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.

Augmented microscopy: real-time overlay of bright-field and near-infrared fluorescence images.

PubMed

Watson, Jeffrey R; Gainer, Christian F; Martirosyan, Nikolay; Skoch, Jesse; Lemole, G Michael; Anton, Rein; Romanowski, Marek

2015-10-01

Intraoperative applications of near-infrared (NIR) fluorescent contrast agents can be aided by instrumentation capable of merging the view of surgical field with that of NIR fluorescence. We demonstrate augmented microscopy, an intraoperative imaging technique in which bright-field (real) and electronically processed NIR fluorescence (synthetic) images are merged within the optical path of a stereomicroscope. Under luminance of 100,000 lx, representing typical illumination of the surgical field, the augmented microscope detects 189 nM concentration of indocyanine green and produces a composite of the real and synthetic images within the eyepiece of the microscope at 20 fps. Augmentation described here can be implemented as an add-on module to visualize NIR contrast agents, laser beams, or various types of electronic data within the surgical microscopes commonly used in neurosurgical, cerebrovascular, otolaryngological, and ophthalmic procedures.

Augmented microscopy: real-time overlay of bright-field and near-infrared fluorescence images

NASA Astrophysics Data System (ADS)

Watson, Jeffrey R.; Gainer, Christian F.; Martirosyan, Nikolay; Skoch, Jesse; Lemole, G. Michael, Jr.; Anton, Rein; Romanowski, Marek

2015-10-01

Intraoperative applications of near-infrared (NIR) fluorescent contrast agents can be aided by instrumentation capable of merging the view of surgical field with that of NIR fluorescence. We demonstrate augmented microscopy, an intraoperative imaging technique in which bright-field (real) and electronically processed NIR fluorescence (synthetic) images are merged within the optical path of a stereomicroscope. Under luminance of 100,000 lx, representing typical illumination of the surgical field, the augmented microscope detects 189 nM concentration of indocyanine green and produces a composite of the real and synthetic images within the eyepiece of the microscope at 20 fps. Augmentation described here can be implemented as an add-on module to visualize NIR contrast agents, laser beams, or various types of electronic data within the surgical microscopes commonly used in neurosurgical, cerebrovascular, otolaryngological, and ophthalmic procedures.

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.

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.

Wide field video-rate two-photon imaging by using spinning disk beam scanner

NASA Astrophysics Data System (ADS)

Maeda, Yasuhiro; Kurokawa, Kazuo; Ito, Yoko; Wada, Satoshi; Nakano, Akihiko

2018-02-01

The microscope technology with wider view field, deeper penetration depth, higher spatial resolution and higher imaging speed are required to investigate the intercellular dynamics or interactions of molecules and organs in cells or a tissue in more detail. The two-photon microscope with a near infrared (NIR) femtosecond laser is one of the technique to improve the penetration depth and spatial resolution. However, the video-rate or high-speed imaging with wide view field is difficult to perform with the conventional two-photon microscope. Because point-to-point scanning method is used in conventional one, so it's difficult to achieve video-rate imaging. In this study, we developed a two-photon microscope with spinning disk beam scanner and femtosecond NIR fiber laser with around 10 W average power for the microscope system to achieve above requirements. The laser is consisted of an oscillator based on mode-locked Yb fiber laser, a two-stage pre-amplifier, a main amplifier based on a Yb-doped photonic crystal fiber (PCF), and a pulse compressor with a pair of gratings. The laser generates a beam with maximally 10 W average power, 300 fs pulse width and 72 MHz repetition rate. And the beam incident to a spinning beam scanner (Yokogawa Electric) optimized for two-photon imaging. By using this system, we achieved to obtain the 3D images with over 1mm-penetration depth and video-rate image with 350 x 350 um view field from the root of Arabidopsis thaliana.

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.

Magnified hard x-ray microtomography: toward tomography with submicron resolution

NASA Astrophysics Data System (ADS)

Schroer, Christian G.; Benner, Boris; Guenzler, Til F.; Kuhlmann, Marion; Lengeler, Bruno; Rau, Christoph; Weitkamp, Timm; Snigirev, Anatoly A.; Snigireva, Irina

2002-01-01

Parabolic compound refractive lenses (PCRLs) are high quality imaging optics for hard x-rays that can be used as an objective lens in a new type of hard x-ray full field microscope. Using an aluminium PCRL, this new type of microscope has been shown to have a resolution of 350 nm. Further improvement of the resolution down to 50 nm can be expected using beryllium as a lens material. The large depth of field (several mm) of the microscope results in sharp projection images for samples that fit into the field of view of about 300 micrometers. This allows to combine magnified imaging with tomographic techniques. First results of magnified microtomography are shown. Contrast formation in the microscope and the consequences for tomographic reconstruction are discussed. An outlook on further developments is given.

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

Augmented microscopy: real-time overlay of bright-field and near-infrared fluorescence images

PubMed Central

Watson, Jeffrey R.; Gainer, Christian F.; Martirosyan, Nikolay; Skoch, Jesse; Lemole, G. Michael; Anton, Rein; Romanowski, Marek

2015-01-01

Abstract. Intraoperative applications of near-infrared (NIR) fluorescent contrast agents can be aided by instrumentation capable of merging the view of surgical field with that of NIR fluorescence. We demonstrate augmented microscopy, an intraoperative imaging technique in which bright-field (real) and electronically processed NIR fluorescence (synthetic) images are merged within the optical path of a stereomicroscope. Under luminance of 100,000 lx, representing typical illumination of the surgical field, the augmented microscope detects 189 nM concentration of indocyanine green and produces a composite of the real and synthetic images within the eyepiece of the microscope at 20 fps. Augmentation described here can be implemented as an add-on module to visualize NIR contrast agents, laser beams, or various types of electronic data within the surgical microscopes commonly used in neurosurgical, cerebrovascular, otolaryngological, and ophthalmic procedures. PMID:26440760

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.

Technique to quantitatively measure magnetic properties of thin structures at <10 NM spatial resolution

DOEpatents

Bajt, Sasa

2003-07-08

A highly sensitive and high resolution magnetic microscope images magnetic properties quantitatively. Imaging is done with a modified transmission electron microscope that allows imaging of the sample in a zero magnetic field. Two images from closely spaced planes, one in focus and one slightly out of focus, are sufficient to calculate the absolute values of the phase change imparted to the electrons, and hence obtain the magnetization vector field distribution.

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.

Trace Element Mapping of a Biological Specimen by a Full-Field X-ray Fluorescence Imaging Microscope with a Wolter Mirror

NASA Astrophysics Data System (ADS)

Hoshino, Masato; Yamada, Norimitsu; Ishino, Toyoaki; Namiki, Takashi; Watanabe, Norio; Aoki, Sadao

2007-01-01

A full-field X-ray fluorescence imaging microscope with a Wolter mirror was applied to the element mapping of alfalfa seeds. The X-ray fluorescence microscope was built at the Photon Factory BL3C2 (KEK). X-ray fluorescence images of several growing stages of the alfalfa seeds were obtained. X-ray fluorescence energy spectra were measured with either a solid state detector or a CCD photon counting method. The element distributions of iron and zinc which were included in the seeds were obtained using a photon counting method.

High-resolution, high-throughput imaging with a multibeam scanning electron microscope

PubMed Central

EBERLE, AL; MIKULA, S; SCHALEK, R; LICHTMAN, J; TATE, ML KNOTHE; ZEIDLER, D

2015-01-01

Electron–electron interactions and detector bandwidth limit the maximal imaging speed of single-beam scanning electron microscopes. We use multiple electron beams in a single column and detect secondary electrons in parallel to increase the imaging speed by close to two orders of magnitude and demonstrate imaging for a variety of samples ranging from biological brain tissue to semiconductor wafers. Lay Description The composition of our world and our bodies on the very small scale has always fascinated people, making them search for ways to make this visible to the human eye. Where light microscopes reach their resolution limit at a certain magnification, electron microscopes can go beyond. But their capability of visualizing extremely small features comes at the cost of a very small field of view. Some of the questions researchers seek to answer today deal with the ultrafine structure of brains, bones or computer chips. Capturing these objects with electron microscopes takes a lot of time – maybe even exceeding the time span of a human being – or new tools that do the job much faster. A new type of scanning electron microscope scans with 61 electron beams in parallel, acquiring 61 adjacent images of the sample at the same time a conventional scanning electron microscope captures one of these images. In principle, the multibeam scanning electron microscope’s field of view is 61 times larger and therefore coverage of the sample surface can be accomplished in less time. This enables researchers to think about large-scale projects, for example in the rather new field of connectomics. A very good introduction to imaging a brain at nanometre resolution can be found within course material from Harvard University on http://www.mcb80x.org/# as featured media entitled ‘connectomics’. PMID:25627873

Real-time terahertz near-field microscope.

PubMed

Blanchard, F; Doi, A; Tanaka, T; Hirori, H; Tanaka, H; Kadoya, Y; Tanaka, K

2011-04-25

We report a terahertz near-field microscope with a high dynamic range that can capture images of a 370 x 740 μm2 area at 35 frames per second. We achieve high spatial resolution (14 μm corresponding to λ/30 for a center frequency at 0.7 THz) on a large area by combining two novel techniques: terahertz generation by tilted-pulse-front excitation and electro-optic balanced imaging detection using a thin crystal. To demonstrate the microscope capability, we reveal the field enhancement at the gap position of a dipole antenna after the irradiation of a terahertz pulse.

Dual-modal three-dimensional imaging of single cells with isometric high resolution using an optical projection tomography microscope

NASA Astrophysics Data System (ADS)

Miao, Qin; Rahn, J. Richard; Tourovskaia, Anna; Meyer, Michael G.; Neumann, Thomas; Nelson, Alan C.; Seibel, Eric J.

2009-11-01

The practice of clinical cytology relies on bright-field microscopy using absorption dyes like hematoxylin and eosin in the transmission mode, while the practice of research microscopy relies on fluorescence microscopy in the epi-illumination mode. The optical projection tomography microscope is an optical microscope that can generate 3-D images of single cells with isometric high resolution both in absorption and fluorescence mode. Although the depth of field of the microscope objective is in the submicron range, it can be extended by scanning the objective's focal plane. The extended depth of field image is similar to a projection in a conventional x-ray computed tomography. Cells suspended in optical gel flow through a custom-designed microcapillary. Multiple pseudoprojection images are taken by rotating the microcapillary. After these pseudoprojection images are further aligned, computed tomography methods are applied to create 3-D reconstruction. 3-D reconstructed images of single cells are shown in both absorption and fluorescence mode. Fluorescence spatial resolution is measured at 0.35 μm in both axial and lateral dimensions. Since fluorescence and absorption images are taken in two different rotations, mechanical error may cause misalignment of 3-D images. This mechanical error is estimated to be within the resolution of the system.

Noise removal in extended depth of field microscope images through nonlinear signal processing.

PubMed

Zahreddine, Ramzi N; Cormack, Robert H; Cogswell, Carol J

2013-04-01

Extended depth of field (EDF) microscopy, achieved through computational optics, allows for real-time 3D imaging of live cell dynamics. EDF is achieved through a combination of point spread function engineering and digital image processing. A linear Wiener filter has been conventionally used to deconvolve the image, but it suffers from high frequency noise amplification and processing artifacts. A nonlinear processing scheme is proposed which extends the depth of field while minimizing background noise. The nonlinear filter is generated via a training algorithm and an iterative optimizer. Biological microscope images processed with the nonlinear filter show a significant improvement in image quality and signal-to-noise ratio over the conventional linear filter.

Wide field of view common-path lateral-shearing digital holographic interference microscope

NASA Astrophysics Data System (ADS)

Vora, Priyanka; Trivedi, Vismay; Mahajan, Swapnil; Patel, Nimit; Joglekar, Mugdha; Chhaniwal, Vani; Moradi, Ali-Reza; Javidi, Bahram; Anand, Arun

2017-12-01

Quantitative three-dimensional (3-D) imaging of living cells provides important information about the cell morphology and its time variation. Off-axis, digital holographic interference microscopy is an ideal tool for 3-D imaging, parameter extraction, and classification of living cells. Two-beam digital holographic microscopes, which are usually employed, provide high-quality 3-D images of micro-objects, albeit with lower temporal stability. Common-path digital holographic geometries, in which the reference beam is derived from the object beam, provide higher temporal stability along with high-quality 3-D images. Self-referencing geometry is the simplest of the common-path techniques, in which a portion of the object beam itself acts as the reference, leading to compact setups using fewer optical elements. However, it has reduced field of view, and the reference may contain object information. Here, we describe the development of a common-path digital holographic microscope, employing a shearing plate and converting one of the beams into a separate reference by employing a pin-hole. The setup is as compact as self-referencing geometry, while providing field of view as wide as that of a two-beam microscope. The microscope is tested by imaging and quantifying the morphology and dynamics of human erythrocytes.

Wide field of view common-path lateral-shearing digital holographic interference microscope.

PubMed

Vora, Priyanka; Trivedi, Vismay; Mahajan, Swapnil; Patel, Nimit; Joglekar, Mugdha; Chhaniwal, Vani; Moradi, Ali-Reza; Javidi, Bahram; Anand, Arun

2017-12-01

Quantitative three-dimensional (3-D) imaging of living cells provides important information about the cell morphology and its time variation. Off-axis, digital holographic interference microscopy is an ideal tool for 3-D imaging, parameter extraction, and classification of living cells. Two-beam digital holographic microscopes, which are usually employed, provide high-quality 3-D images of micro-objects, albeit with lower temporal stability. Common-path digital holographic geometries, in which the reference beam is derived from the object beam, provide higher temporal stability along with high-quality 3-D images. Self-referencing geometry is the simplest of the common-path techniques, in which a portion of the object beam itself acts as the reference, leading to compact setups using fewer optical elements. However, it has reduced field of view, and the reference may contain object information. Here, we describe the development of a common-path digital holographic microscope, employing a shearing plate and converting one of the beams into a separate reference by employing a pin-hole. The setup is as compact as self-referencing geometry, while providing field of view as wide as that of a two-beam microscope. The microscope is tested by imaging and quantifying the morphology and dynamics of human erythrocytes. (2017) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE).

Dark-field imaging with the scanning transmission x-ray microscope

NASA Astrophysics Data System (ADS)

Morrison, G. R.; Browne, M. T.

1992-01-01

The King's College London scanning transmission x-ray microscope in use on beam line 5U2 at the SRS, SERC Daresbury Laboratory, has been modified to allow dark-field images to be formed using only the x rays scattered by the specimen. Experiments have been performed with a number of different detector geometries, and this has confirmed that the strongest scattering arises from edges or thickness gradients in the specimen. Although the dark-field signal is only a small fraction of the normal transmitted bright-field signal, features can be revealed with high contrast, and it has proved possible to detect the presence of features that are below the resolution limit of the microscope.

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

NASA Astrophysics Data System (ADS)

Darafsheh, Arash

2018-02-01

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

Telecentric 3D profilometry based on phase-shifting fringe projection.

PubMed

Li, Dong; Liu, Chunyang; Tian, Jindong

2014-12-29

Three dimensional shape measurement in the microscopic range becomes increasingly important with the development of micro manufacturing technology. Microscopic fringe projection techniques offer a fast, robust, and full-field measurement for field sizes from approximately 1 mm² to several cm². However, the depth of field is very small due to the imaging of non-telecentric microscope, which is often not sufficient to measure the complete depth of a 3D-object. And the calibration of phase-to-depth conversion is complicated which need a precision translation stage and a reference plane. In this paper, we propose a novel telecentric phase-shifting projected fringe profilometry for small and thick objects. Telecentric imaging extends the depth of field approximately to millimeter order, which is much larger than that of microscopy. To avoid the complicated phase-to-depth conversion in microscopic fringe projection, we develop a new system calibration method of camera and projector based on telecentric imaging model. Based on these, a 3D reconstruction of telecentric imaging is presented with stereovision aided by fringe phase maps. Experiments demonstrated the feasibility and high measurement accuracy of the proposed system for thick object.

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

NASA Astrophysics Data System (ADS)

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

2016-06-01

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

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

PubMed Central

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

2016-01-01

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

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

PubMed

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

2016-06-10

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

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.

The plant virus microscope image registration method based on mismatches removing.

PubMed

Wei, Lifang; Zhou, Shucheng; Dong, Heng; Mao, Qianzhuo; Lin, Jiaxiang; Chen, Riqing

2016-01-01

The electron microscopy is one of the major means to observe the virus. The view of virus microscope images is limited by making specimen and the size of the camera's view field. To solve this problem, the virus sample is produced into multi-slice for information fusion and image registration techniques are applied to obtain large field and whole sections. Image registration techniques have been developed in the past decades for increasing the camera's field of view. Nevertheless, these approaches typically work in batch mode and rely on motorized microscopes. Alternatively, the methods are conceived just to provide visually pleasant registration for high overlap ratio image sequence. This work presents a method for virus microscope image registration acquired with detailed visual information and subpixel accuracy, even when overlap ratio of image sequence is 10% or less. The method proposed focus on the correspondence set and interimage transformation. A mismatch removal strategy is proposed by the spatial consistency and the components of keypoint to enrich the correspondence set. And the translation model parameter as well as tonal inhomogeneities is corrected by the hierarchical estimation and model select. In the experiments performed, we tested different registration approaches and virus images, confirming that the translation model is not always stationary, despite the fact that the images of the sample come from the same sequence. The mismatch removal strategy makes building registration of virus microscope images at subpixel accuracy easier and optional parameters for building registration according to the hierarchical estimation and model select strategies make the proposed method high precision and reliable for low overlap ratio image sequence. Copyright © 2015 Elsevier Ltd. All rights reserved.

Holographic pixel super-resolution in portable lensless on-chip microscopy using a fiber-optic array.

PubMed

Bishara, Waheb; Sikora, Uzair; Mudanyali, Onur; Su, Ting-Wei; Yaglidere, Oguzhan; Luckhart, Shirley; Ozcan, Aydogan

2011-04-07

We report a portable lensless on-chip microscope that can achieve <1 µm resolution over a wide field-of-view of ∼ 24 mm(2) without the use of any mechanical scanning. This compact on-chip microscope weighs ∼ 95 g and is based on partially coherent digital in-line holography. Multiple fiber-optic waveguides are butt-coupled to light emitting diodes, which are controlled by a low-cost micro-controller to sequentially illuminate the sample. The resulting lensfree holograms are then captured by a digital sensor-array and are rapidly processed using a pixel super-resolution algorithm to generate much higher resolution holographic images (both phase and amplitude) of the objects. This wide-field and high-resolution on-chip microscope, being compact and light-weight, would be important for global health problems such as diagnosis of infectious diseases in remote locations. Toward this end, we validate the performance of this field-portable microscope by imaging human malaria parasites (Plasmodium falciparum) in thin blood smears. Our results constitute the first-time that a lensfree on-chip microscope has successfully imaged malaria parasites.

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.

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.

High-resolution electron microscope

NASA Technical Reports Server (NTRS)

Nathan, R.

1977-01-01

Employing scanning transmission electron microscope as interferometer, relative phases of diffraction maximums can be determined by analysis of dark field images. Synthetic aperture technique and Fourier-transform computer processing of amplitude and phase information provide high resolution images at approximately one angstrom.

Lensless on-chip imaging of cells provides a new tool for high-throughput cell-biology and medical diagnostics.

PubMed

Mudanyali, Onur; Erlinger, Anthony; Seo, Sungkyu; Su, Ting-Wei; Tseng, Derek; Ozcan, Aydogan

2009-12-14

Conventional optical microscopes image cells by use of objective lenses that work together with other lenses and optical components. While quite effective, this classical approach has certain limitations for miniaturization of the imaging platform to make it compatible with the advanced state of the art in microfluidics. In this report, we introduce experimental details of a lensless on-chip imaging concept termed LUCAS (Lensless Ultra-wide field-of-view Cell monitoring Array platform based on Shadow imaging) that does not require any microscope objectives or other bulky optical components to image a heterogeneous cell solution over an ultra-wide field of view that can span as large as approximately 18 cm(2). Moreover, unlike conventional microscopes, LUCAS can image a heterogeneous cell solution of interest over a depth-of-field of approximately 5 mm without the need for refocusing which corresponds to up to approximately 9 mL sample volume. This imaging platform records the shadows (i.e., lensless digital holograms) of each cell of interest within its field of view, and automated digital processing of these cell shadows can determine the type, the count and the relative positions of cells within the solution. Because it does not require any bulky optical components or mechanical scanning stages it offers a significantly miniaturized platform that at the same time reduces the cost, which is quite important for especially point of care diagnostic tools. Furthermore, the imaging throughput of this platform is orders of magnitude better than conventional optical microscopes, which could be exceedingly valuable for high-throughput cell-biology experiments.

Wide-field computational imaging of pathology slides using lens-free on-chip microscopy.

PubMed

Greenbaum, Alon; Zhang, Yibo; Feizi, Alborz; Chung, Ping-Luen; Luo, Wei; Kandukuri, Shivani R; Ozcan, Aydogan

2014-12-17

Optical examination of microscale features in pathology slides is one of the gold standards to diagnose disease. However, the use of conventional light microscopes is partially limited owing to their relatively high cost, bulkiness of lens-based optics, small field of view (FOV), and requirements for lateral scanning and three-dimensional (3D) focus adjustment. We illustrate the performance of a computational lens-free, holographic on-chip microscope that uses the transport-of-intensity equation, multi-height iterative phase retrieval, and rotational field transformations to perform wide-FOV imaging of pathology samples with comparable image quality to a traditional transmission lens-based microscope. The holographically reconstructed image can be digitally focused at any depth within the object FOV (after image capture) without the need for mechanical focus adjustment and is also digitally corrected for artifacts arising from uncontrolled tilting and height variations between the sample and sensor planes. Using this lens-free on-chip microscope, we successfully imaged invasive carcinoma cells within human breast sections, Papanicolaou smears revealing a high-grade squamous intraepithelial lesion, and sickle cell anemia blood smears over a FOV of 20.5 mm(2). The resulting wide-field lens-free images had sufficient image resolution and contrast for clinical evaluation, as demonstrated by a pathologist's blinded diagnosis of breast cancer tissue samples, achieving an overall accuracy of ~99%. By providing high-resolution images of large-area pathology samples with 3D digital focus adjustment, lens-free on-chip microscopy can be useful in resource-limited and point-of-care settings. Copyright © 2014, American Association for the Advancement of Science.

Lensless On-chip Imaging of Cells Provides a New Tool for High-throughput Cell-Biology and Medical Diagnostics

PubMed Central

Mudanyali, Onur; Erlinger, Anthony; Seo, Sungkyu; Su, Ting-Wei; Tseng, Derek; Ozcan, Aydogan

2009-01-01

Conventional optical microscopes image cells by use of objective lenses that work together with other lenses and optical components. While quite effective, this classical approach has certain limitations for miniaturization of the imaging platform to make it compatible with the advanced state of the art in microfluidics. In this report, we introduce experimental details of a lensless on-chip imaging concept termed LUCAS (Lensless Ultra-wide field-of-view Cell monitoring Array platform based on Shadow imaging) that does not require any microscope objectives or other bulky optical components to image a heterogeneous cell solution over an ultra-wide field of view that can span as large as ~18 cm2. Moreover, unlike conventional microscopes, LUCAS can image a heterogeneous cell solution of interest over a depth-of-field of ~5 mm without the need for refocusing which corresponds to up to ~9 mL sample volume. This imaging platform records the shadows (i.e., lensless digital holograms) of each cell of interest within its field of view, and automated digital processing of these cell shadows can determine the type, the count and the relative positions of cells within the solution. Because it does not require any bulky optical components or mechanical scanning stages it offers a significantly miniaturized platform that at the same time reduces the cost, which is quite important for especially point of care diagnostic tools. Furthermore, the imaging throughput of this platform is orders of magnitude better than conventional optical microscopes, which could be exceedingly valuable for high-throughput cell-biology experiments. PMID:20010542

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.

Camera array based light field microscopy

PubMed Central

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

2015-01-01

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

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.

Advanced MOKE magnetometry in wide-field Kerr-microscopy

NASA Astrophysics Data System (ADS)

Soldatov, I. V.; Schäfer, R.

2017-10-01

The measurement of MOKE (Magneto-Optical Kerr Effect) magnetization loops in a wide-field Kerr microscope offers the advantage that the relevant domain images along the loop can be readily recorded. As the microscope's objective lens is exposed to the magnetic field, the loops are usually strongly distorted by non-linear Faraday rotations of the polarized light that occur in the objective lens and that are superimposed to the MOKE signal. In this paper, an experimental method, based on a motorized analyzer, is introduced which allows to compensate the Faraday contributions, thus leading to pure MOKE loops. A wide field Kerr microscope, equipped with this technology, works well as a laser-based MOKE magnetometer, additionally offering domain images and thus providing the basis for loop interpretation.

Development of a near-field/confocal polarization microscope for local measurements of anisotropy in organic films

NASA Astrophysics Data System (ADS)

Kosterin, Andrey Valentinovich

2000-10-01

Polarization microscopy is a powerful technique for imaging structure and stress distributions in many transparent materials, and has been particularly useful in morphology studies of polymer films. Recently the possibility of combining polarization imaging with near-field scanning optical microscopy (NSOM) has been demonstrated, offering new opportunities for studying molecular organization with better than 50 nm resolution. However, there are challenges associated with near-field polarization experiments on organic films: (1) the films are susceptible to damage by the near-field probe; (2) the phase shift or retardation (80) is small, often <0.1 rad; (3) interpretation of near-field images is complicated by topography and probe-sample coupling. To address these challenges, we have developed a new combined near-field/confocal polarization microscope and tested its sensitivity to linear birefringence in thin polymer films. For near-field imaging, the microscope employs a commercially available scanhead with cantilevered (bent) optical fiber probes. To study soft samples (point 1), we have modified the scanhead for tapping mode feedback, which eliminates probe-sample shear forces and prolongs the lifetime of the probe, while minimizing damage to the sample. To achieve sensitivity to small phase shifts (point 2), we have implemented the phase modulation (PM) technique in the optical path. Enhanced sensitivity relative to the standard crossed polarizers scheme is achieved because of the better signal-to-noise discrimination common to lock-in detection and because the detected first harmonic intensity, Io , is linearly proportional to deltatheta instead of (deltatheta) 2. To facilitate interpretation of near-field contrast (point 3), we have incorporated near-field and confocal channels in one instrument. This allows consecutive acquisition of both near-field and far-field images on the same sample area. Since the far-field images do not suffer from the same artifacts, they can be used as a source of independent information on sample optical properties. The combined near-field/confocal polarization microscope is discussed in this thesis as well as some of its applications. Specifically we consider the results of polyethylene oxide (PEO) and crosslinked polybutadiene (PB) thin film imaging.

New innovations for contrast enhancement in electron microscopy

NASA Astrophysics Data System (ADS)

Mohan, A.

In this study two techniques for producing and improving contrast in Electron Microscopy are discussed. The first technique deals with the production of secondary contrast in a Variable Pressure SEM under poor vacuum conditions using the specimen current signal. A review of the prior work in this field shows that the presence of the gas ions in the microscope column results in the amplification of the specimen current signal which is enriched in secondary content. The focus of this study is to establish practical conditions for imaging samples in the microscope using specimen current with gas amplification. This is done by understanding the different variables in the microscope which affect the image formation process and then finding out optimum conditions for obtaining the best possible image, i.e., the image most enhanced in secondary contrast. A few 'real life' samples analyzed using this technique show that the gas amplified specimen current images contain secondary information and, in some cases, provide clear advantages to imaging with conventional secondary and backscattered detectors. The second technique dealing with the production of phase contrast in the TEM for extremely thin, electron transparent samples, is analyzed. A review of the literature regarding prior work in the field shows that, while the theoretical aspects of production of phase contrast in the TEM using a phase plate are well understood, there have been problems in practically implementing this in the microscope. One major assumption with most of the studies is that a fiber, partially coated with gold, results in the formation of point charges which is an essential requirement for symmetrically shifting the phase of the electron beam. The focus of this portion of the dissertation is to image the type of fields associated with such a phase plate using the technique of electron holography. It is found that there are two types of fields associated with a phase plate of this sort. One is a cylindrical field which extends along the length of the fiber while the other is a localized spherically symmetric field. A series of simulations show that the spherical field can produce phase contrast in the TEM and also improve the contrast transfer properties of the microscope.

Volumetric Light-field Encryption at the Microscopic Scale

PubMed Central

Li, Haoyu; Guo, Changliang; Muniraj, Inbarasan; Schroeder, Bryce C.; Sheridan, John T.; Jia, Shu

2017-01-01

We report a light-field based method that allows the optical encryption of three-dimensional (3D) volumetric information at the microscopic scale in a single 2D light-field image. The system consists of a microlens array and an array of random phase/amplitude masks. The method utilizes a wave optics model to account for the dominant diffraction effect at this new scale, and the system point-spread function (PSF) serves as the key for encryption and decryption. We successfully developed and demonstrated a deconvolution algorithm to retrieve both spatially multiplexed discrete data and continuous volumetric data from 2D light-field images. Showing that the method is practical for data transmission and storage, we obtained a faithful reconstruction of the 3D volumetric information from a digital copy of the encrypted light-field image. The method represents a new level of optical encryption, paving the way for broad industrial and biomedical applications in processing and securing 3D data at the microscopic scale. PMID:28059149

Volumetric Light-field Encryption at the Microscopic Scale

NASA Astrophysics Data System (ADS)

Li, Haoyu; Guo, Changliang; Muniraj, Inbarasan; Schroeder, Bryce C.; Sheridan, John T.; Jia, Shu

2017-01-01

We report a light-field based method that allows the optical encryption of three-dimensional (3D) volumetric information at the microscopic scale in a single 2D light-field image. The system consists of a microlens array and an array of random phase/amplitude masks. The method utilizes a wave optics model to account for the dominant diffraction effect at this new scale, and the system point-spread function (PSF) serves as the key for encryption and decryption. We successfully developed and demonstrated a deconvolution algorithm to retrieve both spatially multiplexed discrete data and continuous volumetric data from 2D light-field images. Showing that the method is practical for data transmission and storage, we obtained a faithful reconstruction of the 3D volumetric information from a digital copy of the encrypted light-field image. The method represents a new level of optical encryption, paving the way for broad industrial and biomedical applications in processing and securing 3D data at the microscopic scale.

FIMic: design for ultimate 3D-integral microscopy of in-vivo biological samples

PubMed Central

Scrofani, G.; Sola-Pikabea, J.; Llavador, A.; Sanchez-Ortiga, E.; Barreiro, J. C.; Saavedra, G.; Garcia-Sucerquia, J.; Martínez-Corral, M.

2017-01-01

In this work, Fourier integral microscope (FIMic), an ultimate design of 3D-integral microscopy, is presented. By placing a multiplexing microlens array at the aperture stop of the microscope objective of the host microscope, FIMic shows extended depth of field and enhanced lateral resolution in comparison with regular integral microscopy. As FIMic directly produces a set of orthographic views of the 3D-micrometer-sized sample, it is suitable for real-time imaging. Following regular integral-imaging reconstruction algorithms, a 2.75-fold enhanced depth of field and 2-time better spatial resolution in comparison with conventional integral microscopy is reported. Our claims are supported by theoretical analysis and experimental images of a resolution test target, cotton fibers, and in-vivo 3D-imaging of biological specimens. PMID:29359107

Sub-micron materials characterization using near-field optics

NASA Astrophysics Data System (ADS)

Blodgett, David Wesley

1998-12-01

High-resolution sub-surface materials characterization and inspection are critical in the microelectronics and thin films industries. To this end, a technique is described that couples the bulk property measurement capabilities of high-frequency ultrasound with the high-resolution surface imaging capabilities of the near-field optical microscope. Sensing bulk microstructure variations in the material, such as grain boundaries, requires a detection footprint smaller than the variation itself. The near-field optical microscope, with the ability to exceed the diffraction limit in optical resolution, meets this requirement. Two apertureless near-field optical microscopes, on-axis and off-axis illumination, have been designed and built. Near-field and far-field approach curves for both microscopes are presented. The sensitivity of the near-field approach curve was 8.3 muV/nm. Resolution studies for the near-field microscope indicate optical resolutions on the order of 50 nm, which exceeds the diffraction limit. The near-field microscope has been adapted to detect both contact-transducer-generated and laser-generated ultrasound. The successful detection of high-frequency ultrasound with the near-field optical microscope demonstrates the potential of this technique.

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.

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.

Bright-field scanning confocal electron microscopy using a double aberration-corrected transmission electron microscope.

PubMed

Wang, Peng; Behan, Gavin; Kirkland, Angus I; Nellist, Peter D; Cosgriff, Eireann C; D'Alfonso, Adrian J; Morgan, Andrew J; Allen, Leslie J; Hashimoto, Ayako; Takeguchi, Masaki; Mitsuishi, Kazutaka; Shimojo, Masayuki

2011-06-01

Scanning confocal electron microscopy (SCEM) offers a mechanism for three-dimensional imaging of materials, which makes use of the reduced depth of field in an aberration-corrected transmission electron microscope. The simplest configuration of SCEM is the bright-field mode. In this paper we present experimental data and simulations showing the form of bright-field SCEM images. We show that the depth dependence of the three-dimensional image can be explained in terms of two-dimensional images formed in the detector plane. For a crystalline sample, this so-called probe image is shown to be similar to a conventional diffraction pattern. Experimental results and simulations show how the diffracted probes in this image are elongated in thicker crystals and the use of this elongation to estimate sample thickness is explored. Copyright © 2010 Elsevier B.V. All rights reserved.

Cellscope Aquatic: a Lab Quality, Portable Cellphone-Based Microscope for On-Site Collection of Algae Images

NASA Astrophysics Data System (ADS)

Steinberg, S. J.; Howard, M. D.

2016-02-01

Collecting algae samples from the field presents issues of specimen damage or degradation caused by preservation methods, handling and transport to laboratory facilities for identification. Traditionally, in-field collection of high quality microscopic images has not been possible due to the size, weight and fragility of high quality instruments and training of field staff in species identification. Scientists at the Southern California Coastal Water Research Project (SCCWRP) in collaboration with the Fletcher Lab, University of California Berkeley, Department of Bioengineering, tested and translated Fletcher's original medical CellScope for use in environmental monitoring applications. Field tests conducted by SCCWRP in 2014 led to modifications of the clinical CellScope to one better suited to in-field microscopic imaging for aquatic organisms. SCCWRP subsequently developed a custom cell-phone application to acquire microscopic imagery using the "CellScope Aquatic "in combination with other cell-phone derived field data (e.g. GPS location, date, time and other field observations). Data and imagery collected in-field may be transmitted in real-time to a web-based data system for tele-taxonomy evaluation and assessment by experts in the office. These hardware and software tools was tested in field in a variety of conditions and settings by multiple algae experts during the spring and summer of 2015 to further test and refine the CellScope Aquatic platform. The CellScope Aquatic provides an easy-to-use, affordable, lightweight, professional quality, data collection platform for environmental monitoring. Our ongoing efforts will focus on development of real-time expert systems for data analysis and image processing, to provide onsite feedback to field scientists.

A method for fast automated microscope image stitching.

PubMed

Yang, Fan; Deng, Zhen-Sheng; Fan, Qiu-Hong

2013-05-01

Image stitching is an important technology to produce a panorama or larger image by combining several images with overlapped areas. In many biomedical researches, image stitching is highly desirable to acquire a panoramic image which represents large areas of certain structures or whole sections, while retaining microscopic resolution. In this study, we develop a fast normal light microscope image stitching algorithm based on feature extraction. At first, an algorithm of scale-space reconstruction of speeded-up robust features (SURF) was proposed to extract features from the images to be stitched with a short time and higher repeatability. Then, the histogram equalization (HE) method was employed to preprocess the images to enhance their contrast for extracting more features. Thirdly, the rough overlapping zones of the images preprocessed were calculated by phase correlation, and the improved SURF was used to extract the image features in the rough overlapping areas. Fourthly, the features were corresponded by matching algorithm and the transformation parameters were estimated, then the images were blended seamlessly. Finally, this procedure was applied to stitch normal light microscope images to verify its validity. Our experimental results demonstrate that the improved SURF algorithm is very robust to viewpoint, illumination, blur, rotation and zoom of the images and our method is able to stitch microscope images automatically with high precision and high speed. Also, the method proposed in this paper is applicable to registration and stitching of common images as well as stitching the microscope images in the field of virtual microscope for the purpose of observing, exchanging, saving, and establishing a database of microscope images. Copyright © 2013 Elsevier Ltd. All rights reserved.

Digital photography for the light microscope: results with a gated, video-rate CCD camera and NIH-image software.

PubMed

Shaw, S L; Salmon, E D; Quatrano, R S

1995-12-01

In this report, we describe a relatively inexpensive method for acquiring, storing and processing light microscope images that combines the advantages of video technology with the powerful medium now termed digital photography. Digital photography refers to the recording of images as digital files that are stored, manipulated and displayed using a computer. This report details the use of a gated video-rate charge-coupled device (CCD) camera and a frame grabber board for capturing 256 gray-level digital images from the light microscope. This camera gives high-resolution bright-field, phase contrast and differential interference contrast (DIC) images but, also, with gated on-chip integration, has the capability to record low-light level fluorescent images. The basic components of the digital photography system are described, and examples are presented of fluorescence and bright-field micrographs. Digital processing of images to remove noise, to enhance contrast and to prepare figures for printing is discussed.

A compact light-sheet microscope for the study of the mammalian central nervous system

PubMed Central

Yang, Zhengyi; Haslehurst, Peter; Scott, Suzanne; Emptage, Nigel; Dholakia, Kishan

2016-01-01

Investigation of the transient processes integral to neuronal function demands rapid and high-resolution imaging techniques over a large field of view, which cannot be achieved with conventional scanning microscopes. Here we describe a compact light sheet fluorescence microscope, featuring a 45° inverted geometry and an integrated photolysis laser, that is optimized for applications in neuroscience, in particular fast imaging of sub-neuronal structures in mammalian brain slices. We demonstrate the utility of this design for three-dimensional morphological reconstruction, activation of a single synapse with localized photolysis, and fast imaging of neuronal Ca2+ signalling across a large field of view. The developed system opens up a host of novel applications for the neuroscience community. PMID:27215692

Apertureless near-field scanning optical microscope working with or without laser source.

PubMed

Formanek, F; De Wilde, Y; Aigouy, L; Chen, Y

2004-01-01

An apertureless near-field scanning optical microscope (ANSOM), used indifferent configurations, is presented. Our versatile home-made setup, based on a sharp tungsten tip glued onto a quartz tuning fork and working in tapping mode, allows to perform imaging over a broad spectral range. We have recorded optical images in the visible (wavelength, lambda = 655 nm) and in the infrared (lambda = 10.6 microm), proving that the setup routinely achieves an optical resolution of <50 nm regardless of the illumination wavelength. We have also shown optical images recorded in the visible (lambda = 655 nm) in an inverted configuration where the tip does not perturb the focused spot of the illumination laser. Approach curves as well as image profiles have revealed that on demodulating the optical signal at higher harmonics, we can obtain an effective probe sharpening which results in an improvement of the resolution. Finally, we have presented optical images recorded in the infrared without any illumination, that is, the usual laser source is replaced by a simple heating of the sample. This has shown that the ANSOM can be used as a near-field thermal optical microscope (NTOM) to probe the near field generated by the thermal emission of the sample.

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.

Chip-scale fluorescence microscope based on a silo-filter complementary metal-oxide semiconductor image sensor.

PubMed

Ah Lee, Seung; Ou, Xiaoze; Lee, J Eugene; Yang, Changhuei

2013-06-01

We demonstrate a silo-filter (SF) complementary metal-oxide semiconductor (CMOS) image sensor for a chip-scale fluorescence microscope. The extruded pixel design with metal walls between neighboring pixels guides fluorescence emission through the thick absorptive filter to the photodiode of a pixel. Our prototype device achieves 13 μm resolution over a wide field of view (4.8 mm × 4.4 mm). We demonstrate bright-field and fluorescence longitudinal imaging of living cells in a compact, low-cost configuration.

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.

Combined reflection and transmission microscope for telemedicine applications in field settings.

PubMed

Biener, Gabriel; Greenbaum, Alon; Isikman, Serhan O; Lee, Kelvin; Tseng, Derek; Ozcan, Aydogan

2011-08-21

We demonstrate a field-portable upright and inverted microscope that can image specimens in both reflection and transmission modes. This compact and cost-effective dual-mode microscope weighs only ∼135 grams (<4.8 ounces) and utilizes a simple light emitting diode (LED) to illuminate the sample of interest using a beam-splitter cube that is positioned above the object plane. This LED illumination is then partially reflected from the sample to be collected by two lenses, creating a reflection image of the specimen onto an opto-electronic sensor-array that is positioned above the beam-splitter cube. In addition to this, the illumination beam is also partially transmitted through the same specimen, which then casts lensfree in-line holograms of the same objects onto a second opto-electronic sensor-array that is positioned underneath the beam-splitter cube. By rapid digital reconstruction of the acquired lensfree holograms, transmission images (both phase and amplitude) of the same specimen are also created. We tested the performance of this field-portable microscope by imaging various micro-particles, blood smears as well as a histopathology slide corresponding to skin tissue. Being compact, light-weight and cost-effective, this combined reflection and transmission microscope might especially be useful for telemedicine applications in resource limited settings. This journal is © The Royal Society of Chemistry 2011

A modular, open-source, slide-scanning microscope for diagnostic applications in resource-constrained settings

PubMed Central

Lu, Qiang; Liu, Guanghui; Xiao, Chuanli; Hu, Chuanzhen; Zhang, Shiwu; Xu, Ronald X.; Chu, Kaiqin; Xu, Qianming

2018-01-01

In this paper we report the development of a cost-effective, modular, open source, and fully automated slide-scanning microscope, composed entirely of easily available off-the-shelf parts, and capable of bright field and fluorescence modes. The automated X-Y stage is composed of two low-cost micrometer stages coupled to stepper motors operated in open-loop mode. The microscope is composed of a low-cost CMOS sensor and low-cost board lenses placed in a 4f configuration. The system has approximately 1 micron resolution, limited by the f/# of available board lenses. The microscope is compact, measuring just 25×25×30 cm, and has an absolute positioning accuracy of ±1 μm in the X and Y directions. A Z-stage enables autofocusing and imaging over large fields of view even on non-planar samples, and custom software enables automatic determination of sample boundaries and image mosaicking. We demonstrate the utility of our device through imaging of fluorescent- and transmission-dye stained blood and fecal smears containing human and animal parasites, as well as several prepared tissue samples. These results demonstrate image quality comparable to high-end commercial microscopes at a cost of less than US$400 for a bright-field system, with an extra US$100 needed for the fluorescence module. PMID:29543835

Near-field microscopy and fluorescence spectroscopy: application to chromosomes labelled with different fluorophores.

PubMed

Mahieu-Williame, L; Falgayrettes, P; Nativel, L; Gall-Borrut, P; Costa, L; Salehzada, T; Bisbal, C

2010-04-01

We have coupled a spectrophotometer with a scanning near-field optical microscope to obtain, with a single scan, simultaneously scanning near-field optical microscope fluorescence images at different wavelengths as well as topography and transmission images. Extraction of the fluorescence spectra enabled us to decompose the different wavelengths of the fluorescence signals which normally overlap. We thus obtained images of the different fluorescence emissions of acridine orange bound to single or double stranded nucleic acids in human metaphase chromosomes before and after DNAse I or RNAse A treatment. The analysis of these images allowed us to visualize some specific chromatin areas where RNA is associated with DNA showing that such a technique could be used to identify multiple components within a cell.

Optically sectioned in vivo imaging with speckle illumination HiLo microscopy

PubMed Central

Lim, Daryl; Ford, Tim N.; Chu, Kengyeh K.; Mertz, Jerome

2011-01-01

We present a simple wide-field imaging technique, called HiLo microscopy, that is capable of producing optically sectioned images in real time, comparable in quality to confocal laser scanning microscopy. The technique is based on the fusion of two raw images, one acquired with speckle illumination and another with standard uniform illumination. The fusion can be numerically adjusted, using a single parameter, to produce optically sectioned images of varying thicknesses with the same raw data. Direct comparison between our HiLo microscope and a commercial confocal laser scanning microscope is made on the basis of sectioning strength and imaging performance. Specifically, we show that HiLo and confocal 3-D imaging of a GFP-labeled mouse brain hippocampus are comparable in quality. Moreover, HiLo microscopy is capable of faster, near video rate imaging over larger fields of view than attainable with standard confocal microscopes. The goal of this paper is to advertise the simplicity, robustness, and versatility of HiLo microscopy, which we highlight with in vivo imaging of common model organisms including planaria, C. elegans, and zebrafish. PMID:21280920

Optically sectioned in vivo imaging with speckle illumination HiLo microscopy.

PubMed

Lim, Daryl; Ford, Tim N; Chu, Kengyeh K; Mertz, Jerome

2011-01-01

We present a simple wide-field imaging technique, called HiLo microscopy, that is capable of producing optically sectioned images in real time, comparable in quality to confocal laser scanning microscopy. The technique is based on the fusion of two raw images, one acquired with speckle illumination and another with standard uniform illumination. The fusion can be numerically adjusted, using a single parameter, to produce optically sectioned images of varying thicknesses with the same raw data. Direct comparison between our HiLo microscope and a commercial confocal laser scanning microscope is made on the basis of sectioning strength and imaging performance. Specifically, we show that HiLo and confocal 3-D imaging of a GFP-labeled mouse brain hippocampus are comparable in quality. Moreover, HiLo microscopy is capable of faster, near video rate imaging over larger fields of view than attainable with standard confocal microscopes. The goal of this paper is to advertise the simplicity, robustness, and versatility of HiLo microscopy, which we highlight with in vivo imaging of common model organisms including planaria, C. elegans, and zebrafish.

Optically sectioned in vivo imaging with speckle illumination HiLo microscopy

NASA Astrophysics Data System (ADS)

Lim, Daryl; Ford, Tim N.; Chu, Kengyeh K.; Mertz, Jerome

2011-01-01

We present a simple wide-field imaging technique, called HiLo microscopy, that is capable of producing optically sectioned images in real time, comparable in quality to confocal laser scanning microscopy. The technique is based on the fusion of two raw images, one acquired with speckle illumination and another with standard uniform illumination. The fusion can be numerically adjusted, using a single parameter, to produce optically sectioned images of varying thicknesses with the same raw data. Direct comparison between our HiLo microscope and a commercial confocal laser scanning microscope is made on the basis of sectioning strength and imaging performance. Specifically, we show that HiLo and confocal 3-D imaging of a GFP-labeled mouse brain hippocampus are comparable in quality. Moreover, HiLo microscopy is capable of faster, near video rate imaging over larger fields of view than attainable with standard confocal microscopes. The goal of this paper is to advertise the simplicity, robustness, and versatility of HiLo microscopy, which we highlight with in vivo imaging of common model organisms including planaria, C. elegans, and zebrafish.

Multi-Contrast Imaging and Digital Refocusing on a Mobile Microscope with a Domed LED Array.

PubMed

Phillips, Zachary F; D'Ambrosio, Michael V; Tian, Lei; Rulison, Jared J; Patel, Hurshal S; Sadras, Nitin; Gande, Aditya V; Switz, Neil A; Fletcher, Daniel A; Waller, Laura

2015-01-01

We demonstrate the design and application of an add-on device for improving the diagnostic and research capabilities of CellScope--a low-cost, smartphone-based point-of-care microscope. We replace the single LED illumination of the original CellScope with a programmable domed LED array. By leveraging recent advances in computational illumination, this new device enables simultaneous multi-contrast imaging with brightfield, darkfield, and phase imaging modes. Further, we scan through illumination angles to capture lightfield datasets, which can be used to recover 3D intensity and phase images without any hardware changes. This digital refocusing procedure can be used for either 3D imaging or software-only focus correction, reducing the need for precise mechanical focusing during field experiments. All acquisition and processing is performed on the mobile phone and controlled through a smartphone application, making the computational microscope compact and portable. Using multiple samples and different objective magnifications, we demonstrate that the performance of our device is comparable to that of a commercial microscope. This unique device platform extends the field imaging capabilities of CellScope, opening up new clinical and research possibilities.

Multi-Contrast Imaging and Digital Refocusing on a Mobile Microscope with a Domed LED Array

PubMed Central

Phillips, Zachary F.; D'Ambrosio, Michael V.; Tian, Lei; Rulison, Jared J.; Patel, Hurshal S.; Sadras, Nitin; Gande, Aditya V.; Switz, Neil A.; Fletcher, Daniel A.; Waller, Laura

2015-01-01

We demonstrate the design and application of an add-on device for improving the diagnostic and research capabilities of CellScope—a low-cost, smartphone-based point-of-care microscope. We replace the single LED illumination of the original CellScope with a programmable domed LED array. By leveraging recent advances in computational illumination, this new device enables simultaneous multi-contrast imaging with brightfield, darkfield, and phase imaging modes. Further, we scan through illumination angles to capture lightfield datasets, which can be used to recover 3D intensity and phase images without any hardware changes. This digital refocusing procedure can be used for either 3D imaging or software-only focus correction, reducing the need for precise mechanical focusing during field experiments. All acquisition and processing is performed on the mobile phone and controlled through a smartphone application, making the computational microscope compact and portable. Using multiple samples and different objective magnifications, we demonstrate that the performance of our device is comparable to that of a commercial microscope. This unique device platform extends the field imaging capabilities of CellScope, opening up new clinical and research possibilities. PMID:25969980

Compact plane illumination plugin device to enable light sheet fluorescence imaging of multi-cellular organisms on an inverted wide-field microscope

PubMed Central

Guan, Zeyi; Lee, Juhyun; Jiang, Hao; Dong, Siyan; Jen, Nelson; Hsiai, Tzung; Ho, Chih-Ming; Fei, Peng

2015-01-01

We developed a compact plane illumination plugin (PIP) device which enabled plane illumination and light sheet fluorescence imaging on a conventional inverted microscope. The PIP device allowed the integration of microscope with tunable laser sheet profile, fast image acquisition, and 3-D scanning. The device is both compact, measuring approximately 15 by 5 by 5 cm, and cost-effective, since we employed consumer electronics and an inexpensive device molding method. We demonstrated that PIP provided significant contrast and resolution enhancement to conventional microscopy through imaging different multi-cellular fluorescent structures, including 3-D branched cells in vitro and live zebrafish embryos. Imaging with the integration of PIP greatly reduced out-of-focus contamination and generated sharper contrast in acquired 2-D plane images when compared with the stand-alone inverted microscope. As a result, the dynamic fluid domain of the beating zebrafish heart was clearly segmented and the functional monitoring of the heart was achieved. Furthermore, the enhanced axial resolution established by thin plane illumination of PIP enabled the 3-D reconstruction of the branched cellular structures, which leads to the improvement on the functionality of the wide field microscopy. PMID:26819828

Compact plane illumination plugin device to enable light sheet fluorescence imaging of multi-cellular organisms on an inverted wide-field microscope.

PubMed

Guan, Zeyi; Lee, Juhyun; Jiang, Hao; Dong, Siyan; Jen, Nelson; Hsiai, Tzung; Ho, Chih-Ming; Fei, Peng

2016-01-01

We developed a compact plane illumination plugin (PIP) device which enabled plane illumination and light sheet fluorescence imaging on a conventional inverted microscope. The PIP device allowed the integration of microscope with tunable laser sheet profile, fast image acquisition, and 3-D scanning. The device is both compact, measuring approximately 15 by 5 by 5 cm, and cost-effective, since we employed consumer electronics and an inexpensive device molding method. We demonstrated that PIP provided significant contrast and resolution enhancement to conventional microscopy through imaging different multi-cellular fluorescent structures, including 3-D branched cells in vitro and live zebrafish embryos. Imaging with the integration of PIP greatly reduced out-of-focus contamination and generated sharper contrast in acquired 2-D plane images when compared with the stand-alone inverted microscope. As a result, the dynamic fluid domain of the beating zebrafish heart was clearly segmented and the functional monitoring of the heart was achieved. Furthermore, the enhanced axial resolution established by thin plane illumination of PIP enabled the 3-D reconstruction of the branched cellular structures, which leads to the improvement on the functionality of the wide field microscopy.

