‘Firefly’ microscope, which achieves these goals in a Ø6 mm FOV. The Firefly optical system is optimized for simultaneous photostimulation and fluorescence imaging in cultured cells. All but one of the optical elements are commercially available, yet the microscope achieves 10-fold higher light collection efficiency at its design magnification than the comparable commercially available microscope using the same objective. The Firefly microscope enables all-optical electrophysiology (‘Optopatch’) in cultured neurons with a throughput and information content unmatched by other neuronal phenotyping systems. This capability opens possibilities in disease modeling and phenotypic drug screening. We also demonstrate applications of the system to voltage and calcium recordings in human induced pluripotent stem cell derived cardiomyocytes. PMID:29296505

Imaging initial formation processes of nanobubbles at the graphite-water interface through high-speed atomic force microscopy

NASA Astrophysics Data System (ADS)

Liao, Hsien-Shun; Yang, Chih-Wen; Ko, Hsien-Chen; Hwu, En-Te; Hwang, Ing-Shouh

2018-03-01

The initial formation process of nanobubbles at solid-water interfaces remains unclear because of the limitations of current imaging techniques. To directly observe the formation process, an astigmatic high-speed atomic force microscope (AFM) was modified to enable imaging in the liquid environment. By using a customized cantilever holder, the resonance of small cantilevers was effectively enhanced in water. The proposed high-speed imaging technique yielded highly dynamic quasi-two-dimensional (2D) gas structures (thickness: 20-30 nm) initially at the graphite-water interface. The 2D structures were laterally mobile mainly within certain areas, but occasionally a gas structure might extensively migrate and settle in a new area. The 2D structures were often confined by substrate step edges in one lateral dimension. Eventually, all quasi-2D gas structures were transformed into cap-shaped nanobubbles of higher heights and reduced lateral dimensions. These nanobubbles were immobile and remained stable under continuous AFM imaging. This study demonstrated that nanobubbles could be stably imaged at a scan rate of 100 lines per second (640 μm/s).

An optical system for detecting 3D high-speed oscillation of a single ultrasound microbubble

PubMed Central

Liu, Yuan; Yuan, Baohong

2013-01-01

As contrast agents, microbubbles have been playing significant roles in ultrasound imaging. Investigation of microbubble oscillation is crucial for microbubble characterization and detection. Unfortunately, 3-dimensional (3D) observation of microbubble oscillation is challenging and costly because of the bubble size—a few microns in diameter—and the high-speed dynamics under MHz ultrasound pressure waves. In this study, a cost-efficient optical confocal microscopic system combined with a gated and intensified charge-coupled device (ICCD) camera were developed to detect 3D microbubble oscillation. The capability of imaging microbubble high-speed oscillation with much lower costs than with an ultra-fast framing or streak camera system was demonstrated. In addition, microbubble oscillations along both lateral (x and y) and axial (z) directions were demonstrated. Accordingly, this system is an excellent alternative for 3D investigation of microbubble high-speed oscillation, especially when budgets are limited. PMID:24049677

Control and acquisition systems for new scanning transmission x-ray microscopes at Advanced Light Source (abstract)

NASA Astrophysics Data System (ADS)

Tyliszczak, T.; Hitchcock, P.; Kilcoyne, A. L. D.; Ade, H.; Hitchcock, A. P.; Fakra, S.; Steele, W. F.; Warwick, T.

2002-03-01

Two new scanning x-ray transmission microscopes are being built at beamline 5.3.2 and beamline 7.0 of the Advanced Light Source that have novel aspects in their control and acquisition systems. Both microscopes use multiaxis laser interferometry to improve the precision of pixel location during imaging and energy scans as well as to remove image distortions. Beam line 5.3.2 is a new beam line where the new microscope will be dedicated to studies of polymers in the 250-600 eV energy range. Since this is a bending magnet beam line with lower x-ray brightness than undulator beam lines, special attention is given to the design not only to minimize distortions and vibrations but also to optimize the controls and acquisition to improve data collection efficiency. 5.3.2 microscope control and acquisition is based on a PC computer running WINDOWS 2000. All mechanical stages are moved by stepper motors with rack mounted controllers. A dedicated counter board is used for counting and timing and a multi-input/output board is used for analog acquisition and control of the focusing mirror. A three axis differential laser interferometer is being used to improve stability and precision by careful tracking of the relative positions of the sample and zone plate. Each axis measures the relative distance between a mirror placed on the sample stage and a mirror attached to the zone plate holder. Agilent Technologies HP 10889A servo-axis interferometer boards are used. While they were designed to control servo motors, our tests show that they can be used to directly control the piezo stage. The use of the interferometer servo-axis boards provides excellent point stability for spectral measurements. The interferometric feedback also provides active vibration isolation which reduces deleterious impact of mechanical vibrations up to 20-30 Hz. It also can improve the speed and precision of image scans. Custom C++ software has been written to provide user friendly control of the microscope and integration with visual light microscopy indexing of the samples. The beam line 7.0 microscope upgrade is a new design which will replace the existing microscope. The design is similar to that of beam line 5.3.2, including interferometric position encoding. However the acquisition and control is based on VXI systems, a Sun computer, and LABVIEW™ software. The main objective of the BL 7.0 microscope upgrade is to achieve precise image scans at very high speed (pixel dwells as short as 10 μs) to take full advantage of the high brightness of the 7.0 undulator beamline. Results of tests and a discussion of the benefits of our scanning microscope designs will be presented.

Portable telepathology: methods and tools.

PubMed

Alfaro, Luis; Roca, Ma José

2008-07-15

Telepathology is becoming easier to implement in most pathology departments. In fact e-mail image transmit can be done from almost any pathologist as a simplistic telepathology system. We tried to develop a way to improve capabilities of communication among pathologists with the idea that the system should be affordable for everybody. We took the premise that any pathology department would have microscopes and computers with Internet connection, and selected a few elements to convert them into a telepathology station. Needs were reduced to a camera to collect images, a universal microscope adapter for the camera, a device to connect the camera to the computer, and a software for the remote image transmit. We found out a microscope adapter (MaxView Plus) that allowed us connect almost any domestic digital camera to any microscope. The video out signal from the camera was sent to the computer through an Aver Media USB connector. At last, we selected a group of portable applications that were assembled into a USB memory device. Portable applications are computer programs that can be carried generally on USB flash drives, but also in any other portable device, and used on any (Windows) computer without installation. Besides, when unplugging the device, none of personal data is left behind. We selected open-source applications, and based the pathology image transmission to VLC Media Player due to its functionality as streaming server, portability and ease of use and configuration. Audio transmission was usually done through normal phone lines. We also employed alternative videoconferencing software, SightSpeed for bi-directional image transmission from microscopes, and conventional cameras allowing visual communication and also image transmit from gross pathology specimens. All these elements allowed us to install and use a telepathology system in a few minutes, fully prepared for real time image broadcast.

Portable telepathology: methods and tools

PubMed Central

Alfaro, Luis; Roca, Ma José

2008-01-01

Telepathology is becoming easier to implement in most pathology departments. In fact e-mail image transmit can be done from almost any pathologist as a simplistic telepathology system. We tried to develop a way to improve capabilities of communication among pathologists with the idea that the system should be affordable for everybody. We took the premise that any pathology department would have microscopes and computers with Internet connection, and selected a few elements to convert them into a telepathology station. Needs were reduced to a camera to collect images, a universal microscope adapter for the camera, a device to connect the camera to the computer, and a software for the remote image transmit. We found out a microscope adapter (MaxView Plus) that allowed us connect almost any domestic digital camera to any microscope. The video out signal from the camera was sent to the computer through an Aver Media USB connector. At last, we selected a group of portable applications that were assembled into a USB memory device. Portable applications are computer programs that can be carried generally on USB flash drives, but also in any other portable device, and used on any (Windows) computer without installation. Besides when unplugging the device, none of personal data is left behind. We selected open-source applications, and based the pathology image transmission to VLC Media Player due to its functionality as streaming server, portability and ease of use and configuration. Audio transmission was usually done through normal phone lines. We also employed alternative videoconferencing software, SightSpeed for bi-directional image transmission from microscopes, and conventional cameras allowing visual communication and also image transmit from gross pathology specimens. All these elements allowed us to install and use a telepathology system in a few minutes, fully prepared for real time image broadcast. PMID:18673507

Compact, cost-effective and field-portable microscope prototype based on MISHELF microscopy

NASA Astrophysics Data System (ADS)

Sanz, Martín; Picazo-Bueno, José Ángel; Granero, Luis; García, Javier; Micó, Vicente

2017-02-01

We report on a reduced cost, portable and compact prototype design of lensless holographic microscope with an illumination/detection scheme based on wavelength multiplexing, working with single hologram acquisition and using a fast convergence algorithm for image processing. All together, MISHELF (initials coming from Multi-Illumination Single-Holographic-Exposure Lensless Fresnel) microscopy allows the recording of three Fresnel domain diffraction patterns in a single camera snap-shot incoming from illuminating the sample with three coherent lights at once. Previous implementations have proposed an illumination/detection procedure based on a tuned (illumination wavelengths centered at the maximum sensitivity of the camera detection channels) configuration but here we report on a detuned (non-centered ones) scheme resulting in prototype miniaturization and cost reduction. Thus, MISHELF microscopy in combination with a novel and fast iterative algorithm allows high-resolution (μm range) phase-retrieved (twin image elimination) quantitative phase imaging of dynamic events (video rate recording speed). The performance of this microscope prototype is validated through experiments using both amplitude (USAF resolution test) and complex (live swine sperm cells and flowing microbeads) samples. The proposed method becomes in an alternative instrument improving some capabilities of existing lensless microscopes.

Compact, cost-effective and field-portable microscope prototype based on MISHELF microscopy

PubMed Central

Sanz, Martín; Picazo-Bueno, José Ángel; Granero, Luis; García, Javier; Micó, Vicente

2017-01-01

We report on a reduced cost, portable and compact prototype design of lensless holographic microscope with an illumination/detection scheme based on wavelength multiplexing, working with single hologram acquisition and using a fast convergence algorithm for image processing. All together, MISHELF (initials coming from Multi-Illumination Single-Holographic-Exposure Lensless Fresnel) microscopy allows the recording of three Fresnel domain diffraction patterns in a single camera snap-shot incoming from illuminating the sample with three coherent lights at once. Previous implementations have proposed an illumination/detection procedure based on a tuned (illumination wavelengths centered at the maximum sensitivity of the camera detection channels) configuration but here we report on a detuned (non-centered ones) scheme resulting in prototype miniaturization and cost reduction. Thus, MISHELF microscopy in combination with a novel and fast iterative algorithm allows high-resolution (μm range) phase-retrieved (twin image elimination) quantitative phase imaging of dynamic events (video rate recording speed). The performance of this microscope prototype is validated through experiments using both amplitude (USAF resolution test) and complex (live swine sperm cells and flowing microbeads) samples. The proposed method becomes in an alternative instrument improving some capabilities of existing lensless microscopes. PMID:28233829

High-speed multi-frame dynamic transmission electron microscope image acquisition system with arbitrary timing

DOEpatents

Reed, Bryan W.; DeHope, William J.; Huete, Glenn; LaGrange, Thomas B.; Shuttlesworth, Richard M.

2016-02-23

An electron microscope is disclosed which has a laser-driven photocathode and an arbitrary waveform generator (AWG) laser system ("laser"). The laser produces a train of temporally-shaped laser pulses each being of a programmable pulse duration, and directs the laser pulses to the laser-driven photocathode to produce a train of electron pulses. An image sensor is used along with a deflector subsystem. The deflector subsystem is arranged downstream of the target but upstream of the image sensor, and has a plurality of plates. A control system having a digital sequencer controls the laser and a plurality of switching components, synchronized with the laser, to independently control excitation of each one of the deflector plates. This allows each electron pulse to be directed to a different portion of the image sensor, as well as to enable programmable pulse durations and programmable inter-pulse spacings.

High-speed multiframe dynamic transmission electron microscope image acquisition system with arbitrary timing

DOEpatents

Reed, Bryan W.; DeHope, William J.; Huete, Glenn; LaGrange, Thomas B.; Shuttlesworth, Richard M.

2015-10-20

An electron microscope is disclosed which has a laser-driven photocathode and an arbitrary waveform generator (AWG) laser system ("laser"). The laser produces a train of temporally-shaped laser pulses of a predefined pulse duration and waveform, and directs the laser pulses to the laser-driven photocathode to produce a train of electron pulses. An image sensor is used along with a deflector subsystem. The deflector subsystem is arranged downstream of the target but upstream of the image sensor, and has two pairs of plates arranged perpendicular to one another. A control system controls the laser and a plurality of switching components synchronized with the laser, to independently control excitation of each one of the deflector plates. This allows each electron pulse to be directed to a different portion of the image sensor, as well as to be provided with an independently set duration and independently set inter-pulse spacings.

High-speed multiframe dynamic transmission electron microscope image acquisition system with arbitrary timing

DOEpatents

Reed, Bryan W.; Dehope, William J; Huete, Glenn; LaGrange, Thomas B.; Shuttlesworth, Richard M

2016-06-21

An electron microscope is disclosed which has a laser-driven photocathode and an arbitrary waveform generator (AWG) laser system ("laser"). The laser produces a train of temporally-shaped laser pulses of a predefined pulse duration and waveform, and directs the laser pulses to the laser-driven photocathode to produce a train of electron pulses. An image sensor is used along with a deflector subsystem. The deflector subsystem is arranged downstream of the target but upstream of the image sensor, and has two pairs of plates arranged perpendicular to one another. A control system controls the laser and a plurality of switching components synchronized with the laser, to independently control excitation of each one of the deflector plates. This allows each electron pulse to be directed to a different portion of the image sensor, as well as to be provided with an independently set duration and independently set inter-pulse spacings.

Development of an imaging system for single droplet characterization using a droplet generator.

PubMed

Minov, S Vulgarakis; Cointault, F; Vangeyte, J; Pieters, J G; Hijazi, B; Nuyttens, D

2012-01-01

The spray droplets generated by agricultural nozzles play an important role in the application accuracy and efficiency of plant protection products. The limitations of the non-imaging techniques and the recent improvements in digital image acquisition and processing increased the interest in using high speed imaging techniques in pesticide spray characterisation. The goal of this study was to develop an imaging technique to evaluate the characteristics of a single spray droplet using a piezoelectric single droplet generator and a high speed imaging technique. Tests were done with different camera settings, lenses, diffusers and light sources. The experiments have shown the necessity for having a good image acquisition and processing system. Image analysis results contributed in selecting the optimal set-up for measuring droplet size and velocity which consisted of a high speed camera with a 6 micros exposure time, a microscope lens at a working distance of 43 cm resulting in a field of view of 1.0 cm x 0.8 cm and a Xenon light source without diffuser used as a backlight. For measuring macro-spray characteristics as the droplet trajectory, the spray angle and the spray shape, a Macro Video Zoom lens at a working distance of 14.3 cm with a bigger field of view of 7.5 cm x 9.5 cm in combination with a halogen spotlight with a diffuser and the high speed camera can be used.

An automatic system to study sperm motility and energetics

PubMed Central

Nascimento, Jaclyn M.; Chandsawangbhuwana, Charlie; Botvinick, Elliot L.; Berns, Michael W.

2012-01-01

An integrated robotic laser and microscope system has been developed to automatically analyze individual sperm motility and energetics. The custom-designed optical system directs near-infrared laser light into an inverted microscope to create a single-point 3-D gradient laser trap at the focal spot of the microscope objective. A two-level computer structure is described that quantifies the sperm motility (in terms of swimming speed and swimming force) and energetics (measuring mid-piece membrane potential) using real-time tracking (done by the upper-level system) and fluorescent ratio imaging (done by the lower-level system). The communication between these two systems is achieved by a gigabit network. The custom-built image processing algorithm identifies the sperm swimming trajectory in real-time using phase contrast images, and then subsequently traps the sperm by automatically moving the microscope stage to relocate the sperm to the laser trap focal plane. Once the sperm is stably trapped (determined by the algorithm), the algorithm can also gradually reduce the laser power by rotating the polarizer in the laser path to measure the trapping power at which the sperm is capable of escaping the trap. To monitor the membrane potential of the mitochondria located in a sperm’s mid-piece, the sperm is treated with a ratiometrically-encoded fluorescent probe. The proposed algorithm can relocate the sperm to the center of the ratio imaging camera and the average ratio value can be measured in real-time. The three parameters, sperm escape power, sperm swimming speed and ratio values of the mid-piece membrane potential of individual sperm can be compared with respect to time. This two-level automatic system to study individual sperm motility and energetics has not only increased experimental throughput by an order of magnitude but also has allowed us to monitor sperm energetics prior to and after exposure to the laser trap. This system should have application in both the human fertility clinic and in animal husbandry. PMID:18299996

An automatic system to study sperm motility and energetics.

PubMed

Shi, Linda Z; Nascimento, Jaclyn M; Chandsawangbhuwana, Charlie; Botvinick, Elliot L; Berns, Michael W

2008-08-01

An integrated robotic laser and microscope system has been developed to automatically analyze individual sperm motility and energetics. The custom-designed optical system directs near-infrared laser light into an inverted microscope to create a single-point 3-D gradient laser trap at the focal spot of the microscope objective. A two-level computer structure is described that quantifies the sperm motility (in terms of swimming speed and swimming force) and energetics (measuring mid-piece membrane potential) using real-time tracking (done by the upper-level system) and fluorescent ratio imaging (done by the lower-level system). The communication between these two systems is achieved by a gigabit network. The custom-built image processing algorithm identifies the sperm swimming trajectory in real-time using phase contrast images, and then subsequently traps the sperm by automatically moving the microscope stage to relocate the sperm to the laser trap focal plane. Once the sperm is stably trapped (determined by the algorithm), the algorithm can also gradually reduce the laser power by rotating the polarizer in the laser path to measure the trapping power at which the sperm is capable of escaping the trap. To monitor the membrane potential of the mitochondria located in a sperm's mid-piece, the sperm is treated with a ratiometrically-encoded fluorescent probe. The proposed algorithm can relocate the sperm to the center of the ratio imaging camera and the average ratio value can be measured in real-time. The three parameters, sperm escape power, sperm swimming speed and ratio values of the mid-piece membrane potential of individual sperm can be compared with respect to time. This two-level automatic system to study individual sperm motility and energetics has not only increased experimental throughput by an order of magnitude but also has allowed us to monitor sperm energetics prior to and after exposure to the laser trap. This system should have application in both the human fertility clinic and in animal husbandry.

Compensator design for improved counterbalancing in high speed atomic force microscopy.

PubMed

Bozchalooi, I S; Youcef-Toumi, K; Burns, D J; Fantner, G E

2011-11-01

High speed atomic force microscopy can provide the possibility of many new scientific observations and applications ranging from nano-manufacturing to the study of biological processes. However, the limited imaging speed has been an imperative drawback of the atomic force microscopes. One of the main reasons behind this limitation is the excitation of the AFM dynamics at high scan speeds, severely undermining the reliability of the acquired images. In this research, we propose a piezo based, feedforward controlled, counter actuation mechanism to compensate for the excited out-of-plane scanner dynamics. For this purpose, the AFM controller output is properly filtered via a linear compensator and then applied to a counter actuating piezo. An effective algorithm for estimating the compensator parameters is developed. The information required for compensator design is extracted from the cantilever deflection signal, hence eliminating the need for any additional sensors. The proposed approach is implemented and experimentally evaluated on the dynamic response of a custom made AFM. It is further assessed by comparing the imaging performance of the AFM with and without the application of the proposed technique and in comparison with the conventional counterbalancing methodology. The experimental results substantiate the effectiveness of the method in significantly improving the imaging performance of AFM at high scan speeds. © 2011 American Institute of Physics

Compensator design for improved counterbalancing in high speed atomic force microscopy

PubMed Central

Bozchalooi, I. S.; Youcef-Toumi, K.; Burns, D. J.; Fantner, G. E.

2011-01-01

High speed atomic force microscopy can provide the possibility of many new scientific observations and applications ranging from nano-manufacturing to the study of biological processes. However, the limited imaging speed has been an imperative drawback of the atomic force microscopes. One of the main reasons behind this limitation is the excitation of the AFM dynamics at high scan speeds, severely undermining the reliability of the acquired images. In this research, we propose a piezo based, feedforward controlled, counter actuation mechanism to compensate for the excited out-of-plane scanner dynamics. For this purpose, the AFM controller output is properly filtered via a linear compensator and then applied to a counter actuating piezo. An effective algorithm for estimating the compensator parameters is developed. The information required for compensator design is extracted from the cantilever deflection signal, hence eliminating the need for any additional sensors. The proposed approach is implemented and experimentally evaluated on the dynamic response of a custom made AFM. It is further assessed by comparing the imaging performance of the AFM with and without the application of the proposed technique and in comparison with the conventional counterbalancing methodology. The experimental results substantiate the effectiveness of the method in significantly improving the imaging performance of AFM at high scan speeds. PMID:22128989

Compensator design for improved counterbalancing in high speed atomic force microscopy

NASA Astrophysics Data System (ADS)

Bozchalooi, I. S.; Youcef-Toumi, K.; Burns, D. J.; Fantner, G. E.

2011-11-01

High speed atomic force microscopy can provide the possibility of many new scientific observations and applications ranging from nano-manufacturing to the study of biological processes. However, the limited imaging speed has been an imperative drawback of the atomic force microscopes. One of the main reasons behind this limitation is the excitation of the AFM dynamics at high scan speeds, severely undermining the reliability of the acquired images. In this research, we propose a piezo based, feedforward controlled, counter actuation mechanism to compensate for the excited out-of-plane scanner dynamics. For this purpose, the AFM controller output is properly filtered via a linear compensator and then applied to a counter actuating piezo. An effective algorithm for estimating the compensator parameters is developed. The information required for compensator design is extracted from the cantilever deflection signal, hence eliminating the need for any additional sensors. The proposed approach is implemented and experimentally evaluated on the dynamic response of a custom made AFM. It is further assessed by comparing the imaging performance of the AFM with and without the application of the proposed technique and in comparison with the conventional counterbalancing methodology. The experimental results substantiate the effectiveness of the method in significantly improving the imaging performance of AFM at high scan speeds.

A high-resolution optical imaging system for obtaining the serial transverse section images of biologic tissue

NASA Astrophysics Data System (ADS)

Wu, Li; Zhang, Bin; Wu, Ping; Liu, Qian; Gong, Hui

2007-05-01

A high-resolution optical imaging system was designed and developed to obtain the serial transverse section images of the biologic tissue, such as the mouse brain, in which new knife-edge imaging technology, high-speed and high-sensitive line-scan CCD and linear air bearing stages were adopted and incorporated with an OLYMPUS microscope. The section images on the tip of the knife-edge were synchronously captured by the reflection imaging in the microscope while cutting the biologic tissue. The biologic tissue can be sectioned at interval of 250 nm with the same resolution of the transverse section images obtained in x and y plane. And the cutting job can be automatically finished based on the control program wrote specially in advance, so we save the mass labor of the registration of the vast images data. In addition, by using this system a larger sample can be cut than conventional ultramicrotome so as to avoid the loss of the tissue structure information because of splitting the tissue sample to meet the size request of the ultramicrotome.

High-speed, two-dimensional synchrotron white-beam x-ray radiography of spray breakup and atomization

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

Halls, Benjamin R.; Radke, Christopher D.; Reuter, Benjamin J.

High-speed, two-dimensional synchrotron x-ray radiography and phase-contrast imaging are demonstrated in propulsion sprays. Measurements are performed at the 7-BM beamline at the Advanced Photon Source user facility at Argonne National Laboratory using a recently developed broadband x-ray white beam. This novel enhancement allows for high speed, high fidelity x-ray imaging for the community at large. Quantitative path-integrated liquid distributions and spatio-temporal dynamics of the sprays were imaged with a LuAG:Ce scintillator optically coupled to a high-speed CMOS camera. Images are collected with a microscope objective at frame rates of 20 kHz and with a macro lens at 120 kHz, achievingmore » spatial resolutions of 12 μm and 65 μm, respectively. Imaging with and without potassium iodide (KI) as a contrast-enhancing agent is compared, and the effects of broadband attenuation and spatial beam characteristics are determined through modeling and experimental calibration. In addition, phase contrast is used to differentiate liquid streams with varying concentrations of KI. The experimental approach is applied to different spray conditions, including quantitative measurements of mass distribution during primary atomization and qualitative visualization of turbulent binary fluid mixing. High-speed, two-dimensional synchrotron white-beam x-ray radiography of spray breakup and atomization. Available from: https://www.researchgate.net/publication/312567827_High-speed_two-dimensional_synchrotron_white-beam_x-ray_radiography_of_spray_breakup_and_atomization [accessed Aug 31, 2017].« less

Thermal diffusivity imaging with the thermal lens microscope.

PubMed

Dada, Oluwatosin O; Feist, Peter E; Dovichi, Norman J

2011-12-01

A coaxial thermal lens microscope was used to generate images based on both the absorbance and thermal diffusivity of histological samples. A pump beam was modulated at frequencies ranging from 50 kHz to 5 MHz using an acousto-optic modulator. The pump and a CW probe beam were combined with a dichroic mirror, directed into an inverted microscope, and focused onto the specimen. The change in the transmitted probe beam's center intensity was detected with a photodiode. The photodiode's signal and a reference signal from the modulator were sent to a high-speed lock-in amplifier. The in-phase and quadrature signals were recorded as a sample was translated through the focused beams and used to generate images based on the amplitude and phase of the lock-in amplifier's signal. The amplitude is related to the absorbance and the phase is related to the thermal diffusivity of the sample. Thin sections of stained liver and bone tissues were imaged; the contrast and signal-to-noise ratio of the phase image was highest at frequencies from 0.1-1 MHz and dropped at higher frequencies. The spatial resolution was 2.5 μm for both amplitude and phase images, limited by the pump beam spot size. © 2011 Optical Society of America

Active mask segmentation of fluorescence microscope images.

PubMed

Srinivasa, Gowri; Fickus, Matthew C; Guo, Yusong; Linstedt, Adam D; Kovacević, Jelena

2009-08-01

We propose a new active mask algorithm for the segmentation of fluorescence microscope images of punctate patterns. It combines the (a) flexibility offered by active-contour methods, (b) speed offered by multiresolution methods, (c) smoothing offered by multiscale methods, and (d) statistical modeling offered by region-growing methods into a fast and accurate segmentation tool. The framework moves from the idea of the "contour" to that of "inside and outside," or masks, allowing for easy multidimensional segmentation. It adapts to the topology of the image through the use of multiple masks. The algorithm is almost invariant under initialization, allowing for random initialization, and uses a few easily tunable parameters. Experiments show that the active mask algorithm matches the ground truth well and outperforms the algorithm widely used in fluorescence microscopy, seeded watershed, both qualitatively, as well as quantitatively.

A handheld MEMS-based line-scanned dual-axis confocal microscope for early cancer detection and surgical guidance (Conference Presentation)

NASA Astrophysics Data System (ADS)

Chen, Ye; Yin, Chengbo; Wei, Linpeng; Glaser, Adam K.; Abeytunge, Sanjee; Peterson, Gary; Mandella, Michael J.; Sanai, Nader; Rajadhyaksha, Milind; Liu, Jonathan T.

2017-02-01

Considerable efforts have been recently undertaken to develop miniature optical-sectioning microscopes for in vivo microendoscopy and point-of-care pathology. These devices enable in vivo interrogation of disease as a real-time and noninvasive alternative to gold-standard histopathology, and therefore could have a transformative impact for the early detection of cancer as well as for guiding tumor-resection procedures. Regardless of the specific modality, various trade-offs in size, speed, field of view, resolution, contrast, and sensitivity are necessary to optimize a device for a particular application. Here, a miniature MEMS-based line-scanned dual-axis confocal (LS-DAC) microscope, with a 12-mm diameter distal tip, has been developed for point-of-care pathology. The dual-axis architecture has demonstrated superior rejection of out-of-focus and multiply scattered photons compared to a conventional single-axis confocal configuration. The use of line scanning enables fast frame rates (≥15 frames/sec), which mitigates motion artifacts of a handheld device during clinical use. We have developed a method to actively align the illumination and collection beams in this miniature LS-DAC microscope through the use of a pair of rotatable alignment mirrors. Incorporation of a custom objective lens, with a small form factor for in vivo application, enables the device to achieve an axial and lateral resolution of 2.0 and 1.1 microns, respectively. Validation measurements with reflective targets, as well as in vivo and ex vivo images of tissues, demonstrate that this high-speed LS-DAC microscope can achieve high-contrast imaging of fluorescently labeled tissues with sufficient sensitivity for applications such as oral cancer detection and guiding brain-tumor resections.

Miniature in vivo MEMS-based line-scanned dual-axis confocal microscope for point-of-care pathology

PubMed Central

Yin, C.; Glaser, A.K.; Leigh, S. Y.; Chen, Y.; Wei, L.; Pillai, P. C. S.; Rosenberg, M. C.; Abeytunge, S.; Peterson, G.; Glazowski, C.; Sanai, N.; Mandella, M. J.; Rajadhyaksha, M.; Liu, J. T. C.

2016-01-01

There is a need for miniature optical-sectioning microscopes to enable in vivo interrogation of tissues as a real-time and noninvasive alternative to gold-standard histopathology. Such devices could have a transformative impact for the early detection of cancer as well as for guiding tumor-resection procedures. Miniature confocal microscopes have been developed by various researchers and corporations to enable optical sectioning of highly scattering tissues, all of which have necessitated various trade-offs in size, speed, depth selectivity, field of view, resolution, image contrast, and sensitivity. In this study, a miniature line-scanned (LS) dual-axis confocal (DAC) microscope, with a 12-mm diameter distal tip, has been developed for clinical point-of-care pathology. The dual-axis architecture has demonstrated an advantage over the conventional single-axis confocal configuration for reducing background noise from out-of-focus and multiply scattered light. The use of line scanning enables fast frame rates (16 frames/sec is demonstrated here, but faster rates are possible), which mitigates motion artifacts of a hand-held device during clinical use. We have developed a method to actively align the illumination and collection beams in a DAC microscope through the use of a pair of rotatable alignment mirrors. Incorporation of a custom objective lens, with a small form factor for in vivo clinical use, enables our device to achieve an optical-sectioning thickness and lateral resolution of 2.0 and 1.1 microns respectively. Validation measurements with reflective targets, as well as in vivo and ex vivo images of tissues, demonstrate the clinical potential of this high-speed optical-sectioning microscopy device. PMID:26977337

Digitally controlled analog proportional-integral-derivative (PID) controller for high-speed scanning probe microscopy

NASA Astrophysics Data System (ADS)

Dukic, Maja; Todorov, Vencislav; Andany, Santiago; Nievergelt, Adrian P.; Yang, Chen; Hosseini, Nahid; Fantner, Georg E.

2017-12-01

Nearly all scanning probe microscopes (SPMs) contain a feedback controller, which is used to move the scanner in the direction of the z-axis in order to maintain a constant setpoint based on the tip-sample interaction. The most frequently used feedback controller in SPMs is the proportional-integral (PI) controller. The bandwidth of the PI controller presents one of the speed limiting factors in high-speed SPMs, where higher bandwidths enable faster scanning speeds and higher imaging resolution. Most SPM systems use digital signal processor-based PI feedback controllers, which require analog-to-digital and digital-to-analog converters. These converters introduce additional feedback delays which limit the achievable imaging speed and resolution. In this paper, we present a digitally controlled analog proportional-integral-derivative (PID) controller. The controller implementation allows tunability of the PID gains over a large amplification and frequency range, while also providing precise control of the system and reproducibility of the gain parameters. By using the analog PID controller, we were able to perform successful atomic force microscopy imaging of a standard silicon calibration grating at line rates up to several kHz.

Digitally controlled analog proportional-integral-derivative (PID) controller for high-speed scanning probe microscopy.

PubMed

Dukic, Maja; Todorov, Vencislav; Andany, Santiago; Nievergelt, Adrian P; Yang, Chen; Hosseini, Nahid; Fantner, Georg E

2017-12-01

Nearly all scanning probe microscopes (SPMs) contain a feedback controller, which is used to move the scanner in the direction of the z-axis in order to maintain a constant setpoint based on the tip-sample interaction. The most frequently used feedback controller in SPMs is the proportional-integral (PI) controller. The bandwidth of the PI controller presents one of the speed limiting factors in high-speed SPMs, where higher bandwidths enable faster scanning speeds and higher imaging resolution. Most SPM systems use digital signal processor-based PI feedback controllers, which require analog-to-digital and digital-to-analog converters. These converters introduce additional feedback delays which limit the achievable imaging speed and resolution. In this paper, we present a digitally controlled analog proportional-integral-derivative (PID) controller. The controller implementation allows tunability of the PID gains over a large amplification and frequency range, while also providing precise control of the system and reproducibility of the gain parameters. By using the analog PID controller, we were able to perform successful atomic force microscopy imaging of a standard silicon calibration grating at line rates up to several kHz.

DURIP: A Confocal Imaging System for Ultra-Fast Three-Dimensional Transport Studies in Thermal Management Applications

DTIC Science & Technology

2011-12-01

Transport Phenomena and Thermal Management Applications,” Proceedings of the XXVIII UIT Heat Transfer Conference, Brescia, Italy, June 21-23, 2010...measurements in microscale systems. The integrated confocal microscope system is a critical component to obtain understanding of fluid- heat ...objective of this work was to develop a high speed three-dimensional (3D) confocal imaging system to study coupled fluidic and heat transport

Implementing an Accurate and Rapid Sparse Sampling Approach for Low-Dose Atomic Resolution STEM Imaging

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

Kovarik, Libor; Stevens, Andrew J.; Liyu, Andrey V.

Aberration correction for scanning transmission electron microscopes (STEM) has dramatically increased spatial image resolution for beam-stable materials, but it is the sample stability rather than the microscope that often limits the practical resolution of STEM images. To extract physical information from images of beam sensitive materials it is becoming clear that there is a critical dose/dose-rate below which the images can be interpreted as representative of the pristine material, while above it the observation is dominated by beam effects. Here we describe an experimental approach for sparse sampling in the STEM and in-painting image reconstruction in order to reduce themore » electron dose/dose-rate to the sample during imaging. By characterizing the induction limited rise-time and hysteresis in scan coils, we show that sparse line-hopping approach to scan randomization can be implemented that optimizes both the speed of the scan and the amount of the sample that needs to be illuminated by the beam. The dose and acquisition time for the sparse sampling is shown to be effectively decreased by factor of 5x relative to conventional acquisition, permitting imaging of beam sensitive materials to be obtained without changing the microscope operating parameters. As a result, the use of sparse line-hopping scan to acquire STEM images is demonstrated with atomic resolution aberration corrected Z-contrast images of CaCO 3, a material that is traditionally difficult to image by TEM/STEM because of dose issues.« less

Implementing an Accurate and Rapid Sparse Sampling Approach for Low-Dose Atomic Resolution STEM Imaging

DOE PAGES

Kovarik, Libor; Stevens, Andrew J.; Liyu, Andrey V.; ...

2016-10-17

Aberration correction for scanning transmission electron microscopes (STEM) has dramatically increased spatial image resolution for beam-stable materials, but it is the sample stability rather than the microscope that often limits the practical resolution of STEM images. To extract physical information from images of beam sensitive materials it is becoming clear that there is a critical dose/dose-rate below which the images can be interpreted as representative of the pristine material, while above it the observation is dominated by beam effects. Here we describe an experimental approach for sparse sampling in the STEM and in-painting image reconstruction in order to reduce themore » electron dose/dose-rate to the sample during imaging. By characterizing the induction limited rise-time and hysteresis in scan coils, we show that sparse line-hopping approach to scan randomization can be implemented that optimizes both the speed of the scan and the amount of the sample that needs to be illuminated by the beam. The dose and acquisition time for the sparse sampling is shown to be effectively decreased by factor of 5x relative to conventional acquisition, permitting imaging of beam sensitive materials to be obtained without changing the microscope operating parameters. The use of sparse line-hopping scan to acquire STEM images is demonstrated with atomic resolution aberration corrected Z-contrast images of CaCO3, a material that is traditionally difficult to image by TEM/STEM because of dose issues.« less

Distance reporting in digital pathology: A study on 950 cases

PubMed Central

Vodovnik, Aleksandar

2015-01-01

Background: Increased workload, case complexity, financial constraints, and staffing shortages justify wider implementations of digital pathology. One of its main advantages is distance reporting. Aim: A feasibility study was conducted at our institution in order to achieve comprehensive pathology services available by distance. Methods: One senior pathologist reported 950 cases (3,650 slides) by distance during 19 weeks. Slides were scanned by ScanScope AT Turbo (Aperio) and digital images accessed through SymPathy (Tieto) on a 14” laptop. Mobile phone, mobile broadband, broadband over Wi-Fi and broadband were used for internet connections along with a virtual private network technology (VPN). Lync (Microsoft) was tested for one case consultation and resident's teaching session. Larger displays were accessed when available. Effects of ergonomics and working flexibility on the user experience were observed. Details on network speed, frequency of technical issues, data usage, scanning, and turnaround, were collected and evaluated. Turnaround was compared to in-office microscopic reporting, measured from the registration to sign off. Results: Network speeds varied 1–80 Mbps (median download speed 8–65 Mbps). 20 Mbps were satisfactory for the instant upload of digital images. VPN, image viewer, and laptop failed on two occasions each. An estimated data usage per digital image was 10 MB (1–50 MB). Two cases (15 slides) were deferred to microscopic slides (0.21/0.41%) due to scanty material and suboptimal slide quality. Additional nine cases (15 slides) needed to be rescanned for various reasons (0.95/0.41%). Average turnaround was shorter, and the percentage of cases reported up to 3 days higher (3.13 days/72.25%) comparing with in-office microscopic reporting (3.90 days/40.56%). Larger displays improved the most user experience at magnifications over ×20. Conclusions: Existing IT solutions at our institution allow efficient and reliable distance reporting for the core pathology services in histology and cytology. Stable network speeds, fully integrated laboratory information management system, technical reliability, working flexibility, larger displays, and shorter turnaround contributed to the overall satisfaction with distance reporting. A further expansion of our pathology services available by distance, diagnostic and educational, rely on gaining experience in digital reporting and marginal IT investment. Adjustments to the organization of pathology services may follow to fully benefit from the implementation of digital pathology. PMID:25969793

Distance reporting in digital pathology: A study on 950 cases.

PubMed

Vodovnik, Aleksandar

2015-01-01

Increased workload, case complexity, financial constraints, and staffing shortages justify wider implementations of digital pathology. One of its main advantages is distance reporting. A feasibility study was conducted at our institution in order to achieve comprehensive pathology services available by distance. One senior pathologist reported 950 cases (3,650 slides) by distance during 19 weeks. Slides were scanned by ScanScope AT Turbo (Aperio) and digital images accessed through SymPathy (Tieto) on a 14" laptop. Mobile phone, mobile broadband, broadband over Wi-Fi and broadband were used for internet connections along with a virtual private network technology (VPN). Lync (Microsoft) was tested for one case consultation and resident's teaching session. Larger displays were accessed when available. Effects of ergonomics and working flexibility on the user experience were observed. Details on network speed, frequency of technical issues, data usage, scanning, and turnaround, were collected and evaluated. Turnaround was compared to in-office microscopic reporting, measured from the registration to sign off. Network speeds varied 1-80 Mbps (median download speed 8-65 Mbps). 20 Mbps were satisfactory for the instant upload of digital images. VPN, image viewer, and laptop failed on two occasions each. An estimated data usage per digital image was 10 MB (1-50 MB). Two cases (15 slides) were deferred to microscopic slides (0.21/0.41%) due to scanty material and suboptimal slide quality. Additional nine cases (15 slides) needed to be rescanned for various reasons (0.95/0.41%). Average turnaround was shorter, and the percentage of cases reported up to 3 days higher (3.13 days/72.25%) comparing with in-office microscopic reporting (3.90 days/40.56%). Larger displays improved the most user experience at magnifications over ×20. Existing IT solutions at our institution allow efficient and reliable distance reporting for the core pathology services in histology and cytology. Stable network speeds, fully integrated laboratory information management system, technical reliability, working flexibility, larger displays, and shorter turnaround contributed to the overall satisfaction with distance reporting. A further expansion of our pathology services available by distance, diagnostic and educational, rely on gaining experience in digital reporting and marginal IT investment. Adjustments to the organization of pathology services may follow to fully benefit from the implementation of digital pathology.

Monitoring synaptic and neuronal activity in 3D with synthetic and genetic indicators using a compact acousto-optic lens two-photon microscope☆

PubMed Central

Fernández-Alfonso, Tomás; Nadella, K.M. Naga Srinivas; Iacaruso, M. Florencia; Pichler, Bruno; Roš, Hana; Kirkby, Paul A.; Silver, R. Angus

2014-01-01

Background Two-photon microscopy is widely used to study brain function, but conventional microscopes are too slow to capture the timing of neuronal signalling and imaging is restricted to one plane. Recent development of acousto-optic-deflector-based random access functional imaging has improved the temporal resolution, but the utility of these technologies for mapping 3D synaptic activity patterns and their performance at the excitation wavelengths required to image genetically encoded indicators have not been investigated. New method Here, we have used a compact acousto-optic lens (AOL) two-photon microscope to make high speed [Ca2+] measurements from spines and dendrites distributed in 3D with different excitation wavelengths (800–920 nm). Results We show simultaneous monitoring of activity from many synaptic inputs distributed over the 3D arborisation of a neuronal dendrite using both synthetic as well as genetically encoded indicators. We confirm the utility of AOL-based imaging for fast in vivo recordings by measuring, simultaneously, visually evoked responses in 100 neurons distributed over a 150 μm focal depth range. Moreover, we explore ways to improve the measurement of timing of neuronal activation by choosing specific regions within the cell soma. Comparison with existing methods These results establish that AOL-based 3D random access two-photon microscopy has a wider range of neuroscience applications than previously shown. Conclusions Our findings show that the compact AOL microscope design has the speed, spatial resolution, sensitivity and wavelength flexibility to measure 3D patterns of synaptic and neuronal activity on individual trials. PMID:24200507

Compact and light-weight automated semen analysis platform using lensfree on-chip microscopy.

PubMed

Su, Ting-Wei; Erlinger, Anthony; Tseng, Derek; Ozcan, Aydogan

2010-10-01

We demonstrate a compact and lightweight platform to conduct automated semen analysis using a lensfree on-chip microscope. This holographic on-chip imaging platform weighs ∼46 g, measures ∼4.2 × 4.2 × 5.8 cm, and does not require any lenses, lasers or other bulky optical components to achieve phase and amplitude imaging of sperms over ∼24 mm(2) field-of-view with an effective numerical aperture of ∼0.2. Using this wide-field lensfree on-chip microscope, semen samples are imaged for ∼10 s, capturing a total of ∼20 holographic frames. Digital subtraction of these consecutive lensfree frames, followed by appropriate processing of the reconstructed images, enables automated quantification of the count, the speed and the dynamic trajectories of motile sperms, while summation of the same frames permits counting of immotile sperms. Such a compact and lightweight automated semen analysis platform running on a wide-field lensfree on-chip microscope could be especially important for fertility clinics, personal male fertility tests, as well as for field use in veterinary medicine such as in stud farming and animal breeding applications.

sideSPIM - selective plane illumination based on a conventional inverted microscope.

PubMed

Hedde, Per Niklas; Malacrida, Leonel; Ahrar, Siavash; Siryaporn, Albert; Gratton, Enrico

2017-09-01

Previously described selective plane illumination microscopy techniques typically offset ease of use and sample handling for maximum imaging performance or vice versa . Also, to reduce cost and complexity while maximizing flexibility, it is highly desirable to implement light sheet microscopy such that it can be added to a standard research microscope instead of setting up a dedicated system. We devised a new approach termed sideSPIM that provides uncompromised imaging performance and easy sample handling while, at the same time, offering new applications of plane illumination towards fluidics and high throughput 3D imaging of multiple specimen. Based on an inverted epifluorescence microscope, all of the previous functionality is maintained and modifications to the existing system are kept to a minimum. At the same time, our implementation is able to take full advantage of the speed of the employed sCMOS camera and piezo stage to record data at rates of up to 5 stacks/s. Additionally, sample handling is compatible with established methods and switching magnification to change the field of view from single cells to whole organisms does not require labor intensive adjustments of the system.

Large area high-speed metrology SPM system.

PubMed

Klapetek, P; Valtr, M; Picco, L; Payton, O D; Martinek, J; Yacoot, A; Miles, M

2015-02-13

We present a large area high-speed measuring system capable of rapidly generating nanometre resolution scanning probe microscopy data over mm(2) regions. The system combines a slow moving but accurate large area XYZ scanner with a very fast but less accurate small area XY scanner. This arrangement enables very large areas to be scanned by stitching together the small, rapidly acquired, images from the fast XY scanner while simultaneously moving the slow XYZ scanner across the region of interest. In order to successfully merge the image sequences together two software approaches for calibrating the data from the fast scanner are described. The first utilizes the low uncertainty interferometric sensors of the XYZ scanner while the second implements a genetic algorithm with multiple parameter fitting during the data merging step of the image stitching process. The basic uncertainty components related to these high-speed measurements are also discussed. Both techniques are shown to successfully enable high-resolution, large area images to be generated at least an order of magnitude faster than with a conventional atomic force microscope.

Large area high-speed metrology SPM system

NASA Astrophysics Data System (ADS)

Klapetek, P.; Valtr, M.; Picco, L.; Payton, O. D.; Martinek, J.; Yacoot, A.; Miles, M.

2015-02-01

We present a large area high-speed measuring system capable of rapidly generating nanometre resolution scanning probe microscopy data over mm2 regions. The system combines a slow moving but accurate large area XYZ scanner with a very fast but less accurate small area XY scanner. This arrangement enables very large areas to be scanned by stitching together the small, rapidly acquired, images from the fast XY scanner while simultaneously moving the slow XYZ scanner across the region of interest. In order to successfully merge the image sequences together two software approaches for calibrating the data from the fast scanner are described. The first utilizes the low uncertainty interferometric sensors of the XYZ scanner while the second implements a genetic algorithm with multiple parameter fitting during the data merging step of the image stitching process. The basic uncertainty components related to these high-speed measurements are also discussed. Both techniques are shown to successfully enable high-resolution, large area images to be generated at least an order of magnitude faster than with a conventional atomic force microscope.

Coherent anti-Stokes Raman scattering microscope with a high-signal-to-noise ratio, high stability, and high-speed imaging for live cell observation

NASA Astrophysics Data System (ADS)

Hayashi, Shinichi; Takimoto, Shinichi; Hashimoto, Takeshi

2007-02-01

Coherent anti-Stokes Raman scattering (CARS) microscopy, which can produce images of specific molecules without staining, has attracted the attention of researchers, as it matches the need for molecular imaging and pathway analysis of live cells. In particular, there have been an increasing number of CARS experimental results regarding lipids in live cells, which cannot be fluorescently tagged while keeping the cells alive. One of the important applications of lipid research is for the metabolic syndrome. Since the metabolic syndrome is said to be related to the lipids in lipocytes, blood, arterial vessels, and so on, the CARS technique is expected to find application in this field. However, CARS microscopy requires a pair of picosecond laser pulses, which overlap both temporally and spatially. This makes the optical adjustments of a CARS microscope challenging. The authors developed a CARS unit that includes optics for easy and stable adjustment of the overlap of these laser pulses. Adding the CARS unit to a laser scanning microscope provides CARS images of a high signal-to-noise ratio, with an acquisition rate as high as 2 microseconds per pixel. Thus, images of fast-moving lipid droplets in Hela cells were obtained.

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).

Motion compensation for in vivo subcellular optical microscopy.

PubMed

Lucotte, B; Balaban, R S

2014-04-01

In this review, we focus on the impact of tissue motion on attempting to conduct subcellular resolution optical microscopy, in vivo. Our position is that tissue motion is one of the major barriers in conducting these studies along with light induced damage, optical probe loading as well as absorbing and scattering effects on the excitation point spread function and collection of emitted light. Recent developments in the speed of image acquisition have reached the limit, in most cases, where the signal from a subcellular voxel limits the speed and not the scanning rate of the microscope. Different schemes for compensating for tissue displacements due to rigid body and deformation are presented from tissue restriction, gating, adaptive gating and active tissue tracking. We argue that methods that minimally impact the natural physiological motion of the tissue are desirable because the major reason to perform in vivo studies is to evaluate normal physiological functions. Towards this goal, active tracking using the optical imaging data itself to monitor tissue displacement and either prospectively or retrospectively correct for the motion without affecting physiological processes is desirable. Critical for this development was the implementation of near real time image processing in conjunction with the control of the microscope imaging parameters. Clearly, the continuing development of methods of motion compensation as well as significant technological solutions to the other barriers to tissue subcellular optical imaging in vivo, including optical aberrations and overall signal-to-noise ratio, will make major contributions to the understanding of cell biology within the body.

A compact Acousto-Optic Lens for 2D and 3D femtosecond based 2-photon microscopy.

PubMed

Kirkby, Paul A; Srinivas Nadella, K M Naga; Silver, R Angus

2010-06-21

We describe a high speed 3D Acousto-Optic Lens Microscope (AOLM) for femtosecond 2-photon imaging. By optimizing the design of the 4 AO Deflectors (AODs) and by deriving new control algorithms, we have developed a compact spherical AOL with a low temporal dispersion that enables 2-photon imaging at 10-fold lower power than previously reported. We show that the AOLM can perform high speed 2D raster-scan imaging (>150 Hz) without scan rate dependent astigmatism. It can deflect and focus a laser beam in a 3D random access sequence at 30 kHz and has an extended focusing range (>137 mum; 40X 0.8NA objective). These features are likely to make the AOLM a useful tool for studying fast physiological processes distributed in 3D space.

Dynamic autofocus for continuous-scanning time-delay-and-integration image acquisition in automated microscopy.

PubMed

Bravo-Zanoguera, Miguel E; Laris, Casey A; Nguyen, Lam K; Oliva, Mike; Price, Jeffrey H

2007-01-01

Efficient image cytometry of a conventional microscope slide means rapid acquisition and analysis of 20 gigapixels of image data (at 0.3-microm sampling). The voluminous data motivate increased acquisition speed to enable many biomedical applications. Continuous-motion time-delay-and-integrate (TDI) scanning has the potential to speed image acquisition while retaining sensitivity, but the challenge of implementing high-resolution autofocus operating simultaneously with acquisition has limited its adoption. We develop a dynamic autofocus system for this need using: 1. a "volume camera," consisting of nine fiber optic imaging conduits to charge-coupled device (CCD) sensors, that acquires images in parallel from different focal planes, 2. an array of mixed analog-digital processing circuits that measure the high spatial frequencies of the multiple image streams to create focus indices, and 3. a software system that reads and analyzes the focus data streams and calculates best focus for closed feedback loop control. Our system updates autofocus at 56 Hz (or once every 21 microm of stage travel) to collect sharply focused images sampled at 0.3x0.3 microm(2)/pixel at a stage speed of 2.3 mms. The system, tested by focusing in phase contrast and imaging long fluorescence strips, achieves high-performance closed-loop image-content-based autofocus in continuous scanning for the first time.

Two-photon microscope for multisite microphotolysis of caged neurotransmitters in acute brain slices

PubMed Central

Losavio, Bradley E.; Iyer, Vijay; Saggau, Peter

2009-01-01

We developed a two-photon microscope optimized for physiologically manipulating single neurons through their postsynaptic receptors. The optical layout fulfills the stringent design criteria required for high-speed, high-resolution imaging in scattering brain tissue with minimal photodamage. We detail the practical compensation of spectral and temporal dispersion inherent in fast laser beam scanning with acousto-optic deflectors, as well as a set of biological protocols for visualizing nearly diffraction-limited structures and delivering physiological synaptic stimuli. The microscope clearly resolves dendritic spines and evokes electrophysiological transients in single neurons that are similar to endogenous responses. This system enables the study of multisynaptic integration and will assist our understanding of single neuron function and dendritic computation. PMID:20059271

Data reduction of digitized images processed from calibrated photographic and spectroscopic films obtained from terrestial, rocket and space shuttle telescopic instruments

NASA Technical Reports Server (NTRS)

Hammond, Ernest C., Jr.

1990-01-01

The Microvax 2 computer, the basic software in VMS, and the Mitsubishi High Speed Disk were received and installed. The digital scanning tunneling microscope is fully installed and operational. A new technique was developed for pseudocolor analysis of the line plot images of a scanning tunneling microscope. Computer studies and mathematical modeling of the empirical data associated with many of the film calibration studies were presented. A gas can follow-up experiment which will be launched in September, on the Space Shuttle STS-50, was prepared and loaded. Papers were presented on the structure of the human hair strand using scanning electron microscopy and x ray analysis and updated research on the annual rings produced by the surf clam of the ocean estuaries of Maryland. Scanning electron microscopic work was conducted by the research team for the study of the Mossbauer and Magnetic Susceptibility Studies on NmNi(4.25)Fe(.85) and its Hydride.

Polarization Imaging Apparatus

NASA Technical Reports Server (NTRS)

Zou, Yingyin K.; Chen, Qiushui

2010-01-01

A polarization imaging apparatus has shown promise as a prototype of instruments for medical imaging with contrast greater than that achievable by use of non-polarized light. The underlying principles of design and operation are derived from observations that light interacts with tissue ultrastructures that affect reflectance, scattering, absorption, and polarization of light. The apparatus utilizes high-speed electro-optical components for generating light properties and acquiring polarization images through aligned polarizers. These components include phase retarders made of OptoCeramic (registered TradeMark) material - a ceramic that has a high electro-optical coefficient. The apparatus includes a computer running a program that implements a novel algorithm for controlling the phase retarders, capturing image data, and computing the Stokes polarization images. Potential applications include imaging of superficial cancers and other skin lesions, early detection of diseased cells, and microscopic analysis of tissues. The high imaging speed of this apparatus could be beneficial for observing live cells or tissues, and could enable rapid identification of moving targets in astronomy and national defense. The apparatus could also be used as an analysis tool in material research and industrial processing.

Sample Preparation and Mounting of Drosophila Embryos for Multiview Light Sheet Microscopy.

PubMed

Schmied, Christopher; Tomancak, Pavel

2016-01-01

Light sheet fluorescent microscopy (LSFM), and in particular its most widespread flavor Selective Plane Illumination Microscopy (SPIM), promises to provide unprecedented insights into developmental dynamics of entire living systems. By combining minimal photo-damage with high imaging speed and sample mounting tailored toward the needs of the specimen, it enables in toto imaging of embryogenesis with high spatial and temporal resolution. Drosophila embryos are particularly well suited for SPIM imaging because the volume of the embryo does not change from the single cell embryo to the hatching larva. SPIM microscopes can therefore image Drosophila embryos embedded in rigid media, such as agarose, from multiple angles every few minutes from the blastoderm stage until hatching. Here, we describe sample mounting strategies to achieve such a recording. We also provide detailed protocols to realize multiview, long-term, time-lapse recording of Drosophila embryos expressing fluorescent markers on the commercially available Zeiss Lightsheet Z.1 microscope and the OpenSPIM.

Light-sheet microscopy by confocal line scanning of dual-Bessel beams

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

Zhang, Pengfei; Phipps, Mary Elizabeth; Goodwin, Peter Marvin

Here, we have developed a light-sheet microscope that uses confocal scanning of dual-Bessel beams for illumination. A digital micromirror device (DMD) is placed in the intermediate image plane of the objective used to collect fluorescence and is programmed with two lines of pixels in the “on” state such that the DMD functions as a spatial filter to reject the out-of-focus background generated by the side-lobes of the Bessel beams. The optical sectioning and out-of-focus background rejection capabilities of this microscope were demonstrated by imaging of fluorescently stained actin in human A431 cells. The dual-Bessel beam system enables twice as manymore » photons to be detected per imaging scan, which is useful for low light applications (e.g., single-molecule localization) or imaging at high speed with a superior signal to noise. While demonstrated for two Bessel beams, this approach is scalable to a larger number of beams.« less

Light-sheet microscopy by confocal line scanning of dual-Bessel beams

DOE PAGES

Zhang, Pengfei; Phipps, Mary Elizabeth; Goodwin, Peter Marvin; ...

2016-10-25

Here, we have developed a light-sheet microscope that uses confocal scanning of dual-Bessel beams for illumination. A digital micromirror device (DMD) is placed in the intermediate image plane of the objective used to collect fluorescence and is programmed with two lines of pixels in the “on” state such that the DMD functions as a spatial filter to reject the out-of-focus background generated by the side-lobes of the Bessel beams. The optical sectioning and out-of-focus background rejection capabilities of this microscope were demonstrated by imaging of fluorescently stained actin in human A431 cells. The dual-Bessel beam system enables twice as manymore » photons to be detected per imaging scan, which is useful for low light applications (e.g., single-molecule localization) or imaging at high speed with a superior signal to noise. While demonstrated for two Bessel beams, this approach is scalable to a larger number of beams.« less

Phase modulation atomic force microscope with true atomic resolution

NASA Astrophysics Data System (ADS)

Fukuma, Takeshi; Kilpatrick, Jason I.; Jarvis, Suzanne P.

2006-12-01

We have developed a dynamic force microscope (DFM) working in a novel operation mode which is referred to as phase modulation atomic force microscopy (PM-AFM). PM-AFM utilizes a fixed-frequency excitation signal to drive a cantilever, which ensures stable imaging even with occasional tip crash and adhesion to the surface. The tip-sample interaction force is detected as a change of the phase difference between the cantilever deflection and excitation signals and hence the time response is not influenced by the Q factor of the cantilever. These features make PM-AFM more suitable for high-speed imaging than existing DFM techniques such as amplitude modulation and frequency modulation atomic force microscopies. Here we present the basic principle of PM-AFM and the theoretical limit of its performance. The design of the developed PM-AFM is described and its theoretically limited noise performance is demonstrated. Finally, we demonstrate the true atomic resolution imaging capability of the developed PM-AFM by imaging atomic-scale features of mica in water.

Imaging with organic indicators and high-speed charge-coupled device cameras in neurons: some applications where these classic techniques have advantages.

PubMed

Ross, William N; Miyazaki, Kenichi; Popovic, Marko A; Zecevic, Dejan

2015-04-01

Dynamic calcium and voltage imaging is a major tool in modern cellular neuroscience. Since the beginning of their use over 40 years ago, there have been major improvements in indicators, microscopes, imaging systems, and computers. While cutting edge research has trended toward the use of genetically encoded calcium or voltage indicators, two-photon microscopes, and in vivo preparations, it is worth noting that some questions still may be best approached using more classical methodologies and preparations. In this review, we highlight a few examples in neurons where the combination of charge-coupled device (CCD) imaging and classical organic indicators has revealed information that has so far been more informative than results using the more modern systems. These experiments take advantage of the high frame rates, sensitivity, and spatial integration of the best CCD cameras. These cameras can respond to the faster kinetics of organic voltage and calcium indicators, which closely reflect the fast dynamics of the underlying cellular events.

Laser based imaging of time depending microscopic scenes with strong light emission

NASA Astrophysics Data System (ADS)

Hahlweg, Cornelius; Wilhelm, Eugen; Rothe, Hendrik

2011-10-01

Investigating volume scatterometry methods based on short range LIDAR devices for non-static objects we achieved interesting results aside the intended micro-LIDAR: the high speed camera recording of the illuminated scene of an exploding wire -intended for Doppler LIDAR tests - delivered a very effective method of observing details of objects with extremely strong light emission. As a side effect a schlieren movie is gathered without any special effort. The fact that microscopic features of short time processes with high emission and material flow might be imaged without endangering valuable equipment makes this technique at least as interesting as the intended one. So we decided to present our results - including latest video and photo material - instead of a more theoretical paper on our progress concerning the primary goal.

Two-dimensional tracking of a motile micro-organism allowing high-resolution observation with various imaging techniques

NASA Astrophysics Data System (ADS)

Oku, H.; Ogawa, N.; Ishikawa, M.; Hashimoto, K.

2005-03-01

In this article, a micro-organism tracking system using a high-speed vision system is reported. This system two dimensionally tracks a freely swimming micro-organism within the field of an optical microscope by moving a chamber of target micro-organisms based on high-speed visual feedback. The system we developed could track a paramecium using various imaging techniques, including bright-field illumination, dark-field illumination, and differential interference contrast, at magnifications of 5 times and 20 times. A maximum tracking duration of 300s was demonstrated. Also, the system could track an object with a velocity of up to 35 000μm/s (175diameters/s), which is significantly faster than swimming micro-organisms.

A two-angle far-field microscope imaging technique for spray flows

NASA Astrophysics Data System (ADS)

Kourmatzis, Agisilaos; Pham, Phuong X.; Masri, Assaad R.

2017-03-01

Backlight imaging is frequently used for the visualization of multiphase flows, where with appropriate microscope lenses, quantitative information on the spray structure can be attained. However, a key issue resides in the nature of the measurement which relies on a single viewing angle, hence preventing imaging of all liquid structures and features, such as those located behind other fragments. This paper presents results from an extensive experimental study aimed as a step forward towards resolving this problem by using a pair of high speed cameras oriented at 90 degrees to each other, and synchronized to two high-speed diode lasers. Both cameras are used with long distance microscope lenses. The images are processed as pairs allowing for identification and classification of the same liquid structure from two perspectives at high temporal (5 kHz) and spatial resolution (∼3 μm). Using a controlled mono-disperse spray, simultaneous, time-resolved visualization of the same spherical object being focused on one plane while de-focused on the other plane 90 degrees to the first has allowed for a quantification of shot-to-shot defocused size measurement error. An extensive error analysis is performed for spheroidal structures imaged from two angles and the dual angle technique is extended to measure the volume of non-spherical fragments for the first time, by ‘discretising’ a fragment into a number of constituent ellipses. Error analysis is performed based on measuring the known volumes of solid arbitrary shapes, and volume estimates were found to be within  ∼11% of the real volume for representative ‘ligament-like’ shapes. The contribution concludes by applying the ellipsoidal method to a real spray consisting of multiple non-spherical fragments. This extended approach clearly demonstrates potential to yield novel volume weighted quantities of non-spherical objects in turbulent multiphase flow applications.

Random-access scanning microscopy for 3D imaging in awake behaving animals

PubMed Central

Nadella, K. M. Naga Srinivas; Roš, Hana; Baragli, Chiara; Griffiths, Victoria A.; Konstantinou, George; Koimtzis, Theo; Evans, Geoffrey J.; Kirkby, Paul A.; Silver, R. Angus

2018-01-01

Understanding how neural circuits process information requires rapid measurements from identified neurons distributed in 3D space. Here we describe an acousto-optic lens two-photon microscope that performs high-speed focussing and line-scanning within a volume spanning hundreds of micrometres. We demonstrate its random access functionality by selectively imaging cerebellar interneurons sparsely distributed in 3D and by simultaneously recording from the soma, proximal and distal dendrites of neocortical pyramidal cells in behaving mice. PMID:27749836

Holographic digital microscopy in on-line process control

NASA Astrophysics Data System (ADS)

Osanlou, Ardeshir

2011-09-01

This article investigates the feasibility of real-time three-dimensional imaging of microscopic objects within various emulsions while being produced in specialized production vessels. The study is particularly relevant to on-line process monitoring and control in chemical, pharmaceutical, food, cleaning, and personal hygiene industries. Such processes are often dynamic and the materials cannot be measured once removed from the production vessel. The technique reported here is applicable to three-dimensional characterization analyses on stirred fluids in small reaction vessels. Relatively expensive pulsed lasers have been avoided through the careful control of the speed of the moving fluid in relation to the speed of the camera exposure and the wavelength of the continuous wave laser used. The ultimate aim of the project is to introduce a fully robust and compact digital holographic microscope as a process control tool in a full size specialized production vessel.

TRIIG - Time-lapse reproduction of images through interactive graphics. [digital processing of quality hard copy

NASA Technical Reports Server (NTRS)

Buckner, J. D.; Council, H. W.; Edwards, T. R.

1974-01-01

Description of the hardware and software implementing the system of time-lapse reproduction of images through interactive graphics (TRIIG). The system produces a quality hard copy of processed images in a fast and inexpensive manner. This capability allows for optimal development of processing software through the rapid viewing of many image frames in an interactive mode. Three critical optical devices are used to reproduce an image: an Optronics photo reader/writer, the Adage Graphics Terminal, and Polaroid Type 57 high speed film. Typical sources of digitized images are observation satellites, such as ERTS or Mariner, computer coupled electron microscopes for high-magnification studies, or computer coupled X-ray devices for medical research.

Effect of time discretization of the imaging process on the accuracy of trajectory estimation in fluorescence microscopy

PubMed Central

Wong, Yau; Chao, Jerry; Lin, Zhiping; Ober, Raimund J.

2014-01-01

In fluorescence microscopy, high-speed imaging is often necessary for the proper visualization and analysis of fast subcellular dynamics. Here, we examine how the speed of image acquisition affects the accuracy with which parameters such as the starting position and speed of a microscopic non-stationary fluorescent object can be estimated from the resulting image sequence. Specifically, we use a Fisher information-based performance bound to investigate the detector-dependent effect of frame rate on the accuracy of parameter estimation. We demonstrate that when a charge-coupled device detector is used, the estimation accuracy deteriorates as the frame rate increases beyond a point where the detector’s readout noise begins to overwhelm the low number of photons detected in each frame. In contrast, we show that when an electron-multiplying charge-coupled device (EMCCD) detector is used, the estimation accuracy improves with increasing frame rate. In fact, at high frame rates where the low number of photons detected in each frame renders the fluorescent object difficult to detect visually, imaging with an EMCCD detector represents a natural implementation of the Ultrahigh Accuracy Imaging Modality, and enables estimation with an accuracy approaching that which is attainable only when a hypothetical noiseless detector is used. PMID:25321248

Velocimetry of fast microscopic liquid jets by nanosecond dual-pulse laser illumination for megahertz X-ray free-electron lasers.

PubMed

Grünbein, Marie Luise; Shoeman, Robert L; Doak, R Bruce

2018-03-19

To conduct X-ray Free-Electron Laser (XFEL) measurements at megahertz (MHz) repetition rates, sample solution must be delivered in a micron-sized liquid free-jet moving at up to 100 m/s. This exceeds by over a factor of two the jet speeds measurable with current high-speed camera techniques. Accordingly we have developed and describe herein an alternative jet velocimetry based on dual-pulse nanosecond laser illumination. Three separate implementations are described, including a small laser-diode system that is inexpensive and highly portable. We have also developed and describe analysis techniques to automatically and rapidly extract jet speed from dual-pulse images.

Ultrahigh speed endoscopic swept source optical coherence tomography using a VCSEL light source and micromotor catheter

NASA Astrophysics Data System (ADS)

Tsai, Tsung-Han; Ahsen, Osman O.; Lee, Hsiang-Chieh; Liang, Kaicheng; Giacomelli, Michael G.; Potsaid, Benjamin M.; Tao, Yuankai K.; Jayaraman, Vijaysekhar; Kraus, Martin F.; Hornegger, Joachim; Figueiredo, Marisa; Huang, Qin; Mashimo, Hiroshi; Cable, Alex E.; Fujimoto, James G.

2014-03-01

We developed an ultrahigh speed endoscopic swept source optical coherence tomography (OCT) system for clinical gastroenterology using a vertical-cavity surface-emitting laser (VCSEL) and micromotor based imaging catheter, which provided an imaging speed of 600 kHz axial scan rate and 8 μm axial resolution in tissue. The micromotor catheter was 3.2 mm in diameter and could be introduced through the 3.7 mm accessory port of an endoscope. Imaging was performed at 400 frames per second with an 8 μm spot size using a pullback to generate volumetric data over 16 mm with a pixel spacing of 5 μm in the longitudinal direction. Three-dimensional OCT (3D-OCT) imaging was performed in patients with a cross section of pathologies undergoing standard upper and lower endoscopy at the Veterans Affairs Boston Healthcare System (VABHS). Patients with Barrett's esophagus, dysplasia, and inflammatory bowel disease were imaged. The use of distally actuated imaging catheters allowed OCT imaging with more flexibility such as volumetric imaging in the terminal ileum and the assessment of the hiatal hernia using retroflex imaging. The high rotational stability of the micromotor enabled 3D volumetric imaging with micron scale volumetric accuracy for both en face and cross-sectional imaging. The ability to perform 3D OCT imaging in the GI tract with microscopic accuracy should enable a wide range of studies to investigate the ability of OCT to detect pathology as well as assess treatment response.

3D real-time visualization of blood flow in cerebral aneurysms by light field particle image velocimetry

NASA Astrophysics Data System (ADS)

Carlsohn, Matthias F.; Kemmling, André; Petersen, Arne; Wietzke, Lennart

2016-04-01

Cerebral aneurysms require endovascular treatment to eliminate potentially lethal hemorrhagic rupture by hemostasis of blood flow within the aneurysm. Devices (e.g. coils and flow diverters) promote homeostasis, however, measurement of blood flow within an aneurysm or cerebral vessel before and after device placement on a microscopic level has not been possible so far. This would allow better individualized treatment planning and improve manufacture design of devices. For experimental analysis, direct measurement of real-time microscopic cerebrovascular flow in micro-structures may be an alternative to computed flow simulations. An application of microscopic aneurysm flow measurement on a regular basis to empirically assess a high number of different anatomic shapes and the corresponding effect of different devices would require a fast and reliable method at low cost with high throughout assessment. Transparent three dimensional 3D models of brain vessels and aneurysms may be used for microscopic flow measurements by particle image velocimetry (PIV), however, up to now the size of structures has set the limits for conventional 3D-imaging camera set-ups. On line flow assessment requires additional computational power to cope with the processing large amounts of data generated by sequences of multi-view stereo images, e.g. generated by a light field camera capturing the 3D information by plenoptic imaging of complex flow processes. Recently, a fast and low cost workflow for producing patient specific three dimensional models of cerebral arteries has been established by stereo-lithographic (SLA) 3D printing. These 3D arterial models are transparent an exhibit a replication precision within a submillimeter range required for accurate flow measurements under physiological conditions. We therefore test the feasibility of microscopic flow measurements by PIV analysis using a plenoptic camera system capturing light field image sequences. Averaging across a sequence of single double or triple shots of flashed images enables reconstruction of the real-time corpuscular flow through the vessel system before and after device placement. This approach could enable 3D-insight of microscopic flow within blood vessels and aneurysms at submillimeter resolution. We present an approach that allows real-time assessment of 3D particle flow by high-speed light field image analysis including a solution that addresses high computational load by image processing. The imaging set-up accomplishes fast and reliable PIV analysis in transparent 3D models of brain aneurysms at low cost. High throughput microscopic flow assessment of different shapes of brain aneurysms may therefore be possibly required for patient specific device designs.

Ultrahigh speed endoscopic optical coherence tomography for gastroenterology.

PubMed

Tsai, Tsung-Han; Lee, Hsiang-Chieh; Ahsen, Osman O; Liang, Kaicheng; Giacomelli, Michael G; Potsaid, Benjamin M; Tao, Yuankai K; Jayaraman, Vijaysekhar; Figueiredo, Marisa; Huang, Qin; Cable, Alex E; Fujimoto, James; Mashimo, Hiroshi

2014-12-01

We describe an ultrahigh speed endoscopic swept source optical coherence tomography (OCT) system for clinical gastroenterology using a vertical-cavity surface-emitting laser (VCSEL) and micromotor imaging catheter. The system had a 600 kHz axial scan rate and 8 µm axial resolution in tissue. Imaging was performed with a 3.2 mm diameter imaging catheter at 400 frames per second with a 12 µm spot size. Three-dimensional OCT (3D-OCT) imaging was performed in patients with a cross section of pathologies undergoing upper and lower endoscopy. The use of distally actuated imaging catheters enabled OCT imaging with more flexibility, such as volumetric imaging in the small intestine and the assessment of hiatal hernia using retroflex imaging. The high rotational scanning stability of the micromotor enabled 3D volumetric imaging with micron scale volumetric accuracy for both en face OCT and cross-sectional imaging, as well as OCT angiography (OCTA) for 3D visualization of subsurface microvasculature. The ability to perform both structural and functional 3D OCT imaging in the GI tract with microscopic accuracy should enable a wide range of studies and enhance the sensitivity and specificity of OCT for detecting pathology.

Ultrahigh speed endoscopic optical coherence tomography for gastroenterology

PubMed Central

Tsai, Tsung-Han; Lee, Hsiang-Chieh; Ahsen, Osman O.; Liang, Kaicheng; Giacomelli, Michael G.; Potsaid, Benjamin M.; Tao, Yuankai K.; Jayaraman, Vijaysekhar; Figueiredo, Marisa; Huang, Qin; Cable, Alex E.; Fujimoto, James; Mashimo, Hiroshi

2014-01-01

We describe an ultrahigh speed endoscopic swept source optical coherence tomography (OCT) system for clinical gastroenterology using a vertical-cavity surface-emitting laser (VCSEL) and micromotor imaging catheter. The system had a 600 kHz axial scan rate and 8 µm axial resolution in tissue. Imaging was performed with a 3.2 mm diameter imaging catheter at 400 frames per second with a 12 µm spot size. Three-dimensional OCT (3D-OCT) imaging was performed in patients with a cross section of pathologies undergoing upper and lower endoscopy. The use of distally actuated imaging catheters enabled OCT imaging with more flexibility, such as volumetric imaging in the small intestine and the assessment of hiatal hernia using retroflex imaging. The high rotational scanning stability of the micromotor enabled 3D volumetric imaging with micron scale volumetric accuracy for both en face OCT and cross-sectional imaging, as well as OCT angiography (OCTA) for 3D visualization of subsurface microvasculature. The ability to perform both structural and functional 3D OCT imaging in the GI tract with microscopic accuracy should enable a wide range of studies and enhance the sensitivity and specificity of OCT for detecting pathology. PMID:25574446

Factors to keep in mind when introducing virtual microscopy.

PubMed

Glatz-Krieger, Katharina; Spornitz, Udo; Spatz, Alain; Mihatsch, Michael J; Glatz, Dieter

2006-03-01

Digitization of glass slides and delivery of so-called virtual slides (VS) emulating a real microscope over the Internet have become reality due to recent improvements in technology. We have implemented a virtual microscope for instruction of medical students and for continuing medical education. Up to 30,000 images per slide are captured using a microscope with an automated stage. The images are post-processed and then served by a plain hypertext transfer protocol (http)-server. A virtual slide client (vMic) based on Macromedia's Flash MX, a highly accepted technology available on every modern Web browser, has been developed. All necessary virtual slide parameters are stored in an XML file together with the image. Evaluation of the courses by questionnaire indicated that most students and many but not all pathologists regard virtual slides as an adequate replacement for traditional slides. All our virtual slides are publicly accessible over the World Wide Web (WWW) at http://vmic.unibas.ch . Recently, several commercially available virtual slide acquisition systems (VSAS) have been developed that use various technologies to acquire and distribute virtual slides. These systems differ in speed, image quality, compatibility, viewer functionalities and price. This paper gives an overview of the factors to keep in mind when introducing virtual microscopy.

Automated detection of fluorescent cells in in-resin fluorescence sections for integrated light and electron microscopy.

PubMed

Delpiano, J; Pizarro, L; Peddie, C J; Jones, M L; Griffin, L D; Collinson, L M

2018-04-26

Integrated array tomography combines fluorescence and electron imaging of ultrathin sections in one microscope, and enables accurate high-resolution correlation of fluorescent proteins to cell organelles and membranes. Large numbers of serial sections can be imaged sequentially to produce aligned volumes from both imaging modalities, thus producing enormous amounts of data that must be handled and processed using novel techniques. Here, we present a scheme for automated detection of fluorescent cells within thin resin sections, which could then be used to drive automated electron image acquisition from target regions via 'smart tracking'. The aim of this work is to aid in optimization of the data acquisition process through automation, freeing the operator to work on other tasks and speeding up the process, while reducing data rates by only acquiring images from regions of interest. This new method is shown to be robust against noise and able to deal with regions of low fluorescence. © 2018 The Authors. Journal of Microscopy published by JohnWiley & Sons Ltd on behalf of Royal Microscopical Society.

sideSPIM – selective plane illumination based on a conventional inverted microscope

PubMed Central

Hedde, Per Niklas; Malacrida, Leonel; Ahrar, Siavash; Siryaporn, Albert; Gratton, Enrico

2017-01-01

Previously described selective plane illumination microscopy techniques typically offset ease of use and sample handling for maximum imaging performance or vice versa. Also, to reduce cost and complexity while maximizing flexibility, it is highly desirable to implement light sheet microscopy such that it can be added to a standard research microscope instead of setting up a dedicated system. We devised a new approach termed sideSPIM that provides uncompromised imaging performance and easy sample handling while, at the same time, offering new applications of plane illumination towards fluidics and high throughput 3D imaging of multiple specimen. Based on an inverted epifluorescence microscope, all of the previous functionality is maintained and modifications to the existing system are kept to a minimum. At the same time, our implementation is able to take full advantage of the speed of the employed sCMOS camera and piezo stage to record data at rates of up to 5 stacks/s. Additionally, sample handling is compatible with established methods and switching magnification to change the field of view from single cells to whole organisms does not require labor intensive adjustments of the system. PMID:29026679

A compact acousto-optic lens for 2D and 3D femtosecond based 2-photon microscopy

PubMed Central

Kirkby, Paul A.; Naga Srinivas, N.K.M.; Silver, R. Angus

2010-01-01

We describe a high speed 3D Acousto-Optic Lens Microscope (AOLM) for femtosecond 2-photon imaging. By optimizing the design of the 4 AO Deflectors (AODs) and by deriving new control algorithms, we have developed a compact spherical AOL with a low temporal dispersion that enables 2-photon imaging at 10-fold lower power than previously reported. We show that the AOLM can perform high speed 2D raster-scan imaging (>150 Hz) without scan rate dependent astigmatism. It can deflect and focus a laser beam in a 3D random access sequence at 30 kHz and has an extended focusing range (>137 μm; 40X 0.8NA objective). These features are likely to make the AOLM a useful tool for studying fast physiological processes distributed in 3D space PMID:20588506

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.

Swept Field Laser Confocal Microscopy for Enhanced Spatial and Temporal Resolution in Live-Cell Imaging

PubMed Central

Castellano-Muñoz, Manuel; Peng, Anthony Wei; Salles, Felipe T.; Ricci, Anthony J.

2013-01-01

Confocal fluorescence microscopy is a broadly used imaging technique that enhances the signal-to-noise ratio by removing out of focal plane fluorescence. Confocal microscopes come with a variety of modifications depending on the particular experimental goals. Microscopes, illumination pathways, and light collection were originally focused upon obtaining the highest resolution image possible, typically on fixed tissue. More recently, live-cell confocal imaging has gained importance. Since measured signals are often rapid or transient, thus requiring higher sampling rates, specializations are included to enhance spatial and temporal resolution while maintaining tissue viability. Thus, a balance between image quality, temporal resolution, and tissue viability is needed. A subtype of confocal imaging, termed swept field confocal (SFC) microscopy, can image live cells at high rates while maintaining confocality. SFC systems can use a pinhole array to obtain high spatial resolution, similar to spinning disc systems. In addition, SFC imaging can achieve faster rates by using a slit to sweep the light across the entire image plane, thus requiring a single scan to generate an image. Coupled to a high-speed charge-coupled device camera and a laser illumination source, images can be obtained at greater than 1,000 frames per second while maintaining confocality. PMID:22831554

Quantifying distortions in two-photon remote focussing microscope images using a volumetric calibration specimen

PubMed Central

Corbett, Alexander D.; Burton, Rebecca A. B.; Bub, Gil; Salter, Patrick S.; Tuohy, Simon; Booth, Martin J.; Wilson, Tony

2014-01-01

Remote focussing microscopy allows sharp, in-focus images to be acquired at high speed from outside of the focal plane of an objective lens without any agitation of the specimen. However, without careful optical alignment, the advantages of remote focussing microscopy could be compromised by the introduction of depth-dependent scaling artifacts. To achieve an ideal alignment in a point-scanning remote focussing microscope, the lateral (XY) scan mirror pair must be imaged onto the back focal plane of both the reference and imaging objectives, in a telecentric arrangement. However, for many commercial objective lenses, it can be difficult to accurately locate the position of the back focal plane. This paper investigates the impact of this limitation on the fidelity of three-dimensional data sets of living cardiac tissue, specifically the introduction of distortions. These distortions limit the accuracy of sarcomere measurements taken directly from raw volumetric data. The origin of the distortion is first identified through simulation of a remote focussing microscope. Using a novel three-dimensional calibration specimen it was then possible to quantify experimentally the size of the distortion as a function of objective misalignment. Finally, by first approximating and then compensating the distortion in imaging data from whole heart rodent studies, the variance of sarcomere length (SL) measurements was reduced by almost 50%. PMID:25339910

High-Speed Fluorescence Microscopy: Lifetime Imaging in the Biomedical Sciences

NASA Astrophysics Data System (ADS)

Periasamy, Ammasi; Wang, Xue F.; Wodnick, Pawel; Gordon, Gerald W.; Kwon, Seongwook; Diliberto, Pamela A.; Herman, Brian

1995-02-01

The ability to observe the behavior of living cells and tissues provides unparalleled access to information regarding the organization and dynamics of complex cellular structures. While great strides have been made over the past 30 to 40 years in the design and application of a variety of novel optical microscopic techniques, until recently, it has not been possible to image biological phenomena that occur over very short time periods (nanosecond to millisecond) or over short distances (10 to 1000 [Angstrom capital A, ring]). However, the recent combination of (1) very rapidly gated and sensitive image intensifiers and (2) the ability to deliver fluorescence excitation energy to intact living biological specimens in a pulsed or sinusoidally modulated fashion has allowed such measurements to become a reality through the imaging of the lifetimes of fluorescent molecules. This capability has resulted in the ability to observe the dynamic organization and interaction of cellular components on a spatial and temporal scale previously not possible using other microscopic techniques. This paper discusses the implementation of a fluorescence lifetime imaging microscope (FLIM) and provides a review of some of the applications of such an instrument. These include measurements of receptor topography and subunit interactions using fluorescence resonance energy transfer (FRET), fluorescence anisotropy of phospholipids in cell membranes, cytosolic free calcium (Ca2+)i and the detection of human papillomavirus (HPV) infection in clinical cervicovaginal smears.

Hyper-spectral imaging in scanning-confocal-fluorescence microscopy using a novel broadband diffractive optic

NASA Astrophysics Data System (ADS)

Wang, Peng; Ebeling, Carl G.; Gerton, Jordan; Menon, Rajesh

In this paper, we demonstrate hyper-spectral imaging of fluorescent microspheres in a scanning-confocal-fluorescence microscope by spatially dispersing the spectra using a novel broadband diffractive optic, and applying a nonlinear optimization technique to extract the full-incident spectra. This broadband diffractive optic has a designed optical efficiency of over 90% across the entire visible spectrum. We used this technique to create two-color images of two fluorophores and also extracted their emission spectra with good fidelity. This method can be extended to image both spatially and spectrally overlapping fluorescent samples. Full control in the number of emission spectra and the feasibility of enhanced imaging speed are demonstrated as well.

Differential high-speed digital micromirror device based fluorescence speckle confocal microscopy.

PubMed

Jiang, Shihong; Walker, John

2010-01-20

We report a differential fluorescence speckle confocal microscope that acquires an image in a fraction of a second by exploiting the very high frame rate of modern digital micromirror devices (DMDs). The DMD projects a sequence of predefined binary speckle patterns to the sample and modulates the intensity of the returning fluorescent light simultaneously. The fluorescent light reflecting from the DMD's "on" and "off" pixels is modulated by correlated speckle and anticorrelated speckle, respectively, to form two images on two CCD cameras in parallel. The sum of the two images recovers a widefield image, but their difference gives a near-confocal image in real time. Experimental results for both low and high numerical apertures are shown.

Simulation study on compressive laminar optical tomography for cardiac action potential propagation

PubMed Central

Harada, Takumi; Tomii, Naoki; Manago, Shota; Kobayashi, Etsuko; Sakuma, Ichiro

2017-01-01

To measure the activity of tissue at the microscopic level, laminar optical tomography (LOT), which is a microscopic form of diffuse optical tomography, has been developed. However, obtaining sufficient recording speed to determine rapidly changing dynamic activity remains major challenges. For a high frame rate of the reconstructed data, we here propose a new LOT method using compressed sensing theory, called compressive laminar optical tomography (CLOT), in which novel digital micromirror device-based illumination and data reduction in a single reconstruction are applied. In the simulation experiments, the reconstructed volumetric images of the action potentials that were acquired from 5 measured images with random pattern featured a wave border at least to a depth of 2.5 mm. Consequently, it was shown that CLOT has potential for over 200 fps required for the cardiac electrophysiological phenomena. PMID:28736675

Femtosecond photography lessons

NASA Astrophysics Data System (ADS)

Fanchenko, S. D.

1999-06-01

Antic scientists, sailors, warriors, physician, etc. were perceiving the space by means of their eye vision system. Nowadays the same people use eyeglasses, telescopes, microscopes, image converters. All these devices fit the necessary magnification, intensification gain and image spectrum to the eyes. The human brain is processing the image data offered to him in a format pertaining to eyes. Hence, the cognition of images can be regarded as a direct measurement. As to the time scale converters, they turned out to be harder done as compared with the spatial scale converters. Hence, the development of the high-speed photography (HSP) continues for more than a hundred and fifty years. The recent pico- femtosecond HSP branch sprang up in 1949 at the Kurchatov Institute -- its cradle. All about the HSP had been advertised. Instead of reprinting what is already well known, it makes sense to emphasize some instructive lessons drawn from past experience. Also it is tempting to look a bit into the high-speed photography future.

Electrochemical wall shear rate microscopy of collapsing bubbles

NASA Astrophysics Data System (ADS)

Reuter, Fabian; Mettin, Robert

2018-06-01

An electrochemical high-speed wall shear raster microscope is presented. It involves chronoamperometric measurements on a microelectrode that is flush-mounted in a submerged test specimen. Wall shear rates are derived from the measured microelectrode signal by numerically solving a convection-diffusion equation with an optimization approach. This way, the unsteady wall shear rates from the collapse of a laser pulse seeded cavitation bubble close to a substrate are measured. By planar scanning, they are resolved in high spatial resolution. The wall shear rates are related to the bubble dynamics via synchronized high-speed imaging of the bubble shape.

A sub-sampled approach to extremely low-dose STEM

DOE PAGES

Stevens, A.; Luzi, L.; Yang, H.; ...

2018-01-22

The inpainting of deliberately and randomly sub-sampled images offers a potential means to image specimens at a high resolution and under extremely low-dose conditions (≤1 e -/Å 2) using a scanning transmission electron microscope. We show that deliberate sub-sampling acquires images at least an order of magnitude faster than conventional low-dose methods for an equivalent electron dose. More importantly, when adaptive sub-sampling is implemented to acquire the images, there is a significant increase in the resolution and sensitivity which accompanies the increase in imaging speed. Lastly, we demonstrate the potential of this method for beam sensitive materials and in-situ observationsmore » by experimentally imaging the node distribution in a metal-organic framework.« less

A sub-sampled approach to extremely low-dose STEM

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

Stevens, A.; Luzi, L.; Yang, H.

The inpainting of deliberately and randomly sub-sampled images offers a potential means to image specimens at a high resolution and under extremely low-dose conditions (≤1 e -/Å 2) using a scanning transmission electron microscope. We show that deliberate sub-sampling acquires images at least an order of magnitude faster than conventional low-dose methods for an equivalent electron dose. More importantly, when adaptive sub-sampling is implemented to acquire the images, there is a significant increase in the resolution and sensitivity which accompanies the increase in imaging speed. Lastly, we demonstrate the potential of this method for beam sensitive materials and in-situ observationsmore » by experimentally imaging the node distribution in a metal-organic framework.« less

Fast processing of microscopic images using object-based extended depth of field.

PubMed

Intarapanich, Apichart; Kaewkamnerd, Saowaluck; Pannarut, Montri; Shaw, Philip J; Tongsima, Sissades

2016-12-22

Microscopic analysis requires that foreground objects of interest, e.g. cells, are in focus. In a typical microscopic specimen, the foreground objects may lie on different depths of field necessitating capture of multiple images taken at different focal planes. The extended depth of field (EDoF) technique is a computational method for merging images from different depths of field into a composite image with all foreground objects in focus. Composite images generated by EDoF can be applied in automated image processing and pattern recognition systems. However, current algorithms for EDoF are computationally intensive and impractical, especially for applications such as medical diagnosis where rapid sample turnaround is important. Since foreground objects typically constitute a minor part of an image, the EDoF technique could be made to work much faster if only foreground regions are processed to make the composite image. We propose a novel algorithm called object-based extended depths of field (OEDoF) to address this issue. The OEDoF algorithm consists of four major modules: 1) color conversion, 2) object region identification, 3) good contrast pixel identification and 4) detail merging. First, the algorithm employs color conversion to enhance contrast followed by identification of foreground pixels. A composite image is constructed using only these foreground pixels, which dramatically reduces the computational time. We used 250 images obtained from 45 specimens of confirmed malaria infections to test our proposed algorithm. The resulting composite images with all in-focus objects were produced using the proposed OEDoF algorithm. We measured the performance of OEDoF in terms of image clarity (quality) and processing time. The features of interest selected by the OEDoF algorithm are comparable in quality with equivalent regions in images processed by the state-of-the-art complex wavelet EDoF algorithm; however, OEDoF required four times less processing time. This work presents a modification of the extended depth of field approach for efficiently enhancing microscopic images. This selective object processing scheme used in OEDoF can significantly reduce the overall processing time while maintaining the clarity of important image features. The empirical results from parasite-infected red cell images revealed that our proposed method efficiently and effectively produced in-focus composite images. With the speed improvement of OEDoF, this proposed algorithm is suitable for processing large numbers of microscope images, e.g., as required for medical diagnosis.

Adaptive AFM scan speed control for high aspect ratio fast structure tracking

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

Ahmad, Ahmad; Schuh, Andreas; Rangelow, Ivo W.

2014-10-15

Improved imaging rates in Atomic Force Microscopes (AFM) are of high interest for disciplines such as life sciences and failure analysis of semiconductor wafers, where the sample topology shows high aspect ratios. Also, fast imaging is necessary to cover a large surface under investigation in reasonable times. Since AFMs are composed of mechanical components, they are associated with comparably low resonance frequencies that undermine the effort to increase the acquisition rates. In particular, high and steep structures are difficult to follow, which causes the cantilever to temporarily loose contact to or crash into the sample. Here, we report on amore » novel approach that does not affect the scanner dynamics, but adapts the lateral scanning speed of the scanner. The controller monitors the control error signal and, only when necessary, decreases the scan speed to allow the z-piezo more time to react to changes in the sample's topography. In this case, the overall imaging rate can be significantly increased, because a general scan speed trade-off decision is not needed and smooth areas are scanned fast. In contrast to methods trying to increase the z-piezo bandwidth, our method is a comparably simple approach that can be easily adapted to standard systems.« less

High-speed and high-SNR photoacoustic microscopy based on a galvanometer mirror in non-conducting liquid

PubMed Central

Kim, Jin Young; Lee, Changho; Park, Kyungjin; Han, Sangyeob; Kim, Chulhong

2016-01-01

Optical-resolution photoacoustic microscopy (OR-PAM), a promising microscopic imaging technique with high ultrasound resolution and superior optical sensitivity, can provide anatomical, functional, and molecular information at scales ranging from the microvasculature to single red blood cells. In particular, real-time OR-PAM imaging with a high signal-to-noise ratio (SNR) is a prerequisite for widespread use in preclinical and clinical applications. Although several technical approaches have been pursued to simultaneously improve the imaging speed and SNR of OR-PAM, they are bulky, complex, not sensitive, and/or not actually real-time. In this paper, we demonstrate a simple and novel OR-PAM technique which is based on a typical galvanometer immersed in non-conducting liquid. Using an opto-ultrasound combiner, this OR-PAM system achieves a high SNR and fast imaging speed. It takes only 2 seconds to acquire a volumetric image with a wide field of view (FOV) of 4 × 8 mm2 along the X and Y axes, respectively. The measured lateral and axial resolutions are 6.0 and 37.7 μm, respectively. Finally, as a demonstration of the system’s capability, we successfully imaged the microvasculature in a mouse ear in vivo. Our new method will contribute substantially to the popularization and commercialization of OR-PAM in various preclinical and clinical applications. PMID:27708379

Mechanical vibration compensation method for 3D+t multi-particle tracking in microscopic volumes.

PubMed

Pimentel, A; Corkidi, G

2009-01-01

The acquisition and analysis of data in microscopic systems with spatiotemporal evolution is a very relevant topic. In this work, we describe a method to optimize an experimental setup for acquiring and processing spatiotemporal (3D+t) data in microscopic systems. The method is applied to a three-dimensional multi-tracking and analysis system of free-swimming sperm trajectories previously developed. The experimental set uses a piezoelectric device making oscillate a large focal-distance objective mounted on an inverted microscope (over its optical axis) to acquire stacks of images at a high frame rate over a depth on the order of 250 microns. A problem arise when the piezoelectric device oscillates, in such a way that a vibration is transmitted to the whole microscope, inducing undesirable 3D vibrations to the whole set. For this reason, as a first step, the biological preparation was isolated from the body of the microscope to avoid modifying the free swimming pattern of the microorganism due to the transmission of these vibrations. Nevertheless, as the image capturing device is mechanically attached to the "vibrating" microscope, the resulting acquired data are contaminated with an undesirable 3D movement that biases the original trajectory of these high speed moving cells. The proposed optimization method determines the functional form of these 3D oscillations to neutralize them from the original acquired data set. Given the spatial scale of the system, the added correction increases significantly the data accuracy. The optimized system may be very useful in a wide variety of 3D+t applications using moving optical devices.

A targeted illumination optical fiber probe for high resolution fluorescence imaging and optical switching

NASA Astrophysics Data System (ADS)

Shinde, Anant; Perinchery, Sandeep Menon; Murukeshan, Vadakke Matham

2017-04-01

An optical imaging probe with targeted multispectral and spatiotemporal illumination features has applications in many diagnostic biomedical studies. However, these systems are mostly adapted in conventional microscopes, limiting their use for in vitro applications. We present a variable resolution imaging probe using a digital micromirror device (DMD) with an achievable maximum lateral resolution of 2.7 μm and an axial resolution of 5.5 μm, along with precise shape selective targeted illumination ability. We have demonstrated switching of different wavelengths to image multiple regions in the field of view. Moreover, the targeted illumination feature allows enhanced image contrast by time averaged imaging of selected regions with different optical exposure. The region specific multidirectional scanning feature of this probe has facilitated high speed targeted confocal imaging.

Time-gated FLIM microscope for corneal metabolic imaging

NASA Astrophysics Data System (ADS)

Silva, Susana F.; Batista, Ana; Domingues, José Paulo; Quadrado, Maria João.; Morgado, António Miguel

2016-03-01

Detecting corneal cells metabolic alterations may prove a valuable tool in the early diagnosis of corneal diseases. Nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) are autofluorescent metabolic co-factors that allow the assessment of metabolic changes through non-invasive optical methods. These co-factors exhibit double-exponential fluorescence decays, with well-separated short and lifetime components, which are related to their protein-bound and free-states. Corneal metabolism can be assessed by measuring the relative contributions of these two components. For that purpose, we have developed a wide-field time-gated fluorescence lifetime microscope based on structured illumination and one-photon excitation to record FAD lifetime images from corneas. NADH imaging was not considered as its UV excitation peak is regarded as not safe for in vivo measurements. The microscope relies on a pulsed blue diode laser (λ=443 nm) as excitation source, an ultra-high speed gated image intensifier coupled to a CCD camera to acquire fluorescence signals and a Digital Micromirror Device (DMD) to implement the Structured Illumination technique. The system has a lateral resolution better than 2.4 μm, a field of view of 160 per 120 μm and an optical sectioning of 6.91 +/- 0.45 μm when used with a 40x, 0.75 NA, Water Immersion Objective. With this setup we were able to measure FAD contributions from ex-vivo chicken corneas collected from a local slaughterhouse..

Micro-Mirrors for Nanoscale Three-Dimensional Microscopy

PubMed Central

Seale, Kevin; Janetopoulos, Chris; Wikswo, John

2013-01-01

A research-grade optical microscope is capable of resolving fine structures in two-dimensional images. However, three-dimensional resolution, or the ability of the microscope to distinguish between objects lying above or below the focal plane from in-focus objects, is not nearly as good as in-plane resolution. In this issue of ACS Nano, McMahon et al. report the use of mirrored pyramidal wells with a conventional microscope for rapid, 3D localization and tracking of nanoparticles. Mirrors have been used in microscopy before, but recent work with MPWs is unique because it enables the rapid determination of the x-, y-, and z-position of freely diffusing nanoparticles and cellular nanostructures with unprecedented speed and spatial accuracy. As inexpensive tools for 3D visualization, mirrored pyramidal wells may prove to be invaluable aids in nanotechnology and engineering of nanomaterials. PMID:19309167

High-speed atomic force microscopy and peak force tapping control

NASA Astrophysics Data System (ADS)

Hu, Shuiqing; Mininni, Lars; Hu, Yan; Erina, Natalia; Kindt, Johannes; Su, Chanmin

2012-03-01

ITRS Roadmap requires defect size measurement below 10 nanometers and challenging classifications for both blank and patterned wafers and masks. Atomic force microscope (AFM) is capable of providing metrology measurement in 3D at sub-nanometer accuracy but has long suffered from drawbacks in throughput and limitation of slow topography imaging without chemical information. This presentation focus on two disruptive technology developments, namely high speed AFM and quantitative nanomechanical mapping, which enables high throughput measurement with capability of identifying components through concurrent physical property imaging. The high speed AFM technology has allowed the imaging speed increase by 10-100 times without loss of the data quality. Such improvement enables the speed of defect review on a wafer to increase from a few defects per hour to nearly 100 defects an hour, approaching the requirements of ITRS Roadmap. Another technology development, Peak Force Tapping, substantially simplified the close loop system response, leading to self-optimization of most challenging samples groups to generate expert quality data. More importantly, AFM also simultaneously provides a series of mechanical property maps with a nanometer spatial resolution during defect review. These nanomechanical maps (including elastic modulus, hardness, and surface adhesion) provide complementary information for elemental analysis, differentiate defect materials by their physical properties, and assist defect classification beyond topographic measurements. This paper will explain the key enabling technologies, namely high speed tip-scanning AFM using innovative flexure design and control algorithm. Another critical element is AFM control using Peak Force Tapping, in which the instantaneous tip-sample interaction force is measured and used to derive a full suite of physical properties at each imaging pixel. We will provide examples of defect review data on different wafers and media disks. The similar AFM-based defect review capacity was also applied to EUV masks.

COMPACT NON-CONTACT TOTAL EMISSION DETECTION FOR IN-VIVO MULTI-PHOTON EXCITATION MICROSCOPY

PubMed Central

Glancy, Brian; Karamzadeh, Nader S.; Gandjbakhche, Amir H.; Redford, Glen; Kilborn, Karl; Knutson, Jay R.; Balaban, Robert S.

2014-01-01

Summary We describe a compact, non-contact design for a Total Emission Detection (c-TED) system for intra-vital multi-photon imaging. To conform to a standard upright two-photon microscope design, this system uses a parabolic mirror surrounding a standard microscope objective in concert with an optical path that does not interfere with normal microscope operation. The non-contact design of this device allows for maximal light collection without disrupting the physiology of the specimen being examined. Tests were conducted on exposed tissues in live animals to examine the emission collection enhancement of the c-TED device compared to heavily optimized objective-based emission collection. The best light collection enhancement was seen from murine fat (5×-2× gains as a function of depth), while murine skeletal muscle and rat kidney showed gains of over two and just under two-fold near the surface, respectively. Gains decreased with imaging depth (particularly in the kidney). Zebrafish imaging on a reflective substrate showed close to a two-fold gain throughout the entire volume of an intact embryo (approximately 150 μm deep). Direct measurement of bleaching rates confirmed that the lower laser powers (enabled by greater light collection efficiency) yielded reduced photobleaching in vivo. The potential benefits of increased light collection in terms of speed of imaging and reduced photo-damage, as well as the applicability of this device to other multi-photon imaging methods is discussed. PMID:24251437

Large scale superres 3D imaging: light-sheet single-molecule localization microscopy (Conference Presentation)

NASA Astrophysics Data System (ADS)

Lu, Chieh Han; Chen, Peilin; Chen, Bi-Chang

2017-02-01

Optical imaging techniques provide much important information in understanding life science especially cellular structure and morphology because "seeing is believing". However, the resolution of optical imaging is limited by the diffraction limit, which is discovered by Ernst Abbe, i.e. λ/2(NA) (NA is the numerical aperture of the objective lens). Fluorescence super-resolution microscopic techniques such as Stimulated emission depletion microscopy (STED), Photoactivated localization microscopy (PALM), and Stochastic optical reconstruction microscopy (STORM) are invented to have the capability of seeing biological entities down to molecular level that are smaller than the diffraction limit (around 200-nm in lateral resolution). These techniques do not physically violate the Abbe limit of resolution but exploit the photoluminescence properties and labelling specificity of fluorescence molecules to achieve super-resolution imaging. However, these super-resolution techniques limit most of their applications to the 2D imaging of fixed or dead samples due to the high laser power needed or slow speed for the localization process. Extended from 2D imaging, light sheet microscopy has been proven to have a lot of applications on 3D imaging at much better spatiotemporal resolutions due to its intrinsic optical sectioning and high imaging speed. Herein, we combine the advantage of localization microscopy and light-sheet microscopy to have super-resolved cellular imaging in 3D across large field of view. With high-density labeled spontaneous blinking fluorophore and wide-field detection of light-sheet microscopy, these allow us to construct 3D super-resolution multi-cellular imaging at high speed ( minutes) by light-sheet single-molecule localization microscopy.

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

Extreme ultraviolet patterned mask inspection performance of advanced projection electron microscope system for 11nm half-pitch generation

NASA Astrophysics Data System (ADS)

Hirano, Ryoichi; Iida, Susumu; Amano, Tsuyoshi; Watanabe, Hidehiro; Hatakeyama, Masahiro; Murakami, Takeshi; Suematsu, Kenichi; Terao, Kenji

2016-03-01

Novel projection electron microscope optics have been developed and integrated into a new inspection system named EBEYE-V30 ("Model EBEYE" is an EBARA's model code) , and the resulting system shows promise for application to half-pitch (hp) 16-nm node extreme ultraviolet lithography (EUVL) patterned mask inspection. To improve the system's inspection throughput for 11-nm hp generation defect detection, a new electron-sensitive area image sensor with a high-speed data processing unit, a bright and stable electron source, and an image capture area deflector that operates simultaneously with the mask scanning motion have been developed. A learning system has been used for the mask inspection tool to meet the requirements of hp 11-nm node EUV patterned mask inspection. Defects are identified by the projection electron microscope system using the "defectivity" from the characteristics of the acquired image. The learning system has been developed to reduce the labor and costs associated with adjustment of the detection capability to cope with newly-defined mask defects. We describe the integration of the developed elements into the inspection tool and the verification of the designed specification. We have also verified the effectiveness of the learning system, which shows enhanced detection capability for the hp 11-nm node.

High-speed, random-access fluorescence microscopy: I. High-resolution optical recording with voltage-sensitive dyes and ion indicators.

PubMed Central

Bullen, A; Patel, S S; Saggau, P

1997-01-01

The design and implementation of a high-speed, random-access, laser-scanning fluorescence microscope configured to record fast physiological signals from small neuronal structures with high spatiotemporal resolution is presented. The laser-scanning capability of this nonimaging microscope is provided by two orthogonal acousto-optic deflectors under computer control. Each scanning point can be randomly accessed and has a positioning time of 3-5 microseconds. Sampling time is also computer-controlled and can be varied to maximize the signal-to-noise ratio. Acquisition rates up to 200k samples/s at 16-bit digitizing resolution are possible. The spatial resolution of this instrument is determined by the minimal spot size at the level of the preparation (i.e., 2-7 microns). Scanning points are selected interactively from a reference image collected with differential interference contrast optics and a video camera. Frame rates up to 5 kHz are easily attainable. Intrinsic variations in laser light intensity and scanning spot brightness are overcome by an on-line signal-processing scheme. Representative records obtained with this instrument by using voltage-sensitive dyes and calcium indicators demonstrate the ability to make fast, high-fidelity measurements of membrane potential and intracellular calcium at high spatial resolution (2 microns) without any temporal averaging. Images FIGURE 6 PMID:9199810

Laser scanning stereomicroscopy for fast volumetric imaging with two-photon excitation and scanned Bessel beams

NASA Astrophysics Data System (ADS)

Yang, Yanlong; Zhou, Xing; Li, Runze; Van Horn, Mark; Peng, Tong; Lei, Ming; Wu, Di; Chen, Xun; Yao, Baoli; Ye, Tong

2015-03-01

Bessel beams have been used in many applications due to their unique optical properties of maintaining their intensity profiles unchanged during propagation. In imaging applications, Bessel beams have been successfully used to provide extended focuses for volumetric imaging and uniformed illumination plane in light-sheet microscopy. Coupled with two-photon excitation, Bessel beams have been successfully used in realizing fluorescence projected volumetric imaging. We demonstrated previously a stereoscopic solution-two-photon fluorescence stereomicroscopy (TPFSM)-for recovering the depth information in volumetric imaging with Bessel beams. In TPFSM, tilted Bessel beams were used to generate stereoscopic images on a laser scanning two-photon fluorescence microscope; upon post image processing we could successfully provide 3D perception of acquired volume images by wearing anaglyph 3D glasses. However, tilted Bessel beams were generated by shifting either an axicon or an objective laterally; the slow imaging speed and severe aberrations made it hard to use in real-time volume imaging. In this article, we report recent improvements of TPFSM with newly designed scanner and imaging software, which allows 3D stereoscopic imaging without moving any of the optical components on the setup. This improvement has dramatically improved focusing qualities and imaging speed so that the TPFSM can be performed potentially in real-time to provide 3D visualization in scattering media without post image processing.

A densitometric analysis of commercial 35mm films

NASA Technical Reports Server (NTRS)

Hammond, Ernest C., Jr.; Ruffin, Christopher, III

1989-01-01

IIaO films have been subjected to various sensitometric tests. The have included thermal and aging effects and reciprocity failure studies. In order to compare the special IIaO film with popular brands of 35 mm films and their possible use in astrophotography, Agfa, Fuji and Kodak print and slide formats, as well as black and white and color formats, were subjected to sensitometric, as well as densitometric analysis. A scanning electron microscope was used to analyze grain structure size, and shape as a function of both speed and brand. Preliminary analysis of the grain structure using an ISI-SS40 scanning electron microscope indicates that the grain sizes for darker densities are much larger than the grain size for lighter densities. Researchers analyze the scanning electron microscope findings of the various grains versus densities as well as enhancement of the grains, using the IP-8500 Digital Image Processor.

In Situ and In Vivo Molecular Analysis by Coherent Raman Scattering Microscopy

PubMed Central

Liao, Chien-Sheng; Cheng, Ji-Xin

2017-01-01

Coherent Raman scattering (CRS) microscopy is a high-speed vibrational imaging platform with the ability to visualize the chemical content of a living specimen by using molecular vibrational fingerprints. We review technical advances and biological applications of CRS microscopy. The basic theory of CRS and the state-of-the-art instrumentation of a CRS microscope are presented. We further summarize and compare the algorithms that are used to separate the Raman signal from the nonresonant background, to denoise a CRS image, and to decompose a hyperspectral CRS image into concentration maps of principal components. Important applications of single-frequency and hyperspectral CRS microscopy are highlighted. Potential directions of CRS microscopy are discussed. PMID:27306307

Portable fiber-optic taper coupled optical microscopy platform

NASA Astrophysics Data System (ADS)

Wang, Weiming; Yu, Yan; Huang, Hui; Ou, Jinping

2017-04-01

The optical fiber taper coupled with CMOS has advantages of high sensitivity, compact structure and low distortion in the imaging platform. So it is widely used in low light, high speed and X-ray imaging systems. In the meanwhile, the peculiarity of the coupled structure can meet the needs of the demand in microscopy imaging. Toward this end, we developed a microscopic imaging platform based on the coupling of cellphone camera module and fiber optic taper for the measurement of the human blood samples and ascaris lumbricoides. The platform, weighing 70 grams, is based on the existing camera module of the smartphone and a fiber-optic array which providing a magnification factor of 6x.The top facet of the taper, on which samples are placed, serves as an irregular sampling grid for contact imaging. The magnified images of the sample, located on the bottom facet of the fiber, are then projected onto the CMOS sensor. This paper introduces the portable medical imaging system based on the optical fiber coupling with CMOS, and theoretically analyzes the feasibility of the system. The image data and process results either can be stored on the memory or transmitted to the remote medical institutions for the telemedicine. We validate the performance of this cell-phone based microscopy platform using human blood samples and test target, achieving comparable results to a standard bench-top microscope.

Remote focusing in confocal microscopy by means of a modified Alvarez lens.

PubMed

Bawart, M; Jesacher, A; Bernet, S; Ritsch-Marte, M

2018-06-22

Alvarez lenses are actuated lens-pairs which allow one to tune the optical power by mechanical displacement of subelements. Here, we show that a recently realized modified Alvarez lens design which does not require mechanical actuation can be integrated into a confocal microscope. Instead of mechanically moving them, the sublenses are imaged onto each other in a 4f-configuration, where the lateral image shift leading to a change in optical power is created by a galvo-mirror. The avoidance of mechanical lens shifts leads to a large speed gain for axial (and hence also 3D) image scans compared to classical Alvarez lenses. We demonstrate that the suggested operation principle is compatible with confocal microscopy. In order to optimize the system, we have drawn advantage of the flexibility a liquid-crystal spatial light modulator offers for the implementation. For given specifications, dedicated diffractive optical elements or freeform elements can be used in combination with resonant galvo-scanners or acousto-optic beam deflectors, to achieve even faster z-scans than reported here, reaching video rate. © 2018 The Authors Journal of Microscopy © 2018 Royal Microscopical Society.

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.

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

Live Cell Imaging and 3D Analysis of Angiotensin Receptor Type 1a Trafficking in Transfected Human Embryonic Kidney Cells Using Confocal Microscopy.

PubMed

Kadam, Parnika; McAllister, Ryan; Urbach, Jeffrey S; Sandberg, Kathryn; Mueller, Susette C

2017-03-27

Live-cell imaging is used to simultaneously capture time-lapse images of angiotensin type 1a receptors (AT1aR) and intracellular compartments in transfected human embryonic kidney-293 (HEK) cells following stimulation with angiotensin II (Ang II). HEK cells are transiently transfected with plasmid DNA containing AT1aR tagged with enhanced green fluorescent protein (EGFP). Lysosomes are identified with a red fluorescent dye. Live-cell images are captured on a laser scanning confocal microscope after Ang II stimulation and analyzed by software in three dimensions (3D, voxels) over time. Live-cell imaging enables investigations into receptor trafficking and avoids confounds associated with fixation, and in particular, the loss or artefactual displacement of EGFP-tagged membrane receptors. Thus, as individual cells are tracked through time, the subcellular localization of receptors can be imaged and measured. Images must be acquired sufficiently rapidly to capture rapid vesicle movement. Yet, at faster imaging speeds, the number of photons collected is reduced. Compromises must also be made in the selection of imaging parameters like voxel size in order to gain imaging speed. Significant applications of live-cell imaging are to study protein trafficking, migration, proliferation, cell cycle, apoptosis, autophagy and protein-protein interaction and dynamics, to name but a few.

Manual stage acquisition and interactive display of digital slides in histopathology.

PubMed

Gherardi, Alessandro; Bevilacqua, Alessandro

2014-07-01

More powerful PC architectures, high-resolution cameras working at increasing frame rates, and more and more accurate motorized microscopes have boosted new applications in the field of biomedicine and medical imaging. In histopathology, the use of digital slides (DSs) imaging through dedicated hardware for digital pathology is increasing for several reasons: digital annotation of suspicious lesions, recorded clinical history, and telepathology as a collaborative environment. In this paper, we propose the first method known in the literature for real-time whole slide acquisition and displaying conceived for conventional nonautomated microscopes. Differently from DS scanner, our software enables biologists and histopathologists to build and view the DS in real time while inspecting the sample, as they are accustomed to. In addition, since our approach is compliant with existing common microscope positions, provided with camera and PC, this could contribute to disseminate the whole slide technology in the majority of small labs not endowed with DS hardware facilities. Experiments performed with different histologic specimens (referring to tumor tissues of different body parts as well as to tumor cells), acquired under different setup conditions and devices, prove the effectiveness of our approach both in terms of quality and speed performances.

Raman imaging of molecular dynamics during cellular events

NASA Astrophysics Data System (ADS)

Fujita, Katsumasa

2017-07-01

To overcome the speed limitation in Raman imaging, we have developed a microscope system that detects Raman spectra from hundreds of points in a sample simultaneously. The sample was illuminated by a line-shaped focus, and Raman scattering from the illuminated positions was measured simultaneously by an imaging spectrophotometer. We applied the line-illumination technique to observe the dynamics of intracellular molecules during cellular events. We found that intracellular cytochrome c can be clearly imaged by resonant Raman scattering. We demonstrated label-free imaging of redistribution of cytochrome c during apoptosis and osteoblastic mineralization. We also proposed alkyne-tagged Raman imaging to observe small molecules in living cells. Due to its small size and the unique Raman band, alkyne can tag molecules without strong perturbation to molecular functions and with the capability to be detected separately from endogenous molecules.

Fast Confocal Raman Imaging Using a 2-D Multifocal Array for Parallel Hyperspectral Detection.

PubMed

Kong, Lingbo; Navas-Moreno, Maria; Chan, James W

2016-01-19

We present the development of a novel confocal hyperspectral Raman microscope capable of imaging at speeds up to 100 times faster than conventional point-scan Raman microscopy under high noise conditions. The microscope utilizes scanning galvomirrors to generate a two-dimensional (2-D) multifocal array at the sample plane, generating Raman signals simultaneously at each focus of the array pattern. The signals are combined into a single beam and delivered through a confocal pinhole before being focused through the slit of a spectrometer. To separate the signals from each row of the array, a synchronized scan mirror placed in front of the spectrometer slit positions the Raman signals onto different pixel rows of the detector. We devised an approach to deconvolve the superimposed signals and retrieve the individual spectra at each focal position within a given row. The galvomirrors were programmed to scan different focal arrays following Hadamard encoding patterns. A key feature of the Hadamard detection is the reconstruction of individual spectra with improved signal-to-noise ratio. Using polystyrene beads as test samples, we demonstrated not only that our system images faster than a conventional point-scan method but that it is especially advantageous under noisy conditions, such as when the CCD detector operates at fast read-out rates and high temperatures. This is the first demonstration of multifocal confocal Raman imaging in which parallel spectral detection is implemented along both axes of the CCD detector chip. We envision this novel 2-D multifocal spectral detection technique can be used to develop faster imaging spontaneous Raman microscopes with lower cost detectors.

Comparison study of five different display modalities for whole slide images in surgical pathology and cytopathology in Europe

NASA Astrophysics Data System (ADS)

D'Haene, Nicky; Maris, Calliope; Rorive, Sandrine; Moles Lopez, Xavier; Rostang, Johan; Marchessoux, Cédric; Pantanowitz, Liron; Parwani, Anil V.; Salmon, Isabelle

2013-03-01

User experience with viewing images in pathology is crucial for accurate interpretation and diagnosis. With digital pathology, images are being read on a display system, and this poses new types of questions: such as what is the difference in terms of pixelation, refresh lag or obscured features compared to an optical microscope. Is there a resultant change in user performance in terms of speed of slide review, perception of adequacy and quality or in diagnostic confidence? A prior psychophysical study was carried out comparing various display modalities on whole slide imaging (WSI) in pathology at the University of Pittsburgh Medical Center (UPMC) in the USA. This prior study compared professional and non-professional grade display modalities and highlighted the importance of using a medical grade display to view pathological digital images. This study was duplicated in Europe at the Department of Pathology in Erasme Hospital (Université Libre de Bruxelles (ULB)) in an attempt to corroborate these findings. Digital WSI with corresponding glass slides of 58 cases including surgical pathology and cytopathology slides of varying difficulty were employed. Similar non-professional and professional grade display modalities were compared to an optical microscope (Olympus BX51). Displays ranged from a laptop (DELL Latitude D620), to a consumer grade display (DELL E248WFPb), to two professional grade monitors (Eizo CG245W and Barco MDCC-6130). Three pathologists were selected from the Department of Pathology in Erasme Hospital (ULB) in Belgium to view and interpret the pathological images on these different displays. The results show that non-professional grade displays (laptop and consumer) have inferior user experience compared to professional grade monitors and the optical microscope.

Full field vertical scanning in short coherence digital holographic microscope.

PubMed

Monemahghdoust, Zahra; Montfort, Frederic; Cuche, Etienne; Emery, Yves; Depeursinge, Christian; Moser, Christophe

2013-05-20

In Digital holography Microscopes (DHM) implemented in the so-called "off axis" configuration, the object and reference wave fronts are not co-planar but form an angle of a few degrees. This results into two main drawbacks. First, the contrast of the interference is not uniform spatially when the light source has low coherence. The interference contrast is optimal along a line, but decreases when moving away from it, resulting in a lower image quality. Second, the non-coplanarity between the coherence plane of both wavefronts impacts the coherence vertical scanning measurement mode: when the optical path difference between the signal and the reference beam is changed, the region of maximum interference contrast shifts laterally in the plane of the objective. This results in more complex calculations to extract the topography of the sample and requires scanning over a much larger vertical range, leading to a longer measurement time. We have previously shown that by placing a volume diffractive optical element (VDOE) in the reference arm, the wavefront can be made coplanar with the object wavefront and the image plane of the microscope objective, resulting in a uniform and optimal interferogram. In this paper, we demonstrate a vertical scanning speed improvement by an order of magnitude. Noise in the phase and intensity images caused by scattering and non-uniform diffraction in the VDOE is analyzed quantitatively. Five VDOEs were fabricated with an identical procedure. We observe that VDOEs introduce a small intensity non-uniformity in the reference beam which results in a 20% noise increase in the extracted phase image as compared to the noise in extracted phase image when the VDOE is removed. However, the VDOE has no impact on the temporal noise measured from extracted phase images.

Applications and challenges of digital pathology and whole slide imaging.