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.

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

Multiscale Imaging of the Mouse Cortex Using Two-Photon Microscopy and Wide-Field Illumination

NASA Astrophysics Data System (ADS)

Bumstead, Jonathan R.

The mouse brain can be studied over vast spatial scales ranging from microscopic imaging of single neurons to macroscopic measurements of hemodynamics acquired over the majority of the mouse cortex. However, most neuroimaging modalities are limited by a fundamental trade-off between the spatial resolution and the field-of-view (FOV) over which the brain can be imaged, making it difficult to fully understand the functional and structural architecture of the healthy mouse brain and its disruption in disease. My dissertation has focused on developing multiscale optical systems capable of imaging the mouse brain at both microscopic and mesoscopic spatial scales, specifically addressing the difference in spatial scales imaged with two-photon microscopy (TPM) and optical intrinsic signal imaging (OISI). Central to this work has been the formulation of a principled design strategy for extending the FOV of the two-photon microscope. Using this design approach, we constructed a TPM system with subcellular resolution and a FOV area 100 times greater than a conventional two-photon microscope. To image the ellipsoidal shape of the mouse cortex, we also developed the microscope to image arbitrary surfaces within a single frame using an electrically tunable lens. Finally, to address the speed limitations of the TPM systems developed during my dissertation, I also conducted research in large-scale neural phenomena occurring in the mouse brain imaged with high-speed OISI. The work conducted during my dissertation addresses some of the fundamental principles in designing and applying optical systems for multiscale imaging of the mouse brain.

Ghost microscope imaging system from the perspective of coherent-mode representation

NASA Astrophysics Data System (ADS)

Shen, Qian; Bai, Yanfeng; Shi, Xiaohui; Nan, Suqin; Qu, Lijie; Li, Hengxing; Fu, Xiquan

2018-03-01

The coherent-mode representation theory of partially coherent fields is firstly used to analyze a two-arm ghost microscope imaging system. It is shown that imaging quality of the generated images depend crucially on the distribution of the decomposition coefficients of the object imaged when the light source is fixed. This theory is also suitable for demonstrating the effects from the distance the object is moved away from the original plane on imaging quality. Our results are verified theoretically and experimentally.

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.

The Athena Pancam and Color Microscopic Imager (CMI)

NASA Technical Reports Server (NTRS)

Bell, J. F., III; Herkenhoff, K. E.; Schwochert, M.; Morris, R. V.; Sullivan, R.

2000-01-01

The Athena Mars rover payload includes two primary science-grade imagers: Pancam, a multispectral, stereo, panoramic camera system, and the Color Microscopic Imager (CMI), a multispectral and variable depth-of-field microscope. Both of these instruments will help to achieve the primary Athena science goals by providing information on the geology, mineralogy, and climate history of the landing site. In addition, Pancam provides important support for rover navigation and target selection for Athena in situ investigations. Here we describe the science goals, instrument designs, and instrument performance of the Pancam and CMI investigations.

Acoustic imaging microscope

DOEpatents

Deason, Vance A.; Telschow, Kenneth L.

2006-10-17

An imaging system includes: an object wavefront source and an optical microscope objective all positioned to direct an object wavefront onto an area of a vibrating subject surface encompassed by a field of view of the microscope objective, and to direct a modulated object wavefront reflected from the encompassed surface area through a photorefractive material; and a reference wavefront source and at least one phase modulator all positioned to direct a reference wavefront through the phase modulator and to direct a modulated reference wavefront from the phase modulator through the photorefractive material to interfere with the modulated object wavefront. The photorefractive material has a composition and a position such that interference of the modulated object wavefront and modulated reference wavefront occurs within the photorefractive material, providing a full-field, real-time image signal of the encompassed surface area.

Wide field-of-view, multi-region two-photon imaging of neuronal activity in the mammalian brain

PubMed Central

Stirman, Jeffrey N.; Smith, Ikuko T.; Kudenov, Michael W.; Smith, Spencer L.

2016-01-01

Two-photon calcium imaging provides an optical readout of neuronal activity in populations of neurons with subcellular resolution. However, conventional two-photon imaging systems are limited in their field of view to ~1 mm2, precluding the visualization of multiple cortical areas simultaneously. Here, we demonstrate a two-photon microscope with an expanded field of view (>9.5 mm2) for rapidly reconfigurable simultaneous scanning of widely separated populations of neurons. We custom designed and assembled an optimized scan engine, objective, and two independently positionable, temporally multiplexed excitation pathways. We used this new microscope to measure activity correlations between two cortical visual areas in mice during visual processing. PMID:27347754

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

Quantitative luminescence imaging system

DOEpatents

Erwin, D.N.; Kiel, J.L.; Batishko, C.R.; Stahl, K.A.

1990-08-14

The QLIS images and quantifies low-level chemiluminescent reactions in an electromagnetic field. It is capable of real time nonperturbing measurement and simultaneous recording of many biochemical and chemical reactions such as luminescent immunoassays or enzyme assays. The system comprises image transfer optics, a low-light level digitizing camera with image intensifying microchannel plates, an image process or, and a control computer. The image transfer optics may be a fiber image guide with a bend, or a microscope, to take the light outside of the RF field. Output of the camera is transformed into a localized rate of cumulative digitalized data or enhanced video display or hard-copy images. The system may be used as a luminescent microdosimetry device for radiofrequency or microwave radiation, as a thermal dosimeter, or in the dosimetry of ultra-sound (sonoluminescence) or ionizing radiation. It provides a near-real-time system capable of measuring the extremely low light levels from luminescent reactions in electromagnetic fields in the areas of chemiluminescence assays and thermal microdosimetry, and is capable of near-real-time imaging of the sample to allow spatial distribution analysis of the reaction. It can be used to instrument three distinctly different irradiation configurations, comprising (1) RF waveguide irradiation of a small Petri-dish-shaped sample cell, (2) RF irradiation of samples in a microscope for the microscopic imaging and measurement, and (3) RF irradiation of small to human body-sized samples in an anechoic chamber. 22 figs.

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.

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

Cell-phone-based platform for biomedical device development and education applications.

PubMed

Smith, Zachary J; Chu, Kaiqin; Espenson, Alyssa R; Rahimzadeh, Mehdi; Gryshuk, Amy; Molinaro, Marco; Dwyre, Denis M; Lane, Stephen; Matthews, Dennis; Wachsmann-Hogiu, Sebastian

2011-03-02

In this paper we report the development of two attachments to a commercial cell phone that transform the phone's integrated lens and image sensor into a 350x microscope and visible-light spectrometer. The microscope is capable of transmission and polarized microscopy modes and is shown to have 1.5 micron resolution and a usable field-of-view of 150 x 50 with no image processing, and approximately 350 x 350 when post-processing is applied. The spectrometer has a 300 nm bandwidth with a limiting spectral resolution of close to 5 nm. We show applications of the devices to medically relevant problems. In the case of the microscope, we image both stained and unstained blood-smears showing the ability to acquire images of similar quality to commercial microscope platforms, thus allowing diagnosis of clinical pathologies. With the spectrometer we demonstrate acquisition of a white-light transmission spectrum through diffuse tissue as well as the acquisition of a fluorescence spectrum. We also envision the devices to have immediate relevance in the educational field.

Cell-Phone-Based Platform for Biomedical Device Development and Education Applications

PubMed Central

Smith, Zachary J.; Chu, Kaiqin; Espenson, Alyssa R.; Rahimzadeh, Mehdi; Gryshuk, Amy; Molinaro, Marco; Dwyre, Denis M.; Lane, Stephen; Matthews, Dennis; Wachsmann-Hogiu, Sebastian

2011-01-01

In this paper we report the development of two attachments to a commercial cell phone that transform the phone's integrated lens and image sensor into a 350× microscope and visible-light spectrometer. The microscope is capable of transmission and polarized microscopy modes and is shown to have 1.5 micron resolution and a usable field-of-view of 150×150 with no image processing, and approximately 350×350 when post-processing is applied. The spectrometer has a 300 nm bandwidth with a limiting spectral resolution of close to 5 nm. We show applications of the devices to medically relevant problems. In the case of the microscope, we image both stained and unstained blood-smears showing the ability to acquire images of similar quality to commercial microscope platforms, thus allowing diagnosis of clinical pathologies. With the spectrometer we demonstrate acquisition of a white-light transmission spectrum through diffuse tissue as well as the acquisition of a fluorescence spectrum. We also envision the devices to have immediate relevance in the educational field. PMID:21399693

A versatile optical microscope for time-dependent single-molecule and single-particle spectroscopy

NASA Astrophysics Data System (ADS)

Li, Hao; Yang, Haw

2018-03-01

This work reports the design and implementation of a multi-function optical microscope for time-dependent spectroscopy on single molecules and single nanoparticles. It integrates the now-routine single-object measurements into one standalone platform so that no reconfiguration is needed when switching between different types of sample or spectroscopy modes. The illumination modes include evanescent field through total internal reflection, dark-field illumination, and epi-excitation onto a diffraction-limited spot suitable for confocal detection. The detection modes include spectrally resolved line imaging, wide-field imaging with dual-color capability, and two-color single-element photon-counting detection. The switch between different spectroscopy and data acquisition modes is fully automated and executed through computer programming. The capability of this microscope is demonstrated through selected proof-of-principle experiments.

A versatile optical microscope for time-dependent single-molecule and single-particle spectroscopy.

PubMed

Li, Hao; Yang, Haw

2018-03-28

This work reports the design and implementation of a multi-function optical microscope for time-dependent spectroscopy on single molecules and single nanoparticles. It integrates the now-routine single-object measurements into one standalone platform so that no reconfiguration is needed when switching between different types of sample or spectroscopy modes. The illumination modes include evanescent field through total internal reflection, dark-field illumination, and epi-excitation onto a diffraction-limited spot suitable for confocal detection. The detection modes include spectrally resolved line imaging, wide-field imaging with dual-color capability, and two-color single-element photon-counting detection. The switch between different spectroscopy and data acquisition modes is fully automated and executed through computer programming. The capability of this microscope is demonstrated through selected proof-of-principle experiments.

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.

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.

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.

Wide-field imaging of birefringent synovial fluid crystals using lens-free polarized microscopy for gout diagnosis

NASA Astrophysics Data System (ADS)

Zhang, Yibo; Lee, Seung Yoon Celine; Zhang, Yun; Furst, Daniel; Fitzgerald, John; Ozcan, Aydogan

2016-06-01

Gout is a form of crystal arthropathy where monosodium urate (MSU) crystals deposit and elicit inflammation in a joint. Diagnosis of gout relies on identification of MSU crystals under a compensated polarized light microscope (CPLM) in synovial fluid aspirated from the patient’s joint. The detection of MSU crystals by optical microscopy is enhanced by their birefringent properties. However, CPLM partially suffers from the high-cost and bulkiness of conventional lens-based microscopy, and its relatively small field-of-view (FOV) limits the efficiency and accuracy of gout diagnosis. Here we present a lens-free polarized microscope which adopts a novel differential and angle-mismatched polarizing optical design achieving wide-field and high-resolution holographic imaging of birefringent objects with a color contrast similar to that of a standard CPLM. The performance of this computational polarization microscope is validated by imaging MSU crystals made from a gout patient’s tophus and steroid crystals used as negative control. This lens-free polarized microscope, with its wide FOV (>20 mm2), cost-effectiveness and field-portability, can significantly improve the efficiency and accuracy of gout diagnosis, reduce costs, and can be deployed even at the point-of-care and in resource-limited clinical settings.

Wide-field imaging of birefringent synovial fluid crystals using lens-free polarized microscopy for gout diagnosis

PubMed Central

Zhang, Yibo; Lee, Seung Yoon Celine; Zhang, Yun; Furst, Daniel; Fitzgerald, John; Ozcan, Aydogan

2016-01-01

Gout is a form of crystal arthropathy where monosodium urate (MSU) crystals deposit and elicit inflammation in a joint. Diagnosis of gout relies on identification of MSU crystals under a compensated polarized light microscope (CPLM) in synovial fluid aspirated from the patient’s joint. The detection of MSU crystals by optical microscopy is enhanced by their birefringent properties. However, CPLM partially suffers from the high-cost and bulkiness of conventional lens-based microscopy, and its relatively small field-of-view (FOV) limits the efficiency and accuracy of gout diagnosis. Here we present a lens-free polarized microscope which adopts a novel differential and angle-mismatched polarizing optical design achieving wide-field and high-resolution holographic imaging of birefringent objects with a color contrast similar to that of a standard CPLM. The performance of this computational polarization microscope is validated by imaging MSU crystals made from a gout patient’s tophus and steroid crystals used as negative control. This lens-free polarized microscope, with its wide FOV (>20 mm2), cost-effectiveness and field-portability, can significantly improve the efficiency and accuracy of gout diagnosis, reduce costs, and can be deployed even at the point-of-care and in resource-limited clinical settings. PMID:27356625

Multistage morphological segmentation of bright-field and fluorescent microscopy images

NASA Astrophysics Data System (ADS)

Korzyńska, A.; Iwanowski, M.

2012-06-01

This paper describes the multistage morphological segmentation method (MSMA) for microscopic cell images. The proposed method enables us to study the cell behaviour by using a sequence of two types of microscopic images: bright field images and/or fluorescent images. The proposed method is based on two types of information: the cell texture coming from the bright field images and intensity of light emission, done by fluorescent markers. The method is dedicated to the image sequences segmentation and it is based on mathematical morphology methods supported by other image processing techniques. The method allows for detecting cells in image independently from a degree of their flattening and from presenting structures which produce the texture. It makes use of some synergic information from the fluorescent light emission image as the support information. The MSMA method has been applied to images acquired during the experiments on neural stem cells as well as to artificial images. In order to validate the method, two types of errors have been considered: the error of cell area detection and the error of cell position using artificial images as the "gold standard".

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.

Single cell magnetic imaging using a quantum diamond microscope

PubMed Central

Park, H.; Weissleder, R.; Yacoby, A.; Lukin, M. D.; Lee, H.; Walsworth, R. L.; Connolly, C. B.

2015-01-01

We apply a quantum diamond microscope to detection and imaging of immunomagnetically labeled cells. This instrument uses nitrogen-vacancy (NV) centers in diamond for correlated magnetic and fluorescence imaging. Our device provides single-cell resolution and two orders of magnitude larger field of view (~1 mm2) than previous NV imaging technologies, enabling practical applications. To illustrate, we quantify cancer biomarkers expressed by rare tumor cells in a large population of healthy cells. PMID:26098019

Calibration of Wide-Field Deconvolution Microscopy for Quantitative Fluorescence Imaging

PubMed Central

Lee, Ji-Sook; Wee, Tse-Luen (Erika); Brown, Claire M.

2014-01-01

Deconvolution enhances contrast in fluorescence microscopy images, especially in low-contrast, high-background wide-field microscope images, improving characterization of features within the sample. Deconvolution can also be combined with other imaging modalities, such as confocal microscopy, and most software programs seek to improve resolution as well as contrast. Quantitative image analyses require instrument calibration and with deconvolution, necessitate that this process itself preserves the relative quantitative relationships between fluorescence intensities. To ensure that the quantitative nature of the data remains unaltered, deconvolution algorithms need to be tested thoroughly. This study investigated whether the deconvolution algorithms in AutoQuant X3 preserve relative quantitative intensity data. InSpeck Green calibration microspheres were prepared for imaging, z-stacks were collected using a wide-field microscope, and the images were deconvolved using the iterative deconvolution algorithms with default settings. Afterwards, the mean intensities and volumes of microspheres in the original and the deconvolved images were measured. Deconvolved data sets showed higher average microsphere intensities and smaller volumes than the original wide-field data sets. In original and deconvolved data sets, intensity means showed linear relationships with the relative microsphere intensities given by the manufacturer. Importantly, upon normalization, the trend lines were found to have similar slopes. In original and deconvolved images, the volumes of the microspheres were quite uniform for all relative microsphere intensities. We were able to show that AutoQuant X3 deconvolution software data are quantitative. In general, the protocol presented can be used to calibrate any fluorescence microscope or image processing and analysis procedure. PMID:24688321

Evaluation of a miniature microscope objective designed for fluorescence array microscopy detection of Mycobacterium tuberculosis.

PubMed

McCall, Brian; Olsen, Randall J; Nelles, Nicole J; Williams, Dawn L; Jackson, Kevin; Richards-Kortum, Rebecca; Graviss, Edward A; Tkaczyk, Tomasz S

2014-03-01

A prototype miniature objective that was designed for a point-of-care diagnostic array microscope for detection of Mycobacterium tuberculosis and previously fabricated and presented in a proof of concept is evaluated for its effectiveness in detecting acid-fast bacteria. To evaluate the ability of the microscope to resolve submicron features and details in the image of acid-fast microorganisms stained with a fluorescent dye, and to evaluate the accuracy of clinical diagnoses made with digital images acquired with the objective. The lens prescription data for the microscope design are presented. A test platform is built by combining parts of a standard microscope, a prototype objective, and a digital single-lens reflex camera. Counts of acid-fast bacteria made with the prototype objective are compared to counts obtained with a standard microscope over matched fields of view. Two sets of 20 smears, positive and negative, are diagnosed by 2 pathologists as sputum smear positive or sputum smear negative, using both a standard clinical microscope and the prototype objective under evaluation. The results are compared to a reference diagnosis of the same sample. More bacteria are counted in matched fields of view in digital images taken with the prototype objective than with the standard clinical microscope. All diagnostic results are found to be highly concordant. An array microscope built with this miniature lens design will be able to detect M tuberculosis with high sensitivity and specificity.

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

NASA Astrophysics Data System (ADS)

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

2017-03-01

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

Custom Super-Resolution Microscope for the Structural Analysis of Nanostructures

DTIC Science & Technology

2018-05-29

research community. As part of our validation of the new design approach, we performed two - color imaging of pairs of adjacent oligo probes hybridized...nanostructures and biological targets. Our microscope features a large field of view and custom optics that facilitate 3D imaging and enhanced contrast in...our imaging throughput by creating two microscopy platforms for high-throughput, super-resolution materials characterization, with the AO set-up being

The influence of the microscope lamp filament colour temperature on the process of digital images of histological slides acquisition standardization.

PubMed

Korzynska, Anna; Roszkowiak, Lukasz; Pijanowska, Dorota; Kozlowski, Wojciech; Markiewicz, Tomasz

2014-01-01

The aim of this study is to compare the digital images of the tissue biopsy captured with optical microscope using bright field technique under various light conditions. The range of colour's variation in immunohistochemically stained with 3,3'-Diaminobenzidine and Haematoxylin tissue samples is immense and coming from various sources. One of them is inadequate setting of camera's white balance to microscope's light colour temperature. Although this type of error can be easily handled during the stage of image acquisition, it can be eliminated with use of colour adjustment algorithms. The examination of the dependence of colour variation from microscope's light temperature and settings of the camera is done as an introductory research to the process of automatic colour standardization. Six fields of view with empty space among the tissue samples have been selected for analysis. Each field of view has been acquired 225 times with various microscope light temperature and camera white balance settings. The fourteen randomly chosen images have been corrected and compared, with the reference image, by the following methods: Mean Square Error, Structural SIMilarity and visual assessment of viewer. For two types of backgrounds and two types of objects, the statistical image descriptors: range, median, mean and its standard deviation of chromaticity on a and b channels from CIELab colour space, and luminance L, and local colour variability for objects' specific area have been calculated. The results have been averaged for 6 images acquired in the same light conditions and camera settings for each sample. The analysis of the results leads to the following conclusions: (1) the images collected with white balance setting adjusted to light colour temperature clusters in certain area of chromatic space, (2) the process of white balance correction for images collected with white balance camera settings not matched to the light temperature moves image descriptors into proper chromatic space but simultaneously the value of luminance changes. So the process of the image unification in a sense of colour fidelity can be solved in separate introductory stage before the automatic image analysis.

A Clinical Wide-Field Fluorescence Endoscopic Device for Molecular Imaging Demonstrating Cathepsin Protease Activity in Colon Cancer.

PubMed

Sensarn, Steven; Zavaleta, Cristina L; Segal, Ehud; Rogalla, Stephan; Lee, Wansik; Gambhir, Sanjiv S; Bogyo, Matthew; Contag, Christopher H

2016-12-01

Early and effective detection of cancers of the gastrointestinal tract will require novel molecular probes and advances in instrumentation that can reveal functional changes in dysplastic and malignant tissues. Here, we describe adaptation of a wide-field clinical fiberscope to perform wide-field fluorescence imaging while preserving its white-light capability for the purpose of providing wide-field fluorescence imaging capability to point-of-care microscopes. We developed and used a fluorescent fiberscope to detect signals from a quenched probe, BMV109, that becomes fluorescent when cleaved by, and covalently bound to, active cathepsin proteases. Cathepsins are expressed in inflammation- and tumor-associated macrophages as well as directly from tumor cells and are a promising target for cancer imaging. The fiberscope has a 1-mm outer diameter enabling validation via endoscopic exams in mice, and therefore we evaluated topically applied BMV109 for the ability to detect colon polyps in an azoxymethane-induced colon tumor model in mice. This wide-field endoscopic imaging device revealed consistent and clear fluorescence signals from BMV109 that specifically localized to the polypoid regions as opposed to the normal adjacent colon tissue (p < 0.004) in the murine colon carcinoma model. The sensitivity of detection of BMV109 with the fluorescence fiberscope suggested utility of these tools for early detection at hard-to-reach sites. The fiberscope was designed to be used in conjunction with miniature, endoscope-compatible fluorescence microscopes for dual wide-field and microscopic cancer detection.

Ultra high frequency imaging acoustic microscope

DOEpatents

Deason, Vance A.; Telschow, Kenneth L.

2006-05-23

An imaging system includes: an object wavefront source and an optical microscope objective all positioned to direct an object wavefront onto an area of a vibrating subject surface encompassed by a field of view of the microscope objective, and to direct a modulated object wavefront reflected from the encompassed surface area through a photorefractive material; and a reference wavefront source and at least one phase modulator all positioned to direct a reference wavefront through the phase modulator and to direct a modulated reference wavefront from the phase modulator through the photorefractive material to interfere with the modulated object wavefront. The photorefractive material has a composition and a position such that interference of the modulated object wavefront and modulated reference wavefront occurs within the photorefractive material, providing a full-field, real-time image signal of the encompassed surface area.

Nanosecond Time-Resolved Microscopic Gate-Modulation Imaging of Polycrystalline Organic Thin-Film Transistors

NASA Astrophysics Data System (ADS)

Matsuoka, Satoshi; Tsutsumi, Jun'ya; Matsui, Hiroyuki; Kamata, Toshihide; Hasegawa, Tatsuo

2018-02-01

We develop a time-resolved microscopic gate-modulation (μ GM ) imaging technique to investigate the temporal evolution of the channel current and accumulated charges in polycrystalline pentacene thin-film transistors (TFTs). A time resolution of as high as 50 ns is achieved by using a fast image-intensifier system that could amplify a series of instantaneous optical microscopic images acquired at various time intervals after the stepped gate bias is switched on. The differential images obtained by subtracting the gate-off image allows us to acquire a series of temporal μ GM images that clearly show the gradual propagation of both channel charges and leaked gate fields within the polycrystalline channel layers. The frontal positions for the propagations of both channel charges and leaked gate fields coincide at all the time intervals, demonstrating that the layered gate dielectric capacitors are successively transversely charged up along the direction of current propagation. The initial μ GM images also indicate that the electric field effect is originally concentrated around a limited area with a width of a few micrometers bordering the channel-electrode interface, and that the field intensity reaches a maximum after 200 ns and then decays. The time required for charge propagation over the whole channel region with a length of 100 μ m is estimated at about 900 ns, which is consistent with the measured field-effect mobility and the temporal-response model for organic TFTs. The effect of grain boundaries can be also visualized by comparison of the μ GM images for the transient and the steady states, which confirms that the potential barriers at the grain boundaries cause the transient shift in the accumulated charges or the transient accumulation of additional charges around the grain boundaries.

Full-field x-ray nano-imaging at SSRF

NASA Astrophysics Data System (ADS)

Deng, Biao; Ren, Yuqi; Wang, Yudan; Du, Guohao; Xie, Honglan; Xiao, Tiqiao

2013-09-01

Full field X-ray nano-imaging focusing on material science is under developing at SSRF. A dedicated full field X-ray nano-imaging beamline based on bending magnet will be built in the SSRF phase-II project. The beamline aims at the 3D imaging of the nano-scale inner structures. The photon energy range is of 5-14keV. The design goals with the field of view (FOV) of 20μm and a spatial resolution of 20nm are proposed at 8 keV, taking a Fresnel zone plate (FZP) with outermost zone width of 25 nm. Futhermore, an X-ray nano-imaging microscope is under developing at the SSRF BL13W beamline, in which a larger FOV will be emphasized. This microscope is based on a beam shaper and a zone plate using both absorption contrast and Zernike phase contrast, with the optimized energy set to 10keV. The detailed design and the progress of the project will be introduced.

Compact Microscope Imaging System With Intelligent Controls Improved

NASA Technical Reports Server (NTRS)

McDowell, Mark

2004-01-01

The Compact Microscope Imaging System (CMIS) with intelligent controls is a diagnostic microscope analysis tool with intelligent controls for use in space, industrial, medical, and security applications. This compact miniature microscope, which can perform tasks usually reserved for conventional microscopes, has unique advantages in the fields of microscopy, biomedical research, inline process inspection, and space science. Its unique approach integrates a machine vision technique with an instrumentation and control technique that provides intelligence via the use of adaptive neural networks. The CMIS system was developed at the NASA Glenn Research Center specifically for interface detection used for colloid hard spheres experiments; biological cell detection for patch clamping, cell movement, and tracking; and detection of anode and cathode defects for laboratory samples using microscope technology.

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.

Imaging of current density distributions with a Nb weak-link scanning nano-SQUID microscope

PubMed Central

Shibata, Yusuke; Nomura, Shintaro; Kashiwaya, Hiromi; Kashiwaya, Satoshi; Ishiguro, Ryosuke; Takayanagi, Hideaki

2015-01-01

Superconducting quantum interference devices (SQUIDs) are accepted as one of the highest magnetic field sensitive probes. There are increasing demands to image local magnetic fields to explore spin properties and current density distributions in a two-dimensional layer of semiconductors or superconductors. Nano-SQUIDs have recently attracting much interest for high spatial resolution measurements in nanometer-scale samples. Whereas weak-link Dayem Josephson junction nano-SQUIDs are suitable to miniaturization, hysteresis in current-voltage (I-V) characteristics that is often observed in Dayem Josephson junction is not desirable for a scanning microscope. Here we report on our development of a weak-link nano-SQUIDs scanning microscope with small hysteresis in I-V curve and on reconstructions of two-dimensional current density vector in two-dimensional electron gas from measured magnetic field. PMID:26459874

Imaging of current density distributions with a Nb weak-link scanning nano-SQUID microscope

NASA Astrophysics Data System (ADS)

Shibata, Yusuke; Nomura, Shintaro; Kashiwaya, Hiromi; Kashiwaya, Satoshi; Ishiguro, Ryosuke; Takayanagi, Hideaki

2015-10-01

Superconducting quantum interference devices (SQUIDs) are accepted as one of the highest magnetic field sensitive probes. There are increasing demands to image local magnetic fields to explore spin properties and current density distributions in a two-dimensional layer of semiconductors or superconductors. Nano-SQUIDs have recently attracting much interest for high spatial resolution measurements in nanometer-scale samples. Whereas weak-link Dayem Josephson junction nano-SQUIDs are suitable to miniaturization, hysteresis in current-voltage (I-V) characteristics that is often observed in Dayem Josephson junction is not desirable for a scanning microscope. Here we report on our development of a weak-link nano-SQUIDs scanning microscope with small hysteresis in I-V curve and on reconstructions of two-dimensional current density vector in two-dimensional electron gas from measured magnetic field.

Aberration correction in wide-field fluorescence microscopy by segmented-pupil image interferometry.

PubMed

Scrimgeour, Jan; Curtis, Jennifer E

2012-06-18

We present a new technique for the correction of optical aberrations in wide-field fluorescence microscopy. Segmented-Pupil Image Interferometry (SPII) uses a liquid crystal spatial light modulator placed in the microscope's pupil plane to split the wavefront originating from a fluorescent object into an array of individual beams. Distortion of the wavefront arising from either system or sample aberrations results in displacement of the images formed from the individual pupil segments. Analysis of image registration allows for the local tilt in the wavefront at each segment to be corrected with respect to a central reference. A second correction step optimizes the image intensity by adjusting the relative phase of each pupil segment through image interferometry. This ensures that constructive interference between all segments is achieved at the image plane. Improvements in image quality are observed when Segmented-Pupil Image Interferometry is applied to correct aberrations arising from the microscope's optical path.

Imaging and identification of waterborne parasites using a chip-scale microscope.

PubMed

Lee, Seung Ah; Erath, Jessey; Zheng, Guoan; Ou, Xiaoze; Willems, Phil; Eichinger, Daniel; Rodriguez, Ana; Yang, Changhuei

2014-01-01

We demonstrate a compact portable imaging system for the detection of waterborne parasites in resource-limited settings. The previously demonstrated sub-pixel sweeping microscopy (SPSM) technique is a lens-less imaging scheme that can achieve high-resolution (<1 µm) bright-field imaging over a large field-of-view (5.7 mm×4.3 mm). A chip-scale microscope system, based on the SPSM technique, can be used for automated and high-throughput imaging of protozoan parasite cysts for the effective diagnosis of waterborne enteric parasite infection. We successfully imaged and identified three major types of enteric parasite cysts, Giardia, Cryptosporidium, and Entamoeba, which can be found in fecal samples from infected patients. We believe that this compact imaging system can serve well as a diagnostic device in challenging environments, such as rural settings or emergency outbreaks.

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.

Improvements in low-cost label-free QPI microscope for live cell imaging

NASA Astrophysics Data System (ADS)

Seniya, C.; Towers, C. E.; Towers, D. P.

2017-07-01

This paper reports an improvement in the development of a low-cost QPI microscope offering new capabilities in term of phase measurement accuracy for label-free live samples in the longer term (i.e., hours to days). The spatially separated scattered and non-scattered image light fields are reshaped in the Fourier plane and modulated to form an interference image at a CCD camera. The apertures that enable these two beams to be generated have been optimised by means of laser-cut apertures placed on the mirrors of a Michelson interferometer and has improved the phase measuring and reconstruction capability of the QPI microscope. The microscope was tested with transparent onion cells as an object of interest.

Portable and cost-effective pixel super-resolution on-chip microscope for telemedicine applications.

PubMed

Bishara, Waheb; Sikora, Uzair; Mudanyali, Onur; Su, Ting-Wei; Yaglidere, Oguzhan; Luckhart, Shirley; Ozcan, Aydogan

2011-01-01

We report a field-portable lensless on-chip microscope with a lateral resolution of <1 μm and a large field-of-view of ~24 mm(2). This microscope is based on digital in-line holography and a pixel super-resolution algorithm to process multiple lensfree holograms and obtain a single high-resolution hologram. In its compact and cost-effective design, we utilize 23 light emitting diodes butt-coupled to 23 multi-mode optical fibers, and a simple optical filter, with no moving parts. Weighing only ~95 grams, we demonstrate the performance of this field-portable microscope by imaging various objects including human malaria parasites in thin blood smears.

Field-portable lensfree tomographic microscope.

PubMed

Isikman, Serhan O; Bishara, Waheb; Sikora, Uzair; Yaglidere, Oguzhan; Yeah, John; Ozcan, Aydogan

2011-07-07

We present a field-portable lensfree tomographic microscope, which can achieve sectional imaging of a large volume (∼20 mm(3)) on a chip with an axial resolution of <7 μm. In this compact tomographic imaging platform (weighing only ∼110 grams), 24 light-emitting diodes (LEDs) that are each butt-coupled to a fibre-optic waveguide are controlled through a cost-effective micro-processor to sequentially illuminate the sample from different angles to record lensfree holograms of the sample that is placed on the top of a digital sensor array. In order to generate pixel super-resolved (SR) lensfree holograms and hence digitally improve the achievable lateral resolution, multiple sub-pixel shifted holograms are recorded at each illumination angle by electromagnetically actuating the fibre-optic waveguides using compact coils and magnets. These SR projection holograms obtained over an angular range of ±50° are rapidly reconstructed to yield projection images of the sample, which can then be back-projected to compute tomograms of the objects on the sensor-chip. The performance of this compact and light-weight lensfree tomographic microscope is validated by imaging micro-beads of different dimensions as well as a Hymenolepis nana egg, which is an infectious parasitic flatworm. Achieving a decent three-dimensional spatial resolution, this field-portable on-chip optical tomographic microscope might provide a useful toolset for telemedicine and high-throughput imaging applications in resource-poor settings. This journal is © The Royal Society of Chemistry 2011

Interior tomography in microscopic CT with image reconstruction constrained by full field of view scan at low spatial resolution

NASA Astrophysics Data System (ADS)

Luo, Shouhua; Shen, Tao; Sun, Yi; Li, Jing; Li, Guang; Tang, Xiangyang

2018-04-01

In high resolution (microscopic) CT applications, the scan field of view should cover the entire specimen or sample to allow complete data acquisition and image reconstruction. However, truncation may occur in projection data and results in artifacts in reconstructed images. In this study, we propose a low resolution image constrained reconstruction algorithm (LRICR) for interior tomography in microscopic CT at high resolution. In general, the multi-resolution acquisition based methods can be employed to solve the data truncation problem if the project data acquired at low resolution are utilized to fill up the truncated projection data acquired at high resolution. However, most existing methods place quite strict restrictions on the data acquisition geometry, which greatly limits their utility in practice. In the proposed LRICR algorithm, full and partial data acquisition (scan) at low and high resolutions, respectively, are carried out. Using the image reconstructed from sparse projection data acquired at low resolution as the prior, a microscopic image at high resolution is reconstructed from the truncated projection data acquired at high resolution. Two synthesized digital phantoms, a raw bamboo culm and a specimen of mouse femur, were utilized to evaluate and verify performance of the proposed LRICR algorithm. Compared with the conventional TV minimization based algorithm and the multi-resolution scout-reconstruction algorithm, the proposed LRICR algorithm shows significant improvement in reduction of the artifacts caused by data truncation, providing a practical solution for high quality and reliable interior tomography in microscopic CT applications. The proposed LRICR algorithm outperforms the multi-resolution scout-reconstruction method and the TV minimization based reconstruction for interior tomography in microscopic CT.

A high-resolution combined scanning laser and widefield polarizing microscope for imaging at temperatures from 4 K to 300 K.

PubMed

Lange, M; Guénon, S; Lever, F; Kleiner, R; Koelle, D

2017-12-01

Polarized light microscopy, as a contrast-enhancing technique for optically anisotropic materials, is a method well suited for the investigation of a wide variety of effects in solid-state physics, as, for example, birefringence in crystals or the magneto-optical Kerr effect (MOKE). We present a microscopy setup that combines a widefield microscope and a confocal scanning laser microscope with polarization-sensitive detectors. By using a high numerical aperture objective, a spatial resolution of about 240 nm at a wavelength of 405 nm is achieved. The sample is mounted on a 4 He continuous flow cryostat providing a temperature range between 4 K and 300 K, and electromagnets are used to apply magnetic fields of up to 800 mT with variable in-plane orientation and 20 mT with out-of-plane orientation. Typical applications of the polarizing microscope are the imaging of the in-plane and out-of-plane magnetization via the longitudinal and polar MOKE, imaging of magnetic flux structures in superconductors covered with a magneto-optical indicator film via the Faraday effect, or imaging of structural features, such as twin-walls in tetragonal SrTiO 3 . The scanning laser microscope furthermore offers the possibility to gain local information on electric transport properties of a sample by detecting the beam-induced voltage change across a current-biased sample. This combination of magnetic, structural, and electric imaging capabilities makes the microscope a viable tool for research in the fields of oxide electronics, spintronics, magnetism, and superconductivity.

A high-resolution combined scanning laser and widefield polarizing microscope for imaging at temperatures from 4 K to 300 K

NASA Astrophysics Data System (ADS)

Lange, M.; Guénon, S.; Lever, F.; Kleiner, R.; Koelle, D.

2017-12-01

Polarized light microscopy, as a contrast-enhancing technique for optically anisotropic materials, is a method well suited for the investigation of a wide variety of effects in solid-state physics, as, for example, birefringence in crystals or the magneto-optical Kerr effect (MOKE). We present a microscopy setup that combines a widefield microscope and a confocal scanning laser microscope with polarization-sensitive detectors. By using a high numerical aperture objective, a spatial resolution of about 240 nm at a wavelength of 405 nm is achieved. The sample is mounted on a 4He continuous flow cryostat providing a temperature range between 4 K and 300 K, and electromagnets are used to apply magnetic fields of up to 800 mT with variable in-plane orientation and 20 mT with out-of-plane orientation. Typical applications of the polarizing microscope are the imaging of the in-plane and out-of-plane magnetization via the longitudinal and polar MOKE, imaging of magnetic flux structures in superconductors covered with a magneto-optical indicator film via the Faraday effect, or imaging of structural features, such as twin-walls in tetragonal SrTiO3. The scanning laser microscope furthermore offers the possibility to gain local information on electric transport properties of a sample by detecting the beam-induced voltage change across a current-biased sample. This combination of magnetic, structural, and electric imaging capabilities makes the microscope a viable tool for research in the fields of oxide electronics, spintronics, magnetism, and superconductivity.

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

Naulleau, Patrick; Mochi, Iacopo; Goldberg, Kenneth A.

Defect free masks remain one of the most significant challenges facing the commercialization of extreme ultraviolet (EUV) lithography. Progress on this front requires high-performance wavelength-specific metrology of EUV masks, including high-resolution and aerial-image microscopy performed near the 13.5 nm wavelength. Arguably the most cost-effective and rapid path to proliferating this capability is through the development of Fresnel zoneplate-based microscopes. Given the relative obscurity of such systems, however, modeling tools are not necessarily optimized to deal with them and their imaging properties are poorly understood. Here we present a modeling methodology to analyze zoneplate microscopes based on commercially available optical modelingmore » software and use the technique to investigate the imaging performance of an off-axis EUV microscope design. The modeling predicts that superior performance can be achieved by tilting the zoneplate, making it perpendicular to the chief ray at the center of the field, while designing the zoneplate to explicitly work in that tilted plane. Although the examples presented here are in the realm of EUV mask inspection, the methods described and analysis results are broadly applicable to zoneplate microscopes in general, including full-field soft-x-ray microscopes rou tinely used in the synchrotron community.« less

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

Naulleau, Patrick P.; Mochi, Iacopo; Goldberg, Kenneth A.

Defect free masks remain one of the most significant challenges facing the commercialization of extreme ultraviolet (EUV) lithography. Progress on this front requires high-performance wavelength-specific metrology of EUV masks, including high-resolution and aerial-image microscopy performed near the 13.5 nm wavelength. Arguably the most cost-effective and rapid path to proliferating this capability is through the development of Fresnel zoneplate-based microscopes. Given the relative obscurity of such systems, however, modeling tools are not necessarily optimized to deal with them and their imaging properties are poorly understood. Here we present a modeling methodology to analyze zoneplate microscopes based on commercially available optical modelingmore » software and use the technique to investigate the imaging performance of an off-axis EUV microscope design. The modeling predicts that superior performance can be achieved by tilting the zoneplate, making it perpendicular to the chief ray at the center of the field, while designing the zoneplate to explicitly work in that tilted plane. Although the examples presented here are in the realm of EUV mask inspection, the methods described and analysis results are broadly applicable to zoneplate microscopes in general, including full-field soft-x-ray microscopes routinely used in the synchrotron community.« less

Nanoscale Optical Imaging and Spectroscopy from Visible to Mid-Infrared

DTIC Science & Technology

2015-11-13

field characterization of nanoscale materials, it also complements the near- field scanning optical microscope currently available in the PI’s lab...field scanning optical microscope currently available in the PI’s lab. This equipment will begin making major impacts on at least three current DoD...SECURITY CLASSIFICATION OF: 1. REPORT DATE (DD-MM-YYYY) 4. TITLE AND SUBTITLE 13. SUPPLEMENTARY NOTES 12. DISTRIBUTION AVAILIBILITY STATEMENT 6

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.

Label-free hyperspectral dark-field microscopy for quantitative scatter imaging

NASA Astrophysics Data System (ADS)

Cheney, Philip; McClatchy, David; Kanick, Stephen; Lemaillet, Paul; Allen, David; Samarov, Daniel; Pogue, Brian; Hwang, Jeeseong

2017-03-01

A hyperspectral dark-field microscope has been developed for imaging spatially distributed diffuse reflectance spectra from light-scattering samples. In this report, quantitative scatter spectroscopy is demonstrated with a uniform scattering phantom, namely a solution of polystyrene microspheres. A Monte Carlo-based inverse model was used to calculate the reduced scattering coefficients of samples of different microsphere concentrations from wavelength-dependent backscattered signal measured by the dark-field microscope. The results are compared to the measurement results from a NIST double-integrating sphere system for validation. Ongoing efforts involve quantitative mapping of scattering and absorption coefficients in samples with spatially heterogeneous optical properties.

X-ray mosaic nanotomography of large microorganisms.

PubMed

Mokso, R; Quaroni, L; Marone, F; Irvine, S; Vila-Comamala, J; Blanke, A; Stampanoni, M

2012-02-01

Full-field X-ray microscopy is a valuable tool for 3D observation of biological systems. In the soft X-ray domain organelles can be visualized in individual cells while hard X-ray microscopes excel in imaging of larger complex biological tissue. The field of view of these instruments is typically 10(3) times the spatial resolution. We exploit the assets of the hard X-ray sub-micrometer imaging and extend the standard approach by widening the effective field of view to match the size of the sample. We show that global tomography of biological systems exceeding several times the field of view is feasible also at the nanoscale with moderate radiation dose. We address the performance issues and limitations of the TOMCAT full-field microscope and more generally for Zernike phase contrast imaging. Two biologically relevant systems were investigated. The first being the largest known bacteria (Thiomargarita namibiensis), the second is a small myriapod species (Pauropoda sp.). Both examples illustrate the capacity of the unique, structured condenser based broad-band full-field microscope to access the 3D structural details of biological systems at the nanoscale while avoiding complicated sample preparation, or even keeping the sample environment close to the natural state. Copyright © 2012 Elsevier Inc. All rights reserved.

High-resolution resonant and nonresonant fiber-scanning confocal microscope.

PubMed

Hendriks, Benno H W; Bierhoff, Walter C J; Horikx, Jeroen J L; Desjardins, Adrien E; Hezemans, Cees A; 't Hooft, Gert W; Lucassen, Gerald W; Mihajlovic, Nenad

2011-02-01

We present a novel, hand-held microscope probe for acquiring confocal images of biological tissue. This probe generates images by scanning a fiber-lens combination with a miniature electromagnetic actuator, which allows it to be operated in resonant and nonresonant scanning modes. In the resonant scanning mode, a circular field of view with a diameter of 190 μm and an angular frequency of 127 Hz can be achieved. In the nonresonant scanning mode, a maximum field of view with a width of 69 μm can be achieved. The measured transverse and axial resolutions are 0.60 and 7.4 μm, respectively. Images of biological tissue acquired in the resonant mode are presented, which demonstrate its potential for real-time tissue differentiation. With an outer diameter of 3 mm, the microscope probe could be utilized to visualize cellular microstructures in vivo across a broad range of minimally-invasive procedures.

First Clinical Applications of a High-Definition Three-Dimensional Exoscope in Pediatric Neurosurgery.

PubMed

Beez, Thomas; Munoz-Bendix, Christopher; Beseoglu, Kerim; Steiger, Hans-Jakob; Ahmadi, Sebastian A

2018-01-24

The ideal visualization tools in microneurosurgery should provide magnification, illumination, wide fields of view, ergonomics, and unobstructed access to the surgical field. The operative microscope was the predominant innovation in modern neurosurgery. Recently, a high-definition three-dimensional (3D) exoscope was developed. We describe the first applications in pediatric neurosurgery. The VITOM 3D exoscope (Karl Storz GmbH, Tuttlingen, Germany) was used in pediatric microneurosurgical operations, along with an OPMI PENTERO operative microscope (Carl Zeiss AG, Jena, Germany). Experiences were retrospectively evaluated with five-level Likert items regarding ease of preparation, image definition, magnification, illumination, field of view, ergonomics, accessibility of the surgical field, and general user-friendliness. Three operations were performed: supratentorial open biopsy in the supine position, infratentorial brain tumor resection in the park bench position, and myelomeningocele closure in the prone position. While preparation and image definition were rated equal for microscope and exoscope, the microscope's field of view, illumination, and user-friendliness were considered superior, while the advantages of the exoscope were seen in ergonomics and the accessibility of the surgical field. No complications attributed to visualization mode occurred. In our experience, the VITOM 3D exoscope is an innovative visualization tool with advantages over the microscope in ergonomics and the accessibility of the surgical field. However, improvements were deemed necessary with regard to field of view, illumination, and user-friendliness. While the debate of a "perfect" visualization modality is influenced by personal preference, this novel visualization device has the potential to become a valuable tool in the neurosurgeon's armamentarium.

Light-sheet enhanced resolution of light field microscopy for rapid imaging of large volumes

NASA Astrophysics Data System (ADS)

Madrid Wolff, Jorge; Castro, Diego; Arbeláez, Pablo; Forero-Shelton, Manu

2018-02-01

Whole-brain imaging is challenging because it demands microscopes with high temporal and spatial resolution, which are often at odds, especially in the context of large fields of view. We have designed and built a light-sheet microscope with digital micromirror illumination and light-field detection. On the one hand, light sheets provide high resolution optical sectioning on live samples without compromising their viability. On the other hand, light field imaging makes it possible to reconstruct full volumes of relatively large fields of view from a single camera exposure; however, its enhanced temporal resolution comes at the expense of spatial resolution, limiting its applicability. We present an approach to increase the resolution of light field images using DMD-based light sheet illumination. To that end, we develop a method to produce synthetic resolution targets for light field microscopy and a procedure to correct the depth at which planes are refocused with rendering software. We measured the axial resolution as a function of depth and show a three-fold potential improvement with structured illumination, albeit by sacrificing some temporal resolution, also three-fold. This results in an imaging system that may be adjusted to specific needs without having to reassemble and realign it. This approach could be used to image relatively large samples at high rates.

Novel instrumentation for multifield time-lapse cinemicrography.

PubMed

Kallman, R F; Blevins, N; Coyne, M A; Prionas, S D

1990-04-01

The most significant feature of the system that is described is its ability to image essentially simultaneously the growth of up to 99 single cells into macroscopic colonies, each in its own microscope field. Operationally, fields are first defined and programmed by a trained observer. All subsequent steps are automatic and under computer control. Salient features of the hardware are stepper motor-controlled movement of the stage and fine adjustment of an inverted microscope, a high-quality 16-mm cine camera with light meter and controls, and a miniature incubator in which cells may be grown under defined conditions directly on the microscope stage. This system, termed MUTLAS, necessitates reordering of the primary images by rephotographing them on fresh film. Software developed for the analysis of cell and colony growth requires frame-by-frame examination of the secondary film and the use of a mouse-driven cursor to trace microscopically visible (4X objective magnification) events.

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.

Thermal radiation scanning tunnelling microscopy

NASA Astrophysics Data System (ADS)

de Wilde, Yannick; Formanek, Florian; Carminati, Rémi; Gralak, Boris; Lemoine, Paul-Arthur; Joulain, Karl; Mulet, Jean-Philippe; Chen, Yong; Greffet, Jean-Jacques

2006-12-01

In standard near-field scanning optical microscopy (NSOM), a subwavelength probe acts as an optical `stethoscope' to map the near field produced at the sample surface by external illumination. This technique has been applied using visible, infrared, terahertz and gigahertz radiation to illuminate the sample, providing a resolution well beyond the diffraction limit. NSOM is well suited to study surface waves such as surface plasmons or surface-phonon polaritons. Using an aperture NSOM with visible laser illumination, a near-field interference pattern around a corral structure has been observed, whose features were similar to the scanning tunnelling microscope image of the electronic waves in a quantum corral. Here we describe an infrared NSOM that operates without any external illumination: it is a near-field analogue of a night-vision camera, making use of the thermal infrared evanescent fields emitted by the surface, and behaves as an optical scanning tunnelling microscope. We therefore term this instrument a `thermal radiation scanning tunnelling microscope' (TRSTM). We show the first TRSTM images of thermally excited surface plasmons, and demonstrate spatial coherence effects in near-field thermal emission.

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

A Clinical Wide-Field Fluorescence Endoscopic Device for Molecular Imaging Demonstrating Cathepsin Protease Activity in Colon Cancer

PubMed Central

Sensarn, Steven; Zavaleta, Cristina L.; Segal, Ehud; Rogalla, Stephan; Lee, Wansik; Gambhir, Sanjiv S.; Bogyo, Matthew; Contag, Christopher H.

2017-01-01

Purpose Early and effective detection of cancers of the gastrointestinal tract will require novel molecular probes and advances in instrumentation that can reveal functional changes in dysplastic and malignant tissues. Here, we describe adaptation of a wide-field clinical fiberscope to perform wide-field fluorescence imaging while preserving its white-light capability for the purpose of providing wide-field fluorescence imaging capability to point-of-care microscopes. Procedures We developed and used a fluorescent fiberscope to detect signals from a quenched probe, BMV109, that becomes fluorescent when cleaved by, and covalently bound to, active cathepsin proteases. Cathepsins are expressed in inflammation- and tumor-associated macrophages as well as directly from tumor cells and are a promising target for cancer imaging. The fiberscope has a 1-mm outer diameter enabling validation via endoscopic exams in mice, and therefore we evaluated topically applied BMV109 for the ability to detect colon polyps in an azoxymethane-induced colon tumor model in mice. Results This wide-field endoscopic imaging device revealed consistent and clear fluorescence signals from BMV109 that specifically localized to the polypoid regions as opposed to the normal adjacent colon tissue (p < 0.004) in the murine colon carcinoma model. Conclusions The sensitivity of detection of BMV109 with the fluorescence fiberscope suggested utility of these tools for early detection at hard-to-reach sites. The fiberscope was designed to be used in conjunction with miniature, endoscope-compatible fluorescence microscopes for dual wide-field and microscopic cancer detection. PMID:27154508

Demonstration of a plenoptic microscope based on laser optical feedback imaging.

PubMed

Glastre, Wilfried; Hugon, Olivier; Jacquin, Olivier; Guillet de Chatellus, Hugues; Lacot, Eric

2013-03-25

A new kind of plenoptic imaging system based on Laser Optical Feedback Imaging (LOFI) is presented and is compared to another previously existing device based on microlens array. Improved photometric performances, resolution and depth of field are obtained at the price of a slow point by point scanning. Main properties of plenoptic microscopes such as numerical refocusing on any curved surface or aberrations compensation are both theoretically and experimentally demonstrated with a LOFI-based device.