PubMed

Higgins, C

2015-07-01

Virtual microscopy is a method for digitizing images of tissue on glass slides and using a computer to view, navigate, change magnification, focus and mark areas of interest. Virtual microscope systems (also called digital pathology or whole slide imaging systems) offer several advantages for biological scientists who use slides as part of their general, pharmaceutical, biotechnology or clinical research. The systems usually are based on one of two methodologies: area scanning or line scanning. Virtual microscope systems enable automatic sample detection, virtual-Z acquisition and creation of focal maps. Virtual slides are layered with multiple resolutions at each location, including the highest resolution needed to allow more detailed review of specific regions of interest. Scans may be acquired at 2, 10, 20, 40, 60 and 100 × or a combination of magnifications to highlight important detail. Digital microscopy starts when a slide collection is put into an automated or manual scanning system. The original slides are archived, then a server allows users to review multilayer digital images of the captured slides either by a closed network or by the internet. One challenge for adopting the technology is the lack of a universally accepted file format for virtual slides. Additional challenges include maintaining focus in an uneven sample, detecting specimens accurately, maximizing color fidelity with optimal brightness and contrast, optimizing resolution and keeping the images artifact-free. There are several manufacturers in the field and each has not only its own approach to these issues, but also its own image analysis software, which provides many options for users to enhance the speed, quality and accuracy of their process through virtual microscopy. Virtual microscope systems are widely used and are trusted to provide high quality solutions for teleconsultation, education, quality control, archiving, veterinary medicine, research and other fields.

Multi-color phase imaging and sickle cell anemia (Conference Presentation)

NASA Astrophysics Data System (ADS)

Hosseini, Poorya; Zhou, Renjie; Yaqoob, Zahid; So, Peter T. C.

2016-03-01

Quantitative phase measurements at multiple wavelengths has created an opportunity for exploring new avenues in phase microscopy such as enhancing imaging-depth (1), measuring hemoglobin concentrations in erythrocytes (2), and more recently in tomographic mapping of the refractive index of live cells (3). To this end, quantitative phase imaging has been demonstrated both at few selected spectral points as well as with high spectral resolution (4,5). However, most of these developed techniques compromise imaging speed, field of view, or the spectral resolution to perform interferometric measurements at multiple colors. In the specific application of quantitative phase in studying blood diseases and red blood cells, current techniques lack the required sensitivity to quantify biological properties of interest at individual cell level. Recently, we have set out to develop a stable quantitative interferometric microscope allowing for measurements of such properties for red cells without compromising field of view or speed of the measurements. The feasibility of the approach will be initially demonstrated in measuring dispersion curves of known solutions, followed by measuring biological properties of red cells in sickle cell anemia. References: 1. Mann CJ, Bingham PR, Paquit VC, Tobin KW. Quantitative phase imaging by three-wavelength digital holography. Opt Express. 2008;16(13):9753-64. 2. Park Y, Yamauchi T, Choi W, Dasari R, Feld MS. Spectroscopic phase microscopy for quantifying hemoglobin concentrations in intact red blood cells. Opt Lett. 2009;34(23):3668-70. 3. Hosseini P, Sung Y, Choi Y, Lue N, Yaqoob Z, So P. Scanning color optical tomography (SCOT). Opt Express. 2015;23(15):19752-62. 4. Jung J-H, Jang J, Park Y. Spectro-refractometry of individual microscopic objects using swept-source quantitative phase imaging. Anal Chem. 2013;85(21):10519-25. 5. Rinehart M, Zhu Y, Wax A. Quantitative phase spectroscopy. Biomed Opt Express. 2012;3(5):958-65.

Dissolution Processes at Step Edges of Calcite in Water Investigated by High-Speed Frequency Modulation Atomic Force Microscopy and Simulation.

PubMed

Miyata, Kazuki; Tracey, John; Miyazawa, Keisuke; Haapasilta, Ville; Spijker, Peter; Kawagoe, Yuta; Foster, Adam S; Tsukamoto, Katsuo; Fukuma, Takeshi

2017-07-12

The microscopic understanding of the crystal growth and dissolution processes have been greatly advanced by the direct imaging of nanoscale step flows by atomic force microscopy (AFM), optical interferometry, and X-ray microscopy. However, one of the most fundamental events that govern their kinetics, namely, atomistic events at the step edges, have not been well understood. In this study, we have developed high-speed frequency modulation AFM (FM-AFM) and enabled true atomic-resolution imaging in liquid at ∼1 s/frame, which is ∼50 times faster than the conventional FM-AFM. With the developed AFM, we have directly imaged subnanometer-scale surface structures around the moving step edges of calcite during its dissolution in water. The obtained images reveal that the transition region with typical width of a few nanometers is formed along the step edges. Building upon insight in previous studies, our simulations suggest that the transition region is most likely to be a Ca(OH) 2 monolayer formed as an intermediate state in the dissolution process. On the basis of this finding, we improve our understanding of the atomistic dissolution model of calcite in water. These results open up a wide range of future applications of the high-speed FM-AFM to the studies on various dynamic processes at solid-liquid interfaces with true atomic resolution.

A statistically harmonized alignment-classification in image space enables accurate and robust alignment of noisy images in single particle analysis.

PubMed

Kawata, Masaaki; Sato, Chikara

2007-06-01

In determining the three-dimensional (3D) structure of macromolecular assemblies in single particle analysis, a large representative dataset of two-dimensional (2D) average images from huge number of raw images is a key for high resolution. Because alignments prior to averaging are computationally intensive, currently available multireference alignment (MRA) software does not survey every possible alignment. This leads to misaligned images, creating blurred averages and reducing the quality of the final 3D reconstruction. We present a new method, in which multireference alignment is harmonized with classification (multireference multiple alignment: MRMA). This method enables a statistical comparison of multiple alignment peaks, reflecting the similarities between each raw image and a set of reference images. Among the selected alignment candidates for each raw image, misaligned images are statistically excluded, based on the principle that aligned raw images of similar projections have a dense distribution around the correctly aligned coordinates in image space. This newly developed method was examined for accuracy and speed using model image sets with various signal-to-noise ratios, and with electron microscope images of the Transient Receptor Potential C3 and the sodium channel. In every data set, the newly developed method outperformed conventional methods in robustness against noise and in speed, creating 2D average images of higher quality. This statistically harmonized alignment-classification combination should greatly improve the quality of single particle analysis.

Lattice dynamics in elemental modulated Sb 2 Te 3 films: Lattice dynamics in elemental modulated Sb 2 Te 3 films

DOE PAGES

Bessas, D.; Winkler, M.; Sergueev, I.; ...

2015-09-03

We investigate the crystallinity and the lattice dynamics in elemental modulated Sbinline imageTeinline image films microscopically using high energy synchrotron radiation diffraction combined with inline imageSb nuclear inelastic scattering. The correlation length is found to be finite but less than 100 . Moreover, the element specific density of phonon states is extracted. A comparison with the element specific density of phonon states in bulk Sbinline imageTeinline image confirms that the main features in the density of phonon states arise from the layered structure. The average speed of sound at inline image inline image, is almost the same compared to bulkmore » Sbinline imageTeinline image at inline image, inline image. Similarly, the change in the acoustic cut-off energy is within the experimental detection limit. Therefore, we suggest that the lattice thermal conductivity in elemental modulated Sbinline imageTeinline image films should not be significantly changed from its bulk value.« less

Digital holographic microscopy applied to measurement of a flow in a T-shaped micromixer

NASA Astrophysics Data System (ADS)

Ooms, T. A.; Lindken, R.; Westerweel, J.

2009-12-01

In this paper, we describe measurements of a three-dimensional (3D) flow in a T-shaped micromixer by means of digital holographic microscopy. Imaging tracer particles in a microscopic flow with conventional microscopy is accompanied by a small depth-of-field, which hinders true volumetric flow measurements. In holographic microscopy, the depth of the measurement domain does not have this limitation because any desired image plane can be reconstructed after recording. Our digital holographic microscope (DHM) consists of a conventional in-line recording system with an added magnifying optical element. The measured flow velocity and the calculated vorticity illustrate four streamwise vortices in the micromixer outflow channel. Because the investigated flow is stationary and strongly 3D, the DHM performance (i.e. accuracy and resolution) can be precisely investigated. The obtained Dynamic spatial range and Dynamic velocity range are larger than 20 and 30, respectively. High-speed multiple-frame measurements illustrate the capability to simultaneously track about 80 particles in a volumetric measurement domain.

Fast volumetric imaging with patterned illumination via digital micro-mirror device-based temporal focusing multiphoton microscopy.

PubMed

Chang, Chia-Yuan; Hu, Yvonne Yuling; Lin, Chun-Yu; Lin, Cheng-Han; Chang, Hsin-Yu; Tsai, Sheng-Feng; Lin, Tzu-Wei; Chen, Shean-Jen

2016-05-01

Temporal focusing multiphoton microscopy (TFMPM) has the advantage of area excitation in an axial confinement of only a few microns; hence, it can offer fast three-dimensional (3D) multiphoton imaging. Herein, fast volumetric imaging via a developed digital micromirror device (DMD)-based TFMPM has been realized through the synchronization of an electron multiplying charge-coupled device (EMCCD) with a dynamic piezoelectric stage for axial scanning. The volumetric imaging rate can achieve 30 volumes per second according to the EMCCD frame rate of more than 400 frames per second, which allows for the 3D Brownian motion of one-micron fluorescent beads to be spatially observed. Furthermore, it is demonstrated that the dynamic HiLo structural multiphoton microscope can reject background noise by way of the fast volumetric imaging with high-speed DMD patterned illumination.

Assessment of incomplete clipping of aneurysms intraoperatively by a near-infrared indocyanine green-video angiography (Niicg-Va) integrated microscope.

PubMed

Imizu, S; Kato, Y; Sangli, A; Oguri, D; Sano, H

2008-08-01

The objective of this article was to assess the clinical use and the completeness of clipping with total occlusion of the aneurysmal lumen, real-time assessment of vascular patency in the parent, branching and perforating vessels, intraoperative assessment of blood flow, image quality, spatial resolution and clinical value in difficult aneurysms using near infrared indocyanine green video angiography integrated on to an operative Pentero neurosurgical microscope (Carl Zeiss, Oberkochen Germany). Thirteen patients with aneurysms were operated upon. An infrared camera with near infrared technology was adapted on to the OPMI Pentero microscope with a special filter and infrared excitation light to illuminate the operating field which was designed to allow passage of the near infrared light required for excitation of indocyanine green (ICG) which was used as the intravascular marker. The intravascular fluorescence was imaged with a video camera attached to the microscope. ICG fluorescence (700-850 nm) from a modified microscope light source on to the surgical field and passage of ICG fluorescence (780-950 nm) from the surgical field, back into the optical path of the microscope was used to detect the completeness of aneurysmal clipping Incomplete clipping in three patients (1 female and 2 males) with unruptured complicated aneurysms was detected using indocyanine green video angiography. There were no adverse effects after injection of indocyanine green. The completeness of clipping was inadequately detected by Doppler ultrasound miniprobe and rigid endoscopy and was thus complemented by indocyanine green video angiography. The operative microscope-integrated ICG video angiography as a new intraoperative method for detecting vascular flow, was found to be quick, reliable, cost-effective and possibly a substitute or adjunct for Doppler ultrasonography or intraoperative DSA, which is presently the gold standard. The simplicity of the method, the speed with which the investigation can be performed, the quality of the images, and the outcome of surgical procedures have all reduced the need for angiography. This technique may be useful during routine aneurysm surgery as an independent form of angiography and/or as an adjunct to intraoperative or postoperative DSA.

Light-sheet microscopy for everyone? Experience of building an OpenSPIM to study flatworm development.

PubMed

Girstmair, Johannes; Zakrzewski, Anne; Lapraz, François; Handberg-Thorsager, Mette; Tomancak, Pavel; Pitrone, Peter Gabriel; Simpson, Fraser; Telford, Maximilian J

2016-06-30

Selective plane illumination microscopy (SPIM a type of light-sheet microscopy) involves focusing a thin sheet of laser light through a specimen at right angles to the objective lens. As only the thin section of the specimen at the focal plane of the lens is illuminated, out of focus light is naturally absent and toxicity due to light (phototoxicity) is greatly reduced enabling longer term live imaging. OpenSPIM is an open access platform (Pitrone et al. 2013 and OpenSPIM.org) created to give new users step-by-step instructions on building a basic configuration of a SPIM microscope, which can in principle be adapted and upgraded to each laboratory's own requirements and budget. Here we describe our own experience with the process of designing, building, configuring and using an OpenSPIM for our research into the early development of the polyclad flatworm Maritigrella crozieri - a non-model animal. Our OpenSPIM builds on the standard design with the addition of two colour laser illumination for simultaneous detection of two probes/molecules and dual sided illumination, which provides more even signal intensity across a specimen. Our OpenSPIM provides high resolution 3d images and time lapse recordings, and we demonstrate the use of two colour lasers and the benefits of two color dual-sided imaging. We used our microscope to study the development of the embryo of the polyclad flatworm M. crozieri. The capabilities of our microscope are demonstrated by our ability to record the stereotypical spiral cleavage pattern of M. crozieri with high-speed multi-view time lapse imaging. 3D and 4D (3D + time) reconstruction of early development from these data is possible using image registration and deconvolution tools provided as part of the open source Fiji platform. We discuss our findings on the pros and cons of a self built microscope. We conclude that home-built microscopes, such as an OpenSPIM, together with the available open source software, such as MicroManager and Fiji, make SPIM accessible to anyone interested in having continuous access to their own light-sheet microscope. However, building an OpenSPIM is not without challenges and an open access microscope is a worthwhile, if significant, investment of time and money. Multi-view 4D microscopy is more challenging than we had expected. We hope that our experience gained during this project will help future OpenSPIM users with similar ambitions.

Gabor domain optical coherence microscopy

NASA Astrophysics Data System (ADS)

Murali, Supraja

Time domain Optical Coherence Tomography (TD-OCT), first reported in 1991, makes use of the low temporal coherence properties of a NIR broadband laser to create depth sectioning of up to 2mm under the surface using optical interferometry and point to point scanning. Prior and ongoing work in OCT in the research community has concentrated on improving axial resolution through the development of broadband sources and speed of image acquisition through new techniques such as Spectral domain OCT (SD-OCT). In SD-OCT, an entire depth scan is acquired at once with a low numerical aperture (NA) objective lens focused at a fixed point within the sample. In this imaging geometry, a longer depth of focus is achieved at the expense of lateral resolution, which is typically limited to 10 to 20 mum. Optical Coherence Microscopy (OCM), introduced in 1994, combined the advantages of high axial resolution obtained in OCT with high lateral resolution obtained by increasing the NA of the microscope placed in the sample arm. However, OCM presented trade-offs caused by the inverse quadratic relationship between the NA and the DOF of the optics used. For applications requiring high lateral resolution, such as cancer diagnostics, several solutions have been proposed including the periodic manual re-focusing of the objective lens in the time domain as well as the spectral domain C-mode configuration in order to overcome the loss in lateral resolution outside the DOF. In this research, we report for the first time, high speed, sub-cellular imaging (lateral resolution of 2 mum) in OCM using a Gabor domain image processing algorithm with a custom designed and fabricated dynamic focus microscope interfaced to a Ti:Sa femtosecond laser centered at 800 nm within an SD-OCM configuration. It is envisioned that this technology will provide a non-invasive replacement for the current practice of multiple biopsies for skin cancer diagnosis. The research reported here presents three important advances to this technology all of which have been demonstrated in full functional hardware conceived and built during the course of this research. First, it has been demonstrated that the coherence gate created by the femtosecond laser can be coupled into a scanning optical microscope using optical design methods to include liquid lens technology that enables scanning below the surface of skin with no moving parts and at high resolution throughout a 2x2x2 mm imaging cube. Second, the integration the variable-focus liquid lens technology within a fixed-optics microscope custom optical design helped increase the working NA by an order of magnitude over the limitation imposed by the liquid lens alone. Thus, this design has enabled homogenous axial and lateral resolution at the micron-level (i.e., 2 mum) while imaging in the spectral domain, and still maintaining in vivo speeds. The latest images in biological specimens clearly demonstrate sub-cellular resolution in all dimensions throughout the imaging volume. Third, this new modality for data collection has been integrated with an automated Gabor domain image registration and fusion algorithm to provide full resolution images across the data cube in real-time. We refer to this overall OCM method as Gabor domain OCM (GD-OCM). These advantages place GD-OCM in a unique position with respect to the diagnosis of cancer, because when fully developed, it promises to enable fast and accurate screening for early symptoms that could lead to prevention. The next step for this technology is to apply it directly, in a clinical environment. This step is underway and is expected to be reported by the next generation of researchers within this group.

Software electron counting for low-dose scanning transmission electron microscopy.

PubMed

Mittelberger, Andreas; Kramberger, Christian; Meyer, Jannik C

2018-05-01

The performance of the detector is of key importance for low-dose imaging in transmission electron microscopy, and counting every single electron can be considered as the ultimate goal. In scanning transmission electron microscopy, low-dose imaging can be realized by very fast scanning, however, this also introduces artifacts and a loss of resolution in the scan direction. We have developed a software approach to correct for artifacts introduced by fast scans, making use of a scintillator and photomultiplier response that extends over several pixels. The parameters for this correction can be directly extracted from the raw image. Finally, the images can be converted into electron counts. This approach enables low-dose imaging in the scanning transmission electron microscope via high scan speeds while retaining the image quality of artifact-free slower scans. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Imaging malaria sporozoites in the dermis of the mammalian host.

PubMed

Amino, Rogerio; Thiberge, Sabine; Blazquez, Samantha; Baldacci, Patricia; Renaud, Olivier; Shorte, Spencer; Ménard, Robert

2007-01-01

The initial phase of malaria infection is the pre-erythrocytic phase, which begins when parasites are injected by the mosquito into the dermis and ends when parasites are released from hepatocytes into the blood. We present here a protocol for the in vivo imaging of GFP-expressing sporozoites in the dermis of rodents, using the combination of a high-speed spinning-disk confocal microscope and a high-speed charge-coupled device (CCD) camera permitting rapid in vivo acquisitions. The steps of this protocol indicate how to infect mice through the bite of infected Anopheles stephensi mosquitoes, record the sporozoites' fate in the mouse ear and to present the data as maximum-fluorescence-intensity projections, time-lapse representations and movie clips. This protocol permits investigating the various aspects of sporozoite behavior in a quantitative manner, such as motility in the matrix, cell traversal, crossing the endothelial barrier of both blood and lymphatic vessels and intravascular gliding. Applied to genetically modified parasites and/or mice, these imaging techniques should be useful for studying the cellular and molecular bases of Plasmodium sporozoite infection in vivo.

Effect of engraving speeds of CO₂ laser irradiation on In-Ceram Alumina roughness: a pilot study.

PubMed

Ersu, Bahadır; Ersoy, Orkun; Yuzugullu, Bulem; Canay, Senay

2015-05-01

The aim of the study was to determine the effect of CO₂ laser on surface roughness of In-Ceram-Alumina-ceramic. Four aluminum-oxide ceramic disc specimens were prepared of In-Ceram Alumina. Discs received CO₂ laser irradiation with different engraving speeds (100, 400, 600 and 800 mm/min) as a surface treatment. The roughness of the surfaces was measured on digital elevation models reconstructed from stereoscopic images acquired by scanning-electron-microscope. Surface roughness data were analyzed with One-Way-Analysis-of-Variance at a significance level of p<0.05. There was no significant difference between the roughness values (p=0.82). Due to higher laser durations, partial melting signs were observed on the surfaces. Tearing, smearing and swelling occurred on melted surfaces. Swelling accompanying melting increased the surface roughness, while laser power was fixed and different laser engraving speeds were applied. Although different laser irradiation speeds did not affect the roughness of ceramic surfaces, swelling was observed which led to changes on surfaces.

Comparative study of image contrast in scanning electron microscope and helium ion microscope.

PubMed

O'Connell, R; Chen, Y; Zhang, H; Zhou, Y; Fox, D; Maguire, P; Wang, J J; Rodenburg, C

2017-12-01

Images of Ga + -implanted amorphous silicon layers in a 110 n-type silicon substrate have been collected by a range of detectors in a scanning electron microscope and a helium ion microscope. The effects of the implantation dose and imaging parameters (beam energy, dwell time, etc.) on the image contrast were investigated. We demonstrate a similar relationship for both the helium ion microscope Everhart-Thornley and scanning electron microscope Inlens detectors between the contrast of the images and the Ga + density and imaging parameters. These results also show that dynamic charging effects have a significant impact on the quantification of the helium ion microscope and scanning electron microscope contrast. © 2017 The Authors Journal of Microscopy © 2017 Royal Microscopical Society.

High temporal and spatial resolution studies of bone cells using real-time confocal reflection microscopy.

PubMed

Boyde, A; Vesely, P; Gray, C; Jones, S J

1994-01-01

Chick and rat bone-derived cells were mounted in sealed coverslip-covered chambers; individual osteoclasts (but also osteoblasts) were selected and studied at 37 degrees C using three different types of high-speed scanning confocal microscopes: (1) A Noran Tandem Scanning Microscope (TSM) was used with a low light level, cooled CCD camera for image transfer to a Noran TN8502 frame store-based image analysing computer to make time lapse movie sequences using 0.1 s exposure periods, thus losing some of the advantage of the high frame rate of the TSM. Rapid focus adjustment using computer controlled piezo drivers permitted two or more focus planes to be imaged sequentially: thus (with additional light-source shuttering) the reflection confocal image could be alternated with the phase contrast image at a different focus. Individual cells were followed for up to 5 days, suggesting no significant irradiation problem. (2) Exceptional temporal and spatial resolution is available in video rate laser confocal scanning microscopes (VRCSLMs). We used the Noran Odyssey unitary beam VRCSLM with an argon ion laser at 488 nm and acousto-optic deflection (AOD) on the line axis: this instrument is truly and adjustably confocal in the reflection mode. (3) We also used the Lasertec 1LM11 line scan instrument, with an He-Ne laser at 633 nm, and AOD for the frame scan. We discuss the technical problems and merits of the different approaches. The VRCSLMs documented rapid, real-time oscillatory motion: all the methods used show rapid net movement of organelles within bone cells. The interference reflection mode gives particularly strong contrasts in confocal instruments. Phase contrast and other interference methods used in the microscopy of living cells can be used simultaneously in the TSM.

A new ball-on-disk vacuum tribometer with in situ measurement of the wear track by digital holographic microscopy

NASA Astrophysics Data System (ADS)

Meylan, B.; Ciani, D.; Zhang, B.; Cuche, E.; Wasmer, K.

2017-12-01

This contribution presents a new ball-on-disk vacuum tribometer with in situ measurement of the wear track by digital holographic microscopy. This new tribometer allows observation of the evolution of the wear track in situ and in real-time. The method combines a high vacuum high temperature ball-on-disk tribometer with a digital holographic microscope (DHM). The machine was tested and validated by taking DHM images during wear tests at room temperature and in vacuum at 2 · 10-6 of polished 100Cr6 steel disks. We demonstrated that the DHM system is well suited to monitor the evolution of the wear track during sliding. We found that, with an acquisition time of 0.1 ms for the DHM, the maximal linear speed is 10 cm s-1 to have reliable images. We proved, via scanning electron microscope (SEM) pictures, that the lines in the sliding direction in all DHM images exist. We also validated the new tribometer by having an excellent correlation between the images and profiles of the wear track taken by the DHM with the ones from a confocal microscope. Finally, the new tribometer combined with the DHM has four advantages. It can test under vacuum and various atmospheric conditions. The evolution of the wear track is measured in situ and in real-time. Hence, the problem of replacing the sample is avoided. Thanks to the DHM technology, the vertical accuracy of the topographical measurement is 4 nm.

New Analysis Method Application in Metallographic Images through the Construction of Mosaics Via Speeded Up Robust Features and Scale Invariant Feature Transform

PubMed Central

Rebouças Filho, Pedro Pedrosa; Moreira, Francisco Diego Lima; Xavier, Francisco Geilson de Lima; Gomes, Samuel Luz; dos Santos, José Ciro; Freitas, Francisco Nélio Costa; Freitas, Rodrigo Guimarães

2015-01-01

In many applications in metallography and analysis, many regions need to be considered and not only the current region. In cases where there are analyses with multiple images, the specialist should also evaluate neighboring areas. For example, in metallurgy, welding technology is derived from conventional testing and metallographic analysis. In welding, these tests allow us to know the features of the metal, especially in the Heat-Affected Zone (HAZ); the region most likely for natural metallurgical problems to occur in welding. The expanse of the Heat-Affected Zone exceeds the size of the area observed through a microscope and typically requires multiple images to be mounted on a larger picture surface to allow for the study of the entire heat affected zone. This image stitching process is performed manually and is subject to all the inherent flaws of the human being due to results of fatigue and distraction. The analyzing of grain growth is also necessary in the examination of multiple regions, although not necessarily neighboring regions, but this analysis would be a useful tool to aid a specialist. In areas such as microscopic metallography, which study metallurgical products with the aid of a microscope, the assembly of mosaics is done manually, which consumes a lot of time and is also subject to failures due to human limitations. The mosaic technique is used in the construct of environment or scenes with corresponding characteristics between themselves. Through several small images, and with corresponding characteristics between themselves, a new model is generated in a larger size. This article proposes the use of Digital Image Processing for the automatization of the construction of these mosaics in metallographic images. The use of this proposed method is meant to significantly reduce the time required to build the mosaic and reduce the possibility of failures in assembling the final image; therefore increasing efficiency in obtaining results and expediting the decision making process. Two different methods are proposed: One using the transformed Scale Invariant Feature Transform (SIFT), and the second using features extractor Speeded Up Robust Features (SURF). Although slower, the SIFT method is more stable and has a better performance than the SURF method and can be applied to real applications. The best results were obtained using SIFT with Peak Signal-to-Noise Ratio = 61.38, Mean squared error = 0.048 and mean-structural-similarity = 0.999, and processing time of 4.91 seconds for mosaic building. The methodology proposed shows be more promissory in aiding specialists during analysis of metallographic images. PMID:28793412

A widefield fluorescence microscope with a linear image sensor for image cytometry of biospecimens: Considerations for image quality optimization

NASA Astrophysics Data System (ADS)

Hutcheson, Joshua A.; Majid, Aneeka A.; Powless, Amy J.; Muldoon, Timothy J.

2015-09-01

Linear image sensors have been widely used in numerous research and industry applications to provide continuous imaging of moving objects. Here, we present a widefield fluorescence microscope with a linear image sensor used to image translating objects for image cytometry. First, a calibration curve was characterized for a custom microfluidic chamber over a span of volumetric pump rates. Image data were also acquired using 15 μm fluorescent polystyrene spheres on a slide with a motorized translation stage in order to match linear translation speed with line exposure periods to preserve the image aspect ratio. Aspect ratios were then calculated after imaging to ensure quality control of image data. Fluorescent beads were imaged in suspension flowing through the microfluidics chamber being pumped by a mechanical syringe pump at 16 μl min-1 with a line exposure period of 150 μs. The line period was selected to acquire images of fluorescent beads with a 40 dB signal-to-background ratio. A motorized translation stage was then used to transport conventional glass slides of stained cellular biospecimens. Whole blood collected from healthy volunteers was stained with 0.02% (w/v) proflavine hemisulfate was imaged to highlight leukocyte morphology with a 1.56 mm × 1.28 mm field of view (1540 ms total acquisition time). Oral squamous cells were also collected from healthy volunteers and stained with 0.01% (w/v) proflavine hemisulfate to demonstrate quantifiable subcellular features and an average nuclear to cytoplasmic ratio of 0.03 (n = 75), with a resolution of 0.31 μm pixels-1.

A widefield fluorescence microscope with a linear image sensor for image cytometry of biospecimens: Considerations for image quality optimization

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

Hutcheson, Joshua A.; Majid, Aneeka A.; Powless, Amy J.

Linear image sensors have been widely used in numerous research and industry applications to provide continuous imaging of moving objects. Here, we present a widefield fluorescence microscope with a linear image sensor used to image translating objects for image cytometry. First, a calibration curve was characterized for a custom microfluidic chamber over a span of volumetric pump rates. Image data were also acquired using 15 μm fluorescent polystyrene spheres on a slide with a motorized translation stage in order to match linear translation speed with line exposure periods to preserve the image aspect ratio. Aspect ratios were then calculated aftermore » imaging to ensure quality control of image data. Fluorescent beads were imaged in suspension flowing through the microfluidics chamber being pumped by a mechanical syringe pump at 16 μl min{sup −1} with a line exposure period of 150 μs. The line period was selected to acquire images of fluorescent beads with a 40 dB signal-to-background ratio. A motorized translation stage was then used to transport conventional glass slides of stained cellular biospecimens. Whole blood collected from healthy volunteers was stained with 0.02% (w/v) proflavine hemisulfate was imaged to highlight leukocyte morphology with a 1.56 mm × 1.28 mm field of view (1540 ms total acquisition time). Oral squamous cells were also collected from healthy volunteers and stained with 0.01% (w/v) proflavine hemisulfate to demonstrate quantifiable subcellular features and an average nuclear to cytoplasmic ratio of 0.03 (n = 75), with a resolution of 0.31 μm pixels{sup −1}.« less

Active Damping of a Piezoelectric Tube Scanner using Self-Sensing Piezo Actuation

PubMed Central

Kuiper, S.; Schitter, G.

2010-01-01

In most Atomic Force Microscopes (AFM), a piezoelectric tube scanner is used to position the sample underneath the measurement probe. Oscillations stemming from the weakly damped resonances of the tube scanner are a major source of image distortion, putting a limitation on the achievable imaging speed. This paper demonstrates active damping of these oscillations in multiple scanning axes without the need for additional position sensors. By connecting the tube scanner in a capacitive bridge circuit the scanner oscillations can be measured in both scanning axes, using the same piezo material as an actuator and sensor simultaneously. In order to compensate for circuit imbalance caused by hysteresis in the piezo element, an adaptive balancing circuit is used. The obtained measurement signal is used for feedback control, reducing the resonance peaks in both scanning axes by 18 dB and the cross-coupling at those frequencies by 30 dB. Experimental results demonstrate a significant reduction in scanner oscillations when applying the typical triangular scanning signals, as well as a strong reduction in coupling induced oscillations. Recorded AFM images show a considerable reduction in image distortion due to the proposed control method, enabling artifact free AFM imaging at a speed of 122 lines per second with a standard piezoelectric tube scanner. PMID:26412944

Detection of immunomagnetically captured 4',6-diamidino-2-phenyl-indole (DAPI)-labeled Escherichia coli 0157:H7 by fluorescent microscopic imaging

NASA Astrophysics Data System (ADS)

Tu, Shu-I.; Uknalis, Joseph; Patterson, Deidre; Gehring, Andrew G.

1999-01-01

Live cells of E. coliO157:H7 were captured by goat anti-E. coliO157 serum coated on the surface of polystyrene based immunomagnetic beads (IMB). The captured bacteria were labeled by 4',6-diamidino-2-phenylindole (DAPI), a nucleic acid stain, for observation by epifluorescent microscopy. The beads with captured bacteria were then concentrated by magnetic separators. The efficiency of this magnetic concentration step was less than that of using high speed centrifugation. The antibody-captured and IMB-immobilized bacteria were then applied on HF-treated, bovine serum albumin (BSA)-coated microscope slides mounted on an automated stage, and magnetically aligned before fluorescence distribution was measured by a cooled CCD attached to an inverted microscope. Since the beads were concentrated and linearly aligned along the edge of the magnetic field, image capture along the edge for a few field widths was sufficient to account for most of captured bacteria. We applied this approach to determine the bacterial counts in spiked beef hamburger patties. The results showed that after a 6-hour enrichment, sufficient number of the bacteria could be detected from the samples spiked with 1 CFU of E. coliO157:H7 per gram of the hamburger.

GPU acceleration towards real-time image reconstruction in 3D tomographic diffractive microscopy

NASA Astrophysics Data System (ADS)

Bailleul, J.; Simon, B.; Debailleul, M.; Liu, H.; Haeberlé, O.

2012-06-01

Phase microscopy techniques regained interest in allowing for the observation of unprepared specimens with excellent temporal resolution. Tomographic diffractive microscopy is an extension of holographic microscopy which permits 3D observations with a finer resolution than incoherent light microscopes. Specimens are imaged by a series of 2D holograms: their accumulation progressively fills the range of frequencies of the specimen in Fourier space. A 3D inverse FFT eventually provides a spatial image of the specimen. Consequently, acquisition then reconstruction are mandatory to produce an image that could prelude real-time control of the observed specimen. The MIPS Laboratory has built a tomographic diffractive microscope with an unsurpassed 130nm resolution but a low imaging speed - no less than one minute. Afterwards, a high-end PC reconstructs the 3D image in 20 seconds. We now expect an interactive system providing preview images during the acquisition for monitoring purposes. We first present a prototype implementing this solution on CPU: acquisition and reconstruction are tied in a producer-consumer scheme, sharing common data into CPU memory. Then we present a prototype dispatching some reconstruction tasks to GPU in order to take advantage of SIMDparallelization for FFT and higher bandwidth for filtering operations. The CPU scheme takes 6 seconds for a 3D image update while the GPU scheme can go down to 2 or > 1 seconds depending on the GPU class. This opens opportunities for 4D imaging of living organisms or crystallization processes. We also consider the relevance of GPU for 3D image interaction in our specific conditions.

A Review of Adaptive Optics Optical Coherence Tomography: Technical Advances, Scientific Applications, and the Future.

PubMed

Jonnal, Ravi S; Kocaoglu, Omer P; Zawadzki, Robert J; Liu, Zhuolin; Miller, Donald T; Werner, John S

2016-07-01

Optical coherence tomography (OCT) has enabled "virtual biopsy" of the living human retina, revolutionizing both basic retina research and clinical practice over the past 25 years. For most of those years, in parallel, adaptive optics (AO) has been used to improve the transverse resolution of ophthalmoscopes to foster in vivo study of the retina at the microscopic level. Here, we review work done over the last 15 years to combine the microscopic transverse resolution of AO with the microscopic axial resolution of OCT, building AO-OCT systems with the highest three-dimensional resolution of any existing retinal imaging modality. We surveyed the literature to identify the most influential antecedent work, important milestones in the development of AO-OCT technology, its applications that have yielded new knowledge, research areas into which it may productively expand, and nascent applications that have the potential to grow. Initial efforts focused on demonstrating three-dimensional resolution. Since then, many improvements have been made in resolution and speed, as well as other enhancements of acquisition and postprocessing techniques. Progress on these fronts has produced numerous discoveries about the anatomy, function, and optical properties of the retina. Adaptive optics OCT continues to evolve technically and to contribute to our basic and clinical knowledge of the retina. Due to its capacity to reveal cellular and microscopic detail invisible to clinical OCT systems, it is an ideal companion to those instruments and has the demonstrable potential to produce images that can guide the interpretation of clinical findings.

Dynamic Torsional and Cyclic Fracture Behavior of ProFile Rotary Instruments at Continuous or Reciprocating Rotation as Visualized with High-speed Digital Video Imaging.

PubMed

Tokita, Daisuke; Ebihara, Arata; Miyara, Kana; Okiji, Takashi

2017-08-01

This study examined the dynamic fracture behavior of nickel-titanium rotary instruments in torsional or cyclic loading at continuous or reciprocating rotation by means of high-speed digital video imaging. The ProFile instruments (size 30, 0.06 taper; Dentsply Maillefer, Ballaigues, Switzerland) were categorized into 4 groups (n = 7 in each group) as follows: torsional/continuous (TC), torsional/reciprocating (TR), cyclic/continuous (CC), and cyclic/reciprocating (CR). Torsional loading was performed by rotating the instruments by holding the tip with a vise. For cyclic loading, a custom-made device with a 38° curvature was used. Dynamic fracture behavior was observed with a high-speed camera. The time to fracture was recorded, and the fractured surface was examined with scanning electron microscopy. The TC group initially exhibited necking of the file followed by the development of an initial crack line. The TR group demonstrated opening and closing of a crack according to its rotation in the cutting and noncutting directions, respectively. The CC group separated without any detectable signs of deformation. In the CR group, initial crack formation was recognized in 5 of 7 samples. The reciprocating rotation exhibited a longer time to fracture in both torsional and cyclic fatigue testing (P < .05). The scanning electron microscopic images showed a severely deformed surface in the TR group. The dynamic fracture behavior of NiTi rotary instruments, as visualized with high-speed digital video imaging, varied between the different modes of rotation and different fatigue testing. Reciprocating rotation induced a slower crack propagation and conferred higher fatigue resistance than continuous rotation in both torsional and cyclic loads. Copyright © 2017 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

Chromatic confocal microscopy for multi-depth imaging of epithelial tissue

PubMed Central

Olsovsky, Cory; Shelton, Ryan; Carrasco-Zevallos, Oscar; Applegate, Brian E.; Maitland, Kristen C.

2013-01-01

We present a novel chromatic confocal microscope capable of volumetric reflectance imaging of microstructure in non-transparent tissue. Our design takes advantage of the chromatic aberration of aspheric lenses that are otherwise well corrected. Strong chromatic aberration, generated by multiple aspheres, longitudinally disperses supercontinuum light onto the sample. The backscattered light detected with a spectrometer is therefore wavelength encoded and each spectrum corresponds to a line image. This approach obviates the need for traditional axial mechanical scanning techniques that are difficult to implement for endoscopy and susceptible to motion artifact. A wavelength range of 590-775 nm yielded a >150 µm imaging depth with ~3 µm axial resolution. The system was further demonstrated by capturing volumetric images of buccal mucosa. We believe these represent the first microstructural images in non-transparent biological tissue using chromatic confocal microscopy that exhibit long imaging depth while maintaining acceptable resolution for resolving cell morphology. Miniaturization of this optical system could bring enhanced speed and accuracy to endomicroscopic in vivo volumetric imaging of epithelial tissue. PMID:23667789

Electron imaging with an EBSD detector.

PubMed

Wright, Stuart I; Nowell, Matthew M; de Kloe, René; Camus, Patrick; Rampton, Travis

2015-01-01

Electron Backscatter Diffraction (EBSD) has proven to be a useful tool for characterizing the crystallographic orientation aspects of microstructures at length scales ranging from tens of nanometers to millimeters in the scanning electron microscope (SEM). With the advent of high-speed digital cameras for EBSD use, it has become practical to use the EBSD detector as an imaging device similar to a backscatter (or forward-scatter) detector. Using the EBSD detector in this manner enables images exhibiting topographic, atomic density and orientation contrast to be obtained at rates similar to slow scanning in the conventional SEM manner. The high-speed acquisition is achieved through extreme binning of the camera-enough to result in a 5 × 5 pixel pattern. At such high binning, the captured patterns are not suitable for indexing. However, no indexing is required for using the detector as an imaging device. Rather, a 5 × 5 array of images is formed by essentially using each pixel in the 5 × 5 pixel pattern as an individual scattered electron detector. The images can also be formed at traditional EBSD scanning rates by recording the image data during a scan or can also be formed through post-processing of patterns recorded at each point in the scan. Such images lend themselves to correlative analysis of image data with the usual orientation data provided by and with chemical data obtained simultaneously via X-Ray Energy Dispersive Spectroscopy (XEDS). Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

Optimized optical clearing method for imaging central nervous system

NASA Astrophysics Data System (ADS)

Yu, Tingting; Qi, Yisong; Gong, Hui; Luo, Qingming; Zhu, Dan

2015-03-01

The development of various optical clearing methods provides a great potential for imaging entire central nervous system by combining with multiple-labelling and microscopic imaging techniques. These methods had made certain clearing contributions with respective weaknesses, including tissue deformation, fluorescence quenching, execution complexity and antibody penetration limitation that makes immunostaining of tissue blocks difficult. The passive clarity technique (PACT) bypasses those problems and clears the samples with simple implementation, excellent transparency with fine fluorescence retention, but the passive tissue clearing method needs too long time. In this study, we not only accelerate the clearing speed of brain blocks but also preserve GFP fluorescence well by screening an optimal clearing temperature. The selection of proper temperature will make PACT more applicable, which evidently broaden the application range of this method.

Panning artifacts in digital pathology images

NASA Astrophysics Data System (ADS)

Avanaki, Ali R. N.; Lanciault, Christian; Espig, Kathryn S.; Xthona, Albert; Kimpe, Tom R. L.

2017-03-01

In making a pathologic diagnosis, a pathologist uses cognitive processes: perception, attention, memory, and search (Pena and Andrade-Filho, 2009). Typically, this involves focus while panning from one region of a slide to another, using either a microscope in a traditional workflow or software program and display in a digital pathology workflow (DICOM Standard Committee, 2010). We theorize that during panning operation, the pathologist receives information important to diagnosis efficiency and/or correctness. As compared to an optical microscope, panning in a digital pathology image involves some visual artifacts due to the following: (i) the frame rate is finite; (ii) time varying visual signals are reconstructed using imperfect zero-order hold. Specifically, after pixel's digital drive is changed, it takes time for a pixel to emit the expected amount of light. Previous work suggests that 49% of navigation is conducted in low-power/overview with digital pathology (Molin et al., 2015), but the influence of display factors has not been measured. We conducted a reader study to establish a relationship between display frame rate, panel response time, and threshold panning speed (above which the artifacts become noticeable). Our results suggest visual tasks that involve tissue structure are more impacted by the simulated panning artifacts than those that only involve color (e.g., staining intensity estimation), and that the panning artifacts versus normalized panning speed has a peak behavior which is surprising and may change for a diagnostic task. This is work in progress and our final findings should be considered in designing future digital pathology systems.

Inverted selective plane illumination microscopy (iSPIM) enables coupled cell identity lineaging and neurodevelopmental imaging in Caenorhabditis elegans

PubMed Central

Wu, Yicong; Ghitani, Alireza; Christensen, Ryan; Santella, Anthony; Du, Zhuo; Rondeau, Gary; Bao, Zhirong; Colón-Ramos, Daniel; Shroff, Hari

2011-01-01

The Caenorhabditis elegans embryo is a powerful model for studying neural development, but conventional imaging methods are either too slow or phototoxic to take full advantage of this system. To solve these problems, we developed an inverted selective plane illumination microscopy (iSPIM) module for noninvasive high-speed volumetric imaging of living samples. iSPIM is designed as a straightforward add-on to an inverted microscope, permitting conventional mounting of specimens and facilitating SPIM use by development and neurobiology laboratories. iSPIM offers a volumetric imaging rate 30× faster than currently used technologies, such as spinning-disk confocal microscopy, at comparable signal-to-noise ratio. This increased imaging speed allows us to continuously monitor the development of C, elegans embryos, scanning volumes every 2 s for the 14-h period of embryogenesis with no detectable phototoxicity. Collecting ∼25,000 volumes over the entirety of embryogenesis enabled in toto visualization of positions and identities of cell nuclei. By merging two-color iSPIM with automated lineaging techniques we realized two goals: (i) identification of neurons expressing the transcription factor CEH-10/Chx10 and (ii) visualization of their neurodevelopmental dynamics. We found that canal-associated neurons use somal translocation and amoeboid movement as they migrate to their final position in the embryo. We also visualized axon guidance and growth cone dynamics as neurons circumnavigate the nerve ring and reach their targets in the embryo. The high-speed volumetric imaging rate of iSPIM effectively eliminates motion blur from embryo movement inside the egg case, allowing characterization of dynamic neurodevelopmental events that were previously inaccessible. PMID:22006307

New insights into mechanisms of sonothrombolysis using ultra-high-speed imaging.

PubMed

Chen, Xucai; Leeman, Jonathan E; Wang, Jianjun; Pacella, John J; Villanueva, Flordeliza S

2014-01-01

Thrombotic arterial occlusion is the principal etiology for acute cardiovascular syndromes such as stroke, myocardial infarction and unstable angina. Exposing the thrombus to ultrasound and microbubbles facilitates thrombus disruption, making "sonothrombolysis" a potentially powerful therapeutic strategy for thromboembolic diseases. However, optimization of such a strategy, and hence clinical translation, is constrained by an incomplete understanding of mechanisms by which ultrasound-induced microbubble vibrations disrupt blood clots. We posit that previously reported sonothrombolytic efficacy using inertial cavitation regimes was due, at least in part, to mechanical clot disruption by oscillating microbubbles. To test this hypothesis, we optically characterized lipid microbubble interactions with thrombus in the presence of ultrasound using a recently developed ultra-high-speed microscopy imaging system to visualize microbubble acoustic behaviors at megahertz frame rates. A microscope/acoustic stage designed for the system allowed an experimentally created thrombus and microbubbles to be insonified at a co-localized acoustic and optical focus during synchronized high-speed imaging. Under inertial cavitation conditions, large-amplitude microbubble oscillations caused thrombus deformation and pitting. Acoustic radiation forces (Bjerknes forces) further augmented microbubble-thrombus interaction. These observations suggest that a direct mechanical effect of oscillating lipid microbubbles on an adjacent thrombus may play a role in mediating clot disruption in the presence of specific ultrasound conditions. Copyright © 2014 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.

Two-photon microscopy of fungal keratitis-affected rabbit cornea ex vivo using moxifloxacin as a labeling agent.

PubMed

Lee, Jun Ho; Le, Viet-Hoan; Lee, Seunghun; Park, Jin Hyoung; Lee, Jin Ah; Tchah, Hungwon; Kim, Sungjee; Kim, Myoung Joon; Kim, Ki Hean

2018-05-19

Two-photon microscopy (TPM) is a three dimensional (3D) microscopic technique based on nonlinear two-photon fluorescence, which has been tested as an alternative to reflectance confocal microscopy (RCM) for detecting fungal keratitis via optical imaging. Although TPM provided images with better contrast than RCM for fungal keratitis, its imaging speed was relatively low because of weak intrinsic signal. Moxifloxacin, a Food and Drug Administration (FDA)-approved antibiotic, was recently used as a cell-labeling agent for TPM. In this study, moxifloxacin was used to label fungal cells for TPM imaging of fungal keratitis models. Fungal cell suspensions and ex vivo fungal keratitis-affected rabbit corneas were prepared using two types of fungal pathogens, Aspergillus fumigatus and Candida albicans, and TPM imaging was performed both with and without moxifloxacin treatment. Fungal cells with enhanced fluorescence were clearly visible by TPM of moxifloxacin-treated fungal cell suspensions. TPM of moxifloxacin-treated fungal keratitis rabbit corneas revealed both the infecting fungal cells and corneal cells similar to those observed in TPM without moxifloxacin treatment, albeit with approximately 10-times enhanced fluorescence. Fungal cells were distinguished from corneal cells on the basis of their distinct morphologies. Thus, TPM with moxifloxacin labeling might be useful for the detection of fungal keratitis at the improved imaging speed. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

Integrated microfluidic probe station.

PubMed

Perrault, C M; Qasaimeh, M A; Brastaviceanu, T; Anderson, K; Kabakibo, Y; Juncker, D

2010-11-01

The microfluidic probe (MFP) consists of a flat, blunt tip with two apertures for the injection and reaspiration of a microjet into a solution--thus hydrodynamically confining the microjet--and is operated atop an inverted microscope that enables live imaging. By scanning across a surface, the microjet can be used for surface processing with the capability of both depositing and removing material; as it operates under immersed conditions, sensitive biological materials and living cells can be processed. During scanning, the MFP is kept immobile and centered over the objective of the inverted microscope, a few micrometers above a substrate that is displaced by moving the microscope stage and that is flushed continuously with the microjet. For consistent and reproducible surface processing, the gap between the MFP and the substrate, the MFP's alignment, the scanning speed, the injection and aspiration flow rates, and the image capture need all to be controlled and synchronized. Here, we present an automated MFP station that integrates all of these functionalities and automates the key operational parameters. A custom software program is used to control an independent motorized Z stage for adjusting the gap, a motorized microscope stage for scanning the substrate, up to 16 syringe pumps for injecting and aspirating fluids, and an inverted fluorescence microscope equipped with a charge-coupled device camera. The parallelism between the MFP and the substrate is adjusted using manual goniometer at the beginning of the experiment. The alignment of the injection and aspiration apertures along the scanning axis is performed using a newly designed MFP screw holder. We illustrate the integrated MFP station by the programmed, automated patterning of fluorescently labeled biotin on a streptavidin-coated surface.

General Mode Scanning Probe Microscopy

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

Somnath, Suhas; Jesse, Stephen

A critical part of SPM measurements is the information transfer from the probe-sample junction to the measurement system. Current information transfer methods heavily compress the information-rich data stream by averaging the data over a time interval, or via heterodyne detection approaches such as lock-in amplifiers and phase-locked loops. As a consequence, highly valuable information at the sub-microsecond time scales or information from frequencies outside the measurement band is lost. We have developed a fundamentally new approach called General Mode (G-mode), where we can capture the complete information stream from the detectors in the microscope. The availability of the complete informationmore » allows the microscope operator to analyze the data via information-theory analysis or comprehensive physical models. Furthermore, the complete data stream enables advanced data-driven filtering algorithms, multi-resolution imaging, ultrafast spectroscropic imaging, spatial mapping of multidimensional variability in material properties, etc. Though we applied this approach to scanning probe microscopy, the general philosophy of G-mode can be applied to many other modes of microscopy. G-mode data is captured by completely custom software written in LabVIEW and Matlab. The software generates the waveforms to electrically, thermally, or mechanically excite the SPM probe. It handles real-time communications with the microscope software for operations such as moving the SPM probe position and also controls other instrumentation hardware. The software also controls multiple variants of high-speed data acquisition cards to excite the SPM probe with the excitation waveform and simultaneously measure multiple channels of information from the microscope detectors at sampling rates of 1-100 MHz. The software also saves the raw data to the computer and allows the microscope operator to visualize processed or filtered data during the experiment. The software performs all these features while offering a user-friendly interface.« less

Fast interactive elastic registration of 12-bit multi-spectral images with subvoxel accuracy using display hardware

NASA Astrophysics Data System (ADS)

Noordmans, Herke Jan; de Roode, Rowland; Verdaasdonk, Rudolf

2007-03-01

Multi-spectral images of human tissue taken in-vivo often contain image alignment problems as patients have difficulty in retaining their posture during the acquisition time of 20 seconds. Previously, it has been attempted to correct motion errors with image registration software developed for MR or CT data but these algorithms have been proven to be too slow and erroneous for practical use with multi-spectral images. A new software package has been developed which allows the user to play a decisive role in the registration process as the user can monitor the progress of the registration continuously and force it in the right direction when it starts to fail. The software efficiently exploits videocard hardware to gain speed and to provide a perfect subvoxel correspondence between registration field and display. An 8 bit graphic card was used to efficiently register and resample 12 bit images using the hardware interpolation modes present on the graphic card. To show the feasibility of this new registration process, the software was applied in clinical practice evaluating the dosimetry for psoriasis and KTP laser treatment. The microscopic differences between images of normal skin and skin exposed to UV light proved that an affine registration step including zooming and slanting is critical for a subsequent elastic match to have success. The combination of user interactive registration software with optimal addressing the potentials of PC video card hardware greatly improves the speed of multi spectral image registration.

Fast interactive registration tool for reproducible multi-spectral imaging for wound healing and treatment evaluation

NASA Astrophysics Data System (ADS)

Noordmans, Herke J.; de Roode, Rowland; Verdaasdonk, Rudolf

2007-02-01

Multi-spectral images of human tissue taken in-vivo often contain image alignment problems as patients have difficulty in retaining their posture during the acquisition time of 20 seconds. Previously, it has been attempted to correct motion errors with image registration software developed for MR or CT data but these algorithms have been proven to be too slow and erroneous for practical use with multi-spectral images. A new software package has been developed which allows the user to play a decisive role in the registration process as the user can monitor the progress of the registration continuously and force it in the right direction when it starts to fail. The software efficiently exploits videocard hardware to gain speed and to provide a perfect subvoxel correspondence between registration field and display. An 8 bit graphic card was used to efficiently register and resample 12 bit images using the hardware interpolation modes present on the graphic card. To show the feasibility of this new registration process, the software was applied in clinical practice evaluating the dosimetry for psoriasis and KTP laser treatment. The microscopic differences between images of normal skin and skin exposed to UV light proved that an affine registration step including zooming and slanting is critical for a subsequent elastic match to have success. The combination of user interactive registration software with optimal addressing the potentials of PC video card hardware greatly improves the speed of multi spectral image registration.

A Parallel Distributed-Memory Particle Method Enables Acquisition-Rate Segmentation of Large Fluorescence Microscopy Images

PubMed Central

Afshar, Yaser; Sbalzarini, Ivo F.

2016-01-01

Modern fluorescence microscopy modalities, such as light-sheet microscopy, are capable of acquiring large three-dimensional images at high data rate. This creates a bottleneck in computational processing and analysis of the acquired images, as the rate of acquisition outpaces the speed of processing. Moreover, images can be so large that they do not fit the main memory of a single computer. We address both issues by developing a distributed parallel algorithm for segmentation of large fluorescence microscopy images. The method is based on the versatile Discrete Region Competition algorithm, which has previously proven useful in microscopy image segmentation. The present distributed implementation decomposes the input image into smaller sub-images that are distributed across multiple computers. Using network communication, the computers orchestrate the collectively solving of the global segmentation problem. This not only enables segmentation of large images (we test images of up to 1010 pixels), but also accelerates segmentation to match the time scale of image acquisition. Such acquisition-rate image segmentation is a prerequisite for the smart microscopes of the future and enables online data compression and interactive experiments. PMID:27046144

A Parallel Distributed-Memory Particle Method Enables Acquisition-Rate Segmentation of Large Fluorescence Microscopy Images.

PubMed

Afshar, Yaser; Sbalzarini, Ivo F

2016-01-01

Modern fluorescence microscopy modalities, such as light-sheet microscopy, are capable of acquiring large three-dimensional images at high data rate. This creates a bottleneck in computational processing and analysis of the acquired images, as the rate of acquisition outpaces the speed of processing. Moreover, images can be so large that they do not fit the main memory of a single computer. We address both issues by developing a distributed parallel algorithm for segmentation of large fluorescence microscopy images. The method is based on the versatile Discrete Region Competition algorithm, which has previously proven useful in microscopy image segmentation. The present distributed implementation decomposes the input image into smaller sub-images that are distributed across multiple computers. Using network communication, the computers orchestrate the collectively solving of the global segmentation problem. This not only enables segmentation of large images (we test images of up to 10(10) pixels), but also accelerates segmentation to match the time scale of image acquisition. Such acquisition-rate image segmentation is a prerequisite for the smart microscopes of the future and enables online data compression and interactive experiments.

Development of an ultrasound microscope combined with optical microscope for multiparametric characterization of a single cell.

PubMed

Arakawa, Mototaka; Shikama, Joe; Yoshida, Koki; Nagaoka, Ryo; Kobayashi, Kazuto; Saijo, Yoshifumi

2015-09-01

Biomechanics of the cell has been gathering much attention because it affects the pathological status in atherosclerosis and cancer. In the present study, an ultrasound microscope system combined with optical microscope for characterization of a single cell with multiple ultrasound parameters was developed. The central frequency of the transducer was 375 MHz and the scan area was 80 × 80 μm with up to 200 × 200 sampling points. An inverted optical microscope was incorporated in the design of the system, allowing for simultaneous optical observations of cultured cells. Two-dimensional mapping of multiple ultrasound parameters, such as sound speed, attenuation, and acoustic impedance, as well as the thickness, density, and bulk modulus of specimen/cell under investigation, etc., was realized by the system. Sound speed and thickness of a 3T3-L1 fibroblast cell were successfully obtained by the system. The ultrasound microscope system combined with optical microscope further enhances our understanding of cellular biomechanics.

Adaptive compensation of aberrations in ultrafast 3D microscopy using a deformable mirror

NASA Astrophysics Data System (ADS)

Sherman, Leah R.; Albert, O.; Schmidt, Christoph F.; Vdovin, Gleb V.; Mourou, Gerard A.; Norris, Theodore B.

2000-05-01

3D imaging using a multiphoton scanning confocal microscope is ultimately limited by aberrations of the system. We describe a system to adaptively compensate the aberrations with a deformable mirror. We have increased the transverse scanning range of the microscope by three with compensation of off-axis aberrations.We have also significantly increased the longitudinal scanning depth with compensation of spherical aberrations from the penetration into the sample. Our correction is based on a genetic algorithm that uses second harmonic or two-photon fluorescence signal excited by femtosecond pulses from the sample as the enhancement parameter. This allows us to globally optimize the wavefront without a wavefront measurement. To improve the speed of the optimization we use Zernike polynomials as the basis for correction. Corrections can be stored in a database for look-up with future samples.

A new scanning system for alpha decay events as calibration sources for range-energy relation in nuclear emulsion

NASA Astrophysics Data System (ADS)

Yoshida, J.; Kinbara, S.; Mishina, A.; Nakazawa, K.; Soe, M. K.; Theint, A. M. M.; Tint, K. T.

2017-03-01

A new scanning system named "Vertex picker" has been developed to rapid collect alpha decay events, which are calibration sources for the range-energy relation in nuclear emulsion. A computer-controlled optical microscope scans emulsion layers exhaustively, and a high-speed and high-resolution camera takes their micrographs. A dedicated image processing picks out vertex-like shapes. Practical operations of alpha decay search were demonstrated by emulsion sheets of the KEK-PS E373 experiment. Alpha decays of nearly 28 events were detected in eye-check work on a PC monitor per hour. This yield is nearly 20 times more effective than that by the conventional eye-scan method. The speed and quality is acceptable for the coming new experiment, J-PARC E07.

Real-Time Dynamic Observation of Micro-Friction on the Contact Interface of Friction Lining

PubMed Central

Zhang, Dekun; Chen, Kai; Guo, Yongbo

2018-01-01

This paper aims to investigate the microscopic friction mechanism based on in situ microscopic observation in order to record the deformation and contact situation of friction lining during the frictional process. The results show that friction coefficient increased with the shear deformation and energy loss of the surfacee, respectively. Furthermore, the friction mechanism mainly included adhesive friction in the high-pressure and high-speed conditions, whereas hysteresis friction was in the low-pressure and low-speed conditions. The mixed-friction mechanism was in the period when the working conditions varied from high pressure and speed to low pressure and speed. PMID:29498677

Advances in Light Microscopy for Neuroscience

PubMed Central

Wilt, Brian A.; Burns, Laurie D.; Ho, Eric Tatt Wei; Ghosh, Kunal K.; Mukamel, Eran A.

2010-01-01

Since the work of Golgi and Cajal, light microscopy has remained a key tool for neuroscientists to observe cellular properties. Ongoing advances have enabled new experimental capabilities using light to inspect the nervous system across multiple spatial scales, including ultrastructural scales finer than the optical diffraction limit. Other progress permits functional imaging at faster speeds, at greater depths in brain tissue, and over larger tissue volumes than previously possible. Portable, miniaturized fluorescence microscopes now allow brain imaging in freely behaving mice. Complementary progress on animal preparations has enabled imaging in head-restrained behaving animals, as well as time-lapse microscopy studies in the brains of live subjects. Mouse genetic approaches permit mosaic and inducible fluorescence-labeling strategies, whereas intrinsic contrast mechanisms allow in vivo imaging of animals and humans without use of exogenous markers. This review surveys such advances and highlights emerging capabilities of particular interest to neuroscientists. PMID:19555292

Video-rate volumetric functional imaging of the brain at synaptic resolution.

PubMed

Lu, Rongwen; Sun, Wenzhi; Liang, Yajie; Kerlin, Aaron; Bierfeld, Jens; Seelig, Johannes D; Wilson, Daniel E; Scholl, Benjamin; Mohar, Boaz; Tanimoto, Masashi; Koyama, Minoru; Fitzpatrick, David; Orger, Michael B; Ji, Na

2017-04-01

Neurons and neural networks often extend hundreds of micrometers in three dimensions. Capturing the calcium transients associated with their activity requires volume imaging methods with subsecond temporal resolution. Such speed is a challenge for conventional two-photon laser-scanning microscopy, because it depends on serial focal scanning in 3D and indicators with limited brightness. Here we present an optical module that is easily integrated into standard two-photon laser-scanning microscopes to generate an axially elongated Bessel focus, which when scanned in 2D turns frame rate into volume rate. We demonstrated the power of this approach in enabling discoveries for neurobiology by imaging the calcium dynamics of volumes of neurons and synapses in fruit flies, zebrafish larvae, mice and ferrets in vivo. Calcium signals in objects as small as dendritic spines could be resolved at video rates, provided that the samples were sparsely labeled to limit overlap in their axially projected images.

Web conferencing systems: Skype and MSN in telepathology

PubMed Central

Klock, Clóvis; Gomes, Regina de Paula Xavier

2008-01-01

Virtual pathology is a very important tool that can be used in several ways, including interconsultations with specialists in many areas and for frozen sections. We considered in this work the use of Windows Live Messenger and Skype for image transmission. The conference was made through wide broad internet using Nikon E 200 microscope and Digital Samsung Colour SCC-131 camera. Internet speed for transmission varied from 400 Kb to 2.0 Mb. Both programs allow voice transmission concomitant to image, so the communication between the involved pathologists was possible using microphones and speakers. Alive image could be seen by the receptor pathologist who was able to ask for moving the field or increase/diminish the augmentation. No phone call or typing required. The programs MSN and Skype can be used in many ways and with different operational systems installed in the computer. The capture system is simple and relatively cheap, what proves the viability of the system to be used in developing countries and in cities where do not exist pathologists. With the improvement of software and the improvement of digital image quality, associated to the use of the high speed broad band Internet this will be able to become a new modality in surgical pathology. PMID:18673501

Web conferencing systems: Skype and MSN in telepathology.

PubMed

Klock, Clóvis; Gomes, Regina de Paula Xavier

2008-07-15

Virtual pathology is a very important tool that can be used in several ways, including interconsultations with specialists in many areas and for frozen sections. We considered in this work the use of Windows Live Messenger and Skype for image transmission. The conference was made through wide broad internet using Nikon E 200 microscope and Digital Samsung Colour SCC-131 camera. Internet speed for transmission varied from 400 Kb to 2.0 Mb. Both programs allow voice transmission concomitant to image, so the communication between the involved pathologists was possible using microphones and speakers. A live image could be seen by the receptor pathologist who was able to ask for moving the field or increase/diminish the augmentation. No phone call or typing required. The programs MSN and Skype can be used in many ways and with different operational systems installed in the computer. The capture system is simple and relatively cheap, what proves the viability of the system to be used in developing countries and in cities where do not exist pathologists. With the improvement of software and the improvement of digital image quality, associated to the use of the high speed broad band Internet this will be able to become a new modality in surgical pathology.

High-throughput microfluidic line scan imaging for cytological characterization

NASA Astrophysics Data System (ADS)

Hutcheson, Joshua A.; Powless, Amy J.; Majid, Aneeka A.; Claycomb, Adair; Fritsch, Ingrid; Balachandran, Kartik; Muldoon, Timothy J.

2015-03-01

Imaging cells in a microfluidic chamber with an area scan camera is difficult due to motion blur and data loss during frame readout causing discontinuity of data acquisition as cells move at relatively high speeds through the chamber. We have developed a method to continuously acquire high-resolution images of cells in motion through a microfluidics chamber using a high-speed line scan camera. The sensor acquires images in a line-by-line fashion in order to continuously image moving objects without motion blur. The optical setup comprises an epi-illuminated microscope with a 40X oil immersion, 1.4 NA objective and a 150 mm tube lens focused on a microfluidic channel. Samples containing suspended cells fluorescently stained with 0.01% (w/v) proflavine in saline are introduced into the microfluidics chamber via a syringe pump; illumination is provided by a blue LED (455 nm). Images were taken of samples at the focal plane using an ELiiXA+ 8k/4k monochrome line-scan camera at a line rate of up to 40 kHz. The system's line rate and fluid velocity are tightly controlled to reduce image distortion and are validated using fluorescent microspheres. Image acquisition was controlled via MATLAB's Image Acquisition toolbox. Data sets comprise discrete images of every detectable cell which may be subsequently mined for morphological statistics and definable features by a custom texture analysis algorithm. This high-throughput screening method, comparable to cell counting by flow cytometry, provided efficient examination including counting, classification, and differentiation of saliva, blood, and cultured human cancer cells.

Characterization of human breast cancer by scanning acoustic microscopy

NASA Astrophysics Data System (ADS)

Chen, Di; Malyarenko, Eugene; Seviaryn, Fedar; Yuan, Ye; Sherman, Mark; Bandyopadhyay, Sudeshna; Gierach, Gretchen; Greenway, Christopher W.; Maeva, Elena; Strumban, Emil; Duric, Neb; Maev, Roman

2013-03-01

Objectives: The purpose of this study was to characterize human breast cancer tissues by the measurement of microacoustic properties. Methods: We investigated eight breast cancer patients using acoustic microscopy. For each patient, seven blocks of tumor tissue were collected from seven different positions around a tumor mass. Frozen sections (10 micrometer, μm) of human breast cancer tissues without staining and fixation were examined in a scanning acoustic microscope with focused transducers at 80 and 200 MHz. Hematoxylin and Eosin (H and E) stained sections from the same frozen breast cancer tissues were imaged by optical microscopy for comparison. Results: The results of acoustic imaging showed that acoustic attenuation and sound speed in cancer cell-rich tissue regions were significantly decreased compared with the surrounding tissue regions, where most components are normal cells/tissues, such as fibroblasts, connective tissue and lymphocytes. Our observation also showed that the ultrasonic properties were influenced by arrangements of cells and tissue patterns. Conclusions: Our data demonstrate that attenuation and sound speed imaging can provide biomechanical information of the tumor and normal tissues. The results also demonstrate the potential of acoustic microscopy as an auxiliary method for operative detection and localization of cancer affected regions.

Grayscale inhomogeneity correction method for multiple mosaicked electron microscope images

NASA Astrophysics Data System (ADS)

Zhou, Fangxu; Chen, Xi; Sun, Rong; Han, Hua

2018-04-01

Electron microscope image stitching is highly desired to acquire microscopic resolution images of large target scenes in neuroscience. However, the result of multiple Mosaicked electron microscope images may exist severe gray scale inhomogeneity due to the instability of the electron microscope system and registration errors, which degrade the visual effect of the mosaicked EM images and aggravate the difficulty of follow-up treatment, such as automatic object recognition. Consequently, the grayscale correction method for multiple mosaicked electron microscope images is indispensable in these areas. Different from most previous grayscale correction methods, this paper designs a grayscale correction process for multiple EM images which tackles the difficulty of the multiple images monochrome correction and achieves the consistency of grayscale in the overlap regions. We adjust overall grayscale of the mosaicked images with the location and grayscale information of manual selected seed images, and then fuse local overlap regions between adjacent images using Poisson image editing. Experimental result demonstrates the effectiveness of our proposed method.

A nano grating tunable MEMS optical filter for high-speed on-chip multispectral fluorescent detection.

PubMed

Truxal, Steven C; Huang, Nien-Tsu; Kurabayashi, Katsuo

2009-01-01

We report a microelectromechanical (MEMS) tunable optical filter and its integration in a fluorescence microscope for high speed on-chip spectral measurements. This integration allows for measurements of any fluorescence sample placed onto the microscope stage. We demonstrate the system capabilities by taking spectral measurements of multicolor fluorescent beads and fluorescently labeled cells passing through a microfluidic cytometer. The system has applications in biological studies where the measurement of multiple fluorescent peaks is restricted by the detection method's speed and sensitivity.

Fast and accurate: high-speed metrological large-range AFM for surface and nanometrology

NASA Astrophysics Data System (ADS)

Dai, Gaoliang; Koenders, Ludger; Fluegge, Jens; Hemmleb, Matthias

2018-05-01

Low measurement speed remains a major shortcoming of the scanning probe microscopic technique. It not only leads to a low measurement throughput, but a significant measurement drift over the long measurement time needed (up to hours or even days). To overcome this challenge, PTB, the national metrology institute of Germany, has developed a high-speed metrological large-range atomic force microscope (HS Met. LR-AFM) capable of measuring speeds up to 1 mm s‑1. This paper has introduced the design concept in detail. After modelling scanning probe microscopic measurements, our results suggest that the signal spectrum of the surface to be measured is the spatial spectrum of the surface scaled by the scanning speed. The higher the scanning speed , the broader the spectrum to be measured. To realise an accurate HS Met. LR-AFM, our solution is to combine different stages/sensors synchronously in measurements, which provide a much larger spectrum area for high-speed measurement capability. Two application examples have been demonstrated. The first is a new concept called reference areal surface metrology. Using the developed HS Met. LR-AFM, surfaces are measured accurately and traceably at a speed of 500 µm s‑1 and the results are applied as a reference 3D data map of the surfaces. By correlating the reference 3D data sets and 3D data sets of tools under calibration, which are measured at the same surface, it has the potential to comprehensively characterise the tools, for instance, the spectrum properties of the tools. The investigation results of two commercial confocal microscopes are demonstrated, indicating very promising results. The second example is the calibration of a kind of 3D nano standard, which has spatially distributed landmarks, i.e. special unique features defined by 3D-coordinates. Experimental investigations confirmed that the calibration accuracy is maintained at a measurement speed of 100 µm s‑1, which improves the calibration efficiency by a factor of 10.

Testing a high-power LED based light source for hyperspectral imaging microscopy

NASA Astrophysics Data System (ADS)

Klomkaew, Phiwat; Mayes, Sam A.; Rich, Thomas C.; Leavesley, Silas J.

2017-02-01

Our lab has worked to develop high-speed hyperspectral imaging systems that scan the fluorescence excitation spectrum for biomedical imaging applications. Hyperspectral imaging can be used in remote sensing, medical imaging, reaction analysis, and other applications. Here, we describe the development of a hyperspectral imaging system that comprised an inverted Nikon Eclipse microscope, sCMOS camera, and a custom light source that utilized a series of high-power LEDs. LED selection was performed to achieve wavelengths of 350-590 nm. To reduce scattering, LEDs with low viewing angles were selected. LEDs were surface-mount soldered and powered by an RCD. We utilized 3D printed mounting brackets to assemble all circuit components. Spectraradiometric calibration was performed using a spectrometer (QE65000, Ocean Optics) and integrating sphere (FOIS-1, Ocean Optics). Optical output and LED driving current were measured over a range of illumination intensities. A normalization algorithm was used to calibrate and optimize the intensity of the light source. The highest illumination power was at 375 nm (3300 mW/cm2), while the lowest illumination power was at 515, 525, and 590 nm (5200 mW/cm2). Comparing the intensities supplied by each LED to the intensities measured at the microscope stage, we found there was a great loss in power output. Future work will focus on using two of the same LEDs to double the power and finding more LED and/or laser diodes and chips around the range. This custom hyperspectral imaging system could be used for the detection of cancer and the identification of biomolecules.

An Automated Method of Scanning Probe Microscopy (SPM) Data Analysis and Reactive Site Tracking for Mineral-Water Interface Reactions Observed at the Nanometer Scale

NASA Astrophysics Data System (ADS)

Campbell, B. D.; Higgins, S. R.

2008-12-01

Developing a method for bridging the gap between macroscopic and microscopic measurements of reaction kinetics at the mineral-water interface has important implications in geological and chemical fields. Investigating these reactions on the nanometer scale with SPM is often limited by image analysis and data extraction due to the large quantity of data usually obtained in SPM experiments. Here we present a computer algorithm for automated analysis of mineral-water interface reactions. This algorithm automates the analysis of sequential SPM images by identifying the kinetically active surface sites (i.e., step edges), and by tracking the displacement of these sites from image to image. The step edge positions in each image are readily identified and tracked through time by a standard edge detection algorithm followed by statistical analysis on the Hough Transform of the edge-mapped image. By quantifying this displacement as a function of time, the rate of step edge displacement is determined. Furthermore, the total edge length, also determined from analysis of the Hough Transform, combined with the computed step speed, yields the surface area normalized rate of the reaction. The algorithm was applied to a study of the spiral growth of the calcite(104) surface from supersaturated solutions, yielding results almost 20 times faster than performing this analysis by hand, with results being statistically similar for both analysis methods. This advance in analysis of kinetic data from SPM images will facilitate the building of experimental databases on the microscopic kinetics of mineral-water interface reactions.

A Review of Adaptive Optics Optical Coherence Tomography: Technical Advances, Scientific Applications, and the Future

PubMed Central

Jonnal, Ravi S.; Kocaoglu, Omer P.; Zawadzki, Robert J.; Liu, Zhuolin; Miller, Donald T.; Werner, John S.

2016-01-01

Purpose Optical coherence tomography (OCT) has enabled “virtual biopsy” of the living human retina, revolutionizing both basic retina research and clinical practice over the past 25 years. For most of those years, in parallel, adaptive optics (AO) has been used to improve the transverse resolution of ophthalmoscopes to foster in vivo study of the retina at the microscopic level. Here, we review work done over the last 15 years to combine the microscopic transverse resolution of AO with the microscopic axial resolution of OCT, building AO-OCT systems with the highest three-dimensional resolution of any existing retinal imaging modality. Methods We surveyed the literature to identify the most influential antecedent work, important milestones in the development of AO-OCT technology, its applications that have yielded new knowledge, research areas into which it may productively expand, and nascent applications that have the potential to grow. Results Initial efforts focused on demonstrating three-dimensional resolution. Since then, many improvements have been made in resolution and speed, as well as other enhancements of acquisition and postprocessing techniques. Progress on these fronts has produced numerous discoveries about the anatomy, function, and optical properties of the retina. Conclusions Adaptive optics OCT continues to evolve technically and to contribute to our basic and clinical knowledge of the retina. Due to its capacity to reveal cellular and microscopic detail invisible to clinical OCT systems, it is an ideal companion to those instruments and has the demonstrable potential to produce images that can guide the interpretation of clinical findings. PMID:27409507

Advanced atomic force microscopy: Development and application

NASA Astrophysics Data System (ADS)

Walters, Deron A.

Over the decade since atomic force microscopy (AFM) was invented, development of new microscopes has been closely intertwined with application of AFM to problems of interest in physics, chemistry, biology, and engineering. New techniques such as tapping mode AFM move quickly in our lab from the designer's bench to the user's table-since this is often the same piece of furniture. In return, designers get ample feedback as to what problems are limiting current instruments, and thus need most urgent attention. Tip sharpness and characterization are such a problem. Chapter 1 describes an AFM designed to operate in a scanning electron microscope, whose electron beam is used to deposit sharp carbonaceous tips. These tips can be tested and used in situ. Another limitation is addressed in Chapter 2: the difficulty of extracting more than just topographic information from a sample. A combined AFM/confocal optical microscope was built to provide simultaneous, independent images of the topography and fluorescence of a sample. In combination with staining or antibody labelling, this could provide submicron information about the composition of a sample. Chapters 3 and 4 discuss two generations of small cantilevers developed for lower-noise, higher-speed AFM of biological samples. In Chapter 4, a 26 mum cantilever is used to image the process of calcite growth from solution at a rate of 1.6 sec/frame. Finally, Chapter 5 explores in detail a biophysics problem that motivates us to develop fast, quiet, and gentle microscopes; namely, the control of crystal growth in seashells by the action of soluble proteins on a growing calcite surface.

Correction of image drift and distortion in a scanning electron microscopy.

PubMed

Jin, P; Li, X

2015-12-01

Continuous research on small-scale mechanical structures and systems has attracted strong demand for ultrafine deformation and strain measurements. Conventional optical microscope cannot meet such requirements owing to its lower spatial resolution. Therefore, high-resolution scanning electron microscope has become the preferred system for high spatial resolution imaging and measurements. However, scanning electron microscope usually is contaminated by distortion and drift aberrations which cause serious errors to precise imaging and measurements of tiny structures. This paper develops a new method to correct drift and distortion aberrations of scanning electron microscope images, and evaluates the effect of correction by comparing corrected images with scanning electron microscope image of a standard sample. The drift correction is based on the interpolation scheme, where a series of images are captured at one location of the sample and perform image correlation between the first image and the consequent images to interpolate the drift-time relationship of scanning electron microscope images. The distortion correction employs the axial symmetry model of charged particle imaging theory to two images sharing with the same location of one object under different imaging fields of view. The difference apart from rigid displacement between the mentioned two images will give distortion parameters. Three-order precision is considered in the model and experiment shows that one pixel maximum correction is obtained for the employed high-resolution electron microscopic system. © 2015 The Authors Journal of Microscopy © 2015 Royal Microscopical Society.

Automated measurement of diatom size

USGS Publications Warehouse

Spaulding, Sarah A.; Jewson, David H.; Bixby, Rebecca J.; Nelson, Harry; McKnight, Diane M.

2012-01-01

Size analysis of diatom populations has not been widely considered, but it is a potentially powerful tool for understanding diatom life histories, population dynamics, and phylogenetic relationships. However, measuring cell dimensions on a light microscope is a time-consuming process. An alternative technique has been developed using digital flow cytometry on a FlowCAM® (Fluid Imaging Technologies) to capture hundreds, or even thousands, of images of a chosen taxon from a single sample in a matter of minutes. Up to 30 morphological measures may be quantified through post-processing of the high resolution images. We evaluated FlowCAM size measurements, comparing them against measurements from a light microscope. We found good agreement between measurement of apical cell length in species with elongated, straight valves, including small Achnanthidium minutissimum (11-21 µm) and largeDidymosphenia geminata (87–137 µm) forms. However, a taxon with curved cells, Hannaea baicalensis (37–96 µm), showed differences of ~ 4 µm between the two methods. Discrepancies appear to be influenced by the choice of feret or geodesic measurement for asymmetric cells. We describe the operating conditions necessary for analysis of size distributions and present suggestions for optimal instrument conditions for size analysis of diatom samples using the FlowCAM. The increased speed of data acquisition through use of imaging flow cytometers like the FlowCAM is an essential step for advancing studies of diatom populations.

Investigation of light scattering characteristics of individual leukocytes using three-dimensional refractive index maps

NASA Astrophysics Data System (ADS)

Sung, Kung-Bin; Lin, Yang-Hsien; Lin, Fong-jheng; Hsieh, Chao-Mao; Wu, Shang-Ju

2017-04-01

Three-dimensional (3D) refractive-index (RI) microscopy is an emerging technique suitable for live-cell imaging due to its label-free and fast 3D imaging capabilities. We have developed a common-path system to acquire 3D RI microscopic images of cells with excellent speed and stability. After obtaining 3D RI distributions of individual leukocytes, we used a 3D finite-difference time-domain tool to study light scattering properties. Backscattering spectra of lymphocytes, monocytes and neutrophils are different from each other. Backscattering spectra of lymphocytes matched well with those of homogeneous spheres as predicted by Mie theory while backscattering spectra of neutrophils are significantly more intense than those of the other two types. This suggests the possibility of classifying the three types of leukocytes based on backscattering.

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

Kang, Hyeonggon; Attota, Ravikiran, E-mail: ravikiran.attota@nist.gov; Tondare, Vipin

We present a method that uses conventional optical microscopes to determine the number of nanoparticles in a cluster, which is typically not possible using traditional image-based optical methods due to the diffraction limit. The method, called through-focus scanning optical microscopy (TSOM), uses a series of optical images taken at varying focus levels to achieve this. The optical images cannot directly resolve the individual nanoparticles, but contain information related to the number of particles. The TSOM method makes use of this information to determine the number of nanoparticles in a cluster. Initial good agreement between the simulations and the measurements ismore » also presented. The TSOM method can be applied to fluorescent and non-fluorescent as well as metallic and non-metallic nano-scale materials, including soft materials, making it attractive for tag-less, high-speed, optical analysis of nanoparticles down to 45 nm diameter.« less

High Speed Photomicrography

NASA Astrophysics Data System (ADS)

Hyzer, William G.

1983-03-01

One of the most challenging areas in applying high-speed photography and videography in the plant and laboratory is in the recording of rapid events at macro and microscopic scales. Depth of field, exposure efficiency, working distance, and required exposure time are all reduced as optical magnification is increased, which severely taxes the skill and ingenuity of workers interested in recording any fast moving phenomena through the microscope or with magnifying lenses. This paper defines the problems inherent in photographing within macro and microscopic ranges and offers a systematic approach to optimizing the selection of equipment and choice of applicable techniques.

Tracking protein dynamics with photoconvertible Dendra2 on spinning disk confocal systems.

PubMed

Woods, Elena; Courtney, Jane; Scholz, Dimitri; Hall, William W; Gautier, Virginie W

2014-12-01

Understanding the dynamic properties of cellular proteins in live cells and in real time is essential to delineate their function. In this context, we introduce the Fluorescence Recovery After Photobleaching-Photoactivation unit (Andor) combined with the Nikon Eclipse Ti E Spinning Disk (Andor) confocal microscope as an advantageous and robust platform to exploit the properties of the Dendra2 photoconvertible fluorescent protein (Evrogen) and analyse protein subcellular trafficking in living cells. A major advantage of the spinning disk confocal is the rapid acquisition speed, enabling high temporal resolution of cellular processes. Furthermore, photoconversion and imaging are less invasive on the spinning disk confocal as the cell exposition to illumination power is reduced, thereby minimizing photobleaching and increasing cell viability. We have tested this commercially available platform using experimental settings adapted to track the migration of fast trafficking proteins such as UBC9, Fibrillarin and have successfully characterized their differential motion between subnuclear structures. We describe here step-by-step procedures, with emphasis on cellular imaging parameters, to successfully perform the dynamic imaging and photoconversion of Dendra2-fused proteins at high spatial and temporal resolutions necessary to characterize the trafficking pathways of proteins. © 2014 The Authors. Journal of Microscopy published by John Wiley & Sons, Ltd on behalf of Royal Microscopical Society.

Swept source optical coherence microscopy using a 1310 nm VCSEL light source

PubMed Central

Ahsen, Osman O.; Tao, Yuankai K.; Potsaid, Benjamin M.; Sheikine, Yuri; Jiang, James; Grulkowski, Ireneusz; Tsai, Tsung-Han; Jayaraman, Vijaysekhar; Kraus, Martin F.; Connolly, James L.; Hornegger, Joachim; Cable, Alex; Fujimoto, James G.

2013-01-01

We demonstrate high speed, swept source optical coherence microscopy (OCM) using a MEMS tunable vertical cavity surface-emitting laser (VCSEL) light source. The light source had a sweep rate of 280 kHz, providing a bidirectional axial scan rate of 560 kHz. The sweep bandwidth was 117 nm centered at 1310 nm, corresponding to an axial resolution of 13.1 µm in air, corresponding to 8.1 µm (9.6 µm spectrally shaped) in tissue. Dispersion mismatch from different objectives was compensated numerically, enabling magnification and field of view to be easily changed. OCM images were acquired with transverse resolutions between 0.86 µm - 3.42 µm using interchangeable 40X, 20X and 10X objectives with ~600 µm x 600 µm, ~1 mm x 1 mm and ~2 mm x 2 mm field-of-view (FOV), respectively. Parasitic variations in path length with beam scanning were corrected numerically. These features enable swept source OCM to be integrated with a wide range of existing scanning microscopes. Large FOV mosaics were generated by serially acquiring adjacent overlapping microscopic fields and combining them in post-processing. Fresh human colon, thyroid and kidney specimens were imaged ex vivo and compared to matching histology sections, demonstrating the ability of OCM to image tissue specimens. PMID:23938673

Automated imaging of cellular spheroids with selective plane illumination microscopy on a chip (Conference Presentation)

NASA Astrophysics Data System (ADS)

Paiè, Petra; Bassi, Andrea; Bragheri, Francesca; Osellame, Roberto

2017-02-01

Selective plane illumination microscopy (SPIM) is an optical sectioning technique that allows imaging of biological samples at high spatio-temporal resolution. Standard SPIM devices require dedicated set-ups, complex sample preparation and accurate system alignment, thus limiting the automation of the technique, its accessibility and throughput. We present a millimeter-scaled optofluidic device that incorporates selective plane illumination and fully automatic sample delivery and scanning. To this end an integrated cylindrical lens and a three-dimensional fluidic network were fabricated by femtosecond laser micromachining into a single glass chip. This device can upgrade any standard fluorescence microscope to a SPIM system. We used SPIM on a CHIP to automatically scan biological samples under a conventional microscope, without the need of any motorized stage: tissue spheroids expressing fluorescent proteins were flowed in the microchannel at constant speed and their sections were acquired while passing through the light sheet. We demonstrate high-throughput imaging of the entire sample volume (with a rate of 30 samples/min), segmentation and quantification in thick (100-300 μm diameter) cellular spheroids. This optofluidic device gives access to SPIM analyses to non-expert end-users, opening the way to automatic and fast screening of a high number of samples at subcellular resolution.

A control approach to cross-coupling compensation of piezotube scanners in tapping-mode atomic force microscope imaging.

PubMed

Wu, Ying; Shi, Jian; Su, Chanmin; Zou, Qingze

2009-04-01

In this article, an approach based on the recently developed inversion-based iterative control (IIC) to cancel the cross-axis coupling effect of piezoelectric tube scanners (piezoscanners) in tapping-mode atomic force microscope (AFM) imaging is proposed. Cross-axis coupling effect generally exists in piezoscanners used for three-dimensional (x-y-z axes) nanopositioning in applications such as AFM, where the vertical z-axis movement can be generated by the lateral x-y axes scanning. Such x/y-to-z cross-coupling becomes pronounced when the scanning is at large range and/or at high speed. In AFM applications, the coupling-caused position errors, when large, can generate various adverse effects, including large imaging and topography distortions, and damage of the cantilever probe and/or the sample. This paper utilizes the IIC technique to obtain the control input to precisely track the coupling-caused x/y-to-z displacement (with sign-flipped). Then the obtained input is augmented as a feedforward control to the existing feedback control in tapping-mode imaging, resulting in the cancellation of the coupling effect. The proposed approach is illustrated through two exemplary applications in industry, the pole-tip recession examination, and the nanoasperity measurement on hard-disk drive. Experimental results show that the x/y-to-z coupling effect in large-range (20 and 45 microm) tapping-mode imaging at both low to high scan rates (2, 12.2 to 24.4 Hz) can be effectively removed.

Telepresence in neurosurgery: the integrated remote neurosurgical system.

PubMed

Kassell, N F; Downs, J H; Graves, B S

1997-01-01

This paper describes the Integrated Remote Neurosurgical System (IRNS), a remotely-operated neurosurgical microscope with high-speed communications and a surgeon-accessible user interface. The IRNS will allow high quality bidirectional mentoring in the neurosurgical suite. The research goals of this effort are twofold: to develop a clinical system allowing a remote neurosurgeon to lend expertise to the OR-based neurosurgical team and to provide an integrated training environment. The IRNS incorporates a generic microscope/transport model, Called SuMIT (Surgical Manipulator Interface Translator). Our system is currently under test using the Zeiss MKM surgical transport. A SuMIT interface is also being constructed for the Robotics Research 1607. The IRNS Remote Planning and Navigation Workstation incorporates surgical planning capabilities, real-time, 30 fps video from the microscope and overhead video camera. The remote workstation includes a force reflecting handcontroller which gives the remote surgeon an intuitive way to position the microscope head. Bidirectional audio, video whiteboarding, and image archiving are also supported by the remote workstation. A simulation mode permits pre-surgical simulation, post-surgical critique, and training for surgeons without access to an actual microscope transport system. The components of the IRNS are integrated using ATM switching to provide low latency data transfer. The research, along with the more sophisticated systems that will follow, will serve as a foundation and test-bed for extending the surgeon's skills without regard to time zone or geographic boundaries.

Integrated circuit authentication using photon-limited x-ray microscopy.

PubMed

Markman, Adam; Javidi, Bahram

2016-07-15

A counterfeit integrated circuit (IC) may contain subtle changes to its circuit configuration. These changes may be observed when imaged using an x-ray; however, the energy from the x-ray can potentially damage the IC. We have investigated a technique to authenticate ICs under photon-limited x-ray imaging. We modeled an x-ray image with lower energy by generating a photon-limited image from a real x-ray image using a weighted photon-counting method. We performed feature extraction on the image using the speeded-up robust features (SURF) algorithm. We then authenticated the IC by comparing the SURF features to a database of SURF features from authentic and counterfeit ICs. Our experimental results with real and counterfeit ICs using an x-ray microscope demonstrate that we can correctly authenticate an IC image captured using orders of magnitude lower energy x-rays. To the best of our knowledge, this Letter is the first one on using a photon-counting x-ray imaging model and relevant algorithms to authenticate ICs to prevent potential damage.

Live Imaging of Shoot Meristems on an Inverted Confocal Microscope Using an Objective Lens Inverter Attachment

PubMed Central

Nimchuk, Zachary L.; Perdue, Tony D.

2017-01-01

Live imaging of above ground meristems can lead to new insights in plant development not possible from static imaging of fixed tissue. The use of an upright confocal microscope offers several technical and biological advantages for live imaging floral or shoot meristems. However, many departments and core facilities possess only inverted confocal microscopes and lack the funding for an additional upright confocal microscope. Here we show that imaging of living apical meristems can be performed on existing inverted confocal microscopes with the use of an affordable and detachable InverterScope accessory. PMID:28579995

Live Imaging of Shoot Meristems on an Inverted Confocal Microscope Using an Objective Lens Inverter Attachment.

PubMed

Nimchuk, Zachary L; Perdue, Tony D

2017-01-01

Live imaging of above ground meristems can lead to new insights in plant development not possible from static imaging of fixed tissue. The use of an upright confocal microscope offers several technical and biological advantages for live imaging floral or shoot meristems. However, many departments and core facilities possess only inverted confocal microscopes and lack the funding for an additional upright confocal microscope. Here we show that imaging of living apical meristems can be performed on existing inverted confocal microscopes with the use of an affordable and detachable InverterScope accessory.

Physics and engineering aspects of cell and tissue imaging systems: microscopic devices and computer assisted diagnosis.

PubMed

Chen, Xiaodong; Ren, Liqiang; Zheng, Bin; Liu, Hong

2013-01-01

The conventional optical microscopes have been used widely in scientific research and in clinical practice. The modern digital microscopic devices combine the power of optical imaging and computerized analysis, archiving and communication techniques. It has a great potential in pathological examinations for improving the efficiency and accuracy of clinical diagnosis. This chapter reviews the basic optical principles of conventional microscopes, fluorescence microscopes and electron microscopes. The recent developments and future clinical applications of advanced digital microscopic imaging methods and computer assisted diagnosis schemes are also discussed.

Whole-central nervous system functional imaging in larval Drosophila

PubMed Central

Lemon, William C.; Pulver, Stefan R.; Höckendorf, Burkhard; McDole, Katie; Branson, Kristin; Freeman, Jeremy; Keller, Philipp J.

2015-01-01

Understanding how the brain works in tight concert with the rest of the central nervous system (CNS) hinges upon knowledge of coordinated activity patterns across the whole CNS. We present a method for measuring activity in an entire, non-transparent CNS with high spatiotemporal resolution. We combine a light-sheet microscope capable of simultaneous multi-view imaging at volumetric speeds 25-fold faster than the state-of-the-art, a whole-CNS imaging assay for the isolated Drosophila larval CNS and a computational framework for analysing multi-view, whole-CNS calcium imaging data. We image both brain and ventral nerve cord, covering the entire CNS at 2 or 5 Hz with two- or one-photon excitation, respectively. By mapping network activity during fictive behaviours and quantitatively comparing high-resolution whole-CNS activity maps across individuals, we predict functional connections between CNS regions and reveal neurons in the brain that identify type and temporal state of motor programs executed in the ventral nerve cord. PMID:26263051

Fast live-cell conventional fluorophore nanoscopy with ImageJ through super-resolution radial fluctuations

PubMed Central

Gustafsson, Nils; Culley, Siân; Ashdown, George; Owen, Dylan M.; Pereira, Pedro Matos; Henriques, Ricardo

2016-01-01

Despite significant progress, high-speed live-cell super-resolution studies remain limited to specialized optical setups, generally requiring intense phototoxic illumination. Here, we describe a new analytical approach, super-resolution radial fluctuations (SRRF), provided as a fast graphics processing unit-enabled ImageJ plugin. In the most challenging data sets for super-resolution, such as those obtained in low-illumination live-cell imaging with GFP, we show that SRRF is generally capable of achieving resolutions better than 150 nm. Meanwhile, for data sets similar to those obtained in PALM or STORM imaging, SRRF achieves resolutions approaching those of standard single-molecule localization analysis. The broad applicability of SRRF and its performance at low signal-to-noise ratios allows super-resolution using modern widefield, confocal or TIRF microscopes with illumination orders of magnitude lower than methods such as PALM, STORM or STED. We demonstrate this by super-resolution live-cell imaging over timescales ranging from minutes to hours. PMID:27514992

Performance evaluation of image segmentation algorithms on microscopic image data.

PubMed

Beneš, Miroslav; Zitová, Barbara

2015-01-01

In our paper, we present a performance evaluation of image segmentation algorithms on microscopic image data. In spite of the existence of many algorithms for image data partitioning, there is no universal and 'the best' method yet. Moreover, images of microscopic samples can be of various character and quality which can negatively influence the performance of image segmentation algorithms. Thus, the issue of selecting suitable method for a given set of image data is of big interest. We carried out a large number of experiments with a variety of segmentation methods to evaluate the behaviour of individual approaches on the testing set of microscopic images (cross-section images taken in three different modalities from the field of art restoration). The segmentation results were assessed by several indices used for measuring the output quality of image segmentation algorithms. In the end, the benefit of segmentation combination approach is studied and applicability of achieved results on another representatives of microscopic data category - biological samples - is shown. © 2014 The Authors Journal of Microscopy © 2014 Royal Microscopical Society.

On-line 3-dimensional confocal imaging in vivo.

PubMed

Li, J; Jester, J V; Cavanagh, H D; Black, T D; Petroll, W M

2000-09-01

In vivo confocal microscopy through focusing (CMTF) can provide a 3-D stack of high-resolution corneal images and allows objective measurements of corneal sublayer thickness and backscattering. However, current systems require time-consuming off-line image processing and analysis on multiple software platforms. Furthermore, there is a trade off between the CMTF speed and measurement precision. The purpose of this study was to develop a novel on-line system for in vivo corneal imaging and analysis that overcomes these limitations. A tandem scanning confocal microscope (TSCM) was used for corneal imaging. The TSCM video camera was interfaced directly to a PC image acquisition board to implement real-time digitization. Software was developed to allow in vivo 2-D imaging, CMTF image acquisition, interactive 3-D reconstruction, and analysis of CMTF data to be performed on line in a single user-friendly environment. A procedure was also incorporated to separate the odd/even video fields, thereby doubling the CMTF sampling rate and theoretically improving the precision of CMTF thickness measurements by a factor of two. In vivo corneal examinations of a normal human and a photorefractive keratectomy patient are presented to demonstrate the capabilities of the new system. Improvements in the convenience, speed, and functionality of in vivo CMTF image acquisition, display, and analysis are demonstrated. This is the first full-featured software package designed for in vivo TSCM imaging of the cornea, which performs both 2-D and 3-D image acquisition, display, and processing as well as CMTF analysis. The use of a PC platform and incorporation of easy to use, on line, and interactive features should help to improve the clinical utility of this technology.

Development of a two photon microscope for tracking Drosophila larvae

NASA Astrophysics Data System (ADS)

Karagyozov, Doycho; Mihovilovic Skanata, Mirna; Gershow, Marc

Current in vivo methods for measuring neural activity in Drosophila larva require immobilization of the animal. Although we can record neural signals while stimulating the sensory organs, we cannot read the behavioral output because we have prevented the animal from moving. Many research questions cannot be answered without observation of neural activity in behaving (freely-moving) animals. We incorporated a Tunable Acoustic Gradient (TAG) lens into a two-photon microscope to achieve a 70kHz axial scan rate, enabling volumetric imaging at tens of hertz. We then implemented a tracking algorithm based on a Kalman filter to maintain the neurons of interest in the field of view and in focus during the rapid three dimensional motion of a free larva. Preliminary results show successful tracking of a neuron moving at speeds reaching 500 μm/s. NIH Grant 1DP2EB022359 and NSF Grant PHY-1455015.

Distinct speed dependence of entorhinal island and ocean cells, including respective grid cells

PubMed Central

Sun, Chen; Kitamura, Takashi; Yamamoto, Jun; Martin, Jared; Pignatelli, Michele; Kitch, Lacey J.; Schnitzer, Mark J.; Tonegawa, Susumu

2015-01-01

Entorhinal–hippocampal circuits in the mammalian brain are crucial for an animal’s spatial and episodic experience, but the neural basis for different spatial computations remain unknown. Medial entorhinal cortex layer II contains pyramidal island and stellate ocean cells. Here, we performed cell type-specific Ca2+ imaging in freely exploring mice using cellular markers and a miniature head-mounted fluorescence microscope. We found that both oceans and islands contain grid cells in similar proportions, but island cell activity, including activity in a proportion of grid cells, is significantly more speed modulated than ocean cell activity. We speculate that this differential property reflects island cells’ and ocean cells’ contribution to different downstream functions: island cells may contribute more to spatial path integration, whereas ocean cells may facilitate contextual representation in downstream circuits. PMID:26170279

Imaging electron motion in graphene

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

Bhandari, Sagar; Westervelt, Robert M.

A cooled scanning probe microscope (SPM) is an ideal tool to image electronic motion in graphene: the SPM tip acts as a scanning gate, which interacts with the electron gas below. We introduce the technique using our group's previous work on imaging electron flow from a quantum point contact in a GaAs 2DEG and tuning an InAs quantum dot in an InAs/InP nanowire. Carriers in graphene have very different characteristics: electrons and holes travel at a constant speed with no bandgap, and they pass through potential barriers via Klein tunneling. In this paper, we review the extension of SPM imagingmore » techniques to graphene. We image the cyclotron orbits passing between two narrow contacts in a single-atomic-layer graphene device in a perpendicular magnetic field. Magnetic focusing produces a peak in transmission between the contacts when the cyclotron diameter is equal to the contact spacing. The charged SPM tip deflects electrons passing from one contact to the other, changing the transmission when it interrupts the flow. By displaying the change in transmission as the tip is raster scanned above the sample, an image of flow is obtained. In addition, we have developed a complementary technique to image electronic charge using a cooled scanning capacitance microscope (SCM) that uses a sensitive charge preamplifier near the SPM tip to achieve a charge noise level 0.13 e Hz -1/2 with high spatial resolution 100 nm. The cooled SPM and SCM can be used to probe the motion of electrons on the nanoscale in graphene devices.« less

Imaging electron motion in graphene

DOE PAGES

Bhandari, Sagar; Westervelt, Robert M.

2017-01-05

A cooled scanning probe microscope (SPM) is an ideal tool to image electronic motion in graphene: the SPM tip acts as a scanning gate, which interacts with the electron gas below. We introduce the technique using our group's previous work on imaging electron flow from a quantum point contact in a GaAs 2DEG and tuning an InAs quantum dot in an InAs/InP nanowire. Carriers in graphene have very different characteristics: electrons and holes travel at a constant speed with no bandgap, and they pass through potential barriers via Klein tunneling. In this paper, we review the extension of SPM imagingmore » techniques to graphene. We image the cyclotron orbits passing between two narrow contacts in a single-atomic-layer graphene device in a perpendicular magnetic field. Magnetic focusing produces a peak in transmission between the contacts when the cyclotron diameter is equal to the contact spacing. The charged SPM tip deflects electrons passing from one contact to the other, changing the transmission when it interrupts the flow. By displaying the change in transmission as the tip is raster scanned above the sample, an image of flow is obtained. In addition, we have developed a complementary technique to image electronic charge using a cooled scanning capacitance microscope (SCM) that uses a sensitive charge preamplifier near the SPM tip to achieve a charge noise level 0.13 e Hz -1/2 with high spatial resolution 100 nm. The cooled SPM and SCM can be used to probe the motion of electrons on the nanoscale in graphene devices.« less

HelioScan: a software framework for controlling in vivo microscopy setups with high hardware flexibility, functional diversity and extendibility.

PubMed

Langer, Dominik; van 't Hoff, Marcel; Keller, Andreas J; Nagaraja, Chetan; Pfäffli, Oliver A; Göldi, Maurice; Kasper, Hansjörg; Helmchen, Fritjof

2013-04-30

Intravital microscopy such as in vivo imaging of brain dynamics is often performed with custom-built microscope setups controlled by custom-written software to meet specific requirements. Continuous technological advancement in the field has created a need for new control software that is flexible enough to support the biological researcher with innovative imaging techniques and provide the developer with a solid platform for quickly and easily implementing new extensions. Here, we introduce HelioScan, a software package written in LabVIEW, as a platform serving this dual role. HelioScan is designed as a collection of components that can be flexibly assembled into microscope control software tailored to the particular hardware and functionality requirements. Moreover, HelioScan provides a software framework, within which new functionality can be implemented in a quick and structured manner. A specific HelioScan application assembles at run-time from individual software components, based on user-definable configuration files. Due to its component-based architecture, HelioScan can exploit synergies of multiple developers working in parallel on different components in a community effort. We exemplify the capabilities and versatility of HelioScan by demonstrating several in vivo brain imaging modes, including camera-based intrinsic optical signal imaging for functional mapping of cortical areas, standard two-photon laser-scanning microscopy using galvanometric mirrors, and high-speed in vivo two-photon calcium imaging using either acousto-optic deflectors or a resonant scanner. We recommend HelioScan as a convenient software framework for the in vivo imaging community. Copyright © 2013 Elsevier B.V. All rights reserved.

Microscope basics.

PubMed

Sluder, Greenfield; Nordberg, Joshua J

2013-01-01

This chapter provides information on how microscopes work and discusses some of the microscope issues to be considered in using a video camera on the microscope. There are two types of microscopes in use today for research in cell biology-the older finite tube-length (typically 160mm mechanical tube length) microscopes and the infinity optics microscopes that are now produced. The objective lens forms a magnified, real image of the specimen at a specific distance from the objective known as the intermediate image plane. All objectives are designed to be used with the specimen at a defined distance from the front lens element of the objective (the working distance) so that the image formed is located at a specific location in the microscope. Infinity optics microscopes differ from the finite tube-length microscopes in that the objectives are designed to project the image of the specimen to infinity and do not, on their own, form a real image of the specimen. Three types of objectives are in common use today-plan achromats, plan apochromats, and plan fluorite lenses. The concept of mounting video cameras on the microscope is also presented in the chapter. Copyright © 2003 Elsevier Inc. All rights reserved.

High-speed, random-access fluorescence microscopy: I. High-resolution optical recording with voltage-sensitive dyes and ion indicators.

PubMed

Bullen, A; Patel, S S; Saggau, P

1997-07-01

The design and implementation of a high-speed, random-access, laser-scanning fluorescence microscope configured to record fast physiological signals from small neuronal structures with high spatiotemporal resolution is presented. The laser-scanning capability of this nonimaging microscope is provided by two orthogonal acousto-optic deflectors under computer control. Each scanning point can be randomly accessed and has a positioning time of 3-5 microseconds. Sampling time is also computer-controlled and can be varied to maximize the signal-to-noise ratio. Acquisition rates up to 200k samples/s at 16-bit digitizing resolution are possible. The spatial resolution of this instrument is determined by the minimal spot size at the level of the preparation (i.e., 2-7 microns). Scanning points are selected interactively from a reference image collected with differential interference contrast optics and a video camera. Frame rates up to 5 kHz are easily attainable. Intrinsic variations in laser light intensity and scanning spot brightness are overcome by an on-line signal-processing scheme. Representative records obtained with this instrument by using voltage-sensitive dyes and calcium indicators demonstrate the ability to make fast, high-fidelity measurements of membrane potential and intracellular calcium at high spatial resolution (2 microns) without any temporal averaging.

Single step high-speed printing of continuous silver lines by laser-induced forward transfer

NASA Astrophysics Data System (ADS)

Puerto, D.; Biver, E.; Alloncle, A.-P.; Delaporte, Ph.

2016-06-01

The development of high-speed ink printing process by Laser-Induced Forward Transfer (LIFT) is of great interest for the printing community. To address the problems and the limitations of this process that have been previously identified, we have performed an experimental study on laser micro-printing of silver nanoparticle inks by LIFT and demonstrated for the first time the printing of continuous conductive lines in a single pass at velocities of 17 m/s using a 1 MHz repetition rate laser. We investigated the printing process by means of a time-resolved imaging technique to visualize the ejection dynamics of single and adjacent jets. The control of the donor film properties is of prime importance to achieve single step printing of continuous lines at high velocities. We use a 30 ps pulse duration laser with a wavelength of 343 nm and a repetition rate from 0.2 to 1 MHz. A galvanometric mirror head controls the distance between two consecutives jets by scanning the focused beam along an ink-coated donor substrate at different velocities. Droplets and lines of silver inks are laser-printed on glass and PET flexible substrates and we characterized their morphological quality by atomic force microscope (AFM) and optical microscope.

Identification of staphylococcus species with hyperspectral microscope imaging and classification algrorithms

USDA-ARS?s Scientific Manuscript database

Hyperspectral microscope imaging is presented as a rapid and efficient tool to classify foodborne bacteria species. The spectral data were obtained from five different species of Staphylococcus spp. with a hyperspectral microscope imaging system that provided a maximum of 89 contiguous spectral imag...

Design of small confocal endo-microscopic probe working under multiwavelength environment

NASA Astrophysics Data System (ADS)

Kim, Young-Duk; Ahn, MyoungKi; Gweon, Dae-Gab

2010-02-01

Recently, optical imaging system is widely used in medical purpose. By using optical imaging system specific diseases can be easily diagnosed at early stage because optical imaging system has high resolution performance and various imaging method. These methods are used to get high resolution image of human body and can be used to verify whether the cell is infected by virus. Confocal microscope is one of the famous imaging systems which is used for in-vivo imaging. Because most of diseases are accompanied with cellular level changes, doctors can diagnosis at early stage by observing the cellular image of human organ. Current research is focused in the development of endo-microscope that has great advantage in accessibility to human body. In this research, I designed small probe that is connected to confocal microscope through optical fiber bundle and work as endo-microscope. And this small probe is mainly designed to correct chromatic aberration to use various laser sources for both fluorescence type and reflection type confocal images. By using two kinds of laser sources at the same time we demonstrated multi-modality confocal endo-microscope.

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

Target identification by image analysis.

PubMed

Fetz, V; Prochnow, H; Brönstrup, M; Sasse, F

2016-05-04

Covering: 1997 to the end of 2015Each biologically active compound induces phenotypic changes in target cells that are characteristic for its mode of action. These phenotypic alterations can be directly observed under the microscope or made visible by labelling structural elements or selected proteins of the cells with dyes. A comparison of the cellular phenotype induced by a compound of interest with the phenotypes of reference compounds with known cellular targets allows predicting its mode of action. While this approach has been successfully applied to the characterization of natural products based on a visual inspection of images, recent studies used automated microscopy and analysis software to increase speed and to reduce subjective interpretation. In this review, we give a general outline of the workflow for manual and automated image analysis, and we highlight natural products whose bacterial and eucaryotic targets could be identified through such approaches.

Towards two-photon excited endogenous fluorescence lifetime imaging microendoscopy

PubMed Central

Hage, C. H.; Leclerc, P.; Brevier, J.; Fabert, M.; Le Nézet, C.; Kudlinski, A.; Héliot, L.; Louradour, F.

2017-01-01

In situ fluorescence lifetime imaging microscopy (FLIM) in an endoscopic configuration of the endogenous biomarker nicotinamide adenine dinucleotide (NADH) has a great potential for malignant tissue diagnosis. Moreover, two-photon nonlinear excitation provides intrinsic optical sectioning along with enhanced imaging depth. We demonstrate, for the first time to our knowledge, nonlinear endogenous FLIM in a fibered microscope with proximal detection, applied to NADH in cultured cells, as a first step to a nonlinear endomicroscope, using a double-clad microstructured fiber with convenient fiber length (> 3 m) and excitation pulse duration (≈50 fs). Fluorescence photons are collected by the fiber inner cladding and we show that its contribution to the impulse response function (IRF), which originates from its intermodal and chromatic dispersions, is small (< 600 ps) and stable for lengths up to 8 m and allows for short lifetime measurements. We use the phasor representation as a quick visualization tool adapted to the endoscopy speed requirements. PMID:29359093

[Diffusion of fluorescent and magnetic molecular probes in brain interstitial space].

PubMed

Li, Huai-ye; Zhao, Yue; Zuo, Long; Fu, Yu; Li, Nan; Yuan, Lan; Zhang, Shu-jia; Han, Hong-bin

2015-08-18

To compare the diffusion properties of fluorescent probes dextran-tetramethylrhodamine (DT) and lucifer yellow CH (LY) and magnetic probe gadolinium-diethylene triamine pentaacetic acid (Gd-DTPA) in porous media and to screen out a suitable fluorescent probe for optical imaging of brain interstitial space (ISS). Agarose gels sample were divided into DT group, LY group and Gd-DTPA group, and the corresponding molecular probes were imported in each group. The dynamic diffusions of DT and LY in agarose gels at different time points (15, 30, 45, 60, 90, and 120 min) were scanned with laser scanning confocal microscope, the dynamic diffusion of Gd-DTPA was imaged with magnetic resonance imaging. The average diffusion speed of LY were demonstrated to be consistent with those of Gd-DTPA. The LY was introduced into caudate putamen of 18 rats, respectively, the diffusion of LY in the sequential slices of rat brain at different time points (0.5, 1, 2, 3, 7, 11 h) were scanned, and the results were compared with those of rats' brain with Gd-DTPA imported and imaged in vivo with magnetic resonance imaging. The diffusions of the three probes were isotropic in the agarose gels, and the average diffusion speeds of DT, LY and Gd-DTPA were: (0.07±0.02)×10(-2) mm2/s, (1.54±0.47)×10(-2) mm2/s, (1.45±0.50)×10(-2) mm2/s, respectively. The speed of DT was more slower than both LY and Gd-DTPA (ANOVA, F=367.15, P<0.001; Post-Hoc LSD, P<0.001), and there was no significant difference between the speeds of LY and Gd-DTPA (Post-Hoc LSD, P=0.091). The variation tendency of diffusion area of DT was different with both that of LY and that of Gd-DTPA (Bonferroni correction, α=0.0125, P<0.001), and there was no significant difference between LY and Gd-DTPA (Bonferroni correction, α=0.0125, P=0.203), in analysis by repeated measures data of ANOVA. The diffusions of LY and Gd-DTPA were anisotropy in rat caudate putamen,and the average diffusion speeds of LY and Gd-DTPA were: (1.03±0.29)×10(-3) mm2/s, (0.81±0.27)×10(-3) mm2/s, respectively, no significant difference was demonstrated (t=0.759, P=0.490); half-time of single intensity of LY and Gd-DTPA was (2.58±0.04) h, (2.46±0.10) h, respectively, no significant difference was found (t=2.025, P=0.113). The diffusion area ratios between LY and Gd-DTPA in rat caudate putamen was not statistically different at hours 0.5, 1, 2, 3 and 7 (t=2.249, P=0.088; t=2.582, P=0.061; t=1.966, P=0.121; t=0.132, P=0.674; t=0.032, P=0.976), while, a slightly difference was found at 11 h (t=2.917, P=0.043,in analysis by t test). LY present the same diffusion property with Gd-DTPA in porous media witch including agarose gels and live rat brain tissue, indicates that LY is a suitable fluorescent probe for optical imaging of brain ISS, and it can be used for microscopic, macro and in vitro measure of brain ISS.

Simultaneous imaging of cellular morphology and multiple biomarkers using an acousto-optic tunable filter-based bright field microscope.

PubMed

Wachman, Elliot S; Geyer, Stanley J; Recht, Joel M; Ward, Jon; Zhang, Bill; Reed, Murray; Pannell, Chris

2014-05-01

An acousto-optic tunable filter (AOTF)-based multispectral imaging microscope system allows the combination of cellular morphology and multiple biomarker stainings on a single microscope slide. We describe advances in AOTF technology that have greatly improved spectral purity, field uniformity, and image quality. A multispectral imaging bright field microscope using these advances demonstrates pathology results that have great potential for clinical use.

Apertureless near-field/far-field CW two-photon microscope for biological and material imaging and spectroscopic applications.

PubMed

Nowak, Derek B; Lawrence, A J; Sánchez, Erik J

2010-12-10

We present the development of a versatile spectroscopic imaging tool to allow for imaging with single-molecule sensitivity and high spatial resolution. The microscope allows for near-field and subdiffraction-limited far-field imaging by integrating a shear-force microscope on top of a custom inverted microscope design. The instrument has the ability to image in ambient conditions with optical resolutions on the order of tens of nanometers in the near field. A single low-cost computer controls the microscope with a field programmable gate array data acquisition card. High spatial resolution imaging is achieved with an inexpensive CW multiphoton excitation source, using an apertureless probe and simplified optical pathways. The high-resolution, combined with high collection efficiency and single-molecule sensitive optical capabilities of the microscope, are demonstrated with a low-cost CW laser source as well as a mode-locked laser source.

Design of a Single-Cell Positioning Controller Using Electroosmotic Flow and Image Processing

PubMed Central

Ay, Chyung; Young, Chao-Wang; Chen, Jhong-Yin

2013-01-01

The objective of the current research was not only to provide a fast and automatic positioning platform for single cells, but also improved biomolecular manipulation techniques. In this study, an automatic platform for cell positioning using electroosmotic flow and image processing technology was designed. The platform was developed using a PCI image acquisition interface card for capturing images from a microscope and then transferring them to a computer using human-machine interface software. This software was designed by the Laboratory Virtual Instrument Engineering Workbench, a graphical language for finding cell positions and viewing the driving trace, and the fuzzy logic method for controlling the voltage or time of an electric field. After experiments on real human leukemic cells (U-937), the success of the cell positioning rate achieved by controlling the voltage factor reaches 100% within 5 s. A greater precision is obtained when controlling the time factor, whereby the success rate reaches 100% within 28 s. Advantages in both high speed and high precision are attained if these two voltage and time control methods are combined. The control speed with the combined method is about 5.18 times greater than that achieved by the time method, and the control precision with the combined method is more than five times greater than that achieved by the voltage method. PMID:23698272

Temporal and spatial binning of TCSPC data to improve signal-to-noise ratio and imaging speed

NASA Astrophysics Data System (ADS)

Walsh, Alex J.; Beier, Hope T.

2016-03-01

Time-correlated single photon counting (TCSPC) is the most robust method for fluorescence lifetime imaging using laser scanning microscopes. However, TCSPC is inherently slow making it ineffective to capture rapid events due to the single photon product per laser pulse causing extensive acquisition time limitations and the requirement of low fluorescence emission efficiency to avoid bias of measurement towards short lifetimes. Furthermore, thousands of photons per pixel are required for traditional instrument response deconvolution and fluorescence lifetime exponential decay estimation. Instrument response deconvolution and fluorescence exponential decay estimation can be performed in several ways including iterative least squares minimization and Laguerre deconvolution. This paper compares the limitations and accuracy of these fluorescence decay analysis techniques to accurately estimate double exponential decays across many data characteristics including various lifetime values, lifetime component weights, signal-to-noise ratios, and number of photons detected. Furthermore, techniques to improve data fitting, including binning data temporally and spatially, are evaluated as methods to improve decay fits and reduce image acquisition time. Simulation results demonstrate that binning temporally to 36 or 42 time bins, improves accuracy of fits for low photon count data. Such a technique reduces the required number of photons for accurate component estimation if lifetime values are known, such as for commercial fluorescent dyes and FRET experiments, and improve imaging speed 10-fold.

Penny for Your Reference

NASA Technical Reports Server (NTRS)

2004-01-01

15 April 2004 This close-up image of a penny shows the degree to which the microscopic imager on the Mars Exploration Rover Spirit can zoom in on a target. The penny is seen exactly as it would be on Mars if it were placed under the microscopic imager. This picture was taken by the imager during testing at JPL.

[figure removed for brevity, see original site] Spirit's Microscopic Vision Demonstrated

This close-up image of a penny shows the power of the microscopic imager onboard the Mars Exploration Rover Spirit to see fine details. The picture was taken by the imager during testing at JPL.

A Comparative Study of Microscopic Images Captured by a Box Type Digital Camera Versus a Standard Microscopic Photography Camera Unit

PubMed Central

Desai, Nandini J.; Gupta, B. D.; Patel, Pratik Narendrabhai

2014-01-01

Introduction: Obtaining images of slides viewed by a microscope can be invaluable for both diagnosis and teaching.They can be transferred among technologically-advanced hospitals for further consultation and evaluation. But a standard microscopic photography camera unit (MPCU)(MIPS-Microscopic Image projection System) is costly and not available in resource poor settings. The aim of our endeavour was to find a comparable and cheaper alternative method for photomicrography. Materials and Methods: We used a NIKON Coolpix S6150 camera (box type digital camera) with Olympus CH20i microscope and a fluorescent microscope for the purpose of this study. Results: We got comparable results for capturing images of light microscopy, but the results were not as satisfactory for fluorescent microscopy. Conclusion: A box type digital camera is a comparable, less expensive and convenient alternative to microscopic photography camera unit. PMID:25478350

Breaking the Time Barrier in Kelvin Probe Force Microscopy: Fast Free Force Reconstruction Using the G-Mode Platform

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

Collins, Liam; Ahmadi, Mahshid; Wu, Ting

The atomic force microscope (AFM) offers unparalleled insight into structure and material functionality across nanometer length scales. However, the spatial resolution afforded by the AFM tip is counterpoised by slow detection speeds compared to other common microscopy techniques (e.g. optical, scanning electron microscopy etc.). In this work, we develop an AFM imaging approach allowing ultrafast reconstruction of the tip-sample forces having ~2 orders of magnitude higher time resolution than standard detection methods. Fast free force recovery (F3R) overcomes the widely-viewed temporal bottleneck in AFM, i.e. the mechanical bandwidth of the cantilever, enabling time-resolved imaging at sub-bandwidth speeds. We demonstrate quantitativemore » recovery of electrostatic forces with ~10 µs temporal resolution, free from cantilever ring-down effects. We further apply the F3R method to Kelvin probe force microscopy (KPFM) measurements. F3R-KPFM is an open loop imaging approach (i.e. no bias feedback), allowing ultrafast surface potential measurements (e.g. < 20 µs) to be performed at regular KPFM scan speeds. F3R-KPFM is demonstrated for exploration of ion migration in organometallic halide perovskites materials and shown to allow spatio-temporal imaging of positively charged ion migration under applied electric field, as well as subsequent formation of accumulated charges at the perovskite/electrode interface. In this work we demonstrate quantitative F3R-KPFM measurements – however, we fully expect the F3R approach to be valid for all modes of non-contact AFM operation, including non-invasive probing of ultrafast electrical and magnetic dynamics.« less

Breaking the Time Barrier in Kelvin Probe Force Microscopy: Fast Free Force Reconstruction Using the G-Mode Platform

DOE PAGES

Collins, Liam; Ahmadi, Mahshid; Wu, Ting; ...