Simple and versatile modifications allowing time gated spectral acquisition, imaging and lifetime profiling on conventional wide-field microscopes

NASA Astrophysics Data System (ADS)

Pal, Robert; Beeby, Andrew

2014-09-01

An inverted microscope has been adapted to allow time-gated imaging and spectroscopy to be carried out on samples containing responsive lanthanide probes. The adaptation employs readily available components, including a pulsed light source, time-gated camera, spectrometer and photon counting detector, allowing imaging, emission spectroscopy and lifetime measurements. Each component is controlled by a suite of software written in LabVIEW and is powered via conventional USB ports.

Soft X-ray microscope with nanometer spatial resolution and its applications

NASA Astrophysics Data System (ADS)

Wachulak, P. W.; Torrisi, A.; Bartnik, A.; Wegrzynski, L.; Fok, T.; Patron, Z.; Fiedorowicz, H.

2016-12-01

A compact size microscope based on nitrogen double stream gas puff target soft X-ray source, which emits radiation in water-window spectral range at the wavelength of λ = 2.88 nm is presented. The microscope employs ellipsoidal grazing incidence condenser mirror for sample illumination and silicon nitride Fresnel zone plate objective for object magnification and imaging. The microscope is capable of capturing water-window images of objects with 60 nm spatial resolution and exposure time as low as a few seconds. Details about the microscopy system as well as some examples of different applications from various fields of science, are presented and discussed.

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.

Performance of bent-crystal x-ray microscopes for high energy density physics research

DOE PAGES

Schollmeier, Marius S.; Geissel, Matthias; Shores, Jonathon E.; ...

2015-05-29

We present calculations for the field of view (FOV), image fluence, image monochromaticity, spectral acceptance, and image aberrations for spherical crystal microscopes, which are used as self-emission imaging or backlighter systems at large-scale high energy density physics facilities. Our analytic results are benchmarked with ray-tracing calculations as well as with experimental measurements from the 6.151 keV backlighter system at Sandia National Laboratories. Furthermore, the analytic expressions can be used for x-ray source positions anywhere between the Rowland circle and object plane. We discovered that this enables quick optimization of the performance of proposed but untested, bent-crystal microscope systems to findmore » the best compromise between FOV, image fluence, and spatial resolution for a particular application.« less

A multiphoton laser scanning microscope setup for transcranial in vivo brain imaging on mice

NASA Astrophysics Data System (ADS)

Nase, Gabriele; Helm, P. Johannes; Reppen, Trond; Ottersen, Ole Petter

2005-12-01

We describe a multiphoton laser scanning microscope setup for transcranial in vivo brain imaging in mice. The modular system is based on a modified industrial standard Confocal Scanning Laser Microscope (CSLM) and is assembled mainly from commercially available components. A special multifunctional stage, which is optimized for both laser scanning microscopic observation and preparative animal surgery, has been developed and built. The detection unit includes a highly efficient photomultiplier tube installed in a Peltier-cooled thermal box shielding the detector from changes in room temperature and from distortions caused by external electromagnetic fields. The images are recorded using a 12-bit analog-to-digital converter. Depending on the characteristics of the staining, individual nerve cells can be imaged down to at least 100μm below the intact cranium and down to at least 200μm below the opened cranium.

Investigation of skin structures based on infrared wave parameter indirect microscopic imaging

NASA Astrophysics Data System (ADS)

Zhao, Jun; Liu, Xuefeng; Xiong, Jichuan; Zhou, Lijuan

2017-02-01

Detailed imaging and analysis of skin structures are becoming increasingly important in modern healthcare and clinic diagnosis. Nanometer resolution imaging techniques such as SEM and AFM can cause harmful damage to the sample and cannot measure the whole skin structure from the very surface through epidermis, dermis to subcutaneous. Conventional optical microscopy has the highest imaging efficiency, flexibility in onsite applications and lowest cost in manufacturing and usage, but its image resolution is too low to be accepted for biomedical analysis. Infrared parameter indirect microscopic imaging (PIMI) uses an infrared laser as the light source due to its high transmission in skins. The polarization of optical wave through the skin sample was modulated while the variation of the optical field was observed at the imaging plane. The intensity variation curve of each pixel was fitted to extract the near field polarization parameters to form indirect images. During the through-skin light modulation and image retrieving process, the curve fitting removes the blurring scattering from neighboring pixels and keeps only the field variations related to local skin structures. By using the infrared PIMI, we can break the diffraction limit, bring the wide field optical image resolution to sub-200nm, in the meantime of taking advantage of high transmission of infrared waves in skin structures.

A pragmatic guide to multiphoton microscope design

PubMed Central

Young, Michael D.; Field, Jeffrey J.; Sheetz, Kraig E.; Bartels, Randy A.; Squier, Jeff

2016-01-01

Multiphoton microscopy has emerged as a ubiquitous tool for studying microscopic structure and function across a broad range of disciplines. As such, the intent of this paper is to present a comprehensive resource for the construction and performance evaluation of a multiphoton microscope that will be understandable to the broad range of scientific fields that presently exploit, or wish to begin exploiting, this powerful technology. With this in mind, we have developed a guide to aid in the design of a multiphoton microscope. We discuss source selection, optical management of dispersion, image-relay systems with scan optics, objective-lens selection, single-element light-collection theory, photon-counting detection, image rendering, and finally, an illustrated guide for building an example microscope. PMID:27182429

Adaptive pixel-super-resolved lensfree in-line digital holography for wide-field on-chip microscopy.

PubMed

Zhang, Jialin; Sun, Jiasong; Chen, Qian; Li, Jiaji; Zuo, Chao

2017-09-18

High-resolution wide field-of-view (FOV) microscopic imaging plays an essential role in various fields of biomedicine, engineering, and physical sciences. As an alternative to conventional lens-based scanning techniques, lensfree holography provides a new way to effectively bypass the intrinsical trade-off between the spatial resolution and FOV of conventional microscopes. Unfortunately, due to the limited sensor pixel-size, unpredictable disturbance during image acquisition, and sub-optimum solution to the phase retrieval problem, typical lensfree microscopes only produce compromised imaging quality in terms of lateral resolution and signal-to-noise ratio (SNR). Here, we propose an adaptive pixel-super-resolved lensfree imaging (APLI) method which can solve, or at least partially alleviate these limitations. Our approach addresses the pixel aliasing problem by Z-scanning only, without resorting to subpixel shifting or beam-angle manipulation. Automatic positional error correction algorithm and adaptive relaxation strategy are introduced to enhance the robustness and SNR of reconstruction significantly. Based on APLI, we perform full-FOV reconstruction of a USAF resolution target (~29.85 mm 2 ) and achieve half-pitch lateral resolution of 770 nm, surpassing 2.17 times of the theoretical Nyquist-Shannon sampling resolution limit imposed by the sensor pixel-size (1.67µm). Full-FOV imaging result of a typical dicot root is also provided to demonstrate its promising potential applications in biologic imaging.

Automated high resolution full-field spatial coherence tomography for quantitative phase imaging of human red blood cells

NASA Astrophysics Data System (ADS)

Singla, Neeru; Dubey, Kavita; Srivastava, Vishal; Ahmad, Azeem; Mehta, D. S.

2018-02-01

We developed an automated high-resolution full-field spatial coherence tomography (FF-SCT) microscope for quantitative phase imaging that is based on the spatial, rather than the temporal, coherence gating. The Red and Green color laser light was used for finding the quantitative phase images of unstained human red blood cells (RBCs). This study uses morphological parameters of unstained RBCs phase images to distinguish between normal and infected cells. We recorded the single interferogram by a FF-SCT microscope for red and green color wavelength and average the two phase images to further reduced the noise artifacts. In order to characterize anemia infected from normal cells different morphological features were extracted and these features were used to train machine learning ensemble model to classify RBCs with high accuracy.

Using Cell-ID 1.4 with R for Microscope-Based Cytometry

PubMed Central

Bush, Alan; Chernomoretz, Ariel; Yu, Richard; Gordon, Andrew

2012-01-01

This unit describes a method for quantifying various cellular features (e.g., volume, total and subcellular fluorescence localization) from sets of microscope images of individual cells. It includes procedures for tracking cells over time. One purposefully defocused transmission image (sometimes referred to as bright-field or BF) is acquired to segment the image and locate each cell. Fluorescent images (one for each of the color channels to be analyzed) are then acquired by conventional wide-field epifluorescence or confocal microscopy. This method uses the image processing capabilities of Cell-ID (Gordon et al., 2007, as updated here) and data analysis by the statistical programming framework R (R-Development-Team, 2008), which we have supplemented with a package of routines for analyzing Cell-ID output. Both Cell-ID and the analysis package are open-source. PMID:23026908

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.

Distortion Correction for a Brewster Angle Microscope Using an Optical Grating.

PubMed

Sun, Zhe; Zheng, Desheng; Baldelli, Steven

2017-02-21

A distortion-corrected Brewster angle microscope (DC-BAM) is designed, constructed, and tested based on the combination of an optical grating and a relay lens. Avoiding the drawbacks of most conventional BAM instruments, this configuration corrects the image propagation direction and consequently provides an image in focus over the entire field of view without any beam scanning or imaging reconstruction. This new BAM can be applied to both liquid and solid subphases with good spatial resolution in static and dynamic studies.

Dark-field imaging based on post-processed electron backscatter diffraction patterns of bulk crystalline materials in a scanning electron microscope.

PubMed

Brodusch, Nicolas; Demers, Hendrix; Gauvin, Raynald

2015-01-01

Dark-field (DF) images were acquired in the scanning electron microscope with an offline procedure based on electron backscatter diffraction (EBSD) patterns (EBSPs). These EBSD-DF images were generated by selecting a particular reflection on the electron backscatter diffraction pattern and by reporting the intensity of one or several pixels around this point at each pixel of the EBSD-DF image. Unlike previous studies, the diffraction information of the sample is the basis of the final image contrast with a pixel scale resolution at the EBSP providing DF imaging in the scanning electron microscope. The offline facility of this technique permits the selection of any diffraction condition available in the diffraction pattern and displaying the corresponding image. The high number of diffraction-based images available allows a better monitoring of deformation structures compared to electron channeling contrast imaging (ECCI) which is generally limited to a few images of the same area. This technique was applied to steel and iron specimens and showed its high capability in describing more rigorously the deformation structures around micro-hardness indents. Due to the offline relation between the reference EBSP and the EBSD-DF images, this new technique will undoubtedly greatly improve our knowledge of deformation mechanism and help to improve our understanding of the ECCI contrast mechanisms. Copyright © 2014 Elsevier B.V. All rights reserved.

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.

CHRONIS: an animal chromosome image database.

PubMed

Toyabe, Shin-Ichi; Akazawa, Kouhei; Fukushi, Daisuke; Fukui, Kiichi; Ushiki, Tatsuo

2005-01-01

We have constructed a database system named CHRONIS (CHROmosome and Nano-Information System) to collect images of animal chromosomes and related nanotechnological information. CHRONIS enables rapid sharing of information on chromosome research among cell biologists and researchers in other fields via the Internet. CHRONIS is also intended to serve as a liaison tool for researchers who work in different centers. The image database contains more than 3,000 color microscopic images, including karyotypic images obtained from more than 1,000 species of animals. Researchers can browse the contents of the database using a usual World Wide Web interface in the following URL: http://chromosome.med.niigata-u.ac.jp/chronis/servlet/chronisservlet. The system enables users to input new images into the database, to locate images of interest by keyword searches, and to display the images with detailed information. CHRONIS has a wide range of applications, such as searching for appropriate probes for fluorescent in situ hybridization, comparing various kinds of microscopic images of a single species, and finding researchers working in the same field of interest.

Use of scanning near-field optical microscope with an aperture probe for detection of luminescent nanodiamonds

NASA Astrophysics Data System (ADS)

Shershulin, V. A.; Samoylenko, S. R.; Shenderova, O. A.; Konov, V. I.; Vlasov, I. I.

2017-02-01

The suitability of scanning near-field optical microscopy (SNOM) to image photoluminescent diamond nanoparticles with nanoscale resolution is demonstrated. Isolated diamond nanocrystals with an average size of 100 nm, containing negatively charged nitrogen-vacancy (NV-) centers, were chosen as tested material. The NV- luminescence was stimulated by continuous 532 nm laser light. Sizes of analyzed crystallites were monitored by an atomic force microscope. The lateral resolution of the order of 100 nm was reached in SNOM imaging of diamond nanoparticles using 150 nm square aperture of the probe.

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

Light field imaging and application analysis in THz

NASA Astrophysics Data System (ADS)

Zhang, Hongfei; Su, Bo; He, Jingsuo; Zhang, Cong; Wu, Yaxiong; Zhang, Shengbo; Zhang, Cunlin

2018-01-01

The light field includes the direction information and location information. Light field imaging can capture the whole light field by single exposure. The four-dimensional light field function model represented by two-plane parameter, which is proposed by Levoy, is adopted in the light field. Acquisition of light field is based on the microlens array, camera array and the mask. We calculate the dates of light-field to synthetize light field image. The processing techniques of light field data include technology of refocusing rendering, technology of synthetic aperture and technology of microscopic imaging. Introducing the technology of light field imaging into THz, the efficiency of 3D imaging is higher than that of conventional THz 3D imaging technology. The advantages compared with visible light field imaging include large depth of field, wide dynamic range and true three-dimensional. It has broad application prospects.

Spectro-refractometry of individual microscopic objects using swept-source quantitative phase imaging.

PubMed

Jung, Jae-Hwang; Jang, Jaeduck; Park, Yongkeun

2013-11-05

We present a novel spectroscopic quantitative phase imaging technique with a wavelength swept-source, referred to as swept-source diffraction phase microscopy (ssDPM), for quantifying the optical dispersion of microscopic individual samples. Employing the swept-source and the principle of common-path interferometry, ssDPM measures the multispectral full-field quantitative phase imaging and spectroscopic microrefractometry of transparent microscopic samples in the visible spectrum with a wavelength range of 450-750 nm and a spectral resolution of less than 8 nm. With unprecedented precision and sensitivity, we demonstrate the quantitative spectroscopic microrefractometry of individual polystyrene beads, 30% bovine serum albumin solution, and healthy human red blood cells.

New design of a cryostat-mounted scanning near-field optical microscope for single molecule spectroscopy

NASA Astrophysics Data System (ADS)

Durand, Yannig; Woehl, Jörg C.; Viellerobe, Bertrand; Göhde, Wolfgang; Orrit, Michel

1999-02-01

Due to the weakness of the fluorescence signal from a single fluorophore, a scanning near-field optical microscope for single molecule spectroscopy requires a very efficient setup for the collection and detection of emitted photons. We have developed a home-built microscope for operation in a l-He cryostat which uses a solid parabolic mirror in order to optimize the fluorescence collection efficiency. This microscope works with Al-coated, tapered optical fibers in illumination mode. The tip-sample separation is probed by an optical shear-force detection. First results demonstrate the capability of the microscope to image single molecules and achieve a topographical resolution of a few nanometers vertically and better than 50 nm laterally.

Chemical imaging of structured SAMs with a novel SFG microscope

NASA Astrophysics Data System (ADS)

Hoffmann, Dominik M. P.; Kuhnke, Klaus; Kern, Klaus

2002-11-01

We present a newly developed microscope for sum frequency generation (SFG) imaging of opaque and reflecting interfaces. The sample is viewed at an angle of 60° with respect to the surface normal in order to increase the collected SFG intensity. Our setup is designed to keep the whole field of view (FOV) in focus and to compensate for the distortion usually related to oblique imaging by means of a blazed grating. The separation of the SFG intensity and the reflected visible beam is accomplished by a suitable combination of spectral filters. The sum frequency microscope (SFM) is capable of in-situ chemically selective imaging by tuning the IR-beam to vibrational transitions of the respective molecules. The SFM is applied to imaging of structured self-assembled monolayers (SAM) of thiol molecules on a gold surface.

Confocal multispot microscope for fast and deep imaging in semicleared tissues

NASA Astrophysics Data System (ADS)

Adam, Marie-Pierre; Müllenbroich, Marie Caroline; Di Giovanna, Antonino Paolo; Alfieri, Domenico; Silvestri, Ludovico; Sacconi, Leonardo; Pavone, Francesco Saverio

2018-02-01

Although perfectly transparent specimens are imaged faster with light-sheet microscopy, less transparent samples are often imaged with two-photon microscopy leveraging its robustness to scattering; however, at the price of increased acquisition times. Clearing methods that are capable of rendering strongly scattering samples such as brain tissue perfectly transparent specimens are often complex, costly, and time intensive, even though for many applications a slightly lower level of tissue transparency is sufficient and easily achieved with simpler and faster methods. Here, we present a microscope type that has been geared toward the imaging of semicleared tissue by combining multispot two-photon excitation with rolling shutter wide-field detection to image deep and fast inside semicleared mouse brain. We present a theoretical and experimental evaluation of the point spread function and contrast as a function of shutter size. Finally, we demonstrate microscope performance in fixed brain slices by imaging dendritic spines up to 400-μm deep.

Identification of malaria infected red blood samples by digital holographic quantitative phase microscope

NASA Astrophysics Data System (ADS)

Patel, Nimit R.; Chhaniwal, Vani K.; Javidi, Bahram; Anand, Arun

2015-07-01

Development of devices for automatic identification of diseases is desired especially in developing countries. In the case of malaria, even today the gold standard is the inspection of chemically treated blood smears through a microscope. This requires a trained technician/microscopist to identify the cells in the field of view, with which the labeling chemicals gets attached. Bright field microscopes provide only low contrast 2D images of red blood cells and cell thickness distribution cannot be obtained. Quantitative phase contrast microscopes can provide both intensity and phase profiles of the cells under study. The phase information can be used to determine thickness profile of the cell. Since cell morphology is available, many parameters pertaining to the 3D shape of the cell can be computed. These parameters in turn could be used to decide about the state of health of the cell leading to disease diagnosis. Here the investigations done on digital holographic microscope, which provides quantitative phase images, for comparison of parameters obtained from the 3D shape profile of objects leading to identification of diseased samples is described.

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).

Capturing and displaying microscopic images used in medical diagnostics and forensic science using 4K video resolution - an application in higher education.

PubMed

Maier, Hans; de Heer, Gert; Ortac, Ajda; Kuijten, Jan

2015-11-01

To analyze, interpret and evaluate microscopic images, used in medical diagnostics and forensic science, video images for educational purposes were made with a very high resolution of 4096 × 2160 pixels (4K), which is four times as many pixels as High-Definition Video (1920 × 1080 pixels). The unprecedented high resolution makes it possible to see details that remain invisible to any other video format. The images of the specimens (blood cells, tissue sections, hair, fibre, etc.) are recorded using a 4K video camera which is attached to a light microscope. After processing, this resulted in very sharp and highly detailed images. This material was then used in education for classroom discussion. Spoken explanation by experts in the field of medical diagnostics and forensic science was also added to the high-resolution video images to make it suitable for self-study. © 2015 The Authors. Journal of Microscopy published by John Wiley & Sons Ltd on behalf of Royal Microscopical Society.

Wide-field and high-resolution optical imaging for early detection of oral neoplasia

NASA Astrophysics Data System (ADS)

Pierce, Mark C.; Schwarz, Richard A.; Rosbach, Kelsey; Roblyer, Darren; Muldoon, Tim; Williams, Michelle D.; El-Naggar, Adel K.; Gillenwater, Ann M.; Richards-Kortum, Rebecca

2010-02-01

Current procedures for oral cancer screening typically involve visual inspection of the entire tissue surface at risk under white light illumination. However, pre-cancerous lesions can be difficult to distinguish from many benign conditions when viewed under these conditions. We have developed wide-field (macroscopic) imaging system which additionally images in cross-polarized white light, narrowband reflectance, and fluorescence imaging modes to reduce specular glare, enhance vascular contrast, and detect disease-related alterations in tissue autofluorescence. We have also developed a portable system to enable high-resolution (microscopic) evaluation of cellular features within the oral mucosa in situ. This system is a wide-field epi-fluorescence microscope coupled to a 1 mm diameter, flexible fiber-optic imaging bundle. Proflavine solution was used to specifically label cell nuclei, enabling the characteristic differences in N/C ratio and nuclear distribution between normal, dysplastic, and cancerous oral mucosa to be quantified. This paper discusses the technical design and performance characteristics of these complementary imaging systems. We will also present data from ongoing clinical studies aimed at evaluating diagnostic performance of these systems for detection of oral neoplasia.

Field-portable lensfree tomographic microscope†

PubMed Central

Isikman, Serhan O.; Bishara, Waheb; Sikora, Uzair; Yaglidere, Oguzhan; Yeah, John; Ozcan, Aydogan

2011-01-01

We present a field-portable lensfree tomographic microscope, which can achieve sectional imaging of a large volume (~20 mm3) on a chip with an axial resolution of <7 μm. In this compact tomographic imaging platform (weighing only ~110 grams), 24 light-emitting diodes (LEDs) that are each butt-coupled to a fibre-optic waveguide are controlled through a cost-effective micro-processor to sequentially illuminate the sample from different angles to record lensfree holograms of the sample that is placed on the top of a digital sensor array. In order to generate pixel super-resolved (SR) lensfree holograms and hence digitally improve the achievable lateral resolution, multiple sub-pixel shifted holograms are recorded at each illumination angle by electromagnetically actuating the fibre-optic waveguides using compact coils and magnets. These SR projection holograms obtained over an angular range of ~50° are rapidly reconstructed to yield projection images of the sample, which can then be back-projected to compute tomograms of the objects on the sensor-chip. The performance of this compact and light-weight lensfree tomographic microscope is validated by imaging micro-beads of different dimensions as well as a Hymenolepis nana egg, which is an infectious parasitic flatworm. Achieving a decent three-dimensional spatial resolution, this field-portable on-chip optical tomographic microscope might provide a useful toolset for telemedicine and high-throughput imaging applications in resource-poor settings. PMID:21573311

Investigation of autofocus algorithms for brightfield microscopy of unstained cells

NASA Astrophysics Data System (ADS)

Wu, Shu Yu; Dugan, Nazim; Hennelly, Bryan M.

2014-05-01

In the past decade there has been significant interest in image processing for brightfield cell microscopy. Much of the previous research on image processing for microscopy has focused on fluorescence microscopy, including cell counting, cell tracking, cell segmentation and autofocusing. Fluorescence microscopy provides functional image information that involves the use of labels in the form of chemical stains or dyes. For some applications, where the biochemical integrity of the cell is required to remain unchanged so that sensitive chemical testing can later be applied, it is necessary to avoid staining. For this reason the challenge of processing images of unstained cells has become a topic of increasing attention. These cells are often effectively transparent and appear to have a homogenous intensity profile when they are in focus. Bright field microscopy is the most universally available and most widely used form of optical microscopy and for this reason we are interested in investigating image processing of unstained cells recorded using a standard bright field microscope. In this paper we investigate the application of a range of different autofocus metrics applied to unstained bladder cancer cell lines using a standard inverted bright field microscope with microscope objectives that have high magnification and numerical aperture. We present a number of conclusions on the optimum metrics and the manner in which they should be applied for this application.

High-contrast 3D image acquisition using HiLo microscopy with an electrically tunable lens

NASA Astrophysics Data System (ADS)

Philipp, Katrin; Smolarski, André; Fischer, Andreas; Koukourakis, Nektarios; Stürmer, Moritz; Wallrabe, Ulricke; Czarske, Jürgen

2016-04-01

We present a HiLo microscope with an electrically tunable lens for high-contrast three-dimensional image acquisition. HiLo microscopy combines wide field and speckled illumination images to create optically sectioned images. Additionally, the depth-of-field is not fixed, but can be adjusted between wide field and confocal-like axial resolution. We incorporate an electrically tunable lens in the HiLo microscope for axial scanning, to obtain three-dimensional data without the need of moving neither the sample nor the objective. The used adaptive lens consists of a transparent polydimethylsiloxane (PDMS) membrane into which an annular piezo bending actuator is embedded. A transparent fluid is filled between the membrane and the glass substrate. When actuated, the piezo generates a pressure in the lens which deflects the membrane and thus changes the refractive power. This technique enables a large tuning range of the refractive power between 1/f = (-24 . . . 25) 1/m. As the NA of the adaptive lens is only about 0.05, a fixed high-NA lens is included in the setup to provide high resolution. In this contribution, the scan properties and capabilities of the tunable lens in the HiLo microscope are analyzed. Eventually, exemplary measurements are presented and discussed.

Micrometer-scale magnetic imaging of geological samples using a quantum diamond microscope

NASA Astrophysics Data System (ADS)

Glenn, D. R.; Fu, R. R.; Kehayias, P.; Le Sage, D.; Lima, E. A.; Weiss, B. P.; Walsworth, R. L.

2017-08-01

Remanent magnetization in geological samples may record the past intensity and direction of planetary magnetic fields. Traditionally, this magnetization is analyzed through measurements of the net magnetic moment of bulk millimeter to centimeter sized samples. However, geological samples are often mineralogically and texturally heterogeneous at submillimeter scales, with only a fraction of the ferromagnetic grains carrying the remanent magnetization of interest. Therefore, characterizing this magnetization in such cases requires a technique capable of imaging magnetic fields at fine spatial scales and with high sensitivity. To address this challenge, we developed a new instrument, based on nitrogen-vacancy centers in diamond, which enables direct imaging of magnetic fields due to both remanent and induced magnetization, as well as optical imaging, of room-temperature geological samples with spatial resolution approaching the optical diffraction limit. We describe the operating principles of this device, which we call the quantum diamond microscope (QDM), and report its optimized image-area-normalized magnetic field sensitivity (20 µTṡµm/Hz1/2), spatial resolution (5 µm), and field of view (4 mm), as well as trade-offs between these parameters. We also perform an absolute magnetic field calibration for the device in different modes of operation, including three-axis (vector) and single-axis (projective) magnetic field imaging. Finally, we use the QDM to obtain magnetic images of several terrestrial and meteoritic rock samples, demonstrating its ability to resolve spatially distinct populations of ferromagnetic carriers.

Common-path digital holographic microscopy based on a beam displacer unit

NASA Astrophysics Data System (ADS)

Di, Jianglei; Zhang, Jiwei; Song, Yu; Wang, Kaiqiang; Wei, Kun; Zhao, Jianlin

2018-02-01

Digital holographic microscopy (DHM) has become a novel tool with advantages of full field, non-destructive, high-resolution and 3D imaging, which captures the quantitative amplitude and phase information of microscopic specimens. It's a well-established method for digital recording and numerical reconstructing the full complex field of wavefront of the samples with a diffraction-limited lateral resolution down to 0.3 μm depending on the numerical aperture of microscope objective. Meanwhile, its axial resolution through axial direction is less than 10 nm due to the interferometric nature in phase imaging. Compared with the typical optical configurations such as Mach-Zehnder interferometer and Michelson interferometer, the common-path DHM has the advantages of simple and compact configuration, high stability, and so on. Here, a simple, compact, and low-cost common-path DHM based on a beam displacer unit is proposed for quantitative phase imaging of biological cells. The beam displacer unit is completely compatible with commercial microscope and can be easily set up in the output port of the microscope as a compact independent device. This technique can be used to achieve the quantitative phase measurement of biological cells with an excellent temporal stability of 0.51 nm, which makes it having a good prospect in the fields of biological and medical science. Living mouse osteoblastic cells are quantitatively measured with the system to demonstrate its capability and applicability.

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.

Transmission environmental scanning electron microscope with scintillation gaseous detection device.

PubMed

Danilatos, Gerasimos; Kollia, Mary; Dracopoulos, Vassileios

2015-03-01

A transmission environmental scanning electron microscope with use of a scintillation gaseous detection device has been implemented. This corresponds to a transmission scanning electron microscope but with addition of a gaseous environment acting both as environmental and detection medium. A commercial type of low vacuum machine has been employed together with appropriate modifications to the detection configuration. This involves controlled screening of various emitted signals in conjunction with a scintillation gaseous detection device already provided with the machine for regular surface imaging. Dark field and bright field imaging has been obtained along with other detection conditions. With a progressive series of modifications and tests, the theory and practice of a novel type of microscopy is briefly shown now ushering further significant improvements and developments in electron microscopy as a whole. Copyright © 2014 Elsevier B.V. All rights reserved.

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

Optical properties of the magnetic monopole field applied to electron microscopy and spectroscopy

NASA Astrophysics Data System (ADS)

Kruit, P.; Lenc, M.

1992-11-01

An analytical treatment of the electron's motion in a magnetic monopole field results in useful expressions for both the lens action and the mirror action of the field. Using an appropriate definition of the magnetic moment of the electron, it is shown that there is an exact conservation of this parameter in the monopole field, implying that the motion is perfectly adiabatic. This property is important when the field is used for directing Auger electrons from a target to a detector; that is, when it is used as a parallelizer in a through-the-lens detection scheme. Regarding the monopole field as an electron lens, the image position and magnification are derived for an arbitrary object position. Expressions for both the axial aberrations (chromatic and spherical) and the image aberrations (coma, field curvature, astigmatism, distortion, and transverse chromatic) are derived for an arbitrary number of intermediate images between object and final image. The chromatic aberration turns out to be independent of the number of intermediate images and the spherical aberration decreases slightly with this number. This property is important when an electron beam must be focused to a small probe in a strong magnetic field. It is shown that if a certain combination of deflectors is used in conjunction with the monopole field, an ideal swinging objective lens is obtained: All image aberrations except field curvature disappear. Designs are presented in which the monopole field is used in the objective lenses of a transmission electron microscope and a scanning electron microscope.

RMB identification based on polarization parameters inversion imaging

NASA Astrophysics Data System (ADS)

Liu, Guoyan; Gao, Kun; Liu, Xuefeng; Ni, Guoqiang

2016-10-01

Social order is threatened by counterfeit money. Conventional anti-counterfeit technology is much too old to identify its authenticity or not. The intrinsic difference between genuine notes and counterfeit notes is its paper tissue. In this paper a new technology of detecting RMB is introduced, the polarization parameter indirect microscopic imaging technique. A conventional reflection microscopic system is used as the basic optical system, and inserting into it with polarization-modulation mechanics. The near-field structural characteristics can be delivered by optical wave and material coupling. According to coupling and conduction physics, calculate the changes of optical wave parameters, then get the curves of the intensity of the image. By analyzing near-field polarization parameters in nanoscale, finally calculate indirect polarization parameter imaging of the fiber of the paper tissue in order to identify its authenticity.

Smartphone based hand-held quantitative phase microscope using the transport of intensity equation method.

PubMed

Meng, Xin; Huang, Huachuan; Yan, Keding; Tian, Xiaolin; Yu, Wei; Cui, Haoyang; Kong, Yan; Xue, Liang; Liu, Cheng; Wang, Shouyu

2016-12-20

In order to realize high contrast imaging with portable devices for potential mobile healthcare, we demonstrate a hand-held smartphone based quantitative phase microscope using the transport of intensity equation method. With a cost-effective illumination source and compact microscope system, multi-focal images of samples can be captured by the smartphone's camera via manual focusing. Phase retrieval is performed using a self-developed Android application, which calculates sample phases from multi-plane intensities via solving the Poisson equation. We test the portable microscope using a random phase plate with known phases, and to further demonstrate its performance, a red blood cell smear, a Pap smear and monocot root and broad bean epidermis sections are also successfully imaged. Considering its advantages as an accurate, high-contrast, cost-effective and field-portable device, the smartphone based hand-held quantitative phase microscope is a promising tool which can be adopted in the future in remote healthcare and medical diagnosis.

Experiments on terahertz 3D scanning microscopic imaging

NASA Astrophysics Data System (ADS)

Zhou, Yi; Li, Qi

2016-10-01

Compared with the visible light and infrared, terahertz (THz) radiation can penetrate nonpolar and nonmetallic materials. There are many studies on the THz coaxial transmission confocal microscopy currently. But few researches on the THz dual-axis reflective confocal microscopy were reported. In this paper, we utilized a dual-axis reflective confocal scanning microscope working at 2.52 THz. In contrast with the THz coaxial transmission confocal microscope, the microscope adopted in this paper can attain higher axial resolution at the expense of reduced lateral resolution, revealing more satisfying 3D imaging capability. Objects such as Chinese characters "Zhong-Hua" written in paper with a pencil and a combined sheet metal which has three layers were scanned. The experimental results indicate that the system can extract two Chinese characters "Zhong," "Hua" or three layers of the combined sheet metal. It can be predicted that the microscope can be applied to biology, medicine and other fields in the future due to its favorable 3D imaging capability.

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.

A diamond-based scanning probe spin sensor operating at low temperature in ultra-high vacuum

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

Schaefer-Nolte, E.; Wrachtrup, J.; 3rd Institute of Physics and Research Center SCoPE, University Stuttgart, 70569 Stuttgart

2014-01-15

We present the design and performance of an ultra-high vacuum (UHV) low temperature scanning probe microscope employing the nitrogen-vacancy color center in diamond as an ultrasensitive magnetic field sensor. Using this center as an atomic-size scanning probe has enabled imaging of nanoscale magnetic fields and single spins under ambient conditions. In this article we describe an experimental setup to operate this sensor in a cryogenic UHV environment. This will extend the applicability to a variety of molecular systems due to the enhanced target spin lifetimes at low temperature and the controlled sample preparation under UHV conditions. The instrument combines amore » tuning-fork based atomic force microscope (AFM) with a high numeric aperture confocal microscope and the facilities for application of radio-frequency (RF) fields for spin manipulation. We verify a sample temperature of <50 K even for strong laser and RF excitation and demonstrate magnetic resonance imaging with a magnetic AFM tip.« less

A full-field transmission x-ray microscope for time-resolved imaging of magnetic nanostructures

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

Ewald, J.; Nisius, T.; Abbati, G.

Sub-nanosecond magnetization dynamics of small permalloy (Ni{sub 80}Fe{sub 20}) elements has been investigated with a new full-field transmission microscope at the soft X-ray beamline P04 of the high brilliance synchrotron radiation source PETRA III. The soft X-ray microscope generates a flat-top illumination field of 20 μm diameter using a grating condenser. A tilted nanostructured magnetic sample can be excited by a picosecond electric current pulse via a coplanar waveguide. The transmitted light of the sample plane is directly imaged by a micro zone plate with < 65 nm resolution onto a 2D gateable X-ray detector to select one particular bunch in themore » storage ring that probes the time evolution of the dynamic information successively via XMCD spectromicroscopy in a pump-probe scheme. In the experiments it was possible to generate a homogeneously magnetized state in patterned magnetic layers by a strong magnetic Oersted field pulse of 200 ps duration and directly observe the recovery to the initial flux-closure vortex patterns.« less

Miniature near-infrared dual-axes confocal microscope utilizing a two-dimensional microelectromechanical systems scanner

PubMed Central

Liu, Jonathan T. C.; Mandella, Michael J.; Ra, Hyejun; Wong, Larry K.; Solgaard, Olav; Kino, Gordon S.; Piyawattanametha, Wibool; Contag, Christopher H.; Wang, Thomas D.

2007-01-01

The first, to our knowledge, miniature dual-axes confocal microscope has been developed, with an outer diameter of 10 mm, for subsurface imaging of biological tissues with 5–7 μm resolution. Depth-resolved en face images are obtained at 30 frames per second, with a field of view of 800 × 100 μm, by employing a two-dimensional scanning microelectromechanical systems mirror. Reflectance and fluorescence images are obtained with a laser source at 785 nm, demonstrating the ability to perform real-time optical biopsy. PMID:17215937

Confocal fluorescence microscope with dual-axis architecture and biaxial postobjective scanning

PubMed Central

Wang, Thomas D.; Contag, Christopher H.; Mandella, Michael J.; Chan, Ning Y.; Kino, Gordon S.

2007-01-01

We present a novel confocal microscope that has dual-axis architecture and biaxial postobjective scanning for the collection of fluorescence images from biological specimens. This design uses two low-numerical-aperture lenses to achieve high axial resolution and long working distance, and the scanning mirror located distal to the lenses rotates along the orthogonal axes to produce arc-surface images over a large field of view (FOV). With fiber optic coupling, this microscope can potentially be scaled down to millimeter dimensions via microelectromechanical systems (MEMS) technology. We demonstrate a benchtop prototype with a spatial resolution ≤4.4 μm that collects fluorescence images with a high SNR and a good contrast ratio from specimens expressing GFP. Furthermore, the scanning mechanism produces only small differences in aberrations over the image FOV. These results demonstrate proof of concept of the dual-axis confocal architecture for in vivo molecular and cellular imaging. PMID:15250760

Cellular Level Brain Imaging in Behaving Mammals: An Engineering Approach

PubMed Central

Hamel, Elizabeth J.O.; Grewe, Benjamin F.; Parker, Jones G.; Schnitzer, Mark J.

2017-01-01

Fluorescence imaging offers expanding capabilities for recording neural dynamics in behaving mammals, including the means to monitor hundreds of cells targeted by genetic type or connectivity, track cells over weeks, densely sample neurons within local microcircuits, study cells too inactive to isolate in extracellular electrical recordings, and visualize activity in dendrites, axons, or dendritic spines. We discuss recent progress and future directions for imaging in behaving mammals from a systems engineering perspective, which seeks holistic consideration of fluorescent indicators, optical instrumentation, and computational analyses. Today, genetically encoded indicators of neural Ca2+ dynamics are widely used, and those of trans-membrane voltage are rapidly improving. Two complementary imaging paradigms involve conventional microscopes for studying head-restrained animals and head-mounted miniature microscopes for imaging in freely behaving animals. Overall, the field has attained sufficient sophistication that increased cooperation between those designing new indicators, light sources, microscopes, and computational analyses would greatly benefit future progress. PMID:25856491

Instant Grainification: Real-Time Grain-Size Analysis from Digital Images in the Field

NASA Astrophysics Data System (ADS)

Rubin, D. M.; Chezar, H.

2007-12-01

Over the past few years, digital cameras and underwater microscopes have been developed to collect in-situ images of sand-sized bed sediment, and software has been developed to measure grain size from those digital images (Chezar and Rubin, 2004; Rubin, 2004; Rubin et al., 2006). Until now, all image processing and grain- size analysis was done back in the office where images were uploaded from cameras and processed on desktop computers. Computer hardware has become small and rugged enough to process images in the field, which for the first time allows real-time grain-size analysis of sand-sized bed sediment. We present such a system consisting of weatherproof tablet computer, open source image-processing software (autocorrelation code of Rubin, 2004, running under Octave and Cygwin), and digital camera with macro lens. Chezar, H., and Rubin, D., 2004, Underwater microscope system: U.S. Patent and Trademark Office, patent number 6,680,795, January 20, 2004. Rubin, D.M., 2004, A simple autocorrelation algorithm for determining grain size from digital images of sediment: Journal of Sedimentary Research, v. 74, p. 160-165. Rubin, D.M., Chezar, H., Harney, J.N., Topping, D.J., Melis, T.S., and Sherwood, C.R., 2006, Underwater microscope for measuring spatial and temporal changes in bed-sediment grain size: USGS Open-File Report 2006-1360.

The impact of the condenser on cytogenetic image quality in digital microscope system.

PubMed

Ren, Liqiang; Li, Zheng; Li, Yuhua; Zheng, Bin; Li, Shibo; Chen, Xiaodong; Liu, Hong

2013-01-01

Optimizing operational parameters of the digital microscope system is an important technique to acquire high quality cytogenetic images and facilitate the process of karyotyping so that the efficiency and accuracy of diagnosis can be improved. This study investigated the impact of the condenser on cytogenetic image quality and system working performance using a prototype digital microscope image scanning system. Both theoretical analysis and experimental validations through objectively evaluating a resolution test chart and subjectively observing large numbers of specimen were conducted. The results show that the optimal image quality and large depth of field (DOF) are simultaneously obtained when the numerical aperture of condenser is set as 60%-70% of the corresponding objective. Under this condition, more analyzable chromosomes and diagnostic information are obtained. As a result, the system shows higher working stability and less restriction for the implementation of algorithms such as autofocusing especially when the system is designed to achieve high throughput continuous image scanning. Although the above quantitative results were obtained using a specific prototype system under the experimental conditions reported in this paper, the presented evaluation methodologies can provide valuable guidelines for optimizing operational parameters in cytogenetic imaging using the high throughput continuous scanning microscopes in clinical practice.

0.4 Microns Spatial Resolution with 1 GHz (lambda = 30 cm) Evanescent Microwave Probe

NASA Technical Reports Server (NTRS)

Tabib-Azar, M.; Su, D.-P.; Pohar, A.; LeClair, S. R.; Ponchak, George E.

1999-01-01

In this article we describe evanescent field imaging of material nonuniformities with a record resolution of 0.4 microns at 1 GHz (lambda(sub g)/750000), using a resonant stripline scanning microwave probe. A chemically etched tip is used as a point-like evanescent field emitter and a probe-sample distance modulation is employed to improve the signal-to-noise ratio. Images obtained by evanescent microwave probe, by optical microscope, and by scanning tunneling microscope are presented for comparison. Probe was calibrated to perform quantitative conductivity measurements. The principal factors affecting the ultimate resolution of evanescent microwave probe are also discussed.

A milliKelvin scanning Hall probe microscope for high resolution magnetic imaging

NASA Astrophysics Data System (ADS)

Khotkevych, V. V.; Bending, S. J.

2009-02-01

The design and performance of a novel scanning Hall probe microscope for milliKelvin magnetic imaging with submicron lateral resolution is presented. The microscope head is housed in the vacuum chamber of a commercial 3He-refrigerator and operates between room temperature and 300 mK in magnetic fields up to 10 T. Mapping of the local magnetic induction at the sample surface is performed by a micro-fabricated 2DEG Hall probe equipped with an integrated STM tip. The latter provides a reliable mechanism of surface tracking by sensing and controlling the tunnel currents. We discuss the results of tests of the system and illustrate its potential with images of suitable reference samples captured in different modes of operation.

SIL-STED microscopy technique enhancing super-resolution of fluorescence microscopy

NASA Astrophysics Data System (ADS)

Park, No-Cheol; Lim, Geon; Lee, Won-sup; Moon, Hyungbae; Choi, Guk-Jong; Park, Young-Pil

2017-08-01

We have characterized a new type STED microscope which combines a high numerical aperture (NA) optical head with a solid immersion lens (SIL), and we call it as SIL-STED microscope. The advantage of a SIL-STED microscope is that its high NA of the SIL makes it superior to a general STED microscope in lateral resolution, thus overcoming the optical diffraction limit at the macromolecular level and enabling advanced super-resolution imaging of cell surface or cell membrane structure and function Do. This study presents the first implementation of higher NA illumination in a STED microscope limiting the fluorescence lateral resolution to about 40 nm. The refractive index of the SIL which is made of material KTaO3 is about 2.23 and 2.20 at a wavelength of 633 nm and 780 nm which are used for excitation and depletion in STED imaging, respectively. Based on the vector diffraction theory, the electric field focused by the SILSTED microscope is numerically calculated so that the numerical results of the point dispersion function of the microscope and the expected resolution could be analyzed. For further investigation, fluorescence imaging of nano size fluorescent beads is fulfilled to show improved performance of the technique.

Learning a cost function for microscope image segmentation.

PubMed

Nilufar, Sharmin; Perkins, Theodore J

2014-01-01

Quantitative analysis of microscopy images is increasingly important in clinical researchers' efforts to unravel the cellular and molecular determinants of disease, and for pathological analysis of tissue samples. Yet, manual segmentation and measurement of cells or other features in images remains the norm in many fields. We report on a new system that aims for robust and accurate semi-automated analysis of microscope images. A user interactively outlines one or more examples of a target object in a training image. We then learn a cost function for detecting more objects of the same type, either in the same or different images. The cost function is incorporated into an active contour model, which can efficiently determine optimal boundaries by dynamic programming. We validate our approach and compare it to some standard alternatives on three different types of microscopic images: light microscopy of blood cells, light microscopy of muscle tissue sections, and electron microscopy cross-sections of axons and their myelin sheaths.

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

PubMed Central

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

2013-01-01

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

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

PubMed

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

2013-06-07

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

First Clinical Applications of a High-Definition Three-Dimensional Exoscope in Pediatric Neurosurgery

PubMed Central

Munoz-Bendix, Christopher; Beseoglu, Kerim; Steiger, Hans-Jakob; Ahmadi, Sebastian A

2018-01-01

The ideal visualization tools in microneurosurgery should provide magnification, illumination, wide fields of view, ergonomics, and unobstructed access to the surgical field. The operative microscope was the predominant innovation in modern neurosurgery. Recently, a high-definition three-dimensional (3D) exoscope was developed. We describe the first applications in pediatric neurosurgery. The VITOM 3D exoscope (Karl Storz GmbH, Tuttlingen, Germany) was used in pediatric microneurosurgical operations, along with an OPMI PENTERO operative microscope (Carl Zeiss AG, Jena, Germany). Experiences were retrospectively evaluated with five-level Likert items regarding ease of preparation, image definition, magnification, illumination, field of view, ergonomics, accessibility of the surgical field, and general user-friendliness. Three operations were performed: supratentorial open biopsy in the supine position, infratentorial brain tumor resection in the park bench position, and myelomeningocele closure in the prone position. While preparation and image definition were rated equal for microscope and exoscope, the microscope’s field of view, illumination, and user-friendliness were considered superior, while the advantages of the exoscope were seen in ergonomics and the accessibility of the surgical field. No complications attributed to visualization mode occurred. In our experience, the VITOM 3D exoscope is an innovative visualization tool with advantages over the microscope in ergonomics and the accessibility of the surgical field. However, improvements were deemed necessary with regard to field of view, illumination, and user-friendliness. While the debate of a “perfect” visualization modality is influenced by personal preference, this novel visualization device has the potential to become a valuable tool in the neurosurgeon’s armamentarium. PMID:29581920

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.

Hard X-ray full field microscopy and magnifying microtomography using compound refractive lenses

NASA Astrophysics Data System (ADS)

Schroer, Christian G.; Günzler, Til Florian; Benner, Boris; Kuhlmann, Marion; Tümmler, Johannes; Lengeler, Bruno; Rau, Christoph; Weitkamp, Timm; Snigirev, Anatoly; Snigireva, Irina

2001-07-01

For hard X-rays, parabolic compound refractive lenses (PCRLs) are genuine imaging devices like glass lenses for visible light. Based on these new lenses, a hard X-ray full field microscope has been constructed that is ideally suited to image the interior of opaque samples with a minimum of sample preparation. As a result of a large depth of field, CRL micrographs are sharp projection images of most samples. To obtain 3D information about a sample, tomographic techniques are combined with magnified imaging.

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.

Atomic imaging using secondary electrons in a scanning transmission electron microscope: experimental observations and possible mechanisms.

PubMed

Inada, H; Su, D; Egerton, R F; Konno, M; Wu, L; Ciston, J; Wall, J; Zhu, Y

2011-06-01

We report detailed investigation of high-resolution imaging using secondary electrons (SE) with a sub-nanometer probe in an aberration-corrected transmission electron microscope, Hitachi HD2700C. This instrument also allows us to acquire the corresponding annular dark-field (ADF) images both simultaneously and separately. We demonstrate that atomic SE imaging is achievable for a wide range of elements, from uranium to carbon. Using the ADF images as a reference, we studied the SE image intensity and contrast as functions of applied bias, atomic number, crystal tilt, and thickness to shed light on the origin of the unexpected ultrahigh resolution in SE imaging. We have also demonstrated that the SE signal is sensitive to the terminating species at a crystal surface. A possible mechanism for atomic-scale SE imaging is proposed. The ability to image both the surface and bulk of a sample at atomic-scale is unprecedented, and can have important applications in the field of electron microscopy and materials characterization. Copyright © 2010 Elsevier B.V. All rights reserved.

Visualizing individual microtubules by bright field microscopy

NASA Astrophysics Data System (ADS)

Gutiérrez-Medina, Braulio; Block, Steven M.

2010-11-01

Microtubules are slender (˜25 nm diameter), filamentous polymers involved in cellular structure and organization. Individual microtubules have been visualized via fluorescence imaging of dye-labeled tubulin subunits and by video-enhanced, differential interference-contrast microscopy of unlabeled polymers using sensitive CCD cameras. We demonstrate the imaging of unstained microtubules using a microscope with conventional bright field optics in conjunction with a webcam-type camera and a light-emitting diode illuminator. The light scattered by microtubules is image-processed to remove the background, reduce noise, and enhance contrast. The setup is based on a commercial microscope with a minimal set of inexpensive components, suitable for implementation in a student laboratory. We show how this approach can be used in a demonstration motility assay, tracking the gliding motions of microtubules driven by the motor protein kinesin.

Construction and characterization of the fringe field monochromator for a field emission gun

PubMed

Mook; Kruit

2000-04-01

Although some microscopes have shown stabilities sufficient to attain below 0.1 eV spectral resolution in high-resolution electron energy loss spectroscopy, the intrinsic energy width of the high brightness source (0.3-0.6 eV) has been limiting the resolution. To lower the energy width of the source to 50 meV without unnecessary loss of brightness, a monochromator has been designed consisting of a short (4 mm) fringe field Wien filter and a 150 nm energy selection slit (nanoslit) both to be incorporated in the gun area of the microscope. A prototype has been built and tested in an ultra-high-vacuum setup (10(-9) mbar). The monochromator, operating on a Schottky field emission gun, showed stable and reproducible operation. The nanoslits did not contaminate and the structure remained stable. By measuring the current through the slit structure a direct image of the beam in the monochromator could be attained and the monochromator could be aligned without the use of a microscope. Good dispersed imaging conditions were found indicating an ultimate resolution of 55 meV. A Mark II fringe field monochromator (FFM) was designed and constructed compatible with the cold tungsten field emitter of the VG scanning transmission microscope. The monochromator was incorporated in the gun area of the microscope at IBM T.J. Watson research center, New York. The monochromator was aligned on 100 kV and the energy distribution measured using the monochromator displayed a below 50 meV filtering capability. The retarding Wien filter spectrometer was used to show a 61 meV EELS system resolution. The FFM is shown to be a monochromator which can be aligned without the use of the electron microscope. This makes it directly applicable for scanning transmission microscopy and low-voltage scanning electron microscopy, where it can lower the resolution loss which is caused by chromatic blur of the spot.

An interchangeable scanning Hall probe/scanning SQUID microscope

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

Tang, Chiu-Chun; Lin, Hui-Ting; Wu, Sing-Lin

2014-08-15

We have constructed a scanning probe microscope for magnetic imaging, which can function as a scanning Hall probe microscope (SHPM) and as a scanning SQUID microscope (SSM). The scanning scheme, applicable to SHPM and SSM, consists of a mechanical positioning (sub) micron-XY stage and a flexible direct contact to the sample without a feedback control system for the Z-axis. With the interchangeable capability of operating two distinct scanning modes, our microscope can incorporate the advantageous functionalities of the SHPM and SSM with large scan range up to millimeter, high spatial resolution (⩽4 μm), and high field sensitivity in a widemore » range of temperature (4.2 K-300 K) and magnetic field (10{sup −7} T-1 T). To demonstrate the capabilities of the system, we present magnetic images scanned with SHPM and SSM, including a RbFeB magnet and a nickel grid pattern at room temperature, surface magnetic domain structures of a La{sub 2/3}Ca{sub 1/3}MnO{sub 3} thin film at 77 K, and superconducting vortices in a striped niobium film at 4.2 K.« less

Automatic measurement of images on astrometric plates

NASA Astrophysics Data System (ADS)

Ortiz Gil, A.; Lopez Garcia, A.; Martinez Gonzalez, J. M.; Yershov, V.

1994-04-01

We present some results on the process of automatic detection and measurement of objects in overlapped fields of astrometric plates. The main steps of our algorithm are the following: determination of the Scale and Tilt between charge coupled devices (CCD) and microscope coordinate systems and estimation of signal-to-noise ratio in each field;--image identification and improvement of its position and size;--image final centering;--image selection and storage. Several parameters allow the use of variable criteria for image identification, characterization and selection. Problems related with faint images and crowded fields will be approached by special techniques (morphological filters, histogram properties and fitting models).