2017-08-06

The atomic force microscope (AFM) offers unparalleled insight into structure and material functionality across nanometer length scales. However, the spatial resolution afforded by the AFM tip is counterpoised by slow detection speeds compared to other common microscopy techniques (e.g. optical, scanning electron microscopy etc.). In this work, we develop an AFM imaging approach allowing ultrafast reconstruction of the tip-sample forces having ~2 orders of magnitude higher time resolution than standard detection methods. Fast free force recovery (F3R) overcomes the widely-viewed temporal bottleneck in AFM, i.e. the mechanical bandwidth of the cantilever, enabling time-resolved imaging at sub-bandwidth speeds. We demonstrate quantitativemore » recovery of electrostatic forces with ~10 µs temporal resolution, free from cantilever ring-down effects. We further apply the F3R method to Kelvin probe force microscopy (KPFM) measurements. F3R-KPFM is an open loop imaging approach (i.e. no bias feedback), allowing ultrafast surface potential measurements (e.g. < 20 µs) to be performed at regular KPFM scan speeds. F3R-KPFM is demonstrated for exploration of ion migration in organometallic halide perovskites materials and shown to allow spatio-temporal imaging of positively charged ion migration under applied electric field, as well as subsequent formation of accumulated charges at the perovskite/electrode interface. In this work we demonstrate quantitative F3R-KPFM measurements – however, we fully expect the F3R approach to be valid for all modes of non-contact AFM operation, including non-invasive probing of ultrafast electrical and magnetic dynamics.« less

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.

Simplified Automated Image Analysis for Detection and Phenotyping of Mycobacterium tuberculosis on Porous Supports by Monitoring Growing Microcolonies

PubMed Central

den Hertog, Alice L.; Visser, Dennis W.; Ingham, Colin J.; Fey, Frank H. A. G.; Klatser, Paul R.; Anthony, Richard M.

2010-01-01

Background Even with the advent of nucleic acid (NA) amplification technologies the culture of mycobacteria for diagnostic and other applications remains of critical importance. Notably microscopic observed drug susceptibility testing (MODS), as opposed to traditional culture on solid media or automated liquid culture, has shown potential to both speed up and increase the provision of mycobacterial culture in high burden settings. Methods Here we explore the growth of Mycobacterial tuberculosis microcolonies, imaged by automated digital microscopy, cultured on a porous aluminium oxide (PAO) supports. Repeated imaging during colony growth greatly simplifies “computer vision” and presumptive identification of microcolonies was achieved here using existing publically available algorithms. Our system thus allows the growth of individual microcolonies to be monitored and critically, also to change the media during the growth phase without disrupting the microcolonies. Transfer of identified microcolonies onto selective media allowed us, within 1-2 bacterial generations, to rapidly detect the drug susceptibility of individual microcolonies, eliminating the need for time consuming subculturing or the inoculation of multiple parallel cultures. Significance Monitoring the phenotype of individual microcolonies as they grow has immense potential for research, screening, and ultimately M. tuberculosis diagnostic applications. The method described is particularly appealing with respect to speed and automation. PMID:20544033

Microscopic simulation model calibration and validation handbook.

DOT National Transportation Integrated Search

2006-01-01

Microscopic traffic simulation models are widely used in the transportation engineering field. Because of their cost-effectiveness, risk-free nature, and high-speed benefits, areas of use include transportation system design, traffic operations, and ...

HoloGondel: in situ cloud observations on a cable car in the Swiss Alps using a holographic imager

NASA Astrophysics Data System (ADS)

Beck, Alexander; Henneberger, Jan; Schöpfer, Sarah; Fugal, Jacob; Lohmann, Ulrike

2017-02-01

In situ observations of cloud properties in complex alpine terrain where research aircraft cannot sample are commonly conducted at mountain-top research stations and limited to single-point measurements. The HoloGondel platform overcomes this limitation by using a cable car to obtain vertical profiles of the microphysical and meteorological cloud parameters. The main component of the HoloGondel platform is the HOLographic Imager for Microscopic Objects (HOLIMO 3G), which uses digital in-line holography to image cloud particles. Based on two-dimensional images the microphysical cloud parameters for the size range from small cloud particles to large precipitation particles are obtained for the liquid and ice phase. The low traveling velocity of a cable car on the order of 10 m s-1 allows measurements with high spatial resolution; however, at the same time it leads to an unstable air speed towards the HoloGondel platform. Holographic cloud imagers, which have a sample volume that is independent of the air speed, are therefore well suited for measurements on a cable car. Example measurements of the vertical profiles observed in a liquid cloud and a mixed-phase cloud at the Eggishorn in the Swiss Alps in the winters 2015 and 2016 are presented. The HoloGondel platform reliably observes cloud droplets larger than 6.5 µm, partitions between cloud droplets and ice crystals for a size larger than 25 µm and obtains a statistically significantly size distribution for every 5 m in vertical ascent.

Dynamic speckle illumination wide-field reflection phase microscopy

PubMed Central

Choi, Youngwoon; Hosseini, Poorya; Choi, Wonshik; Dasari, Ramachandra R.; So, Peter T. C.; Yaqoob, Zahid

2014-01-01

We demonstrate a quantitative reflection-phase microscope based on time-varying speckle-field illumination. Due to the short spatial coherence length of the speckle field, the proposed imaging system features superior lateral resolution, 520 nm, as well as high-depth selectivity, 1.03 µm. Off-axis interferometric detection enables wide-field and single-shot imaging appropriate for high-speed measurements. In addition, the measured phase sensitivity of this method, which is the smallest measurable axial motion, is more than 40 times higher than that available using a transmission system. We demonstrate the utility of our method by successfully distinguishing the motion of the top surface from that of the bottom in red blood cells. The proposed method will be useful for studying membrane dynamics in complex eukaryotic cells. PMID:25361156

Scanning Microscopes Using X Rays and Microchannels

NASA Technical Reports Server (NTRS)

Wang, Yu

2003-01-01

Scanning microscopes that would be based on microchannel filters and advanced electronic image sensors and that utilize x-ray illumination have been proposed. Because the finest resolution attainable in a microscope is determined by the wavelength of the illumination, the xray illumination in the proposed microscopes would make it possible, in principle, to achieve resolutions of the order of nanometers about a thousand times as fine as the resolution of a visible-light microscope. Heretofore, it has been necessary to use scanning electron microscopes to obtain such fine resolution. In comparison with scanning electron microscopes, the proposed microscopes would likely be smaller, less massive, and less expensive. Moreover, unlike in scanning electron microscopes, it would not be necessary to place specimens under vacuum. The proposed microscopes are closely related to the ones described in several prior NASA Tech Briefs articles; namely, Miniature Microscope Without Lenses (NPO-20218), NASA Tech Briefs, Vol. 22, No. 8 (August 1998), page 43; and Reflective Variants of Miniature Microscope Without Lenses (NPO-20610), NASA Tech Briefs, Vol. 26, No. 9 (September 2002) page 6a. In all of these microscopes, the basic principle of design and operation is the same: The focusing optics of a conventional visible-light microscope are replaced by a combination of a microchannel filter and a charge-coupled-device (CCD) image detector. A microchannel plate containing parallel, microscopic-cross-section holes much longer than they are wide is placed between a specimen and an image sensor, which is typically the CCD. The microchannel plate must be made of a material that absorbs the illuminating radiation reflected or scattered from the specimen. The microchannels must be positioned and dimensioned so that each one is registered with a pixel on the image sensor. Because most of the radiation incident on the microchannel walls becomes absorbed, the radiation that reaches the image sensor consists predominantly of radiation that was launched along the longitudinal direction of the microchannels. Therefore, most of the radiation arriving at each pixel on the sensor must have traveled along a straight line from a corresponding location on the specimen. Thus, there is a one-to-one mapping from a point on a specimen to a pixel in the image sensor, so that the output of the image sensor contains image information equivalent to that from a microscope.

A remote operating slit lamp microscope system. Development and its utility in ophthalmologic examinations.

PubMed

Tanabe, N; Go, K; Sakurada, Y; Imasawa, M; Mabuchi, F; Chiba, T; Abe, K; Kashiwagi, K

2011-01-01

To develop a remote-operating slit lamp microscope system (the remote slit lamp) as the core for highly specialized ophthalmology diagnoses, and to compare the utility of this system with the conventional slit lamp microscope system (the conventional slit lamp) in making a diagnosis. The remote slit lamp system was developed. Three factors were evaluated in comparison to the conventional slit lamp. The ability to acquire skills was investigated using a task loading system among specialists and residents in ophthalmology. Participants repeated a task up to ten times and the time required for each task was analyzed. The consistency of the two systems in making a diagnosis was investigated using eyes of patients with ocular diseases as well as healthy volunteers. The remote slit lamp is composed of a patient's unit and ophthalmologist's unit connected by high-speed internet. The two units share images acquired by the slit lamp in addition to the images and voices of patients and ophthalmologists. Both ophthalmology specialists and residents could minimize the completion times after several trials. The remote slit lamp took more time than the conventional slit lamp. Both systems showed a high consistency in evaluations among eyes with healthy eyes or those with ocular diseases. The remote slit lamp has a similar diagnostic ability, but required more examination time in comparison to the conventional slit lamp. The currently developed remote slit lamp has the potential to be employed for tele-medicine purposes in the field of ophthalmology.

Openstage: A Low-Cost Motorized Microscope Stage with Sub-Micron Positioning Accuracy

PubMed Central

Campbell, Robert A. A.; Eifert, Robert W.; Turner, Glenn C.

2014-01-01

Recent progress in intracellular calcium sensors and other fluorophores has promoted the widespread adoption of functional optical imaging in the life sciences. Home-built multiphoton microscopes are easy to build, highly customizable, and cost effective. For many imaging applications a 3-axis motorized stage is critical, but commercially available motorization hardware (motorized translators, controller boxes, etc) are often very expensive. Furthermore, the firmware on commercial motor controllers cannot easily be altered and is not usually designed with a microscope stage in mind. Here we describe an open-source motorization solution that is simple to construct, yet far cheaper and more customizable than commercial offerings. The cost of the controller and motorization hardware are under $1000. Hardware costs are kept low by replacing linear actuators with high quality stepper motors. Electronics are assembled from commonly available hobby components, which are easy to work with. Here we describe assembly of the system and quantify the positioning accuracy of all three axes. We obtain positioning repeatability of the order of in X/Y and in Z. A hand-held control-pad allows the user to direct stage motion precisely over a wide range of speeds ( to ), rapidly store and return to different locations, and execute “jumps” of a fixed size. In addition, the system can be controlled from a PC serial port. Our “OpenStage” controller is sufficiently flexible that it could be used to drive other devices, such as micro-manipulators, with minimal modifications. PMID:24586468

Design of a normal incidence multilayer imaging X-ray microscope

NASA Astrophysics Data System (ADS)

Shealy, David L.; Gabardi, David R.; Hoover, Richard B.; Walker, Arthur B. C., Jr.; Lindblom, Joakim F.

Normal incidence multilayer Cassegrain X-ray telescopes were flown on the Stanford/MSFC Rocket X-ray Spectroheliograph. These instruments produced high spatial resolution images of the sun and conclusively demonstrated that doubly reflecting multilayer X-ray optical systems are feasible. The images indicated that aplanatic imaging soft X-ray/EUV microscopes should be achievable using multilayer optics technology. A doubly reflecting normal incidence multilayer imaging X-ray microscope based on the Schwarzschild configuration has been designed. The design of the microscope and the results of the optical system ray trace analysis are discussed. High resolution aplanatic imaging X-ray microscopes using normal incidence multilayer X-ray mirrors should have many important applications in advanced X-ray astronomical instrumentation, X-ray lithography, biological, biomedical, metallurgical, and laser fusion research.

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.

Femtosecond electron imaging of defect-modulated phonon dynamics

PubMed Central

Cremons, Daniel R.; Plemmons, Dayne A.; Flannigan, David J.

2016-01-01

Precise manipulation and control of coherent lattice oscillations via nanostructuring and phonon-wave interference has the potential to significantly impact a broad array of technologies and research areas. Resolving the dynamics of individual phonons in defect-laden materials presents an enormous challenge, however, owing to the interdependent nanoscale and ultrafast spatiotemporal scales. Here we report direct, real-space imaging of the emergence and evolution of acoustic phonons at individual defects in crystalline WSe2 and Ge. Via bright-field imaging with an ultrafast electron microscope, we are able to image the sub-picosecond nucleation and the launch of wavefronts at step edges and resolve dispersion behaviours during propagation and scattering. We discover that the appearance of speed-of-sound (for example, 6 nm ps−1) wavefronts are influenced by spatially varying nanoscale strain fields, taking on the appearance of static bend contours during propagation. These observations provide unprecedented insight into the roles played by individual atomic and nanoscale features on acoustic-phonon dynamics. PMID:27079790

Methyl green and nitrotetrazolium blue chloride co-expression in colon tissue: A hyperspectral microscopic imaging analysis

NASA Astrophysics Data System (ADS)

Li, Qingli; Liu, Hongying; Wang, Yiting; Sun, Zhen; Guo, Fangmin; Zhu, Jianzhong

2014-12-01

Histological observation of dual-stained colon sections is usually performed by visual observation under a light microscope, or by viewing on a computer screen with the assistance of image processing software in both research and clinical settings. These traditional methods are usually not sufficient to reliably differentiate spatially overlapping chromogens generated by different dyes. Hyperspectral microscopic imaging technology offers a solution for these constraints as the hyperspectral microscopic images contain information that allows differentiation between spatially co-located chromogens with similar but different spectra. In this paper, a hyperspectral microscopic imaging (HMI) system is used to identify methyl green and nitrotetrazolium blue chloride in dual-stained colon sections. Hyperspectral microscopic images are captured and the normalized score algorithm is proposed to identify the stains and generate the co-expression results. Experimental results show that the proposed normalized score algorithm can generate more accurate co-localization results than the spectral angle mapper algorithm. The hyperspectral microscopic imaging technology can enhance the visualization of dual-stained colon sections, improve the contrast and legibility of each stain using their spectral signatures, which is helpful for pathologist performing histological analyses.

Automatic Focus Adjustment of a Microscope

NASA Technical Reports Server (NTRS)

Huntsberger, Terrance

2005-01-01

AUTOFOCUS is a computer program for use in a control system that automatically adjusts the position of an instrument arm that carries a microscope equipped with an electronic camera. In the original intended application of AUTOFOCUS, the imaging microscope would be carried by an exploratory robotic vehicle on a remote planet, but AUTOFOCUS could also be adapted to similar applications on Earth. Initially control software other than AUTOFOCUS brings the microscope to a position above a target to be imaged. Then the instrument arm is moved to lower the microscope toward the target: nominally, the target is approached from a starting distance of 3 cm in 10 steps of 3 mm each. After each step, the image in the camera is subjected to a wavelet transform, which is used to evaluate the texture in the image at multiple scales to determine whether and by how much the microscope is approaching focus. A focus measure is derived from the transform and used to guide the arm to bring the microscope to the focal height. When the analysis reveals that the microscope is in focus, image data are recorded and transmitted.

A neotropical Miocene pollen database employing image-based search and semantic modeling.

PubMed

Han, Jing Ginger; Cao, Hongfei; Barb, Adrian; Punyasena, Surangi W; Jaramillo, Carlos; Shyu, Chi-Ren

2014-08-01

Digital microscopic pollen images are being generated with increasing speed and volume, producing opportunities to develop new computational methods that increase the consistency and efficiency of pollen analysis and provide the palynological community a computational framework for information sharing and knowledge transfer. • Mathematical methods were used to assign trait semantics (abstract morphological representations) of the images of neotropical Miocene pollen and spores. Advanced database-indexing structures were built to compare and retrieve similar images based on their visual content. A Web-based system was developed to provide novel tools for automatic trait semantic annotation and image retrieval by trait semantics and visual content. • Mathematical models that map visual features to trait semantics can be used to annotate images with morphology semantics and to search image databases with improved reliability and productivity. Images can also be searched by visual content, providing users with customized emphases on traits such as color, shape, and texture. • Content- and semantic-based image searches provide a powerful computational platform for pollen and spore identification. The infrastructure outlined provides a framework for building a community-wide palynological resource, streamlining the process of manual identification, analysis, and species discovery.

Atomic force microscopy contact, tapping, and jumping modes for imaging biological samples in liquids

NASA Astrophysics Data System (ADS)

Moreno-Herrero, F.; Colchero, J.; Gómez-Herrero, J.; Baró, A. M.

2004-03-01

The capabilities of the atomic force microscope for imaging biomolecules under physiological conditions has been systematically investigated. Contact, dynamic, and jumping modes have been applied to four different biological systems: DNA, purple membrane, Alzheimer paired helical filaments, and the bacteriophage φ29. These samples have been selected to cover a wide variety of biological systems in terms of sizes and substrate contact area, which make them very appropriate for the type of comparative studies carried out in the present work. Although dynamic mode atomic force microscopy is clearly the best choice for imaging soft samples in air, in liquids there is not a leading technique. In liquids, the most appropriate imaging mode depends on the sample characteristics and preparation methods. Contact or dynamic modes are the best choices for imaging molecular assemblies arranged as crystals such as the purple membrane. In this case, the advantage of image acquisition speed predominates over the disadvantage of high lateral or normal force. For imaging individual macromolecules, which are weakly bonded to the substrate, lateral and normal forces are the relevant factors, and hence the jumping mode, an imaging mode which minimizes lateral and normal forces, is preferable to other imaging modes.

Characterization and Effects of Fiber Pull-Outs in Hole Quality of Carbon Fiber Reinforced Plastics Composite.

PubMed

Alizadeh Ashrafi, Sina; Miller, Peter W; Wandro, Kevin M; Kim, Dave

2016-10-13

Hole quality plays a crucial role in the production of close-tolerance holes utilized in aircraft assembly. Through drilling experiments of carbon fiber-reinforced plastic composites (CFRP), this study investigates the impact of varying drilling feed and speed conditions on fiber pull-out geometries and resulting hole quality parameters. For this study, hole quality parameters include hole size variance, hole roundness, and surface roughness. Fiber pull-out geometries are quantified by using scanning electron microscope (SEM) images of the mechanically-sectioned CFRP-machined holes, to measure pull-out length and depth. Fiber pull-out geometries and the hole quality parameter results are dependent on the drilling feed and spindle speed condition, which determines the forces and undeformed chip thickness during the process. Fiber pull-out geometries influence surface roughness parameters from a surface profilometer, while their effect on other hole quality parameters obtained from a coordinate measuring machine is minimal.

fastSIM: a practical implementation of fast structured illumination microscopy.

PubMed

Lu-Walther, Hui-Wen; Kielhorn, Martin; Förster, Ronny; Jost, Aurélie; Wicker, Kai; Heintzmann, Rainer

2015-01-16

A significant improvement in acquisition speed of structured illumination microscopy (SIM) opens a new field of applications to this already well-established super-resolution method towards 3D scanning real-time imaging of living cells. We demonstrate a method of increased acquisition speed on a two-beam SIM fluorescence microscope with a lateral resolution of ~100 nm at a maximum raw data acquisition rate of 162 frames per second (fps) with a region of interest of 16.5  ×  16.5 µm 2 , free of mechanically moving components. We use a programmable spatial light modulator (ferroelectric LCOS) which promises precise and rapid control of the excitation pattern in the sample plane. A passive Fourier filter and a segmented azimuthally patterned polarizer are used to perform structured illumination with maximum contrast. Furthermore, the free running mode in a modern sCMOS camera helps to achieve faster data acquisition.

Macroindentation hardness measurement-Modernization and applications.

PubMed

Patel, Sarsvat; Sun, Changquan Calvin

2016-06-15

In this study, we first developed a modernized indentation technique for measuring tablet hardness. This technique is featured by rapid digital image capture, using a calibrated light microscope, and precise area-determination. We then systematically studied effects of key experimental parameters, including indentation force, speed, and holding time, on measured hardness of a very soft material, hydroxypropyl cellulose, and a very hard material, dibasic calcium phosphate, to cover a wide range of material properties. Based on the results, a holding period of 3min at the peak indentation load is recommended to minimize the effect of testing speed on H. Using this method, we show that an exponential decay function well describes the relationship between tablet hardness and porosity for seven commonly used pharmaceutical powders investigated in this work. We propose that H and H at zero porosity may be used to quantify the tablet deformability and powder plasticity, respectively. Copyright © 2016 Elsevier B.V. All rights reserved.

fastSIM: a practical implementation of fast structured illumination microscopy

NASA Astrophysics Data System (ADS)

Lu-Walther, Hui-Wen; Kielhorn, Martin; Förster, Ronny; Jost, Aurélie; Wicker, Kai; Heintzmann, Rainer

2015-03-01

A significant improvement in acquisition speed of structured illumination microscopy (SIM) opens a new field of applications to this already well-established super-resolution method towards 3D scanning real-time imaging of living cells. We demonstrate a method of increased acquisition speed on a two-beam SIM fluorescence microscope with a lateral resolution of ~100 nm at a maximum raw data acquisition rate of 162 frames per second (fps) with a region of interest of 16.5  ×  16.5 µm2, free of mechanically moving components. We use a programmable spatial light modulator (ferroelectric LCOS) which promises precise and rapid control of the excitation pattern in the sample plane. A passive Fourier filter and a segmented azimuthally patterned polarizer are used to perform structured illumination with maximum contrast. Furthermore, the free running mode in a modern sCMOS camera helps to achieve faster data acquisition.

Smartphone adapters for digital photomicrography.

PubMed

Roy, Somak; Pantanowitz, Liron; Amin, Milon; Seethala, Raja R; Ishtiaque, Ahmed; Yousem, Samuel A; Parwani, Anil V; Cucoranu, Ioan; Hartman, Douglas J

2014-01-01

Photomicrographs in Anatomic Pathology provide a means of quickly sharing information from a glass slide for consultation, education, documentation and publication. While static image acquisition historically involved the use of a permanently mounted camera unit on a microscope, such cameras may be expensive, need to be connected to a computer, and often require proprietary software to acquire and process images. Another novel approach for capturing digital microscopic images is to use smartphones coupled with the eyepiece of a microscope. Recently, several smartphone adapters have emerged that allow users to attach mobile phones to the microscope. The aim of this study was to test the utility of these various smartphone adapters. We surveyed the market for adapters to attach smartphones to the ocular lens of a conventional light microscope. Three adapters (Magnifi, Skylight and Snapzoom) were tested. We assessed the designs of these adapters and their effectiveness at acquiring static microscopic digital images. All adapters facilitated the acquisition of digital microscopic images with a smartphone. The optimal adapter was dependent on the type of phone used. The Magnifi adapters for iPhone were incompatible when using a protective case. The Snapzoom adapter was easiest to use with iPhones and other smartphones even with protective cases. Smartphone adapters are inexpensive and easy to use for acquiring digital microscopic images. However, they require some adjustment by the user in order to optimize focus and obtain good quality images. Smartphone microscope adapters provide an economically feasible method of acquiring and sharing digital pathology photomicrographs.

A light field microscope imaging spectrometer based on the microlens array

NASA Astrophysics Data System (ADS)

Yao, Yu-jia; Xu, Feng; Xia, Yin-xiang

2017-10-01

A new light field spectrometry microscope imaging system, which was composed by microscope objective, microlens array and spectrometry system was designed in this paper. 5-D information (4-D light field and 1-D spectrometer) of the sample could be captured by the snapshot system in only one exposure, avoiding the motion blur and aberration caused by the scanning imaging process of the traditional imaging spectrometry. Microscope objective had been used as the former group while microlens array used as the posterior group. The optical design of the system was simulated by Zemax, the parameter matching condition between microscope objective and microlens array was discussed significantly during the simulation process. The result simulated in the image plane was analyzed and discussed.

A Mach-Zender Holographic Microscope for Quantifying Bacterial Motility

NASA Astrophysics Data System (ADS)

Niraula, B.; Nadeau, J. L.; Serabyn, E.; Wallace, J. K.; Liewer, K.; Kuhn, J.; Graff, E.; Lindensmith, C.

2014-12-01

New microscopic techniques have revolutionized cell biology over the past two decades. However, there are still biological processes whose details elude us, especially those involving motility: e.g. feeding behavior of microorganisms in the ocean, or migration of cancer cells to form metastases. Imaging prokaryotes, which range in size from several hundred nm to a few microns, is especially challenging. An emerging technique to address these issues is Digital Holographic Microscopy (DHM). DHM is an imaging technique that uses the interference of light to record and reproduce three-dimensional magnified images of objects. This approach has several advantages over ordinary brightfield microscopy for fieldwork: a larger depth of field, hands-off operation, robustness regarding environmental conditions, and large sampling volumes with quantitative 3D records of motility behavior. Despite these promising features, real-time DHM was thought to be impractical for technological and computational reasons until recently, and there has so far been very limited application of DHM to biology. Most existing instruments are limited in performance by their particular (e.g. in-line, lens-less, phase-shifting) approach to holography. These limitations can be mitigated with an off-axis dual-path configuration. Here we describe the design and implementation of a design for a Mach-Zehnder-type holographic microscope with diffraction-limited lateral resolution, with intended applications in environmental microbiology. We have achieved sub-micron resolution and three-dimensional tracking of prokaryotic and eukaryotic test strains designed to represent different modes and speeds of microbial motility. Prokaryotes are Escherichia coli, Vibrio alginolyticus, and Bacillus subtilis. Each shows a characteristic motility pattern, as we illustrate in holographic videos in sample chambers 0.6 mm in depth. The ability to establish gradients of attractants with bacterial taxis towards the attractant is also established. The eukaryotic strains are Euglena gracilis, which demonstrates both phototaxis and geotaxis, and Paramecium micromultinucleatum. The challenges of optimizing resolution vs. field of view, and of handling the large volumes of data generated during holographic imaging, are discussed.

A Monochromatic, Aberration-Corrected, Dual-Beam Low Energy Electron Microscope

PubMed Central

Mankos, Marian; Shadman, Khashayar

2013-01-01

The monochromatic, aberration-corrected, dual-beam low energy electron microscope (MAD-LEEM) is a novel instrument aimed at imaging of nanostructures and surfaces at sub-nanometer resolution that includes a monochromator, aberration corrector and dual beam illumination. The monochromator reduces the energy spread of the illuminating electron beam, which significantly improves spectroscopic and spatial resolution. The aberration corrector utilizes an electron mirror with negative aberrations that can be used to compensate the aberrations of the LEEM objective lens for a range of electron energies. Dual flood illumination eliminates charging generated when a conventional LEEM is used to image insulating specimens. MAD-LEEM is designed for the purpose of imaging biological and insulating specimens, which are difficult to image with conventional LEEM, Low-Voltage SEM, and TEM instruments. The MAD-LEEM instrument can also be used as a general purpose LEEM with significantly improved resolution. The low impact energy of the electrons is critical for avoiding beam damage, as high energy electrons with keV kinetic energies used in SEMs and TEMs cause irreversible change to many specimens, in particular biological materials. A potential application for MAD-LEEM is in DNA sequencing, which demands imaging techniques that enable DNA sequencing at high resolution and speed, and at low cost. The key advantages of the MAD-LEEM approach for this application are the low electron impact energies, the long read lengths, and the absence of heavy-atom DNA labeling. Image contrast simulations of the detectability of individual nucleotides in a DNA strand have been developed in order to refine the optics blur and DNA base contrast requirements for this application. PMID:23582636

A monochromatic, aberration-corrected, dual-beam low energy electron microscope.

PubMed

Mankos, Marian; Shadman, Khashayar

2013-07-01

The monochromatic, aberration-corrected, dual-beam low energy electron microscope (MAD-LEEM) is a novel instrument aimed at imaging of nanostructures and surfaces at sub-nanometer resolution that includes a monochromator, aberration corrector and dual beam illumination. The monochromator reduces the energy spread of the illuminating electron beam, which significantly improves spectroscopic and spatial resolution. The aberration corrector utilizes an electron mirror with negative aberrations that can be used to compensate the aberrations of the LEEM objective lens for a range of electron energies. Dual flood illumination eliminates charging generated when a conventional LEEM is used to image insulating specimens. MAD-LEEM is designed for the purpose of imaging biological and insulating specimens, which are difficult to image with conventional LEEM, Low-Voltage SEM, and TEM instruments. The MAD-LEEM instrument can also be used as a general purpose LEEM with significantly improved resolution. The low impact energy of the electrons is critical for avoiding beam damage, as high energy electrons with keV kinetic energies used in SEMs and TEMs cause irreversible change to many specimens, in particular biological materials. A potential application for MAD-LEEM is in DNA sequencing, which demands imaging techniques that enable DNA sequencing at high resolution and speed, and at low cost. The key advantages of the MAD-LEEM approach for this application are the low electron impact energies, the long read lengths, and the absence of heavy-atom DNA labeling. Image contrast simulations of the detectability of individual nucleotides in a DNA strand have been developed in order to refine the optics blur and DNA base contrast requirements for this application. Copyright © 2013 Elsevier B.V. All rights reserved.

First Atomic Force Microscope Image from Mars

NASA Technical Reports Server (NTRS)

2008-01-01

This calibration image presents three-dimensional data from the atomic force microscope on NASA's Phoenix Mars Lander, showing surface details of a substrate on the microscope station's sample wheel. It will be used as an aid for interpreting later images that will show shapes of minuscule Martian soil particles.

The area imaged by the microscope is 40 microns by 40 microns, small enough to fit on an eyelash. The grooves in this substrate are 14 microns (0.00055 inch) apart, from center to center. The vertical dimension is exaggerated in the image to make surface details more visible. The grooves are 300 nanometers (0.00001 inch) deep.

This is the first atomic force microscope image recorded on another planet. It was taken on July 9, 2008, during the 44th Martian day, or sol, of the Phoenix mission since landing.

Phoenix's Swiss-made atomic force microscope builds an image of the surface shape of a particle by sensing it with a sharp tip at the end of a spring, all microfabricated out of a silicon wafer. A strain gauge records how far the spring flexes to follow the contour of the surface. It can provide details of soil-particle shapes smaller than one-hundredth the width of a human hair. This is about 20 times smaller than what can be resolved with Phoenix's optical microscope, which has provided much higher-magnification imaging than anything seen on Mars previously. Both microscopes are part of Phoenix's Microscopy, Electrochemistry and Conductivity Analyzer.

Eight-channel Kirkpatrick-Baez microscope for multiframe x-ray imaging diagnostics in laser plasma experiments.

PubMed

Yi, Shengzhen; Zhang, Zhe; Huang, Qiushi; Zhang, Zhong; Mu, Baozhong; Wang, Zhanshan; Fang, Zhiheng; Wang, Wei; Fu, Sizu

2016-10-01

Because grazing-incidence Kirkpatrick-Baez (KB) microscopes have better resolution and collection efficiency than pinhole cameras, they have been widely used for x-ray imaging diagnostics of laser inertial confinement fusion. The assembly and adjustment of a multichannel KB microscope must meet stringent requirements for image resolution and reproducible alignment. In the present study, an eight-channel KB microscope was developed for diagnostics by imaging self-emission x-rays with a framing camera at the Shenguang-II Update (SGII-Update) laser facility. A consistent object field of view is ensured in the eight channels using an assembly method based on conical reference cones, which also allow the intervals between the eight images to be tuned to couple with the microstrips of the x-ray framing camera. The eight-channel KB microscope was adjusted via real-time x-ray imaging experiments in the laboratory. This paper describes the details of the eight-channel KB microscope, its optical and multilayer design, the assembly and alignment methods, and results of imaging in the laboratory and at the SGII-Update.

Imaging properties and its improvements of scanning/imaging x-ray microscope

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

Takeuchi, Akihisa, E-mail: take@spring8.or.jp; Uesugi, Kentaro; Suzuki, Yoshio

A scanning / imaging X-ray microscope (SIXM) system has been developed at SPring-8. The SIXM consists of a scanning X-ray microscope with a one-dimensional (1D) X-ray focusing device and an imaging (full-field) X-ray microscope with a 1D X-ray objective. The motivation of the SIXM system is to realize a quantitative and highly-sensitive multimodal 3D X-ray tomography by taking advantages of both the scanning X-ray microscope using multi-pixel detector and the imaging X-ray microscope. Data acquisition process of a 2D image is completely different between in the horizontal direction and in the vertical direction; a 1D signal is obtained with themore » linear-scanning while the other dimensional signal is obtained with the imaging optics. Such condition have caused a serious problem on the imaging properties that the imaging quality in the vertical direction has been much worse than that in the horizontal direction. In this paper, two approaches to solve this problem will be presented. One is introducing a Fourier transform method for phase retrieval from one phase derivative image, and the other to develop and employ a 1D diffuser to produce an asymmetrical coherent illumination.« less

Automated micromanipulation desktop station based on mobile piezoelectric microrobots

NASA Astrophysics Data System (ADS)

Fatikow, Sergej

1996-12-01

One of the main problems of present-day research on microsystem technology (MST) is to assemble a whole micro- system from different microcomponents. This paper presents a new concept of an automated micromanipulation desktop- station including piezoelectrically driven microrobots placed on a high-precise x-y-stage of a light microscope, a CCD-camera as a local sensor subsystem, a laser sensor unit as a global sensor subsystem, a parallel computer system with C167 microcontrollers, and a Pentium PC equipped additionally with an optical grabber. The microrobots can perform high-precise manipulations (with an accuracy of up to 10 nm) and a nondestructive transport (at a speed of about 3 cm/sec) of very small objects under the microscope. To control the desktop-station automatically, an advanced control system that includes a task planning level and a real-time execution level is being developed. The main function of the task planning sub-system is to interpret the implicit action plan and to generate a sequence of explicit operations which are sent to the execution level of the control system. The main functions of the execution control level are the object recognition, image processing and feedback position control of the microrobot and the microscope stage.

Hyperspectral microscope imaging methods to classify gram-positive and gram-negative foodborne pathogenic bacteria

USDA-ARS?s Scientific Manuscript database

An acousto-optic tunable filter-based hyperspectral microscope imaging method has potential for identification of foodborne pathogenic bacteria from microcolony rapidly with a single cell level. We have successfully developed the method to acquire quality hyperspectral microscopic images from variou...

Optical design and system characterization of an imaging microscope at 121.6 nm

NASA Astrophysics Data System (ADS)

Gao, Weichuan; Finan, Emily; Kim, Geon-Hee; Kim, Youngsik; Milster, Thomas D.

2018-03-01

We present the optical design and system characterization of an imaging microscope prototype at 121.6 nm. System engineering processes are demonstrated through the construction of a Schwarzschild microscope objective, including tolerance analysis, fabrication, alignment, and testing. Further improvements on the as-built system with a correction phase plate are proposed and analyzed. Finally, the microscope assembly and the imaging properties of the prototype are demonstrated.

Scanning electron microscope observation of dislocations in semiconductor and metal materials.

PubMed

Kuwano, Noriyuki; Itakura, Masaru; Nagatomo, Yoshiyuki; Tachibana, Shigeaki

2010-08-01

Scanning electron microscope (SEM) image contrasts have been investigated for dislocations in semiconductor and metal materials. It is revealed that single dislocations can be observed in a high contrast in SEM images formed by backscattered electrons (BSE) under the condition of a normal configuration of SEM. The BSE images of dislocations were compared with those of the transmission electron microscope and scanning transmission electron microscope (STEM) and the dependence of BSE image contrast on the tilting of specimen was examined to discuss the origin of image contrast. From the experimental results, it is concluded that the BSE images of single dislocations are attributed to the diffraction effect and related with high-angle dark-field images of STEM.

CHAMP (Camera, Handlens, and Microscope Probe)

NASA Technical Reports Server (NTRS)

Mungas, Greg S.; Boynton, John E.; Balzer, Mark A.; Beegle, Luther; Sobel, Harold R.; Fisher, Ted; Klein, Dan; Deans, Matthew; Lee, Pascal; Sepulveda, Cesar A.

2005-01-01

CHAMP (Camera, Handlens And Microscope Probe)is a novel field microscope capable of color imaging with continuously variable spatial resolution from infinity imaging down to diffraction-limited microscopy (3 micron/pixel). As a robotic arm-mounted imager, CHAMP supports stereo imaging with variable baselines, can continuously image targets at an increasing magnification during an arm approach, can provide precision rangefinding estimates to targets, and can accommodate microscopic imaging of rough surfaces through a image filtering process called z-stacking. CHAMP was originally developed through the Mars Instrument Development Program (MIDP) in support of robotic field investigations, but may also find application in new areas such as robotic in-orbit servicing and maintenance operations associated with spacecraft and human operations. We overview CHAMP'S instrument performance and basic design considerations below.

Scanning electron microscope automatic defect classification of process induced defects

NASA Astrophysics Data System (ADS)

Wolfe, Scott; McGarvey, Steve

2017-03-01

With the integration of high speed Scanning Electron Microscope (SEM) based Automated Defect Redetection (ADR) in both high volume semiconductor manufacturing and Research and Development (R and D), the need for reliable SEM Automated Defect Classification (ADC) has grown tremendously in the past few years. In many high volume manufacturing facilities and R and D operations, defect inspection is performed on EBeam (EB), Bright Field (BF) or Dark Field (DF) defect inspection equipment. A comma separated value (CSV) file is created by both the patterned and non-patterned defect inspection tools. The defect inspection result file contains a list of the inspection anomalies detected during the inspection tools' examination of each structure, or the examination of an entire wafers surface for non-patterned applications. This file is imported into the Defect Review Scanning Electron Microscope (DRSEM). Following the defect inspection result file import, the DRSEM automatically moves the wafer to each defect coordinate and performs ADR. During ADR the DRSEM operates in a reference mode, capturing a SEM image at the exact position of the anomalies coordinates and capturing a SEM image of a reference location in the center of the wafer. A Defect reference image is created based on the Reference image minus the Defect image. The exact coordinates of the defect is calculated based on the calculated defect position and the anomalies stage coordinate calculated when the high magnification SEM defect image is captured. The captured SEM image is processed through either DRSEM ADC binning, exporting to a Yield Analysis System (YAS), or a combination of both. Process Engineers, Yield Analysis Engineers or Failure Analysis Engineers will manually review the captured images to insure that either the YAS defect binning is accurately classifying the defects or that the DRSEM defect binning is accurately classifying the defects. This paper is an exploration of the feasibility of the utilization of a Hitachi RS4000 Defect Review SEM to perform Automatic Defect Classification with the objective of the total automated classification accuracy being greater than human based defect classification binning when the defects do not require multiple process step knowledge for accurate classification. The implementation of DRSEM ADC has the potential to improve the response time between defect detection and defect classification. Faster defect classification will allow for rapid response to yield anomalies that will ultimately reduce the wafer and/or the die yield.

Design of a normal incidence multilayer imaging x-ray microscope.

PubMed

Shealy, D L; Gabardi, D R; Hoover, R B; Walker, A B; Lindblom, J F; Barbee, T W

1989-01-01

Normal incidence multilayer Cassegrain x-ray telescopes were flown on the Stanford/MSFC Rocket X-Ray Spectroheliograph. These instruments produced high spatial resolution images of the Sun and conclusively demonstrated that doubly reflecting multilayer x-ray optical systems are feasible. The images indicated that aplanatic imaging soft x-ray /EUV microscopes should be achievable using multilayer optics technology. We have designed a doubly reflecting normal incidence multilayer imaging x-ray microscope based on the Schwarzschild configuration. The Schwarzschild microscope utilizes two spherical mirrors with concentric radii of curvature which are chosen such that the third-order spherical aberration and coma are minimized. We discuss the design of the microscope and the results of the optical system ray trace analysis which indicates that diffraction-limited performance with 600 Å spatial resolution should be obtainable over a 1 mm field of view at a wavelength of 100 Å. Fabrication of several imaging soft x-ray microscopes based upon these designs, for use in conjunction with x-ray telescopes and laser fusion research, is now in progress. High resolution aplanatic imaging x-ray microscopes using normal incidence multilayer x-ray mirrors should have many important applications in advanced x-ray astronomical instrumentation, x-ray lithography, biological, biomedical, metallurgical, and laser fusion research.

Telecytology: Is it possible with smartphone images?

PubMed

Sahin, Davut; Hacisalihoglu, Uguray Payam; Kirimlioglu, Saime Hale

2018-01-01

This study aimed to discuss smartphone usage in telecytology and determine intraobserver concordance between microscopic cytopathological diagnoses and diagnoses derived via static smartphone images. The study was conducted with 172 cytologic material. A pathologist captured static images of the cytology slides from the ocular lens of a microscope using a smartphone. The images were transferred via WhatsApp® to a cytopathologist working in another center who made all the microscopic cytopathological diagnoses 5-27 months ago. The cytopathologist diagnosed images on a smartphone without knowledge of their previous microscopic diagnoses. The Kappa agreement between microscopic cytopathological diagnoses and smartphone image diagnoses was determined. The average image capturing, transfer, and remote cytopathological diagnostic time for one case was 6.20 minutes. The percentage of cases whose microscopic and smartphone image diagnoses were concordant was 84.30%, and the percentage of those whose diagnoses were discordant was 15.69%. The highest Kappa agreement was observed in endoscopic ultrasound-guided fine needle aspiration (1.000), and the lowest agreement was observed in urine cytology (0.665). Patient management changed with smart phone image diagnoses at 11.04%. This study showed that easy, fast, and high-quality image capturing and transfer is possible from cytology slides using smartphones. The intraobserver Kappa agreement between the microscopic cytopathological diagnoses and remote smartphone image diagnoses was high. It was found that remote diagnosis due to difficulties in telecytology might change patient management. The developments in the smartphone camera technology and transfer software make them efficient telepathology and telecytology tools. © 2017 Wiley Periodicals, Inc.

Smartphone adapters for digital photomicrography

PubMed Central

Roy, Somak; Pantanowitz, Liron; Amin, Milon; Seethala, Raja R.; Ishtiaque, Ahmed; Yousem, Samuel A.; Parwani, Anil V.; Cucoranu, Ioan; Hartman, Douglas J.

2014-01-01

Background: Photomicrographs in Anatomic Pathology provide a means of quickly sharing information from a glass slide for consultation, education, documentation and publication. While static image acquisition historically involved the use of a permanently mounted camera unit on a microscope, such cameras may be expensive, need to be connected to a computer, and often require proprietary software to acquire and process images. Another novel approach for capturing digital microscopic images is to use smartphones coupled with the eyepiece of a microscope. Recently, several smartphone adapters have emerged that allow users to attach mobile phones to the microscope. The aim of this study was to test the utility of these various smartphone adapters. Materials and Methods: We surveyed the market for adapters to attach smartphones to the ocular lens of a conventional light microscope. Three adapters (Magnifi, Skylight and Snapzoom) were tested. We assessed the designs of these adapters and their effectiveness at acquiring static microscopic digital images. Results: All adapters facilitated the acquisition of digital microscopic images with a smartphone. The optimal adapter was dependent on the type of phone used. The Magnifi adapters for iPhone were incompatible when using a protective case. The Snapzoom adapter was easiest to use with iPhones and other smartphones even with protective cases. Conclusions: Smartphone adapters are inexpensive and easy to use for acquiring digital microscopic images. However, they require some adjustment by the user in order to optimize focus and obtain good quality images. Smartphone microscope adapters provide an economically feasible method of acquiring and sharing digital pathology photomicrographs. PMID:25191623

Portable oral cancer detection using a miniature confocal imaging probe with a large field of view

NASA Astrophysics Data System (ADS)

Wang, Youmin; Raj, Milan; McGuff, H. Stan; Bhave, Gauri; Yang, Bin; Shen, Ting; Zhang, Xiaojing

2012-06-01

We demonstrate a MEMS micromirror enabled handheld confocal imaging probe for portable oral cancer detection, where a comparatively large field of view (FOV) was generated through the programmable Lissajous scanning pattern of the MEMS micromirror. Miniaturized handheld MEMS confocal imaging probe was developed, and further compared with the desktop confocal prototype under clinical setting. For the handheld confocal imaging system, optical design simulations using CODE VR® shows the lateral and axial resolution to be 0.98 µm and 4.2 µm, where experimental values were determined to be 3 µm and 5.8 µm, respectively, with a FOV of 280 µm×300 µm. Fast Lissajous imaging speed up to 2 fps was realized with improved Labview and Java based real-time imaging software. Properties such as 3D imaging through autofocusing and mosaic imaging for extended lateral view (6 mm × 8 mm) were examined for carcinoma real-time pathology. Neoplastic lesion tissues of giant cell fibroma and peripheral ossifying fibroma, the fibroma inside the paraffin box and ex vivo gross tissues were imaged by the bench-top and handheld imaging modalities, and further compared with commercial microscope imaging results. The MEMS scanner-based handheld confocal imaging probe shows great promise as a potential clinical tool for oral cancer diagnosis and treatment.

Real-time Fluorescence Image-Guided Oncologic Surgery

PubMed Central

Mondal, Suman B.; Gao, Shengkui; Zhu, Nan; Liang, Rongguang; Gruev, Viktor; Achilefu, Samuel

2014-01-01

Medical imaging plays a critical role in cancer diagnosis and planning. Many of these patients rely on surgical intervention for curative outcomes. This requires a careful identification of the primary and microscopic tumors, and the complete removal of cancer. Although there have been efforts to adapt traditional imaging modalities for intraoperative image guidance, they suffer from several constraints such as large hardware footprint, high operation cost, and disruption of the surgical workflow. Because of the ease of image acquisition, relatively low cost devices and intuitive operation, optical imaging methods have received tremendous interests for use in real-time image-guided surgery. To improve imaging depth under low interference by tissue autofluorescence, many of these applications utilize light in the near-infra red (NIR) wavelengths, which is invisible to human eyes. With the availability of a wide selection of tumor-avid contrast agents, advancements in imaging sensors, electronic and optical designs, surgeons are able to combine different attributes of NIR optical imaging techniques to improve treatment outcomes. The emergence of diverse commercial and experimental image guidance systems, which are in various stages of clinical translation, attests to the potential high impact of intraoperative optical imaging methods to improve speed of oncologic surgery with high accuracy and minimal margin positivity. PMID:25287689

Integrated telemedicine workstation for intercontinental grand rounds

NASA Astrophysics Data System (ADS)

Willis, Charles E.; Leckie, Robert G.; Brink, Linda; Goeringer, Fred

1995-04-01

The Telemedicine Spacebridge to Moscow was a series of intercontinental sessions sponsored jointly by NASA and the Moscow Academy of Medicine. To improve the quality of medical images presented, the MDIS Project developed a workstation for acquisition, storage, and interactive display of radiology and pathology images. The workstation was based on a Macintosh IIfx platform with a laser digitizer for radiographs and video capture capability for microscope images. Images were transmitted via the Russian Lyoutch Satellite which had only a single video channel available and no high speed data channels. Two workstations were configured -- one for use at the Uniformed Services University of Health Sciences in Bethesda, MD. and the other for use at the Hospital of the Interior in Moscow, Russia. The two workstations were used may times during 16 sessions. As clinicians used the systems, we modified the original configuration to improve interactive use. This project demonstrated that numerous acquisition and output devices could be brought together in a single interactive workstation. The video images were satisfactory for remote consultation in a grand rounds format.

Adaptive optical microscope for brain imaging in vivo

NASA Astrophysics Data System (ADS)

Wang, Kai

2017-04-01

The optical heterogeneity of biological tissue imposes a major limitation to acquire detailed structural and functional information deep in the biological specimens using conventional microscopes. To restore optimal imaging performance, we developed an adaptive optical microscope based on direct wavefront sensing technique. This microscope can reliably measure and correct biological samples induced aberration. We demonstrated its performance and application in structural and functional brain imaging in various animal models, including fruit fly, zebrafish and mouse.

Development of High-Speed Copper Chemical Mechanical Polishing Slurry for Through Silicon Via Application Based on Friction Analysis Using Atomic Force Microscope

NASA Astrophysics Data System (ADS)

Amanokura, Jin; Ono, Hiroshi; Hombo, Kyoko

2011-05-01

In order to obtain a high-speed copper chemical mechanical polishing (CMP) process for through silicon vias (TSV) application, we developed a new Cu CMP slurry through friction analysis of Cu reaction layer by an atomic force microscope (AFM) technique. A lateral modulation friction force microscope (LM-FFM) is able to measure the friction value properly giving a vibration to the layer. We evaluated the torsional displacement between the probe of the LM-FFM and the Cu reaction layer under a 5 nm vibration to cancel the shape effect of the Cu reaction layer. The developed Cu CMP slurry forms a frictionally easy-removable Cu reaction layer.

Visible-to-visible four-photon ultrahigh resolution microscopic imaging with 730-nm diode laser excited nanocrystals.

PubMed

Wang, Baoju; Zhan, Qiuqiang; Zhao, Yuxiang; Wu, Ruitao; Liu, Jing; He, Sailing

2016-01-25

Further development of multiphoton microscopic imaging is confronted with a number of limitations, including high-cost, high complexity and relatively low spatial resolution due to the long excitation wavelength. To overcome these problems, for the first time, we propose visible-to-visible four-photon ultrahigh resolution microscopic imaging by using a common cost-effective 730-nm laser diode to excite the prepared Nd(3+)-sensitized upconversion nanoparticles (Nd(3+)-UCNPs). An ordinary multiphoton scanning microscope system was built using a visible CW diode laser and the lateral imaging resolution as high as 161-nm was achieved via the four-photon upconversion process. The demonstrated large saturation excitation power for Nd(3+)-UCNPs would be more practical and facilitate the four-photon imaging in the application. A sample with fine structure was imaged to demonstrate the advantages of visible-to-visible four-photon ultrahigh resolution microscopic imaging with 730-nm diode laser excited nanocrystals. Combining the uniqueness of UCNPs, the proposed visible-to-visible four-photon imaging would be highly promising and attractive in the field of multiphoton imaging.

A flexible and rapid frequency selective scheme for SRS microscopy

NASA Astrophysics Data System (ADS)

Li, Jingting; Yue, Yuankai; Shih, Wei-Chuan

2017-02-01

Stimulated Raman scattering (SRS) is a label-free imaging technique suitable for studying biological systems. Due to stimulated nature by ultrafast laser pulses, SRS microscopy has the advantage of significantly higher sensitivity but often reduced spectroscopic information. In this paper, we present a newly constructed femtosecond SRS microscope with a high-speed dynamic micromirror device based pulse shaper to achieve flexible and rapid frequency selection within the C-H stretch region near 2800 to 3100 cm-1 with spectral width of 30 cm-1. This technique is applicable to lipid profiling such as cell activity mapping, lipid distribution mapping and distinction among subclasses.

AtomicJ: An open source software for analysis of force curves

NASA Astrophysics Data System (ADS)

Hermanowicz, Paweł; Sarna, Michał; Burda, Kvetoslava; Gabryś, Halina

2014-06-01

We present an open source Java application for analysis of force curves and images recorded with the Atomic Force Microscope. AtomicJ supports a wide range of contact mechanics models and implements procedures that reduce the influence of deviations from the contact model. It generates maps of mechanical properties, including maps of Young's modulus, adhesion force, and sample height. It can also calculate stacks, which reveal how sample's response to deformation changes with indentation depth. AtomicJ analyzes force curves concurrently on multiple threads, which allows for high speed of analysis. It runs on all popular operating systems, including Windows, Linux, and Macintosh.

Remote Histology Learning from Static versus Dynamic Microscopic Images

ERIC Educational Resources Information Center

Mione, Sylvia; Valcke, Martin; Cornelissen, Maria

2016-01-01

Histology is the study of microscopic structures in normal tissue sections. Curriculum redesign in medicine has led to a decrease in the use of optical microscopes during practical classes. Other imaging solutions have been implemented to facilitate remote learning. With advancements in imaging technologies, learning material can now be digitized.…

A frameless stereotaxic operating microscope for neurosurgery.

PubMed

Friets, E M; Strohbehn, J W; Hatch, J F; Roberts, D W

1989-06-01

A new system, which we call the frameless stereotaxic operating microscope, is discussed. Its purpose is to display CT or other image data in the operating microscope in the correct scale, orientation, and position without the use of a stereotaxic frame. A nonimaging ultrasonic rangefinder allows the position of the operating microscope and the position of the patient to be determined. Discrete fiducial points on the patient's external anatomy are located in both image space and operating room space, linking the image data and the operating room. Physician-selected image information, e.g., tumor contours or guidance to predetermined targets, is projected through the optics of the operating microscope using a miniature cathode ray tube and a beam splitter. Projected images superpose the surgical field, reconstructed from image data to match the focal plane of the operating microscope. The algorithms on which the system is based are described, and the sources and effects of errors are discussed. The system's performance is simulated, providing an estimate of accuracy. Two phantoms are used to measure accuracy experimentally. Clinical results and observations are given.

Low cost light-sheet microscopy for whole brain imaging

NASA Astrophysics Data System (ADS)

Kumar, Manish; Nasenbeny, Jordan; Kozorovitskiy, Yevgenia

2018-02-01

Light-sheet microscopy has evolved as an indispensable tool in imaging biological samples. It can image 3D samples at fast speed, with high-resolution optical sectioning, and with reduced photobleaching effects. These properties make light-sheet microscopy ideal for imaging fluorophores in a variety of biological samples and organisms, e.g. zebrafish, drosophila, cleared mouse brains, etc. While most commercial turnkey light-sheet systems are expensive, the existing lower cost implementations, e.g. OpenSPIM, are focused on achieving high-resolution imaging of small samples or organisms like zebrafish. In this work, we substantially reduce the cost of light-sheet microscope system while targeting to image much larger samples, i.e. cleared mouse brains, at single-cell resolution. The expensive components of a lightsheet system - excitation laser, water-immersion objectives, and translation stage - are replaced with an incoherent laser diode, dry objectives, and a custom-built Arduino-controlled translation stage. A low-cost CUBIC protocol is used to clear fixed mouse brain samples. The open-source platforms of μManager and Fiji support image acquisition, processing, and visualization. Our system can easily be extended to multi-color light-sheet microscopy.

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.

Development of an ultralow-light-level luminescence image analysis system for dynamic measurements of transcriptional activity in living and migrating cells.

PubMed

Maire, E; Lelièvre, E; Brau, D; Lyons, A; Woodward, M; Fafeur, V; Vandenbunder, B

2000-04-10

We have developed an approach to study in single living epithelial cells both cell migration and transcriptional activation, which was evidenced by the detection of luminescence emission from cells transfected with luciferase reporter vectors. The image acquisition chain consists of an epifluorescence inverted microscope, connected to an ultralow-light-level photon-counting camera and an image-acquisition card associated to specialized image analysis software running on a PC computer. Using a simple method based on a thin calibrated light source, the image acquisition chain has been optimized following comparisons of the performance of microscopy objectives and photon-counting cameras designed to observe luminescence. This setup allows us to measure by image analysis the luminescent light emitted by individual cells stably expressing a luciferase reporter vector. The sensitivity of the camera was adjusted to a high value, which required the use of a segmentation algorithm to eliminate the background noise. Following mathematical morphology treatments, kinetic changes of luminescent sources were analyzed and then correlated with the distance and speed of migration. Our results highlight the usefulness of our image acquisition chain and mathematical morphology software to quantify the kinetics of luminescence changes in migrating cells.

Lateral resolution testing of a novel developed confocal microscopic imaging system

NASA Astrophysics Data System (ADS)

Zhang, Xin; Zhang, Yunhai; Chang, Jian; Huang, Wei; Xue, Xiaojun; Xiao, Yun

2015-10-01

Laser scanning confocal microscope has been widely used in biology, medicine and material science owing to its advantages of high resolution and tomographic imaging. Based on a set of confirmatory experiments and system design, a novel confocal microscopic imaging system is developed. The system is composed of a conventional fluorescence microscope and a confocal scanning unit. In the scanning unit a laser beam coupling module provides four different wavelengths 405nm 488nm 561nm and 638nm which can excite a variety of dyes. The system works in spot-to-spot scanning mode with a two-dimensional galvanometer. A 50 microns pinhole is used to guarantee that stray light is blocked and only the fluorescence signal from the focal point can be received . The three-channel spectral splitter is used to perform fluorescence imaging at three different working wavelengths simultaneously. The rat kidney tissue slice is imaged using the developed confocal microscopic imaging system. Nucleues labeled by DAPI and kidney spherule curved pipe labeled by Alexa Fluor 488 can be imaged clearly and respectively, realizing the distinction between the different components of mouse kidney tissue. The three-dimensional tomographic imaging of mouse kidney tissue is reconstructed by several two-dimensional images obtained in different depths. At last the resolution of the confocal microscopic imaging system is tested quantitatively. The experimental result shows that the system can achieve lateral resolution priority to 230nm.

Dynamic compressive behavior of Pr-Nd alloy at high strain rates and temperatures

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

Wang Huanran; Cai Canyuan; Chen Danian

2012-07-01

Based on compressive tests, static on 810 material test system and dynamic on the first compressive loading in split Hopkinson pressure bar (SHPB) tests for Pr-Nd alloy cylinder specimens at high strain rates and temperatures, this study determined a J-C type [G. R. Johnson and W. H. Cook, in Proceedings of Seventh International Symposium on Ballistics (The Hague, The Netherlands, 1983), pp. 541-547] compressive constitutive equation of Pr-Nd alloy. It was recorded by a high speed camera that the Pr-Nd alloy cylinder specimens fractured during the first compressive loading in SHPB tests at high strain rates and temperatures. From highmore » speed camera images, the critical strains of the dynamic shearing instability for Pr-Nd alloy in SHPB tests were determined, which were consistent with that estimated by using Batra and Wei's dynamic shearing instability criterion [R. C. Batra and Z. G. Wei, Int. J. Impact Eng. 34, 448 (2007)] and the determined compressive constitutive equation of Pr-Nd alloy. The transmitted and reflected pulses of SHPB tests for Pr-Nd alloy cylinder specimens computed with the determined compressive constitutive equation of Pr-Nd alloy and Batra and Wei's dynamic shearing instability criterion could be consistent with the experimental data. The fractured Pr-Nd alloy cylinder specimens of compressive tests were investigated by using 3D supper depth digital microscope and scanning electron microscope.« less

Field-Portable Pixel Super-Resolution Colour Microscope

PubMed Central

Greenbaum, Alon; Akbari, Najva; Feizi, Alborz; Luo, Wei; Ozcan, Aydogan

2013-01-01

Based on partially-coherent digital in-line holography, we report a field-portable microscope that can render lensfree colour images over a wide field-of-view of e.g., >20 mm2. This computational holographic microscope weighs less than 145 grams with dimensions smaller than 17×6×5 cm, making it especially suitable for field settings and point-of-care use. In this lensfree imaging design, we merged a colorization algorithm with a source shifting based multi-height pixel super-resolution technique to mitigate ‘rainbow’ like colour artefacts that are typical in holographic imaging. This image processing scheme is based on transforming the colour components of an RGB image into YUV colour space, which separates colour information from brightness component of an image. The resolution of our super-resolution colour microscope was characterized using a USAF test chart to confirm sub-micron spatial resolution, even for reconstructions that employ multi-height phase recovery to handle dense and connected objects. To further demonstrate the performance of this colour microscope Papanicolaou (Pap) smears were also successfully imaged. This field-portable and wide-field computational colour microscope could be useful for tele-medicine applications in resource poor settings. PMID:24086742

Field-portable pixel super-resolution colour microscope.