Wide-field FTIR microscopy using mid-IR pulse shaping

PubMed Central

Serrano, Arnaldo L.; Ghosh, Ayanjeet; Ostrander, Joshua S.; Zanni, Martin T.

2015-01-01

We have developed a new table-top technique for collecting wide-field Fourier transform infrared (FTIR) microscopic images by combining a femtosecond pulse shaper with a mid-IR focal plane array. The pulse shaper scans the delay between a pulse pair extremely rapidly for high signal-to-noise, while also enabling phase control of the individual pulses to under-sample the interferograms and subtract background. Infrared absorption images were collected for a mixture of W(CO)6 or Mn2(CO)10 absorbed polystyrene beads, demonstrating that this technique can spatially resolve chemically distinct species. The images are sub-diffraction limited, as measured with a USAF test target patterned on CaF2 and verified with scalar wave simulations. We also find that refractive, rather than reflective, objectives are preferable for imaging with coherent radiation. We discuss this method with respect to conventional FTIR microscopes. PMID:26191843

Preparation of Murine Submandibular Salivary Gland for Upright Intravital Microscopy.

PubMed

Ficht, Xenia; Thelen, Flavian; Stolp, Bettina; Stein, Jens V

2018-05-07

The submandibular salivary gland (SMG) is one of the three major salivary glands, and is of interest for many different fields of biological research, including cell biology, oncology, dentistry, and immunology. The SMG is an exocrine gland comprised of secretory epithelial cells, myofibroblasts, endothelial cells, nerves, and extracellular matrix. Dynamic cellular processes in the rat and mouse SMG have previously been imaged, mostly using inverted multi-photon microscope systems. Here, we describe a straightforward protocol for the surgical preparation and stabilization of the murine SMG in anesthetized mice for in vivo imaging with upright multi-photon microscope systems. We present representative intravital image sets of endogenous and adoptively transferred fluorescent cells, including the labeling of blood vessels or salivary ducts and second harmonic generation to visualize fibrillar collagen. In sum, our protocol allows for surgical preparation of mouse salivary glands in upright microscopy systems, which are commonly used for intravital imaging in the field of immunology.

Microscopic validation of whole mouse micro-metastatic tumor imaging agents using cryo-imaging and sliding organ image registration.

PubMed

Liu, Yiqiao; Zhou, Bo; Qutaish, Mohammed; Wilson, David L

2016-01-01

We created a metastasis imaging, analysis platform consisting of software and multi-spectral cryo-imaging system suitable for evaluating emerging imaging agents targeting micro-metastatic tumor. We analyzed CREKA-Gd in MRI, followed by cryo-imaging which repeatedly sectioned and tiled microscope images of the tissue block face, providing anatomical bright field and molecular fluorescence, enabling 3D microscopic imaging of the entire mouse with single metastatic cell sensitivity. To register MRI volumes to the cryo bright field reference, we used our standard mutual information, non-rigid registration which proceeded: preprocess → affine → B-spline non-rigid 3D registration. In this report, we created two modified approaches: mask where we registered locally over a smaller rectangular solid, and sliding organ . Briefly, in sliding organ , we segmented the organ, registered the organ and body volumes separately and combined results. Though s liding organ required manual annotation, it provided the best result as a standard to measure other registration methods. Regularization parameters for standard and mask methods were optimized in a grid search. Evaluations consisted of DICE, and visual scoring of a checkerboard display. Standard had accuracy of 2 voxels in all regions except near the kidney, where there were 5 voxels sliding. After mask and sliding organ correction, kidneys sliding were within 2 voxels, and Dice overlap increased 4%-10% in mask compared to standard . Mask generated comparable results with sliding organ and allowed a semi-automatic process.

Microscopic validation of whole mouse micro-metastatic tumor imaging agents using cryo-imaging and sliding organ image registration

NASA Astrophysics Data System (ADS)

Liu, Yiqiao; Zhou, Bo; Qutaish, Mohammed; Wilson, David L.

2016-03-01

We created a metastasis imaging, analysis platform consisting of software and multi-spectral cryo-imaging system suitable for evaluating emerging imaging agents targeting micro-metastatic tumor. We analyzed CREKA-Gd in MRI, followed by cryo-imaging which repeatedly sectioned and tiled microscope images of the tissue block face, providing anatomical bright field and molecular fluorescence, enabling 3D microscopic imaging of the entire mouse with single metastatic cell sensitivity. To register MRI volumes to the cryo bright field reference, we used our standard mutual information, non-rigid registration which proceeded: preprocess --> affine --> B-spline non-rigid 3D registration. In this report, we created two modified approaches: mask where we registered locally over a smaller rectangular solid, and sliding organ. Briefly, in sliding organ, we segmented the organ, registered the organ and body volumes separately and combined results. Though sliding organ required manual annotation, it provided the best result as a standard to measure other registration methods. Regularization parameters for standard and mask methods were optimized in a grid search. Evaluations consisted of DICE, and visual scoring of a checkerboard display. Standard had accuracy of 2 voxels in all regions except near the kidney, where there were 5 voxels sliding. After mask and sliding organ correction, kidneys sliding were within 2 voxels, and Dice overlap increased 4%-10% in mask compared to standard. Mask generated comparable results with sliding organ and allowed a semi-automatic process.

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

Schollmeier, Marius S.; Geissel, Matthias; Shores, Jonathon E.

We present calculations for the field of view (FOV), image fluence, image monochromaticity, spectral acceptance, and image aberrations for spherical crystal microscopes, which are used as self-emission imaging or backlighter systems at large-scale high energy density physics facilities. Our analytic results are benchmarked with ray-tracing calculations as well as with experimental measurements from the 6.151 keV backlighter system at Sandia National Laboratories. Furthermore, the analytic expressions can be used for x-ray source positions anywhere between the Rowland circle and object plane. We discovered that this enables quick optimization of the performance of proposed but untested, bent-crystal microscope systems to findmore » the best compromise between FOV, image fluence, and spatial resolution for a particular application.« less

Thin laser light sheet microscope for microbial oceanography

NASA Astrophysics Data System (ADS)

Fuchs, Eran; Jaffe, Jules S.; Long, Richard A.; Azam, Farooq

2002-01-01

Despite a growing need, oceanographers are limited by existing technological constrains and are unable to observe aquatic microbes in their natural setting. In order to provide a simple and easy to implement solution for such studies, a new Thin Light Sheet Microscope (TLSM) has been developed. The TLSM utilizes a well-defined sheet of laser light, which has a narrow (23 micron) axial dimension over a 1 mm x 1 mm field of view. This light sheet is positioned precisely within the depth of field of the microscope’s objective lens. The technique thus utilizes conventional microscope optics but replaces the illumination system. The advantages of the TLSM are two-fold: First, it concentrates light only where excitation is needed, thus maximizing the efficiency of the illumination source. Secondly, the TLSM maximizes image sharpness while at the same time minimizing the level of background noise. Particles that are not located within the objective's depth of field are not illuminated and therefore do not contribute to an out-of-focus image. Images from a prototype system that used SYBR Green I fluorescence stain in order to localize single bacteria are reported. The bacteria were in a relatively large and undisturbed volume of 4ml, which contained natural seawater. The TLSM can be used for fresh water studies of bacteria with no modification. The microscope permits the observation of interactions at the microscale and has potential to yield insights into how microbes structure pelagic ecosystems.

Servo-controlled intravital microscope system

NASA Technical Reports Server (NTRS)

Mansour, M. N.; Wayland, H. J.; Chapman, C. P. (Inventor)

1975-01-01

A microscope system is described for viewing an area of a living body tissue that is rapidly moving, by maintaining the same area in the field-of-view and in focus. A focus sensing portion of the system includes two video cameras at which the viewed image is projected, one camera being slightly in front of the image plane and the other slightly behind it. A focus sensing circuit for each camera differentiates certain high frequency components of the video signal and then detects them and passes them through a low pass filter, to provide dc focus signal whose magnitudes represent the degree of focus. An error signal equal to the difference between the focus signals, drives a servo that moves the microscope objective so that an in-focus view is delivered to an image viewing/recording camera.

Quasi-microscope concept for planetary missions.

PubMed

Huck, F O; Arvidson, R E; Burcher, E E; Giat, O; Wall, S D

1977-09-01

Viking lander cameras have returned stereo and multispectral views of the Martian surface with a resolution that approaches 2 mm/lp in the near field. A two-orders-of-magnitude increase in resolution could be obtained for collected surface samples by augmenting these cameras with auxiliary optics that would neither impose special camera design requirements nor limit the cameras field of view of the terrain. Quasi-microscope images would provide valuable data on the physical and chemical characteristics of planetary regoliths.

Dual-axis confocal microscope for high-resolution in vivo imaging

PubMed Central

Wang, Thomas D.; Mandella, Michael J.; Contag, Christopher H.; Kino, Gordon S.

2007-01-01

We describe a novel confocal microscope that uses separate low-numerical-aperture objectives with the illumination and collection axes crossed at angle θ from the midline. This architecture collects images in scattering media with high transverse and axial resolution, long working distance, large field of view, and reduced noise from scattered light. We measured transverse and axial (FWHM) resolution of 1.3 and 2.1 μm, respectively, in free space, and confirm subcellular resolution in excised esophageal mucosa. The optics may be scaled to millimeter dimensions and fiber coupled for collection of high-resolution images in vivo. PMID:12659264

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-compact fiber-optic two-photon microscope for functional fluorescence imaging in vivo.

PubMed

Engelbrecht, Christoph J; Johnston, Richard S; Seibel, Eric J; Helmchen, Fritjof

2008-04-14

We present a small, lightweight two-photon fiberscope and demonstrate its suitability for functional imaging in the intact brain. Our device consists of a hollow-core photonic crystal fiber for efficient delivery of near-IR femtosecond laser pulses, a spiral fiber-scanner for resonant beam steering, and a gradient-index lens system for fluorescence excitation, dichroic beam splitting, and signal collection. Fluorescence light is remotely detected using a standard photomultiplier tube. All optical components have 1 mm dimensions and the microscope's headpiece weighs only 0.6 grams. The instrument achieves micrometer resolution at frame rates of typically 25 Hz with a field-of-view of up to 200 microns. We demonstrate functional imaging of calcium signals in Purkinje cell dendrites in the cerebellum of anesthetized rats. The microscope will be easily portable by a rat or mouse and thus should enable functional imaging in freely behaving animals.

Characterization of a subwavelength-scale 3D void structure using the FDTD-based confocal laser scanning microscopic image mapping technique.

PubMed

Choi, Kyongsik; Chon, James W; Gu, Min; Lee, Byoungho

2007-08-20

In this paper, a simple confocal laser scanning microscopic (CLSM) image mapping technique based on the finite-difference time domain (FDTD) calculation has been proposed and evaluated for characterization of a subwavelength-scale three-dimensional (3D) void structure fabricated inside polymer matrix. The FDTD simulation method adopts a focused Gaussian beam incident wave, Berenger's perfectly matched layer absorbing boundary condition, and the angular spectrum analysis method. Through the well matched simulation and experimental results of the xz-scanned 3D void structure, we first characterize the exact position and the topological shape factor of the subwavelength-scale void structure, which was fabricated by a tightly focused ultrashort pulse laser. The proposed CLSM image mapping technique based on the FDTD can be widely applied from the 3D near-field microscopic imaging, optical trapping, and evanescent wave phenomenon to the state-of-the-art bio- and nanophotonics.

Laboratory-size three-dimensional x-ray microscope with Wolter type I mirror optics and an electron-impact water window x-ray source

NASA Astrophysics Data System (ADS)

Ohsuka, Shinji; Ohba, Akira; Onoda, Shinobu; Nakamoto, Katsuhiro; Nakano, Tomoyasu; Miyoshi, Motosuke; Soda, Keita; Hamakubo, Takao

2014-09-01

We constructed a laboratory-size three-dimensional water window x-ray microscope that combines wide-field transmission x-ray microscopy with tomographic reconstruction techniques, and observed bio-medical samples to evaluate its applicability to life science research fields. It consists of a condenser and an objective grazing incidence Wolter type I mirror, an electron-impact type oxygen Kα x-ray source, and a back-illuminated CCD for x-ray imaging. A spatial resolution limit of around 1.0 line pairs per micrometer was obtained for two-dimensional transmission images, and 1-μm scale three-dimensional fine structures were resolved.

Laboratory-size three-dimensional x-ray microscope with Wolter type I mirror optics and an electron-impact water window x-ray source.

PubMed

Ohsuka, Shinji; Ohba, Akira; Onoda, Shinobu; Nakamoto, Katsuhiro; Nakano, Tomoyasu; Miyoshi, Motosuke; Soda, Keita; Hamakubo, Takao

2014-09-01

We constructed a laboratory-size three-dimensional water window x-ray microscope that combines wide-field transmission x-ray microscopy with tomographic reconstruction techniques, and observed bio-medical samples to evaluate its applicability to life science research fields. It consists of a condenser and an objective grazing incidence Wolter type I mirror, an electron-impact type oxygen Kα x-ray source, and a back-illuminated CCD for x-ray imaging. A spatial resolution limit of around 1.0 line pairs per micrometer was obtained for two-dimensional transmission images, and 1-μm scale three-dimensional fine structures were resolved.

Time-resolved wide-field optically sectioned fluorescence microscopy

NASA Astrophysics Data System (ADS)

Dupuis, Guillaume; Benabdallah, Nadia; Chopinaud, Aurélien; Mayet, Céline; Lévêque-Fort, Sandrine

2013-02-01

We present the implementation of a fast wide-field optical sectioning technique called HiLo microscopy on a fluorescence lifetime imaging microscope. HiLo microscopy is based on the fusion of two images, one with structured illumination and another with uniform illumination. Optically sectioned images are then digitally generated thanks to a fusion algorithm. HiLo images are comparable in quality with confocal images but they can be acquired faster over larger fields of view. We obtain 4D imaging by combining HiLo optical sectioning, time-gated detection, and z-displacement. We characterize the performances of this set-up in terms of 3D spatial resolution and time-resolved capabilities in both fixed- and live-cell imaging modes.

Multilevel Space-Time Aggregation for Bright Field Cell Microscopy Segmentation and Tracking

PubMed Central

Inglis, Tiffany; De Sterck, Hans; Sanders, Geoffrey; Djambazian, Haig; Sladek, Robert; Sundararajan, Saravanan; Hudson, Thomas J.

2010-01-01

A multilevel aggregation method is applied to the problem of segmenting live cell bright field microscope images. The method employed is a variant of the so-called “Segmentation by Weighted Aggregation” technique, which itself is based on Algebraic Multigrid methods. The variant of the method used is described in detail, and it is explained how it is tailored to the application at hand. In particular, a new scale-invariant “saliency measure” is proposed for deciding when aggregates of pixels constitute salient segments that should not be grouped further. It is shown how segmentation based on multilevel intensity similarity alone does not lead to satisfactory results for bright field cells. However, the addition of multilevel intensity variance (as a measure of texture) to the feature vector of each aggregate leads to correct cell segmentation. Preliminary results are presented for applying the multilevel aggregation algorithm in space time to temporal sequences of microscope images, with the goal of obtaining space-time segments (“object tunnels”) that track individual cells. The advantages and drawbacks of the space-time aggregation approach for segmentation and tracking of live cells in sequences of bright field microscope images are presented, along with a discussion on how this approach may be used in the future work as a building block in a complete and robust segmentation and tracking system. PMID:20467468

Fourier transform infrared spectroscopy microscopic imaging classification based on spatial-spectral features

NASA Astrophysics Data System (ADS)

Liu, Lian; Yang, Xiukun; Zhong, Mingliang; Liu, Yao; Jing, Xiaojun; Yang, Qin

2018-04-01

The discrete fractional Brownian incremental random (DFBIR) field is used to describe the irregular, random, and highly complex shapes of natural objects such as coastlines and biological tissues, for which traditional Euclidean geometry cannot be used. In this paper, an anisotropic variable window (AVW) directional operator based on the DFBIR field model is proposed for extracting spatial characteristics of Fourier transform infrared spectroscopy (FTIR) microscopic imaging. Probabilistic principal component analysis first extracts spectral features, and then the spatial features of the proposed AVW directional operator are combined with the former to construct a spatial-spectral structure, which increases feature-related information and helps a support vector machine classifier to obtain more efficient distribution-related information. Compared to Haralick’s grey-level co-occurrence matrix, Gabor filters, and local binary patterns (e.g. uniform LBPs, rotation-invariant LBPs, uniform rotation-invariant LBPs), experiments on three FTIR spectroscopy microscopic imaging datasets show that the proposed AVW directional operator is more advantageous in terms of classification accuracy, particularly for low-dimensional spaces of spatial characteristics.

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

PubMed

Lee, Seung Ah; Yang, Changhuei

2014-08-21

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

A smartphone-based chip-scale microscope using ambient illumination

PubMed Central

Lee, Seung Ah; Yang, Changhuei

2014-01-01

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

Computational-optical microscopy for 3D biological imaging beyond the diffraction limit

NASA Astrophysics Data System (ADS)

Grover, Ginni

In recent years, super-resolution imaging has become an important fluorescent microscopy tool. It has enabled imaging of structures smaller than the optical diffraction limit with resolution less than 50 nm. Extension to high-resolution volume imaging has been achieved by integration with various optical techniques. In this thesis, development of a fluorescent microscope to enable high resolution, extended depth, three dimensional (3D) imaging is discussed; which is achieved by integration of computational methods with optical systems. In the first part of the thesis, point spread function (PSF) engineering for volume imaging is discussed. A class of PSFs, referred to as double-helix (DH) PSFs, is generated. The PSFs exhibit two focused spots in the image plane which rotate about the optical axis, encoding depth in rotation of the image. These PSFs extend the depth-of-field up to a factor of ˜5. Precision performance of the DH-PSFs, based on an information theoretical analysis, is compared with other 3D methods with conclusion that the DH-PSFs provide the best precision and the longest depth-of-field. Out of various possible DH-PSFs, a suitable PSF is obtained for super-resolution microscopy. The DH-PSFs are implemented in imaging systems, such as a microscope, with a special phase modulation at the pupil plane. Surface-relief elements which are polarization-insensitive and ˜90% light efficient are developed for phase modulation. The photon-efficient DH-PSF microscopes thus developed are used, along with optimal position estimation algorithms, for tracking and super-resolution imaging in 3D. Imaging at depths-of-field of up to 2.5 microm is achieved without focus scanning. Microtubules were imaged with 3D resolution of (6, 9, 39) nm, which is in close agreement with the theoretical limit. A quantitative study of co-localization of two proteins in volume was conducted in live bacteria. In the last part of the thesis practical aspects of the DH-PSF microscope are discussed. A method to stabilize it, for extended periods of time, with 3-4 nm precision in 3D is developed. 3D Super-resolution is demonstrated without drift. A PSF correction algorithm is demonstrated to improve characteristics of the DH-PSF in an experiment, where it is implemented with a polarization-insensitive liquid crystal spatial light modulator.

Thimble microscope system

NASA Astrophysics Data System (ADS)

Kamal, Tahseen; Rubinstein, Jaden; Watkins, Rachel; Cen, Zijian; Kong, Gary; Lee, W. M.

2016-12-01

Wearable computing devices, e.g. Google Glass, Smart watch, embodies the new human design frontier, where technology interfaces seamlessly with human gestures. During examination of any subject in the field (clinic, surgery, agriculture, field survey, water collection), our sensory peripherals (touch and vision) often go hand-in-hand. The sensitivity and maneuverability of the human fingers are guided with tight distribution of biological nerve cells, which perform fine motor manipulation over a range of complex surfaces that is often out of sight. Our sight (or naked vision), on the other hand, is generally restricted to line of sight that is ill-suited to view around corner. Hence, conventional imaging methods are often resort to complex light guide designs (periscope, endoscopes etc) to navigate over obstructed surfaces. Using modular design strategies, we constructed a prototype miniature microscope system that is incorporated onto a wearable fixture (thimble). This unique platform allows users to maneuver around a sample and take high resolution microscopic images. In this paper, we provide an exposition of methods to achieve a thimble microscopy; microscope lens fabrication, thimble design, integration of miniature camera and liquid crystal display.

The Impact of the Condenser on Cytogenetic Image Quality in Digital Microscope System

PubMed Central

Ren, Liqiang; Li, Zheng; Li, Yuhua; Zheng, Bin; Li, Shibo; Chen, Xiaodong; Liu, Hong

2013-01-01

Background: Optimizing operational parameters of the digital microscope system is an important technique to acquire high quality cytogenetic images and facilitate the process of karyotyping so that the efficiency and accuracy of diagnosis can be improved. OBJECTIVE: This study investigated the impact of the condenser on cytogenetic image quality and system working performance using a prototype digital microscope image scanning system. Methods: Both theoretical analysis and experimental validations through objectively evaluating a resolution test chart and subjectively observing large numbers of specimen were conducted. Results: The results show that the optimal image quality and large depth of field (DOF) are simultaneously obtained when the numerical aperture of condenser is set as 60%–70% of the corresponding objective. Under this condition, more analyzable chromosomes and diagnostic information are obtained. As a result, the system shows higher working stability and less restriction for the implementation of algorithms such as autofocusing especially when the system is designed to achieve high throughput continuous image scanning. Conclusions: Although the above quantitative results were obtained using a specific prototype system under the experimental conditions reported in this paper, the presented evaluation methodologies can provide valuable guidelines for optimizing operational parameters in cytogenetic imaging using the high throughput continuous scanning microscopes in clinical practice. PMID:23676284

High-contrast 3D microscopic imaging of deep layers in a biological medium

NASA Astrophysics Data System (ADS)

Faridian, Ahmad; Pedrini, Giancarlo; Osten, Wolfgang

2014-03-01

Multilayer imaging of biological specimens is a demanding field of research, but scattering is one of the major obstacles in imaging the internal layers of a specimen. Although in many studies the biological object is assumed to be a weak scatterer, this condition is hardly satisfied for sub-millimeter sized organisms. The scattering medium is inhomogeneously distributed inside the specimen. Therefore, the scattering which occurs in the upper layers of a given internal layer of interest is different from the lower layers. That results in a different amount of collectable information for a specific point in the layer from each view. An opposed view dark-field digital holographic microscope (DHM) has been implemented in this work to collect the information concurrently from both views and increase the image quality. Implementing a DHM system gives the possibility to perform digital refocusing process and obtain multilayer images from each side without depth scanning of the object. The results have been presented and discussed here for a Drosophila embryo.

Correlative super-resolution fluorescence microscopy combined with optical coherence microscopy

NASA Astrophysics Data System (ADS)

Kim, Sungho; Kim, Gyeong Tae; Jang, Soohyun; Shim, Sang-Hee; Bae, Sung Chul

2015-03-01

Recent development of super-resolution fluorescence imaging technique such as stochastic optical reconstruction microscopy (STORM) and photoactived localization microscope (PALM) has brought us beyond the diffraction limits. It allows numerous opportunities in biology because vast amount of formerly obscured molecular structures, due to lack of spatial resolution, now can be directly observed. A drawback of fluorescence imaging, however, is that it lacks complete structural information. For this reason, we have developed a super-resolution multimodal imaging system based on STORM and full-field optical coherence microscopy (FF-OCM). FF-OCM is a type of interferometry systems based on a broadband light source and a bulk Michelson interferometer, which provides label-free and non-invasive visualization of biological samples. The integration between the two systems is simple because both systems use a wide-field illumination scheme and a conventional microscope. This combined imaging system gives us both functional information at a molecular level (~20nm) and structural information at the sub-cellular level (~1μm). For thick samples such as tissue slices, while FF-OCM is readily capable of imaging the 3D architecture, STORM suffer from aberrations and high background fluorescence that substantially degrade the resolution. In order to correct the aberrations in thick tissues, we employed an adaptive optics system in the detection path of the STORM microscope. We used our multimodal system to obtain images on brain tissue samples with structural and functional information.

Open-source image reconstruction of super-resolution structured illumination microscopy data in ImageJ

PubMed Central

Müller, Marcel; Mönkemöller, Viola; Hennig, Simon; Hübner, Wolfgang; Huser, Thomas

2016-01-01

Super-resolved structured illumination microscopy (SR-SIM) is an important tool for fluorescence microscopy. SR-SIM microscopes perform multiple image acquisitions with varying illumination patterns, and reconstruct them to a super-resolved image. In its most frequent, linear implementation, SR-SIM doubles the spatial resolution. The reconstruction is performed numerically on the acquired wide-field image data, and thus relies on a software implementation of specific SR-SIM image reconstruction algorithms. We present fairSIM, an easy-to-use plugin that provides SR-SIM reconstructions for a wide range of SR-SIM platforms directly within ImageJ. For research groups developing their own implementations of super-resolution structured illumination microscopy, fairSIM takes away the hurdle of generating yet another implementation of the reconstruction algorithm. For users of commercial microscopes, it offers an additional, in-depth analysis option for their data independent of specific operating systems. As a modular, open-source solution, fairSIM can easily be adapted, automated and extended as the field of SR-SIM progresses. PMID:26996201

Multimodal ophthalmic imaging using spectrally encoded scanning laser ophthalmoscopy and optical coherence tomography

NASA Astrophysics Data System (ADS)

El-Haddad, Mohamed T.; Malone, Joseph D.; Li, Jianwei D.; Bozic, Ivan; Arquitola, Amber M.; Joos, Karen M.; Patel, Shriji N.; Tao, Yuankai K.

2017-08-01

Ophthalmic surgery involves manipulation of delicate, layered tissue structures on milli- to micrometer scales. Traditional surgical microscopes provide an inherently two-dimensional view of the surgical field with limited depth perception which precludes accurate depth-resolved visualization of these tissue layers, and limits the development of novel surgical techniques. We demonstrate multimodal swept-source spectrally encoded scanning laser ophthalmoscopy and optical coherence tomography (SS-SESLO-OCT) to address current limitations of image-guided ophthalmic microsurgery. SS-SESLO-OCT provides inherently co-registered en face and cross-sectional field-of-views (FOVs) at a line rate of 400 kHz and >2 GPix/s throughput. We show in vivo imaging of the anterior segment and retinal fundus of a healthy volunteer, and preliminary results of multi-volumetric mosaicking for ultrawide-field retinal imaging with 90° FOV. Additionally, a scan-head was rapid-prototyped with a modular architecture which enabled integration of SS-SESLO-OCT with traditional surgical microscope and slit-lamp imaging optics. Ex vivo surgical maneuvers were simulated in cadaveric porcine eyes. The system throughput enabled volumetric acquisition at 10 volumes-per-second (vps) and allowed visualization of surgical dynamics in corneal sweeps, compressions, and dissections, and retinal sweeps, compressions, and elevations. SESLO en face images enabled simple real-time co-registration with the surgical microscope FOV, and OCT cross-sections provided depth-resolved visualization of instrument-tissue interactions. Finally, we demonstrate novel augmented-reality integration with the surgical view using segmentation overlays to aid surgical guidance. SS-SESLO-OCT may benefit clinical diagnostics by enabling aiming, registration, and mosaicking; and intraoperative imaging by allowing for real-time surgical feedback, instrument tracking, and overlays of computationally extracted biomarkers of disease.

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

Optical detection of Trypanosoma cruzi in blood samples for diagnosis purpose

NASA Astrophysics Data System (ADS)

Alanis, Elvio; Romero, Graciela; Alvarez, Liliana; Martinez, Carlos C.; Basombrio, Miguel A.

2004-10-01

An optical method for detection of Trypanosoma Cruzi (T. cruzi) parasites in blood samples of mice infected with Chagas disease is presented. The method is intended for use in human blood, for diagnosis purposes. A thin layer of blood infected by T. cruzi parasites, in small concentrations, is examined in an interferometric microscope in which the images of the vision field are taken by a CCD camera and temporarily stored in the memory of a host computer. The whole sample is scanned displacing the microscope plate by means of step motors driven by the computer. Several consecutive images of the same field are taken and digitally processed by means of image temporal differentiation in order to detect if a parasite is eventually present in the field. Each field of view is processed in the same fashion, until the full area of the sample is covered or until a parasite is detected, in which case an acoustical warning is activated and the corresponding image is displayed permitting the technician to corroborate the result visually. A discussion of the reliability of the method as well as a comparison with other well established techniques are presented.

Quantitative luminescence imaging system

DOEpatents

Erwin, David N.; Kiel, Johnathan L.; Batishko, Charles R.; Stahl, Kurt A.

1990-01-01

The QLIS images and quantifies low-level chemiluminescent reactions in an electromagnetic field. It is capable of real time nonperturbing measurement and simultaneous recording of many biochemical and chemical reactions such as luminescent immunoassays or enzyme assays. The system comprises image transfer optics, a low-light level digitizing camera with image intensifying microchannel plates, an image process or, and a control computer. The image transfer optics may be a fiber image guide with a bend, or a microscope, to take the light outside of the RF field. Output of the camera is transformed into a localized rate of cumulative digitalized data or enhanced video display or hard-copy images. The system may be used as a luminescent microdosimetry device for radiofrequency or microwave radiation, as a thermal dosimeter, or in the dosimetry of ultra-sound (sonoluminescence) or ionizing radiation. It provides a near-real-time system capable of measuring the extremely low light levels from luminescent reactions in electromagnetic fields in the areas of chemiluminescence assays and thermal microdosimetry, and is capable of near-real-time imaging of the sample to allow spatial distribution analysis of the reaction. It can be used to instrument three distinctly different irradiation configurations, comprising (1) RF waveguide irradiation of a small Petri-dish-shaped sample cell, (2) RF irradiation of samples in a microscope for the microscopie imaging and measurement, and (3) RF irradiation of small to human body-sized samples in an anechoic chamber.

Design and performance of an ultra-high vacuum spin-polarized scanning tunneling microscope operating at 30 mK and in a vector magnetic field

NASA Astrophysics Data System (ADS)

von Allwörden, Henning; Eich, Andreas; Knol, Elze J.; Hermenau, Jan; Sonntag, Andreas; Gerritsen, Jan W.; Wegner, Daniel; Khajetoorians, Alexander A.

2018-03-01

We describe the design and performance of a scanning tunneling microscope (STM) that operates at a base temperature of 30 mK in a vector magnetic field. The cryogenics is based on an ultra-high vacuum (UHV) top-loading wet dilution refrigerator that contains a vector magnet allowing for fields up to 9 T perpendicular and 4 T parallel to the sample. The STM is placed in a multi-chamber UHV system, which allows in situ preparation and exchange of samples and tips. The entire system rests on a 150-ton concrete block suspended by pneumatic isolators, which is housed in an acoustically isolated and electromagnetically shielded laboratory optimized for extremely low noise scanning probe measurements. We demonstrate the overall performance by illustrating atomic resolution and quasiparticle interference imaging and detail the vibrational noise of both the laboratory and microscope. We also determine the electron temperature via measurement of the superconducting gap of Re(0001) and illustrate magnetic field-dependent measurements of the spin excitations of individual Fe atoms on Pt(111). Finally, we demonstrate spin resolution by imaging the magnetic structure of the Fe double layer on W(110).

Design and performance of an ultra-high vacuum spin-polarized scanning tunneling microscope operating at 30 mK and in a vector magnetic field.

PubMed

von Allwörden, Henning; Eich, Andreas; Knol, Elze J; Hermenau, Jan; Sonntag, Andreas; Gerritsen, Jan W; Wegner, Daniel; Khajetoorians, Alexander A

2018-03-01

We describe the design and performance of a scanning tunneling microscope (STM) that operates at a base temperature of 30 mK in a vector magnetic field. The cryogenics is based on an ultra-high vacuum (UHV) top-loading wet dilution refrigerator that contains a vector magnet allowing for fields up to 9 T perpendicular and 4 T parallel to the sample. The STM is placed in a multi-chamber UHV system, which allows in situ preparation and exchange of samples and tips. The entire system rests on a 150-ton concrete block suspended by pneumatic isolators, which is housed in an acoustically isolated and electromagnetically shielded laboratory optimized for extremely low noise scanning probe measurements. We demonstrate the overall performance by illustrating atomic resolution and quasiparticle interference imaging and detail the vibrational noise of both the laboratory and microscope. We also determine the electron temperature via measurement of the superconducting gap of Re(0001) and illustrate magnetic field-dependent measurements of the spin excitations of individual Fe atoms on Pt(111). Finally, we demonstrate spin resolution by imaging the magnetic structure of the Fe double layer on W(110).

New solutions for standardization, monitoring and quality management of fluorescence-based imaging systems (Conference Presentation)

NASA Astrophysics Data System (ADS)

Royon, Arnaud; Papon, Gautier

2016-03-01

Fluorescence microscopes have become ubiquitous in life sciences laboratories, including those focused on pharmaceuticals, diagnosis, and forensics. For the past few years, the need for both performance guarantees and quantifiable results has driven development in this area. However, the lack of appropriate standards and reference materials makes it difficult or impossible to compare the results of two fluorescence microscopes, or to measure performance fluctuations of one microscope over time. Therefore, the operation of fluorescence microscopes is not monitored as often as their use warrants - an issue that is recognized by both systems manufacturers and national metrology institutes. We have developed a new process that enables the etching of long-term stable fluorescent patterns with sub-micrometer sizes in three dimensions inside glass. In this paper, we present, based on this new process, a fluorescent multi-dimensional ruler and a dedicated software that are suitable for monitoring and quality management of fluorescence-based imaging systems (wide-field, confocal, multiphoton, high content machines). In addition to fluorescence, the same patterns exhibit bright- and dark-field contrast, DIC, and phase contrast, which make them also relevant to monitor these types of microscopes. Non-exhaustively, this new solution enables the measurement of: The stage repositioning accuracy; The illumination and detection homogeneities; The field flatness; The detectors' characteristics; The lateral and axial spatial resolutions; The spectral response (spectrum, intensity and lifetime) of the system. Thanks to the stability of the patterns, microscope performance assessment can be carried out as well in a daily basis as in the long term.

The ALBA spectroscopic LEEM-PEEM experimental station: layout and performance

PubMed Central

Aballe, Lucia; Foerster, Michael; Pellegrin, Eric; Nicolas, Josep; Ferrer, Salvador

2015-01-01

The spectroscopic LEEM-PEEM experimental station at the CIRCE helical undulator beamline, which started user operation at the ALBA Synchrotron Light Facility in 2012, is presented. This station, based on an Elmitec LEEM III microscope with electron imaging energy analyzer, permits surfaces to be imaged with chemical, structural and magnetic sensitivity down to a lateral spatial resolution better than 20 nm with X-ray excited photoelectrons and 10 nm in LEEM and UV-PEEM modes. Rotation around the surface normal and application of electric and (weak) magnetic fields are possible in the microscope chamber. In situ surface preparation capabilities include ion sputtering, high-temperature flashing, exposure to gases, and metal evaporation with quick evaporator exchange. Results from experiments in a variety of fields and imaging modes will be presented in order to illustrate the ALBA XPEEM capabilities. PMID:25931092

High definition infrared chemical imaging of colorectal tissue using a Spero QCL microscope.

PubMed

Bird, B; Rowlette, J

2017-04-10

Mid-infrared microscopy has become a key technique in the field of biomedical science and spectroscopy. This label-free, non-destructive technique permits the visualisation of a wide range of intrinsic biochemical markers in tissues, cells and biofluids by detection of the vibrational modes of the constituent molecules. Together, infrared microscopy and chemometrics is a widely accepted method that can distinguish healthy and diseased states with high accuracy. However, despite the exponential growth of the field and its research world-wide, several barriers currently exist for its full translation into the clinical sphere, namely sample throughput and data management. The advent and incorporation of quantum cascade lasers (QCLs) into infrared microscopes could help propel the field over these remaining hurdles. Such systems offer several advantages over their FT-IR counterparts, a simpler instrument architecture, improved photon flux, use of room temperature camera systems, and the flexibility of a tunable illumination source. In this current study we explore the use of a QCL infrared microscope to produce high definition, high throughput chemical images useful for the screening of biopsied colorectal tissue.

Identification of mycobacterium tuberculosis in sputum smear slide using automatic scanning microscope

NASA Astrophysics Data System (ADS)

Rulaningtyas, Riries; Suksmono, Andriyan B.; Mengko, Tati L. R.; Saptawati, Putri

2015-04-01

Sputum smear observation has an important role in tuberculosis (TB) disease diagnosis, because it needs accurate identification to avoid high errors diagnosis. In development countries, sputum smear slide observation is commonly done with conventional light microscope from Ziehl-Neelsen stained tissue and it doesn't need high cost to maintain the microscope. The clinicians do manual screening process for sputum smear slide which is time consuming and needs highly training to detect the presence of TB bacilli (mycobacterium tuberculosis) accurately, especially for negative slide and slide with less number of TB bacilli. For helping the clinicians, we propose automatic scanning microscope with automatic identification of TB bacilli. The designed system modified the field movement of light microscope with stepper motor which was controlled by microcontroller. Every sputum smear field was captured by camera. After that some image processing techniques were done for the sputum smear images. The color threshold was used for background subtraction with hue canal in HSV color space. Sobel edge detection algorithm was used for TB bacilli image segmentation. We used feature extraction based on shape for bacilli analyzing and then neural network classified TB bacilli or not. The results indicated identification of TB bacilli that we have done worked well and detected TB bacilli accurately in sputum smear slide with normal staining, but not worked well in over staining and less staining tissue slide. However, overall the designed system can help the clinicians in sputum smear observation becomes more easily.

Puzzling Little Martian Spheres That Dont Taste Like Blueberries

NASA Image and Video Library

2012-09-14

Small spherical objects fill the field in this mosaic combining four images from the Microscopic Imager on NASA Mars Exploration Rover Opportunity at an outcrop called Kirkwood in the Cape York segment of the western rim of Endeavour Crater.

Review of current progress in nanometrology with the helium ion microscope

NASA Astrophysics Data System (ADS)

Postek, Michael T.; Vladár, András; Archie, Charles; Ming, Bin

2011-02-01

Scanning electron microscopy has been employed as an imaging and measurement tool for more than 50 years and it continues as a primary tool in many research and manufacturing facilities across the world. A new challenger to this work is the helium ion microscope (HIM). The HIM is a new imaging and metrology technology. Essentially, substitution of the electron source with a helium ion source yields a tool visually similar in function to the scanning electron microscope, but very different in the fundamental imaging and measurement process. The imaged and measured signal originates differently than in the scanning electron microscope and that fact and its single atom source diameter may be able to push the obtainable resolution lower, provide greater depth-of-field and ultimately improve the metrology. Successful imaging and metrology with this instrument entails understanding and modeling of new ion beam/specimen interaction physics. As a new methodology, HIM is beginning to show promise and the abundance of potentially advantageous applications for nanometrology has yet to be fully exploited. This paper discusses some of the progress made at NIST in collaboration with IBM to understand the science behind this new technology.

Sub-micrometer resolution proximity X-ray microscope with digital image registration

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

Chkhalo, N. I.; Salashchenko, N. N.; Sherbakov, A. V., E-mail: SherbakovAV@ipm.sci-nnov.ru

A compact laboratory proximity soft X-ray microscope providing submicrometer spatial resolution and digital image registration is described. The microscope consists of a laser-plasma soft X-ray radiation source, a Schwarzschild objective to illuminate the test sample, and a two-coordinate detector for image registration. Radiation, which passes through the sample under study, generates an absorption image on the front surface of the detector. Optical ceramic YAG:Ce was used to convert the X-rays into visible light. An image was transferred from the scintillator to a charge-coupled device camera with a Mitutoyo Plan Apo series lens. The detector’s design allows the use of lensesmore » with numerical apertures of NA = 0.14, 0.28, and 0.55 without changing the dimensions and arrangement of the elements of the device. This design allows one to change the magnification, spatial resolution, and field of view of the X-ray microscope. A spatial resolution better than 0.7 μm and an energy conversion efficiency of the X-ray radiation with a wavelength of 13.5 nm into visible light collected by the detector of 7.2% were achieved with the largest aperture lens.« less

Field and laboratory guide to freshwater cyanobacteria harmful algal blooms for Native American and Alaska Native communities

USGS Publications Warehouse

Rosen, Barry H.; St. Amand, Ann

2015-09-14

Cyanobacteria can produce toxins and form harmful algal blooms. The Native American and Alaska Native communities that are dependent on subsistence fishing have an increased risk of exposure to these cyanotoxins. It is important to recognize the presence of an algal bloom in a waterbody and to distinguish a potentially toxic harmful algal bloom from a non-toxic bloom. This guide provides field images that show cyanobacteria blooms, some of which can be toxin producers, as well as other non-toxic algae blooms and floating plants that might be confused with algae. After recognition of a potential toxin-producing cyanobacterial bloom in the field, the type(s) of cyanobacteria present needs to be identified. Species identification, which requires microscopic examination, may help distinguish a toxin-producer from a non-toxin producer. This guide also provides microscopic images of the common cyanobacteria that are known to produce toxins, as well as images of algae that form blooms but do not produce toxins.

Chip-based wide field-of-view nanoscopy

NASA Astrophysics Data System (ADS)

Diekmann, Robin; Helle, Øystein I.; Øie, Cristina I.; McCourt, Peter; Huser, Thomas R.; Schüttpelz, Mark; Ahluwalia, Balpreet S.

2017-04-01

Present optical nanoscopy techniques use a complex microscope for imaging and a simple glass slide to hold the sample. Here, we demonstrate the inverse: the use of a complex, but mass-producible optical chip, which hosts the sample and provides a waveguide for the illumination source, and a standard low-cost microscope to acquire super-resolved images via two different approaches. Waveguides composed of a material with high refractive-index contrast provide a strong evanescent field that is used for single-molecule switching and fluorescence excitation, thus enabling chip-based single-molecule localization microscopy. Additionally, multimode interference patterns induce spatial fluorescence intensity variations that enable fluctuation-based super-resolution imaging. As chip-based nanoscopy separates the illumination and detection light paths, total-internal-reflection fluorescence excitation is possible over a large field of view, with up to 0.5 mm × 0.5 mm being demonstrated. Using multicolour chip-based nanoscopy, we visualize fenestrations in liver sinusoidal endothelial cells.

Panoramic view of human corneal endothelial cell layer observed by a prototype slit-scanning wide-field contact specular microscope.

PubMed

Tanaka, Hiroshi; Okumura, Naoki; Koizumi, Noriko; Sotozono, Chie; Sumii, Yasuhiro; Kinoshita, Shigeru

2017-05-01

To observe the most peripheral region of the corneal endothelial cell (CEC) layer as long as optically recordable by use of a prototype slit-scanning wide-field contact specular microscope and produce a panoramic image to evaluate the variation of CEC density with ageing. Observational case series study. This study involved 15 eyes of 15 normal healthy subjects divided into three groups according to age: A (20-40 years), B (41-60 years) and C (>60 years). The corneal endothelial layer of each eye was recorded in a horizontal direction, from nasal to temporal, with a slit-scanning wide-field contact specular microscope (Konan) and endothelial cell density (ECD) in three specific regions (central, mid-peripheral, and peripheral) was automatically calculated via built-in analysis software. Corneal endothelial images from near the surgical limbus to limbus in all eyes were clearly recorded and panoramic images were made by combining still images. ECD in groups A, B and C were 2809±186, 2717±91 and 2580±129 cells/mm 2 at the centre, 2902±242, 2772±97 and 2604±187 cells/mm 2 at the mid-periphery and 2893±308, 2691±99 and 2533±112 cells/mm 2 at the periphery. Significance differences in ECD was found between groups A and C in all regions and groups between B and C at mid-peripheral region. A prototype slit-scanning wide-field contact specular microscope enabled us to record the endothelial layer from the surgical limbus to limbus of the cornea and compare specific areas among subjects, and showed that ECD in each region of the cornea decreases with ageing. UMIN000021264, Results. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

Cryo X-ray microscope with flat sample geometry for correlative fluorescence and nanoscale tomographic imaging.

PubMed

Schneider, Gerd; Guttmann, Peter; Rehbein, Stefan; Werner, Stephan; Follath, Rolf

2012-02-01

X-ray imaging offers a new 3-D view into cells. With its ability to penetrate whole hydrated cells it is ideally suited for pairing fluorescence light microscopy and nanoscale X-ray tomography. In this paper, we describe the X-ray optical set-up and the design of the cryo full-field transmission X-ray microscope (TXM) at the electron storage ring BESSY II. Compared to previous TXM set-ups with zone plate condenser monochromator, the new X-ray optical layout employs an undulator source, a spherical grating monochromator and an elliptically shaped glass capillary mirror as condenser. This set-up improves the spectral resolution by an order of magnitude. Furthermore, the partially coherent object illumination improves the contrast transfer of the microscope compared to incoherent conditions. With the new TXM, cells grown on flat support grids can be tilted perpendicular to the optical axis without any geometrical restrictions by the previously required pinhole for the zone plate monochromator close to the sample plane. We also developed an incorporated fluorescence light microscope which permits to record fluorescence, bright field and DIC images of cryogenic cells inside the TXM. For TXM tomography, imaging with multi-keV X-rays is a straightforward approach to increase the depth of focus. Under these conditions phase contrast imaging is necessary. For soft X-rays with shrinking depth of focus towards 10nm spatial resolution, thin optical sections through a thick specimen might be obtained by deconvolution X-ray microscopy. As alternative 3-D X-ray imaging techniques, the confocal cryo-STXM and the dual beam cryo-FIB/STXM with photoelectron detection are proposed. Copyright © 2012 Elsevier Inc. All rights reserved.

[Quantitative data analysis for live imaging of bone.

PubMed

Seno, Shigeto

Bone tissue is a hard tissue, it was difficult to observe the interior of the bone tissue alive. With the progress of microscopic technology and fluorescent probe technology in recent years, it becomes possible to observe various activities of various cells forming bone society. On the other hand, the quantitative increase in data and the diversification and complexity of the images makes it difficult to perform quantitative analysis by visual inspection. It has been expected to develop a methodology for processing microscopic images and data analysis. In this article, we introduce the research field of bioimage informatics which is the boundary area of biology and information science, and then outline the basic image processing technology for quantitative analysis of live imaging data of bone.

Mirrored pyramidal wells for simultaneous multiple vantage point microscopy.

PubMed

Seale, K T; Reiserer, R S; Markov, D A; Ges, I A; Wright, C; Janetopoulos, C; Wikswo, J P

2008-10-01

We report a novel method for obtaining simultaneous images from multiple vantage points of a microscopic specimen using size-matched microscopic mirrors created from anisotropically etched silicon. The resulting pyramidal wells enable bright-field and fluorescent side-view images, and when combined with z-sectioning, provide additional information for 3D reconstructions of the specimen. We have demonstrated the 3D localization and tracking over time of the centrosome of a live Dictyostelium discoideum. The simultaneous acquisition of images from multiple perspectives also provides a five-fold increase in the theoretical collection efficiency of emitted photons, a property which may be useful for low-light imaging modalities such as bioluminescence, or low abundance surface-marker labelling.

Performance of low-voltage STEM/TEM with delta corrector and cold field emission gun.

PubMed

Sasaki, Takeo; Sawada, Hidetaka; Hosokawa, Fumio; Kohno, Yuji; Tomita, Takeshi; Kaneyama, Toshikatsu; Kondo, Yukihito; Kimoto, Koji; Sato, Yuta; Suenaga, Kazu

2010-08-01

To reduce radiation damage caused by the electron beam and to obtain high-contrast images of specimens, we have developed a highly stabilized transmission electron microscope equipped with a cold field emission gun and spherical aberration correctors for image- and probe-forming systems, which operates at lower acceleration voltages than conventional transmission electron microscopes. A delta-type aberration corrector is designed to simultaneously compensate for third-order spherical aberration and fifth-order 6-fold astigmatism. Both were successfully compensated in both scanning transmission electron microscopy (STEM) and transmission electron microscopy (TEM) modes in the range 30-60 kV. The Fourier transforms of raw high-angle annular dark field (HAADF) images of a Si[110] sample revealed spots corresponding to lattice spacings of 111 and 96 pm at 30 and 60 kV, respectively, and those of raw TEM images of an amorphous Ge film with gold particles showed spots corresponding to spacings of 91 and 79 pm at 30 and 60 kV, respectively. Er@C(82)-doped single-walled carbon nanotubes, which are carbon-based samples, were successfully observed by HAADF-STEM imaging with an atomic-level resolution.

Ultra-fast bright field and fluorescence imaging of the dynamics of micrometer-sized objects

NASA Astrophysics Data System (ADS)

Chen, Xucai; Wang, Jianjun; Versluis, Michel; de Jong, Nico; Villanueva, Flordeliza S.

2013-06-01

High speed imaging has application in a wide area of industry and scientific research. In medical research, high speed imaging has the potential to reveal insight into mechanisms of action of various therapeutic interventions. Examples include ultrasound assisted thrombolysis, drug delivery, and gene therapy. Visual observation of the ultrasound, microbubble, and biological cell interaction may help the understanding of the dynamic behavior of microbubbles and may eventually lead to better design of such delivery systems. We present the development of a high speed bright field and fluorescence imaging system that incorporates external mechanical waves such as ultrasound. Through collaborative design and contract manufacturing, a high speed imaging system has been successfully developed at the University of Pittsburgh Medical Center. We named the system "UPMC Cam," to refer to the integrated imaging system that includes the multi-frame camera and its unique software control, the customized modular microscope, the customized laser delivery system, its auxiliary ultrasound generator, and the combined ultrasound and optical imaging chamber for in vitro and in vivo observations. This system is capable of imaging microscopic bright field and fluorescence movies at 25 × 106 frames per second for 128 frames, with a frame size of 920 × 616 pixels. Example images of microbubble under ultrasound are shown to demonstrate the potential application of the system.

Ultra-fast bright field and fluorescence imaging of the dynamics of micrometer-sized objects

PubMed Central

Chen, Xucai; Wang, Jianjun; Versluis, Michel; de Jong, Nico; Villanueva, Flordeliza S.

2013-01-01

High speed imaging has application in a wide area of industry and scientific research. In medical research, high speed imaging has the potential to reveal insight into mechanisms of action of various therapeutic interventions. Examples include ultrasound assisted thrombolysis, drug delivery, and gene therapy. Visual observation of the ultrasound, microbubble, and biological cell interaction may help the understanding of the dynamic behavior of microbubbles and may eventually lead to better design of such delivery systems. We present the development of a high speed bright field and fluorescence imaging system that incorporates external mechanical waves such as ultrasound. Through collaborative design and contract manufacturing, a high speed imaging system has been successfully developed at the University of Pittsburgh Medical Center. We named the system “UPMC Cam,” to refer to the integrated imaging system that includes the multi-frame camera and its unique software control, the customized modular microscope, the customized laser delivery system, its auxiliary ultrasound generator, and the combined ultrasound and optical imaging chamber for in vitro and in vivo observations. This system is capable of imaging microscopic bright field and fluorescence movies at 25 × 106 frames per second for 128 frames, with a frame size of 920 × 616 pixels. Example images of microbubble under ultrasound are shown to demonstrate the potential application of the system. PMID:23822346

Evaluation of methods for detection of fluorescence labeled subcellular objects in microscope images.