PubMed

Greenbaum, Alon; Akbari, Najva; Feizi, Alborz; Luo, Wei; Ozcan, Aydogan

2013-01-01

Based on partially-coherent digital in-line holography, we report a field-portable microscope that can render lensfree colour images over a wide field-of-view of e.g., >20 mm(2). This computational holographic microscope weighs less than 145 grams with dimensions smaller than 17×6×5 cm, making it especially suitable for field settings and point-of-care use. In this lensfree imaging design, we merged a colorization algorithm with a source shifting based multi-height pixel super-resolution technique to mitigate 'rainbow' like colour artefacts that are typical in holographic imaging. This image processing scheme is based on transforming the colour components of an RGB image into YUV colour space, which separates colour information from brightness component of an image. The resolution of our super-resolution colour microscope was characterized using a USAF test chart to confirm sub-micron spatial resolution, even for reconstructions that employ multi-height phase recovery to handle dense and connected objects. To further demonstrate the performance of this colour microscope Papanicolaou (Pap) smears were also successfully imaged. This field-portable and wide-field computational colour microscope could be useful for tele-medicine applications in resource poor settings.

Nanoimaging using soft X-ray and EUV laser-plasma sources

NASA Astrophysics Data System (ADS)

Wachulak, Przemyslaw; Torrisi, Alfio; Ayele, Mesfin; Bartnik, Andrzej; Czwartos, Joanna; Węgrzyński, Łukasz; Fok, Tomasz; Fiedorowicz, Henryk

2018-01-01

In this work we present three experimental, compact desk-top imaging systems: SXR and EUV full field microscopes and the SXR contact microscope. The systems are based on laser-plasma EUV and SXR sources based on a double stream gas puff target. The EUV and SXR full field microscopes, operating at 13.8 nm and 2.88 nm wavelengths are capable of imaging nanostructures with a sub-50 nm spatial resolution and short (seconds) exposure times. The SXR contact microscope operates in the "water-window" spectral range and produces an imprint of the internal structure of the imaged sample in a thin layer of SXR sensitive photoresist. Applications of such desk-top EUV and SXR microscopes, mostly for biological samples (CT26 fibroblast cells and Keratinocytes) are also presented. Details about the sources, the microscopes as well as the imaging results for various objects will be presented and discussed. The development of such compact imaging systems may be important to the new research related to biological, material science and nanotechnology applications.

Experimental study of hot cracking at circular welding joints of 42CrMo steel

NASA Astrophysics Data System (ADS)

Zhang, Yan; Chen, Genyu; Chen, Binghua; Wang, Jinhai; Zhou, Cong

2017-12-01

The hot cracking at circular welding joints of quenched and tempered 42CrMo steel were studied. The flow of the molten pool and the solidification process of weld were observed with a high-speed video camera. The information on the variations in the weld temperature was collected using an infrared (IR) thermal imaging system. The metallurgical factors of hot cracking were analyzed via metallographic microscope and scanning electron microscope (SEM). The result shows that leading laser laser-metal active gas (MAG) hybrid welding process has a smaller solid-liquid boundary movement rate (VSL) and a smaller solid-liquid boundary temperature gradient (GSL) compared with leading arc laser-MAG hybrid welding process and laser welding process. Additionally, the metal in the molten pool has superior permeability while flowing toward the dendritic roots and can compensate for the inner-dendritic pressure balance. Therefore, leading laser laser-MAG hybrid welding process has the lowest hot cracking susceptibility.

Modular Scanning Confocal Microscope with Digital Image Processing.

PubMed

Ye, Xianjun; McCluskey, Matthew D

2016-01-01

In conventional confocal microscopy, a physical pinhole is placed at the image plane prior to the detector to limit the observation volume. In this work, we present a modular design of a scanning confocal microscope which uses a CCD camera to replace the physical pinhole for materials science applications. Experimental scans were performed on a microscope resolution target, a semiconductor chip carrier, and a piece of etched silicon wafer. The data collected by the CCD were processed to yield images of the specimen. By selecting effective pixels in the recorded CCD images, a virtual pinhole is created. By analyzing the image moments of the imaging data, a lateral resolution enhancement is achieved by using a 20 × / NA = 0.4 microscope objective at 532 nm laser wavelength.

CHAMP - Camera, Handlens, and Microscope Probe

NASA Technical Reports Server (NTRS)

Mungas, G. S.; Beegle, L. W.; Boynton, J.; Sepulveda, C. A.; Balzer, M. A.; Sobel, H. R.; Fisher, T. A.; Deans, M.; Lee, P.

2005-01-01

CHAMP (Camera, Handlens And Microscope Probe) is a novel field microscope capable of color imaging with continuously variable spatial resolution from infinity imaging down to diffraction-limited microscopy (3 micron/pixel). As an arm-mounted imager, CHAMP supports stereo-imaging with variable baselines, can continuously image targets at an increasing magnification during an arm approach, can provide precision range-finding estimates to targets, and can accommodate microscopic imaging of rough surfaces through a image filtering process called z-stacking. Currently designed with a filter wheel with 4 different filters, so that color and black and white images can be obtained over the entire Field-of-View, future designs will increase the number of filter positions to include 8 different filters. Finally, CHAMP incorporates controlled white and UV illumination so that images can be obtained regardless of sun position, and any potential fluorescent species can be identified so the most astrobiologically interesting samples can be identified.

An Evolutionary Method for Financial Forecasting in Microscopic High-Speed Trading Environment.

PubMed

Huang, Chien-Feng; Li, Hsu-Chih

2017-01-01

The advancement of information technology in financial applications nowadays have led to fast market-driven events that prompt flash decision-making and actions issued by computer algorithms. As a result, today's markets experience intense activity in the highly dynamic environment where trading systems respond to others at a much faster pace than before. This new breed of technology involves the implementation of high-speed trading strategies which generate significant portion of activity in the financial markets and present researchers with a wealth of information not available in traditional low-speed trading environments. In this study, we aim at developing feasible computational intelligence methodologies, particularly genetic algorithms (GA), to shed light on high-speed trading research using price data of stocks on the microscopic level. Our empirical results show that the proposed GA-based system is able to improve the accuracy of the prediction significantly for price movement, and we expect this GA-based methodology to advance the current state of research for high-speed trading and other relevant financial applications.

Development of a SEM-based low-energy in-line electron holography microscope for individual particle imaging.

PubMed

Adaniya, Hidehito; Cheung, Martin; Cassidy, Cathal; Yamashita, Masao; Shintake, Tsumoru

2018-05-01

A new SEM-based in-line electron holography microscope has been under development. The microscope utilizes conventional SEM and BF-STEM functionality to allow for rapid searching of the specimen of interest, seamless interchange between SEM, BF-STEM and holographic imaging modes, and makes use of coherent low-energy in-line electron holography to obtain low-dose, high-contrast images of light element materials. We report here an overview of the instrumentation and first experimental results on gold nano-particles and carbon nano-fibers for system performance tests. Reconstructed images obtained from the holographic imaging mode of the new microscope show substantial image contrast and resolution compared to those acquired by SEM and BF-STEM modes, demonstrating the feasibility of high-contrast imaging via low-energy in-line electron holography. The prospect of utilizing the new microscope to image purified biological specimens at the individual particle level is discussed and electron optical issues and challenges to further improve resolution and contrast are considered. Copyright © 2018 Elsevier B.V. All rights reserved.

A novel intravital multi-harmonic generation microscope for early diagnosis of oral cancer

NASA Astrophysics Data System (ADS)

Cheng, Yu-Hsiang; Lin, Chih-Feng; Shih, Ting-Fang; Sun, Chi-Kuang

2013-03-01

Oral cancer is one of the most frequently diagnosed human cancers and leading causes of cancer death all over the world, but the prognosis and overall survival rate are still poor because of delay in diagnosis and lack of early intervention. The failure of early diagnosis is due to insufficiency of proper diagnostic and screening tools and most patients are reluctant to undergo biopsy. Optical virtual biopsy techniques, for imaging cells and tissues at microscopic details capable of differentiating benign from malignant lesions non-invasively, are thus highly desirable. A novel multi-harmonic generation microscope, excited by a 1260 nm Cr:forsterite laser, with second and third harmonic signals demonstrating collagen fiber distribution and cell morphology in a sub-micron resolution, was developed for clinical use. To achieve invivo observation inside the human oral cavity, a small objective probe with a suction capability was carefully designed for patients' comfort and stability. By remotely changing its focus point, the same objective can image the mucosa surface with a low magnification, illuminated by side light-emitting diodes, with a charge-coupled device (CCD) for site location selection before the harmonic generation biopsy was applied. Furthermore, the slow galvanometer mirror and the fast resonant mirror provide a 30 fps frame rate for high-speed real-time observation and the z-motor of this system is triggered at the same rate to provide fast 3D scanning, again ensuring patients' comfort. Focusing on the special cytological and morphological changes of the oral epithelial cells, our preliminary result disclosed excellent consistency with traditional histopathology studies.

The Atmospheric Scanning Electron Microscope with open sample space observes dynamic phenomena in liquid or gas.

PubMed

Suga, Mitsuo; Nishiyama, Hidetoshi; Konyuba, Yuji; Iwamatsu, Shinnosuke; Watanabe, Yoshiyuki; Yoshiura, Chie; Ueda, Takumi; Sato, Chikara

2011-12-01

Although conventional electron microscopy (EM) requires samples to be in vacuum, most chemical and physical reactions occur in liquid or gas. The Atmospheric Scanning Electron Microscope (ASEM) can observe dynamic phenomena in liquid or gas under atmospheric pressure in real time. An electron-permeable window made of pressure-resistant 100 nm-thick silicon nitride (SiN) film, set into the bottom of the open ASEM sample dish, allows an electron beam to be projected from underneath the sample. A detector positioned below captures backscattered electrons. Using the ASEM, we observed the radiation-induced self-organization process of particles, as well as phenomena accompanying volume change, including evaporation-induced crystallization. Using the electrochemical ASEM dish, we observed tree-like electrochemical depositions on the cathode. In silver nitrate solution, we observed silver depositions near the cathode forming incidental internal voids. The heated ASEM dish allowed observation of patterns of contrast in melting and solidifying solder. Finally, to demonstrate its applicability for monitoring and control of industrial processes, silver paste and solder paste were examined at high throughput. High resolution, imaging speed, flexibility, adaptability, and ease of use facilitate the observation of previously difficult-to-image phenomena, and make the ASEM applicable to various fields. Copyright © 2011 Elsevier B.V. All rights reserved.

Analysis on the grinding quality of palm oil fibers by using combined grinding equipment

NASA Astrophysics Data System (ADS)

Gan, H. L.; Gan, L. M.; Law, H. C.

2015-12-01

As known, Malaysia is the second largest palm oil producer worldwide after Indonesia, therefore indicating the abundance of its wastes within the country. The plantation would be seen to increase to at least 5.2 million ha by 2020, and the waste generation would be 50-70 times the plantation. However, the efficiency of bulk density is reduced. This is one of the main reasons of the initiation of this size reduction/ grinding research. With appropriate parameters, grinding will be seen to be helping in enhancing the inter-particle bindings, subsequently increasing the quality of final products. This paper focuses on the grinding quality involving palm oil wastes by using the Scanning Electron Microscope (SEM). The samples would first be ground to powder at varying grinding speed and finally got the randomly chosen particles measured to obtain the size range. The grinding speed was manipulated from 15 Hz to 40 Hz. From the data obtained, it was found the particles fineness increased with increasing grinding speed. In general, the size ranged from 45 μm to about 600 μm, where the finest was recorded at the speed of 40 Hz. It was also found that the binding was not so encouraging at very low speeds. Therefore, the optimum grinding speed for oil palm residues lied in the range of 25 Hz to 30 Hz. However, there were still limitations to be overcome if the accuracy of the image clarity is to be enhanced.

Applications of virtual reality technology in pathology.

PubMed

Grimes, G J; McClellan, S A; Goldman, J; Vaughn, G L; Conner, D A; Kujawski, E; McDonald, J; Winokur, T; Fleming, W

1997-01-01

TelePath(SM) a telerobotic system utilizing virtual microscope concepts based on high quality still digital imaging and aimed at real-time support for surgery by remote diagnosis of frozen sections. Many hospitals and clinics have an application for the remote practice of pathology, particularly in the area of reading frozen sections in support of surgery, commonly called anatomic pathology. The goal is to project the expertise of the pathologist into the remote setting by giving the pathologist access to the microscope slides with an image quality and human interface comparable to what the pathologist would experience at a real rather than a virtual microscope. A working prototype of a virtual microscope has been defined and constructed which has the needed performance in both the image quality and human interface areas for a pathologist to work remotely. This is accomplished through the use of telerobotics and an image quality which provides the virtual microscope the same diagnostic capabilities as a real microscope. The examination of frozen sections is performed a two-dimensional world. The remote pathologist is in a virtual world with the same capabilities as a "real" microscope, but response times may be slower depending on the specific computing and telecommunication environments. The TelePath system has capabilities far beyond a normal biological microscope, such as the ability to create a low power image of the entire sample using multiple images digitally matched together; the ability to digitally retrace a viewing trajectory; and the ability to archive images using CD ROM and other mass storage devices.

Comprehensive optical and data management infrastructure for high-throughput light-sheet microscopy of whole mouse brains.

PubMed

Müllenbroich, M Caroline; Silvestri, Ludovico; Onofri, Leonardo; Costantini, Irene; Hoff, Marcel Van't; Sacconi, Leonardo; Iannello, Giulio; Pavone, Francesco S

2015-10-01

Comprehensive mapping and quantification of neuronal projections in the central nervous system requires high-throughput imaging of large volumes with microscopic resolution. To this end, we have developed a confocal light-sheet microscope that has been optimized for three-dimensional (3-D) imaging of structurally intact clarified whole-mount mouse brains. We describe the optical and electromechanical arrangement of the microscope and give details on the organization of the microscope management software. The software orchestrates all components of the microscope, coordinates critical timing and synchronization, and has been written in a versatile and modular structure using the LabVIEW language. It can easily be adapted and integrated to other microscope systems and has been made freely available to the light-sheet community. The tremendous amount of data routinely generated by light-sheet microscopy further requires novel strategies for data handling and storage. To complete the full imaging pipeline of our high-throughput microscope, we further elaborate on big data management from streaming of raw images up to stitching of 3-D datasets. The mesoscale neuroanatomy imaged at micron-scale resolution in those datasets allows characterization and quantification of neuronal projections in unsectioned mouse brains.

Scanning Miniature Microscopes without Lenses

NASA Technical Reports Server (NTRS)

Wang, Yu

2009-01-01

The figure schematically depicts some alternative designs of proposed compact, lightweight optoelectronic microscopes that would contain no lenses and would generate magnified video images of specimens. Microscopes of this type were described previously in Miniature Microscope Without Lenses (NPO - 20218), NASA Tech Briefs, Vol. 22, No. 8 (August 1998), page 43 and Reflective Variants of Miniature Microscope Without Lenses (NPO 20610), NASA Tech Briefs, Vol. 26, No. 9 (September 1999), page 6a. To recapitulate: In the design and construction of a microscope of this type, the focusing optics of a conventional microscope are replaced by a combination of a microchannel filter and a charge-coupled-device (CCD) image detector. Elimination of focusing optics reduces the size and weight of the instrument and eliminates the need for the time-consuming focusing operation. The microscopes described in the cited prior articles contained two-dimensional CCDs registered with two-dimensional arrays of microchannels and, as such, were designed to produce full two-dimensional images, without need for scanning. The microscopes of the present proposal would contain one-dimensional (line image) CCDs registered with linear arrays of microchannels. In the operation of such a microscope, one would scan a specimen along a line perpendicular to the array axis (in other words, one would scan in pushbroom fashion). One could then synthesize a full two-dimensional image of the specimen from the line-image data acquired at one-pixel increments of position along the scan. In one of the proposed microscopes, a beam of unpolarized light for illuminating the specimen would enter from the side. This light would be reflected down onto the specimen by a nonpolarizing beam splitter attached to the microchannels at their lower ends. A portion of the light incident on the specimen would be reflected upward, through the beam splitter and along the microchannels, to form an image on the CCD. If the nonpolarizing beam splitter were replaced by a polarizing one, then the specimen would be illuminated by s-polarized light. Upon reflection from the specimen, some of the s-polarized light would become p-polarized. Only the p-polarized light would contribute to the image on the CCD; in other words, the image would contain information on the polarization rotating characteristic of the specimen.

Modular Scanning Confocal Microscope with Digital Image Processing

PubMed Central

McCluskey, Matthew D.

2016-01-01

In conventional confocal microscopy, a physical pinhole is placed at the image plane prior to the detector to limit the observation volume. In this work, we present a modular design of a scanning confocal microscope which uses a CCD camera to replace the physical pinhole for materials science applications. Experimental scans were performed on a microscope resolution target, a semiconductor chip carrier, and a piece of etched silicon wafer. The data collected by the CCD were processed to yield images of the specimen. By selecting effective pixels in the recorded CCD images, a virtual pinhole is created. By analyzing the image moments of the imaging data, a lateral resolution enhancement is achieved by using a 20 × / NA = 0.4 microscope objective at 532 nm laser wavelength. PMID:27829052

High-speed digital imaging of cytosolic Ca2+ and contraction in single cardiomyocytes.

PubMed

O'Rourke, B; Reibel, D K; Thomas, A P

1990-07-01

A charge-coupled device (CCD) camera, with the capacity for simultaneous spatially resolved photon counting and rapid frame transfer, was utilized for high-speed digital image collection from an inverted epifluorescence microscope. The unique properties of the CCD detector were applied to an analysis of cell shortening and the Ca2+ transient from fluorescence images of fura-2-loaded [corrected] cardiomyocytes. On electrical stimulation of the cell, a series of sequential subimages was collected and used to create images of Ca2+ within the cell during contraction. The high photosensitivity of the camera, combined with a detector-based frame storage technique, permitted collection of fluorescence images 10 ms apart. This rate of image collection was sufficient to resolve the rapid events of contraction, e.g., the upstroke of the Ca2+ transient (less than 40 ms) and the time to peak shortening (less than 80 ms). The technique was used to examine the effects of beta-adrenoceptor activation, fura-2 load, and stimulus frequency on cytosolic Ca2+ transients and contractions of single cardiomyocytes. beta-Adrenoceptor stimulation resulted in pronounced increases in peak Ca2+, maximal rates of rise and decay of Ca2+, extent of shortening, and maximal velocities of shortening and relaxation. Raising the intracellular load of fura-2 had little effect on the rising phase of Ca2+ or the extent of shortening but extended the duration of the Ca2+ transient and contraction. In related experiments utilizing differential-interference contrast microscopy, the same technique was applied to visualize sarcomere dynamics in contracting cells. This newly developed technique is a versatile tool for analyzing the Ca2+ transient and mechanical events in studies of excitation-contraction coupling in cardiomyocytes.

Role of phase instabilities in the early response of bulk fused silica during laser-induced breakdown

NASA Astrophysics Data System (ADS)

Demange, P.; Negres, R. A.; Raman, R. N.; Colvin, J. D.; Demos, S. G.

2011-08-01

We report on the experimental and hydrocode modeling investigation of the early material response to localized energy deposition via nanosecond laser pulses in bulk fused silica. A time-resolved microscope system was used to acquire transient images with adequate spatial and temporal resolution to resolve the material behavior from the onset of the process. These images revealed a high-pressure shock front propagating at twice the speed of sound at ambient conditions and bounding a region of modified material at delays up to one nanosecond. Hydrocode simulations matching the experimental conditions were also performed and indicated initial pressures of ˜40 GPa and temperatures of ˜1 eV at the absorption region. Both the simulations and the image data show a clear boundary between distinct material phases, a hot plasma and solid silica, with a suggestion that growth of perturbations at the Rayleigh-Taylor unstable interface between the two phases is the seed mechanism for the growth of cracks into the stressed solid.

A simple water-immersion condenser for imaging living brain slices on an inverted microscope.

PubMed

Prusky, G T

1997-09-05

Due to some physical limitations of conventional condensers, inverted compound microscopes are not optimally suited for imaging living brain slices with transmitted light. Herein is described a simple device that converts an inverted microscope into an effective tool for this application by utilizing an objective as a condenser. The device is mounted on a microscope in place of the condenser, is threaded to accept a water immersion objective, and has a slot for a differential interference contrast (DIC) slider. When combined with infrared video techniques, this device allows an inverted microscope to effectively image living cells within thick brain slices in an open perfusion chamber.

A neotropical Miocene pollen database employing image-based search and semantic modeling1

PubMed Central

Han, Jing Ginger; Cao, Hongfei; Barb, Adrian; Punyasena, Surangi W.; Jaramillo, Carlos; Shyu, Chi-Ren

2014-01-01

• Premise of the study: Digital microscopic pollen images are being generated with increasing speed and volume, producing opportunities to develop new computational methods that increase the consistency and efficiency of pollen analysis and provide the palynological community a computational framework for information sharing and knowledge transfer. • Methods: Mathematical methods were used to assign trait semantics (abstract morphological representations) of the images of neotropical Miocene pollen and spores. Advanced database-indexing structures were built to compare and retrieve similar images based on their visual content. A Web-based system was developed to provide novel tools for automatic trait semantic annotation and image retrieval by trait semantics and visual content. • Results: Mathematical models that map visual features to trait semantics can be used to annotate images with morphology semantics and to search image databases with improved reliability and productivity. Images can also be searched by visual content, providing users with customized emphases on traits such as color, shape, and texture. • Discussion: Content- and semantic-based image searches provide a powerful computational platform for pollen and spore identification. The infrastructure outlined provides a framework for building a community-wide palynological resource, streamlining the process of manual identification, analysis, and species discovery. PMID:25202648

Preclinical evaluation and intraoperative human retinal imaging with a high-resolution microscope-integrated spectral domain optical coherence tomography device.

PubMed

Hahn, Paul; Migacz, Justin; O'Donnell, Rachelle; Day, Shelley; Lee, Annie; Lin, Phoebe; Vann, Robin; Kuo, Anthony; Fekrat, Sharon; Mruthyunjaya, Prithvi; Postel, Eric A; Izatt, Joseph A; Toth, Cynthia A

2013-01-01

The authors have recently developed a high-resolution microscope-integrated spectral domain optical coherence tomography (MIOCT) device designed to enable OCT acquisition simultaneous with surgical maneuvers. The purpose of this report is to describe translation of this device from preclinical testing into human intraoperative imaging. Before human imaging, surgical conditions were fully simulated for extensive preclinical MIOCT evaluation in a custom model eye system. Microscope-integrated spectral domain OCT images were then acquired in normal human volunteers and during vitreoretinal surgery in patients who consented to participate in a prospective institutional review board-approved study. Microscope-integrated spectral domain OCT images were obtained before and at pauses in surgical maneuvers and were compared based on predetermined diagnostic criteria to images obtained with a high-resolution spectral domain research handheld OCT system (HHOCT; Bioptigen, Inc) at the same time point. Cohorts of five consecutive patients were imaged. Successful end points were predefined, including ≥80% correlation in identification of pathology between MIOCT and HHOCT in ≥80% of the patients. Microscope-integrated spectral domain OCT was favorably evaluated by study surgeons and scrub nurses, all of whom responded that they would consider participating in human intraoperative imaging trials. The preclinical evaluation identified significant improvements that were made before MIOCT use during human surgery. The MIOCT transition into clinical human research was smooth. Microscope-integrated spectral domain OCT imaging in normal human volunteers demonstrated high resolution comparable to tabletop scanners. In the operating room, after an initial learning curve, surgeons successfully acquired human macular MIOCT images before and after surgical maneuvers. Microscope-integrated spectral domain OCT imaging confirmed preoperative diagnoses, such as full-thickness macular hole and vitreomacular traction, and demonstrated postsurgical changes in retinal morphology. Two cohorts of five patients were imaged. In the second cohort, the predefined end points were exceeded with ≥80% correlation between microscope-mounted OCT and HHOCT imaging in 100% of the patients. This report describes high-resolution MIOCT imaging using the prototype device in human eyes during vitreoretinal surgery, with successful achievement of predefined end points for imaging. Further refinements and investigations will be directed toward fully integrating MIOCT with vitreoretinal and other ocular surgery to image surgical maneuvers in real time.

Highest Resolution Image of Dust and Sand Yet Acquired on Mars

NASA Technical Reports Server (NTRS)

2008-01-01

[figure removed for brevity, see original site] [figure removed for brevity, see original site] [figure removed for brevity, see original site] Click on image for Figure 1Click on image for Figure 2Click on image for Figure 3

This mosaic of four side-by-side microscope images (one a color composite) was acquired by the Optical Microscope, a part of the Microscopy, Electrochemistry, and Conductivity Analyzer (MECA) instrument suite on NASA's Phoenix Mars Lander. Taken on the ninth Martian day of the mission, or Sol 9 (June 3, 2008), the image shows a 3 millimeter (0.12 inch) diameter silicone target after it has been exposed to dust kicked up by the landing. It is the highest resolution image of dust and sand ever acquired on Mars. The silicone substrate provides a sticky surface for holding the particles to be examined by the microscope.

Martian Particles on Microscope's Silicone Substrate In figure 1, the particles are on a silcone substrate target 3 millimeters (0.12 inch) in diameter, which provides a sticky surface for holding the particles while the microscope images them. Blow-ups of four of the larger particles are shown in the center. These particles range in size from about 30 microns to 150 microns (from about one one-thousandth of an inch to six one-thousandths of an inch).

Possible Nature of Particles Viewed by Mars Lander's Optical Microscope In figure 2, the color composite on the right was acquired to examine dust that had fallen onto an exposed surface. The translucent particle highlighted at bottom center is of comparable size to white particles in a Martian soil sample (upper pictures) seen two sols earlier inside the scoop of Phoenix's Robotic Arm as imaged by the lander's Robotic Arm Camera. The white particles may be examples of the abundant salts that have been found in the Martian soil by previous missions. Further investigations will be needed to determine the white material's composition and whether translucent particles like the one in this microscopic image are found in Martian soil samples.

Scale of Phoenix Optical Microscope Images This set of pictures in figure 3 gives context for the size of individual images from the Optical Microscope on NASA's Mars Phoenix Lander.

The picture in the upper left was taken on Mars by the Surface Stereo Imager on Phoenix. It shows a portion of the microscope's sample stage exposed to accept a sample. In this case, the sample was of dust kicked up by the spacecraft thrusters during landers. Later samples will include soil delivered by the Robotic Arm.

The other pictures were taken on Earth. They show close-ups of circular substrates on which the microscopic samples rest when the microscope images them. Each circular substrate target is 3 millimeters (about one-tenth of an inch) in diameter. Each image taken by the microscope covers and area 2 millimeters by 1 millimeter (0.08 inch by 0.04 inch), the size of a large grain of sand.

The Phoenix Mission is led by the University of Arizona, Tucson, on behalf of NASA. Project management of the mission is by NASA's Jet Propulsion Laboratory, Pasadena, Calif. Spacecraft development is by Lockheed Martin Space Systems, Denver.

A mini-microscope for in situ monitoring of cells.

PubMed

Kim, Sang Bok; Koo, Kyo-in; Bae, Hojae; Dokmeci, Mehmet R; Hamilton, Geraldine A; Bahinski, Anthony; Kim, Sun Min; Ingber, Donald E; Khademhosseini, Ali

2012-10-21

A mini-microscope was developed for in situ monitoring of cells by modifying off-the-shelf components of a commercial webcam. The mini-microscope consists of a CMOS imaging module, a small plastic lens and a white LED illumination source. The CMOS imaging module was connected to a laptop computer through a USB port for image acquisition and analysis. Due to its compact size, 8 × 10 × 9 cm, the present microscope is portable and can easily fit inside a conventional incubator, and enables real-time monitoring of cellular behaviour. Moreover, the mini-microscope can be used for imaging cells in conventional cell culture flasks, such as Petri dishes and multi-well plates. To demonstrate the operation of the mini-microscope, we monitored the cellular migration of mouse 3T3 fibroblasts in a scratch assay in medium containing three different concentrations of fetal bovine serum (5, 10, and 20%) and demonstrated differential responses depending on serum levels. In addition, we seeded embryonic stem cells inside poly(ethylene glycol) microwells and monitored the formation of stem cell aggregates in real time using the mini-microscope. Furthermore, we also combined a lab-on-a-chip microfluidic device for microdroplet generation and analysis with the mini-microscope and observed the formation of droplets under different flow conditions. Given its cost effectiveness, robust imaging and portability, the presented platform may be useful for a range of applications for real-time cellular imaging using lab-on-a-chip devices at low cost.

A mini-microscope for in situ monitoring of cells†‡

PubMed Central

Kim, Sang Bok; Koo, Kyo-in; Bae, Hojae; Dokmeci, Mehmet R.; Hamilton, Geraldine A.; Bahinski, Anthony; Kim, Sun Min; Ingber, Donald E.

2013-01-01

A mini-microscope was developed for in situ monitoring of cells by modifying off-the-shelf components of a commercial webcam. The mini-microscope consists of a CMOS imaging module, a small plastic lens and a white LED illumination source. The CMOS imaging module was connected to a laptop computer through a USB port for image acquisition and analysis. Due to its compact size, 8 × 10 × 9 cm, the present microscope is portable and can easily fit inside a conventional incubator, and enables real-time monitoring of cellular behaviour. Moreover, the mini-microscope can be used for imaging cells in conventional cell culture flasks, such as Petri dishes and multi-well plates. To demonstrate the operation of the mini-microscope, we monitored the cellular migration of mouse 3T3 fibroblasts in a scratch assay in medium containing three different concentrations of fetal bovine serum (5, 10, and 20%) and demonstrated differential responses depending on serum levels. In addition, we seeded embryonic stem cells inside poly(ethylene glycol) microwells and monitored the formation of stem cell aggregates in real time using the mini-microscope. Furthermore, we also combined a lab-on-a-chip microfluidic device for microdroplet generation and analysis with the mini-microscope and observed the formation of droplets under different flow conditions. Given its cost effectiveness, robust imaging and portability, the presented platform may be useful for a range of applications for real-time cellular imaging using lab-on-a-chip devices at low cost. PMID:22911426

Cryotomography x-ray microscopy state

DOEpatents

Le Gros, Mark; Larabell, Carolyn A.

2010-10-26

An x-ray microscope stage enables alignment of a sample about a rotation axis to enable three dimensional tomographic imaging of the sample using an x-ray microscope. A heat exchanger assembly provides cooled gas to a sample during x-ray microscopic imaging.

Lensless high-resolution on-chip optofluidic microscopes for Caenorhabditis elegans and cell imaging

PubMed Central

Cui, Xiquan; Lee, Lap Man; Heng, Xin; Zhong, Weiwei; Sternberg, Paul W.; Psaltis, Demetri; Yang, Changhuei

2008-01-01

Low-cost and high-resolution on-chip microscopes are vital for reducing cost and improving efficiency for modern biomedicine and bioscience. Despite the needs, the conventional microscope design has proven difficult to miniaturize. Here, we report the implementation and application of two high-resolution (≈0.9 μm for the first and ≈0.8 μm for the second), lensless, and fully on-chip microscopes based on the optofluidic microscopy (OFM) method. These systems abandon the conventional microscope design, which requires expensive lenses and large space to magnify images, and instead utilizes microfluidic flow to deliver specimens across array(s) of micrometer-size apertures defined on a metal-coated CMOS sensor to generate direct projection images. The first system utilizes a gravity-driven microfluidic flow for sample scanning and is suited for imaging elongate objects, such as Caenorhabditis elegans; and the second system employs an electrokinetic drive for flow control and is suited for imaging cells and other spherical/ellipsoidal objects. As a demonstration of the OFM for bioscience research, we show that the prototypes can be used to perform automated phenotype characterization of different Caenorhabditis elegans mutant strains, and to image spores and single cellular entities. The optofluidic microscope design, readily fabricable with existing semiconductor and microfluidic technologies, offers low-cost and highly compact imaging solutions. More functionalities, such as on-chip phase and fluorescence imaging, can also be readily adapted into OFM systems. We anticipate that the OFM can significantly address a range of biomedical and bioscience needs, and engender new microscope applications. PMID:18663227

Augmented microscopy with near-infrared fluorescence detection

NASA Astrophysics Data System (ADS)

Watson, Jeffrey R.; Martirosyan, Nikolay; Skoch, Jesse; Lemole, G. Michael; Anton, Rein; Romanowski, Marek

2015-03-01

Near-infrared (NIR) fluorescence has become a frequently used intraoperative technique for image-guided surgical interventions. In procedures such as cerebral angiography, surgeons use the optical surgical microscope for the color view of the surgical field, and then switch to an electronic display for the NIR fluorescence images. However, the lack of stereoscopic, real-time, and on-site coregistration adds time and uncertainty to image-guided surgical procedures. To address these limitations, we developed the augmented microscope, whereby the electronically processed NIR fluorescence image is overlaid with the anatomical optical image in real-time within the optical path of the microscope. In vitro, the augmented microscope can detect and display indocyanine green (ICG) concentrations down to 94.5 nM, overlaid with the anatomical color image. We prepared polyacrylamide tissue phantoms with embedded polystyrene beads, yielding scattering properties similar to brain matter. In this model, 194 μM solution of ICG was detectable up to depths of 5 mm. ICG angiography was then performed in anesthetized rats. A dynamic process of ICG distribution in the vascular system overlaid with anatomical color images was observed and recorded. In summary, the augmented microscope demonstrates NIR fluorescence detection with superior real-time coregistration displayed within the ocular of the stereomicroscope. In comparison to other techniques, the augmented microscope retains full stereoscopic vision and optical controls including magnification and focus, camera capture, and multiuser access. Augmented microscopy may find application in surgeries where the use of traditional microscopes can be enhanced by contrast agents and image guided delivery of therapeutics, including oncology, neurosurgery, and ophthalmology.

Video-Rate Confocal Microscopy for Single-Molecule Imaging in Live Cells and Superresolution Fluorescence Imaging

PubMed Central

Lee, Jinwoo; Miyanaga, Yukihiro; Ueda, Masahiro; Hohng, Sungchul

2012-01-01

There is no confocal microscope optimized for single-molecule imaging in live cells and superresolution fluorescence imaging. By combining the swiftness of the line-scanning method and the high sensitivity of wide-field detection, we have developed a, to our knowledge, novel confocal fluorescence microscope with a good optical-sectioning capability (1.0 μm), fast frame rates (<33 fps), and superior fluorescence detection efficiency. Full compatibility of the microscope with conventional cell-imaging techniques allowed us to do single-molecule imaging with a great ease at arbitrary depths of live cells. With the new microscope, we monitored diffusion motion of fluorescently labeled cAMP receptors of Dictyostelium discoideum at both the basal and apical surfaces and obtained superresolution fluorescence images of microtubules of COS-7 cells at depths in the range 0–85 μm from the surface of a coverglass. PMID:23083712

Automatic segementation of histological structures in normal and neoplastic mammary gland tissue sections

NASA Astrophysics Data System (ADS)

Fernandez-Gonzalez, Rodrigo; Deschamps, Thomas; Idica, Adam; Malladi, Ravikanth; Ortiz de Solorzano, Carlos

2003-07-01

In this paper we present a scheme for real time segmentation of histological structures in microscopic images of normal and neoplastic mammary gland sections. Paraffin embedded or frozen tissue blocks are sliced, and sections are stained with hematoxylin and eosin (H&E). The sections are then imaged using conventional bright field microscopy. The background of the images is corrected by arithmetic manipulation using a "phantom." Then we use the fast marching method with a speed function that depends on the brightness gradient of the image to obtain a preliminary approximation to the boundaries of the structures of interest within a region of interest (ROI) of the entire section manually selected by the user. We use the result of the fast marching method as the initial condition for the level set motion equation. We run this last method for a few steps and obtain the final result of the segmentation. These results can be connected from section to section to build a three-dimensional reconstruction of the entire tissue block that we are studying.

Three-dimensional mapping of microcircuit correlation structure

PubMed Central

Cotton, R. James; Froudarakis, Emmanouil; Storer, Patrick; Saggau, Peter; Tolias, Andreas S.

2013-01-01

Great progress has been made toward understanding the properties of single neurons, yet the principles underlying interactions between neurons remain poorly understood. Given that connectivity in the neocortex is locally dense through both horizontal and vertical connections, it is of particular importance to characterize the activity structure of local populations of neurons arranged in three dimensions. However, techniques for simultaneously measuring microcircuit activity are lacking. We developed an in vivo 3D high-speed, random-access two-photon microscope that is capable of simultaneous 3D motion tracking. This allows imaging from hundreds of neurons at several hundred Hz, while monitoring tissue movement. Given that motion will induce common artifacts across the population, accurate motion tracking is absolutely necessary for studying population activity with random-access based imaging methods. We demonstrate the potential of this imaging technique by measuring the correlation structure of large populations of nearby neurons in the mouse visual cortex, and find that the microcircuit correlation structure is stimulus-dependent. Three-dimensional random access multiphoton imaging with concurrent motion tracking provides a novel, powerful method to characterize the microcircuit activity in vivo. PMID:24133414

Membrane dynamics of dividing cells imaged by lattice light-sheet microscopy

PubMed Central

Aguet, François; Upadhyayula, Srigokul; Gaudin, Raphaël; Chou, Yi-ying; Cocucci, Emanuele; He, Kangmin; Chen, Bi-Chang; Mosaliganti, Kishore; Pasham, Mithun; Skillern, Wesley; Legant, Wesley R.; Liu, Tsung-Li; Findlay, Greg; Marino, Eric; Danuser, Gaudenz; Megason, Sean; Betzig, Eric; Kirchhausen, Tom

2016-01-01

Membrane remodeling is an essential part of transferring components to and from the cell surface and membrane-bound organelles and for changes in cell shape, which are particularly critical during cell division. Earlier analyses, based on classical optical live-cell imaging and mostly restricted by technical necessity to the attached bottom surface, showed persistent formation of endocytic clathrin pits and vesicles during mitosis. Taking advantage of the resolution, speed, and noninvasive illumination of the newly developed lattice light-sheet fluorescence microscope, we reexamined their assembly dynamics over the entire cell surface and found that clathrin pits form at a lower rate during late mitosis. Full-cell imaging measurements of cell surface area and volume throughout the cell cycle of single cells in culture and in zebrafish embryos showed that the total surface increased rapidly during the transition from telophase to cytokinesis, whereas cell volume increased slightly in metaphase and was relatively constant during cytokinesis. These applications demonstrate the advantage of lattice light-sheet microscopy and enable a new standard for imaging membrane dynamics in single cells and multicellular assemblies. PMID:27535432

The Filament Sensor for Near Real-Time Detection of Cytoskeletal Fiber Structures

PubMed Central

Eltzner, Benjamin; Wollnik, Carina; Gottschlich, Carsten; Huckemann, Stephan; Rehfeldt, Florian

2015-01-01

A reliable extraction of filament data from microscopic images is of high interest in the analysis of acto-myosin structures as early morphological markers in mechanically guided differentiation of human mesenchymal stem cells and the understanding of the underlying fiber arrangement processes. In this paper, we propose the filament sensor (FS), a fast and robust processing sequence which detects and records location, orientation, length, and width for each single filament of an image, and thus allows for the above described analysis. The extraction of these features has previously not been possible with existing methods. We evaluate the performance of the proposed FS in terms of accuracy and speed in comparison to three existing methods with respect to their limited output. Further, we provide a benchmark dataset of real cell images along with filaments manually marked by a human expert as well as simulated benchmark images. The FS clearly outperforms existing methods in terms of computational runtime and filament extraction accuracy. The implementation of the FS and the benchmark database are available as open source. PMID:25996921

Thermal-Wave Microscope

NASA Technical Reports Server (NTRS)

Jones, Robert E.; Kramarchuk, Ihor; Williams, Wallace D.; Pouch, John J.; Gilbert, Percy

1989-01-01

Computer-controlled thermal-wave microscope developed to investigate III-V compound semiconductor devices and materials. Is nondestructive technique providing information on subsurface thermal features of solid samples. Furthermore, because this is subsurface technique, three-dimensional imaging also possible. Microscope uses intensity-modulated electron beam of modified scanning electron microscope to generate thermal waves in sample. Acoustic waves generated by thermal waves received by transducer and processed in computer to form images displayed on video display of microscope or recorded on magnetic disk.

Optical Coherence Tomography–Enhanced Microlaryngoscopy: Preliminary Report of a Noncontact Optical Coherence Tomography System Integrated With a Surgical Microscope

PubMed Central

Vokes, David E.; Jackson, Ryan; Guo, Shuguang; Perez, Jorge A.; Su, Jianping; Ridgway, James M.; Armstrong, William B.; Chen, Zhongping; Wong, Brian J. F.

2014-01-01

Objectives Optical coherence tomography (OCT) is a new imaging modality that uses near-infrared light to produce cross-sectional images of tissue with a resolution approaching that of light microscopy. We have previously reported use of OCT imaging of the vocal folds (VFs) during direct laryngoscopy with a probe held in contact or near-contact with the VFs. This aim of this study was to develop and evaluate a novel OCT system integrated with a surgical microscope to allow hands-free OCT imaging of the VFs, which could be performed simultaneously with microscopic visualization. Methods We performed a prospective evaluation of a new method of acquiring OCT images of the VFs. Results An OCT system was successfully integrated with a surgical microscope to permit noncontact OCT imaging of the VFs of 10 patients. With this novel device we were able to identify VF epithelium and lamina propria; however, the resolution was reduced compared to that achieved with the standard contact or near-contact OCT. Conclusions Optical coherence tomography is able to produce high-resolution images of vocal fold mucosa to a maximum depth of 1.6 mm. It may be used in the diagnosis of VF lesions, particularly early squamous cell carcinoma, in which OCT can show disruption of the basement membrane. Mounting the OCT device directly onto the operating microscope allows hands-free noncontact OCT imaging and simultaneous conventional microscopic visualization of the VFs. However, the lateral resolution of the OCT microscope system is 50 µm, in contrast to the conventional handheld probe system (10 µm). Although such images at this resolution are still useful clinically, improved resolution would enhance the system’s performance, potentially enabling real-time OCT-guided microsurgery of the larynx. PMID:18700431

Mark of the Moessbauer

NASA Technical Reports Server (NTRS)

2004-01-01

This image, taken by an instrument called the microscopic imager on the Mars Exploration Rover Spirit, reveals an imprint left by another instrument, the Moessbauer spectrometer. The imprint is at a location within the rover wheel track named 'Middle of Road.' Both instruments are located on the rover's instrument deployment device, or 'arm.'

Not only was the Moessbauer spectrometer able to gain important mineralogical information about this site, it also aided in the placement of the microscopic imager. On hard rocks, the microscopic imager uses its tiny metal sensor to determine proper placement for best possible focus. However, on the soft martian soil this guide would sink, prohibiting proper placement of the microscopic imager. After the Moessbauer spectrometer's much larger, donut-shaped plate touches the surface, Spirit can correctly calculate where to position the microscopic imager.

Scientists find this image particularly interesting because of the compacted nature of the soil that was underneath the Moessbauer spectrometer plate. Also of interest are the embedded, round grains and the fractured appearance of the material disturbed within the hole. The material appears to be slightly cohesive. The field of view in this image, taken on Sol 43 (February 16, 2004), measures approximately 3 centimeters (1.2 inches) across.

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.

Computational scalability of large size image dissemination

NASA Astrophysics Data System (ADS)

Kooper, Rob; Bajcsy, Peter

2011-01-01

We have investigated the computational scalability of image pyramid building needed for dissemination of very large image data. The sources of large images include high resolution microscopes and telescopes, remote sensing and airborne imaging, and high resolution scanners. The term 'large' is understood from a user perspective which means either larger than a display size or larger than a memory/disk to hold the image data. The application drivers for our work are digitization projects such as the Lincoln Papers project (each image scan is about 100-150MB or about 5000x8000 pixels with the total number to be around 200,000) and the UIUC library scanning project for historical maps from 17th and 18th century (smaller number but larger images). The goal of our work is understand computational scalability of the web-based dissemination using image pyramids for these large image scans, as well as the preservation aspects of the data. We report our computational benchmarks for (a) building image pyramids to be disseminated using the Microsoft Seadragon library, (b) a computation execution approach using hyper-threading to generate image pyramids and to utilize the underlying hardware, and (c) an image pyramid preservation approach using various hard drive configurations of Redundant Array of Independent Disks (RAID) drives for input/output operations. The benchmarks are obtained with a map (334.61 MB, JPEG format, 17591x15014 pixels). The discussion combines the speed and preservation objectives.

Remote microscopy and volumetric imaging on the surface of icy satellites

NASA Astrophysics Data System (ADS)

Soto, Alejandro; Nowicki, Keith; Howett, Carly; Feldkhun, Daniel; Retherford, Kurt D.

2017-10-01

With NASA PIDDP support we have applied recent advancements in Fourier-domain microscopy to develop an instrument capable of microscopic imaging from meter-scale distances for use on a planetary lander on the surface of an icy satellite or other planetary bodies. Without moving parts, our instrument projects dynamic patterns of laser light onto a distant target using a lightweight large-aperture reflector, which then collects the light scattered or fluoresced by the target on a fast photon-bucket detector. Using Fourier Transform based techniques, we reconstruct an image from the detected light. The remote microscope has been demonstrated to produce 2D images with better than 15 micron lateral resolution for targets at a distance of 5 meters and is capable of linearly proportionally higher resolution at shorter distances. The remote microscope is also capable of providing three-dimensional (3D) microscopic imaging capabilities, allowing future surface scientists to explore the morphology of microscopic features in surface ices, for example. The instrument enables microscopic in-situ imaging during day or night without the use of a robotic arm, greatly facilitating the surface operations for a lander or rover while expanding the area of investigation near a landing site for improved science targeting. We are developing this remote microscope for in-situ planetary exploration as a collaboration between the Southwest Research Institute, LambdaMetrics, and the University of Colorado.

Failure Analysis of Heavy-Ion-Irradiated Schottky Diodes

NASA Technical Reports Server (NTRS)

Casey, Megan C.; Lauenstein, Jean-Marie; Wilcox, Edward P.; Topper, Alyson D.; Campola, Michael J.; Label, Kenneth A.

2017-01-01

In this work, we use high- and low-magnitude optical microscope images, infrared camera images, and scanning electron microscope images to identify and describe the failure locations in heavy-ion-irradiated Schottky diodes.

Imaging Schwarzschild multilayer X-ray microscope

NASA Technical Reports Server (NTRS)

Hoover, Richard B.; Baker, Phillip C.; Shealy, David L.; Core, David B.; Walker, Arthur B. C., Jr.; Barbee, Troy W., Jr.; Kerstetter, Ted

1993-01-01

We have designed, analyzed, fabricated, and tested Schwarzschild multilayer X-ray microscopes. These instruments use flow-polished Zerodur mirror substrates which have been coated with multilayers optimized for maximum reflectivity at normal incidence at 135 A. They are being developed as prototypes for the Water Window Imaging X-Ray Microscope. Ultrasmooth mirror sets of hemlite grade sapphire have been fabricated and they are now being coated with multilayers to reflect soft X-rays at 38 A, within the biologically important 'water window'. In this paper, we discuss the fabrication of the microscope optics and structural components as well as the mounting of the optics and assembly of the microscopes. We also describe the optical alignment, interferometric and visible light testing of the microscopes, present interferometrically measured performance data, and provide the first results of optical imaging tests.

Full-field OCT: ex vivo and in vivo biological imaging applications

NASA Astrophysics Data System (ADS)

Grieve, Katharine; Dubois, Arnaud; Moneron, Gael; Guyot, Elvire; Boccara, Albert C.

2005-04-01

We present results of studies in embryology and ophthalmology performed using our ultrahigh-resolution full-field OCT system. We also discuss recent developments to our ultrashort acquisition time full-field optical coherence tomography system designed to allow in vivo biological imaging. Preliminary results of high-speed imaging in biological samples are presented. The core of the experimental setup is the Linnik interferometer, illuminated by a white light source. En face tomographic images are obtained in real-time without scanning by computing the difference of two phase-opposed interferometric images recorded by high-resolution CCD cameras. An isotropic spatial resolution of ~1 μm is achieved thanks to the short source coherence length and the use of high numerical aperture microscope objectives. A detection sensitivity of ~90 dB is obtained by means of image averaging and pixel binning. In ophthalmology, reconstructed xz images from rat ocular tissue are presented, where cellular-level structures in the retina are revealed, demonstrating the unprecedented resolution of our instrument. Three-dimensional reconstructions of the mouse embryo allowing the study of the establishment of the anterior-posterior axis are shown. Finally we present the first results of embryonic imaging using the new rapid acquisition full-field OCT system, which offers an acquisition time of 10 μs per frame.

Design, Fabrication and Testing of Multilayer Coated X-Ray Optics for the Water Window Imaging X-Ray Microscope

NASA Technical Reports Server (NTRS)

Spencer, Dwight C.

1996-01-01

Hoover et. al. built and tested two imaging Schwarzschild multilayer microscopes. These instruments were constructed as prototypes for the "Water Window Imaging X-Ray Microscope," which is a doubly reflecting, multilayer x-ray microscope configured to operate within the "water window." The "water window" is the narrow region of the x-ray spectrum between the K absorption edges of oxygen (lamda = 23.3 Angstroms) and of carbon (lamda = 43.62 Angstroms), where water is relatively highly transmissive and carbon is highly absorptive. This property of these materials, thus permits the use of high resolution multilayer x-ray microscopes for producing high contrast images of carbon-based structures within the aqueous physiological environments of living cells. We report the design, fabrication and testing of multilayer optics that operate in this regime.

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

Sabau, Adrian S.; Greer, Clayton M.; Chen, Jian

Here, the increasing use of Carbon Fiber-reinforced Polymer matrix Composites (CFPC) and aluminum alloys as lightweight materials in the automotive and aerospace industries demands enhanced surface preparation and control of surface morphology prior to joining. In this study, surfaces of both composite and aluminum were prepared for joining using an Nd:YAG laser in a two-beam interference setup, enabling the (a) structuring of the AL 5182 surface, (b) removal of the resin layer on top of carbon fibers, and (c) structuring of the carbon fibers. CFPC specimens of T700S carbon fiber, Prepreg - T8 3 epoxy, 5 ply thick, 0/90o plaquesmore » were used. The effect of laser fluence, scanning speed, and number of shots-per-spot was investigated on the removal rate of the resin without an excessive damage of the fibers. Optical micrographs, 3D imaging, and scanning electron microscope (SEM) imaging were used to study the effect of the laser processing on surface morphology.« less

Long-distance super-resolution imaging assisted by enhanced spatial Fourier transform.

PubMed

Tang, Heng-He; Liu, Pu-Kun

2015-09-07

A new gradient-index (GRIN) lens that can realize enhanced spatial Fourier transform (FT) over optically long distances is demonstrated. By using an anisotropic GRIN metamaterial with hyperbolic dispersion, evanescent wave in free space can be transformed into propagating wave in the metamaterial and then focused outside due to negative-refraction. Both the results based on the ray tracing and the finite element simulation show that the spatial frequency bandwidth of the spatial FT can be extended to 2.7k(0) (k(0) is the wave vector in free space). Furthermore, assisted by the enhanced spatial FT, a new long-distance (in the optical far-field region) super-resolution imaging scheme is also proposed and the super resolved capability of λ/5 (λ is the wavelength in free space) is verified. The work may provide technical support for designing new-type high-speed microscopes with long working distances.

Imaging high-speed friction at the nanometer scale

PubMed Central

Thorén, Per-Anders; de Wijn, Astrid S.; Borgani, Riccardo; Forchheimer, Daniel; Haviland, David B.

2016-01-01

Friction is a complicated phenomenon involving nonlinear dynamics at different length and time scales. Understanding its microscopic origin requires methods for measuring force on nanometer-scale asperities sliding at velocities reaching centimetres per second. Despite enormous advances in experimental technique, this combination of small length scale and high velocity remain elusive. We present a technique for rapidly measuring the frictional forces on a single asperity over a velocity range from zero to several centimetres per second. At each image pixel we obtain the velocity dependence of both conservative and dissipative forces, revealing the transition from stick-slip to smooth sliding friction. We explain measurements on graphite using a modified Prandtl–Tomlinson model, including the damped elastic deformation of the asperity. With its improved force sensitivity and small sliding amplitude, our method enables rapid and detailed surface mapping of the velocity dependence of frictional forces with less than 10 nm spatial resolution. PMID:27958267

Microscopic Analysis of Bacterial Motility at High Pressure

PubMed Central

Nishiyama, Masayoshi; Sowa, Yoshiyuki

2012-01-01

The bacterial flagellar motor is a molecular machine that converts an ion flux to the rotation of a helical flagellar filament. Counterclockwise rotation of the filaments allows them to join in a bundle and propel the cell forward. Loss of motility can be caused by environmental factors such as temperature, pH, and solvation. Hydrostatic pressure is also a physical inhibitor of bacterial motility, but the detailed mechanism of this inhibition is still unknown. Here, we developed a high-pressure microscope that enables us to acquire high-resolution microscopic images, regardless of applied pressures. We also characterized the pressure dependence of the motility of swimming Escherichia coli cells and the rotation of single flagellar motors. The fraction and speed of swimming cells decreased with increased pressure. At 80 MPa, all cells stopped swimming and simply diffused in solution. After the release of pressure, most cells immediately recovered their initial motility. Direct observation of the motility of single flagellar motors revealed that at 80 MPa, the motors generate torque that should be sufficient to join rotating filaments in a bundle. The discrepancy in the behavior of free swimming cells and individual motors could be due to the applied pressure inhibiting the formation of rotating filament bundles that can propel the cell body in an aqueous environment. PMID:22768943

Adaptive optics plug-and-play setup for high-resolution microscopes with multi-actuator adaptive lens

NASA Astrophysics Data System (ADS)

Quintavalla, M.; Pozzi, P.; Verhaegen, Michelle; Bijlsma, Hielke; Verstraete, Hans; Bonora, S.

2018-02-01

Adaptive Optics (AO) has revealed as a very promising technique for high-resolution microscopy, where the presence of optical aberrations can easily compromise the image quality. Typical AO systems however, are almost impossible to implement on commercial microscopes. We propose a simple approach by using a Multi-actuator Adaptive Lens (MAL) that can be inserted right after the objective and works in conjunction with an image optimization software allowing for a wavefront sensorless correction. We presented the results obtained on several commercial microscopes among which a confocal microscope, a fluorescence microscope, a light sheet microscope and a multiphoton microscope.

Colloidal Microworms Propelling via a Cooperative Hydrodynamic Conveyor Belt.

PubMed

Martinez-Pedrero, Fernando; Ortiz-Ambriz, Antonio; Pagonabarraga, Ignacio; Tierno, Pietro

2015-09-25

We study propulsion arising from microscopic colloidal rotors dynamically assembled and driven in a viscous fluid upon application of an elliptically polarized rotating magnetic field. Close to a confining plate, the motion of this self-assembled microscopic worm results from the cooperative flow generated by the spinning particles which act as a hydrodynamic "conveyor belt." Chains of rotors propel faster than individual ones, until reaching a saturation speed at distances where induced-flow additivity vanishes. By combining experiments and theoretical arguments, we elucidate the mechanism of motion and fully characterize the propulsion speed in terms of the field parameters.

An open source, wireless capable miniature microscope system

NASA Astrophysics Data System (ADS)

Liberti, William A., III; Perkins, L. Nathan; Leman, Daniel P.; Gardner, Timothy J.

2017-08-01

Objective. Fluorescence imaging through head-mounted microscopes in freely behaving animals is becoming a standard method to study neural circuit function. Flexible, open-source designs are needed to spur evolution of the method. Approach. We describe a miniature microscope for single-photon fluorescence imaging in freely behaving animals. The device is made from 3D printed parts and off-the-shelf components. These microscopes weigh less than 1.8 g, can be configured to image a variety of fluorophores, and can be used wirelessly or in conjunction with active commutators. Microscope control software, based in Swift for macOS, provides low-latency image processing capabilities for closed-loop, or BMI, experiments. Main results. Miniature microscopes were deployed in the songbird premotor region HVC (used as a proper name), in singing zebra finches. Individual neurons yield temporally precise patterns of calcium activity that are consistent over repeated renditions of song. Several cells were tracked over timescales of weeks and months, providing an opportunity to study learning related changes in HVC. Significance. 3D printed miniature microscopes, composed completely of consumer grade components, are a cost-effective, modular option for head-mounting imaging. These easily constructed and customizable tools provide access to cell-type specific neural ensembles over timescales of weeks.

Smartphone confocal microscopy for imaging cellular structures in human skin in vivo.

PubMed

Freeman, Esther E; Semeere, Aggrey; Osman, Hany; Peterson, Gary; Rajadhyaksha, Milind; González, Salvador; Martin, Jeffery N; Anderson, R Rox; Tearney, Guillermo J; Kang, Dongkyun

2018-04-01

We report development of a low-cost smartphone confocal microscope and its first demonstration of in vivo human skin imaging. The smartphone confocal microscope uses a slit aperture and diffraction grating to conduct two-dimensional confocal imaging without using any beam scanning devices. Lateral and axial resolutions of the smartphone confocal microscope were measured as 2 and 5 µm, respectively. In vivo confocal images of human skin revealed characteristic cellular structures, including spinous and basal keratinocytes and papillary dermis. Results suggest that the smartphone confocal microscope has a potential to examine cellular details in vivo and may help disease diagnosis in resource-poor settings, where conducting standard histopathologic analysis is challenging.

Smartphone confocal microscopy for imaging cellular structures in human skin in vivo

PubMed Central

Freeman, Esther E.; Semeere, Aggrey; Osman, Hany; Peterson, Gary; Rajadhyaksha, Milind; González, Salvador; Martin, Jeffery N.; Anderson, R. Rox; Tearney, Guillermo J.; Kang, Dongkyun

2018-01-01

We report development of a low-cost smartphone confocal microscope and its first demonstration of in vivo human skin imaging. The smartphone confocal microscope uses a slit aperture and diffraction grating to conduct two-dimensional confocal imaging without using any beam scanning devices. Lateral and axial resolutions of the smartphone confocal microscope were measured as 2 and 5 µm, respectively. In vivo confocal images of human skin revealed characteristic cellular structures, including spinous and basal keratinocytes and papillary dermis. Results suggest that the smartphone confocal microscope has a potential to examine cellular details in vivo and may help disease diagnosis in resource-poor settings, where conducting standard histopathologic analysis is challenging. PMID:29675328

Scanning evanescent electro-magnetic microscope

DOEpatents

Xiang, Xiao-Dong; Gao, Chen; Schultz, Peter G.; Wei, Tao

2003-01-01

A novel scanning microscope is described that uses near-field evanescent electromagnetic waves to probe sample properties. The novel microscope is capable of high resolution imaging and quantitative measurements of the electrical properties of the sample. The inventive scanning evanescent wave electromagnetic microscope (SEMM) can map dielectric constant, tangent loss, conductivity, complex electrical impedance, and other electrical parameters of materials. The quantitative map corresponds to the imaged detail. The novel microscope can be used to measure electrical properties of both dielectric and electrically conducting materials.

Scanning evanescent electro-magnetic microscope

DOEpatents

Xiang, Xiao-Dong; Gao, Chen

2001-01-01

A novel scanning microscope is described that uses near-field evanescent electromagnetic waves to probe sample properties. The novel microscope is capable of high resolution imaging and quantitative measurements of the electrical properties of the sample. The inventive scanning evanescent wave electromagnetic microscope (SEMM) can map dielectric constant, tangent loss, conductivity, complex electrical impedance, and other electrical parameters of materials. The quantitative map corresponds to the imaged detail. The novel microscope can be used to measure electrical properties of both dielectric and electrically conducting materials.