PubMed

Ruusuvuori, Pekka; Aijö, Tarmo; Chowdhury, Sharif; Garmendia-Torres, Cecilia; Selinummi, Jyrki; Birbaumer, Mirko; Dudley, Aimée M; Pelkmans, Lucas; Yli-Harja, Olli

2010-05-13

Several algorithms have been proposed for detecting fluorescently labeled subcellular objects in microscope images. Many of these algorithms have been designed for specific tasks and validated with limited image data. But despite the potential of using extensive comparisons between algorithms to provide useful information to guide method selection and thus more accurate results, relatively few studies have been performed. To better understand algorithm performance under different conditions, we have carried out a comparative study including eleven spot detection or segmentation algorithms from various application fields. We used microscope images from well plate experiments with a human osteosarcoma cell line and frames from image stacks of yeast cells in different focal planes. These experimentally derived images permit a comparison of method performance in realistic situations where the number of objects varies within image set. We also used simulated microscope images in order to compare the methods and validate them against a ground truth reference result. Our study finds major differences in the performance of different algorithms, in terms of both object counts and segmentation accuracies. These results suggest that the selection of detection algorithms for image based screens should be done carefully and take into account different conditions, such as the possibility of acquiring empty images or images with very few spots. Our inclusion of methods that have not been used before in this context broadens the set of available detection methods and compares them against the current state-of-the-art methods for subcellular particle detection.

The Athena Microscopic Imager on the Mars Exploration Rovers

NASA Astrophysics Data System (ADS)

Herkenhoff, K. E.; Squyres, S. W.; Bell, J. F.; Maki, J. N.; Schwochert, M. A.

2002-12-01

The Athena science payload on the Mars Exploration Rovers (MER) includes the Microscopic Imager (MI). The MI is a fixed-focus camera mounted on the end of the Instrument Deployment Device (IDD). The MI was designed to acquire images at a spatial resolution of 30 microns/pixel over a broad spectral range (400-700 nm). Technically speaking, the ''microscopic'' imager is not a microscope: it has a fixed magnification of 0.4, and is intended to produce images that simulate a geologist's view when using a common hand lens. The MI uses the same electronics design as the other MER cameras, but has optics that yield a field of view of 31 x 31 mm. The MI will acquire images using only solar or skylight illumination of the target surface. A contact sensor will be used to place the MI slightly closer to the target surface than its best focus distance (about 66 mm), allowing concave surfaces to be imaged in good focus. Because the MI has a relatively small depth of field (+/- 3 mm), a single MI image of a rough surface will contain both focused and unfocused areas. Coarse (~2 mm precision) focusing will be achieved by moving the IDD away from a target after the contact sensor is activated. Multiple images taken at various distances will be acquired to ensure good focus on all parts of rough surfaces. By combining a set of images acquired in this way, a completely focused image will be assembled. The MI optics will be protected from the martian environment by a dust cover. The dust cover includes a polycarbonate window that is tinted yellow to restrict the spectral bandpass to 500-700 nm and allow color information to be obtained by taking images with the dust cover open and closed. The MI will be used to image the same materials measured by other Athena instruments, as well as targets of opportunity (before rover traverses). The resulting images will be used to place other instrumental data in context and to aid in the petrologic interpretation of rocks and soils on Mars.

Angularly-selective transmission imaging in a scanning electron microscope.

PubMed

Holm, Jason; Keller, Robert R

2016-08-01

This work presents recent advances in transmission scanning electron microscopy (t-SEM) imaging control capabilities. A modular aperture system and a cantilever-style sample holder that enable comprehensive angular selectivity of forward-scattered electrons are described. When combined with a commercially available solid-state transmission detector having only basic bright-field and dark-field imaging capabilities, the advances described here enable numerous transmission imaging modes. Several examples are provided that demonstrate how contrast arising from diffraction to mass-thickness can be obtained. Unanticipated image contrast at some imaging conditions is also observed and addressed. Published by Elsevier B.V.

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

PubMed

Pandiyan, Vimal Prabhu; John, Renu

2016-01-20

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

3D imaging of optically cleared tissue using a simplified CLARITY method and on-chip microscopy

PubMed Central

Zhang, Yibo; Shin, Yoonjung; Sung, Kevin; Yang, Sam; Chen, Harrison; Wang, Hongda; Teng, Da; Rivenson, Yair; Kulkarni, Rajan P.; Ozcan, Aydogan

2017-01-01

High-throughput sectioning and optical imaging of tissue samples using traditional immunohistochemical techniques can be costly and inaccessible in resource-limited areas. We demonstrate three-dimensional (3D) imaging and phenotyping in optically transparent tissue using lens-free holographic on-chip microscopy as a low-cost, simple, and high-throughput alternative to conventional approaches. The tissue sample is passively cleared using a simplified CLARITY method and stained using 3,3′-diaminobenzidine to target cells of interest, enabling bright-field optical imaging and 3D sectioning of thick samples. The lens-free computational microscope uses pixel super-resolution and multi-height phase recovery algorithms to digitally refocus throughout the cleared tissue and obtain a 3D stack of complex-valued images of the sample, containing both phase and amplitude information. We optimized the tissue-clearing and imaging system by finding the optimal illumination wavelength, tissue thickness, sample preparation parameters, and the number of heights of the lens-free image acquisition and implemented a sparsity-based denoising algorithm to maximize the imaging volume and minimize the amount of the acquired data while also preserving the contrast-to-noise ratio of the reconstructed images. As a proof of concept, we achieved 3D imaging of neurons in a 200-μm-thick cleared mouse brain tissue over a wide field of view of 20.5 mm2. The lens-free microscope also achieved more than an order-of-magnitude reduction in raw data compared to a conventional scanning optical microscope imaging the same sample volume. Being low cost, simple, high-throughput, and data-efficient, we believe that this CLARITY-enabled computational tissue imaging technique could find numerous applications in biomedical diagnosis and research in low-resource settings. PMID:28819645

High-pressure freezing for scanning transmission electron tomography analysis of cellular organelles.

PubMed

Walther, Paul; Schmid, Eberhard; Höhn, Katharina

2013-01-01

Using an electron microscope's scanning transmission mode (STEM) for collection of tomographic datasets is advantageous compared to bright field transmission electron microscopic (TEM). For image formation, inelastic scattering does not cause chromatic aberration, since in STEM mode no image forming lenses are used after the beam has passed the sample, in contrast to regular TEM. Therefore, thicker samples can be imaged. It has been experimentally demonstrated that STEM is superior to TEM and energy filtered TEM for tomography of samples as thick as 1 μm. Even when using the best electron microscope, adequate sample preparation is the key for interpretable results. We adapted protocols for high-pressure freezing of cultivated cells from a physiological state. In this chapter, we describe optimized high-pressure freezing and freeze substitution protocols for STEM tomography in order to obtain high membrane contrast.

Development and Optical Testing of the Camera, Hand Lens, and Microscope Probe with Scannable Laser Spectroscopy (CHAMP-SLS)

NASA Technical Reports Server (NTRS)

Mungas, Greg S.; Gursel, Yekta; Sepulveda, Cesar A.; Anderson, Mark; La Baw, Clayton; Johnson, Kenneth R.; Deans, Matthew; Beegle, Luther; Boynton, John

2008-01-01

Conducting high resolution field microscopy with coupled laser spectroscopy that can be used to selectively analyze the surface chemistry of individual pixels in a scene is an enabling capability for next generation robotic and manned spaceflight missions, civil, and military applications. In the laboratory, we use a range of imaging and surface preparation tools that provide us with in-focus images, context imaging for identifying features that we want to investigate at high magnification, and surface-optical coupling that allows us to apply optical spectroscopic analysis techniques for analyzing surface chemistry particularly at high magnifications. The camera, hand lens, and microscope probe with scannable laser spectroscopy (CHAMP-SLS) is an imaging/spectroscopy instrument capable of imaging continuously from infinity down to high resolution microscopy (resolution of approx. 1 micron/pixel in a final camera format), the closer CHAMP-SLS is placed to a feature, the higher the resultant magnification. At hand lens to microscopic magnifications, the imaged scene can be selectively interrogated with point spectroscopic techniques such as Raman spectroscopy, microscopic Laser Induced Breakdown Spectroscopy (micro-LIBS), laser ablation mass-spectrometry, Fluorescence spectroscopy, and/or Reflectance spectroscopy. This paper summarizes the optical design, development, and testing of the CHAMP-SLS optics.

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.

Four-probe measurements with a three-probe scanning tunneling microscope.

PubMed

Salomons, Mark; Martins, Bruno V C; Zikovsky, Janik; Wolkow, Robert A

2014-04-01

We present an ultrahigh vacuum (UHV) three-probe scanning tunneling microscope in which each probe is capable of atomic resolution. A UHV JEOL scanning electron microscope aids in the placement of the probes on the sample. The machine also has a field ion microscope to clean, atomically image, and shape the probe tips. The machine uses bare conductive samples and tips with a homebuilt set of pliers for heating and loading. Automated feedback controlled tip-surface contacts allow for electrical stability and reproducibility while also greatly reducing tip and surface damage due to contact formation. The ability to register inter-tip position by imaging of a single surface feature by multiple tips is demonstrated. Four-probe material characterization is achieved by deploying two tips as fixed current probes and the third tip as a movable voltage probe.

AUTOMATED CELL SEGMENTATION WITH 3D FLUORESCENCE MICROSCOPY IMAGES.

PubMed

Kong, Jun; Wang, Fusheng; Teodoro, George; Liang, Yanhui; Zhu, Yangyang; Tucker-Burden, Carol; Brat, Daniel J

2015-04-01

A large number of cell-oriented cancer investigations require an effective and reliable cell segmentation method on three dimensional (3D) fluorescence microscopic images for quantitative analysis of cell biological properties. In this paper, we present a fully automated cell segmentation method that can detect cells from 3D fluorescence microscopic images. Enlightened by fluorescence imaging techniques, we regulated the image gradient field by gradient vector flow (GVF) with interpolated and smoothed data volume, and grouped voxels based on gradient modes identified by tracking GVF field. Adaptive thresholding was then applied to voxels associated with the same gradient mode where voxel intensities were enhanced by a multiscale cell filter. We applied the method to a large volume of 3D fluorescence imaging data of human brain tumor cells with (1) small cell false detection and missing rates for individual cells; and (2) trivial over and under segmentation incidences for clustered cells. Additionally, the concordance of cell morphometry structure between automated and manual segmentation was encouraging. These results suggest a promising 3D cell segmentation method applicable to cancer studies.

Design and performance of an ultra-high vacuum scanning tunneling microscope operating at dilution refrigerator temperatures and high magnetic fields.

PubMed

Misra, S; Zhou, B B; Drozdov, I K; Seo, J; Urban, L; Gyenis, A; Kingsley, S C J; Jones, H; Yazdani, A

2013-10-01

We describe the construction and performance of a scanning tunneling microscope capable of taking maps of the tunneling density of states with sub-atomic spatial resolution at dilution refrigerator temperatures and high (14 T) magnetic fields. The fully ultra-high vacuum system features visual access to a two-sample microscope stage at the end of a bottom-loading dilution refrigerator, which facilitates the transfer of in situ prepared tips and samples. The two-sample stage enables location of the best area of the sample under study and extends the experiment lifetime. The successful thermal anchoring of the microscope, described in detail, is confirmed through a base temperature reading of 20 mK, along with a measured electron temperature of 250 mK. Atomically resolved images, along with complementary vibration measurements, are presented to confirm the effectiveness of the vibration isolation scheme in this instrument. Finally, we demonstrate that the microscope is capable of the same level of performance as typical machines with more modest refrigeration by measuring spectroscopic maps at base temperature both at zero field and in an applied magnetic field.

Evaluation environment for digital and analog pathology: a platform for validation studies

PubMed Central

Gallas, Brandon D.; Gavrielides, Marios A.; Conway, Catherine M.; Ivansky, Adam; Keay, Tyler C.; Cheng, Wei-Chung; Hipp, Jason; Hewitt, Stephen M.

2014-01-01

Abstract. We present a platform for designing and executing studies that compare pathologists interpreting histopathology of whole slide images (WSIs) on a computer display to pathologists interpreting glass slides on an optical microscope. eeDAP is an evaluation environment for digital and analog pathology. The key element in eeDAP is the registration of the WSI to the glass slide. Registration is accomplished through computer control of the microscope stage and a camera mounted on the microscope that acquires real-time images of the microscope field of view (FOV). Registration allows for the evaluation of the same regions of interest (ROIs) in both domains. This can reduce or eliminate disagreements that arise from pathologists interpreting different areas and focuses on the comparison of image quality. We reduced the pathologist interpretation area from an entire glass slide (10 to 30  mm2) to small ROIs (<50  μm2). We also made possible the evaluation of individual cells. We summarize eeDAP’s software and hardware and provide calculations and corresponding images of the microscope FOV and the ROIs extracted from the WSIs. The eeDAP software can be downloaded from the Google code website (project: eeDAP) as a MATLAB source or as a precompiled stand-alone license-free application. PMID:26158076

Evaluation environment for digital and analog pathology: a platform for validation studies.

PubMed

Gallas, Brandon D; Gavrielides, Marios A; Conway, Catherine M; Ivansky, Adam; Keay, Tyler C; Cheng, Wei-Chung; Hipp, Jason; Hewitt, Stephen M

2014-10-01

We present a platform for designing and executing studies that compare pathologists interpreting histopathology of whole slide images (WSIs) on a computer display to pathologists interpreting glass slides on an optical microscope. eeDAP is an evaluation environment for digital and analog pathology. The key element in eeDAP is the registration of the WSI to the glass slide. Registration is accomplished through computer control of the microscope stage and a camera mounted on the microscope that acquires real-time images of the microscope field of view (FOV). Registration allows for the evaluation of the same regions of interest (ROIs) in both domains. This can reduce or eliminate disagreements that arise from pathologists interpreting different areas and focuses on the comparison of image quality. We reduced the pathologist interpretation area from an entire glass slide (10 to [Formula: see text]) to small ROIs ([Formula: see text]). We also made possible the evaluation of individual cells. We summarize eeDAP's software and hardware and provide calculations and corresponding images of the microscope FOV and the ROIs extracted from the WSIs. The eeDAP software can be downloaded from the Google code website (project: eeDAP) as a MATLAB source or as a precompiled stand-alone license-free application.

Scanning Electron Microscopy (SEM) Procedure for HE Powders on a Zeiss Sigma HD VP SEM

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

Zaka, F.

This method describes the characterization of inert and HE materials by the Zeiss Sigma HD VP field emission Scanning Electron Microscope (SEM). The SEM uses an accelerated electron beam to generate high-magnification images of explosives and other materials. It is fitted with five detectors (SE, Inlens, STEM, VPSE, HDBSD) to enable imaging of the sample via different secondary electron signatures, angles, and energies. In addition to imaging through electron detection, the microscope is also fitted with two Oxford Instrument Energy Dispersive Spectrometer (EDS) 80 mm detectors to generate elemental constituent spectra and two-dimensional maps of the material being scanned.

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

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

Rosch, R.; Boutin, J. Y.; Le Breton, J. P.

This article describes x-ray imaging with grazing-incidence microscopes, developed for the experimental program carried out on the Ligne d'Integration Laser (LIL) facility [J. P. Le Breton et al., Inertial Fusion Sciences and Applications 2001 (Elsevier, Paris, 2002), pp. 856-862] (24 kJ, UV--0.35 nm). The design includes a large target-to-microscope (400-700 mm) distance required by the x-ray ablation issues anticipated on the Laser MegaJoule facility [P. A. Holstein et al., Laser Part. Beams 17, 403 (1999)] (1.8 MJ) which is under construction. Two eight-image Kirkpatrick-Baez microscopes [P. Kirkpatrick and A. V. Baez J. Opt. Soc. Am. 38, 766 (1948)] with differentmore » spectral wavelength ranges and with a 400 mm source-to-mirror distance image the target on a custom-built framing camera (time resolution of {approx}80 ps). The soft x-ray version microscope is sensitive below 1 keV and its spatial resolution is better than 30 {mu}m over a 2-mm-diam region. The hard x-ray version microscope has a 10 {mu}m resolution over an 800-{mu}m-diam region and is sensitive in the 1-5 keV energy range. Two other x-ray microscopes based on an association of toroidal/spherical surfaces (T/S microscopes) produce an image on a streak camera with a spatial resolution better than 30 {mu}m over a 3 mm field of view in the direction of the camera slit. Both microscopes have been designed to have, respectively, a maximum sensitivity in the 0.1-1 and 1-5 keV energy range. We present the original design of these four microscopes and their test on a dc x-ray tube in the laboratory. The diagnostics were successfully used on LIL first experiments early in 2005. Results of soft x-ray imaging of a radiative jet during conical shaped laser interaction are shown.« less

A modular designed ultra-high-vacuum spin-polarized scanning tunneling microscope with controllable magnetic fields for investigating epitaxial thin films.

PubMed

Wang, Kangkang; Lin, Wenzhi; Chinchore, Abhijit V; Liu, Yinghao; Smith, Arthur R

2011-05-01

A room-temperature ultra-high-vacuum scanning tunneling microscope for in situ scanning freshly grown epitaxial films has been developed. The core unit of the microscope, which consists of critical components including scanner and approach motors, is modular designed. This enables easy adaptation of the same microscope units to new growth systems with different sample-transfer geometries. Furthermore the core unit is designed to be fully compatible with cryogenic temperatures and high magnetic field operations. A double-stage spring suspension system with eddy current damping has been implemented to achieve ≤5 pm z stability in a noisy environment and in the presence of an interconnected growth chamber. Both tips and samples can be quickly exchanged in situ; also a tunable external magnetic field can be introduced using a transferable permanent magnet shuttle. This allows spin-polarized tunneling with magnetically coated tips. The performance of this microscope is demonstrated by atomic-resolution imaging of surface reconstructions on wide band-gap GaN surfaces and spin-resolved experiments on antiferromagnetic Mn(3)N(2)(010) surfaces.

Wide-field high spatial frequency domain imaging of tissue microstructure

NASA Astrophysics Data System (ADS)

Lin, Weihao; Zeng, Bixin; Cao, Zili; Zhu, Danfeng; Xu, M.

2018-02-01

Wide-field tissue imaging is usually not capable of resolving tissue microstructure. We present High Spatial Frequency Domain Imaging (HSFDI) - a noncontact imaging modality that spatially maps the tissue microscopic scattering structures over a large field of view. Based on an analytical reflectance model of sub-diffusive light from forward-peaked highly scattering media, HSFDI quantifies the spatially-resolved parameters of the light scattering phase function from the reflectance of structured light modulated at high spatial frequencies. We have demonstrated with ex vivo cancerous tissue to validate the robustness of HSFDI in significant contrast and differentiation of the microstructutral parameters between different types and disease states of tissue.

Fluorescence microscopy.

PubMed

Sanderson, Michael J; Smith, Ian; Parker, Ian; Bootman, Martin D

2014-10-01

Fluorescence microscopy is a major tool with which to monitor cell physiology. Although the concepts of fluorescence and its optical separation using filters remain similar, microscope design varies with the aim of increasing image contrast and spatial resolution. The basics of wide-field microscopy are outlined to emphasize the selection, advantages, and correct use of laser scanning confocal microscopy, two-photon microscopy, scanning disk confocal microscopy, total internal reflection, and super-resolution microscopy. In addition, the principles of how these microscopes form images are reviewed to appreciate their capabilities, limitations, and constraints for operation. © 2014 Cold Spring Harbor Laboratory Press.

Laboratory-size three-dimensional water-window x-ray microscope with Wolter type I mirror optics

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

Ohsuka, Shinji; The Graduate School for the Creation of New Photonics Industries, 1955-1 Kurematsu-cho, Nishi-ku, Hamamatsu-City, 431-1202; Ohba, Akira

2016-01-28

We constructed a laboratory-size three-dimensional water-window x-ray microscope that combines wide-field transmission x-ray microscopy with tomographic reconstruction techniques. It consists of an electron-impact x-ray source emitting oxygen Kα x-rays, Wolter type I grazing incidence mirror optics, and a back-illuminated CCD for x-ray imaging. A spatial resolution limit better than 1.0 line pairs per micrometer was obtained for two-dimensional transmission images, and 1-μm-scale three-dimensional fine structures were resolved.

Fluorescence Microscopy

PubMed Central

Sanderson, Michael J.; Smith, Ian; Parker, Ian; Bootman, Martin D.

2016-01-01

Fluorescence microscopy is a major tool with which to monitor cell physiology. Although the concepts of fluorescence and its optical separation using filters remain similar, microscope design varies with the aim of increasing image contrast and spatial resolution. The basics of wide-field microscopy are outlined to emphasize the selection, advantages, and correct use of laser scanning confocal microscopy, two-photon microscopy, scanning disk confocal microscopy, total internal reflection, and super-resolution microscopy. In addition, the principles of how these microscopes form images are reviewed to appreciate their capabilities, limitations, and constraints for operation. PMID:25275114

SQCRAMscope imaging of transport in an iron-pnictide superconductor

NASA Astrophysics Data System (ADS)

Yang, Fan; Kollar, Alicia; Taylor, Stephen; Palmstrom, Johanna; Chu, Jiun-Haw; Fisher, Ian; Lev, Benjamin

2017-04-01

Microscopic imaging of local magnetic fields provides a window into the organizing principles of complex and technologically relevant condensed matter materials. However, a wide variety of intriguing strongly correlated and topologically nontrivial materials exhibit poorly understood phenomena outside the detection capability of state-of-the-art high-sensitivity, high-resolution scanning probe magnetometers. We have recently introduced a quantum-noise-limited scanning probe magnetometer that can operate from room-to-cryogenic temperatures with unprecedented DC-field sensitivity and micron-scale resolution. The Scanning Quantum Cryogenic Atom Microscope (SQCRAMscope) employs a magnetically levitated atomic Bose-Einstein condensate (BEC), thereby providing immunity to conductive and blackbody radiative heating. We will report on the first use of the SQCRAMscope for imaging a strongly correlated material. Specifically, we will present measurements of electron transport in iron-pnictide superconductors across the electron nematic phase transition at T = 135 K.

A "two-objective, one-area" procedure in absorption microphotometry and its application using an inverted microscope.

PubMed

Chaubal, K A

1988-08-01

A 'two-objective, one-area' method and related equations are suggested to measure absorbance of microscopic stained objects. In such work, the measuring field invariably includes an image of the object and some clear area surrounding the image. The total intensity in the two areas is measured photometrically, using two different objectives, and substituted in the equation for absorbance. The equation is independent of the term representing intensity from the clear area and hence the error in the measurement of absorbance is reduced. The limitations of the 'two-objective, one-area' method are discussed and its pragmatic operation described with an experimental setup involving an inverted microscope. The method permits measurement of intensity in a part of a stained cell while the rest of the cell remains in the field of view. The method is applied to measure absorbance in Giemsa stained ascites cells and Feulgen stained liver and Human Amnion cells.

Portable microscopy platform for the clinical and environmental monitoring

NASA Astrophysics Data System (ADS)

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

2016-04-01

Light microscopy can not only address various diagnosis needs such as aquatic parasites and bacteria such as E. coli in water, but also provide a method for the screening of red tide. Traditional microscope based on the smartphone created by adding lens couldn't keep the tradeoff between field-of-view(FOV) and the resolution. In this paper, we demonstrate a non-contact, light and cost-effective microscope platform, that can image highly dense samples with a spatial resolution of ~0.8um over a field-of-view(FOV) of >1mm2. After captured the direct images, we performed the pixel super-resolution algorithm to improve the image resolution and overcome the hardware interference. The system would be a good point-of-care diagnostic solution in resource limited settings. We validated the performance of the system by imaging resolution test targets, the squamous cell cancer(SqCC) and green algae that necessary to detect the squamous carcinoma and red tide

Coherence and diffraction limited resolution in microscopic OCT by a unified approach for the correction of dispersion and aberrations

NASA Astrophysics Data System (ADS)

Schulz-Hildebrandt, H.; Münter, Michael; Ahrens, M.; Spahr, H.; Hillmann, D.; König, P.; Hüttmann, G.

2018-03-01

Optical coherence tomography (OCT) images scattering tissues with 5 to 15 μm resolution. This is usually not sufficient for a distinction of cellular and subcellular structures. Increasing axial and lateral resolution and compensation of artifacts caused by dispersion and aberrations is required to achieve cellular and subcellular resolution. This includes defocus which limit the usable depth of field at high lateral resolution. OCT gives access the phase of the scattered light and hence correction of dispersion and aberrations is possible by numerical algorithms. Here we present a unified dispersion/aberration correction which is based on a polynomial parameterization of the phase error and an optimization of the image quality using Shannon's entropy. For validation, a supercontinuum light sources and a costume-made spectrometer with 400 nm bandwidth were combined with a high NA microscope objective in a setup for tissue and small animal imaging. Using this setup and computation corrections, volumetric imaging at 1.5 μm resolution is possible. Cellular and near cellular resolution is demonstrated in porcine cornea and the drosophila larva, when computational correction of dispersion and aberrations is used. Due to the excellent correction of the used microscope objective, defocus was the main contribution to the aberrations. In addition, higher aberrations caused by the sample itself were successfully corrected. Dispersion and aberrations are closely related artifacts in microscopic OCT imaging. Hence they can be corrected in the same way by optimization of the image quality. This way microscopic resolution is easily achieved in OCT imaging of static biological tissues.

Flexible non-diffractive vortex microscope for three-dimensional depth-enhanced super-localization of dielectric, metal and fluorescent nanoparticles

NASA Astrophysics Data System (ADS)

Bouchal, Petr; Bouchal, Zdeněk

2017-10-01

In the past decade, probe-based super-resolution using temporally resolved localization of emitters became a groundbreaking imaging strategy in fluorescence microscopy. Here we demonstrate a non-diffractive vortex microscope (NVM), enabling three-dimensional super-resolution fluorescence imaging and localization and tracking of metal and dielectric nanoparticles. The NVM benefits from vortex non-diffractive beams (NBs) creating a double-helix point spread function that rotates under defocusing while maintaining its size and shape unchanged. Using intrinsic properties of the NBs, the dark-field localization of weakly scattering objects is achieved in a large axial range exceeding the depth of field of the microscope objective up to 23 times. The NVM was developed using an upright microscope Nikon Eclipse E600 operating with a spiral lithographic mask optimized using Fisher information and built into an add-on imaging module or microscope objective. In evaluation of the axial localization accuracy the root mean square error below 18 nm and 280 nm was verified over depth ranges of 3.5 μm and 13.6 μm, respectively. Subwavelength gold and polystyrene beads were localized with isotropic precision below 10 nm in the axial range of 3.5 μm and the axial precision reduced to 30 nm in the extended range of 13.6 μm. In the fluorescence imaging, the localization with isotropic precision below 15 nm was demonstrated in the range of 2.5 μm, whereas in the range of 8.3 μm, the precision of 15 nm laterally and 30-50 nm axially was achieved. The tracking of nanoparticles undergoing Brownian motion was demonstrated in the volume of 14 × 10 × 16 μm3. Applicability of the NVM was tested by fluorescence imaging of LW13K2 cells and localization of cellular proteins.

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

Recent progress in understanding the imaging and metrology using the helium ion microscope

NASA Astrophysics Data System (ADS)

Postek, Michael T.; Vladar, Andras E.; Ming, Bin

2009-05-01

Nanotechnology is pushing imaging and measurement instrument technology to high levels of required performance. As this continues, new barriers confronting innovation in this field are encountered. Particle beam instrument resolution remains one of these barriers. A new tool for imaging and metrology for nanotechnology is the scanning Helium Ion Microscope (HIM). The HIM is a new approach to imaging and metrology for nanotechnology which may be able to push this barrier lower. As a new methodology, it is just beginning to show promise and the number of potentially advantageous applications for nanotechnology and nanometrology has yet to be fully exploited. This presentation will discuss some of the progress made at NIST in collaboration with the manufacturing community in understanding the imaging and metrology for this new technology.

Use of a night vision intensifier for direct visualization by eye of far-red and near-infrared fluorescence through an optical microscope.

PubMed

Siddiqi, M A; Kilduff, G M; Gearhart, J D

2003-11-01

We describe the design, construction and testing of a prototype device that allows the direct visualization by eye of far-red and near-infrared (NIR) fluorescence through an optical microscope. The device incorporates a gallium arsenide (GaAs) image intensifier, typically utilized in low-light or 'night vision' applications. The intensifier converts far-red and NIR light into electrons and then into green light, which is visible to the human eye. The prototype makes possible the direct, real-time viewing by eye of normally invisible far-red and NIR fluorescence from a wide variety of fluorophores, using the full field of view of the microscope to which it is applied. The high sensitivity of the image intensifier facilitates the viewing of a wide variety of photosensitive specimens, including live cells and embryos, at vastly reduced illumination levels in both fluorescence and bright-field microscopy. Modifications to the microscope are not required in order to use the prototype, which is fully compatible with all current fluorescence techniques. Refined versions of the prototype device will have broad research and clinical applications.

Scanning Transmission Electron Microscopy at High Resolution

PubMed Central

Wall, J.; Langmore, J.; Isaacson, M.; Crewe, A. V.

1974-01-01

We have shown that a scanning transmission electron microscope with a high brightness field emission source is capable of obtaining better than 3 Å resolution using 30 to 40 keV electrons. Elastic dark field images of single atoms of uranium and mercury are shown which demonstrate this fact as determined by a modified Rayleigh criterion. Point-to-point micrograph resolution between 2.5 and 3.0 Å is found in dark field images of micro-crystallites of uranium and thorium compounds. Furthermore, adequate contrast is available to observe single atoms as light as silver. Images PMID:4521050

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

PubMed Central

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

2012-01-01

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

Camera, handlens, and microscope optical system for imaging and coupled optical spectroscopy

NASA Technical Reports Server (NTRS)

Mungas, Greg S. (Inventor); Boynton, John (Inventor); Sepulveda, Cesar A. (Inventor); Nunes de Sepulveda, legal representative, Alicia (Inventor); Gursel, Yekta (Inventor)

2012-01-01

An optical system comprising two lens cells, each lens cell comprising multiple lens elements, to provide imaging over a very wide image distance and within a wide range of magnification by changing the distance between the two lens cells. An embodiment also provides scannable laser spectroscopic measurements within the field-of-view of the instrument.

Camera, handlens, and microscope optical system for imaging and coupled optical spectroscopy

NASA Technical Reports Server (NTRS)

Mungas, Greg S. (Inventor); Boynton, John (Inventor); Sepulveda, Cesar A. (Inventor); Nunes de Sepulveda, Alicia (Inventor); Gursel, Yekta (Inventor)

2011-01-01

An optical system comprising two lens cells, each lens cell comprising multiple lens elements, to provide imaging over a very wide image distance and within a wide range of magnification by changing the distance between the two lens cells. An embodiment also provides scannable laser spectroscopic measurements within the field-of-view of the instrument.

Mirrored pyramidal wells for simultaneous multiple vantage point microscopy

PubMed Central

Seale, K.T.; Reiserer, R.S.; Markov, D.A.; Ges, I.A.; Wright, C.; Janetopoulos, C.; Wikswo, J.P.

2013-01-01

Summary We report a novel method for obtaining simultaneous images from multiple vantage points of a microscopic specimen using size-matched microscopic mirrors created from anisotropically etched silicon. The resulting pyramidal wells enable bright-field and fluorescent side-view images, and when combined with z-sectioning, provide additional information for 3D reconstructions of the specimen. We have demonstrated the 3D localization and tracking over time of the centrosome of a live Dictyostelium discoideum. The simultaneous acquisition of images from multiple perspectives also provides a five-fold increase in the theoretical collection efficiency of emitted photons, a property which may be useful for low-light imaging modalities such as bioluminescence, or low abundance surface-marker labelling. PMID:19017196

Miniaturized fiber-coupled confocal fluorescence microscope with an electrowetting variable focus lens using no moving parts

PubMed Central

Ozbay, Baris N.; Losacco, Justin T.; Cormack, Robert; Weir, Richard; Bright, Victor M.; Gopinath, Juliet T.; Restrepo, Diego; Gibson, Emily A.

2015-01-01

We report a miniature, lightweight fiber-coupled confocal fluorescence microscope that incorporates an electrowetting variable focus lens to provide axial scanning for full three-dimensional (3D) imaging. Lateral scanning is accomplished by coupling our device to a laser-scanning confocal microscope through a coherent imaging fiber-bundle. The optical components of the device are combined in a custom 3D-printed adapter with an assembled weight of <2 g that can be mounted onto the head of a mouse. Confocal sectioning provides an axial resolution of ~12 µm and an axial scan range of ~80 µm. The lateral field-of-view is 300 µm, and the lateral resolution is 1.8 µm. We determined these parameters by imaging fixed sections of mouse neuronal tissue labeled with green fluorescent protein (GFP) and fluorescent bead samples in agarose gel. To demonstrate viability for imaging intact tissue, we resolved multiple optical sections of ex vivo mouse olfactory nerve fibers expressing yellow fluorescent protein (YFP). PMID:26030555

Dynamic nano-imaging of label-free living cells using electron beam excitation-assisted optical microscope

PubMed Central

Fukuta, Masahiro; Kanamori, Satoshi; Furukawa, Taichi; Nawa, Yasunori; Inami, Wataru; Lin, Sheng; Kawata, Yoshimasa; Terakawa, Susumu

2015-01-01

Optical microscopes are effective tools for cellular function analysis because biological cells can be observed non-destructively and non-invasively in the living state in either water or atmosphere condition. Label-free optical imaging technique such as phase-contrast microscopy has been analysed many cellular functions, and it is essential technology for bioscience field. However, the diffraction limit of light makes it is difficult to image nano-structures in a label-free living cell, for example the endoplasmic reticulum, the Golgi body and the localization of proteins. Here we demonstrate the dynamic imaging of a label-free cell with high spatial resolution by using an electron beam excitation-assisted optical (EXA) microscope. We observed the dynamic movement of the nucleus and nano-scale granules in living cells with better than 100 nm spatial resolution and a signal-to-noise ratio (SNR) around 10. Our results contribute to the development of cellular function analysis and open up new bioscience applications. PMID:26525841

Dynamic nano-imaging of label-free living cells using electron beam excitation-assisted optical microscope.

PubMed

Fukuta, Masahiro; Kanamori, Satoshi; Furukawa, Taichi; Nawa, Yasunori; Inami, Wataru; Lin, Sheng; Kawata, Yoshimasa; Terakawa, Susumu

2015-11-03

Optical microscopes are effective tools for cellular function analysis because biological cells can be observed non-destructively and non-invasively in the living state in either water or atmosphere condition. Label-free optical imaging technique such as phase-contrast microscopy has been analysed many cellular functions, and it is essential technology for bioscience field. However, the diffraction limit of light makes it is difficult to image nano-structures in a label-free living cell, for example the endoplasmic reticulum, the Golgi body and the localization of proteins. Here we demonstrate the dynamic imaging of a label-free cell with high spatial resolution by using an electron beam excitation-assisted optical (EXA) microscope. We observed the dynamic movement of the nucleus and nano-scale granules in living cells with better than 100 nm spatial resolution and a signal-to-noise ratio (SNR) around 10. Our results contribute to the development of cellular function analysis and open up new bioscience applications.

Dynamic nano-imaging of label-free living cells using electron beam excitation-assisted optical microscope

NASA Astrophysics Data System (ADS)

Fukuta, Masahiro; Kanamori, Satoshi; Furukawa, Taichi; Nawa, Yasunori; Inami, Wataru; Lin, Sheng; Kawata, Yoshimasa; Terakawa, Susumu

2015-11-01

Optical microscopes are effective tools for cellular function analysis because biological cells can be observed non-destructively and non-invasively in the living state in either water or atmosphere condition. Label-free optical imaging technique such as phase-contrast microscopy has been analysed many cellular functions, and it is essential technology for bioscience field. However, the diffraction limit of light makes it is difficult to image nano-structures in a label-free living cell, for example the endoplasmic reticulum, the Golgi body and the localization of proteins. Here we demonstrate the dynamic imaging of a label-free cell with high spatial resolution by using an electron beam excitation-assisted optical (EXA) microscope. We observed the dynamic movement of the nucleus and nano-scale granules in living cells with better than 100 nm spatial resolution and a signal-to-noise ratio (SNR) around 10. Our results contribute to the development of cellular function analysis and open up new bioscience applications.

Dynamic imaging with electron microscopy

ScienceCinema

Campbell, Geoffrey; McKeown, Joe; Santala, Melissa

2018-02-13

Livermore researchers have perfected an electron microscope to study fast-evolving material processes and chemical reactions. By applying engineering, microscopy, and laser expertise to the decades-old technology of electron microscopy, the dynamic transmission electron microscope (DTEM) team has developed a technique that can capture images of phenomena that are both very small and very fast. DTEM uses a precisely timed laser pulse to achieve a short but intense electron beam for imaging. When synchronized with a dynamic event in the microscope's field of view, DTEM allows scientists to record and measure material changes in action. A new movie-mode capability, which earned a 2013 R&D 100 Award from R&D Magazine, uses up to nine laser pulses to sequentially capture fast, irreversible, even one-of-a-kind material changes at the nanometer scale. DTEM projects are advancing basic and applied materials research, including such areas as nanostructure growth, phase transformations, and chemical reactions.

Epi-Fluorescence Microscopy

PubMed Central

Webb, Donna J.; Brown, Claire M.

2012-01-01

Epi-fluorescence microscopy is available in most life sciences research laboratories, and when optimized can be a central laboratory tool. In this chapter, the epi-fluorescence light path is introduced and the various components are discussed in detail. Recommendations are made for incident lamp light sources, excitation and emission filters, dichroic mirrors, objective lenses, and charge-coupled device (CCD) cameras in order to obtain the most sensitive epi-fluorescence microscope. The even illumination of metal-halide lamps combined with new “hard” coated filters and mirrors, a high resolution monochrome CCD camera, and a high NA objective lens are all recommended for high resolution and high sensitivity fluorescence imaging. Recommendations are also made for multicolor imaging with the use of monochrome cameras, motorized filter turrets, individual filter cubes, and corresponding dyes that are the best choice for sensitive, high resolution multicolor imaging. Images should be collected using Nyquist sampling and should be corrected for background intensity contributions and nonuniform illumination across the field of view. Photostable fluorescent probes and proteins that absorb a lot of light (i.e., high extinction co-efficients) and generate a lot of fluorescence signal (i.e., high quantum yields) are optimal. A neuronal immune-fluorescence labeling protocol is also presented. Finally, in order to maximize the utility of sensitive wide-field microscopes and generate the highest resolution images with high signal-to-noise, advice for combining wide-field epi-fluorescence imaging with restorative image deconvolution is presented. PMID:23026996

Optical diffraction tomography with fully and partially coherent illumination in high numerical aperture label-free microscopy [Invited].

PubMed

Soto, Juan M; Rodrigo, José A; Alieva, Tatiana

2018-01-01

Quantitative label-free imaging is an important tool for the study of living microorganisms that, during the last decade, has attracted wide attention from the optical community. Optical diffraction tomography (ODT) is probably the most relevant technique for quantitative label-free 3D imaging applied in wide-field microscopy in the visible range. The ODT is usually performed using spatially coherent light illumination and specially designed holographic microscopes. Nevertheless, the ODT is also compatible with partially coherent illumination and can be realized in conventional wide-field microscopes by applying refocusing techniques, as it has been recently demonstrated. Here, we compare these two ODT modalities, underlining their pros and cons and discussing the optical setups for their implementation. In particular, we pay special attention to a system that is compatible with a conventional wide-field microscope that can be used for both ODT modalities. It consists of two easily attachable modules: the first for sample illumination engineering based on digital light processing technology; the other for focus scanning by using an electrically driven tunable lens. This hardware allows for a programmable selection of the wavelength and the illumination design, and provides fast data acquisition as well. Its performance is experimentally demonstrated in the case of ODT with partially coherent illumination providing speckle-free 3D quantitative imaging.

Four-probe measurements with a three-probe scanning tunneling microscope

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

Salomons, Mark; Martins, Bruno V. C.; Zikovsky, Janik

2014-04-15

We present an ultrahigh vacuum (UHV) three-probe scanning tunneling microscope in which each probe is capable of atomic resolution. A UHV JEOL scanning electron microscope aids in the placement of the probes on the sample. The machine also has a field ion microscope to clean, atomically image, and shape the probe tips. The machine uses bare conductive samples and tips with a homebuilt set of pliers for heating and loading. Automated feedback controlled tip-surface contacts allow for electrical stability and reproducibility while also greatly reducing tip and surface damage due to contact formation. The ability to register inter-tip position bymore » imaging of a single surface feature by multiple tips is demonstrated. Four-probe material characterization is achieved by deploying two tips as fixed current probes and the third tip as a movable voltage probe.« less

Focal depth measurement of scanning helium ion microscope

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

Guo, Hongxuan, E-mail: Guo.hongxuan@nims.go.jp; Itoh, Hiroshi; Wang, Chunmei

2014-07-14

When facing the challenges of critical dimension measurement of complicated nanostructures, such as of the three dimension integrated circuit, characterization of the focal depth of microscopes is important. In this Letter, we developed a method for characterizing the focal depth of a scanning helium ion microscope (HIM) by using an atomic force microscope tip characterizer (ATC). The ATC was tilted in a sample chamber at an angle to the scanning plan. Secondary electron images (SEIs) were obtained at different positions of the ATC. The edge resolution of the SEIs shows the nominal diameters of the helium ion beam at differentmore » focal levels. With this method, the nominal shapes of the helium ion beams were obtained with different apertures. Our results show that a small aperture is necessary to get a high spatial resolution and high depth of field images with HIM. This work provides a method for characterizing and improving the performance of HIM.« less

Focal depth measurement of scanning helium ion microscope

NASA Astrophysics Data System (ADS)

Guo, Hongxuan; Itoh, Hiroshi; Wang, Chunmei; Zhang, Han; Fujita, Daisuke

2014-07-01

When facing the challenges of critical dimension measurement of complicated nanostructures, such as of the three dimension integrated circuit, characterization of the focal depth of microscopes is important. In this Letter, we developed a method for characterizing the focal depth of a scanning helium ion microscope (HIM) by using an atomic force microscope tip characterizer (ATC). The ATC was tilted in a sample chamber at an angle to the scanning plan. Secondary electron images (SEIs) were obtained at different positions of the ATC. The edge resolution of the SEIs shows the nominal diameters of the helium ion beam at different focal levels. With this method, the nominal shapes of the helium ion beams were obtained with different apertures. Our results show that a small aperture is necessary to get a high spatial resolution and high depth of field images with HIM. This work provides a method for characterizing and improving the performance of HIM.

Detection of blob objects in microscopic zebrafish images based on gradient vector diffusion.

PubMed

Li, Gang; Liu, Tianming; Nie, Jingxin; Guo, Lei; Malicki, Jarema; Mara, Andrew; Holley, Scott A; Xia, Weiming; Wong, Stephen T C

2007-10-01

The zebrafish has become an important vertebrate animal model for the study of developmental biology, functional genomics, and disease mechanisms. It is also being used for drug discovery. Computerized detection of blob objects has been one of the important tasks in quantitative phenotyping of zebrafish. We present a new automated method that is able to detect blob objects, such as nuclei or cells in microscopic zebrafish images. This method is composed of three key steps. The first step is to produce a diffused gradient vector field by a physical elastic deformable model. In the second step, the flux image is computed on the diffused gradient vector field. The third step performs thresholding and nonmaximum suppression based on the flux image. We report the validation and experimental results of this method using zebrafish image datasets from three independent research labs. Both sensitivity and specificity of this method are over 90%. This method is able to differentiate closely juxtaposed or connected blob objects, with high sensitivity and specificity in different situations. It is characterized by a good, consistent performance in blob object detection.

Near-Field Acoustical Imaging using Lateral Bending Mode of Atomic Force Microscope Cantilevers

NASA Astrophysics Data System (ADS)

Caron, A.; Rabe, U.; Rödel, J.; Arnold, W.

Scanning probe microscopy techniques enable one to investigate surface properties such as contact stiffness and friction between the probe tip and a sample with nm resolution. So far the bending and the torsional eigenmodes of an atomic force microscope cantilever have been used to image variations of elasticity and shear elasticity, respectively. Such images are near-field images with the resolution given by the contact radius typically between 10 nm and 50 nm. We show that the flexural modes of a cantilever oscillating in the width direction and parallel to the sample surface can also be used for imaging. Additional to the dominant in-plane component of the oscillation, the lateral modes exhibit a vertical component as well, provided there is an asymmetry in the cross-section of the cantilever or in its suspension. The out-of-plane deflection renders the lateral modes detectable by the optical position sensors used in atomic force microscopes. We studied cracks which were generated by Vickers indents, in submicro- and nanocrystalline ZrO2. Images of the lateral contact stiffness were obtained by vibrating the cantilever close to a contact-resonance frequency. A change in contact stiffness causes a shift of the resonant frequency and hence a change of the cantilever vibration amplitude. The lateral contact-stiffness images close to the crack faces display a contrast that we attribute to altered elastic properties indicating a process zone. This could be caused by a stress-induced phase transformation during crack propagation. Using the contact mode of an atomic force microscope, we measured the crack-opening displacement as a function of distance from the crack tip, and we determined the crack-tip toughness Ktip. Furthermore, K1c was inferred from the length of radial cracks of Vickers indents that were measured using classical scanning acoustic microscopy

Dielectrophoretic immobilization of proteins: Quantification by atomic force microscopy.

PubMed

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

2015-09-01

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

Total variation based image deconvolution for extended depth-of-field microscopy images

NASA Astrophysics Data System (ADS)

Hausser, F.; Beckers, I.; Gierlak, M.; Kahraman, O.

2015-03-01

One approach for a detailed understanding of dynamical cellular processes during drug delivery is the use of functionalized biocompatible nanoparticles and fluorescent markers. An appropriate imaging system has to detect these moving particles so as whole cell volumes in real time with high lateral resolution in a range of a few 100 nm. In a previous study Extended depth-of-field microscopy (EDF-microscopy) has been applied to fluorescent beads and tradiscantia stamen hair cells and the concept of real-time imaging has been proved in different microscopic modes. In principle a phase retardation system like a programmable space light modulator or a static waveplate is incorporated in the light path and modulates the wavefront of light. Hence the focal ellipsoid is smeared out and images seem to be blurred in a first step. An image restoration by deconvolution using the known point-spread-function (PSF) of the optical system is necessary to achieve sharp microscopic images of an extended depth-of-field. This work is focused on the investigation and optimization of deconvolution algorithms to solve this restoration problem satisfactorily. This inverse problem is challenging due to presence of Poisson distributed noise and Gaussian noise, and since the PSF used for deconvolution exactly fits in just one plane within the object. We use non-linear Total Variation based image restoration techniques, where different types of noise can be treated properly. Various algorithms are evaluated for artificially generated 3D images as well as for fluorescence measurements of BPAE cells.

Transportable and vibration-free full-field low-coherent quantitative phase microscope

NASA Astrophysics Data System (ADS)

Yamauchi, Toyohiko; Yamada, Hidenao; Goto, Kentaro; Matsui, Hisayuki; Yasuhiko, Osamu; Ueda, Yukio

2018-02-01

We developed a transportable Linnik-type full-field low-coherent quantitative phase microscope that is able to compensate for optical path length (OPL) disturbance due to environmental mechanical noises. Though two-beam interferometers such as Linnik ones suffer from unstable OPL difference, we overcame this problem with a mechanical feedback system based on digital signal-processing that controls the OPL difference in sub-nanometer resolution precisely with a feedback bandwidth of 4 kHz. The developed setup has a footprint of 200 mm by 200 mm, a height of 500 mm, and a weight of 4.5 kilograms. In the transmission imaging mode, cells were cultured on a reflection-enhanced glass-bottom dish, and we obtained interference images sequentially while performing stepwise quarter-wavelength phase-shifting. Real-time image processing, including retrieval of the unwrapped phase from interference images and its background correction, along with the acquisition of interference images, was performed on a laptop computer. Emulation of the phase contrast (PhC) images and the differential interference contrast (DIC) images was also performed in real time. Moreover, our setup was applied for full-field cell membrane imaging in the reflection mode, where the cells were cultured on an anti-reflection (AR)-coated glass-bottom dish. The phase and intensity of the light reflected by the membrane revealed the outer shape of the cells independent of the refractive index. In this paper, we show imaging results on cultured cells in both transmission and reflection modes.

Optics of wide-angle panoramic viewing system-assisted vitreous surgery.

PubMed

Chalam, Kakarla V; Shah, Vinay A

2004-01-01

The purpose of the article is to describe the optics of the contact wide-angle lens system with stereo-reinverter for vitreous surgery. A panoramic viewing system is made up of two components; an indirect ophthalmoscopy lens system for fundus image viewing, which is placed on the patient's cornea as a contact lens, and a separate removable prism system for reinversion of the image mounted on the microscope above the zooming system. The system provides a 104 degrees field of view in a phakic emmetropic eye with minification, which can be magnified by the operating microscope. It permits a binocular stereoptic view even through a small pupil (3 mm) or larger. In an air-filled phakic eye, field of view increases to approximately 130 degrees. The obtained image of the patient's fundus is reinverted to form true, erect, stereoscopic image by the reinversion system. In conclusion, this system permits wide-angle panoramic view of the surgical field. The contact lens neutralizes the optical irregularities of the corneal surface and allows improved visualization in eyes with irregular astigmatism induced by corneal scars. Excellent visualization is achieved in complex clinical situations such as miotic pupils, lenticular opacities, and in air-filled phakic eyes.

eeDAP: An Evaluation Environment for Digital and Analog Pathology.

PubMed

Gallas, Brandon D; Cheng, Wei-Chung; Gavrielides, Marios A; Ivansky, Adam; Keay, Tyler; Wunderlich, Adam; Hipp, Jason; Hewitt, Stephen M

2014-01-01

The purpose of this work is to present a platform for designing and executing studies that compare pathologists interpreting histopathology of whole slide images (WSI) on a computer display to pathologists interpreting glass slides on an optical microscope. Here we present eeDAP, an evaluation environment for digital and analog pathology. The key element in eeDAP is the registration of the WSI to the glass slide. Registration is accomplished through computer control of the microscope stage and a camera mounted on the microscope that acquires images of the real time microscope view. Registration allows for the evaluation of the same regions of interest (ROIs) in both domains. This can reduce or eliminate disagreements that arise from pathologists interpreting different areas and focuses the comparison on image quality. We reduced the pathologist interpretation area from an entire glass slide (≈10-30 mm) 2 to small ROIs <(50 um) 2 . We also made possible the evaluation of individual cells. We summarize eeDAP's software and hardware and provide calculations and corresponding images of the microscope field of view and the ROIs extracted from the WSIs. These calculations help provide a sense of eeDAP's functionality and operating principles, while the images provide a sense of the look and feel of studies that can be conducted in the digital and analog domains. The eeDAP software can be downloaded from code.google.com (project: eeDAP) as Matlab source or as a precompiled stand-alone license-free application.

In vivo imaging of the Drosophila Melanogaster heart using a novel optical coherence tomography microscope

NASA Astrophysics Data System (ADS)

Izatt, Susan D.; Choma, Michael A.; Israel, Steven; Wessells, Robert J.; Bodmer, Rolf; Izatt, Joseph A.

2005-03-01

Real time in vivo optical coherence tomography (OCT) imaging of the adult fruit fly Drosophila melanogaster heart using a newly designed OCT microscope allows accurate assessment of cardiac anatomy and function. D. melanogaster has been used extensively in genetic research for over a century, but in vivo evaluation of the heart has been limited by available imaging technology. The ability to assess phenotypic changes with micrometer-scale resolution noninvasively in genetic models such as D. melanogaster is needed in the advancing fields of developmental biology and genetics. We have developed a dedicated small animal OCT imaging system incorporating a state-of-the-art, real time OCT scanner integrated into a standard stereo zoom microscope which allows for simultaneous OCT and video imaging. System capabilities include A-scan, B-scan, and M-scan imaging as well as automated 3D volumetric acquisition and visualization. Transverse and sagittal B-mode scans of the four chambered D. melanogaster heart have been obtained with the OCT microscope and are consistent with detailed anatomical studies from the literature. Further analysis by M-mode scanning is currently under way to assess cardiac function as a function of age and sex by determination of shortening fraction and ejection fraction. These studies create control cardiac data on the wild type D. melanogaster, allowing subsequent evaluation of phenotypic cardiac changes in this model after regulated genetic mutation.