Characterization and Effects of Fiber Pull-Outs in Hole Quality of Carbon Fiber Reinforced Plastics Composite

PubMed Central

Alizadeh Ashrafi, Sina; Miller, Peter W.; Wandro, Kevin M.; Kim, Dave

2016-01-01

Hole quality plays a crucial role in the production of close-tolerance holes utilized in aircraft assembly. Through drilling experiments of carbon fiber-reinforced plastic composites (CFRP), this study investigates the impact of varying drilling feed and speed conditions on fiber pull-out geometries and resulting hole quality parameters. For this study, hole quality parameters include hole size variance, hole roundness, and surface roughness. Fiber pull-out geometries are quantified by using scanning electron microscope (SEM) images of the mechanically-sectioned CFRP-machined holes, to measure pull-out length and depth. Fiber pull-out geometries and the hole quality parameter results are dependent on the drilling feed and spindle speed condition, which determines the forces and undeformed chip thickness during the process. Fiber pull-out geometries influence surface roughness parameters from a surface profilometer, while their effect on other hole quality parameters obtained from a coordinate measuring machine is minimal. PMID:28773950

Method of mechanical holding of cantilever chip for tip-scan high-speed atomic force microscope

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

Fukuda, Shingo; Uchihashi, Takayuki; Ando, Toshio

In tip-scan atomic force microscopy (AFM) that scans a cantilever chip in the three dimensions, the chip body is held on the Z-scanner with a holder. However, this holding is not easy for high-speed (HS) AFM because the holder that should have a small mass has to be able to clamp the cantilever chip firmly without deteriorating the Z-scanner’s fast performance, and because repeated exchange of cantilever chips should not damage the Z-scanner. This is one of the reasons that tip-scan HS-AFM has not been established, despite its advantages over sample stage-scan HS-AFM. Here, we present a novel method ofmore » cantilever chip holding which meets all conditions required for tip-scan HS-AFM. The superior performance of this novel chip holding mechanism is demonstrated by imaging of the α{sub 3}β{sub 3} subcomplex of F{sub 1}-ATPase in dynamic action at ∼7 frames/s.« less

DMD-based LED-illumination super-resolution and optical sectioning microscopy.

PubMed

Dan, Dan; Lei, Ming; Yao, Baoli; Wang, Wen; Winterhalder, Martin; Zumbusch, Andreas; Qi, Yujiao; Xia, Liang; Yan, Shaohui; Yang, Yanlong; Gao, Peng; Ye, Tong; Zhao, Wei

2013-01-01

Super-resolution three-dimensional (3D) optical microscopy has incomparable advantages over other high-resolution microscopic technologies, such as electron microscopy and atomic force microscopy, in the study of biological molecules, pathways and events in live cells and tissues. We present a novel approach of structured illumination microscopy (SIM) by using a digital micromirror device (DMD) for fringe projection and a low-coherence LED light for illumination. The lateral resolution of 90 nm and the optical sectioning depth of 120 μm were achieved. The maximum acquisition speed for 3D imaging in the optical sectioning mode was 1.6×10(7) pixels/second, which was mainly limited by the sensitivity and speed of the CCD camera. In contrast to other SIM techniques, the DMD-based LED-illumination SIM is cost-effective, ease of multi-wavelength switchable and speckle-noise-free. The 2D super-resolution and 3D optical sectioning modalities can be easily switched and applied to either fluorescent or non-fluorescent specimens.

DMD-based LED-illumination Super-resolution and optical sectioning microscopy

PubMed Central

Dan, Dan; Lei, Ming; Yao, Baoli; Wang, Wen; Winterhalder, Martin; Zumbusch, Andreas; Qi, Yujiao; Xia, Liang; Yan, Shaohui; Yang, Yanlong; Gao, Peng; Ye, Tong; Zhao, Wei

2013-01-01

Super-resolution three-dimensional (3D) optical microscopy has incomparable advantages over other high-resolution microscopic technologies, such as electron microscopy and atomic force microscopy, in the study of biological molecules, pathways and events in live cells and tissues. We present a novel approach of structured illumination microscopy (SIM) by using a digital micromirror device (DMD) for fringe projection and a low-coherence LED light for illumination. The lateral resolution of 90 nm and the optical sectioning depth of 120 μm were achieved. The maximum acquisition speed for 3D imaging in the optical sectioning mode was 1.6×107 pixels/second, which was mainly limited by the sensitivity and speed of the CCD camera. In contrast to other SIM techniques, the DMD-based LED-illumination SIM is cost-effective, ease of multi-wavelength switchable and speckle-noise-free. The 2D super-resolution and 3D optical sectioning modalities can be easily switched and applied to either fluorescent or non-fluorescent specimens. PMID:23346373

Stimulated penetrating keratoplasty using real-time virtual intraoperative surgical optical coherence tomography

PubMed Central

Lee, Changho; Kim, Kyungun; Han, Seunghoon; Kim, Sehui; Lee, Jun Hoon; Kim, Hong kyun; Kim, Chulhong; Jung, Woonggyu; Kim, Jeehyun

2014-01-01

Abstract. An intraoperative surgical microscope is an essential tool in a neuro- or ophthalmological surgical environment. Yet, it has an inherent limitation to classify subsurface information because it only provides the surface images. To compensate for and assist in this problem, combining the surgical microscope with optical coherence tomography (OCT) has been adapted. We developed a real-time virtual intraoperative surgical OCT (VISOCT) system by adapting a spectral-domain OCT scanner with a commercial surgical microscope. Thanks to our custom-made beam splitting and image display subsystems, the OCT images and microscopic images are simultaneously visualized through an ocular lens or the eyepiece of the microscope. This improvement helps surgeons to focus on the operation without distraction to view OCT images on another separate display. Moreover, displaying the OCT live images on the eyepiece helps surgeon’s depth perception during the surgeries. Finally, we successfully processed stimulated penetrating keratoplasty in live rabbits. We believe that these technical achievements are crucial to enhance the usability of the VISOCT system in a real surgical operating condition. PMID:24604471

High-Speed Automatic Microscopy for Real Time Tracks Reconstruction in Nuclear Emulsion

NASA Astrophysics Data System (ADS)

D'Ambrosio, N.

2006-06-01

The Oscillation Project with Emulsion-tRacking Apparatus (OPERA) experiment will use a massive nuclear emulsion detector to search for /spl nu//sub /spl mu///spl rarr//spl nu//sub /spl tau// oscillation by identifying /spl tau/ leptons through the direct detection of their decay topology. The feasibility of experiments using a large mass emulsion detector is linked to the impressive progress under way in the development of automatic emulsion analysis. A new generation of scanning systems requires the development of fast automatic microscopes for emulsion scanning and image analysis to reconstruct tracks of elementary particles. The paper presents the European Scanning System (ESS) developed in the framework of OPERA collaboration.

75 FR 23272 - Government-Owned Inventions; Availability for Licensing

Federal Register 2010, 2011, 2012, 2013, 2014

2010-05-03

...) Protection in Sunscreen Products Description of Invention: There are different types of ultraviolet (UV) rays..., PhD at 301-435-3131 or [email protected] for more information. Laser Scanning Microscopy for Three... data from a high-speed laser-scanning microscope and compute motion of the sample under the microscope...

Seamless stitching of tile scan microscope images.

PubMed

Legesse, F B; Chernavskaia, O; Heuke, S; Bocklitz, T; Meyer, T; Popp, J; Heintzmann, R

2015-06-01

For diagnostic purposes, optical imaging techniques need to obtain high-resolution images of extended biological specimens in reasonable time. The field of view of an objective lens, however, is often smaller than the sample size. To image the whole sample, laser scanning microscopes acquire tile scans that are stitched into larger mosaics. The appearance of such image mosaics is affected by visible edge artefacts that arise from various optical aberrations which manifest in grey level jumps across tile boundaries. In this contribution, a technique for stitching tiles into a seamless mosaic is presented. The stitching algorithm operates by equilibrating neighbouring edges and forcing the brightness at corners to a common value. The corrected image mosaics appear to be free from stitching artefacts and are, therefore, suited for further image analysis procedures. The contribution presents a novel method to seamlessly stitch tiles captured by a laser scanning microscope into a large mosaic. The motivation for the work is the failure of currently existing methods for stitching nonlinear, multimodal images captured by our microscopic setups. Our method eliminates the visible edge artefacts that appear between neighbouring tiles by taking into account the overall illumination differences among tiles in such mosaics. The algorithm first corrects the nonuniform brightness that exists within each of the tiles. It then compensates for grey level differences across tile boundaries by equilibrating neighbouring edges and forcing the brightness at the corners to a common value. After these artefacts have been removed further image analysis procedures can be applied on the microscopic images. Even though the solution presented here is tailored for the aforementioned specific case, it could be easily adapted to other contexts where image tiles are assembled into mosaics such as in astronomical or satellite photos. © 2015 The Authors Journal of Microscopy © 2015 Royal Microscopical Society.

Interfacial Microstructure and Mechanical Properties of Friction Stir Welded Joints of Commercially Pure Aluminum and 304 Stainless Steel

NASA Astrophysics Data System (ADS)

Murugan, Balamagendiravarman; Thirunavukarasu, Gopinath; Kundu, Sukumar; Kailas, Satish V.; Chatterjee, Subrata

2018-05-01

In the present investigation, friction stir welding of commercially pure aluminum and 304 stainless steel was carried out at varying tool rotational speeds from 200 to 1000 rpm in steps of 200 rpm using 60 mm/min traverse speed at 2 (degree) tool tilt angle. Microstructural characterization of the interfacial zone was carried out using optical microscope and scanning electron microscope. Energy-dispersive spectroscopy indicated the presence of FeAl3 intermetallic phase. Thickness of the intermetallic layer increased with the increase in tool rotational speed. X-ray diffraction studies indicated the formation of intermetallic phases like FeAl2, Fe4Al13, Fe2Al5, and FeAl3. A maximum tensile strength of 90% that of aluminum along with 4.5% elongation was achieved with the welded sample at tool rotational speed of 400 rpm. The stir zone showed higher hardness as compared to base metals, heat affected zone, and thermo-mechanically affected zone due to the presence of intermetallics. The maximum hardness value at the stir zone was achieved at 1000 rpm tool rotational speed.

An Evolutionary Method for Financial Forecasting in Microscopic High-Speed Trading Environment

PubMed Central

Li, Hsu-Chih

2017-01-01

The advancement of information technology in financial applications nowadays have led to fast market-driven events that prompt flash decision-making and actions issued by computer algorithms. As a result, today's markets experience intense activity in the highly dynamic environment where trading systems respond to others at a much faster pace than before. This new breed of technology involves the implementation of high-speed trading strategies which generate significant portion of activity in the financial markets and present researchers with a wealth of information not available in traditional low-speed trading environments. In this study, we aim at developing feasible computational intelligence methodologies, particularly genetic algorithms (GA), to shed light on high-speed trading research using price data of stocks on the microscopic level. Our empirical results show that the proposed GA-based system is able to improve the accuracy of the prediction significantly for price movement, and we expect this GA-based methodology to advance the current state of research for high-speed trading and other relevant financial applications. PMID:28316618

Evaluation of breast tissue with confocal strip-mosaicking microscopy: a test approach emulating pathology-like examination

PubMed Central

Abeytunge, Sanjee; Larson, Bjorg; Peterson, Gary; Morrow, Monica; Rajadhyaksha, Milind

2017-01-01

Abstract. Confocal microscopy is an emerging technology for rapid imaging of freshly excised tissue without the need for frozen- or fixed-section processing. Initial studies have described imaging of breast tissue using fluorescence confocal microscopy with small regions of interest, typically 750×750  μm2. We present exploration with a microscope, termed confocal strip-mosaicking microscope (CSM microscope), which images an area of 2×2  cm2 of tissue with cellular-level resolution in 10 min of excision. Using the CSM microscope, we imaged 34 fresh, human, large breast tissue specimens from 18 patients, blindly analyzed by a board-certified pathologist and subsequently correlated with the corresponding standard fixed histopathology. Invasive tumors and benign tissue were clearly identified in CSM strip-mosaic images. Thirty specimens were concordant for image-to-histopathology correlation while four were discordant. PMID:28327961

DIY: "Do Imaging Yourself" - Conventional microscopes as powerful tools for in vivo investigation.

PubMed

Antunes, Maísa Mota; Carvalho, Érika de; Menezes, Gustavo Batista

2018-01-01

Intravital imaging has been increasingly employed in cell biology studies and it is becoming one of the most powerful tools for in vivo investigation. Although some protocols can be extremely complex, most intravital imaging procedures can be performed using basic surgery and animal maintenance techniques. More importantly, regular confocal microscopes - the same that are used for imaging immunofluorescence slides - can also acquire high quality intravital images and movies after minor adaptations. Here we propose minimal adaptations in stock microscopes that allow major improvements in different fields of scientific investigation. Copyright © 2017 Elsevier Ltd. All rights reserved.

Microscope mode secondary ion mass spectrometry imaging with a Timepix detector.

PubMed

Kiss, Andras; Jungmann, Julia H; Smith, Donald F; Heeren, Ron M A

2013-01-01

In-vacuum active pixel detectors enable high sensitivity, highly parallel time- and space-resolved detection of ions from complex surfaces. For the first time, a Timepix detector assembly was combined with a secondary ion mass spectrometer for microscope mode secondary ion mass spectrometry (SIMS) imaging. Time resolved images from various benchmark samples demonstrate the imaging capabilities of the detector system. The main advantages of the active pixel detector are the higher signal-to-noise ratio and parallel acquisition of arrival time and position. Microscope mode SIMS imaging of biomolecules is demonstrated from tissue sections with the Timepix detector.

Martian Microscope

NASA Technical Reports Server (NTRS)

2004-01-01

The microscopic imager (circular device in center) is in clear view above the surface at Meridiani Planum, Mars, in this approximate true-color image taken by the panoramic camera on the Mars Exploration Rover Opportunity. The image was taken on the 9th sol of the rover's journey. The microscopic imager is located on the rover's instrument deployment device, or arm. The arrow is pointing to the lens of the instrument. Note the dust cover, which flips out to the left of the lens, is open. This approximated color image was created using the camera's violet and infrared filters as blue and red.

Predictive value of magnetic resonance imaging determined tumor contact length for extracapsular extension of prostate cancer.

PubMed

Baco, Eduard; Rud, Erik; Vlatkovic, Ljiljana; Svindland, Aud; Eggesbø, Heidi B; Hung, Andrew J; Matsugasumi, Toru; Bernhard, Jean-Christophe; Gill, Inderbir S; Ukimura, Osamu

2015-02-01

Tumor contact length is defined as the amount of prostate cancer in contact with the prostatic capsule. We evaluated the ability of magnetic resonance imaging determined tumor contact length to predict microscopic extracapsular extension compared to existing predictors of extracapsular extension. We retrospectively analyzed the records of 111 consecutive patients with magnetic resonance imaging/ultrasound fusion targeted, biopsy proven prostate cancer who underwent radical prostatectomy from January 2010 to July 2013. Median patient age was 64 years and median prostate specific antigen was 8.9 ng/ml. Clinical stage was cT1 in 93 cases (84%) and cT2 in 18 (16%). Postoperative pathological analysis confirmed pT2 in 71 patients (64%) and pT3 in 40 (36%). We evaluated 1) in the radical prostatectomy specimen the correlation of microscopic extracapsular extension with pathological cancer volume, pathological tumor contact length and Gleason score, 2) the correlation between microscopic extracapsular extension and magnetic resonance imaging tumor contact length, and 3) the ability of preoperative variables to predict microscopic extracapsular extension. Logistic regression analysis revealed that pathological tumor contact length correlated better with microscopic extracapsular extension than the predictive power of pathological cancer volume (0.821 vs 0.685). The Spearman correlation between pathological and magnetic resonance imaging tumor contact length was r = 0.839 (p <0.0001). ROC AUC analysis revealed that magnetic resonance imaging tumor contact length outperformed cancer core involvement on targeted biopsy and the Partin tables to predict microscopic extracapsular extension (0.88 vs 0.70 and 0.63, respectively). At a magnetic resonance imaging tumor contact length threshold of 20 mm the accuracy for diagnosing microscopic extracapsular extension was superior to that of conventional magnetic resonance imaging criteria (82% vs 67%, p = 0.015). We developed a predicted probability plot curve of extracapsular extension according to magnetic resonance imaging tumor contact length. Magnetic resonance imaging determined tumor contact length could be a promising quantitative predictor of microscopic extracapsular extension. Copyright © 2015 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

Optical Imaging of Flow Pattern and Phantom

NASA Technical Reports Server (NTRS)

Galland, Pierre A.; Liang, X.; Wang, L.; Ho, P. P.; Alfano, R. R.; Breisacher, K.

1999-01-01

Time-resolved optical imaging technique has been used to image the spatial distribution of small droplets and jet sprays in a highly scattering environment. The snake and ballistic components of the transmitted pulse are less scattered, and contain direct information about the sample to facilitate image formation as opposed to the diffusive components which are due to multiple collisions as a light pulse propagates through a scattering medium. In a time-gated imaging scheme, these early-arriving, image-bearing components of the incident pulse are selected by opening a gate for an ultrashort period of time and a shadowgram image is detected. Using a single shot cooled CCD camera system, the formation of water droplets is monitored as a function of time. Picosecond time-gated image of drop in scattering cells, spray droplets as a function of let speed and gas pressure, and model calcification samples consisted of calcium carbonate particles of irregular shapes ranging in size from 0. 1 to 1.5 mm affixed to a microscope slide have been measured. Formation produced by an impinging jet will be further monitored using a CCD with 1 kHz framing illuminated with pulsed light. The desired image resolution of the fuel droplets is on the 20 pm scale using early light through a highly scattering medium. A 10(exp -6)m displacement from a jet spray with a flow speed of 100 m/sec introduced by the ns grating pulse used in the imaging is negligible. Early ballistic/snake light imaging offers nondestructive and noninvasive method to observe the spatial distribution of hidden objects inside a highly scattering environment for space, biomedical, and materials applications. In this paper, the techniques we will present are time-resolved K-F transillumination imaging and time-gated scattered light imaging. With a large dynamic range and high resolution, time-gated early light imaging has the potential for improving rocket/aircraft design by determining jets shape and particle sizes. Refinements to these techniques may enable drop size measurements in the highly scattering, optically dense region of multi-element rocket injectors. These types of measurements should greatly enhance the design of stable, and higher performing rocket engines.

Snapshot 3D tracking of insulin granules in live cells

NASA Astrophysics Data System (ADS)

Wang, Xiaolei; Huang, Xiang; Gdor, Itay; Daddysman, Matthew; Yi, Hannah; Selewa, Alan; Haunold, Theresa; Hereld, Mark; Scherer, Norbert F.

2018-02-01

Rapid and accurate volumetric imaging remains a challenge, yet has the potential to enhance understanding of cell function. We developed and used a multifocal microscope (MFM) for 3D snapshot imaging to allow 3D tracking of insulin granules labeled with mCherry in MIN6 cells. MFM employs a special diffractive optical element (DOE) to simultaneously image multiple focal planes. This simultaneous acquisition of information determines the 3D location of single objects at a speed only limited by the array detector's frame rate. We validated the accuracy of MFM imaging/tracking with fluorescence beads; the 3D positions and trajectories of single fluorescence beads can be determined accurately over a wide range of spatial and temporal scales. The 3D positions and trajectories of single insulin granules in a 3.2um deep volume were determined with imaging processing that combines 3D decovolution, shift correction, and finally tracking using the Imaris software package. We find that the motion of the granules is superdiffusive, but less so in 3D than 2D for cells grown on coverslip surfaces, suggesting an anisotropy in the cytoskeleton (e.g. microtubules and action).

TiO2 nanoparticles as exogenous contrast agent for 1 µm swept source optical coherence tomography: an in vitro study

NASA Astrophysics Data System (ADS)

Kumar, Atul; Mondal, Indranil; Roy, Poulomi; Poddar, Raju

2018-03-01

Optical coherence tomography (OCT) is a rapidly evolving, robust technology that has profoundly changed the practice of medical imaging. Swept source OCT (SSOCT) combines the standard time domain and the spatially encoded frequency domain OCT. We have employed a high-speed SSOCT system that utilizes a swept source laser with an A-scan rate of 100 kHz and a central wavelength of 1060 nm for the imaging of the tissue. SSOCT at 1060 nm allows for high penetration in the tissue. TiO2 nanoparticles (NPs) are mostly used for various experimental purposes as an exogenous imaging contrast agent. The in vitro imaging of chicken breast tissue is performed with and without the application of TiO2 NPs for exogenous contrast. Characterization of the chemically synthesized TiO2 NPs was done with dynamic light scattering and a scanning electron microscope method. The effect of TiO2 is studied at different exposure times. A significant improvement in the contrast to noise ratio has been observed through the in vitro imaging of a TiO2 treated tissue.

Video-rate hyperspectral two-photon fluorescence microscopy for in vivo imaging

NASA Astrophysics Data System (ADS)

Deng, Fengyuan; Ding, Changqin; Martin, Jerald C.; Scarborough, Nicole M.; Song, Zhengtian; Eakins, Gregory S.; Simpson, Garth J.

2018-02-01

Fluorescence hyperspectral imaging is a powerful tool for in vivo biological studies. The ability to recover the full spectra of the fluorophores allows accurate classification of different structures and study of the dynamic behaviors during various biological processes. However, most existing methods require significant instrument modifications and/or suffer from image acquisition rates too low for compatibility with in vivo imaging. In the present work, a fast (up to 18 frames per second) hyperspectral two-photon fluorescence microscopy approach was demonstrated. Utilizing the beamscanning hardware inherent in conventional multi-photon microscopy, the angle dependence of the generated fluorescence signal as a function beam's position allowed the system to probe of a different potion of the spectrum at every single scanning line. An iterative algorithm to classify the fluorophores recovered spectra with up to 2,400 channels using a custom high-speed 16-channel photon multiplier tube array. Several dynamic samples including live fluorescent labeled C. elegans were imaged at video rate. Fluorescence spectra recovered using no a priori spectral information agreed well with those obtained by fluorimetry. This system required minimal changes to most existing beam-scanning multi-photon fluorescence microscopes, already accessible in many research facilities.

Surface imaging microscope

NASA Astrophysics Data System (ADS)

Rogala, Eric W.; Bankman, Isaac N.

2008-04-01

The three-dimensional shapes of microscopic objects are becoming increasingly important for battlespace CBRNE sensing. Potential applications of microscopic 3D shape observations include characterization of biological weapon particles and manufacturing of micromechanical components. Aerosol signatures of stand-off lidar systems, using elastic backscatter or polarization, are dictated by the aerosol particle shapes and sizes that must be well characterized in the lab. A low-cost, fast instrument for 3D surface shape microscopy will be a valuable point sensor for biological particle sensing applications. Both the cost and imaging durations of traditional techniques such as confocal microscopes, atomic force microscopes, and electron scanning microscopes are too high. We investigated the feasibility of a low-cost, fast interferometric technique for imaging the 3D surface shape of microscopic objects at frame rates limited only by the camera in the system. The system operates at two laser wavelengths producing two fringe images collected simultaneously by a digital camera, and a specialized algorithm we developed reconstructs the surface map of the microscopic object. The current implementation assembled to test the concept and develop the new 3D reconstruction algorithm has 0.25 micron resolution in the x and y directions, and about 0.1 micron accuracy in the z direction, as tested on a microscopic glass test object manufactured with etching techniques. We describe the interferometric instrument, present the reconstruction algorithm, and discuss further development.

X ray imaging microscope for cancer research

NASA Technical Reports Server (NTRS)

Hoover, Richard B.; Shealy, David L.; Brinkley, B. R.; Baker, Phillip C.; Barbee, Troy W., Jr.; Walker, Arthur B. C., Jr.

1991-01-01

The NASA technology employed during the Stanford MSFC LLNL Rocket X Ray Spectroheliograph flight established that doubly reflecting, normal incidence multilayer optics can be designed, fabricated, and used for high resolution x ray imaging of the Sun. Technology developed as part of the MSFC X Ray Microscope program, showed that high quality, high resolution multilayer x ray imaging microscopes are feasible. Using technology developed at Stanford University and at the DOE Lawrence Livermore National Laboratory (LLNL), Troy W. Barbee, Jr. has fabricated multilayer coatings with near theoretical reflectivities and perfect bandpass matching for a new rocket borne solar observatory, the Multi-Spectral Solar Telescope Array (MSSTA). Advanced Flow Polishing has provided multilayer mirror substrates with sub-angstrom (rms) smoothnesss for the astronomical x ray telescopes and x ray microscopes. The combination of these important technological advancements has paved the way for the development of a Water Window Imaging X Ray Microscope for cancer research.

Correlative imaging across microscopy platforms using the fast and accurate relocation of microscopic experimental regions (FARMER) method

NASA Astrophysics Data System (ADS)

Huynh, Toan; Daddysman, Matthew K.; Bao, Ying; Selewa, Alan; Kuznetsov, Andrey; Philipson, Louis H.; Scherer, Norbert F.

2017-05-01

Imaging specific regions of interest (ROIs) of nanomaterials or biological samples with different imaging modalities (e.g., light and electron microscopy) or at subsequent time points (e.g., before and after off-microscope procedures) requires relocating the ROIs. Unfortunately, relocation is typically difficult and very time consuming to achieve. Previously developed techniques involve the fabrication of arrays of features, the procedures for which are complex, and the added features can interfere with imaging the ROIs. We report the Fast and Accurate Relocation of Microscopic Experimental Regions (FARMER) method, which only requires determining the coordinates of 3 (or more) conspicuous reference points (REFs) and employs an algorithm based on geometric operators to relocate ROIs in subsequent imaging sessions. The 3 REFs can be quickly added to various regions of a sample using simple tools (e.g., permanent markers or conductive pens) and do not interfere with the ROIs. The coordinates of the REFs and the ROIs are obtained in the first imaging session (on a particular microscope platform) using an accurate and precise encoded motorized stage. In subsequent imaging sessions, the FARMER algorithm finds the new coordinates of the ROIs (on the same or different platforms), using the coordinates of the manually located REFs and the previously recorded coordinates. FARMER is convenient, fast (3-15 min/session, at least 10-fold faster than manual searches), accurate (4.4 μm average error on a microscope with a 100x objective), and precise (almost all errors are <8 μm), even with deliberate rotating and tilting of the sample well beyond normal repositioning accuracy. We demonstrate this versatility by imaging and re-imaging a diverse set of samples and imaging methods: live mammalian cells at different time points; fixed bacterial cells on two microscopes with different imaging modalities; and nanostructures on optical and electron microscopes. FARMER can be readily adapted to any imaging system with an encoded motorized stage and can facilitate multi-session and multi-platform imaging experiments in biology, materials science, photonics, and nanoscience.

Automated and unsupervised detection of malarial parasites in microscopic images.

PubMed

Purwar, Yashasvi; Shah, Sirish L; Clarke, Gwen; Almugairi, Areej; Muehlenbachs, Atis

2011-12-13

Malaria is a serious infectious disease. According to the World Health Organization, it is responsible for nearly one million deaths each year. There are various techniques to diagnose malaria of which manual microscopy is considered to be the gold standard. However due to the number of steps required in manual assessment, this diagnostic method is time consuming (leading to late diagnosis) and prone to human error (leading to erroneous diagnosis), even in experienced hands. The focus of this study is to develop a robust, unsupervised and sensitive malaria screening technique with low material cost and one that has an advantage over other techniques in that it minimizes human reliance and is, therefore, more consistent in applying diagnostic criteria. A method based on digital image processing of Giemsa-stained thin smear image is developed to facilitate the diagnostic process. The diagnosis procedure is divided into two parts; enumeration and identification. The image-based method presented here is designed to automate the process of enumeration and identification; with the main advantage being its ability to carry out the diagnosis in an unsupervised manner and yet have high sensitivity and thus reducing cases of false negatives. The image based method is tested over more than 500 images from two independent laboratories. The aim is to distinguish between positive and negative cases of malaria using thin smear blood slide images. Due to the unsupervised nature of method it requires minimal human intervention thus speeding up the whole process of diagnosis. Overall sensitivity to capture cases of malaria is 100% and specificity ranges from 50-88% for all species of malaria parasites. Image based screening method will speed up the whole process of diagnosis and is more advantageous over laboratory procedures that are prone to errors and where pathological expertise is minimal. Further this method provides a consistent and robust way of generating the parasite clearance curves.

Contrast-enhanced and targeted ultrasound.

PubMed

Postema, Michiel; Gilja, Odd Helge

2011-01-07

Ultrasonic imaging is becoming the most popular medical imaging modality, owing to the low price per examination and its safety. However, blood is a poor scatterer of ultrasound waves at clinical diagnostic transmit frequencies. For perfusion imaging, markers have been designed to enhance the contrast in B-mode imaging. These so-called ultrasound contrast agents consist of microscopically small gas bubbles encapsulated in biodegradable shells. In this review, the physical principles of ultrasound contrast agent microbubble behavior and their adjustment for drug delivery including sonoporation are described. Furthermore, an outline of clinical imaging applications of contrast-enhanced ultrasound is given. It is a challenging task to quantify and predict which bubble phenomenon occurs under which acoustic condition, and how these phenomena may be utilized in ultrasonic imaging. Aided by high-speed photography, our improved understanding of encapsulated microbubble behavior will lead to more sophisticated detection and delivery techniques. More sophisticated methods use quantitative approaches to measure the amount and the time course of bolus or reperfusion curves, and have shown great promise in revealing effective tumor responses to anti-angiogenic drugs in humans before tumor shrinkage occurs. These are beginning to be accepted into clinical practice. In the long term, targeted microbubbles for molecular imaging and eventually for directed anti-tumor therapy are expected to be tested.

Contrast-enhanced and targeted ultrasound

PubMed Central

Postema, Michiel; Gilja, Odd Helge

2011-01-01

Ultrasonic imaging is becoming the most popular medical imaging modality, owing to the low price per examination and its safety. However, blood is a poor scatterer of ultrasound waves at clinical diagnostic transmit frequencies. For perfusion imaging, markers have been designed to enhance the contrast in B-mode imaging. These so-called ultrasound contrast agents consist of microscopically small gas bubbles encapsulated in biodegradable shells. In this review, the physical principles of ultrasound contrast agent microbubble behavior and their adjustment for drug delivery including sonoporation are described. Furthermore, an outline of clinical imaging applications of contrast-enhanced ultrasound is given. It is a challenging task to quantify and predict which bubble phenomenon occurs under which acoustic condition, and how these phenomena may be utilized in ultrasonic imaging. Aided by high-speed photography, our improved understanding of encapsulated microbubble behavior will lead to more sophisticated detection and delivery techniques. More sophisticated methods use quantitative approaches to measure the amount and the time course of bolus or reperfusion curves, and have shown great promise in revealing effective tumor responses to anti-angiogenic drugs in humans before tumor shrinkage occurs. These are beginning to be accepted into clinical practice. In the long term, targeted microbubbles for molecular imaging and eventually for directed anti-tumor therapy are expected to be tested. PMID:21218081

Ultrafast superresolution fluorescence imaging with spinning disk confocal microscope optics.

PubMed

Hayashi, Shinichi; Okada, Yasushi

2015-05-01

Most current superresolution (SR) microscope techniques surpass the diffraction limit at the expense of temporal resolution, compromising their applications to live-cell imaging. Here we describe a new SR fluorescence microscope based on confocal microscope optics, which we name the spinning disk superresolution microscope (SDSRM). Theoretically, the SDSRM is equivalent to a structured illumination microscope (SIM) and achieves a spatial resolution of 120 nm, double that of the diffraction limit of wide-field fluorescence microscopy. However, the SDSRM is 10 times faster than a conventional SIM because SR signals are recovered by optical demodulation through the stripe pattern of the disk. Therefore a single SR image requires only a single averaged image through the rotating disk. On the basis of this theory, we modified a commercial spinning disk confocal microscope. The improved resolution around 120 nm was confirmed with biological samples. The rapid dynamics of micro-tubules, mitochondria, lysosomes, and endosomes were observed with temporal resolutions of 30-100 frames/s. Because our method requires only small optical modifications, it will enable an easy upgrade from an existing spinning disk confocal to a SR microscope for live-cell imaging. © 2015 Hayashi and Okada. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

A Painter's View of the Cosmos In the Twenty-first Century

NASA Astrophysics Data System (ADS)

Cro-Ken, K.

2016-01-01

I am an ecosystem artist who uses paint to bring nature's “invisible forces” into view. My eco-sensitive palette recreates the push-pull forces that shape and mold all things. As a result, I create microscopic and telescopic views of earth and places scattered throughout our universe. Self-similarity led me to realize that if I want my mind to wonder into the far reaches of the universe, I must draw closer to nature. I show how space looks and appears and, more importantly, how it moves. My speed element palette is a portal through which I peer into the universe at scales great and small using paint as my lens. Microscopes, telescopes, the Internet, and even eyeglasses are portals through which technology affords us the ability to see that which is unseen to the unaided eye. Rather than see the world and then paint, the opposite is true for me. My work is revelatory, not representational and, as such, seeks similar occurrences in nature. Just as a planet's surface is a visual record of past events, so too do speed element experiments reveal traces of the past. It would be more accurate to call a painting that comes to rest a “painted.” It is video that captures images that eluded capture by the canvas and could more accurately be called a “painting. ” Simply put, I manipulate space, time, and matter—and the matter is never just paint.

Simulation of electrokinetic flow in microfluidic channels

NASA Astrophysics Data System (ADS)

Sabur, Romena; Matin, M.

2005-08-01

Electrokinetic phenomena become an increasingly efficient fluid transport mechanism in micro- and nano-fluidic fields. These phenomena have also been applied successfully in microfluidic devices to achieve particle separation, pre-concentration and mixing. Electrokinetic is the flow produced by the action of an electric field on a fluid with a net charge, where the charged ions of fluid are able to drag the whole solution through the channels in the microfluidic device from one analyzing point to the other. We will present the simulation results of electrokinetic transports of fluid in various typical micro-channel geometries such as T-channel, Y-channel, cross channel and straight channel. In practice, high-speed micro-PIV technique is used to measure transient fluidic phenomena in a microfluidic channel. Particle Image Velocimetry (PIV) systems provide two- or three-dimensional velocity maps in flows using whole field techniques based on imaging the light scattered by small particles in the flow illuminated by a laser light sheet. The system generally consists of an epifluorescent microscope, CW laser and a high-speed CMOS of CCD camera. The flow of a liquid, (water for example), containing fluorescent particle is then analyzed in a counter microchannel by the highly accurate PIV method. One can then compare the simulated and experimental microfluidic flow due to electroosmotic effect.

Automating fruit fly Drosophila embryo injection for high throughput transgenic studies

NASA Astrophysics Data System (ADS)

Cornell, E.; Fisher, W. W.; Nordmeyer, R.; Yegian, D.; Dong, M.; Biggin, M. D.; Celniker, S. E.; Jin, J.

2008-01-01

To decipher and manipulate the 14 000 identified Drosophila genes, there is a need to inject a large number of embryos with transgenes. We have developed an automated instrument for high throughput injection of Drosophila embryos. It was built on an inverted microscope, equipped with a motorized xy stage, autofocus, a charge coupled device camera, and an injection needle mounted on a high speed vertical stage. A novel, micromachined embryo alignment device was developed to facilitate the arrangement of a large number of eggs. The control system included intelligent and dynamic imaging and analysis software and an embryo injection algorithm imitating a human operator. Once the injection needle and embryo slide are loaded, the software automatically images and characterizes each embryo and subsequently injects DNA into all suitable embryos. The ability to program needle flushing and monitor needle status after each injection ensures reliable delivery of biomaterials. Using this instrument, we performed a set of transformation injection experiments. The robot achieved injection speeds and transformation efficiencies comparable to those of a skilled human injector. Because it can be programed to allow injection at various locations in the embryo, such as the anterior pole or along the dorsal or ventral axes, this system is also suitable for injection of general biochemicals, including drugs and RNAi.

Microstructure of wave propagation during combustion synthesis of advanced materials: Experiments and theory

NASA Astrophysics Data System (ADS)

Hwang, Stephen

Combustion synthesis (CS) is an attractive method for producing advanced materials, including ceramics, intermetallics, and composites. In this process, after initiation by an external heat source, a highly exothermic reaction propagates through the sample in a self-sustained combustion wave. The process offers the possibility of producing materials with novel structures and properties. At conventional magnifications and imaging rates, the combustion wave appears to propagate in a planar, steady manner. However, using higher magnifications (>400X) and imaging rates (1000 frames/sec), fluctuations in the shape and propagation of the combustion front were observed. These variations in local conditions (i.e., the microstructure of the combustion wave) can influence the microstructure and properties of materials produced by combustion synthesis. In this work, the microstructure of wave propagation during combustion synthesis is investigated experimentally and theoretically. Using microscopic high-speed imaging, the spatial and temporal fluctuations of the combustion front shape and propagation were investigated. New image analysis methods were developed to characterize the heterogeneity of the combustion front quantitatively. The initial organization of the reaction medium was found to affect the heterogeneity of the combustion wave. Moreover, at the microscopic level, two different regimes of combustion propagation were observed. In the quasihomogeneous mechanism, the microstructure of the combustion wave resembles what is viewed macroscopically, and steady, planar propagation is observed. In the relay-race mechanism, while planar at the macroscopic level, the combustion front profiles are irregularly shaped, with arc-shaped convexities and concavities at the microscopic level. Also, the reaction front propagates as a series of rapid jumps and hesitations. Based on the combustion wave microstructure, new criteria were developed to determine the boundaries between quasihomogeneous and relay-race mechanisms, as functions of the initial organization of the reaction medium (i.e. particle size and porosity). In conjunction with the experiments, a microheterogeneous cell model was developed that simulates the local propagation of the combustion wave. Accounting for the stochastically organized medium with non-uniform properties, calculated results for the microstructural parameters of the combustion wave, and their dependence on density and reactant particle size, were in good qualitative agreement with experimental data.

Soft x-ray imaging with incoherent sources

NASA Astrophysics Data System (ADS)

Wachulak, P.; Torrisi, A.; Ayele, M.; Bartnik, A.; Czwartos, J.; Wegrzyński, Ł.; Fok, T.; Parkman, T.; Vondrová, Š.; Turnová, J.; Odstrcil, M.; Fiedorowicz, H.

2017-05-01

In this work we present experimental, compact desk-top SXR microscope, the EUV microscope which is at this stage a technology demonstrator, and finally, the SXR contact microscope. The systems are based on laser-plasma EUV and SXR sources, employing a double stream gas puff target. The EUV and SXR full field microscopes, operating at 13.8 nm and 2.88 nm wavelengths, respectively, are capable of imaging nanostructures with a sub-50 nm spatial resolution with relatively short (seconds) exposure times. The SXR contact microscope operates in the "water-window" spectral range, to produce an imprint of the internal structure of the sample in a thin layer of SXR light sensitive photoresist. Applications of such desk-top EUV and SXR microscopes for studies of variety of different samples - test objects for resolution assessment and other objects such as carbon membranes, DNA plasmid samples, organic and inorganic thin layers, diatoms, algae and carcinoma cells, are also presented. Details about the sources, the microscopes as well as the imaging results for various objects will be presented and discussed. The development of such compact imaging systems may be important to the new research related to biological, material science and nanotechnology applications.

Water window imaging x ray microscope

NASA Technical Reports Server (NTRS)

Hoover, Richard B. (Inventor)

1992-01-01

A high resolution x ray microscope for imaging microscopic structures within biological specimens has an optical system including a highly polished primary and secondary mirror coated with identical multilayer coatings, the mirrors acting at normal incidence. The coatings have a high reflectivity in the narrow wave bandpass between 23.3 and 43.7 angstroms and have low reflectivity outside of this range. The primary mirror has a spherical concave surface and the secondary mirror has a spherical convex surface. The radii of the mirrors are concentric about a common center of curvature on the optical axis of the microscope extending from the object focal plane to the image focal plane. The primary mirror has an annular configuration with a central aperture and the secondary mirror is positioned between the primary mirror and the center of curvature for reflecting radiation through the aperture to a detector. An x ray filter is mounted at the stage end of the microscope, and film sensitive to x rays in the desired band width is mounted in a camera at the image plane of the optical system. The microscope is mounted within a vacuum chamber for minimizing the absorption of x rays in air from a source through the microscope.

SlideJ: An ImageJ plugin for automated processing of whole slide images.

PubMed

Della Mea, Vincenzo; Baroni, Giulia L; Pilutti, David; Di Loreto, Carla

2017-01-01

The digital slide, or Whole Slide Image, is a digital image, acquired with specific scanners, that represents a complete tissue sample or cytological specimen at microscopic level. While Whole Slide image analysis is recognized among the most interesting opportunities, the typical size of such images-up to Gpixels- can be very demanding in terms of memory requirements. Thus, while algorithms and tools for processing and analysis of single microscopic field images are available, Whole Slide images size makes the direct use of such tools prohibitive or impossible. In this work a plugin for ImageJ, named SlideJ, is proposed with the objective to seamlessly extend the application of image analysis algorithms implemented in ImageJ for single microscopic field images to a whole digital slide analysis. The plugin has been complemented by examples of macro in the ImageJ scripting language to demonstrate its use in concrete situations.

Video-rate confocal microscopy for single-molecule imaging in live cells and superresolution fluorescence imaging.

PubMed

Lee, Jinwoo; Miyanaga, Yukihiro; Ueda, Masahiro; Hohng, Sungchul

2012-10-17

There is no confocal microscope optimized for single-molecule imaging in live cells and superresolution fluorescence imaging. By combining the swiftness of the line-scanning method and the high sensitivity of wide-field detection, we have developed a, to our knowledge, novel confocal fluorescence microscope with a good optical-sectioning capability (1.0 μm), fast frame rates (<33 fps), and superior fluorescence detection efficiency. Full compatibility of the microscope with conventional cell-imaging techniques allowed us to do single-molecule imaging with a great ease at arbitrary depths of live cells. With the new microscope, we monitored diffusion motion of fluorescently labeled cAMP receptors of Dictyostelium discoideum at both the basal and apical surfaces and obtained superresolution fluorescence images of microtubules of COS-7 cells at depths in the range 0-85 μm from the surface of a coverglass. Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.

In vivo imaging of oral neoplasia using a miniaturized fiber optic confocal reflectance microscope.

PubMed

Maitland, Kristen C; Gillenwater, Ann M; Williams, Michelle D; El-Naggar, Adel K; Descour, Michael R; Richards-Kortum, Rebecca R

2008-11-01

The purpose of this study was to determine whether in vivo images of oral mucosa obtained with a fiber optic confocal reflectance microscope could be used to differentiate normal and neoplastic tissues. We imaged 20 oral sites in eight patients undergoing surgery for squamous cell carcinoma. Normal and abnormal areas within the oral cavity were identified clinically, and real-time videos of each site were obtained in vivo using a fiber optic confocal reflectance microscope. Following imaging, each site was biopsied and submitted for histopathologic examination. We identified distinct features, such as nuclear irregularity and spacing, which can be used to qualitatively differentiate between normal and abnormal tissue. Representative confocal images of normal, pre-neoplastic, and neoplastic oral tissue are presented. Previous work using much larger microscopes has demonstrated the ability of confocal reflectance microscopy to image cellular and tissue architecture in situ. New advances in technology have enabled miniaturization of imaging systems for in vivo use.

Spatial-spectral blood cell classification with microscopic hyperspectral imagery

NASA Astrophysics Data System (ADS)

Ran, Qiong; Chang, Lan; Li, Wei; Xu, Xiaofeng

2017-10-01

Microscopic hyperspectral images provide a new way for blood cell examination. The hyperspectral imagery can greatly facilitate the classification of different blood cells. In this paper, the microscopic hyperspectral images are acquired by connecting the microscope and the hyperspectral imager, and then tested for blood cell classification. For combined use of the spectral and spatial information provided by hyperspectral images, a spatial-spectral classification method is improved from the classical extreme learning machine (ELM) by integrating spatial context into the image classification task with Markov random field (MRF) model. Comparisons are done among ELM, ELM-MRF, support vector machines(SVM) and SVMMRF methods. Results show the spatial-spectral classification methods(ELM-MRF, SVM-MRF) perform better than pixel-based methods(ELM, SVM), and the proposed ELM-MRF has higher precision and show more accurate location of cells.

Long-Term Live Cell Imaging of Cell Migration: Effects of Pathogenic Fungi on Human Epithelial Cell Migration.

PubMed

Wöllert, Torsten; Langford, George M

2016-01-01

Long-term live cell imaging was used in this study to determine the responses of human epithelial cells to pathogenic biofilms formed by Candida albicans. Epithelial cells of the skin represent the front line of defense against invasive pathogens such as C. albicans but under certain circumstances, especially when the host's immune system is compromised, the skin barrier is breached. The mechanisms by which the fungal pathogen penetrates the skin and invade the deeper layers are not fully understood. In this study we used keratinocytes grown in culture as an in vitro model system to determine changes in host cell migration and the actin cytoskeleton in response to virulence factors produced by biofilms of pathogenic C. albicans. It is clear that changes in epithelial cell migration are part of the response to virulence factors secreted by biofilms of C. albicans and the actin cytoskeleton is the downstream effector that mediates cell migration. Our goal is to understand the mechanism by which virulence factors hijack the signaling pathways of the actin cytoskeleton to alter cell migration and thereby invade host tissues. To understand the dynamic changes of the actin cytoskeleton during infection, we used long-term live cell imaging to obtain spatial and temporal information of actin filament dynamics and to identify signal transduction pathways that regulate the actin cytoskeleton and its associated proteins. Long-term live cell imaging was achieved using a high resolution, multi-mode epifluorescence microscope equipped with specialized light sources, high-speed cameras with high sensitivity detectors, and specific biocompatible fluorescent markers. In addition to the multi-mode epifluorescence microscope, a spinning disk confocal long-term live cell imaging system (Olympus CV1000) equipped with a stage incubator to create a stable in vitro environment for long-term real-time and time-lapse microscopy was used. Detailed descriptions of these two long-term live cell imaging systems are provided.

Videoexoscopic real-time intraoperative navigation for spinal neurosurgery: a novel co-adaptation of two existing technology platforms, technical note.

PubMed

Huang, Meng; Barber, Sean Michael; Steele, William James; Boghani, Zain; Desai, Viren Rajendrakumar; Britz, Gavin Wayne; West, George Alexander; Trask, Todd Wilson; Holman, Paul Joseph

2018-06-01

Image-guided approaches to spinal instrumentation and interbody fusion have been widely popularized in the last decade [1-5]. Navigated pedicle screws are significantly less likely to breach [2, 3, 5, 6]. Navigation otherwise remains a point reference tool because the projection is off-axis to the surgeon's inline loupe or microscope view. The Synaptive robotic brightmatter drive videoexoscope monitor system represents a new paradigm for off-axis high-definition (HD) surgical visualization. It has many advantages over the traditional microscope and loupes, which have already been demonstrated in a cadaveric study [7]. An auxiliary, but powerful capability of this system is projection of a second, modifiable image in a split-screen configuration. We hypothesized that integration of both Medtronic and Synaptive platforms could permit the visualization of reconstructed navigation and surgical field images simultaneously. By utilizing navigated instruments, this configuration has the ability to support live image-guided surgery or real-time navigation (RTN). Medtronic O-arm/Stealth S7 navigation, MetRx, NavLock, and SureTrak spinal systems were implemented on a prone cadaveric specimen with a stream output to the Synaptive Display. Surgical visualization was provided using a Storz Image S1 platform and camera mounted to the Synaptive robotic brightmatter drive. We were able to successfully technically co-adapt both platforms. A minimally invasive transforaminal lumbar interbody fusion (MIS TLIF) and an open pedicle subtraction osteotomy (PSO) were performed using a navigated high-speed drill under RTN. Disc Shaver and Trials under RTN were implemented on the MIS TLIF. The synergy of Synaptive HD videoexoscope robotic drive and Medtronic Stealth platforms allow for live image-guided surgery or real-time navigation (RTN). Off-axis projection also allows upright neutral cervical spine operative ergonomics for the surgeons and improved surgical team visualization and education compared to traditional means. This technique has the potential to augment existing minimally invasive and open approaches, but will require long-term outcome measurements for efficacy.

Electrically tunable lens speeds up 3D orbital tracking

PubMed Central

Annibale, Paolo; Dvornikov, Alexander; Gratton, Enrico

2015-01-01

3D orbital particle tracking is a versatile and effective microscopy technique that allows following fast moving fluorescent objects within living cells and reconstructing complex 3D shapes using laser scanning microscopes. We demonstrated notable improvements in the range, speed and accuracy of 3D orbital particle tracking by replacing commonly used piezoelectric stages with Electrically Tunable Lens (ETL) that eliminates mechanical movement of objective lenses. This allowed tracking and reconstructing shape of structures extending 500 microns in the axial direction. Using the ETL, we tracked at high speed fluorescently labeled genomic loci within the nucleus of living cells with unprecedented temporal resolution of 8ms using a 1.42NA oil-immersion objective. The presented technology is cost effective and allows easy upgrade of scanning microscopes for fast 3D orbital tracking. PMID:26114037

Image Montaging for Creating a Virtual Pathology Slide: An Innovative and Economical Tool to Obtain a Whole Slide Image

PubMed Central

Pandurangappa, Rohit; Annavajjula, Saileela; Rajashekaraiah, Premalatha Bidadi

2016-01-01

Background. Microscopes are omnipresent throughout the field of biological research. With microscopes one can see in detail what is going on at the cellular level in tissues. Though it is a ubiquitous tool, the limitation is that with high magnification there is a small field of view. It is often advantageous to see an entire sample at high magnification. Over the years technological advancements in optics have helped to provide solutions to this limitation of microscopes by creating the so-called dedicated “slide scanners” which can provide a “whole slide digital image.” These scanners can provide seamless, large-field-of-view, high resolution image of entire tissue section. The only disadvantage of such complete slide imaging system is its outrageous cost, thereby hindering their practical use by most laboratories, especially in developing and low resource countries. Methods. In a quest for their substitute, we tried commonly used image editing software Adobe Photoshop along with a basic image capturing device attached to a trinocular microscope to create a digital pathology slide. Results. The seamless image created using Adobe Photoshop maintained its diagnostic quality. Conclusion. With time and effort photomicrographs obtained from a basic camera-microscope set up can be combined and merged in Adobe Photoshop to create a whole slide digital image of practically usable quality at a negligible cost. PMID:27747147

Image Montaging for Creating a Virtual Pathology Slide: An Innovative and Economical Tool to Obtain a Whole Slide Image.

PubMed

Banavar, Spoorthi Ravi; Chippagiri, Prashanthi; Pandurangappa, Rohit; Annavajjula, Saileela; Rajashekaraiah, Premalatha Bidadi

2016-01-01

Background . Microscopes are omnipresent throughout the field of biological research. With microscopes one can see in detail what is going on at the cellular level in tissues. Though it is a ubiquitous tool, the limitation is that with high magnification there is a small field of view. It is often advantageous to see an entire sample at high magnification. Over the years technological advancements in optics have helped to provide solutions to this limitation of microscopes by creating the so-called dedicated "slide scanners" which can provide a "whole slide digital image." These scanners can provide seamless, large-field-of-view, high resolution image of entire tissue section. The only disadvantage of such complete slide imaging system is its outrageous cost, thereby hindering their practical use by most laboratories, especially in developing and low resource countries. Methods . In a quest for their substitute, we tried commonly used image editing software Adobe Photoshop along with a basic image capturing device attached to a trinocular microscope to create a digital pathology slide. Results . The seamless image created using Adobe Photoshop maintained its diagnostic quality. Conclusion . With time and effort photomicrographs obtained from a basic camera-microscope set up can be combined and merged in Adobe Photoshop to create a whole slide digital image of practically usable quality at a negligible cost.

SlideJ: An ImageJ plugin for automated processing of whole slide images

PubMed Central

Baroni, Giulia L.; Pilutti, David; Di Loreto, Carla

2017-01-01

The digital slide, or Whole Slide Image, is a digital image, acquired with specific scanners, that represents a complete tissue sample or cytological specimen at microscopic level. While Whole Slide image analysis is recognized among the most interesting opportunities, the typical size of such images—up to Gpixels- can be very demanding in terms of memory requirements. Thus, while algorithms and tools for processing and analysis of single microscopic field images are available, Whole Slide images size makes the direct use of such tools prohibitive or impossible. In this work a plugin for ImageJ, named SlideJ, is proposed with the objective to seamlessly extend the application of image analysis algorithms implemented in ImageJ for single microscopic field images to a whole digital slide analysis. The plugin has been complemented by examples of macro in the ImageJ scripting language to demonstrate its use in concrete situations. PMID:28683129

Cryogenic immersion microscope

DOEpatents

Le Gros, Mark; Larabell, Carolyn A.

2010-12-14

A cryogenic immersion microscope whose objective lens is at least partially in contact with a liquid reservoir of a cryogenic liquid, in which reservoir a sample of interest is immersed is disclosed. When the cryogenic liquid has an index of refraction that reduces refraction at interfaces between the lens and the sample, overall resolution and image quality are improved. A combination of an immersion microscope and x-ray microscope, suitable for imaging at cryogenic temperatures is also disclosed.

Images from Phoenix's MECA Instruments

NASA Technical Reports Server (NTRS)

2008-01-01

The image on the upper left is from NASA's Phoenix Mars Lander's Optical Microscope after a sample informally called 'Sorceress' was delivered to its silicon substrate on the 38th Martian day, or sol, of the mission (July 2, 2008).

A 3D representation of the same sample is on the right, as seen by Phoenix's Atomic Force Microscope. This is 100 times greater magnification than the view from the Optical Microscope, and the most highly magnified image ever seen from another world.

The Optical Microscope and the Atomic Force Microscope are part of Phoenix's Microscopy, Electrochemistry and Conductivity Analyzer instrument.

The Atomic Force Microscope was developed by a Swiss-led consortium in collaboration with Imperial College London.

The Phoenix Mission is led by the University of Arizona, Tucson, on behalf of NASA. Project management of the mission is by NASA's Jet Propulsion Laboratory, Pasadena, Calif. Spacecraft development is by Lockheed Martin Space Systems, Denver.

Near real-time digital holographic microscope based on GPU parallel computing

NASA Astrophysics Data System (ADS)

Zhu, Gang; Zhao, Zhixiong; Wang, Huarui; Yang, Yan

2018-01-01

A transmission near real-time digital holographic microscope with in-line and off-axis light path is presented, in which the parallel computing technology based on compute unified device architecture (CUDA) and digital holographic microscopy are combined. Compared to other holographic microscopes, which have to implement reconstruction in multiple focal planes and are time-consuming the reconstruction speed of the near real-time digital holographic microscope can be greatly improved with the parallel computing technology based on CUDA, so it is especially suitable for measurements of particle field in micrometer and nanometer scale. Simulations and experiments show that the proposed transmission digital holographic microscope can accurately measure and display the velocity of particle field in micrometer scale, and the average velocity error is lower than 10%.With the graphic processing units(GPU), the computing time of the 100 reconstruction planes(512×512 grids) is lower than 120ms, while it is 4.9s using traditional reconstruction method by CPU. The reconstruction speed has been raised by 40 times. In other words, it can handle holograms at 8.3 frames per second and the near real-time measurement and display of particle velocity field are realized. The real-time three-dimensional reconstruction of particle velocity field is expected to achieve by further optimization of software and hardware. Keywords: digital holographic microscope,

Optimal resolution in Fresnel incoherent correlation holographic fluorescence microscopy

PubMed Central

Brooker, Gary; Siegel, Nisan; Wang, Victor; Rosen, Joseph

2011-01-01

Fresnel Incoherent Correlation Holography (FINCH) enables holograms and 3D images to be created from incoherent light with just a camera and spatial light modulator (SLM). We previously described its application to microscopic incoherent fluorescence wherein one complex hologram contains all the 3D information in the microscope field, obviating the need for scanning or serial sectioning. We now report experiments which have led to the optimal optical, electro-optic, and computational conditions necessary to produce holograms which yield high quality 3D images from fluorescent microscopic specimens. An important improvement from our previous FINCH configurations capitalizes on the polarization sensitivity of the SLM so that the same SLM pixels which create the spherical wave simulating the microscope tube lens, also pass the plane waves from the infinity corrected microscope objective, so that interference between the two wave types at the camera creates a hologram. This advance dramatically improves the resolution of the FINCH system. Results from imaging a fluorescent USAF pattern and a pollen grain slide reveal resolution which approaches the Rayleigh limit by this simple method for 3D fluorescent microscopic imaging. PMID:21445140

Assessing delivery and quantifying efficacy of small interfering ribonucleic acid therapeutics in the skin using a dual-axis confocal microscope

NASA Astrophysics Data System (ADS)

Ra, Hyejun; Gonzalez-Gonzalez, Emilio; Smith, Bryan R.; Gambhir, Sanjiv S.; Kino, Gordon S.; Solgaard, Olav; Kaspar, Roger L.; Contag, Christopher H.

2010-05-01

Transgenic reporter mice and advances in imaging instrumentation are enabling real-time visualization of cellular mechanisms in living subjects and accelerating the development of novel therapies. Innovative confocal microscope designs are improving their utility for microscopic imaging of fluorescent reporters in living animals. We develop dual-axis confocal (DAC) microscopes for such in vivo studies and create mouse models where fluorescent proteins are expressed in the skin for the purpose of advancing skin therapeutics and transdermal delivery tools. Three-dimensional image volumes, through the different skin compartments of the epidermis and dermis, can be acquired in several seconds with the DAC microscope in living mice, and are comparable to histologic analyses of reporter protein expression patterns in skin sections. Intravital imaging with the DAC microscope further enables visualization of green fluorescent protein (GFP) reporter gene expression in the skin over time, and quantification of transdermal delivery of small interfering RNA (siRNA) and therapeutic efficacy. Visualization of transdermal delivery of nucleic acids will play an important role in the development of innovative strategies for treating skin pathologies.

Sub-nanosecond time-resolved near-field scanning magneto-optical microscope.

PubMed

Rudge, J; Xu, H; Kolthammer, J; Hong, Y K; Choi, B C

2015-02-01

We report on the development of a new magnetic microscope, time-resolved near-field scanning magneto-optical microscope, which combines a near-field scanning optical microscope and magneto-optical contrast. By taking advantage of the high temporal resolution of time-resolved Kerr microscope and the sub-wavelength spatial resolution of a near-field microscope, we achieved a temporal resolution of ∼50 ps and a spatial resolution of <100 nm. In order to demonstrate the spatiotemporal magnetic imaging capability of this microscope, the magnetic field pulse induced gyrotropic vortex dynamics occurring in 1 μm diameter, 20 nm thick CoFeB circular disks has been investigated. The microscope provides sub-wavelength resolution magnetic images of the gyrotropic motion of the vortex core at a resonance frequency of ∼240 MHz.