Automated in-chamber specimen coating for serial block-face electron microscopy.

PubMed

Titze, B; Denk, W

2013-05-01

When imaging insulating specimens in a scanning electron microscope, negative charge accumulates locally ('sample charging'). The resulting electric fields distort signal amplitude, focus and image geometry, which can be avoided by coating the specimen with a conductive film prior to introducing it into the microscope chamber. This, however, is incompatible with serial block-face electron microscopy (SBEM), where imaging and surface removal cycles (by diamond knife or focused ion beam) alternate, with the sample remaining in place. Here we show that coating the sample after each cutting cycle with a 1-2 nm metallic film, using an electron beam evaporator that is integrated into the microscope chamber, eliminates charging effects for both backscattered (BSE) and secondary electron (SE) imaging. The reduction in signal-to-noise ratio (SNR) caused by the film is smaller than that caused by the widely used low-vacuum method. Sample surfaces as large as 12 mm across were coated and imaged without charging effects at beam currents as high as 25 nA. The coatings also enabled the use of beam deceleration for non-conducting samples, leading to substantial SNR gains for BSE contrast. We modified and automated the evaporator to enable the acquisition of SBEM stacks, and demonstrated the acquisition of stacks of over 1000 successive cut/coat/image cycles and of stacks using beam deceleration or SE contrast. © 2013 The Authors Journal of Microscopy © 2013 Royal Microscopical Society.

Wide-field synovial fluid imaging using polarized lens-free on-chip microscopy for point-of-care diagnostics of gout (Conference Presentation)

NASA Astrophysics Data System (ADS)

Zhang, Yibo; Lee, Seung Yoon; Zhang, Yun; Furst, Daniel; Fitzgerald, John; Ozcan, Aydogan

2016-03-01

Gout and pseudogout are forms of crystal arthropathy caused by monosodium urate (MSU) and calcium pyrophosphate dehydrate (CPPD) crystals in the joint, respectively, that can result in painful joints. Detecting the unique-shaped, birefringent MSU/CPPD crystals in a synovial fluid sample using a compensated polarizing microscope has been the gold-standard for diagnosis since the 1960's. However, this can be time-consuming and inaccurate, especially if there are only few crystals in the fluid. The high-cost and bulkiness of conventional microscopes can also be limiting for point-of-care diagnosis. Lens-free on-chip microscopy based on digital holography routinely achieves high-throughput and high-resolution imaging in a cost-effective and field-portable design. Here we demonstrate, for the first time, polarized lens-free on-chip imaging of MSU and CPPD crystals over a wide field-of-view (FOV ~ 20.5 mm2, i.e., <20-fold larger compared a typical 20X objective-lens FOV) for point-of-care diagnostics of gout and pseudogout. Circularly polarizer partially-coherent light is used to illuminate the synovial fluid sample on a glass slide, after which a quarter-wave-plate and an angle-mismatched linear polarizer are used to analyze the transmitted light. Two lens-free holograms of the MSU/CPPD sample are taken, with the sample rotated by 90°, to rule out any non-birefringent objects within the specimen. A phase-recovery algorithm is also used to improve the reconstruction quality, and digital pseudo-coloring is utilized to match the color and contrast of the lens-free image to that of a gold-standard microscope image to ease the examination by a rheumatologist or a laboratory technician, and to facilitate computerized analysis.

Transmission X-ray microscopy for full-field nano-imaging of biomaterials

PubMed Central

ANDREWS, JOY C; MEIRER, FLORIAN; LIU, YIJIN; MESTER, ZOLTAN; PIANETTA, PIERO

2010-01-01

Imaging of cellular structure and extended tissue in biological materials requires nanometer resolution and good sample penetration, which can be provided by current full-field transmission X-ray microscopic techniques in the soft and hard X-ray regions. The various capabilities of full-field transmission X-ray microscopy (TXM) include 3D tomography, Zernike phase contrast, quantification of absorption, and chemical identification via X-ray fluorescence and X-ray absorption near edge structure (XANES) imaging. These techniques are discussed and compared in light of results from imaging of biological materials including microorganisms, bone and mineralized tissue and plants, with a focus on hard X-ray TXM at ≤ 40 nm resolution. PMID:20734414

Remotely sensed geology from lander-based to orbital perspectives: Results of FIDO rover May 2000 field tests

USGS Publications Warehouse

Jolliff, B.; Knoll, A.; Morris, R.V.; Moersch, J.; McSween, H.; Gilmore, M.; Arvidson, R.; Greeley, R.; Herkenhoff, K.; Squyres, S.

2002-01-01

Blind field tests of the Field Integration Design and Operations (FIDO) prototype Mars rover were carried out 7-16 May 2000. A Core Operations Team (COT), sequestered at the Jet Propulsion Laboratory without knowledge of test site location, prepared command sequences and interpreted data acquired by the rover. Instrument sensors included a stereo panoramic camera, navigational and hazard-avoidance cameras, a color microscopic imager, an infrared point spectrometer, and a rock coring drill. The COT designed command sequences, which were relayed by satellite uplink to the rover, and evaluated instrument data. Using aerial photos and Airborne Visible and Infrared Imaging Spectrometer (AVIRIS) data, and information from the rover sensors, the COT inferred the geology of the landing site during the 18 sol mission, including lithologic diversity, stratigraphic relationships, environments of deposition, and weathering characteristics. Prominent lithologic units were interpreted to be dolomite-bearing rocks, kaolinite-bearing altered felsic volcanic materials, and basalt. The color panoramic camera revealed sedimentary layering and rock textures, and geologic relationships seen in rock exposures. The infrared point spectrometer permitted identification of prominent carbonate and kaolinite spectral features and permitted correlations to outcrops that could not be reached by the rover. The color microscopic imager revealed fine-scale rock textures, soil components, and results of coring experiments. Test results show that close-up interrogation of rocks is essential to investigations of geologic environments and that observations must include scales ranging from individual boulders and outcrops (microscopic, macroscopic) to orbital remote sensing, with sufficient intermediate steps (descent images) to connect in situ and remote observations.

Magnetic microscopic imaging with an optically pumped magnetometer and flux guides

DOE PAGES

Kim, Young Jin; Savukov, Igor Mykhaylovich; Huang, Jen -Huang; ...

2017-01-23

Here, by combining an optically pumped magnetometer (OPM) with flux guides (FGs) and by installing a sample platform on automated translation stages, we have implemented an ultra-sensitive FG-OPM scanning magnetic imaging system that is capable of detecting magnetic fields of ~20 pT with spatial resolution better than 300 μm (expected to reach ~10 pT sensitivity and ~100 μm spatial resolution with optimized FGs). As a demonstration of one possible application of the FG-OPM device, we conducted magnetic imaging of micron-size magnetic particles. Magnetic imaging of such particles, including nano-particles and clusters, is very important for many fields, especially for medicalmore » cancer diagnostics and biophysics applications. For rapid, precise magnetic imaging, we constructed an automatic scanning system, which holds and moves a target sample containing magnetic particles at a given stand-off distance from the FG tips. We show that the device was able to produce clear microscopic magnetic images of 10 μm-size magnetic particles. In addition, we also numerically investigated how the magnetic flux from a target sample at a given stand-off distance is transmitted to the OPM vapor cell.« less

Multicolor probe-based confocal laser endomicroscopy: a new world for in vivo and real-time cellular imaging

NASA Astrophysics Data System (ADS)

Vercauteren, Tom; Doussoux, François; Cazaux, Matthieu; Schmid, Guillaume; Linard, Nicolas; Durin, Marie-Amélie; Gharbi, Hédi; Lacombe, François

2013-03-01

Since its inception in the field of in vivo imaging, endomicroscopy through optical fiber bundles, or probe-based Confocal Laser Endomicroscopy (pCLE), has extensively proven the benefit of in situ and real-time examination of living tissues at the microscopic scale. By continuously increasing image quality, reducing invasiveness and improving system ergonomics, Mauna Kea Technologies has turned pCLE not only into an irreplaceable research instrument for small animal imaging, but also into an accurate clinical decision making tool with applications as diverse as gastrointestinal endoscopy, pulmonology and urology. The current implementation of pCLE relies on a single fluorescence spectral band making different sources of in vivo information challenging to distinguish. Extending the pCLE approach to multi-color endomicroscopy therefore appears as a natural plan. Coupling simultaneous multi-laser excitation with minimally invasive, microscopic resolution, thin and flexible optics, allows the fusion of complementary and valuable biological information, thus paving the way to a combination of morphological and functional imaging. This paper will detail the architecture of a new system, Cellvizio Dual Band, capable of video rate in vivo and in situ multi-spectral fluorescence imaging with a microscopic resolution. In its standard configuration, the system simultaneously operates at 488 and 660 nm, where it automatically performs the necessary spectral, photometric and geometric calibrations to provide unambiguously co-registered images in real-time. The main hardware and software features, including calibration procedures and sub-micron registration algorithms, will be presented as well as a panorama of its current applications, illustrated with recent results in the field of pre-clinical imaging.

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.

Near-field transport imaging applied to photovoltaic materials

DOE PAGES

Xiao, Chuanxiao; Jiang, Chun -Sheng; Moseley, John; ...

2017-05-26

We developed and applied a new analytical technique - near-field transport imaging (NF-TI or simply TI) - to photovoltaic materials. Charge-carrier transport is an important factor in solar cell performance, and TI is an innovative approach that integrates a scanning electron microscope with a near-field scanning optical microscope, providing the possibility to study luminescence associated with recombination and transport with high spatial resolution. In this paper, we describe in detail the technical barriers we had to overcome to develop the technique for routine application and the data-fitting procedure used to calculate minority-carrier diffusion length values. The diffusion length measured bymore » TI agrees well with the results calculated by time-resolved photoluminescence on well-controlled gallium arsenide (GaAs) thin-film samples. We report for the first time on measurements on thin-film cadmium telluride using this technique, including the determination of effective carrier diffusion length, as well as the first near-field imaging of the effect of a single localized defect on carrier transport and recombination in a GaAs heterostructure. Furthermore, by changing the scanning setup, we were able to demonstrate near-field cathodoluminescence (CL), and correlated the results with standard CL measurements. In conclusion, the TI technique shows great potential for mapping transport properties in solar cell materials with high spatial resolution.« less

Overview of Athena Microscopic Imager Results

NASA Technical Reports Server (NTRS)

Herkenhoff, K.; Squyres, S.; Arvidson, R.; Bass, D.; Bell, J., III; Bertelsen, P.; Cabrol, N.; Ehlmann, B.; Farrand, W.; Gaddis, L.

2005-01-01

The Athena science payload on the Mars Exploration Rovers (MER) includes the Microscopic Imager (MI). The MI is a fixed-focus camera mounted on an extendable arm, the Instrument Deployment Device (IDD). The MI acquires images at a spatial resolution of 31 microns/pixel over a broad spectral range (400 - 700 nm). The MI uses the same electronics design as the other MER cameras but its optics yield a field of view of 32 32 mm across a 1024 1024 pixel CCD image. The MI acquires images using only solar or skylight illumination of the target surface. The MI science objectives, instrument design and calibration, operation, and data processing were described by Herkenhoff et al. Initial results of the MI experiment on both MER rovers (Spirit and Opportunity) have been published previously. Highlights of these and more recent results are described.

A versatile LabVIEW and field-programmable gate array-based scanning probe microscope for in operando electronic device characterization.

PubMed

Berger, Andrew J; Page, Michael R; Jacob, Jan; Young, Justin R; Lewis, Jim; Wenzel, Lothar; Bhallamudi, Vidya P; Johnston-Halperin, Ezekiel; Pelekhov, Denis V; Hammel, P Chris

2014-12-01

Understanding the complex properties of electronic and spintronic devices at the micro- and nano-scale is a topic of intense current interest as it becomes increasingly important for scientific progress and technological applications. In operando characterization of such devices by scanning probe techniques is particularly well-suited for the microscopic study of these properties. We have developed a scanning probe microscope (SPM) which is capable of both standard force imaging (atomic, magnetic, electrostatic) and simultaneous electrical transport measurements. We utilize flexible and inexpensive FPGA (field-programmable gate array) hardware and a custom software framework developed in National Instrument's LabVIEW environment to perform the various aspects of microscope operation and device measurement. The FPGA-based approach enables sensitive, real-time cantilever frequency-shift detection. Using this system, we demonstrate electrostatic force microscopy of an electrically biased graphene field-effect transistor device. The combination of SPM and electrical transport also enables imaging of the transport response to a localized perturbation provided by the scanned cantilever tip. Facilitated by the broad presence of LabVIEW in the experimental sciences and the openness of our software solution, our system permits a wide variety of combined scanning and transport measurements by providing standardized interfaces and flexible access to all aspects of a measurement (input and output signals, and processed data). Our system also enables precise control of timing (synchronization of scanning and transport operations) and implementation of sophisticated feedback protocols, and thus should be broadly interesting and useful to practitioners in the field.

A versatile LabVIEW and field-programmable gate array-based scanning probe microscope for in operando electronic device characterization

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

Berger, Andrew J., E-mail: berger.156@osu.edu; Page, Michael R.; Young, Justin R.

Understanding the complex properties of electronic and spintronic devices at the micro- and nano-scale is a topic of intense current interest as it becomes increasingly important for scientific progress and technological applications. In operando characterization of such devices by scanning probe techniques is particularly well-suited for the microscopic study of these properties. We have developed a scanning probe microscope (SPM) which is capable of both standard force imaging (atomic, magnetic, electrostatic) and simultaneous electrical transport measurements. We utilize flexible and inexpensive FPGA (field-programmable gate array) hardware and a custom software framework developed in National Instrument's LabVIEW environment to perform themore » various aspects of microscope operation and device measurement. The FPGA-based approach enables sensitive, real-time cantilever frequency-shift detection. Using this system, we demonstrate electrostatic force microscopy of an electrically biased graphene field-effect transistor device. The combination of SPM and electrical transport also enables imaging of the transport response to a localized perturbation provided by the scanned cantilever tip. Facilitated by the broad presence of LabVIEW in the experimental sciences and the openness of our software solution, our system permits a wide variety of combined scanning and transport measurements by providing standardized interfaces and flexible access to all aspects of a measurement (input and output signals, and processed data). Our system also enables precise control of timing (synchronization of scanning and transport operations) and implementation of sophisticated feedback protocols, and thus should be broadly interesting and useful to practitioners in the field.« less

Resolution enhancement of wide-field interferometric microscopy by coupled deep autoencoders.

PubMed

Işil, Çağatay; Yorulmaz, Mustafa; Solmaz, Berkan; Turhan, Adil Burak; Yurdakul, Celalettin; Ünlü, Selim; Ozbay, Ekmel; Koç, Aykut

2018-04-01

Wide-field interferometric microscopy is a highly sensitive, label-free, and low-cost biosensing imaging technique capable of visualizing individual biological nanoparticles such as viral pathogens and exosomes. However, further resolution enhancement is necessary to increase detection and classification accuracy of subdiffraction-limited nanoparticles. In this study, we propose a deep-learning approach, based on coupled deep autoencoders, to improve resolution of images of L-shaped nanostructures. During training, our method utilizes microscope image patches and their corresponding manual truth image patches in order to learn the transformation between them. Following training, the designed network reconstructs denoised and resolution-enhanced image patches for unseen input.

A Scanning Quantum Cryogenic Atom Microscope

NASA Astrophysics Data System (ADS)

Lev, Benjamin

Microscopic imaging of local magnetic fields provides a window into the organizing principles of complex and technologically relevant condensed matter materials. However, a wide variety of intriguing strongly correlated and topologically nontrivial materials exhibit poorly understood phenomena outside the detection capability of state-of-the-art high-sensitivity, high-resolution scanning probe magnetometers. We introduce a quantum-noise-limited scanning probe magnetometer that can operate from room-to-cryogenic temperatures with unprecedented DC-field sensitivity and micron-scale resolution. The Scanning Quantum Cryogenic Atom Microscope (SQCRAMscope) employs a magnetically levitated atomic Bose-Einstein condensate (BEC), thereby providing immunity to conductive and blackbody radiative heating. The SQCRAMscope has a field sensitivity of 1.4 nT per resolution-limited point (2 um), or 6 nT / Hz1 / 2 per point at its duty cycle. Compared to point-by-point sensors, the long length of the BEC provides a naturally parallel measurement, allowing one to measure nearly one-hundred points with an effective field sensitivity of 600 pT / Hz1 / 2 each point during the same time as a point-by-point scanner would measure these points sequentially. Moreover, it has a noise floor of 300 pT and provides nearly two orders of magnitude improvement in magnetic flux sensitivity (down to 10- 6 Phi0 / Hz1 / 2) over previous atomic probe magnetometers capable of scanning near samples. These capabilities are for the first time carefully benchmarked by imaging magnetic fields arising from microfabricated wire patterns and done so using samples that may be scanned, cryogenically cooled, and easily exchanged. We anticipate the SQCRAMscope will provide charge transport images at temperatures from room to \\x9D4K in unconventional superconductors and topologically nontrivial materials.

Scanning Quantum Cryogenic Atom Microscope

NASA Astrophysics Data System (ADS)

Yang, Fan; Kollár, Alicia J.; Taylor, Stephen F.; Turner, Richard W.; Lev, Benjamin L.

2017-03-01

Microscopic imaging of local magnetic fields provides a window into the organizing principles of complex and technologically relevant condensed-matter materials. However, a wide variety of intriguing strongly correlated and topologically nontrivial materials exhibit poorly understood phenomena outside the detection capability of state-of-the-art high-sensitivity high-resolution scanning probe magnetometers. We introduce a quantum-noise-limited scanning probe magnetometer that can operate from room-to-cryogenic temperatures with unprecedented dc-field sensitivity and micron-scale resolution. The Scanning Quantum Cryogenic Atom Microscope (SQCRAMscope) employs a magnetically levitated atomic Bose-Einstein condensate (BEC), thereby providing immunity to conductive and blackbody radiative heating. The SQCRAMscope has a field sensitivity of 1.4 nT per resolution-limited point (approximately 2 μ m ) or 6 nT /√{Hz } per point at its duty cycle. Compared to point-by-point sensors, the long length of the BEC provides a naturally parallel measurement, allowing one to measure nearly 100 points with an effective field sensitivity of 600 pT /√{Hz } for each point during the same time as a point-by-point scanner measures these points sequentially. Moreover, it has a noise floor of 300 pT and provides nearly 2 orders of magnitude improvement in magnetic flux sensitivity (down to 10-6 Φ0/√{Hz } ) over previous atomic probe magnetometers capable of scanning near samples. These capabilities are carefully benchmarked by imaging magnetic fields arising from microfabricated wire patterns in a system where samples may be scanned, cryogenically cooled, and easily exchanged. We anticipate the SQCRAMscope will provide charge-transport images at temperatures from room temperature to 4 K in unconventional superconductors and topologically nontrivial materials.

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..

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.

Real-Time Nanoscopy by Using Blinking Enhanced Quantum Dots

PubMed Central

Watanabe, Tomonobu M.; Fukui, Shingo; Jin, Takashi; Fujii, Fumihiko; Yanagida, Toshio

2010-01-01

Superresolution optical microscopy (nanoscopy) is of current interest in many biological fields. Superresolution optical fluctuation imaging, which utilizes higher-order cumulant of fluorescence temporal fluctuations, is an excellent method for nanoscopy, as it requires neither complicated optics nor illuminations. However, it does need an impractical number of images for real-time observation. Here, we achieved real-time nanoscopy by modifying superresolution optical fluctuation imaging and enhancing the fluctuation of quantum dots. Our developed quantum dots have higher blinking than commercially available ones. The fluctuation of the blinking improved the resolution when using a variance calculation for each pixel instead of a cumulant calculation. This enabled us to obtain microscopic images with 90-nm and 80-ms spatial-temporal resolution by using a conventional fluorescence microscope without any optics or devices. PMID:20923631

Microsphere-aided optical microscopy and its applications for super-resolution imaging

NASA Astrophysics Data System (ADS)

Upputuri, Paul Kumar; Pramanik, Manojit

2017-12-01

The spatial resolution of a standard optical microscope (SOM) is limited by diffraction. In visible spectrum, SOM can provide ∼ 200 nm resolution. To break the diffraction limit several approaches were developed including scanning near field microscopy, metamaterial super-lenses, nanoscale solid immersion lenses, super-oscillatory lenses, confocal fluorescence microscopy, techniques that exploit non-linear response of fluorophores like stimulated emission depletion microscopy, stochastic optical reconstruction microscopy, etc. Recently, photonic nanojet generated by a dielectric microsphere was used to break the diffraction limit. The microsphere-approach is simple, cost-effective and can be implemented under a standard microscope, hence it has gained enormous attention for super-resolution imaging. In this article, we briefly review the microsphere approach and its applications for super-resolution imaging in various optical imaging modalities.

Imaging Research With Non-Periodic Multilayers for Inertial Confinement Fusion Diagnostic Experiments

NASA Astrophysics Data System (ADS)

L. Wang, F.; Mu, B. Z.; Wang, Z. S.; Gu, C. S.; Zhang, Z.; Qin, S. J.; Chen, L. Y.

A grazing Kirkpatrick-Baez (K-B) microscope was designed for hard x-ray (8keV; Cu Ka radiation) imaging in Inertial Confinement Fusion (ICF) diagnostic experiments. Ray tracing software was used to simulate optical system performance. The optimized theoretical resolution of K-B microscope was about 2 micron and better than 10 micron in 200 micron field of view. Tungsten and boron carbide were chosen as multilayer materials and the multilayer was deposited onto the silicon wafer substrate and the reflectivity was measured by x-ray diffraction (XRD). The reflectivity of supermirror was about 20 % in 0.3 % of bandwidth. 8keV Cu target x-ray tube source was used in x-ray imaging experiments and the magnification of 1x and 2x x-ray images were obtained.

Photonic-crystal membranes for optical detection of single nano-particles, designed for biosensor application.

PubMed

Grepstad, Jon Olav; Kaspar, Peter; Solgaard, Olav; Johansen, Ib-Rune; Sudbø, Aasmund S

2012-03-26

A sensor designed to detect bio-molecules is presented. The sensor exploits a planar 2D photonic crystal (PC) membrane with sub-micron thickness and through holes, to induce high optical fields that allow detection of nano-particles smaller than the diffraction limit of an optical microscope. We report on our design and fabrication of a PC membrane with a nano-particle trapped inside. We have also designed and built an imaging system where an optical microscope and a CCD camera are used to take images of the PC membrane. Results show how the trapped nano-particle appears as a bright spot in the image. In a first experimental realization of the imaging system, single particles with a radius of 75 nm can be detected.

Single-Shot Optical Sectioning Using Two-Color Probes in HiLo Fluorescence Microscopy

PubMed Central

Muro, Eleonora; Vermeulen, Pierre; Ioannou, Andriani; Skourides, Paris; Dubertret, Benoit; Fragola, Alexandra; Loriette, Vincent

2011-01-01

We describe a wide-field fluorescence microscope setup which combines HiLo microscopy technique with the use of a two-color fluorescent probe. It allows one-shot fluorescence optical sectioning of thick biological moving sample which is illuminated simultaneously with a flat and a structured pattern at two different wavelengths. Both homogenous and structured fluorescence images are spectrally separated at detection and combined similarly with the HiLo microscopy technique. We present optically sectioned full-field images of Xenopus laevis embryos acquired at 25 images/s frame rate. PMID:21641327

Large-field high-contrast hard x-ray Zernike phase-contrast nano-imaging beamline at Pohang Light Source.

PubMed

Lim, Jun; Park, So Yeong; Huang, Jung Yun; Han, Sung Mi; Kim, Hong-Tae

2013-01-01

We developed an off-axis-illuminated zone-plate-based hard x-ray Zernike phase-contrast microscope beamline at Pohang Light Source. Owing to condenser optics-free and off-axis illumination, a large field of view was achieved. The pinhole-type Zernike phase plate affords high-contrast images of a cell with minimal artifacts such as the shade-off and halo effects. The setup, including the optics and the alignment, is simple and easy, and allows faster and easier imaging of large bio-samples.

A simple approach to spectrally resolved fluorescence and bright field microscopy over select regions of interest

NASA Astrophysics Data System (ADS)

Dahlberg, Peter D.; Boughter, Christopher T.; Faruk, Nabil F.; Hong, Lu; Koh, Young Hoon; Reyer, Matthew A.; Shaiber, Alon; Sherani, Aiman; Zhang, Jiacheng; Jureller, Justin E.; Hammond, Adam T.

2016-11-01

A standard wide field inverted microscope was converted to a spatially selective spectrally resolved microscope through the addition of a polarizing beam splitter, a pair of polarizers, an amplitude-mode liquid crystal-spatial light modulator, and a USB spectrometer. The instrument is capable of simultaneously imaging and acquiring spectra over user defined regions of interest. The microscope can also be operated in a bright-field mode to acquire absorption spectra of micron scale objects. The utility of the instrument is demonstrated on three different samples. First, the instrument is used to resolve three differently labeled fluorescent beads in vitro. Second, the instrument is used to recover time dependent bleaching dynamics that have distinct spectral changes in the cyanobacteria, Synechococcus leopoliensis UTEX 625. Lastly, the technique is used to acquire the absorption spectra of CH3NH3PbBr3 perovskites and measure differences between nanocrystal films and micron scale crystals.

A simple approach to spectrally resolved fluorescence and bright field microscopy over select regions of interest.

PubMed

Dahlberg, Peter D; Boughter, Christopher T; Faruk, Nabil F; Hong, Lu; Koh, Young Hoon; Reyer, Matthew A; Shaiber, Alon; Sherani, Aiman; Zhang, Jiacheng; Jureller, Justin E; Hammond, Adam T

2016-11-01

A standard wide field inverted microscope was converted to a spatially selective spectrally resolved microscope through the addition of a polarizing beam splitter, a pair of polarizers, an amplitude-mode liquid crystal-spatial light modulator, and a USB spectrometer. The instrument is capable of simultaneously imaging and acquiring spectra over user defined regions of interest. The microscope can also be operated in a bright-field mode to acquire absorption spectra of micron scale objects. The utility of the instrument is demonstrated on three different samples. First, the instrument is used to resolve three differently labeled fluorescent beads in vitro. Second, the instrument is used to recover time dependent bleaching dynamics that have distinct spectral changes in the cyanobacteria, Synechococcus leopoliensis UTEX 625. Lastly, the technique is used to acquire the absorption spectra of CH 3 NH 3 PbBr 3 perovskites and measure differences between nanocrystal films and micron scale crystals.

In vivo and ex vivo imaging with ultrahigh resolution full-field OCT

NASA Astrophysics Data System (ADS)

Grieve, Kate; Moneron, Gael; Schwartz, Wilfrid; Boccara, Albert C.; Dubois, Arnaud

2005-08-01

Imaging of in vivo and ex vivo biological samples using full-field optical coherence tomography is demonstrated. Three variations on the original full-field optical coherence tomography instrument are presented, and evaluated in terms of performance. The instruments are based on the Linnik interferometer illuminated by a white light source. Images in the en face orientation are obtained in real-time without scanning by using a two-dimensional parallel detector array. An isotropic resolution capability better than 1 μm is achieved thanks to the use of a broad spectrum source and high numerical aperture microscope objectives. Detection sensitivity up to 90 dB is demonstrated. Image acquisition times as short as 10 μs per en face image are possible. A variety of in vivo and ex vivo imaging applications is explored, particularly in the fields of embryology, ophthalmology and botany.

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.

Light Microscopy's New Jobs

NASA Astrophysics Data System (ADS)

Ritsch-Marte, Monika

2009-04-01

300 years since the first glimpse through the earliest microscopes, light microscopy is still an active field of research, breaking new frontiers in optical imaging and even becoming a means of mechanical manipulation of microparticles.

Plasmonic Imaging of Electrochemical Reactions of Single Nanoparticles.

PubMed

Fang, Yimin; Wang, Hui; Yu, Hui; Liu, Xianwei; Wang, Wei; Chen, Hong-Yuan; Tao, N J

2016-11-15

Electrochemical reactions are involved in many natural phenomena, and are responsible for various applications, including energy conversion and storage, material processing and protection, and chemical detection and analysis. An electrochemical reaction is accompanied by electron transfer between a chemical species and an electrode. For this reason, it has been studied by measuring current, charge, or related electrical quantities. This approach has led to the development of various electrochemical methods, which have played an essential role in the understanding and applications of electrochemistry. While powerful, most of the traditional methods lack spatial and temporal resolutions desired for studying heterogeneous electrochemical reactions on electrode surfaces and in nanoscale materials. To overcome the limitations, scanning probe microscopes have been invented to map local electrochemical reactions with nanometer resolution. Examples include the scanning electrochemical microscope and scanning electrochemical cell microscope, which directly image local electrochemical reaction current using a scanning electrode or pipet. The use of a scanning probe in these microscopes provides high spatial resolution, but at the expense of temporal resolution and throughput. This Account discusses an alternative approach to study electrochemical reactions. Instead of measuring electron transfer electrically, it detects the accompanying changes in the reactant and product concentrations on the electrode surface optically via surface plasmon resonance (SPR). SPR is highly surface sensitive, and it provides quantitative information on the surface concentrations of reactants and products vs time and electrode potential, from which local reaction kinetics can be analyzed and quantified. The plasmonic approach allows imaging of local electrochemical reactions with high temporal resolution and sensitivity, making it attractive for studying electrochemical reactions in biological systems and nanoscale materials with high throughput. The plasmonic approach has two imaging modes: electrochemical current imaging and interfacial impedance imaging. The former images local electrochemical current associated with electrochemical reactions (faradic current), and the latter maps local interfacial impedance, including nonfaradic contributions (e.g., double layer charging). The plasmonic imaging technique can perform voltammetry (cyclic or square wave) in an analogous manner to the traditional electrochemical methods. It can also be integrated with bright field, dark field, and fluorescence imaging capabilities in one optical setup to provide additional capabilities. To date the plasmonic imaging technique has found various applications, including mapping of heterogeneous surface reactions, analysis of trace substances, detection of catalytic reactions, and measurement of graphene quantum capacitance. The plasmonic and other emerging optical imaging techniques (e.g., dark field and fluorescence microscopy), together with the scanning probe-based electrochemical imaging and single nanoparticle analysis techniques, provide new capabilities for one to study single nanoparticle electrochemistry with unprecedented spatial and temporal resolutions. In this Account, we focus on imaging of electrochemical reactions at single nanoparticles.

Bridging Philosophy of Technology and Neurobiological Research: Interpreting Images from the "Slam Freezer"

ERIC Educational Resources Information Center

Rosenberger, Robert

2005-01-01

The swiftly growing field of neurobiological research utilizes highly advanced technologies (e.g., magnetic resonance imaging, electron microscopy) to mediate between investigators and the brains they investigate. Here, the author analyzes a device called the "slam freezer" that quick-freezes neurons to be studied under the microscope. Employing…

Multispectral Microscopic Imager (MMI): Multispectral Imaging of Geological Materials at a Handlens Scale

NASA Astrophysics Data System (ADS)

Farmer, J. D.; Nunez, J. I.; Sellar, R. G.; Gardner, P. B.; Manatt, K. S.; Dingizian, A.; Dudik, M. J.; McDonnell, G.; Le, T.; Thomas, J. A.; Chu, K.

2011-12-01

The Multispectral Microscopic Imager (MMI) is a prototype instrument presently under development for future astrobiological missions to Mars. The MMI is designed to be a arm-mounted rover instrument for use in characterizing the microtexture and mineralogy of materials along geological traverses [1,2,3]. Such geological information is regarded as essential for interpreting petrogenesis and geological history, and when acquired in near real-time, can support hypothesis-driven exploration and optimize science return. Correlated microtexure and mineralogy also provides essential data for selecting samples for analysis with onboard lab instruments, and for prioritizing samples for potential Earth return. The MMI design employs multispectral light-emitting diodes (LEDs) and an uncooled focal plane array to achieve the low-mass (<1kg), low-cost, and high reliability (no moving parts) required for an arm-mounted instrument on a planetary rover [2,3]. The MMI acquires multispectral, reflectance images at 62 μm/pixel, in which each image pixel is comprised of a 21-band VNIR spectrum (0.46 to 1.73 μm). This capability enables the MMI to discriminate and resolve the spatial distribution of minerals and textures at the microscale [2, 3]. By extending the spectral range into the infrared, and increasing the number of spectral bands, the MMI exceeds the capabilities of current microimagers, including the MER Microscopic Imager (MI); 4, the Phoenix mission Robotic Arm Camera (RAC; 5) and the Mars Science Laboratory's Mars Hand Lens Imager (MAHLI; 6). In this report we will review the capabilities of the MMI by highlighting recent lab and field applications, including: 1) glove box deployments in the Astromaterials lab at Johnson Space Center to analyze Apollo lunar samples; 2) GeoLab glove box deployments during the 2011 Desert RATS field trials in northern AZ to characterize analog materials collected by astronauts during simulated EVAs; 3) field deployments on Mauna Kea Volcano, Hawaii, during NASA's 2010 ISRU field trials, to analyze materials at the primary feedstock mining site; 4) lab characterization of geological samples from a complex, volcanic-hydrothermal terrain in the Cady Mts., SE Mojave Desert, California. We will show how field and laboratory applications have helped drive the development and refinement of MMI capabilities, while identifying synergies with other potential payload instruments (e.g. X-ray Diffraction) for solving real geological problems.

3D Cryo-Imaging: A Very High-Resolution View of the Whole Mouse

PubMed Central

Roy, Debashish; Steyer, Grant J.; Gargesha, Madhusudhana; Stone, Meredith E.; Wilson, David L.

2009-01-01

We developed the Case Cryo-imaging system that provides information rich, very high-resolution, color brightfield, and molecular fluorescence images of a whole mouse using a section-and-image block-face imaging technology. The system consists of a mouse-sized, motorized cryo-microtome with special features for imaging, a modified, brightfield/ fluorescence microscope, and a robotic xyz imaging system positioner, all of which is fully automated by a control system. Using the robotic system, we acquired microscopic tiled images at a pixel size of 15.6 µm over the block face of a whole mouse sectioned at 40 µm, with a total data volume of 55 GB. Viewing 2D images at multiple resolutions, we identified small structures such as cardiac vessels, muscle layers, villi of the small intestine, the optic nerve, and layers of the eye. Cryo-imaging was also suitable for imaging embryo mutants in 3D. A mouse, in which enhanced green fluorescent protein was expressed under gamma actin promoter in smooth muscle cells, gave clear 3D views of smooth muscle in the urogenital and gastrointestinal tracts. With cryo-imaging, we could obtain 3D vasculature down to 10 µm, over very large regions of mouse brain. Software is fully automated with fully programmable imaging/sectioning protocols, email notifications, and automatic volume visualization. With a unique combination of field-of-view, depth of field, contrast, and resolution, the Case Cryo-imaging system fills the gap between whole animal in vivo imaging and histology. PMID:19248166

Hard X-ray Full Field Nano-imaging of Bone and Nanowires at SSRL

NASA Astrophysics Data System (ADS)

Andrews, Joy C.; Pianetta, Piero; Meirer, Florian; Chen, Jie; Almeida, Eduardo; van der Meulen, Marjolein C. H.; Alwood, Joshua S.; Lee, Cathy; Zhu, Jia; Cui, Yi

2010-06-01

A hard X-ray full field microscope from Xradia Inc. has been installed at SSRL on a 54-pole wiggler end station at beam line 6-2. It has been optimized to operate from 5-14 keV with resolution as high as 30 nm. High quality images are achieved using a vertical beam stabilizer and condenser scanner with high efficiency zone plates with 30 nm outermost zone width. The microscope has been used in Zernike phase contrast, available at 5.4 keV and 8 keV, as well as absorption contrast to image a variety of biological, environmental and materials samples. Calibration of the X-ray attenuation with crystalline apatite enabled quantification of bone density of plate-like and rod-like regions of mouse bone trabecula. 3D tomography of individual lacuna revealed the surrounding cell canaliculi and processes. 3D tomography of chiral branched PbSe nanowires showed orthogonal branches around a central nanowire.

Hard X-ray Full Field Nano-imaging of Bone and Nanowires at SSRL.

PubMed

Andrews, Joy C; Pianetta, Piero; Meirer, Florian; Chen, Jie; Almeida, Eduardo; van der Meulen, Marjolein C H; Alwood, Joshua S; Lee, Cathy; Zhu, Jia; Cui, Yi

2010-06-23

A hard X-ray full field microscope from Xradia Inc. has been installed at SSRL on a 54-pole wiggler end station at beam line 6-2. It has been optimized to operate from 5-14 keV with resolution as high as 30 nm. High quality images are achieved using a vertical beam stabilizer and condenser scanner with high efficiency zone plates with 30 nm outermost zone width. The microscope has been used in Zernike phase contrast, available at 5.4 keV and 8 keV, as well as absorption contrast to image a variety of biological, environmental and materials samples. Calibration of the X-ray attenuation with crystalline apatite enabled quantification of bone density of plate-like and rod-like regions of mouse bone trabecula. 3D tomography of individual lacuna revealed the surrounding cell canaliculi and processes. 3D tomography of chiral branched PbSe nanowires showed orthogonal branches around a central nanowire.

Surface defects evaluation system based on electromagnetic model simulation and inverse-recognition calibration method

NASA Astrophysics Data System (ADS)

Yang, Yongying; Chai, Huiting; Li, Chen; Zhang, Yihui; Wu, Fan; Bai, Jian; Shen, Yibing

2017-05-01

Digitized evaluation of micro sparse defects on large fine optical surfaces is one of the challenges in the field of optical manufacturing and inspection. The surface defects evaluation system (SDES) for large fine optical surfaces is developed based on our previously reported work. In this paper, the electromagnetic simulation model based on Finite-Difference Time-Domain (FDTD) for vector diffraction theory is firstly established to study the law of microscopic scattering dark-field imaging. Given the aberration in actual optical systems, point spread function (PSF) approximated by a Gaussian function is introduced in the extrapolation from the near field to the far field and the scatter intensity distribution in the image plane is deduced. Analysis shows that both diffraction-broadening imaging and geometrical imaging should be considered in precise size evaluation of defects. Thus, a novel inverse-recognition calibration method is put forward to avoid confusion caused by diffraction-broadening effect. The evaluation method is applied to quantitative evaluation of defects information. The evaluation results of samples of many materials by SDES are compared with those by OLYMPUS microscope to verify the micron-scale resolution and precision. The established system has been applied to inspect defects on large fine optical surfaces and can achieve defects inspection of surfaces as large as 850 mm×500 mm with the resolution of 0.5 μm.

Method for stitching microbial images using a neural network

NASA Astrophysics Data System (ADS)

Semenishchev, E. A.; Voronin, V. V.; Marchuk, V. I.; Tolstova, I. V.

2017-05-01

Currently an analog microscope has a wide distribution in the following fields: medicine, animal husbandry, monitoring technological objects, oceanography, agriculture and others. Automatic method is preferred because it will greatly reduce the work involved. Stepper motors are used to move the microscope slide and allow to adjust the focus in semi-automatic or automatic mode view with transfer images of microbiological objects from the eyepiece of the microscope to the computer screen. Scene analysis allows to locate regions with pronounced abnormalities for focusing specialist attention. This paper considers the method for stitching microbial images, obtained of semi-automatic microscope. The method allows to keep the boundaries of objects located in the area of capturing optical systems. Objects searching are based on the analysis of the data located in the area of the camera view. We propose to use a neural network for the boundaries searching. The stitching image boundary is held of the analysis borders of the objects. To auto focus, we use the criterion of the minimum thickness of the line boundaries of object. Analysis produced the object located in the focal axis of the camera. We use method of recovery of objects borders and projective transform for the boundary of objects which are based on shifted relative to the focal axis. Several examples considered in this paper show the effectiveness of the proposed approach on several test images.

Compact Microscope Imaging System Developed

NASA Technical Reports Server (NTRS)

McDowell, Mark

2001-01-01

The Compact Microscope Imaging System (CMIS) is a diagnostic tool with intelligent controls for use in space, industrial, medical, and security applications. The CMIS can be used in situ with a minimum amount of user intervention. This system, which was developed at the NASA Glenn Research Center, can scan, find areas of interest, focus, and acquire images automatically. Large numbers of multiple cell experiments require microscopy for in situ observations; this is only feasible with compact microscope systems. CMIS is a miniature machine vision system that combines intelligent image processing with remote control capabilities. The software also has a user-friendly interface that can be used independently of the hardware for post-experiment analysis. CMIS has potential commercial uses in the automated online inspection of precision parts, medical imaging, security industry (examination of currency in automated teller machines and fingerprint identification in secure entry locks), environmental industry (automated examination of soil/water samples), biomedical field (automated blood/cell analysis), and microscopy community. CMIS will improve research in several ways: It will expand the capabilities of MSD experiments utilizing microscope technology. It may be used in lunar and Martian experiments (Rover Robot). Because of its reduced size, it will enable experiments that were not feasible previously. It may be incorporated into existing shuttle orbiter and space station experiments, including glove-box-sized experiments as well as ground-based experiments.

Compact Wireless Microscope for In-Situ Time Course Study of Large Scale Cell Dynamics within an Incubator

NASA Astrophysics Data System (ADS)

Jin, Di; Wong, Dennis; Li, Junxiang; Luo, Zhang; Guo, Yiran; Liu, Bifeng; Wu, Qiong; Ho, Chih-Ming; Fei, Peng

2015-12-01

Imaging of live cells in a region of interest is essential to life science research. Unlike the traditional way that mounts CO2 incubator onto a bulky microscope for observation, here we propose a wireless microscope (termed w-SCOPE) that is based on the “microscope-in-incubator” concept and can be easily housed into a standard CO2 incubator for prolonged on-site observation of the cells. The w-SCOPE is capable of tunable magnification, remote control and wireless image transmission. At the same time, it is compact, measuring only ~10 cm in each dimension, and cost-effective. With the enhancement of compressive sensing computation, the acquired images can achieve a wide field of view (FOV) of ~113 mm2 as well as a cellular resolution of ~3 μm, which enables various forms of follow-up image-based cell analysis. We performed 12 hours time-lapse study on paclitaxel-treated MCF-7 and HEK293T cell lines using w-SCOPE. The analytic results, such as the calculated viability and therapeutic window, from our device were validated by standard cell detection assays and imaging-based cytometer. In addition to those end-point detection methods, w-SCOPE further uncovered the time course of the cell’s response to the drug treatment over the whole period of drug exposure.

Compact Wireless Microscope for In-Situ Time Course Study of Large Scale Cell Dynamics within an Incubator

PubMed Central

Jin, Di; Wong, Dennis; Li, Junxiang; Luo, Zhang; Guo, Yiran; Liu, Bifeng; Wu, Qiong; Ho, Chih-Ming; Fei, Peng

2015-01-01

Imaging of live cells in a region of interest is essential to life science research. Unlike the traditional way that mounts CO2 incubator onto a bulky microscope for observation, here we propose a wireless microscope (termed w-SCOPE) that is based on the “microscope-in-incubator” concept and can be easily housed into a standard CO2 incubator for prolonged on-site observation of the cells. The w-SCOPE is capable of tunable magnification, remote control and wireless image transmission. At the same time, it is compact, measuring only ~10 cm in each dimension, and cost-effective. With the enhancement of compressive sensing computation, the acquired images can achieve a wide field of view (FOV) of ~113 mm2 as well as a cellular resolution of ~3 μm, which enables various forms of follow-up image-based cell analysis. We performed 12 hours time-lapse study on paclitaxel-treated MCF-7 and HEK293T cell lines using w-SCOPE. The analytic results, such as the calculated viability and therapeutic window, from our device were validated by standard cell detection assays and imaging-based cytometer. In addition to those end-point detection methods, w-SCOPE further uncovered the time course of the cell’s response to the drug treatment over the whole period of drug exposure. PMID:26681552

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

Segmentation of White Blood Cells From Microscopic Images Using a Novel Combination of K-Means Clustering and Modified Watershed Algorithm.

PubMed

Ghane, Narjes; Vard, Alireza; Talebi, Ardeshir; Nematollahy, Pardis

2017-01-01

Recognition of white blood cells (WBCs) is the first step to diagnose some particular diseases such as acquired immune deficiency syndrome, leukemia, and other blood-related diseases that are usually done by pathologists using an optical microscope. This process is time-consuming, extremely tedious, and expensive and needs experienced experts in this field. Thus, a computer-aided diagnosis system that assists pathologists in the diagnostic process can be so effective. Segmentation of WBCs is usually a first step in developing a computer-aided diagnosis system. The main purpose of this paper is to segment WBCs from microscopic images. For this purpose, we present a novel combination of thresholding, k-means clustering, and modified watershed algorithms in three stages including (1) segmentation of WBCs from a microscopic image, (2) extraction of nuclei from cell's image, and (3) separation of overlapping cells and nuclei. The evaluation results of the proposed method show that similarity measures, precision, and sensitivity respectively were 92.07, 96.07, and 94.30% for nucleus segmentation and 92.93, 97.41, and 93.78% for cell segmentation. In addition, statistical analysis presents high similarity between manual segmentation and the results obtained by the proposed method.

Fine Metal Mask 3-Dimensional Measurement by using Scanning Digital Holographic Microscope

NASA Astrophysics Data System (ADS)

Shin, Sanghoon; Yu, Younghun

2018-04-01

For three-dimensional microscopy, fast and high axial resolution are very important. Extending the depth of field for digital holographic is necessary for three-dimensional measurements of thick samples. We propose an optical sectioning method for optical scanning digital holography that is performed in the frequency domain by spatial filtering of a reconstructed amplitude image. We established a scanning dual-wavelength off-axis digital holographic microscope to measure samples that exhibit a large amount of coherent noise and a thickness larger than the depth of focus of the objective lens. As a demonstration, we performed a three-dimensional measurement of a fine metal mask with a reconstructed sectional phase image and filtering with a reconstructed amplitude image.

Periscope for noninvasive two-photon imaging of murine retina in vivo

PubMed Central

Stremplewski, Patrycjusz; Komar, Katarzyna; Palczewski, Krzysztof; Wojtkowski, Maciej; Palczewska, Grazyna

2015-01-01

Two-photon microscopy allows visualization of subcellular structures in the living animal retina. In previously reported experiments it was necessary to apply a contact lens to each subject. Extending this technology to larger animals would require fitting a custom contact lens to each animal and cumbersome placement of the living animal head on microscope stage. Here we demonstrate a new device, periscope, for coupling light energy into mouse eye and capturing emitted fluorescence. Using this periscope we obtained images of the RPE and their subcellular organelles, retinosomes, with larger field of view than previously reported. This periscope provides an interface with a commercial microscope, does not require contact lens and its design could be modified to image retina in larger animals. PMID:26417507

Long working distance incoherent interference microscope

DOEpatents

Sinclair, Michael B [Albuquerque, NM; De Boer, Maarten P [Albuquerque, NM

2006-04-25

A full-field imaging, long working distance, incoherent interference microscope suitable for three-dimensional imaging and metrology of MEMS devices and test structures on a standard microelectronics probe station. A long working distance greater than 10 mm allows standard probes or probe cards to be used. This enables nanometer-scale 3-dimensional height profiles of MEMS test structures to be acquired across an entire wafer while being actively probed, and, optionally, through a transparent window. An optically identical pair of sample and reference arm objectives is not required, which reduces the overall system cost, and also the cost and time required to change sample magnifications. Using a LED source, high magnification (e.g., 50.times.) can be obtained having excellent image quality, straight fringes, and high fringe contrast.

Speckle-free and halo-free low coherent Mach-Zehnder quantitative-phase-imaging module as a replacement of objective lens in conventional inverted microscopes

NASA Astrophysics Data System (ADS)

Yamauchi, Toyohiko; Yamada, Hidenao; Matsui, Hisayuki; Yasuhiko, Osamu; Ueda, Yukio

2018-02-01

We developed a compact Mach-Zehnder interferometer module to be used as a replacement of the objective lens in a conventional inverted microscope (Nikon, TS100-F) in order to make them quantitative phase microscopes. The module has a 90-degree-flipped U-shape; the dimensions of the module are 160 mm by 120 mm by 40 mm and the weight is 380 grams. The Mach-Zehnder interferometer equipped with the separate reference and sample arms was implemented in this U-shaped housing and the path-length difference between the two arms was manually adjustable. The sample under test was put on the stage of the microscope and a sample light went through it. Both arms had identical achromatic lenses for image formation and the lateral positions of them were also manually adjustable. Therefore, temporally and spatially low coherent illumination was applicable because the users were able to balance precisely the path length of the two arms and to overlap the two wavefronts. In the experiment, spectrally filtered LED light for illumination (wavelength = 633 nm and bandwidth = 3 nm) was input to the interferometer module via a 50 micrometer core optical fiber. We have successfully captured full-field interference images by a camera put on the trinocular tube of the microscope and constructed quantitative phase images of the cultured cells by means of the quarter-wavelength phase shifting algorithm. The resultant quantitative phase images were speckle-free and halo-free due to spectrally and spatially low coherent illumination.

eeDAP: An Evaluation Environment for Digital and Analog Pathology

PubMed Central

Gallas, Brandon D.; Cheng, Wei-Chung; Gavrielides, Marios A.; Ivansky, Adam; Keay, Tyler; Wunderlich, Adam; Hipp, Jason; Hewitt, Stephen M.

2017-01-01

Purpose The purpose of this work is to present a platform for designing and executing studies that compare pathologists interpreting histopathology of whole slide images (WSI) on a computer display to pathologists interpreting glass slides on an optical microscope. Methods Here we present eeDAP, an evaluation environment for digital and analog pathology. The key element in eeDAP is the registration of the WSI to the glass slide. Registration is accomplished through computer control of the microscope stage and a camera mounted on the microscope that acquires images of the real time microscope view. Registration allows for the evaluation of the same regions of interest (ROIs) in both domains. This can reduce or eliminate disagreements that arise from pathologists interpreting different areas and focuses the comparison on image quality. Results We reduced the pathologist interpretation area from an entire glass slide (≈10–30 mm)2 to small ROIs <(50 um)2. We also made possible the evaluation of individual cells. Conclusions We summarize eeDAP’s software and hardware and provide calculations and corresponding images of the microscope field of view and the ROIs extracted from the WSIs. These calculations help provide a sense of eeDAP’s functionality and operating principles, while the images provide a sense of the look and feel of studies that can be conducted in the digital and analog domains. The eeDAP software can be downloaded from code.google.com (project: eeDAP) as Matlab source or as a precompiled stand-alone license-free application. PMID:28845079

Imaging microscopic structures in pathological retinas using a flood-illumination adaptive optics retinal camera

NASA Astrophysics Data System (ADS)

Viard, Clément; Nakashima, Kiyoko; Lamory, Barbara; Pâques, Michel; Levecq, Xavier; Château, Nicolas

2011-03-01

This research is aimed at characterizing in vivo differences between healthy and pathological retinal tissues at the microscopic scale using a compact adaptive optics (AO) retinal camera. Tests were performed in 120 healthy eyes and 180 eyes suffering from 19 different pathological conditions, including age-related maculopathy (ARM), glaucoma and rare diseases such as inherited retinal dystrophies. Each patient was first examined using SD-OCT and infrared SLO. Retinal areas of 4°x4° were imaged using an AO flood-illumination retinal camera based on a large-stroke deformable mirror. Contrast was finally enhanced by registering and averaging rough images using classical algorithms. Cellular-resolution images could be obtained in most cases. In ARM, AO images revealed granular contents in drusen, which were invisible in SLO or OCT images, and allowed the observation of the cone mosaic between drusen. In glaucoma cases, visual field was correlated to changes in cone visibility. In inherited retinal dystrophies, AO helped to evaluate cone loss across the retina. Other microstructures, slightly larger in size than cones, were also visible in several retinas. AO provided potentially useful diagnostic and prognostic information in various diseases. In addition to cones, other microscopic structures revealed by AO images may also be of interest in monitoring retinal diseases.