Single-frame 3D fluorescence microscopy with ultraminiature lensless FlatScope

PubMed Central

Adams, Jesse K.; Boominathan, Vivek; Avants, Benjamin W.; Vercosa, Daniel G.; Ye, Fan; Baraniuk, Richard G.; Robinson, Jacob T.; Veeraraghavan, Ashok

2017-01-01

Modern biology increasingly relies on fluorescence microscopy, which is driving demand for smaller, lighter, and cheaper microscopes. However, traditional microscope architectures suffer from a fundamental trade-off: As lenses become smaller, they must either collect less light or image a smaller field of view. To break this fundamental trade-off between device size and performance, we present a new concept for three-dimensional (3D) fluorescence imaging that replaces lenses with an optimized amplitude mask placed a few hundred micrometers above the sensor and an efficient algorithm that can convert a single frame of captured sensor data into high-resolution 3D images. The result is FlatScope: perhaps the world’s tiniest and lightest microscope. FlatScope is a lensless microscope that is scarcely larger than an image sensor (roughly 0.2 g in weight and less than 1 mm thick) and yet able to produce micrometer-resolution, high–frame rate, 3D fluorescence movies covering a total volume of several cubic millimeters. The ability of FlatScope to reconstruct full 3D images from a single frame of captured sensor data allows us to image 3D volumes roughly 40,000 times faster than a laser scanning confocal microscope while providing comparable resolution. We envision that this new flat fluorescence microscopy paradigm will lead to implantable endoscopes that minimize tissue damage, arrays of imagers that cover large areas, and bendable, flexible microscopes that conform to complex topographies. PMID:29226243

Microscopic neural image registration based on the structure of mitochondria

NASA Astrophysics Data System (ADS)

Cao, Huiwen; Han, Hua; Rao, Qiang; Xiao, Chi; Chen, Xi

2017-02-01

Microscopic image registration is a key component of the neural structure reconstruction with serial sections of neural tissue. The goal of microscopic neural image registration is to recover the 3D continuity and geometrical properties of specimen. During image registration, various distortions need to be corrected, including image rotation, translation, tissue deformation et.al, which come from the procedure of sample cutting, staining and imaging. Furthermore, there is only certain similarity between adjacent sections, and the degree of similarity depends on local structure of the tissue and the thickness of the sections. These factors make the microscopic neural image registration a challenging problem. To tackle the difficulty of corresponding landmarks extraction, we introduce a novel image registration method for Scanning Electron Microscopy (SEM) images of serial neural tissue sections based on the structure of mitochondria. The ellipsoidal shape of mitochondria ensures that the same mitochondria has similar shape between adjacent sections, and its characteristic of broad distribution in the neural tissue guarantees that landmarks based on the mitochondria distributed widely in the image. The proposed image registration method contains three parts: landmarks extraction between adjacent sections, corresponding landmarks matching and image deformation based on the correspondences. We demonstrate the performance of our method with SEM images of drosophila brain.

Light field creating and imaging with different order intensity derivatives

NASA Astrophysics Data System (ADS)

Wang, Yu; Jiang, Huan

2014-10-01

Microscopic image restoration and reconstruction is a challenging topic in the image processing and computer vision, which can be widely applied to life science, biology and medicine etc. A microscopic light field creating and three dimensional (3D) reconstruction method is proposed for transparent or partially transparent microscopic samples, which is based on the Taylor expansion theorem and polynomial fitting. Firstly the image stack of the specimen is divided into several groups in an overlapping or non-overlapping way along the optical axis, and the first image of every group is regarded as reference image. Then different order intensity derivatives are calculated using all the images of every group and polynomial fitting method based on the assumption that the structure of the specimen contained by the image stack in a small range along the optical axis are possessed of smooth and linear property. Subsequently, new images located any position from which to reference image the distance is Δz along the optical axis can be generated by means of Taylor expansion theorem and the calculated different order intensity derivatives. Finally, the microscopic specimen can be reconstructed in 3D form using deconvolution technology and all the images including both the observed images and the generated images. The experimental results show the effectiveness and feasibility of our method.

A high-resolution multimode digital microscope system.

PubMed

Salmon, Edward D; Shaw, Sidney L; Waters, Jennifer C; Waterman-Storer, Clare M; Maddox, Paul S; Yeh, Elaine; Bloom, Kerry

2013-01-01

This chapter describes the development of a high-resolution, multimode digital imaging system based on a wide-field epifluorescent and transmitted light microscope, and a cooled charge-coupled device (CCD) camera. The three main parts of this imaging system are Nikon FXA microscope, Hamamatsu C4880 cooled CCD camera, and MetaMorph digital imaging system. This chapter presents various design criteria for the instrument and describes the major features of the microscope components-the cooled CCD camera and the MetaMorph digital imaging system. The Nikon FXA upright microscope can produce high resolution images for both epifluorescent and transmitted light illumination without switching the objective or moving the specimen. The functional aspects of the microscope set-up can be considered in terms of the imaging optics, the epi-illumination optics, the transillumination optics, the focus control, and the vibration isolation table. This instrument is somewhat specialized for microtubule and mitosis studies, and it is also applicable to a variety of problems in cellular imaging, including tracking proteins fused to the green fluorescent protein in live cells. The instrument is also valuable for correlating the assembly dynamics of individual cytoplasmic microtubules (labeled by conjugating X-rhodamine to tubulin) with the dynamics of membranes of the endoplasmic reticulum (labeled with DiOC6) and the dynamics of the cell cortex (by differential interference contrast) in migrating vertebrate epithelial cells. This imaging system also plays an important role in the analysis of mitotic mutants in the powerful yeast genetic system Saccharomyces cerevisiae. Copyright © 1998 Elsevier Inc. All rights reserved.

Compact Video Microscope Imaging System Implemented in Colloid Studies

NASA Technical Reports Server (NTRS)

McDowell, Mark

2002-01-01

Long description Photographs showing fiber-optic light source, microscope and charge-coupled discharge (CCD) camera head connected to camera body, CCD camera body feeding data to image acquisition board in PC, and Cartesian robot controlled via PC board. The Compact Microscope Imaging System (CMIS) is a diagnostic tool with intelligent controls for use in space, industrial, medical, and security applications. CMIS can be used in situ with a minimum amount of user intervention. This system can scan, find areas of interest in, focus on, and acquire images automatically. Many multiple-cell experiments require microscopy for in situ observations; this is feasible only with compact microscope systems. CMIS is a miniature machine vision system that combines intelligent image processing with remote control. The software also has a user-friendly interface, which can be used independently of the hardware for further post-experiment analysis. CMIS has been successfully developed in the SML Laboratory at the NASA Glenn Research Center and adapted for use for colloid studies and is available for telescience experiments. The main innovations this year are an improved interface, optimized algorithms, and the ability to control conventional full-sized microscopes in addition to compact microscopes. The CMIS software-hardware interface is being integrated into our SML Analysis package, which will be a robust general-purpose image-processing package that can handle over 100 space and industrial applications.

Scanning-electron-microscope study of normal-impingement erosion of ductile metals

NASA Technical Reports Server (NTRS)

Brainard, W. A.; Salik, J.

1980-01-01

Scanning electron microscopy was used to characterize the erosion of annealed copper and aluminum surfaces produced by both single- and multiple-particle impacts. Macroscopic 3.2 mm diameter steel balls and microscopic, brittle erodant particles were projected by a gas gun system so as to impact at normal incidence at speeds up to 140 m/sec. During the impacts by the brittle erodant particles, at lower speeds the erosion behavior was similar to that observed for the larger steel balls. At higher velocities, particle fragmentation and the subsequent cutting by the radial wash of debris created a marked change in the erosion mechanism.

A Submersible, Off-Axis Holographic Microscope for Detection of Microbial Motility and Morphology in Aqueous and Icy Environments

PubMed Central

Lindensmith, Christian A.; Rider, Stephanie; Bedrossian, Manuel; Wallace, J. Kent; Serabyn, Eugene; Showalter, G. Max; Deming, Jody W.; Nadeau, Jay L.

2016-01-01

Sea ice is an analog environment for several of astrobiology’s near-term targets: Mars, Europa, Enceladus, and perhaps other Jovian or Saturnian moons. Microorganisms, both eukaryotic and prokaryotic, remain active within brine channels inside the ice, making it unnecessary to penetrate through to liquid water below in order to detect life. We have developed a submersible digital holographic microscope (DHM) that is capable of resolving individual bacterial cells, and demonstrated its utility for immediately imaging samples taken directly from sea ice at several locations near Nuuk, Greenland. In all samples, the appearance and motility of eukaryotes were conclusive signs of life. The appearance of prokaryotic cells alone was not sufficient to confirm life, but when prokaryotic motility occurred, it was rapid and conclusive. Warming the samples to above-freezing temperatures or supplementing with serine increased the number of motile cells and the speed of motility; supplementing with serine also stimulated chemotaxis. These results show that DHM is a useful technique for detection of active organisms in extreme environments, and that motility may be used as a biosignature in the liquid brines that persist in ice. These findings have important implications for the design of missions to icy environments and suggest ways in which DHM imaging may be integrated with chemical life-detection suites in order to create more conclusive life detection packages. PMID:26812683

A Submersible, Off-Axis Holographic Microscope for Detection of Microbial Motility and Morphology in Aqueous and Icy Environments.

PubMed

Lindensmith, Christian A; Rider, Stephanie; Bedrossian, Manuel; Wallace, J Kent; Serabyn, Eugene; Showalter, G Max; Deming, Jody W; Nadeau, Jay L

2016-01-01

Sea ice is an analog environment for several of astrobiology's near-term targets: Mars, Europa, Enceladus, and perhaps other Jovian or Saturnian moons. Microorganisms, both eukaryotic and prokaryotic, remain active within brine channels inside the ice, making it unnecessary to penetrate through to liquid water below in order to detect life. We have developed a submersible digital holographic microscope (DHM) that is capable of resolving individual bacterial cells, and demonstrated its utility for immediately imaging samples taken directly from sea ice at several locations near Nuuk, Greenland. In all samples, the appearance and motility of eukaryotes were conclusive signs of life. The appearance of prokaryotic cells alone was not sufficient to confirm life, but when prokaryotic motility occurred, it was rapid and conclusive. Warming the samples to above-freezing temperatures or supplementing with serine increased the number of motile cells and the speed of motility; supplementing with serine also stimulated chemotaxis. These results show that DHM is a useful technique for detection of active organisms in extreme environments, and that motility may be used as a biosignature in the liquid brines that persist in ice. These findings have important implications for the design of missions to icy environments and suggest ways in which DHM imaging may be integrated with chemical life-detection suites in order to create more conclusive life detection packages.

Development of a networked four-million-pixel pathological and radiological digital image presentation system and its application to medical conferences

NASA Astrophysics Data System (ADS)

Sakano, Toshikazu; Furukawa, Isao; Okumura, Akira; Yamaguchi, Takahiro; Fujii, Tetsuro; Ono, Sadayasu; Suzuki, Junji; Matsuya, Shoji; Ishihara, Teruo

2001-08-01

The wide spread of digital technology in the medical field has led to a demand for the high-quality, high-speed, and user-friendly digital image presentation system in the daily medical conferences. To fulfill this demand, we developed a presentation system for radiological and pathological images. It is composed of a super-high-definition (SHD) imaging system, a radiological image database (R-DB), a pathological image database (P-DB), and the network interconnecting these three. The R-DB consists of a 270GB RAID, a database server workstation, and a film digitizer. The P-DB includes an optical microscope, a four-million-pixel digital camera, a 90GB RAID, and a database server workstation. A 100Mbps Ethernet LAN interconnects all the sub-systems. The Web-based system operation software was developed for easy operation. We installed the whole system in NTT East Kanto Hospital to evaluate it in the weekly case conferences. The SHD system could display digital full-color images of 2048 x 2048 pixels on a 28-inch CRT monitor. The doctors evaluated the image quality and size, and found them applicable to the actual medical diagnosis. They also appreciated short image switching time that contributed to smooth presentation. Thus, we confirmed that its characteristics met the requirements.

Development of an adaptable coherent x-ray diffraction microscope with the emphasis on imaging hydrated specimens.

PubMed

Nam, Daewoong; Park, Jaehyun; Gallagher-Jones, Marcus; Shimada, Hiroki; Kim, Sangsoo; Kim, Sunam; Kohmura, Yoshiki; Ishikawa, Tetsuya; Song, Changyong

2013-11-01

This paper describes the development of a versatile coherent x-ray diffraction microscope capable of imaging biological specimens in solution. The microscope is a flexible platform accommodating various conditions, from low vacuum (10(-2) Pa) to helium gas filled ambient pressure. This flexibility greatly expands the application area, from in situ materials science to biology systems in their native state, by significantly relaxing restrictions to the sample environment. The coherent diffraction microscope has been used successfully to image a yeast cell immersed in buffer solution. We believe that the design of this coherent diffraction microscope can be directly adapted to various platforms such as table top soft x-ray laser, synchrotron x-ray sources, and x-ray free electron laser with minor relevant adjustments.

Development of an adaptable coherent x-ray diffraction microscope with the emphasis on imaging hydrated specimens

NASA Astrophysics Data System (ADS)

Nam, Daewoong; Park, Jaehyun; Gallagher-Jones, Marcus; Shimada, Hiroki; Kim, Sangsoo; Kim, Sunam; Kohmura, Yoshiki; Ishikawa, Tetsuya; Song, Changyong

2013-11-01

This paper describes the development of a versatile coherent x-ray diffraction microscope capable of imaging biological specimens in solution. The microscope is a flexible platform accommodating various conditions, from low vacuum (10-2 Pa) to helium gas filled ambient pressure. This flexibility greatly expands the application area, from in situ materials science to biology systems in their native state, by significantly relaxing restrictions to the sample environment. The coherent diffraction microscope has been used successfully to image a yeast cell immersed in buffer solution. We believe that the design of this coherent diffraction microscope can be directly adapted to various platforms such as table top soft x-ray laser, synchrotron x-ray sources, and x-ray free electron laser with minor relevant adjustments.

Programmable Colored Illumination Microscopy (PCIM): A practical and flexible optical staining approach for microscopic contrast enhancement

NASA Astrophysics Data System (ADS)

Zuo, Chao; Sun, Jiasong; Feng, Shijie; Hu, Yan; Chen, Qian

2016-03-01

Programmable colored illumination microscopy (PCIM) has been proposed as a flexible optical staining technique for microscopic contrast enhancement. In this method, we replace the condenser diaphragm of a conventional microscope with a programmable thin film transistor-liquid crystal display (TFT-LCD). By displaying different patterns on the LCD, numerous established imaging modalities can be realized, such as bright field, dark field, phase contrast, oblique illumination, and Rheinberg illuminations, which conventionally rely on intricate alterations in the respective microscope setups. Furthermore, the ease of modulating both the color and the intensity distribution at the aperture of the condenser opens the possibility to combine multiple microscopic techniques, or even realize completely new methods for optical color contrast staining, such as iridescent dark-field and iridescent phase-contrast imaging. The versatility and effectiveness of PCIM is demonstrated by imaging of several transparent colorless specimens, such as unstained lung cancer cells, diatom, textile fibers, and a cryosection of mouse kidney. Finally, the potentialities of PCIM for RGB-splitting imaging with stained samples are also explored by imaging stained red blood cells and a histological section.

A portable confocal hyperspectral microscope without any scan or tube lens and its application in fluorescence and Raman spectral imaging

NASA Astrophysics Data System (ADS)

Li, Jingwei; Cai, Fuhong; Dong, Yongjiang; Zhu, Zhenfeng; Sun, Xianhe; Zhang, Hequn; He, Sailing

2017-06-01

In this study, a portable confocal hyperspectral microscope is developed. In traditional confocal laser scanning microscopes, scan lens and tube lens are utilized to achieve a conjugate relationship between the galvanometer and the back focal plane of the objective, in order to achieve a better resolution. However, these lenses make it difficult to scale down the volume of the system. In our portable confocal hyperspectral microscope (PCHM), the objective is placed directly next to the galvomirror. Thus, scan lens and tube lens are not included in our system and the size of this system is greatly reduced. Furthermore, the resolution is also acceptable in many biomedical and food-safety applications. Through reducing the optical length of the system, the signal detection efficiency is enhanced. This is conducive to realizing both the fluorescence and Raman hyperspectral imaging. With a multimode fiber as a pinhole, an improved image contrast is also achieved. Fluorescent spectral images for HeLa cells/fingers and Raman spectral images of kumquat pericarp are present. The spectral resolution and spatial resolutions are about 0.4 nm and 2.19 μm, respectively. These results demonstrate that this portable hyperspectral microscope can be used in in-vivo fluorescence imaging and in situ Raman spectral imaging.

Design and analysis of multilayer x ray/XUV microscope

NASA Technical Reports Server (NTRS)

Shealy, David L.

1990-01-01

The design and analysis of a large number of normal incidence multilayer x ray microscopes based on the spherical mirror Schwarzschild configuration is examined. Design equations for the spherical mirror Schwarzschild microscopes are summarized and used to evaluate mirror parameters for microscopes with magnifications ranging from 2 to 50x. Ray tracing and diffraction analyses are carried out for many microscope configurations to determine image resolution as a function of system parameters. The results are summarized in three publication included herein. A preliminary study of advanced reflecting microscope configurations, where aspherics are used in place of the spherical microscope mirror elements, has indicated that the aspherical elements will improve off-axis image resolution and increase the effective field of view.

Comparisons between conventional optical imaging and parametric indirect microscopic imaging on human skin detection

NASA Astrophysics Data System (ADS)

Liu, Guoyan; Gao, Kun; Liu, Xuefeng; Ni, Guoqiang

2016-10-01

We report a new method, polarization parameters indirect microscopic imaging with a high transmission infrared light source, to detect the morphology and component of human skin. A conventional reflection microscopic system is used as the basic optical system, into which a polarization-modulation mechanics is inserted and a high transmission infrared light source is utilized. The near-field structural characteristics of human skin can be delivered by infrared waves and material coupling. According to coupling and conduction physics, changes of the optical wave parameters can be calculated and curves of the intensity of the image can be obtained. By analyzing the near-field polarization parameters in nanoscale, we can finally get the inversion images of human skin. Compared with the conventional direct optical microscope, this method can break diffraction limit and achieve a super resolution of sub-100nm. Besides, the method is more sensitive to the edges, wrinkles, boundaries and impurity particles.

Tracking of Cells with a Compact Microscope Imaging System with Intelligent Controls

NASA Technical Reports Server (NTRS)

McDowell, Mark (Inventor)

2007-01-01

A Microscope Imaging System (CMIS) with intelligent controls is disclosed that provides techniques for scanning, identifying, detecting and tracking microscopic changes in selected characteristics or features of various surfaces including, but not limited to, cells, spheres, and manufactured products subject to difficult-to-see imperfections. The practice of the present invention provides applications that include colloidal hard spheres experiments, biological cell detection for patch clamping, cell movement and tracking, as well as defect identification in products, such as semiconductor devices, where surface damage can be significant, but difficult to detect. The CMIS system is a machine vision system, which combines intelligent image processing with remote control capabilities and provides the ability to autofocus on a microscope sample, automatically scan an image, and perform machine vision analysis on multiple samples simultaneously

Tracking of cells with a compact microscope imaging system with intelligent controls

NASA Technical Reports Server (NTRS)

McDowell, Mark (Inventor)

2007-01-01

A Microscope Imaging System (CMIS) with intelligent controls is disclosed that provides techniques for scanning, identifying, detecting and tracking microscopic changes in selected characteristics or features of various surfaces including, but not limited to, cells, spheres, and manufactured products subject to difficult-to-see imperfections. The practice of the present invention provides applications that include colloidal hard spheres experiments, biological cell detection for patch clamping, cell movement and tracking, as well as defect identification in products, such as semiconductor devices, where surface damage can be significant, but difficult to detect. The CMIS system is a machine vision system, which combines intelligent image processing with remote control capabilities and provides the ability to auto-focus on a microscope sample, automatically scan an image, and perform machine vision analysis on multiple samples simultaneously.

Laser speckle contrast imaging using light field microscope approach

NASA Astrophysics Data System (ADS)

Ma, Xiaohui; Wang, Anting; Ma, Fenghua; Wang, Zi; Ming, Hai

2018-01-01

In this paper, a laser speckle contrast imaging (LSCI) system using light field (LF) microscope approach is proposed. As far as we known, it is first time to combine LSCI with LF. To verify this idea, a prototype consists of a modified LF microscope imaging system and an experimental device was built. A commercially used Lytro camera was modified for microscope imaging. Hollow glass tubes with different depth fixed in glass dish were used to simulate the vessels in brain and test the performance of the system. Compared with conventional LSCI, three new functions can be realized by using our system, which include refocusing, extending the depth of field (DOF) and gathering 3D information. Experiments show that the principle is feasible and the proposed system works well.

A multi-modal stereo microscope based on a spatial light modulator.

PubMed

Lee, M P; Gibson, G M; Bowman, R; Bernet, S; Ritsch-Marte, M; Phillips, D B; Padgett, M J

2013-07-15

Spatial Light Modulators (SLMs) can emulate the classic microscopy techniques, including differential interference (DIC) contrast and (spiral) phase contrast. Their programmability entails the benefit of flexibility or the option to multiplex images, for single-shot quantitative imaging or for simultaneous multi-plane imaging (depth-of-field multiplexing). We report the development of a microscope sharing many of the previously demonstrated capabilities, within a holographic implementation of a stereo microscope. Furthermore, we use the SLM to combine stereo microscopy with a refocusing filter and with a darkfield filter. The instrument is built around a custom inverted microscope and equipped with an SLM which gives various imaging modes laterally displaced on the same camera chip. In addition, there is a wide angle camera for visualisation of a larger region of the sample.

Scanning laser microscope for imaging nanostructured superconductors

NASA Astrophysics Data System (ADS)

Ishida, Takekazu; Arai, Kohei; Akita, Yukio; Miyanari, Mitsunori; Minami, Yusuke; Yotsuya, Tsutomu; Kato, Masaru; Satoh, Kazuo; Uno, Mayumi; Shimakage, Hisashi; Miki, Shigehito; Wang, Zhen

2010-10-01

The nanofabrication of superconductors yields various interesting features in superconducting properties. A variety of different imaging techniques have been developed for probing the local superconducting profiles. A scanning pulsed laser microscope has been developed by the combination of the XYZ piezo-driven stages and an optical fiber with an aspheric focusing lens. The scanning laser microscope is used to understand the position-dependent properties of a superconducting MgB 2 stripline of length 100 μm and width of 3 μm under constant bias current. Our results show that the superconducting stripline can clearly be seen in the contour image of the scanning laser microscope on the signal voltage. It is suggested from the observed image that the inhomogeneity is relevant in specifying the operating conditions such as detection efficiency of the sensor.

Polarization properties of amyloid-beta plaques in Alzheimer's disease (Conference Presentation)

NASA Astrophysics Data System (ADS)

Baumann, Bernhard; Wöhrer, Adelheid; Ricken, Gerda; Pircher, Michael; Kovacs, Gabor G.; Hitzenberger, Christoph K.

2016-03-01

In histopathological practice, birefringence is used for the identification of amyloidosis in numerous tissues. Amyloid birefringence is caused by the parallel arrangement of fibrous protein aggregates. Since neurodegenerative processes in Alzheimer's disease (AD) are also linked to the formation of amyloid-beta (Aβ) plaques, optical methods sensitive to birefringence may act as non-invasive tools for Aβ identification. At last year's Photonics West, we demonstrated polarization-sensitive optical coherence tomography (PS-OCT) imaging of ex vivo cerebral tissue of advanced stage AD patients. PS-OCT provides volumetric, structural imaging based on both backscatter contrast and tissue polarization properties. In this presentation, we report on polarization-sensitive neuroimaging along with numerical simulations of three-dimensional Aβ plaques. High speed PS-OCT imaging was performed using a spectral domain approach based on polarization maintaining fiber optics. The sample beam was interfaced to a confocal scanning microscope arrangement. Formalin-fixed tissue samples as well as thin histological sections were imaged. For comparison to the PS-OCT results, ray propagation through plaques was modeled using Jones analysis and various illumination geometries and plaque sizes. Characteristic polarization patterns were found. The results of this study may not only help to understand PS-OCT imaging of neuritic Aβ plaques but may also have implications for polarization-sensitive imaging of other fibrillary structures.

Chromatically encoded high-speed photography of cavitation bubble dynamics inside inhomogeneous ophthalmic tissue

NASA Astrophysics Data System (ADS)

Tinne, N.; Matthias, B.; Kranert, F.; Wetzel, C.; Krüger, A.; Ripken, T.

2016-03-01

The interaction effect of photodisruption, which is used for dissection of biological tissue with fs-laser pulses, has been intensively studied inside water as prevalent sample medium. In this case, the single effect is highly reproducible and, hence, the method of time-resolved photography is sufficiently applicable. In contrast, the reproducibility significantly decreases analyzing more solid and anisotropic media like biological tissue. Therefore, a high-speed photographic approach is necessary in this case. The presented study introduces a novel technique for high-speed photography based on the principle of chromatic encoding. For illumination of the region of interest within the sample medium, the light paths of up to 12 LEDs with various emission wavelengths are overlaid via optical filters. Here, MOSFET-electronics provide a LED flash with a duration <100 ns; the diodes are externally triggered with a distinct delay for every LED. Furthermore, the different illumination wavelengths are chromatically separated again for detection via camera chip. Thus, the experimental setup enables the generation of a time-sequence of <= 12 images of a single cavitation bubble dynamics. In comparison to conventional time-resolved photography, images in sample media like water and HEMA show the significant advantages of this novel illumination technique. In conclusion, the results of this study are of great importance for the fundamental evaluation of the laser-tissue interaction inside anisotropic biological tissue and for the optimization of the surgical process with high-repetition rate fs-lasers. Additionally, this application is also suitable for the investigation of other microscopic, ultra-fast events in transparent inhomogeneous materials.

Microscope on Mars

NASA Technical Reports Server (NTRS)

2004-01-01

This image taken at Meridiani Planum, Mars by the panoramic camera on the Mars Exploration Rover Opportunity shows the rover's microscopic imager (circular device in center), located on its instrument deployment device, or 'arm.' The image was acquired on the ninth martian day or sol of the rover's mission.

Super-resolution imaging of ciliary microdomains in isolated olfactory sensory neurons using a custom two-color stimulated emission depletion microscope

NASA Astrophysics Data System (ADS)

Meyer, Stephanie A.; Ozbay, Baris N.; Potcoava, Mariana; Salcedo, Ernesto; Restrepo, Diego; Gibson, Emily A.

2016-06-01

We performed stimulated emission depletion (STED) imaging of isolated olfactory sensory neurons (OSNs) using a custom-built microscope. The STED microscope uses a single pulsed laser to excite two separate fluorophores, Atto 590 and Atto 647N. A gated timing circuit combined with temporal interleaving of the different color excitation/STED laser pulses filters the two channel detection and greatly minimizes crosstalk. We quantified the instrument resolution to be ˜81 and ˜44 nm, for the Atto 590 and Atto 647N channels. The spatial separation between the two channels was measured to be under 10 nm, well below the resolution limit. The custom-STED microscope is incorporated onto a commercial research microscope allowing brightfield, differential interference contrast, and epifluorescence imaging on the same field of view. We performed immunolabeling of OSNs in mice to image localization of ciliary membrane proteins involved in olfactory transduction. We imaged Ca2+-permeable cyclic nucleotide gated (CNG) channel (Atto 594) and adenylyl cyclase type III (ACIII) (Atto 647N) in distinct cilia. STED imaging resolved well-separated subdiffraction limited clusters for each protein. We quantified the size of each cluster to have a mean value of 88±48 nm and 124±43 nm, for CNG and ACIII, respectively. STED imaging showed separated clusters that were not resolvable in confocal images.

Designed Er(3+)-singly doped NaYF4 with double excitation bands for simultaneous deep macroscopic and microscopic upconverting bioimaging.

PubMed

Wen, Xuanyuan; Wang, Baoju; Wu, Ruitao; Li, Nana; He, Sailing; Zhan, Qiuqiang

2016-06-01

Simultaneous deep macroscopic imaging and microscopic imaging is in urgent demand, but is challenging to achieve experimentally due to the lack of proper fluorescent probes. Herein, we have designed and successfully synthesized simplex Er(3+)-doped upconversion nanoparticles (UCNPs) with double excitation bands for simultaneous deep macroscopic and microscopic imaging. The material structure and the excitation wavelength of Er(3+)-singly doped UCNPs were further optimized to enhance the upconversion emission efficiency. After optimization, we found that NaYF4:30%Er(3+)@NaYF4:2%Er(3+) could simultaneously achieve efficient two-photon excitation (2PE) macroscopic tissue imaging and three-photon excitation (3PE) deep microscopic when excited by 808 nm continuous wave (CW) and 1480 nm CW lasers, respectively. In vitro cell imaging and in vivo imaging have also been implemented to demonstrate the feasibility and potential of the proposed simplex Er(3+)-doped UCNPs as bioprobe.

Evidence from Opportunity's Microscopic Imager for water on Meridiani Planum.

PubMed

Herkenhoff, K E; Squyres, S W; Arvidson, R; Bass, D S; Bell, J F; Bertelsen, P; Ehlmann, B L; Farrand, W; Gaddis, L; Greeley, R; Grotzinger, J; Hayes, A G; Hviid, S F; Johnson, J R; Jolliff, B; Kinch, K M; Knoll, A H; Madsen, M B; Maki, J N; McLennan, S M; McSween, H Y; Ming, D W; Rice, J W; Richter, L; Sims, M; Smith, P H; Soderblom, L A; Spanovich, N; Sullivan, R; Thompson, S; Wdowiak, T; Weitz, C; Whelley, P

2004-12-03

The Microscopic Imager on the Opportunity rover analyzed textures of soils and rocks at Meridiani Planum at a scale of 31 micrometers per pixel. The uppermost millimeter of some soils is weakly cemented, whereas other soils show little evidence of cohesion. Rock outcrops are laminated on a millimeter scale; image mosaics of cross-stratification suggest that some sediments were deposited by flowing water. Vugs in some outcrop faces are probably molds formed by dissolution of relatively soluble minerals during diagenesis. Microscopic images support the hypothesis that hematite-rich spherules observed in outcrops and soils also formed diagenetically as concretions.

Microscopic Materials on a Magnet

NASA Technical Reports Server (NTRS)

2008-01-01

These images show a comparison of the weak magnet OM7 from the Optical Microscope on NASA's Phoenix Mars Lander before (left) and after (right) soil deposition.

The microscope took the left image during Phoenix's Sol 15 (June 10, 2008) and the right image during Sol 21 (Jun 16, 2008).

The Phoenix Mission is led by the University of Arizona, Tucson, on behalf of NASA. Project management of the mission is by NASA's Jet Propulsion Laboratory, Pasadena, Calif. Spacecraft development is by Lockheed Martin Space Systems, Denver.

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.

Optical second harmonic images of 90 deg domain structure in BaTiO3 and periodically inverted antiparallel domains in LiTaO3

NASA Astrophysics Data System (ADS)

Uesu, Y.; Kurimura, S.; Yamamoto, Y.

1995-04-01

Applied is a microscope to observations of 90 deg ferroelectric domain structure in BaTiO3 and inverted periodically are ferroelectric domains in LiTaO3. It is founded that the second harmonic generation microscope gives information which cannot be obtained by ordinary optical microscopes. The developed nonlinear optical microscope builds two dimensional second harmonic image of a specimen with inhomogenous distribution of d(sub ijk) and applied the microscope to observations of inhomogeneity in some nonlinear-optical organic microcrystals.

ultraLM and miniLM: Locator tools for smart tracking of fluorescent cells in correlative light and electron microscopy.

PubMed

Brama, Elisabeth; Peddie, Christopher J; Wilkes, Gary; Gu, Yan; Collinson, Lucy M; Jones, Martin L

2016-12-13

In-resin fluorescence (IRF) protocols preserve fluorescent proteins in resin-embedded cells and tissues for correlative light and electron microscopy, aiding interpretation of macromolecular function within the complex cellular landscape. Dual-contrast IRF samples can be imaged in separate fluorescence and electron microscopes, or in dual-modality integrated microscopes for high resolution correlation of fluorophore to organelle. IRF samples also offer a unique opportunity to automate correlative imaging workflows. Here we present two new locator tools for finding and following fluorescent cells in IRF blocks, enabling future automation of correlative imaging. The ultraLM is a fluorescence microscope that integrates with an ultramicrotome, which enables 'smart collection' of ultrathin sections containing fluorescent cells or tissues for subsequent transmission electron microscopy or array tomography. The miniLM is a fluorescence microscope that integrates with serial block face scanning electron microscopes, which enables 'smart tracking' of fluorescent structures during automated serial electron image acquisition from large cell and tissue volumes.

ScanImage: flexible software for operating laser scanning microscopes.

PubMed

Pologruto, Thomas A; Sabatini, Bernardo L; Svoboda, Karel

2003-05-17

Laser scanning microscopy is a powerful tool for analyzing the structure and function of biological specimens. Although numerous commercial laser scanning microscopes exist, some of the more interesting and challenging applications demand custom design. A major impediment to custom design is the difficulty of building custom data acquisition hardware and writing the complex software required to run the laser scanning microscope. We describe a simple, software-based approach to operating a laser scanning microscope without the need for custom data acquisition hardware. Data acquisition and control of laser scanning are achieved through standard data acquisition boards. The entire burden of signal integration and image processing is placed on the CPU of the computer. We quantitate the effectiveness of our data acquisition and signal conditioning algorithm under a variety of conditions. We implement our approach in an open source software package (ScanImage) and describe its functionality. We present ScanImage, software to run a flexible laser scanning microscope that allows easy custom design.

Algorithms for differentiating between images of heterogeneous tissue across fluorescence microscopes.

PubMed

Chitalia, Rhea; Mueller, Jenna; Fu, Henry L; Whitley, Melodi Javid; Kirsch, David G; Brown, J Quincy; Willett, Rebecca; Ramanujam, Nimmi

2016-09-01

Fluorescence microscopy can be used to acquire real-time images of tissue morphology and with appropriate algorithms can rapidly quantify features associated with disease. The objective of this study was to assess the ability of various segmentation algorithms to isolate fluorescent positive features (FPFs) in heterogeneous images and identify an approach that can be used across multiple fluorescence microscopes with minimal tuning between systems. Specifically, we show a variety of image segmentation algorithms applied to images of stained tumor and muscle tissue acquired with 3 different fluorescence microscopes. Results indicate that a technique called maximally stable extremal regions followed by thresholding (MSER + Binary) yielded the greatest contrast in FPF density between tumor and muscle images across multiple microscopy systems.

Measurement of Microscopic Growth Rates in Float-Zone Silicon Crystals

NASA Technical Reports Server (NTRS)

Dold, P.; Schweizer, M.; Benz, K. W.; Rose, M. Franklin (Technical Monitor)

2001-01-01

Time dependent convective flows during crystal growth of doped semiconductors lead to fluctuations of the composition, so called dopant striations. In general, it is difficult to decide which is the main mechanism for the generation of these striations, it might be either the fluctuation of the concentration field in the melt and the extent of the solute boundary layer ahead of the solid-liquid interface or a variation of the growth velocity. Direct access to the concentration field is rather complicated to achieve, especially considering the high process temperature and the chemical activity of liquid silicon. The contribution of growth rate fluctuations to the formation of compositional fluctuations can be determined by measuring microscopic growth rates. The classical method of current pulses requires electrical feed-throughs and good electrical contacts, both are critical issues for the growth of high purity silicon crystals. Using a radiation based heating system, the heating power can be modulated very fast and effectively. We added to the normal heater power a sinusoidal off-set in the frequency range of 1 to 10 Hz, generating a narrow spaced weak rippling in the grown crystals which are superposed to the dopant striations caused by natural and by thermocapillary convection. The pulling speed was varied between 1 and 4mm/min. The microscope images of etched crystals slices have been analyzed by peak-search algorithms (measuring the spacing between each artificially induced marker) and by FFT. Performing growth experiments under a time-dependent flow regime, fluctuations of the microscopic growth velocity of Delta(v)/v(sub average) up to 50% have been measured. Damping the time-dependent convection by the use of an axial, static magnetic field of 500mT, the microscopic growth rate became constant within the resolution limit of this method. The results will be discussed using analytical methods for the calculation of microscopic growth velocity and by comparing them with measurements of temperature fluctuations in the melt during growth experiments itself.

LIPS database with LIPService: a microscopic image database of intracellular structures in Arabidopsis guard cells.

PubMed

Higaki, Takumi; Kutsuna, Natsumaro; Hasezawa, Seiichiro

2013-05-16

Intracellular configuration is an important feature of cell status. Recent advances in microscopic imaging techniques allow us to easily obtain a large number of microscopic images of intracellular structures. In this circumstance, automated microscopic image recognition techniques are of extreme importance to future phenomics/visible screening approaches. However, there was no benchmark microscopic image dataset for intracellular organelles in a specified plant cell type. We previously established the Live Images of Plant Stomata (LIPS) database, a publicly available collection of optical-section images of various intracellular structures of plant guard cells, as a model system of environmental signal perception and transduction. Here we report recent updates to the LIPS database and the establishment of a database table, LIPService. We updated the LIPS dataset and established a new interface named LIPService to promote efficient inspection of intracellular structure configurations. Cell nuclei, microtubules, actin microfilaments, mitochondria, chloroplasts, endoplasmic reticulum, peroxisomes, endosomes, Golgi bodies, and vacuoles can be filtered using probe names or morphometric parameters such as stomatal aperture. In addition to the serial optical sectional images of the original LIPS database, new volume-rendering data for easy web browsing of three-dimensional intracellular structures have been released to allow easy inspection of their configurations or relationships with cell status/morphology. We also demonstrated the utility of the new LIPS image database for automated organelle recognition of images from another plant cell image database with image clustering analyses. The updated LIPS database provides a benchmark image dataset for representative intracellular structures in Arabidopsis guard cells. The newly released LIPService allows users to inspect the relationship between organellar three-dimensional configurations and morphometrical parameters.

In vivo cellular imaging with microscopes enabled by MEMS scanners

NASA Astrophysics Data System (ADS)

Ra, Hyejun

High-resolution optical imaging plays an important role in medical diagnosis and biomedical research. Confocal microscopy is a widely used imaging method for obtaining cellular and sub-cellular images of biological tissue in reflectance and fluorescence modes. Its characteristic optical sectioning capability also enables three-dimensional (3-D) image reconstruction. However, its use has mostly been limited to excised tissues due to the requirement of high numerical aperture (NA) lenses for cellular resolution. Microscope miniaturization can enable in vivo imaging to make possible early cancer diagnosis and biological studies in the innate environment. In this dissertation, microscope miniaturization for in vivo cellular imaging is presented. The dual-axes confocal (DAC) architecture overcomes limitations of the conventional single-axis confocal (SAC) architecture to allow for miniaturization with high resolution. A microelectromechanical systems (MEMS) scanner is the central imaging component that is key in miniaturization of the DAC architecture. The design, fabrication, and characterization of the two-dimensional (2-D) MEMS scanner are presented. The gimbaled MEMS scanner is fabricated on a double silicon-on-insulator (SOI) wafer and is actuated by self-aligned vertical electrostatic combdrives. The imaging performance of the MEMS scanner in a DAC configuration is shown in a breadboard microscope setup, where reflectance and fluorescence imaging is demonstrated. Then, the MEMS scanner is integrated into a miniature DAC microscope. The whole imaging system is integrated into a portable unit for research in small animal models of human biology and disease. In vivo 3-D imaging is demonstrated on mouse skin models showing gene transfer and siRNA silencing. The siRNA silencing process is sequentially imaged in one mouse over time.

Thrombus segmentation by texture dynamics from microscopic image sequences

NASA Astrophysics Data System (ADS)

Brieu, Nicolas; Serbanovic-Canic, Jovana; Cvejic, Ana; Stemple, Derek; Ouwehand, Willem; Navab, Nassir; Groher, Martin

2010-03-01

The genetic factors of thrombosis are commonly explored by microscopically imaging the coagulation of blood cells induced by injuring a vessel of mice or of zebrafish mutants. The latter species is particularly interesting since skin transparency permits to non-invasively acquire microscopic images of the scene with a CCD camera and to estimate the parameters characterizing the thrombus development. These parameters are currently determined by manual outlining, which is both error prone and extremely time consuming. Even though a technique for automatic thrombus extraction would be highly valuable for gene analysts, little work can be found, which is mainly due to very low image contrast and spurious structures. In this work, we propose to semi-automatically segment the thrombus over time from microscopic image sequences of wild-type zebrafish larvae. To compensate the lack of valuable spatial information, our main idea consists of exploiting the temporal information by modeling the variations of the pixel intensities over successive temporal windows with a linear Markov-based dynamic texture formalization. We then derive an image from the estimated model parameters, which represents the probability of a pixel to belong to the thrombus. We employ this probability image to accurately estimate the thrombus position via an active contour segmentation incorporating also prior and spatial information of the underlying intensity images. The performance of our approach is tested on three microscopic image sequences. We show that the thrombus is accurately tracked over time in each sequence if the respective parameters controlling prior influence and contour stiffness are correctly chosen.

Microscopic Image of Martian Surface Material on a Silicone Substrate

NASA Technical Reports Server (NTRS)

2008-01-01

[figure removed for brevity, see original site] Click on image for larger version of Figure 1

This image taken by the Optical Microscope on NASA's Phoenix Mars Lander shows soil sprinkled from the lander's Robot Arm scoop onto a silicone substrate. The substrate was then rotated in front of the microscope. This is the first sample collected and delivered for instrumental analysis onboard a planetary lander since NASA's Viking Mars missions of the 1970s. It is also the highest resolution image yet seen of Martian soil.

The image is dominated by fine particles close to the resolution of the microscope. These particles have formed clumps, which may be a smaller scale version of what has been observed by Phoenix during digging of the surface material.

The microscope took this image during Phoenix's Sol 17 (June 11), or the 17th Martian day after landing. The scale bar is 1 millimeter (0.04 inch).

Zooming in on the Martian Soil

In figure 1, three zoomed-in portions are shown with an image of Martian soil particles taken by the Optical Microscope on NASA's Phoenix Mars Lander.

The left zoom box shows a composite particle. The top of the particle has a green tinge, possibly indicating olivine. The bottom of the particle has been reimaged at a different focus position in black and white (middle zoom box), showing that this is a clump of finer particles.

The right zoom box shows a rounded, glassy particle, similar to those which have also been seen in an earlier sample of airfall dust collected on a surface exposed during landing.

The shadows at the bottom of image are of the beams of the Atomic Force Microscope.

The Phoenix Mission is led by the University of Arizona, Tucson, on behalf of NASA. Project management of the mission is by NASA's Jet Propulsion Laboratory, Pasadena, Calif. Spacecraft development is by Lockheed Martin Space Systems, Denver.

Multidirectional Image Sensing for Microscopy Based on a Rotatable Robot.

PubMed

Shen, Yajing; Wan, Wenfeng; Zhang, Lijun; Yong, Li; Lu, Haojian; Ding, Weili

2015-12-15

Image sensing at a small scale is essentially important in many fields, including microsample observation, defect inspection, material characterization and so on. However, nowadays, multi-directional micro object imaging is still very challenging due to the limited field of view (FOV) of microscopes. This paper reports a novel approach for multi-directional image sensing in microscopes by developing a rotatable robot. First, a robot with endless rotation ability is designed and integrated with the microscope. Then, the micro object is aligned to the rotation axis of the robot automatically based on the proposed forward-backward alignment strategy. After that, multi-directional images of the sample can be obtained by rotating the robot within one revolution under the microscope. To demonstrate the versatility of this approach, we view various types of micro samples from multiple directions in both optical microscopy and scanning electron microscopy, and panoramic images of the samples are processed as well. The proposed method paves a new way for the microscopy image sensing, and we believe it could have significant impact in many fields, especially for sample detection, manipulation and characterization at a small scale.

Multiparallel Three-Dimensional Optical Microscopy

NASA Technical Reports Server (NTRS)

Nguyen, Lam K.; Price, Jeffrey H.; Kellner, Albert L.; Bravo-Zanoquera, Miguel

2010-01-01

Multiparallel three-dimensional optical microscopy is a method of forming an approximate three-dimensional image of a microscope sample as a collection of images from different depths through the sample. The imaging apparatus includes a single microscope plus an assembly of beam splitters and mirrors that divide the output of the microscope into multiple channels. An imaging array of photodetectors in each channel is located at a different distance along the optical path from the microscope, corresponding to a focal plane at a different depth within the sample. The optical path leading to each photodetector array also includes lenses to compensate for the variation of magnification with distance so that the images ultimately formed on all the photodetector arrays are of the same magnification. The use of optical components common to multiple channels in a simple geometry makes it possible to obtain high light-transmission efficiency with an optically and mechanically simple assembly. In addition, because images can be read out simultaneously from all the photodetector arrays, the apparatus can support three-dimensional imaging at a high scanning rate.

Full-field OCT: applications in ophthalmology

NASA Astrophysics Data System (ADS)

Grieve, Kate; Dubois, Arnaud; Paques, Michel; Le Gargasson, Jean-Francois; Boccara, Albert C.

2005-04-01

We present images of ocular tissues obtained using ultrahigh resolution full-field OCT. The experimental setup is based on the Linnik interferometer, illuminated by a tungsten halogen lamp. En face tomographic images are obtained in real-time without scanning by computing the difference of two phase-opposed interferometric images recorded by a high-resolution CCD camera. A spatial resolution of 0.7 μm × 0.9 μm (axial × transverse) is achieved thanks to the short source coherence length and the use of high numerical aperture microscope objectives. A detection sensitivity of 90 dB is obtained by means of image averaging and pixel binning. Whole unfixed eyes and unstained tissue samples (cornea, lens, retina, choroid and sclera) of ex vivo rat, mouse, rabbit and porcine ocular tissues were examined. The unprecedented resolution of our instrument allows cellular-level resolution in the cornea and retina, and visualization of individual fibers in the lens. Transcorneal lens imaging was possible in all animals, and in albino animals, transscleral retinal imaging was achieved. We also introduce our rapid acquisition full-field optical coherence tomography system designed to accommodate in vivo ophthalmologic imaging. The variations on the original system technology include the introduction of a xenon arc lamp as source, and rapid image acquisition performed by a high-speed CMOS camera, reducing acquisition time to 5 ms per frame.

In situ high-resolution thermal microscopy on integrated circuits.

PubMed

Zhuo, Guan-Yu; Su, Hai-Ching; Wang, Hsien-Yi; Chan, Ming-Che

2017-09-04

The miniaturization of metal tracks in integrated circuits (ICs) can cause abnormal heat dissipation, resulting in electrostatic discharge, overvoltage breakdown, and other unwanted issues. Unfortunately, locating areas of abnormal heat dissipation is limited either by the spatial resolution or imaging acquisition speed of current thermal analytical techniques. A rapid, non-contact approach to the thermal imaging of ICs with sub-μm resolution could help to alleviate this issue. In this work, based on the intensity of the temperature-dependent two-photon fluorescence (TPF) of Rhodamine 6G (R6G) material, we developed a novel fast and non-invasive thermal microscopy with a sub-μm resolution. Its application to the location of hotspots that may evolve into thermally induced defects in ICs was also demonstrated. To the best of our knowledge, this is the first study to present high-resolution 2D thermal microscopic images of ICs, showing the generation, propagation, and distribution of heat during its operation. According to the demonstrated results, this scheme has considerable potential for future in situ hotspot analysis during the optimization stage of IC development.

Microscopy imaging device with advanced imaging properties

DOEpatents

Ghosh, Kunal; Burns, Laurie; El Gamal, Abbas; Schnitzer, Mark J.; Cocker, Eric; Ho, Tatt Wei

2015-11-24

Systems, methods and devices are implemented for microscope imaging solutions. One embodiment of the present disclosure is directed toward an epifluorescence microscope. The microscope includes an image capture circuit including an array of optical sensor. An optical arrangement is configured to direct excitation light of less than about 1 mW to a target object in a field of view of that is at least 0.5 mm.sup.2 and to direct epi-fluorescence emission caused by the excitation light to the array of optical sensors. The optical arrangement and array of optical sensors are each sufficiently close to the target object to provide at least 2.5 .mu.m resolution for an image of the field of view.

Microdose fluorescence imaging of ABY-029 on an operating microscope adapted by custom illumination and imaging modules.

PubMed

Elliott, Jonathan T; Dsouza, Alisha V; Marra, Kayla; Pogue, Brian W; Roberts, David W; Paulsen, Keith D

2016-09-01

Fluorescence guided surgery has the potential to positively impact surgical oncology; current operating microscopes and stand-alone imaging systems are too insensitive or too cumbersome to maximally take advantage of new tumor-specific agents developed through the microdose pathway. To this end, a custom-built illumination and imaging module enabling picomolar-sensitive near-infrared fluorescence imaging on a commercial operating microscope is described. The limits of detection and system specifications are characterized, and in vivo efficacy of the system in detecting ABY-029 is evaluated in a rat orthotopic glioma model following microdose injections, showing the suitability of the device for microdose phase 0 clinical trials.

Microdose fluorescence imaging of ABY-029 on an operating microscope adapted by custom illumination and imaging modules

PubMed Central

Dsouza, Alisha V.; Marra, Kayla; Pogue, Brian W.; Roberts, David W.; Paulsen, Keith D.

2016-01-01

Fluorescence guided surgery has the potential to positively impact surgical oncology; current operating microscopes and stand-alone imaging systems are too insensitive or too cumbersome to maximally take advantage of new tumor-specific agents developed through the microdose pathway. To this end, a custom-built illumination and imaging module enabling picomolar-sensitive near-infrared fluorescence imaging on a commercial operating microscope is described. The limits of detection and system specifications are characterized, and in vivo efficacy of the system in detecting ABY-029 is evaluated in a rat orthotopic glioma model following microdose injections, showing the suitability of the device for microdose phase 0 clinical trials. PMID:27699098

Microscopy imaging device with advanced imaging properties

DOEpatents

Ghosh, Kunal; Burns, Laurie; El Gamal, Abbas; Schnitzer, Mark J.; Cocker, Eric; Ho, Tatt Wei

2016-10-25

Systems, methods and devices are implemented for microscope imaging solutions. One embodiment of the present disclosure is directed toward an epifluorescence microscope. The microscope includes an image capture circuit including an array of optical sensor. An optical arrangement is configured to direct excitation light of less than about 1 mW to a target object in a field of view of that is at least 0.5 mm.sup.2 and to direct epi-fluorescence emission caused by the excitation light to the array of optical sensors. The optical arrangement and array of optical sensors are each sufficiently close to the target object to provide at least 2.5 .mu.m resolution for an image of the field of view.

Microscopy imaging device with advanced imaging properties

DOEpatents

Ghosh, Kunal; Burns, Laurie; El Gamal, Abbas; Schnitzer, Mark J.; Cocker, Eric; Ho, Tatt Wei

2016-11-22

Systems, methods and devices are implemented for microscope imaging solutions. One embodiment of the present disclosure is directed toward an epifluorescence microscope. The microscope includes an image capture circuit including an array of optical sensor. An optical arrangement is configured to direct excitation light of less than about 1 mW to a target object in a field of view of that is at least 0.5 mm.sup.2 and to direct epi-fluorescence emission caused by the excitation light to the array of optical sensors. The optical arrangement and array of optical sensors are each sufficiently close to the target object to provide at least 2.5 .mu.m resolution for an image of the field of view.

Microscopy imaging device with advanced imaging properties

DOEpatents

Ghosh, Kunal; Burns, Laurie; El Gamal, Abbas; Schnitzer, Mark J.; Cocker, Eric; Ho, Tatt Wei

2017-04-25

Systems, methods and devices are implemented for microscope imaging solutions. One embodiment of the present disclosure is directed toward an epifluorescence microscope. The microscope includes an image capture circuit including an array of optical sensor. An optical arrangement is configured to direct excitation light of less than about 1 mW to a target object in a field of view of that is at least 0.5 mm.sup.2 and to direct epi-fluorescence emission caused by the excitation light to the array of optical sensors. The optical arrangement and array of optical sensors are each sufficiently close to the target object to provide at least 2.5 .mu.m resolution for an image of the field of view.

The different ways to obtain digital images of urine microscopy findings: Their advantages and limitations.

PubMed

Fogazzi, G B; Garigali, G

2017-03-01

We describe three ways to take digital images of urine sediment findings. Way 1 encompasses a digital camera permanently mounted on the microscope and connected with a computer equipped with a proprietary software to acquire, process and store the images. Way 2 is based on the use of inexpensive compact digital cameras, held by hands - or mounted on a tripod - close to one eyepiece of the microscope. Way 3 is based on the use of smartphones, held by hands close to one eyepiece of the microscope or connected to the microscope by an adapter. The procedures, advantages and limitations of each way are reported. Copyright © 2017. Published by Elsevier B.V.

Handy Microscopic Close-Range Videogrammetry

NASA Astrophysics Data System (ADS)

Esmaeili, F.; Ebadi, H.

2017-09-01

The modeling of small-scale objects is used in different applications such as medicine, industry, and cultural heritage. The capability of modeling small-scale objects using imaging with the help of hand USB digital microscopes and use of videogrammetry techniques has been implemented and evaluated in this paper. Use of this equipment and convergent imaging of the environment for modeling, provides an appropriate set of images for generation of three-dimensional models. The results of the measurements made with the help of a microscope micrometer calibration ruler have demonstrated that self-calibration of a hand camera-microscope set can help obtain a three-dimensional detail extraction precision of about 0.1 millimeters on small-scale environments.

Actinic inspection of EUV reticles with arbitrary pattern design

NASA Astrophysics Data System (ADS)

Mochi, Iacopo; Helfenstein, Patrick; Rajeev, Rajendran; Fernandez, Sara; Kazazis, Dimitrios; Yoshitake, Shusuke; Ekinci, Yasin

2017-10-01

The re ective-mode EUV mask scanning lensless imaging microscope (RESCAN) is being developed to provide actinic mask inspection capabilities for defects and patterns with high resolution and high throughput, for 7 nm node and beyond. Here we, will report on our progress and present the results on programmed defect detection on random, logic-like patterns. The defects we investigated range from 200 nm to 50 nm size on the mask. We demonstrated the ability of RESCAN to detect these defects in die-to-die and die-to-database mode with a high signal to noise ratio. We also describe future plans for the upgrades to increase the resolution, the sensitivity, and the inspection speed of the demo tool.

Moving Toward the Light: Using New Technology to Answer Old Questions

PubMed Central

LUCITTI, JENNIFER L.; DICKINSON, MARY E.

2006-01-01

Fluorescence microscopy has become a principle methodology in the field of developmental biology. Recent technological advances have led to the design of high-speed and high-resolution confocal and multiphoton microscopes that enable researchers to obtain three- and four-dimensional information in living cells and whole embryos. Paralleling this progress, the development of stable and bright vital fluorescent probes has revolutionized the ability to track individual cells in vitro and in vivo and to visualize intercellular and subcellular molecular interactions in real time. Combining imaging modalities and labeling techniques that are increasingly unobtrusive to cell and whole animal function, our understanding of how proteins interact, tissues take form, and organs synchronize to create a functioning animal is reaching a whole new level. PMID:16690954

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.

Image Analysis, Microscopic, and Spectrochemical Study of the PVC Dry Blending Process,

DTIC Science & Technology

The dry blending process used in the production of electrical grade pvc formulations has been studies using a combination of image analysis , microscopic...by image analysis techniques. Optical and scanning electron microscopy were used to assess morphological differences. Spectrochemical techniques were used to indicate chemical changes.

Compact Microscope Imaging System with Intelligent Controls

NASA Technical Reports Server (NTRS)

McDowell, Mark

2004-01-01

The figure presents selected views of a compact microscope imaging system (CMIS) that includes a miniature video microscope, a Cartesian robot (a computer- controlled three-dimensional translation stage), and machine-vision and control subsystems. The CMIS was built from commercial off-the-shelf instrumentation, computer hardware and software, and custom machine-vision software. The machine-vision and control subsystems include adaptive neural networks that afford a measure of artificial intelligence. The CMIS can perform several automated tasks with accuracy and repeatability . tasks that, heretofore, have required the full attention of human technicians using relatively bulky conventional microscopes. In addition, the automation and control capabilities of the system inherently include a capability for remote control. Unlike human technicians, the CMIS is not at risk of becoming fatigued or distracted: theoretically, it can perform continuously at the level of the best human technicians. In its capabilities for remote control and for relieving human technicians of tedious routine tasks, the CMIS is expected to be especially useful in biomedical research, materials science, inspection of parts on industrial production lines, and space science. The CMIS can automatically focus on and scan a microscope sample, find areas of interest, record the resulting images, and analyze images from multiple samples simultaneously. Automatic focusing is an iterative process: The translation stage is used to move the microscope along its optical axis in a succession of coarse, medium, and fine steps. A fast Fourier transform (FFT) of the image is computed at each step, and the FFT is analyzed for its spatial-frequency content. The microscope position that results in the greatest dispersal of FFT content toward high spatial frequencies (indicating that the image shows the greatest amount of detail) is deemed to be the focal position.

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.

Defocus and magnification dependent variation of TEM image astigmatism.

PubMed

Yan, Rui; Li, Kunpeng; Jiang, Wen

2018-01-10

Daily alignment of the microscope is a prerequisite to reaching optimal lens conditions for high resolution imaging in cryo-EM. In this study, we have investigated how image astigmatism varies with the imaging conditions (e.g. defocus, magnification). We have found that the large change of defocus/magnification between visual correction of astigmatism and subsequent data collection tasks, or during data collection, will inevitably result in undesirable astigmatism in the final images. The dependence of astigmatism on the imaging conditions varies significantly from time to time, so that it cannot be reliably compensated by pre-calibration of the microscope. Based on these findings, we recommend that the same magnification and the median defocus of the intended defocus range for final data collection are used in the objective lens astigmatism correction task during microscope alignment and in the focus mode of the iterative low-dose imaging. It is also desirable to develop a fast, accurate method that can perform dynamic correction of the astigmatism for different intended defocuses during automated imaging. Our findings also suggest that the slope of astigmatism changes caused by varying defocuses can be used as a convenient measurement of objective lens rotation symmetry and potentially an acceptance test of new electron microscopes.

Long-working-distance fluorescence microscope with high-numerical-aperture objectives for variable-magnification imaging in live mice from macro- to subcellular

NASA Astrophysics Data System (ADS)

Kimura, Hiroaki; Momiyama, Masashi; Tomita, Katsuro; Tsuchiya, Hiroyuki; Hoffman, Robert M.

2010-11-01

We demonstrate the development of a long-working-distance fluorescence microscope with high-numerical-aperture objectives for variable-magnification imaging in live mice from macro- to subcellular. To observe cytoplasmic and nuclear dynamics of cancer cells in the living mouse, 143B human osteosarcoma cells are labeled with green fluorescent protein in the nucleus and red fluorescent protein in the cytoplasm. These dual-color cells are injected by a vascular route in an abdominal skin flap in nude mice. The mice are then imaged with the Olympus MVX10 macroview fluorescence microscope. With the MVX10, the nuclear and cytoplasmic behavior of cancer cells trafficking in blood vessels of live mice is observed. We also image lung metastases in live mice from the macro- to the subcellular level by opening the chest wall and imaging the exposed lung in live mice. Injected splenocytes, expressing cyan fluorescent protein, could also be imaged on the lung of live mice. We demonstrate that the MVX10 microscope offers the possibility of full-range in vivo fluorescence imaging from macro- to subcellular and should enable widespread use of powerful imaging technologies enabled by genetic reporters and other fluorophores.