Transmission X-ray microscopy for full-field nano imaging of biomaterials.

PubMed

Andrews, Joy C; Meirer, Florian; Liu, Yijin; Mester, Zoltan; Pianetta, Piero

2011-07-01

Imaging of cellular structure and extended tissue in biological materials requires nanometer resolution and good sample penetration, which can be provided by current full-field transmission X-ray microscopic techniques in the soft and hard X-ray regions. The various capabilities of full-field transmission X-ray microscopy (TXM) include 3D tomography, Zernike phase contrast, quantification of absorption, and chemical identification via X-ray fluorescence and X-ray absorption near edge structure imaging. These techniques are discussed and compared in light of results from the imaging of biological materials including microorganisms, bone and mineralized tissue, and plants, with a focus on hard X-ray TXM at ≤ 40-nm resolution. Copyright © 2010 Wiley-Liss, Inc.

Automatic detection of the hippocampal region associated with Alzheimer's disease from microscopic images of mice brain

NASA Astrophysics Data System (ADS)

Albaidhani, Tahseen; Hawkes, Cheryl; Jassim, Sabah; Al-Assam, Hisham

2016-05-01

The hippocampus is the region of the brain that is primarily associated with memory and spatial navigation. It is one of the first brain regions to be damaged when a person suffers from Alzheimer's disease. Recent research in this field has focussed on the assessment of damage to different blood vessels within the hippocampal region from a high throughput brain microscopic images. The ultimate aim of our research is the creation of an automatic system to count and classify different blood vessels such as capillaries, veins, and arteries in the hippocampus region. This work should provide biologists with efficient and accurate tools in their investigation of the causes of Alzheimer's disease. Locating the boundary of the Region of Interest in the hippocampus from microscopic images of mice brain is the first essential stage towards developing such a system. This task benefits from the variation in colour channels and texture between the two sides of the hippocampus and the boundary region. Accordingly, the developed initial step of our research to locating the hippocampus edge uses a colour-based segmentation of the brain image followed by Hough transforms on the colour channel that isolate the hippocampus region. The output is then used to split the brain image into two sides of the detected section of the boundary: the inside region and the outside region. Experimental results on a sufficiently number of microscopic images demonstrate the effectiveness of the developed solution.

Dark-field microscopic image stitching method for surface defects evaluation of large fine optics.

PubMed

Liu, Dong; Wang, Shitong; Cao, Pin; Li, Lu; Cheng, Zhongtao; Gao, Xin; Yang, Yongying

2013-03-11

One of the challenges in surface defects evaluation of large fine optics is to detect defects of microns on surfaces of tens or hundreds of millimeters. Sub-aperture scanning and stitching is considered to be a practical and efficient method. But since there are usually few defects on the large aperture fine optics, resulting in no defects or only one run-through line feature in many sub-aperture images, traditional stitching methods encounter with mismatch problem. In this paper, a feature-based multi-cycle image stitching algorithm is proposed to solve the problem. The overlapping areas of sub-apertures are categorized based on the features they contain. Different types of overlapping areas are then stitched in different cycles with different methods. The stitching trace is changed to follow the one that determined by the features. The whole stitching procedure is a region-growing like process. Sub-aperture blocks grow bigger after each cycle and finally the full aperture image is obtained. Comparison experiment shows that the proposed method is very suitable to stitch sub-apertures that very few feature information exists in the overlapping areas and can stitch the dark-field microscopic sub-aperture images very well.

Computational microscopy: illumination coding and nonlinear optimization enables gigapixel 3D phase imaging

NASA Astrophysics Data System (ADS)

Tian, Lei; Waller, Laura

2017-05-01

Microscope lenses can have either large field of view (FOV) or high resolution, not both. Computational microscopy based on illumination coding circumvents this limit by fusing images from different illumination angles using nonlinear optimization algorithms. The result is a Gigapixel-scale image having both wide FOV and high resolution. We demonstrate an experimentally robust reconstruction algorithm based on a 2nd order quasi-Newton's method, combined with a novel phase initialization scheme. To further extend the Gigapixel imaging capability to 3D, we develop a reconstruction method to process the 4D light field measurements from sequential illumination scanning. The algorithm is based on a 'multislice' forward model that incorporates both 3D phase and diffraction effects, as well as multiple forward scatterings. To solve the inverse problem, an iterative update procedure that combines both phase retrieval and 'error back-propagation' is developed. To avoid local minimum solutions, we further develop a novel physical model-based initialization technique that accounts for both the geometric-optic and 1st order phase effects. The result is robust reconstructions of Gigapixel 3D phase images having both wide FOV and super resolution in all three dimensions. Experimental results from an LED array microscope were demonstrated.

Microscopic image processing systems for measuring nonuniform film thickness profiles

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

Liu, A.H.; Plawsky, J.L.; DasGupta, S.

1994-01-01

In very thin liquid films. transport processes are controlled by the temperature and the interfacial intermolecular force field which is a function of the film thickness profile and interfacial properties. The film thickness profile and interfacial properties can be measured most efficiently using a microscopic image processing system. IPS, to record the intensity pattern of the reflected light from the film. There are two types of IPS: an image analyzing interferometer (IAI) and/or an image scanning ellipsometer (ISE). The ISE is a novel technique to measure the two dimensional thickness profile of a nonuniform, thin film, from 1 nm upmore » to several {mu}m, in a steady state as well as in a transient state. It is a full field imaging technique which can study every point on the surface simultaneously with high spatial resolution and thickness sensitivity, i.e., it can measure and map the 2-D film thickness profile. Using the ISE, the transient thickness profile of a draining thin liquid film was measured and modeled. The interfacial conditions were determined in situ by measuring the Hamaker constant. The ISE and IAI systems are compared.« less

Single shot, three-dimensional fluorescence microscopy with a spatially rotating point spread function

PubMed Central

Wang, Zhaojun; Cai, Yanan; Liang, Yansheng; Zhou, Xing; Yan, Shaohui; Dan, Dan; Bianco, Piero R.; Lei, Ming; Yao, Baoli

2017-01-01

A wide-field fluorescence microscope with a double-helix point spread function (PSF) is constructed to obtain the specimen’s three-dimensional distribution with a single snapshot. Spiral-phase-based computer-generated holograms (CGHs) are adopted to make the depth-of-field of the microscope adjustable. The impact of system aberrations on the double-helix PSF at high numerical aperture is analyzed to reveal the necessity of the aberration correction. A modified cepstrum-based reconstruction scheme is promoted in accordance with properties of the new double-helix PSF. The extended depth-of-field images and the corresponding depth maps for both a simulated sample and a tilted section slice of bovine pulmonary artery endothelial (BPAE) cells are recovered, respectively, verifying that the depth-of-field is properly extended and the depth of the specimen can be estimated at a precision of 23.4nm. This three-dimensional fluorescence microscope with a framerate-rank time resolution is suitable for studying the fast developing process of thin and sparsely distributed micron-scale cells in extended depth-of-field. PMID:29296483

Gradient descent algorithm applied to wavefront retrieval from through-focus images by an extreme ultraviolet microscope with partially coherent source

DOE PAGES

Yamazoe, Kenji; Mochi, Iacopo; Goldberg, Kenneth A.

2014-12-01

The wavefront retrieval by gradient descent algorithm that is typically applied to coherent or incoherent imaging is extended to retrieve a wavefront from a series of through-focus images by partially coherent illumination. For accurate retrieval, we modeled partial coherence as well as object transmittance into the gradient descent algorithm. However, this modeling increases the computation time due to the complexity of partially coherent imaging simulation that is repeatedly used in the optimization loop. To accelerate the computation, we incorporate not only the Fourier transform but also an eigenfunction decomposition of the image. As a demonstration, the extended algorithm is appliedmore » to retrieve a field-dependent wavefront of a microscope operated at extreme ultraviolet wavelength (13.4 nm). The retrieved wavefront qualitatively matches the expected characteristics of the lens design.« less

Gradient descent algorithm applied to wavefront retrieval from through-focus images by an extreme ultraviolet microscope with partially coherent source

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

Yamazoe, Kenji; Mochi, Iacopo; Goldberg, Kenneth A.

The wavefront retrieval by gradient descent algorithm that is typically applied to coherent or incoherent imaging is extended to retrieve a wavefront from a series of through-focus images by partially coherent illumination. For accurate retrieval, we modeled partial coherence as well as object transmittance into the gradient descent algorithm. However, this modeling increases the computation time due to the complexity of partially coherent imaging simulation that is repeatedly used in the optimization loop. To accelerate the computation, we incorporate not only the Fourier transform but also an eigenfunction decomposition of the image. As a demonstration, the extended algorithm is appliedmore » to retrieve a field-dependent wavefront of a microscope operated at extreme ultraviolet wavelength (13.4 nm). The retrieved wavefront qualitatively matches the expected characteristics of the lens design.« less

A low cost mobile phone dark-field microscope for nanoparticle-based quantitative studies.

PubMed

Sun, Dali; Hu, Tony Y

2018-01-15

Dark-field microscope (DFM) analysis of nanoparticle binding signal is highly useful for a variety of research and biomedical applications, but current applications for nanoparticle quantification rely on expensive DFM systems. The cost, size, limited robustness of these DFMs limits their utility for non-laboratory settings. Most nanoparticle analyses use high-magnification DFM images, which are labor intensive to acquire and subject to operator bias. Low-magnification DFM image capture is faster, but is subject to background from surface artifacts and debris, although image processing can partially compensate for background signal. We thus mated an LED light source, a dark-field condenser and a 20× objective lens with a mobile phone camera to create an inexpensive, portable and robust DFM system suitable for use in non-laboratory conditions. This proof-of-concept mobile DFM device weighs less than 400g and costs less than $2000, but analysis of images captured with this device reveal similar nanoparticle quantitation results to those acquired with a much larger and more expensive desktop DFMM system. Our results suggest that similar devices may be useful for quantification of stable, nanoparticle-based activity and quantitation assays in resource-limited areas where conventional assay approaches are not practical. Copyright © 2017 Elsevier B.V. All rights reserved.

Multicolor fluorescence microscopic imaging of cancer cells on the plasmonic chip (Presentation Recording)

NASA Astrophysics Data System (ADS)

Tawa, Keiko; Sasakawa, Chisato; Yamamura, Shohei; Shibata, Izumi; Kataoka, Masatoshi

2015-09-01

A plasmonic chip which is a metal coated substrate with grating structure can provide the enhanced fluorescence by the grating-coupled surface plasmon field. In our previous studies, bright epi-fluorescence microscopic imaging of neuron cells and sensitive immunosesnsing have been reported. In this study, two kinds of breast cancer cells, MCF-7 and MDA-MB231, were observed with epi-fluorescence microscope on the plasmonic chip with 2D hole-arrays . They were multicolor stained with 4', 6-diamidino-2-phenylindole (DAPI) and allophycocyanin (APC)-labeled anti-epithelial cell adhesion molecule (EpCAM) antibody. Our plasmonic chip provided the brighter fluorescence images of these cells compared with the glass slide. Even in the cells including few EpCAM, the distribution of EpCAM was clearly observed in the cell membrane. It was found that the plasmonic chip can be one of the powerful tools to detect the marker protein existing around the chip surface even at low concentration.

High-performance imaging of stem cells using single-photon emissions

NASA Astrophysics Data System (ADS)

Wagenaar, Douglas J.; Moats, Rex A.; Hartsough, Neal E.; Meier, Dirk; Hugg, James W.; Yang, Tang; Gazit, Dan; Pelled, Gadi; Patt, Bradley E.

2011-10-01

Radiolabeled cells have been imaged for decades in the field of autoradiography. Recent advances in detector and microelectronics technologies have enabled the new field of "digital autoradiography" which remains limited to ex vivo specimens of thin tissue slices. The 3D field-of-view (FOV) of single cell imaging can be extended to millimeters if the low energy (10-30 keV) photon emissions of radionuclides are used for single-photon nuclear imaging. This new microscope uses a coded aperture foil made of highly attenuating elements such as gold or platinum to form the image as a kind of "lens". The detectors used for single-photon emission microscopy are typically silicon detectors with a pixel pitch less than 60 μm. The goal of this work is to image radiolabeled mesenchymal stem cells in vivo in an animal model of tendon repair processes. Single-photon nuclear imaging is an attractive modality for translational medicine since the labeled cells can be imaged simultaneously with the reparative processes by using the dual-isotope imaging technique. The details our microscope's two-layer gold aperture and the operation of the energy-dispersive, pixellated silicon detector are presented along with the first demonstration of energy discrimination with a 57Co source. Cell labeling techniques have been augmented by genetic engineering with the sodium-iodide symporter, a type of reporter gene imaging method that enables in vivo uptake of free 99mTc or an iodine isotope at a time point days or weeks after the insertion of the genetically modified stem cells into the animal model. This microscopy work in animal research may expand to the imaging of reporter-enabled stem cells simultaneously with the expected biological repair process in human clinical trials of stem cell therapies.

Microscopy illumination engineering using a low-cost liquid crystal display.

PubMed

Guo, Kaikai; Bian, Zichao; Dong, Siyuan; Nanda, Pariksheet; Wang, Ying Min; Zheng, Guoan

2015-02-01

Illumination engineering is critical for obtaining high-resolution, high-quality images in microscope settings. In a typical microscope, the condenser lens provides sample illumination that is uniform and free from glare. The associated condenser diaphragm can be manually adjusted to obtain the optimal illumination numerical aperture. In this paper, we report a programmable condenser lens for active illumination control. In our prototype setup, we used a $15 liquid crystal display as a transparent spatial light modulator and placed it at the back focal plane of the condenser lens. By setting different binary patterns on the display, we can actively control the illumination and the spatial coherence of the microscope platform. We demonstrated the use of such a simple scheme for multimodal imaging, including bright-field microscopy, darkfield microscopy, phase-contrast microscopy, polarization microscopy, 3D tomographic imaging, and super-resolution Fourier ptychographic imaging. The reported illumination engineering scheme is cost-effective and compatible with most existing platforms. It enables a turnkey solution with high flexibility for researchers in various communities. From the engineering point-of-view, the reported illumination scheme may also provide new insights for the development of multimodal microscopy and Fourier ptychographic imaging.

Quantitative label-free sperm imaging by means of transport of intensity

NASA Astrophysics Data System (ADS)

Poola, Praveen Kumar; Pandiyan, Vimal Prabhu; Jayaraman, Varshini; John, Renu

2016-03-01

Most living cells are optically transparent which makes it difficult to visualize them under bright field microscopy. Use of contrast agents or markers and staining procedures are often followed to observe these cells. However, most of these staining agents are toxic and not applicable for live cell imaging. In the last decade, quantitative phase imaging has become an indispensable tool for morphological characterization of the phase objects without any markers. In this paper, we report noninterferometric quantitative phase imaging of live sperm cells by solving transport of intensity equations with recorded intensity measurements along optical axis on a commercial bright field microscope.

EVALUATION OF CONFOCAL MICROSCOPY SYSTEM PERFORMANCE: APPLICATIONS FOR IMAGING MORPHOLOGY AND DEATH IN EMBRYOS AND REPRODUCTIVE TISSUE/ORGANS

EPA Science Inventory

The confocal laser-scanning microscope (CLSM) has enormous potential in many biological fields. It is remarkable that procedures to test the performance of these machines are not done routinely by most investigators and thus many of the machines in the field are working at level...

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.

Tunneling magnetic force microscopy

NASA Technical Reports Server (NTRS)

Burke, Edward R.; Gomez, Romel D.; Adly, Amr A.; Mayergoyz, Isaak D.

1993-01-01

We have developed a powerful new tool for studying the magnetic patterns on magnetic recording media. This was accomplished by modifying a conventional scanning tunneling microscope. The fine-wire probe that is used to image surface topography was replaced with a flexible magnetic probe. Images obtained with these probes reveal both the surface topography and the magnetic structure. We have made a thorough theoretical analysis of the interaction between the probe and the magnetic fields emanating from a typical recorded surface. Quantitative data about the constituent magnetic fields can then be obtained. We have employed these techniques in studies of two of the most important issues of magnetic record: data overwrite and maximizing data-density. These studies have shown: (1) overwritten data can be retrieved under certain conditions; and (2) improvements in data-density will require new magnetic materials. In the course of these studies we have developed new techniques to analyze magnetic fields of recorded media. These studies are both theoretical and experimental and combined with the use of our magnetic force scanning tunneling microscope should lead to further breakthroughs in the field of magnetic recording.

A 30 mK, 13.5 T scanning tunneling microscope with two independent tips.

PubMed

Roychowdhury, Anita; Gubrud, M A; Dana, R; Anderson, J R; Lobb, C J; Wellstood, F C; Dreyer, M

2014-04-01

We describe the design, construction, and performance of an ultra-low temperature, high-field scanning tunneling microscope (STM) with two independent tips. The STM is mounted on a dilution refrigerator and operates at a base temperature of 30 mK with magnetic fields of up to 13.5 T. We focus on the design of the two-tip STM head, as well as the sample transfer mechanism, which allows in situ transfer from an ultra high vacuum preparation chamber while the STM is at 1.5 K. Other design details such as the vibration isolation and rf-filtered wiring are also described. Their effectiveness is demonstrated via spectral current noise characteristics and the root mean square roughness of atomic resolution images. The high-field capability is shown by the magnetic field dependence of the superconducting gap of CuxBi2Se3. Finally, we present images and spectroscopy taken with superconducting Nb tips with the refrigerator at 35 mK that indicate that the effective temperature of our tips/sample is approximately 184 mK, corresponding to an energy resolution of 16 μeV.

Phase from defocus

NASA Astrophysics Data System (ADS)

Mandula, Ondrej; Allier, Cédric; Hervé, Lionel; Denarier, Eric; Fourest-Lieuvin, Anne; Gory-Fauré, Sylvie; Vinit, Angélique; Morales, Sophie

2018-02-01

We present a simple and compact phase imaging microscope for long-term observation of non-absorbing biological samples such as unstained cells in nutritive media. The phase image is obtained from a single defocused image taken with a standard wide-field microscope. Using a semi-coherent light source allows us to computationally re-focus image post-acquisition and recover both phase and transmission of the complex specimen. The simplicity of the system reduces both the cost and its physical size and allows a long-term observation of samples directly in a standard biological incubator. The low cost of the system can contribute to the democratization of science by allowing to perform complex long-term biological experiments to the laboratories with constrained budget. In this proceeding we present several results taken with our prototype and discuss the possibilities and limitations of our system.

Single DNA imaging and length quantification through a mobile phone microscope

NASA Astrophysics Data System (ADS)

Wei, Qingshan; Luo, Wei; Chiang, Samuel; Kappel, Tara; Mejia, Crystal; Tseng, Derek; Chan, Raymond Yan L.; Yan, Eddie; Qi, Hangfei; Shabbir, Faizan; Ozkan, Haydar; Feng, Steve; Ozcan, Aydogan

2016-03-01

The development of sensitive optical microscopy methods for the detection of single DNA molecules has become an active research area which cultivates various promising applications including point-of-care (POC) genetic testing and diagnostics. Direct visualization of individual DNA molecules usually relies on sophisticated optical microscopes that are mostly available in well-equipped laboratories. For POC DNA testing/detection, there is an increasing need for the development of new single DNA imaging and sensing methods that are field-portable, cost-effective, and accessible for diagnostic applications in resource-limited or field-settings. For this aim, we developed a mobile-phone integrated fluorescence microscopy platform that allows imaging and sizing of single DNA molecules that are stretched on a chip. This handheld device contains an opto-mechanical attachment integrated onto a smartphone camera module, which creates a high signal-to-noise ratio dark-field imaging condition by using an oblique illumination/excitation configuration. Using this device, we demonstrated imaging of individual linearly stretched λ DNA molecules (48 kilobase-pair, kbp) over 2 mm2 field-of-view. We further developed a robust computational algorithm and a smartphone app that allowed the users to quickly quantify the length of each DNA fragment imaged using this mobile interface. The cellphone based device was tested by five different DNA samples (5, 10, 20, 40, and 48 kbp), and a sizing accuracy of <1 kbp was demonstrated for DNA strands longer than 10 kbp. This mobile DNA imaging and sizing platform can be very useful for various diagnostic applications including the detection of disease-specific genes and quantification of copy-number-variations at POC settings.

Optical Coherence Tomography for Retinal Surgery: Perioperative Analysis to Real-Time Four-Dimensional Image-Guided Surgery.

PubMed

Carrasco-Zevallos, Oscar M; Keller, Brenton; Viehland, Christian; Shen, Liangbo; Seider, Michael I; Izatt, Joseph A; Toth, Cynthia A

2016-07-01

Magnification of the surgical field using the operating microscope facilitated profound innovations in retinal surgery in the 1970s, such as pars plana vitrectomy. Although surgical instrumentation and illumination techniques are continually developing, the operating microscope for vitreoretinal procedures has remained essentially unchanged and currently limits the surgeon's depth perception and assessment of subtle microanatomy. Optical coherence tomography (OCT) has revolutionized clinical management of retinal pathology, and its introduction into the operating suite may have a similar impact on surgical visualization and treatment. In this article, we review the evolution of OCT for retinal surgery, from perioperative analysis to live volumetric (four-dimensional, 4D) image-guided surgery. We begin by briefly addressing the benefits and limitations of the operating microscope, the progression of OCT technology, and OCT applications in clinical/perioperative retinal imaging. Next, we review intraoperative OCT (iOCT) applications using handheld probes during surgical pauses, two-dimensional (2D) microscope-integrated OCT (MIOCT) of live surgery, and volumetric MIOCT of live surgery. The iOCT discussion focuses on technological advancements, applications during human retinal surgery, translational difficulties and limitations, and future directions.

In vivo observation of age-related structural changes of dermal collagen in human facial skin using collagen-sensitive second harmonic generation microscope equipped with 1250-nm mode-locked Cr:Forsterite laser

NASA Astrophysics Data System (ADS)

Yasui, Takeshi; Yonetsu, Makoto; Tanaka, Ryosuke; Tanaka, Yuji; Fukushima, Shu-ichiro; Yamashita, Toyonobu; Ogura, Yuki; Hirao, Tetsuji; Murota, Hiroyuki; Araki, Tsutomu

2013-03-01

In vivo visualization of human skin aging is demonstrated using a Cr:Forsterite (Cr:F) laser-based, collagen-sensitive second harmonic generation (SHG) microscope. The deep penetration into human skin, as well as the specific sensitivity to collagen molecules, achieved by this microscope enables us to clearly visualize age-related structural changes of collagen fiber in the reticular dermis. Here we investigated intrinsic aging and/or photoaging in the male facial skin. Young subjects show dense distributions of thin collagen fibers, whereas elderly subjects show coarse distributions of thick collagen fibers. Furthermore, a comparison of SHG images between young and elderly subjects with and without a recent life history of excessive sun exposure show that a combination of photoaging with intrinsic aging significantly accelerates skin aging. We also perform image analysis based on two-dimensional Fourier transformation of the SHG images and extracted an aging parameter for human skin. The in vivo collagen-sensitive SHG microscope will be a powerful tool in fields such as cosmeceutical sciences and anti-aging dermatology.

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.

Optical Coherence Tomography for Retinal Surgery: Perioperative Analysis to Real-Time Four-Dimensional Image-Guided Surgery

PubMed Central

Carrasco-Zevallos, Oscar M.; Keller, Brenton; Viehland, Christian; Shen, Liangbo; Seider, Michael I.; Izatt, Joseph A.; Toth, Cynthia A.

2016-01-01

Magnification of the surgical field using the operating microscope facilitated profound innovations in retinal surgery in the 1970s, such as pars plana vitrectomy. Although surgical instrumentation and illumination techniques are continually developing, the operating microscope for vitreoretinal procedures has remained essentially unchanged and currently limits the surgeon's depth perception and assessment of subtle microanatomy. Optical coherence tomography (OCT) has revolutionized clinical management of retinal pathology, and its introduction into the operating suite may have a similar impact on surgical visualization and treatment. In this article, we review the evolution of OCT for retinal surgery, from perioperative analysis to live volumetric (four-dimensional, 4D) image-guided surgery. We begin by briefly addressing the benefits and limitations of the operating microscope, the progression of OCT technology, and OCT applications in clinical/perioperative retinal imaging. Next, we review intraoperative OCT (iOCT) applications using handheld probes during surgical pauses, two-dimensional (2D) microscope-integrated OCT (MIOCT) of live surgery, and volumetric MIOCT of live surgery. The iOCT discussion focuses on technological advancements, applications during human retinal surgery, translational difficulties and limitations, and future directions. PMID:27409495

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

Whole slide imaging of unstained tissue using lensfree microscopy

NASA Astrophysics Data System (ADS)

Morel, Sophie Nhu An; Hervé, Lionel; Bordy, Thomas; Cioni, Olivier; Delon, Antoine; Fromentin, Catherine; Dinten, Jean-Marc; Allier, Cédric

2016-04-01

Pathologist examination of tissue slides provides insightful information about a patient's disease. Traditional analysis of tissue slides is performed under a binocular microscope, which requires staining of the sample and delays the examination. We present a simple cost-effective lensfree imaging method to record 2-4μm resolution wide-field (10 mm2 to 6 cm2) images of unstained tissue slides. The sample processing time is reduced as there is no need for staining. A wide field of view (10 mm2) lensfree hologram is recorded in a single shot and the image is reconstructed in 2s providing a very fast acquisition chain. The acquisition is multispectral, i.e. multiple holograms are recorded simultaneously at three different wavelengths, and a dedicated holographic reconstruction algorithm is used to retrieve both amplitude and phase. Whole tissue slides imaging is obtained by recording 130 holograms with X-Y translation stages and by computing the mosaic of a 25 x 25 mm2 reconstructed image. The reconstructed phase provides a phase-contrast-like image of the unstained specimen, revealing structures of healthy and diseased tissue. Slides from various organs can be reconstructed, e.g. lung, colon, ganglion, etc. To our knowledge, our method is the first technique that enables fast wide-field lensfree imaging of such unlabeled dense samples. This technique is much cheaper and compact than a conventional phase contrast microscope and could be made portable. In sum, we present a new methodology that could quickly provide useful information when a rapid diagnosis is needed, such as tumor margin identification on frozen section biopsies during surgery.

Optically Sectioned Imaging of Microvasculature of In-Vivo and Ex-Vivo Thick Tissue Models with Speckle-illumination HiLo Microscopy and HiLo Image Processing Implementation in MATLAB Architecture

NASA Astrophysics Data System (ADS)

Suen, Ricky Wai

The work described in this thesis covers the conversion of HiLo image processing into MATLAB architecture and the use of speckle-illumination HiLo microscopy for use of ex-vivo and in-vivo imaging of thick tissue models. HiLo microscopy is a wide-field fluorescence imaging technique and has been demonstrated to produce optically sectioned images comparable to confocal in thin samples. The imaging technique was developed by Jerome Mertz and the Boston University Biomicroscopy Lab and has been implemented in our lab as a stand-alone optical setup and a modification to a conventional fluorescence microscope. Speckle-illumination HiLo microscopy combines two images taken under speckle-illumination and standard uniform-illumination to generate an optically sectioned image that reject out-of-focus fluorescence. The evaluated speckle contrast in the images is used as a weighting function where elements that move out-of-focus have a speckle contrast that decays to zero. The experiments shown here demonstrate the capability of our HiLo microscopes to produce optically-sectioned images of the microvasculature of ex-vivo and in-vivo thick tissue models. The HiLo microscope were used to image the microvasculature of ex-vivo mouse heart sections prepared for optical histology and the microvasculature of in-vivo rodent dorsal window chamber models. Studies in label-free surface profiling with HiLo microscopy is also presented.

Imaging Josephson vortices on the surface superconductor Si(111)-(√7×√3)-In using a scanning tunneling microscope.

PubMed

Yoshizawa, Shunsuke; Kim, Howon; Kawakami, Takuto; Nagai, Yuki; Nakayama, Tomonobu; Hu, Xiao; Hasegawa, Yukio; Uchihashi, Takashi

2014-12-12

We have studied the superconducting Si(111)-(√7×√3)-In surface using a ³He-based low-temperature scanning tunneling microscope. Zero-bias conductance images taken over a large surface area reveal that vortices are trapped at atomic steps after magnetic fields are applied. The crossover behavior from Pearl to Josephson vortices is clearly identified from their elongated shapes along the steps and significant recovery of superconductivity within the cores. Our numerical calculations combined with experiments clarify that these characteristic features are determined by the relative strength of the interterrace Josephson coupling at the atomic step.

Neural imaging in songbirds using fiber optic fluorescence microscopy

NASA Astrophysics Data System (ADS)

Nooshabadi, Fatemeh; Hearn, Gentry; Lints, Thierry; Maitland, Kristen C.

2012-02-01

The song control system of juvenile songbirds is an important model for studying the developmental acquisition and generation of complex learned vocal motor sequences, two processes that are fundamental to human speech and language. To understand the neural mechanisms underlying song production, it is critical to characterize the activity of identified neurons in the song control system when the bird is singing. Neural imaging in unrestrained singing birds, although technically challenging, will advance our understanding of neural ensemble coding mechanisms in this system. We are exploring the use of a fiber optic microscope for functional imaging in the brain of behaving and singing birds in order to better understand the contribution of a key brain nucleus (high vocal center nucleus; HVC) to temporal aspects of song motor control. We have constructed a fluorescence microscope with LED illumination, a fiber bundle for transmission of fluorescence excitation and emission light, a ~2x GRIN lens, and a CCD for image acquisition. The system has 2 μm resolution, 375 μm field of view, 200 μm working distance, and 1 mm outer diameter. As an initial characterization of this setup, neurons in HVC were imaged using the fiber optic microscope after injection of quantum dots or fluorescent retrograde tracers into different song nuclei. A Lucid Vivascope confocal microscope was used to confirm the imaging results. Long-term imaging of the activity of these neurons in juvenile birds during singing may lead us to a better understanding of the central motor codes for song and the central mechanism by which auditory experience modifies song motor commands to enable vocal learning and imitation.

4D microscope-integrated OCT improves accuracy of ophthalmic surgical maneuvers

NASA Astrophysics Data System (ADS)

Carrasco-Zevallos, Oscar; Keller, Brenton; Viehland, Christian; Shen, Liangbo; Todorich, Bozho; Shieh, Christine; Kuo, Anthony; Toth, Cynthia; Izatt, Joseph A.

2016-03-01

Ophthalmic surgeons manipulate micron-scale tissues using stereopsis through an operating microscope and instrument shadowing for depth perception. While ophthalmic microsurgery has benefitted from rapid advances in instrumentation and techniques, the basic principles of the stereo operating microscope have not changed since the 1930's. Optical Coherence Tomography (OCT) has revolutionized ophthalmic imaging and is now the gold standard for preoperative and postoperative evaluation of most retinal and many corneal procedures. We and others have developed initial microscope-integrated OCT (MIOCT) systems for concurrent OCT and operating microscope imaging, but these are limited to 2D real-time imaging and require offline post-processing for 3D rendering and visualization. Our previously presented 4D MIOCT system can record and display the 3D surgical field stereoscopically through the microscope oculars using a dual-channel heads-up display (HUD) at up to 10 micron-scale volumes per second. In this work, we show that 4D MIOCT guidance improves the accuracy of depth-based microsurgical maneuvers (with statistical significance) in mock surgery trials in a wet lab environment. Additionally, 4D MIOCT was successfully performed in 38/45 (84%) posterior and 14/14 (100%) anterior eye human surgeries, and revealed previously unrecognized lesions that were invisible through the operating microscope. These lesions, such as residual and potentially damaging retinal deformation during pathologic membrane peeling, were visualized in real-time by the surgeon. Our integrated system provides an enhanced 4D surgical visualization platform that can improve current ophthalmic surgical practice and may help develop and refine future microsurgical techniques.

Designs for a quantum electron microscope.

PubMed

Kruit, P; Hobbs, R G; Kim, C-S; Yang, Y; Manfrinato, V R; Hammer, J; Thomas, S; Weber, P; Klopfer, B; Kohstall, C; Juffmann, T; Kasevich, M A; Hommelhoff, P; Berggren, K K

2016-05-01

One of the astounding consequences of quantum mechanics is that it allows the detection of a target using an incident probe, with only a low probability of interaction of the probe and the target. This 'quantum weirdness' could be applied in the field of electron microscopy to generate images of beam-sensitive specimens with substantially reduced damage to the specimen. A reduction of beam-induced damage to specimens is especially of great importance if it can enable imaging of biological specimens with atomic resolution. Following a recent suggestion that interaction-free measurements are possible with electrons, we now analyze the difficulties of actually building an atomic resolution interaction-free electron microscope, or "quantum electron microscope". A quantum electron microscope would require a number of unique components not found in conventional transmission electron microscopes. These components include a coherent electron beam-splitter or two-state-coupler, and a resonator structure to allow each electron to interrogate the specimen multiple times, thus supporting high success probabilities for interaction-free detection of the specimen. Different system designs are presented here, which are based on four different choices of two-state-couplers: a thin crystal, a grating mirror, a standing light wave and an electro-dynamical pseudopotential. Challenges for the detailed electron optical design are identified as future directions for development. While it is concluded that it should be possible to build an atomic resolution quantum electron microscope, we have also identified a number of hurdles to the development of such a microscope and further theoretical investigations that will be required to enable a complete interpretation of the images produced by such a microscope. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Enhanced visualization of inner ear structures

NASA Astrophysics Data System (ADS)

Niemczyk, Kazimierz; Kucharski, Tomasz; Kujawinska, Malgorzata; Bruzgielewicz, Antoni

2004-07-01

Recently surgery requires extensive support from imaging technologies in order to increase effectiveness and safety of operations. One of important tasks is to enhance visualisation of quasi-phase (transparent) 3d structures. Those structures are characterized by very low contrast. It makes differentiation of tissues in field of view very difficult. For that reason the surgeon may be extremly uncertain during operation. This problem is connected with supporting operations of inner ear during which physician has to perform cuts at specific places of quasi-transparent velums. Conventionally during such operations medical doctor views the operating field through stereoscopic microscope. In the paper we propose a 3D visualisation system based on Helmet Mounted Display. Two CCD cameras placed at the output of microscope perform acquisition of stereo pairs of images. The images are processed in real-time with the goal of enhancement of quasi-phased structures. The main task is to create algorithm that is not sensitive to changes in intensity distribution. The disadvantages of existing algorithms is their lack of adaptation to occuring reflexes and shadows in field of view. The processed images from both left and right channels are overlaid on the actual images exported and displayed at LCD's of Helmet Mounted Display. A physician observes by HMD (Helmet Mounted Display) a stereoscopic operating scene with indication of the places of special interest. The authors present the hardware ,procedures applied and initial results of inner ear structure visualisation. Several problems connected with processing of stereo-pair images are discussed.

Field portable mobile phone based fluorescence microscopy for detection of Giardia lamblia cysts in water samples

NASA Astrophysics Data System (ADS)

Ceylan Koydemir, Hatice; Gorocs, Zoltan; McLeod, Euan; Tseng, Derek; Ozcan, Aydogan

2015-03-01

Giardia lamblia is a waterborne parasite that causes an intestinal infection, known as giardiasis, and it is found not only in countries with inadequate sanitation and unsafe water but also streams and lakes of developed countries. Simple, sensitive, and rapid detection of this pathogen is important for monitoring of drinking water. Here we present a cost-effective and field portable mobile-phone based fluorescence microscopy platform designed for automated detection of Giardia lamblia cysts in large volume water samples (i.e., 10 ml) to be used in low-resource field settings. This fluorescence microscope is integrated with a disposable water-sampling cassette, which is based on a flow-through porous polycarbonate membrane and provides a wide surface area for fluorescence imaging and enumeration of the captured Giardia cysts on the membrane. Water sample of interest, containing fluorescently labeled Giardia cysts, is introduced into the absorbent pads that are in contact with the membrane in the cassette by capillary action, which eliminates the need for electrically driven flow for sample processing. Our fluorescence microscope weighs ~170 grams in total and has all the components of a regular microscope, capable of detecting individual fluorescently labeled cysts under light-emitting-diode (LED) based excitation. Including all the sample preparation, labeling and imaging steps, the entire measurement takes less than one hour for a sample volume of 10 ml. This mobile phone based compact and cost-effective fluorescent imaging platform together with its machine learning based cyst counting interface is easy to use and can even work in resource limited and field settings for spatio-temporal monitoring of water quality.

Proposed alteration of images of molecular orbitals obtained using a scanning tunneling microscope as a probe of electron correlation.

PubMed

Toroz, Dimitrios; Rontani, Massimo; Corni, Stefano

2013-01-04

Scanning tunneling spectroscopy (STS) allows us to image single molecules decoupled from the supporting substrate. The obtained images are routinely interpreted as the square moduli of molecular orbitals, dressed by the mean-field electron-electron interaction. Here we demonstrate that the effect of electron correlation beyond the mean field qualitatively alters the uncorrelated STS images. Our evidence is based on the ab initio many-body calculation of STS images of planar molecules with metal centers. We find that many-body correlations alter significantly the image spectral weight close to the metal center of the molecules. This change is large enough to be accessed experimentally, surviving to molecule-substrate interactions.

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.

Coherent imaging with incoherent light in digital holographic microscopy

NASA Astrophysics Data System (ADS)

Chmelik, Radim

2012-01-01

Digital holographic microscope (DHM) allows for imaging with a quantitative phase contrast. In this way it becomes an important instrument, a completely non-invasive tool for a contrast intravital observation of living cells and a cell drymass density distribution measurement. A serious drawback of current DHMs is highly coherent illumination which makes the lateral resolution worse and impairs the image quality by a coherence noise and a parasitic interference. An uncompromising solution to this problem can be found in the Leith concept of incoherent holography. An off-axis hologram can be formed with arbitrary degree of light coherence in systems equipped with an achromatic interferometer and thus the resolution and the image quality typical for an incoherent-light wide-field microscopy can be achieved. In addition, advanced imaging modes based on limited coherence can be utilized. The typical example is a coherence-gating effect which provides a finite axial resolution and makes DHM image similar to that of a confocal microscope. These possibilities were described theoretically using the formalism of three-dimensional coherent transfer functions and proved experimentally by the coherence-controlled holographic microscope which is DHM based on the Leith achromatic interferometer. Quantitative-phase-contrast imaging is demonstrated with incoherent light by the living cancer cells observation and their motility evaluation. The coherence-gating effect was proved by imaging of model samples through a scattering layer and living cells inside an opalescent medium.

FPscope: a field-portable high-resolution microscope using a cellphone lens.

PubMed

Dong, Siyuan; Guo, Kaikai; Nanda, Pariksheet; Shiradkar, Radhika; Zheng, Guoan

2014-10-01

The large consumer market has made cellphone lens modules available at low-cost and in high-quality. In a conventional cellphone camera, the lens module is used to demagnify the scene onto the image plane of the camera, where image sensor is located. In this work, we report a 3D-printed high-resolution Fourier ptychographic microscope, termed FPscope, which uses a cellphone lens in a reverse manner. In our platform, we replace the image sensor with sample specimens, and use the cellphone lens to project the magnified image to the detector. To supersede the diffraction limit of the lens module, we use an LED array to illuminate the sample from different incident angles and synthesize the acquired images using the Fourier ptychographic algorithm. As a demonstration, we use the reported platform to acquire high-resolution images of resolution target and biological specimens, with a maximum synthetic numerical aperture (NA) of 0.5. We also show that, the depth-of-focus of the reported platform is about 0.1 mm, orders of magnitude longer than that of a conventional microscope objective with a similar NA. The reported platform may enable healthcare accesses in low-resource settings. It can also be used to demonstrate the concept of computational optics for educational purposes.

Quantitative Imaging of Microwave Electric Fields through Near-Field Scanning Microwave Microscopy

NASA Astrophysics Data System (ADS)

Dutta, S. K.; Vlahacos, C. P.; Steinhauer, D. E.; Thanawalla, A.; Feenstra, B. J.; Wellstood, F. C.; Anlage, Steven M.; Newman, H. S.

1998-03-01

The ability to non-destructively image electric field patterns generated by operating microwave devices (e.g. filters, antennas, circulators, etc.) would greatly aid in the design and testing of these structures. Such detailed information can be used to reconcile discrepancies between simulated behavior and experimental data (such as scattering parameters). The near-field scanning microwave microscope we present uses a coaxial probe to provide a simple, broadband method of imaging electric fields.(S. M. Anlage, et al.) IEEE Trans. Appl. Supercond. 7, 3686 (1997).^,(See http://www.csr.umd.edu/research/hifreq/micr_microscopy.html) The signal that is measured is related to the incident electric flux normal to the face of the center conductor of the probe, allowing different components of the field to be measured by orienting the probe appropriately. By using a simple model of the system, we can also convert raw data to absolute electric field. Detailed images of standing waves on copper microstrip will be shown and compared to theory.

Review of intraoperative optical coherence tomography: technology and applications [Invited

PubMed Central

Carrasco-Zevallos, Oscar M.; Viehland, Christian; Keller, Brenton; Draelos, Mark; Kuo, Anthony N.; Toth, Cynthia A.; Izatt, Joseph A.

2017-01-01

During microsurgery, en face imaging of the surgical field through the operating microscope limits the surgeon’s depth perception and visualization of instruments and sub-surface anatomy. Surgical procedures outside microsurgery, such as breast tumor resections, may also benefit from visualization of the sub-surface tissue structures. The widespread clinical adoption of optical coherence tomography (OCT) in ophthalmology and its growing prominence in other fields, such as cancer imaging, has motivated the development of intraoperative OCT for real-time tomographic visualization of surgical interventions. This article reviews key technological developments in intraoperative OCT and their applications in human surgery. We focus on handheld OCT probes, microscope-integrated OCT systems, and OCT-guided laser treatment platforms designed for intraoperative use. Moreover, we discuss intraoperative OCT adjuncts and processing techniques currently under development to optimize the surgical feedback derivable from OCT data. Lastly, we survey salient clinical studies of intraoperative OCT for human surgery. PMID:28663853

Optical method for high magnification imaging and video recording of live cells at sub-micron resolution

NASA Astrophysics Data System (ADS)

Romo, Jaime E., Jr.

Optical microscopy, the most common technique for viewing living microorganisms, is limited in resolution by Abbe's criterion. Recent microscopy techniques focus on circumnavigating the light diffraction limit by using different methods to obtain the topography of the sample. Systems like the AFM and SEM provide images with fields of view in the nanometer range with high resolvable detail, however these techniques are expensive, and limited in their ability to document live cells. The Dino-Lite digital microscope coupled with the Zeiss Axiovert 25 CFL microscope delivers a cost-effective method for recording live cells. Fields of view ranging from 8 microns to 300 microns with fair resolution provide a reliable method for discovering native cell structures at the nanoscale. In this report, cultured HeLa cells are recorded using different optical configurations resulting in documentation of cell dynamics at high magnification and resolution.

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

Video-mosaicking of in vivo reflectance confocal microscopy images for noninvasive examination of skin lesion (Conference Presentation)

NASA Astrophysics Data System (ADS)

Kose, Kivanc; Gou, Mengran; Yelamos, Oriol; Cordova, Miguel A.; Rossi, Anthony; Nehal, Kishwer S.; Camps, Octavia I.; Dy, Jennifer G.; Brooks, Dana H.; Rajadhyaksha, Milind

2017-02-01

In this report we describe a computer vision based pipeline to convert in-vivo reflectance confocal microscopy (RCM) videos collected with a handheld system into large field of view (FOV) mosaics. For many applications such as imaging of hard to access lesions, intraoperative assessment of MOHS margins, or delineation of lesion margins beyond clinical borders, raster scan based mosaicing techniques have clinically significant limitations. In such cases, clinicians often capture RCM videos by freely moving a handheld microscope over the area of interest, but the resulting videos lose large-scale spatial relationships. Videomosaicking is a standard computational imaging technique to register, and stitch together consecutive frames of videos into large FOV high resolution mosaics. However, mosaicing RCM videos collected in-vivo has unique challenges: (i) tissue may deform or warp due to physical contact with the microscope objective lens, (ii) discontinuities or "jumps" between consecutive images and motion blur artifacts may occur, due to manual operation of the microscope, and (iii) optical sectioning and resolution may vary between consecutive images due to scattering and aberrations induced by changes in imaging depth and tissue morphology. We addressed these challenges by adapting or developing new algorithmic methods for videomosaicking, specifically by modeling non-rigid deformations, followed by automatically detecting discontinuities (cut locations) and, finally, applying a data-driven image stitching approach that fully preserves resolution and tissue morphologic detail without imposing arbitrary pre-defined boundaries. We will present example mosaics obtained by clinical imaging of both melanoma and non-melanoma skin cancers. The ability to combine freehand mosaicing for handheld microscopes with preserved cellular resolution will have high impact application in diverse clinical settings, including low-resource healthcare systems.

Novel approaches for near and far field super-resolved imaging

NASA Astrophysics Data System (ADS)

Zalevsky, Zeev; Gur, Aviram; Aharoni, Ran; Kutchoukov, Vladimir G.; Garini, Yuval; Beiderman, Yevgeny; Micó, Vicente; García, Javier

2011-09-01

In this paper we start by presenting one recent development in the field of near field imaging where a lensless microscope is introduced. Its operation principle is based upon wavelength encoding of the spatial information through non periodic holes array and right after decoding the spatial information using a spectrometer. In the second part of the paper we demonstrate a remote super sensing technique allowing monitoring, from a distance, the glucose level in the blood stream of a patient by tracking the trajectory of secondary speckle patterns reflected from the skin of the wrist.

Visualizing Electric Fields at Au(111) Step Edges via Tip-Enhanced Raman Scattering

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

None, None

Tip-enhanced Raman scattering (TERS) can be used to image plasmon-enhanced local electric fields on the nanoscale. This is illustrated through ambient TERS measurements recorded using silver atomic force microscope tips coated with 4-mercaptobenzonitrile molecules and used to image step edges on an Au(111) surface. The observed 2D TERS images uniquely map electric fields localized at Au(111) step edges following 671-nm excitation. We establish that our measurements are not only sensitive to spatial variations in the enhanced electric fields but also to their vector components. We also experimentally demonstrate that (i) few nanometer precision is attainable in TERS nanoscopy using corrugatedmore » tips with nominally radii on the order of 100-200 nm, and (ii) TERS signals do not necessarily exhibit the expected E4 dependence. Overall, we illustrate the concept of electric field imaging via TERS and establish the connections between our observations and conventional TERS chemical imaging measurements.« less

Construction of imaging system for wide-field-range ESR spectra using localized microwave field and its case study of crystal orientation in suspension of copper sulfate pentahydrate (CuSO4 . 5H2O).

PubMed

Tani, Atsushi; Ueno, Takehiro; Yamanaka, Chihiro; Katsura, Makoto; Ikeya, Motoji

2005-02-01

A scanning electron spin resonance (ESR) microscope using a localized microwave field was redesigned to measure ESR spectra from 0 to 400 mT using electromagnets. Divalent copper ion (Cu2+) in copper sulfate pentahydrate (CuSO4 . 5H2O) was imaged, after the powdered samples were cemented in silicone rubber under a magnetic field. The ratio of the two signal intensities at g=2.27 and 2.08 clearly indicates the orientation of the particles. This method can be used for mapping the local magnetic field and its direction.

Optical method for distance and displacement measurements of the probe-sample separation in a scanning near-field optical microscope

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

Santamaria, L.; Siller, H. R.; Garcia-Ortiz, C. E., E-mail: cegarcia@cicese.mx

In this work, we present an alternative optical method to determine the probe-sample separation distance in a scanning near-field optical microscope. The experimental method is based in a Lloyd’s mirror interferometer and offers a measurement precision deviation of ∼100 nm using digital image processing and numerical analysis. The technique can also be strategically combined with the characterization of piezoelectric actuators and stability evaluation of the optical system. It also opens the possibility for the development of an automatic approximation control system valid for probe-sample distances from 5 to 500 μm.

Development of tapered silver-halide fiber tips for a scanning near-field microscope operating in the middle infrared

NASA Astrophysics Data System (ADS)

Platkov, Max; Tsun, Alexander; Nagli, Lev; Katzir, Abraham

2006-12-01

We have constructed a scanning near-field infrared microscope (SNIM) which was based on a AgClBr fiber probe whose end was etched to form an aperture of a subwavelength diameter. A detailed study of the mechanical properties of a vibrating AgClBr probe was required for proper operation of the SNIM system. We have demonstrated that the system can be used for imaging and for topographic mapping of samples with a subwavelength resolution in the middle infrared. Such a SNIM will be a powerful tool for the study of microelectronic components or subcellular structures in biological cells.

Fast and Adaptive Auto-focusing Microscope

NASA Astrophysics Data System (ADS)

Obara, Takeshi; Igarashi, Yasunobu; Hashimoto, Koichi

Optical microscopes are widely used in biological and medical researches. By using the microscope, we can observe cellular movements including intracellular ions and molecules tagged with fluorescent dyes at a high magnification. However, a freely motile cell easily escapes from a 3D field of view of the typical microscope. Therefore, we propose a novel auto-focusing algorithm and develop a auto-focusing and tracking microscope. XYZ positions of a microscopic stage are feedback controlled to focus and track the cell automatically. A bright-field image is used to estimate a cellular position. XY centroids are used to estimate XY positions of the tracked cell. To estimate Z position, we use a diffraction pattern around the cell membrane. This estimation method is so-called Depth from Diffraction (DFDi). However, this method is not robust for individual differences between cells because the diffraction pattern depends on each cellular shape. Therefore, in this study, we propose a real-time correction of DFDi by using 2D Laplacian of an intracellular area as a goodness of the focus. To evaluate the performance of our developed algorithm and microscope, we auto-focus and track a freely moving paramecium. In this experimental result, the paramecium is auto-focused and kept inside the scope of the microscope during 45s. The evaluated focal error is within 5µm, while a length and a thickness of the paramecium are about 200µm and 50µm, respectively.

Diamond-Based Magnetic Imaging with Fourier Optical Processing

NASA Astrophysics Data System (ADS)

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

2017-11-01

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

Wavefront image sensor chip

PubMed Central

Cui, Xiquan; Ren, Jian; Tearney, Guillermo J.; Yang, Changhuei

2010-01-01

We report the implementation of an image sensor chip, termed wavefront image sensor chip (WIS), that can measure both intensity/amplitude and phase front variations of a light wave separately and quantitatively. By monitoring the tightly confined transmitted light spots through a circular aperture grid in a high Fresnel number regime, we can measure both intensity and phase front variations with a high sampling density (11 µm) and high sensitivity (the sensitivity of normalized phase gradient measurement is 0.1 mrad under the typical working condition). By using WIS in a standard microscope, we can collect both bright-field (transmitted light intensity) and normalized phase gradient images. Our experiments further demonstrate that the normalized phase gradient images of polystyrene microspheres, unstained and stained starfish embryos, and strongly birefringent potato starch granules are improved versions of their corresponding differential interference contrast (DIC) microscope images in that they are artifact-free and quantitative. Besides phase microscopy, WIS can benefit machine recognition, object ranging, and texture assessment for a variety of applications. PMID:20721059

Intraoperative imaging using intravascular contrast agent

NASA Astrophysics Data System (ADS)

Watson, Jeffrey R.; Martirosyan, Nikolay; Garland, Summer; Lemole, G. Michael; Romanowski, Marek

2016-03-01

Near-infrared (NIR) contrast agents are becoming more frequently studied in medical imaging due to their advantageous characteristics, most notably the ability to capture near-infrared signal across the tissue and the safety of the technique. This produces a need for imaging technology that can be specific for both the NIR dye and medical application. Indocyanine green (ICG) is currently the primary NIR dye used in neurosurgery. Here we report on using the augmented microscope we described previously for image guidance in a rat glioma resection. Luc-C6 cells were implanted in a rat in the left-frontal lobe and grown for 22 days. Surgical resection was performed by a neurosurgeon using augmented microscopy guidance with ICG contrast. Videos and images were acquired to evaluate image quality and resection margins. ICG accumulated in the tumor tissue due to enhanced permeation and retention from the compromised bloodbrain- barrier. The augmented microscope was capable of guiding the rat glioma resection and intraoperatively highlighted tumor tissue regions via ICG fluorescence under normal illumination of the surgical field.

Near-common-path interferometer for imaging Fourier-transform spectroscopy in wide-field microscopy

PubMed Central

Wadduwage, Dushan N.; Singh, Vijay Raj; Choi, Heejin; Yaqoob, Zahid; Heemskerk, Hans; Matsudaira, Paul; So, Peter T. C.

2017-01-01

Imaging Fourier-transform spectroscopy (IFTS) is a powerful method for biological hyperspectral analysis based on various imaging modalities, such as fluorescence or Raman. Since the measurements are taken in the Fourier space of the spectrum, it can also take advantage of compressed sensing strategies. IFTS has been readily implemented in high-throughput, high-content microscope systems based on wide-field imaging modalities. However, there are limitations in existing wide-field IFTS designs. Non-common-path approaches are less phase-stable. Alternatively, designs based on the common-path Sagnac interferometer are stable, but incompatible with high-throughput imaging. They require exhaustive sequential scanning over large interferometric path delays, making compressive strategic data acquisition impossible. In this paper, we present a novel phase-stable, near-common-path interferometer enabling high-throughput hyperspectral imaging based on strategic data acquisition. Our results suggest that this approach can improve throughput over those of many other wide-field spectral techniques by more than an order of magnitude without compromising phase stability. PMID:29392168

A simple approach to spectrally resolved fluorescence and bright field microscopy over select regions of interest

PubMed Central

Dahlberg, Peter D.; Boughter, Christopher T.; Faruk, Nabil F.; Hong, Lu; Koh, Young Hoon; Reyer, Matthew A.; Sherani, Aiman; Hammond, Adam T.

2016-01-01

A standard wide field inverted microscope was converted to a spatially selective spectrally resolved microscope through the addition of a polarizing beam splitter, a pair of polarizers, an amplitude-mode liquid crystal-spatial light modulator, and a USB spectrometer. The instrument is capable of simultaneously imaging and acquiring spectra over user defined regions of interest. The microscope can also be operated in a bright-field mode to acquire absorption spectra of micron scale objects. The utility of the instrument is demonstrated on three different samples. First, the instrument is used to resolve three differently labeled fluorescent beads in vitro. Second, the instrument is used to recover time dependent bleaching dynamics that have distinct spectral changes in the cyanobacteria, Synechococcus leopoliensis UTEX 625. Lastly, the technique is used to acquire the absorption spectra of CH3NH3PbBr3 perovskites and measure differences between nanocrystal films and micron scale crystals. PMID:27910631

Applications of holographic on-chip microscopy (Conference Presentation)

NASA Astrophysics Data System (ADS)

Ozcan, Aydogan

2017-02-01

My research focuses on the use of computation/algorithms to create new optical microscopy, sensing, and diagnostic techniques, significantly improving existing tools for probing micro- and nano-objects while also simplifying the designs of these analysis tools. In this presentation, I will introduce a set of computational microscopes which use lens-free on-chip imaging to replace traditional lenses with holographic reconstruction algorithms. Basically, 3D images of specimens are reconstructed from their "shadows" providing considerably improved field-of-view (FOV) and depth-of-field, thus enabling large sample volumes to be rapidly imaged, even at nanoscale. These new computational microscopes routinely generate <1-2 billion pixels (giga-pixels), where even single viruses can be detected with a FOV that is <100 fold wider than other techniques. At the heart of this leapfrog performance lie self-assembled liquid nano-lenses that are computationally imaged on a chip. The field-of-view of these computational microscopes is equal to the active-area of the sensor-array, easily reaching, for example, <20 mm^2 or <10 cm^2 by employing state-of-the-art CMOS or CCD imaging chips, respectively. In addition to this remarkable increase in throughput, another major benefit of this technology is that it lends itself to field-portable and cost-effective designs which easily integrate with smartphones to conduct giga-pixel tele-pathology and microscopy even in resource-poor and remote settings where traditional techniques are difficult to implement and sustain, thus opening the door to various telemedicine applications in global health. Through the development of similar computational imagers, I will also report the discovery of new 3D swimming patterns observed in human and animal sperm. One of this newly discovered and extremely rare motion is in the form of "chiral ribbons" where the planar swings of the sperm head occur on an osculating plane creating in some cases a helical ribbon and in some others a twisted ribbon. Shedding light onto the statistics and biophysics of various micro-swimmers' 3D motion, these results provide an important example of how biomedical imaging significantly benefits from emerging computational algorithms/theories, revolutionizing existing tools for observing various micro- and nano-scale phenomena in innovative, high-throughput, and yet cost-effective ways.

The National Ignition Facility modular Kirkpatrick-Baez microscope

DOE PAGES

Pickworth, L. A.; Ayers, J.; Bell, P.; ...

2016-08-10

Current two-dimensional X-ray imaging at the National Ignition Facility (NIF) uses time resolved pinhole cameras with ~10-25µm pinholes. This method has limitations in the smallest resolvable features that can be imaged with reasonable photon statistics for inertial confinement fusion (ICF) applications. ICF sources have a broadband self-emission spectrum that causes the pinhole images obtained, through thin foil filters, to contain a similarly broadband spectrum complicating the interpretation of structure in the source. In order to study phenomena on the scale of ~5 µm, such as dopant mix in the ICF capsule, a narrow energy band, higher spatial resolution microscope systemmore » with improved signal/noise has been developed using X-ray optics. Utilizing grazing incidence mirrors in a Kirkpatrick-Baez microscope (KBM) configuration, an X-ray microscope has been designed and fielded on NIF with four imaging channels. The KBM has ~12x magnification, <8 µm resolution and higher throughput in comparison to similar pinhole systems. The first KBM mirrors are coated with a multilayer mirror to allow a ‘narrow band’ energy response at 10.2keV with ΔE~3keV. By adjusting the mirror coating only, the energy response can be matched to future experimental requirements. Here, several mirror packs have been commissioned and are interchangeable in the diagnostic snout.« less

The National Ignition Facility modular Kirkpatrick-Baez microscope

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

Pickworth, L. A.; Ayers, J.; Bell, P.

Current two-dimensional X-ray imaging at the National Ignition Facility (NIF) uses time resolved pinhole cameras with ~10-25µm pinholes. This method has limitations in the smallest resolvable features that can be imaged with reasonable photon statistics for inertial confinement fusion (ICF) applications. ICF sources have a broadband self-emission spectrum that causes the pinhole images obtained, through thin foil filters, to contain a similarly broadband spectrum complicating the interpretation of structure in the source. In order to study phenomena on the scale of ~5 µm, such as dopant mix in the ICF capsule, a narrow energy band, higher spatial resolution microscope systemmore » with improved signal/noise has been developed using X-ray optics. Utilizing grazing incidence mirrors in a Kirkpatrick-Baez microscope (KBM) configuration, an X-ray microscope has been designed and fielded on NIF with four imaging channels. The KBM has ~12x magnification, <8 µm resolution and higher throughput in comparison to similar pinhole systems. The first KBM mirrors are coated with a multilayer mirror to allow a ‘narrow band’ energy response at 10.2keV with ΔE~3keV. By adjusting the mirror coating only, the energy response can be matched to future experimental requirements. Here, several mirror packs have been commissioned and are interchangeable in the diagnostic snout.« less

The National Ignition Facility modular Kirkpatrick-Baez microscope.

PubMed

Pickworth, L A; Ayers, J; Bell, P; Brejnholt, N F; Buscho, J G; Bradley, D; Decker, T; Hau-Riege, S; Kilkenny, J; McCarville, T; Pardini, T; Vogel, J; Walton, C

2016-11-01

Current two-dimensional X-ray imaging at the National Ignition Facility (NIF) uses time resolved pinhole cameras with ∼10-25 μm pinholes. This method has limitations in the smallest resolvable features that can be imaged with reasonable photon statistics for inertial confinement fusion (ICF) applications. ICF sources have a broadband self-emission spectrum that causes the pinhole images obtained, through thin foil filters, to contain a similarly broadband spectrum complicating the interpretation of structure in the source. In order to study phenomena on the scale of ∼5 μm, such as dopant mix in the ICF capsule, a narrow energy band, higher spatial resolution microscope system with improved signal/noise has been developed using X-ray optics. Utilizing grazing incidence mirrors in a Kirkpatrick-Baez microscope (KBM) configuration [P. Kirkpatrick and A. V. Baez, J. Opt. Soc. Am. 38, 766-774 (1948)], an X-ray microscope has been designed and fielded on NIF with four imaging channels. The KBM has ∼12 × magnification, <8 μm resolution, and higher throughput in comparison to similar pinhole systems. The first KBM mirrors are coated with a multilayer mirror to allow a "narrow band" energy response at 10.2 keV with ΔE ∼ 3 keV. By adjusting the mirror coating only, the energy response can be matched to the future experimental requirements. Several mirror packs have been commissioned and are interchangeable in the diagnostic snout.

Wide-band acousto-optic deflectors for large field of view two-photon microscope.

PubMed

Jiang, Runhua; Zhou, Zhenqiao; Lv, Xiaohua; Zeng, Shaoqun

2012-04-01

Acousto-optic deflector (AOD) is an attractive scanner for two-photon microscopy because it can provide fast and versatile laser scanning and does not involve any mechanical movements. However, due to the small scan range of available AOD, the field of view (FOV) of the AOD-based microscope is typically smaller than that of the conventional galvanometer-based microscope. Here, we developed a novel wide-band AOD to enlarge the scan angle. Considering the maximum acceptable acoustic attenuation in the acousto-optic crystal, relatively lower operating frequencies and moderate aperture were adopted. The custom AOD was able to provide 60 MHz 3-dB bandwidth and 80% peak diffraction efficiency at 840 nm wavelength. Based on a pair of such AOD, a large FOV two-photon microscope was built with a FOV up to 418.5 μm (40× objective). The spatiotemporal dispersion was compensated simultaneously with a single custom-made prism. By means of dynamic power modulation, the variation of laser intensity within the FOV was reduced below 5%. The lateral and axial resolution of the system were 0.58-2.12 μm and 2.17-3.07 μm, respectively. Pollen grain images acquired by this system were presented to demonstrate the imaging capability at different positions across the entire FOV. © 2012 American Institute of Physics

Atmospheric scanning electron microscope observes cells and tissues in open medium through silicon nitride film.

PubMed

Nishiyama, Hidetoshi; Suga, Mitsuo; Ogura, Toshihiko; Maruyama, Yuusuke; Koizumi, Mitsuru; Mio, Kazuhiro; Kitamura, Shinichi; Sato, Chikara

2010-03-01

Direct observation of subcellular structures and their characterization is essential for understanding their physiological functions. To observe them in open environment, we have developed an inverted scanning electron microscope with a detachable, open-culture dish, capable of 8 nm resolution, and combined with a fluorescence microscope quasi-simultaneously observing the same area from the top. For scanning electron microscopy from the bottom, a silicon nitride film window in the base of the dish maintains a vacuum between electron gun and open sample dish while allowing electrons to pass through. Electrons are backscattered from the sample and captured by a detector under the dish. Cells cultured on the open dish can be externally manipulated under optical microscopy, fixed, and observed using scanning electron microscopy. Once fine structures have been revealed by scanning electron microscopy, their component proteins may be identified by comparison with separately prepared fluorescence-labeled optical microscopic images of the candidate proteins, with their heavy-metal-labeled or stained ASEM images. Furthermore, cell nuclei in a tissue block stained with platinum-blue were successfully observed without thin-sectioning, which suggests the applicability of this inverted scanning electron microscope to cancer diagnosis. This microscope visualizes mesoscopic-scale structures, and is also applicable to non-bioscience fields including polymer chemistry. (c) 2010 Elsevier Inc. All rights reserved.

Fiber laser-microscope system for femtosecond photodisruption of biological samples

PubMed Central

Yavaş, Seydi; Erdogan, Mutlu; Gürel, Kutan; Ilday, F. Ömer; Eldeniz, Y. Burak; Tazebay, Uygar H.

2012-01-01

We report on the development of a ultrafast fiber laser-microscope system for femtosecond photodisruption of biological targets. A mode-locked Yb-fiber laser oscillator generates few-nJ pulses at 32.7 MHz repetition rate, amplified up to ∼125 nJ at 1030 nm. Following dechirping in a grating compressor, ∼240 fs-long pulses are delivered to the sample through a diffraction-limited microscope, which allows real-time imaging and control. The laser can generate arbitrary pulse patterns, formed by two acousto-optic modulators (AOM) controlled by a custom-developed field-programmable gate array (FPGA) controller. This capability opens the route to fine optimization of the ablation processes and management of thermal effects. Sample position, exposure time and imaging are all computerized. The capability of the system to perform femtosecond photodisruption is demonstrated through experiments on tissue and individual cells. PMID:22435105

A review of cellphone microscopy for disease detection.

PubMed

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

2015-12-01

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

The Ames MER microscopic imager toolkit

USGS Publications Warehouse

Sargent, R.; Deans, Matthew; Kunz, C.; Sims, M.; Herkenhoff, K.

2005-01-01

12The Mars Exploration Rovers, Spirit and Opportunity, have spent several successful months on Mars, returning gigabytes of images and spectral data to scientists on Earth. One of the instruments on the MER rovers, the Athena Microscopic Imager (MI), is a fixed focus, megapixel camera providing a ??3mm depth of field and a 31??31mm field of view at a working distance of 63 mm from the lens to the object being imaged. In order to maximize the science return from this instrument, we developed the Ames MI Toolkit and supported its use during the primary mission. The MI Toolkit is a set of programs that operate on collections of MI images, with the goal of making the data more understandable to the scientists on the ground. Because of the limited depth of field of the camera, and the often highly variable topography of the terrain being imaged, MI images of a given rock are often taken as a stack, with the Instrument Deployment Device (IDD) moving along a computed normal vector, pausing every few millimeters for the MI to acquire an image. The MI Toolkit provides image registration and focal section merging, which combine these images to form a single, maximally in-focus image, while compensating for changes in lighting as well as parallax due to the motion of the camera. The MI Toolkit also provides a 3-D reconstruction of the surface being imaged using stereo and can embed 2-D MI images as texture maps into 3-D meshes produced by other imagers on board the rover to provide context. The 2-D images and 3-D meshes output from the Toolkit are easily viewed by scientists using other mission tools, such as Viz or the MI Browser.This paper describes the MI Toolkit in detail, as well as our experience using it with scientists at JPL during the primary MER mission. ?? 2005 IEEE.

The Ames MER Microscopic Imager Toolkit

NASA Technical Reports Server (NTRS)

Sargent, Randy; Deans, Matthew; Kunz, Clayton; Sims, Michael; Herkenhoff, Ken

2005-01-01

The Mars Exploration Rovers, Spirit and Opportunity, have spent several successful months on Mars, returning gigabytes of images and spectral data to scientists on Earth. One of the instruments on the MER rovers, the Athena Microscopic Imager (MI), is a fixed focus, megapixel camera providing a plus or minus mm depth of field and a 3lx31mm field of view at a working distance of 63 mm from the lens to the object being imaged. In order to maximize the science return from this instrument, we developed the Ames MI Toolkit and supported its use during the primary mission. The MI Toolkit is a set of programs that operate on collections of MI images, with the goal of making the data more understandable to the scientists on the ground. Because of the limited depth of field of the camera, and the often highly variable topography of the terrain being imaged, MI images of a given rock are often taken as a stack, with the Instrument Deployment Device (IDD) moving along a computed normal vector, pausing every few millimeters for the MI to acquire an image. The MI Toolkit provides image registration and focal section merging, which combine these images to form a single, maximally in-focus image, while compensating for changes in lighting as well as parallax due to the motion of the camera. The MI Toolkit also provides a 3-D reconstruction of the surface being imaged using stereo and can embed 2-D MI images as texture maps into 3-D meshes produced by other imagers on board the rover to provide context. The 2-D images and 3-D meshes output from the Toolkit are easily viewed by scientists using other mission tools, such as Viz or the MI Browser. This paper describes the MI Toolkit in detail, as well as our experience using it with scientists at JPL during the primary MER mission.

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.

Curiosity's Mars Hand Lens Imager (MAHLI): Inital Observations and Activities

NASA Technical Reports Server (NTRS)

Edgett, K. S.; Yingst, R. A.; Minitti, M. E.; Robinson, M. L.; Kennedy, M. R.; Lipkaman, L. J.; Jensen, E. H.; Anderson, R. C.; Bean, K. M.; Beegle, L. W.;

Photon counting phosphorescence lifetime imaging with TimepixCam

DOE PAGES

Hirvonen, Liisa M.; Fisher-Levine, Merlin; Suhling, Klaus; ...

2017-01-12

TimepixCam is a novel fast optical imager based on an optimized silicon pixel sensor with a thin entrance window, and read out by a Timepix ASIC. The 256 x 256 pixel sensor has a time resolution of 15 ns at a sustained frame rate of 10 Hz. We used this sensor in combination with an image intensifier for wide-field time-correlated single photon counting (TCSPC) imaging. We have characterised the photon detection capabilities of this detector system, and employed it on a wide-field epifluorescence microscope to map phosphorescence decays of various iridium complexes with lifetimes of about 1 μs in 200more » μm diameter polystyrene beads.« less

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

Hirvonen, Liisa M.; Le Marois, Alix; Suhling, Klaus, E-mail: klaus.suhling@kcl.ac.uk

We perform wide-field time-correlated single photon counting-based fluorescence lifetime imaging (FLIM) with a crossed delay line anode image intensifier, where the pulse propagation time yields the photon position. This microchannel plate-based detector was read out with conventional fast timing electronics and mounted on a fluorescence microscope with total internal reflection (TIR) illumination. The picosecond time resolution of this detection system combines low illumination intensity of microwatts with wide-field data collection. This is ideal for fluorescence lifetime imaging of cell membranes using TIR. We show that fluorescence lifetime images of living HeLa cells stained with membrane dye di-4-ANEPPDHQ exhibit a reducedmore » lifetime near the coverslip in TIR compared to epifluorescence FLIM.« less

Photon counting phosphorescence lifetime imaging with TimepixCam.

PubMed

Hirvonen, Liisa M; Fisher-Levine, Merlin; Suhling, Klaus; Nomerotski, Andrei

2017-01-01

TimepixCam is a novel fast optical imager based on an optimized silicon pixel sensor with a thin entrance window and read out by a Timepix Application Specific Integrated Circuit. The 256 × 256 pixel sensor has a time resolution of 15 ns at a sustained frame rate of 10 Hz. We used this sensor in combination with an image intensifier for wide-field time-correlated single photon counting imaging. We have characterised the photon detection capabilities of this detector system and employed it on a wide-field epifluorescence microscope to map phosphorescence decays of various iridium complexes with lifetimes of about 1 μs in 200 μm diameter polystyrene beads.

Photon counting phosphorescence lifetime imaging with TimepixCam

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

Hirvonen, Liisa M.; Fisher-Levine, Merlin; Suhling, Klaus

TimepixCam is a novel fast optical imager based on an optimized silicon pixel sensor with a thin entrance window, and read out by a Timepix ASIC. The 256 x 256 pixel sensor has a time resolution of 15 ns at a sustained frame rate of 10 Hz. We used this sensor in combination with an image intensifier for wide-field time-correlated single photon counting (TCSPC) imaging. We have characterised the photon detection capabilities of this detector system, and employed it on a wide-field epifluorescence microscope to map phosphorescence decays of various iridium complexes with lifetimes of about 1 μs in 200more » μm diameter polystyrene beads.« less

Photon counting phosphorescence lifetime imaging with TimepixCam

NASA Astrophysics Data System (ADS)

Hirvonen, Liisa M.; Fisher-Levine, Merlin; Suhling, Klaus; Nomerotski, Andrei

2017-01-01

TimepixCam is a novel fast optical imager based on an optimized silicon pixel sensor with a thin entrance window and read out by a Timepix Application Specific Integrated Circuit. The 256 × 256 pixel sensor has a time resolution of 15 ns at a sustained frame rate of 10 Hz. We used this sensor in combination with an image intensifier for wide-field time-correlated single photon counting imaging. We have characterised the photon detection capabilities of this detector system and employed it on a wide-field epifluorescence microscope to map phosphorescence decays of various iridium complexes with lifetimes of about 1 μs in 200 μm diameter polystyrene beads.

Optoelectronic tweezers integrated with lensfree holographic microscopy for wide-field interactive cell and particle manipulation on a chip.

PubMed

Huang, Kuo-Wei; Su, Ting-Wei; Ozcan, Aydogan; Chiou, Pei-Yu

2013-06-21

We demonstrate an optoelectronic tweezer (OET) coupled to a lensfree holographic microscope for real-time interactive manipulation of cells and micro-particles over a large field-of-view (FOV). This integrated platform can record the holographic images of cells and particles over the entire active area of a CCD sensor array, perform digital image reconstruction to identify target cells, dynamically track the positions of cells and particles, and project light beams to trigger light-induced dielectrophoretic forces to pattern and sort cells on a chip. OET technology has been previously shown to be capable of performing parallel single cell manipulation over a large area. However, its throughput has been bottlenecked by the number of cells that can be imaged within the limited FOV of a conventional microscope objective lens. Integrating lensfree holographic imaging with OET solves this fundamental FOV barrier, while also creating a compact on-chip cell/particle manipulation platform. Using this unique platform, we have successfully demonstrated real-time interactive manipulation of thousands of single cells and micro-particles over an ultra-large area of e.g., 240 mm(2) (i.e. 17.96 mm × 13.52 mm).

SEM technique for imaging and measuring electronic transport in nanocomposites based on electric field induced contrast

DOEpatents

Jesse, Stephen [Knoxville, TN; Geohegan, David B [Knoxville, TN; Guillorn, Michael [Brooktondale, NY

2009-02-17

Methods and apparatus are described for SEM imaging and measuring electronic transport in nanocomposites based on electric field induced contrast. A method includes mounting a sample onto a sample holder, the sample including a sample material; wire bonding leads from the sample holder onto the sample; placing the sample holder in a vacuum chamber of a scanning electron microscope; connecting leads from the sample holder to a power source located outside the vacuum chamber; controlling secondary electron emission from the sample by applying a predetermined voltage to the sample through the leads; and generating an image of the secondary electron emission from the sample. An apparatus includes a sample holder for a scanning electron microscope having an electrical interconnect and leads on top of the sample holder electrically connected to the electrical interconnect; a power source and a controller connected to the electrical interconnect for applying voltage to the sample holder to control the secondary electron emission from a sample mounted on the sample holder; and a computer coupled to a secondary electron detector to generate images of the secondary electron emission from the sample.

Super-resolution imaging based on the temperature-dependent electron-phonon collision frequency effect of metal thin films

NASA Astrophysics Data System (ADS)

Ding, Chenliang; Wei, Jingsong; Xiao, Mufei

2018-05-01

We herein propose a far-field super-resolution imaging with metal thin films based on the temperature-dependent electron-phonon collision frequency effect. In the proposed method, neither fluorescence labeling nor any special properties are required for the samples. The 100 nm lands and 200 nm grooves on the Blu-ray disk substrates were clearly resolved and imaged through a laser scanning microscope of wavelength 405 nm. The spot size was approximately 0.80 μm , and the imaging resolution of 1/8 of the laser spot size was experimentally obtained. This work can be applied to the far-field super-resolution imaging of samples with neither fluorescence labeling nor any special properties.

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.

An Innovative Method for Obtaining Consistent Images and Quantification of Histochemically Stained Specimens

PubMed Central

Sedgewick, Gerald J.; Ericson, Marna

2015-01-01

Obtaining digital images of color brightfield microscopy is an important aspect of biomedical research and the clinical practice of diagnostic pathology. Although the field of digital pathology has had tremendous advances in whole-slide imaging systems, little effort has been directed toward standardizing color brightfield digital imaging to maintain image-to-image consistency and tonal linearity. Using a single camera and microscope to obtain digital images of three stains, we show that microscope and camera systems inherently produce image-to-image variation. Moreover, we demonstrate that post-processing with a widely used raster graphics editor software program does not completely correct for session-to-session inconsistency. We introduce a reliable method for creating consistent images with a hardware/software solution (ChromaCal™; Datacolor Inc., NJ) along with its features for creating color standardization, preserving linear tonal levels, providing automated white balancing and setting automated brightness to consistent levels. The resulting image consistency using this method will also streamline mean density and morphometry measurements, as images are easily segmented and single thresholds can be used. We suggest that this is a superior method for color brightfield imaging, which can be used for quantification and can be readily incorporated into workflows. PMID:25575568

Snow crystal imaging using scanning electron microscopy: III. Glacier ice, snow and biota

USGS Publications Warehouse

Rango, A.; Wergin, W.P.; Erbe, E.F.; Josberger, E.G.

2000-01-01

Low-temperature scanning electron microscopy (SEM) was used to observe metamorphosed snow, glacial firn, and glacial ice obtained from South Cascade Glacier in Washington State, USA. Biotic samples consisting of algae (Chlamydomonas nivalis) and ice worms (a species of oligochaetes) were also collected and imaged. In the field, the snow and biological samples were mounted on copper plates, cooled in liquid nitrogen, and stored in dry shipping containers which maintain a temperature of -196??C. The firn and glacier ice samples were obtained by extracting horizontal ice cores, 8 mm in diameter, at different levels from larger standard glaciological (vertical) ice cores 7.5 cm in diameter. These samples were cooled in liquid nitrogen and placed in cryotubes, were stored in the same dry shipping container, and sent to the SEM facility. In the laboratory, the samples were sputter coated with platinum and imaged by a low-temperature SEM. To image the firn and glacier ice samples, the cores were fractured in liquid nitrogen, attached to a specimen holder, and then imaged. While light microscope images of snow and ice are difficult to interpret because of internal reflection and refraction, the SEM images provide a clear and unique view of the surface of the samples because they are generated from electrons emitted or reflected only from the surface of the sample. In addition, the SEM has a great depth of field with a wide range of magnifying capabilities. The resulting images clearly show the individual grains of the seasonal snowpack and the bonding between the snow grains. Images of firn show individual ice crystals, the bonding between the crystals, and connected air spaces. Images of glacier ice show a crystal structure on a scale of 1-2 mm which is considerably smaller than the expected crystal size. Microscopic air bubbles, less than 15 ??m in diameter, clearly marked the boundaries between these crystal-like features. The life forms associated with the glacier were easily imaged and studied. The low-temperature SEM sample collecting and handling methods proved to be operable in the field; the SEM analysis is applicable to glaciological studies and reveals details unattainable by conventional light microscopic methods.Low temperature scanning electron microscopy (SEM) was used to observe metamorphosed snow, glacial firn, and glacial ice obtained from South Cascade Glacier in Washington State, USA. Biotic samples consisting of algae and ice worms were also collected and imaged. The SEM images provide a clear and unique view of the surface of the samples because they are generated from electrons emitted or reflected only from the surface of the sample. The SEM has a great depth of field with a wide range of magnifying capabilities.

High resolution computational on-chip imaging of biological samples using sparsity constraint (Conference Presentation)

NASA Astrophysics Data System (ADS)

Rivenson, Yair; Wu, Chris; Wang, Hongda; Zhang, Yibo; Ozcan, Aydogan

2017-03-01

Microscopic imaging of biological samples such as pathology slides is one of the standard diagnostic methods for screening various diseases, including cancer. These biological samples are usually imaged using traditional optical microscopy tools; however, the high cost, bulkiness and limited imaging throughput of traditional microscopes partially restrict their deployment in resource-limited settings. In order to mitigate this, we previously demonstrated a cost-effective and compact lens-less on-chip microscopy platform with a wide field-of-view of >20-30 mm^2. The lens-less microscopy platform has shown its effectiveness for imaging of highly connected biological samples, such as pathology slides of various tissue samples and smears, among others. This computational holographic microscope requires a set of super-resolved holograms acquired at multiple sample-to-sensor distances, which are used as input to an iterative phase recovery algorithm and holographic reconstruction process, yielding high-resolution images of the samples in phase and amplitude channels. Here we demonstrate that in order to reconstruct clinically relevant images with high resolution and image contrast, we require less than 50% of the previously reported nominal number of holograms acquired at different sample-to-sensor distances. This is achieved by incorporating a loose sparsity constraint as part of the iterative holographic object reconstruction. We demonstrate the success of this sparsity-based computational lens-less microscopy platform by imaging pathology slides of breast cancer tissue and Papanicolaou (Pap) smears.

The Athena Microscopic Imager Investigation

NASA Technical Reports Server (NTRS)

Herkenhoff, K. E.; Aquyres, S. W.; Bell, J. F., III; Maki, J. N.; Arneson, H. M.; Brown, D. I.; Collins, S. A.; Dingizian, A.; Elliot, S. T.; Geotz, W.

2003-01-01

The Athena science payload on the Mars Exploration Rovers (MER) includes the Microscopic Imager (MI) [1]. The MI is a fixed-focus camera mounted on the end of an extendable instrument arm, the Instrument Deployment Device (IDD; see Figure 1).The MI was designed to acquire images at a spatial resolution of 30 microns/pixel over a broad spectral range (400 - 700 nm; see Table 1). Technically, the microscopic imager is not a microscope: it has a fixed magnification of 0.4 and is intended to produce images that simulate a geologist s view through a common hand lens. In photographers parlance, the system makes use of a macro lens. The MI uses the same electronics design as the other MER cameras [2, 3] but has optics that yield a field of view of 31 31 mm across a 1024 1024 pixel CCD image (Figure 2). The MI acquires images using only solar or skylightillumination of the target surface. A contact sensor is used to place the MI slightly closer to the target surface than its best focus distance (about 66 mm), allowing concave surfaces to be imaged in good focus. Because the MI has a relatively small depth of field (3 mm), a single MI image of a rough surface will contain both focused and unfocused areas. Coarse focusing will be achieved by moving the IDD away from a rock target after the contact sensor is activated. Multiple images taken at various distances will be acquired to ensure good focus on all parts of rough surfaces. By combining a set of images acquired in this way, a completely focused image can be assembled. Stereoscopic observations can be obtained by moving the MI laterally relative to its boresight. Estimates of the position and orientation of the MI for each acquired image will be stored in the rover computer and returned to Earth with the image data. The MI optics will be protected from the Martian environment by a retractable dust cover. The dust cover includes a Kapton window that is tinted orange to restrict the spectral bandpass to 500-700 nm, allowing color information to be obtained by taking images with the dust cover open and closed. The MI will image the same materials measured by other Athena instruments (including surfaces prepared by the Rock Abrasion Tool), as well as rock and soil targets of opportunity. Subsets of the full image array can be selected and/or pixels can be binned to reduce data volume. Image compression will be used to maximize the information contained in the data returned to Earth. The resulting MI data will place other MER instrument data in context and aid in petrologic and geologic interpretations of rocks and soils on Mars.

Biological tissue imaging with a position and time sensitive pixelated detector.

PubMed

Jungmann, Julia H; Smith, Donald F; MacAleese, Luke; Klinkert, Ivo; Visser, Jan; Heeren, Ron M A

2012-10-01

We demonstrate the capabilities of a highly parallel, active pixel detector for large-area, mass spectrometric imaging of biological tissue sections. A bare Timepix assembly (512 × 512 pixels) is combined with chevron microchannel plates on an ion microscope matrix-assisted laser desorption time-of-flight mass spectrometer (MALDI TOF-MS). The detector assembly registers position- and time-resolved images of multiple m/z species in every measurement frame. We prove the applicability of the detection system to biomolecular mass spectrometry imaging on biologically relevant samples by mass-resolved images from Timepix measurements of a peptide-grid benchmark sample and mouse testis tissue slices. Mass-spectral and localization information of analytes at physiologic concentrations are measured in MALDI-TOF-MS imaging experiments. We show a high spatial resolution (pixel size down to 740 × 740 nm(2) on the sample surface) and a spatial resolving power of 6 μm with a microscope mode laser field of view of 100-335 μm. Automated, large-area imaging is demonstrated and the Timepix' potential for fast, large-area image acquisition is highlighted.

Detection of early osteoarthritis in the centrodistal joints of Icelandic horses: Evaluation of radiography and low-field magnetic resonance imaging.

PubMed

Ley, C J; Björnsdóttir, S; Ekman, S; Boyde, A; Hansson, K

2016-01-01

Validated noninvasive detection methods for early osteoarthritis (OA) are required for OA prevention and early intervention treatment strategies. To evaluate radiography and low-field magnetic resonance imaging (MRI) for the detection of early stage OA osteochondral lesions in equine centrodistal joints using microscopy as the reference standard. Prospective imaging of live horses and imaging and microscopy of cadaver tarsal joints. Centrodistal (distal intertarsal) joints of 38 Icelandic research horses aged 27-29 months were radiographed. Horses were subjected to euthanasia approximately 2 months later and cadaver joints examined with low-field MRI. Osteochondral joint specimens were classified as negative or positive for OA using light microscopy histology or scanning electron microscopy. Radiographs and MRIs were evaluated for osteochondral lesions and results compared with microscopy. Forty-two joints were classified OA positive with microscopy. Associations were detected between microscopic OA and the radiography lesion categories; mineralisation front defect (P<0.0001), joint margin lesion (P<0.0001), central osteophyte (P = 0.03) and the low-field MRI lesion categories; mineralisation front defect (P = 0.01), joint margin lesion (P = 0.02) and articular cartilage lesion (P = 0.0003). The most frequent lesion category detected in microscopic OA positive joints was the mineralisation front defect in radiographs (28/42 OA positive joints, specificity 97%, sensitivity 67%). No significant differences were detected between the sensitivity and specificity of radiography and low-field MRI pooled lesion categories, but radiography was often superior when individual lesion categories were compared. Early stage centrodistal joint OA changes may be detected with radiography and low-field MRI. Detection of mineralisation front defects in radiographs may be a useful screening method for detection of early OA in centrodistal joints of young Icelandic horses. © 2015 EVJ Ltd.

Development of and Clinical Experience with a Simple Device for Performing Intraoperative Fluorescein Fluorescence Cerebral Angiography: Technical Notes.

PubMed

Ichikawa, Tsuyoshi; Suzuki, Kyouichi; Watanabe, Yoichi; Sato, Taku; Sakuma, Jun; Saito, Kiyoshi

2016-01-01

To perform intraoperative fluorescence angiography (FAG) under a microscope without an integrated FAG function with reasonable cost and sufficient quality for evaluation, we made a small and easy to use device for fluorescein FAG (FAG filter). We investigated the practical use of this FAG filter during aneurysm surgery, revascularization surgery, and brain tumor surgery. The FAG filter consists of two types of filters: an excitatory filter and a barrier filter. The excitatory filter excludes all wavelengths except for blue light and the barrier filter passes long waves except for blue light. By adding this FAG filter to a microscope without an integrated FAG function, light from the microscope illuminating the surgical field becomes blue, which is blocked by the barrier filter. We put the FAG filter on the objective lens of the operating microscope correctly and fluorescein sodium was injected intravenously or intra-arterially. Fluorescence (green light) from vessels in the surgical field and the dyed tumor were clearly observed through the microscope and recorded by a memory device. This method was easy and could be performed in a short time (about 10 seconds). Blood flow of small vessels deep in the surgical field could be observed. Blood flow stagnation could be evaluated. However, images from this method were inferior to those obtained by currently commercially available microscopes with an integrated FAG function. In brain tumor surgery, a stained tumor on the brain surface could be observed using this method. FAG could be performed with a microscope without an integrated FAG function easily with only this FAG filter.

Development of and Clinical Experience with a Simple Device for Performing Intraoperative Fluorescein Fluorescence Cerebral Angiography: Technical Notes

PubMed Central

ICHIKAWA, Tsuyoshi; SUZUKI, Kyouichi; WATANABE, Yoichi; SATO, Taku; SAKUMA, Jun; SAITO, Kiyoshi

2016-01-01

To perform intraoperative fluorescence angiography (FAG) under a microscope without an integrated FAG function with reasonable cost and sufficient quality for evaluation, we made a small and easy to use device for fluorescein FAG (FAG filter). We investigated the practical use of this FAG filter during aneurysm surgery, revascularization surgery, and brain tumor surgery. The FAG filter consists of two types of filters: an excitatory filter and a barrier filter. The excitatory filter excludes all wavelengths except for blue light and the barrier filter passes long waves except for blue light. By adding this FAG filter to a microscope without an integrated FAG function, light from the microscope illuminating the surgical field becomes blue, which is blocked by the barrier filter. We put the FAG filter on the objective lens of the operating microscope correctly and fluorescein sodium was injected intravenously or intra-arterially. Fluorescence (green light) from vessels in the surgical field and the dyed tumor were clearly observed through the microscope and recorded by a memory device. This method was easy and could be performed in a short time (about 10 seconds). Blood flow of small vessels deep in the surgical field could be observed. Blood flow stagnation could be evaluated. However, images from this method were inferior to those obtained by currently commercially available microscopes with an integrated FAG function. In brain tumor surgery, a stained tumor on the brain surface could be observed using this method. FAG could be performed with a microscope without an integrated FAG function easily with only this FAG filter. PMID:26597335

Automated Diatom Analysis Applied to Traditional Light Microscopy: A Proof-of-Concept Study

NASA Astrophysics Data System (ADS)

Little, Z. H. L.; Bishop, I.; Spaulding, S. A.; Nelson, H.; Mahoney, C.

2017-12-01

Diatom identification and enumeration by high resolution light microscopy is required for many areas of research and water quality assessment. Such analyses, however, are both expertise and labor-intensive. These challenges motivate the need for an automated process to efficiently and accurately identify and enumerate diatoms. Improvements in particle analysis software have increased the likelihood that diatom enumeration can be automated. VisualSpreadsheet software provides a possible solution for automated particle analysis of high-resolution light microscope diatom images. We applied the software, independent of its complementary FlowCam hardware, to automated analysis of light microscope images containing diatoms. Through numerous trials, we arrived at threshold settings to correctly segment 67% of the total possible diatom valves and fragments from broad fields of view. (183 light microscope images were examined containing 255 diatom particles. Of the 255 diatom particles present, 216 diatoms valves and fragments of valves were processed, with 170 properly analyzed and focused upon by the software). Manual analysis of the images yielded 255 particles in 400 seconds, whereas the software yielded a total of 216 particles in 68 seconds, thus highlighting that the software has an approximate five-fold efficiency advantage in particle analysis time. As in past efforts, incomplete or incorrect recognition was found for images with multiple valves in contact or valves with little contrast. The software has potential to be an effective tool in assisting taxonomists with diatom enumeration by completing a large portion of analyses. Benefits and limitations of the approach are presented to allow for development of future work in image analysis and automated enumeration of traditional light microscope images containing diatoms.

Construction and performance of a dilution-refrigerator based spectroscopic-imaging scanning tunneling microscope.

PubMed

Singh, U R; Enayat, M; White, S C; Wahl, P

2013-01-01

We report on the set-up and performance of a dilution-refrigerator based spectroscopic imaging scanning tunneling microscope. It operates at temperatures below 10 mK and in magnetic fields up to 14T. The system allows for sample transfer and in situ cleavage. We present first-results demonstrating atomic resolution and the multi-gap structure of the superconducting gap of NbSe(2) at base temperature. To determine the energy resolution of our system we have measured a normal metal/vacuum/superconductor tunneling junction consisting of an aluminum tip on a gold sample. Our system allows for continuous measurements at base temperature on time scales of up to ≈170 h.

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

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.

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.

Single-shot optical sectioning using two-color probes in HiLo fluorescence microscopy.

PubMed

Muro, Eleonora; Vermeulen, Pierre; Ioannou, Andriani; Skourides, Paris; Dubertret, Benoit; Fragola, Alexandra; Loriette, Vincent

2011-06-08

We describe a wide-field fluorescence microscope setup which combines HiLo microscopy technique with the use of a two-color fluorescent probe. It allows one-shot fluorescence optical sectioning of thick biological moving sample which is illuminated simultaneously with a flat and a structured pattern at two different wavelengths. Both homogenous and structured fluorescence images are spectrally separated at detection and combined similarly with the HiLo microscopy technique. We present optically sectioned full-field images of Xenopus laevis embryos acquired at 25 images/s frame rate. Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Low-power laser effects at the single-cell level: a confocal microscopy study

NASA Astrophysics Data System (ADS)

Alexandratou, Eleni; Yova, Dido M.; Atlamazoglou, Vassilis; Handris, Panagiotis; Kletsas, Dimitris; Loukas, Spyros

2000-11-01

Confocal microscopy was used for irradiation and observation of the same area of interest, allowing the imaging of low power laser effects in subcellular components and functions, at the single cell level. Coverslips cultures of human fetal foreskin fibroblasts (HFFF2) were placed in a small incubation chamber for in vivo microscopic observation. Cells were stimulated by the 647 nm line of the Argon- Krypton laser of the confocal microscope (0.1 mW/cm2). Membrane permeability, mitochondrial membrane potential ((delta) Psim), intracellular pHi, calcium alterations and nuclear chromatin accessibility were monitored, at different times after irradiation, using specific fluorescent vital probes. Images were stored to the computer and quantitative evaluation was performed using image- processing software. After irradiation, influx and efflux of the appropriate dyes monitored changes in cell membrane permeability. Laser irradiation caused alkalizatoin of the cytosolic pHi and increase of the mitochondrial membrane potential ((delta) Psim). Temporary global Ca2+ responses were also observed. No such effects were noted in microscopic fields other than the irradiated ones. No toxic effects were observed, during time course of the experiment.

Live cell imaging of cytoskeletal and organelle dynamics in gravity-sensing cells in plant gravitropism.

PubMed

Nakamura, Moritaka; Toyota, Masatsugu; Tasaka, Masao; Morita, Miyo Terao

2015-01-01

Plants sense gravity and change their morphology/growth direction accordingly (gravitropism). The early process of gravitropism, gravity sensing, is supposed to be triggered by sedimentation of starch-filled plastids (amyloplasts) in statocytes such as root columella cells and shoot endodermal cells. For several decades, many scientists have focused on characterizing the role of the amyloplasts and observed their intracellular sedimentation in various plants. Recently, it has been discovered that the complex sedimentary movements of the amyloplasts are created not only by gravity but also by cytoskeletal/organelle dynamics, such as those of actin filaments and the vacuolar membrane. Thus, to understand how plants sense gravity, we need to analyze both amyloplast movements and their regulatory systems in statocytes. We have developed a vertical-stage confocal microscope that allows multicolor fluorescence imaging of amyloplasts, actin filaments and vacuolar membranes in vertically oriented plant tissues. We also developed a centrifuge microscope that allows bright-field imaging of amyloplasts during centrifugation. These microscope systems provide new insights into gravity-sensing mechanisms in Arabidopsis.

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.

Localized Surface Plasmon Resonance of Metal Nanodot Nanowire Arrays Studied by Far-Field and Near-Field Optical

DTIC Science & Technology

2007-09-05

microscope, nanoholes or nanogrooves can be created on the film. After coating a thin Au film by electron beam evaporation and soaking the sample in acetone...SNOM. III. Results and Discussion: (a) LSPR of Au Nanodots With the use of an indentation force of 3.8 μN, a nanohole array was generated on the...images of (a) a nanohole array on PMMA and (b) the corresponding Au nanodot array after lift-off. SEM images of (c) a Au nanodot pattern “NANO” on

Target-locking acquisition with real-time confocal (TARC) microscopy.

PubMed

Lu, Peter J; Sims, Peter A; Oki, Hidekazu; Macarthur, James B; Weitz, David A

2007-07-09

We present a real-time target-locking confocal microscope that follows an object moving along an arbitrary path, even as it simultaneously changes its shape, size and orientation. This Target-locking Acquisition with Realtime Confocal (TARC) microscopy system integrates fast image processing and rapid image acquisition using a Nipkow spinning-disk confocal microscope. The system acquires a 3D stack of images, performs a full structural analysis to locate a feature of interest, moves the sample in response, and then collects the next 3D image stack. In this way, data collection is dynamically adjusted to keep a moving object centered in the field of view. We demonstrate the system's capabilities by target-locking freely-diffusing clusters of attractive colloidal particles, and activelytransported quantum dots (QDs) endocytosed into live cells free to move in three dimensions, for several hours. During this time, both the colloidal clusters and live cells move distances several times the length of the imaging volume.

A high resolution study of the glycocalyx of rat uterine epithelial cells during early pregnancy with the field emission gun scanning electron microscope.

PubMed Central

Jones, B J; Murphy, C R

1994-01-01

The field emission gun scanning electron microscope has been used to investigate morphological changes at the macromolecular level in the glycocalyx of rat uterine luminal epithelial cells during early pregnancy. This very high resolution microscope has allowed visualisation at a level previously unobtainable and has enabled us to establish that dramatic alterations occur in this glycocalyx at the time of blastocyst attachment. On d 1 of pregnancy a prominent, filamentous glycocalyx radiates from the microvilli. However, by d 6 of pregnancy when the microvilli have been replaced by irregular cell surface protrusions, the glycocalyceal filaments are completely lost and the plasma membrane appears smooth and covered with a felt-like coating. These morphological observations suggest a major reorganisation in surface carbohydrates during early pregnancy and extend histochemical observations on the uterine epithelial glycocalyx. Images Fig. 1 Fig. 2 Figs. 3 and 4 PMID:7961152

A Simple low-cost device enables four epi-illumination techniques on standard light microscopes.

PubMed

Ishmukhametov, Robert R; Russell, Aidan N; Wheeler, Richard J; Nord, Ashley L; Berry, Richard M

2016-02-08

Back-scattering darkfield (BSDF), epi-fluorescence (EF), interference reflection contrast (IRC), and darkfield surface reflection (DFSR) are advanced but expensive light microscopy techniques with limited availability. Here we show a simple optical design that combines these four techniques in a simple low-cost miniature epi-illuminator, which inserts into the differential interference-contrast (DIC) slider bay of a commercial microscope, without further additions required. We demonstrate with this device: 1) BSDF-based detection of Malarial parasites inside unstained human erythrocytes; 2) EF imaging with and without dichroic components, including detection of DAPI-stained Leishmania parasite without using excitation or emission filters; 3) RIC of black lipid membranes and other thin films, and 4) DFSR of patterned opaque and transparent surfaces. We believe that our design can expand the functionality of commercial bright field microscopes, provide easy field detection of parasites and be of interest to many users of light microscopy.

A Simple low-cost device enables four epi-illumination techniques on standard light microscopes

NASA Astrophysics Data System (ADS)

Ishmukhametov, Robert R.; Russell, Aidan N.; Wheeler, Richard J.; Nord, Ashley L.; Berry, Richard M.

2016-02-01

Back-scattering darkfield (BSDF), epi-fluorescence (EF), interference reflection contrast (IRC), and darkfield surface reflection (DFSR) are advanced but expensive light microscopy techniques with limited availability. Here we show a simple optical design that combines these four techniques in a simple low-cost miniature epi-illuminator, which inserts into the differential interference-contrast (DIC) slider bay of a commercial microscope, without further additions required. We demonstrate with this device: 1) BSDF-based detection of Malarial parasites inside unstained human erythrocytes; 2) EF imaging with and without dichroic components, including detection of DAPI-stained Leishmania parasite without using excitation or emission filters; 3) RIC of black lipid membranes and other thin films, and 4) DFSR of patterned opaque and transparent surfaces. We believe that our design can expand the functionality of commercial bright field microscopes, provide easy field detection of parasites and be of interest to many users of light microscopy.

Collaborative Research and Development (CR&D). Delivery Order 0051: Atomic Scale Transmission Electron Microscope Image Modeling and Application to Semiconductor Heterointerface Characterization

DTIC Science & Technology

2008-01-01

information if it does not display a currently valid OMB control number. PLEASE DO NOT RETURN YOUR FORM TO THE ABOVE ADDRESS. 1. REPORT DATE (DD...microscopy ( AEM ), to characterize a variety of III-V semiconductor thin films. The materials investigated include superlattices based on the InAs- GaSb...technique. TEM observations were performed using a Philips-CM 200 FEG transmission electron microscope equipped with a field emission gun, operated at an

Electric Field Controlled Magnetism in BiFeO3/Ferromagnet Films

NASA Astrophysics Data System (ADS)

Barry, M.; Lee, K.; Chu, Y. H.; Yang, P. L.; Martin, L. W.; Jenkins, C. A.; Ramesh, R.; Scholl, A.; Doran, A.

2007-03-01

BiFeO3 is the only single phase room temperature multiferroic that is currently known. Not only does it have applications as a lead-free replacement for ferroelectric memory cells and piezoelectric sensors, but its interactions with other materials are now attracting a great deal of attention. Its multiferroic nature has potential in the field of exchange bias, where it could allow electric-field control of the ferromagnetic (FM) magnetization. In order to understand this coupling, an understanding of the magnetization in BiFeO3 is necessary. X-ray linear and circular dichroism images were obtained using a high spatial resolution photoelectron emission microscope (PEEM), allowing elemental specificity and surface sensitivity. A piezoelectric force microscope (PFM) was used to map the ferroelectric state in micron-sized regions of the films, which were then probed using crystallographic measurements and temperature dependent PEEM measurements. Temperature dependent structural measurements allow decoupling of the two order parameters, ferroelectric and magnetic, contributing to the photoemission signal. Careful analysis of linear and circular dichroism images allows determination of magnetic directions in BiFeO3 and FM layers.

A 30 mK, 13.5 T scanning tunneling microscope with two independent tips

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

Roychowdhury, Anita; Center for Nanophysics and Advanced Materials, Department of Physics, University of Maryland, College Park, Maryland 20740; Gubrud, M. A.

We describe the design, construction, and performance of an ultra-low temperature, high-field scanning tunneling microscope (STM) with two independent tips. The STM is mounted on a dilution refrigerator and operates at a base temperature of 30 mK with magnetic fields of up to 13.5 T. We focus on the design of the two-tip STM head, as well as the sample transfer mechanism, which allows in situ transfer from an ultra high vacuum preparation chamber while the STM is at 1.5 K. Other design details such as the vibration isolation and rf-filtered wiring are also described. Their effectiveness is demonstrated viamore » spectral current noise characteristics and the root mean square roughness of atomic resolution images. The high-field capability is shown by the magnetic field dependence of the superconducting gap of Cu{sub x}Bi{sub 2}Se{sub 3}. Finally, we present images and spectroscopy taken with superconducting Nb tips with the refrigerator at 35 mK that indicate that the effective temperature of our tips/sample is approximately 184 mK, corresponding to an energy resolution of 16 μeV.« less

NMR Microscopy - Micron-Level Resolution.

NASA Astrophysics Data System (ADS)

Kwok, Wing-Chi Edmund

1990-01-01

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

Development of a high brightness ultrafast Transmission Electron Microscope based on a laser-driven cold field emission source.

PubMed

Houdellier, F; Caruso, G M; Weber, S; Kociak, M; Arbouet, A

2018-03-01

We report on the development of an ultrafast Transmission Electron Microscope based on a cold field emission source which can operate in either DC or ultrafast mode. Electron emission from a tungsten nanotip is triggered by femtosecond laser pulses which are tightly focused by optical components integrated inside a cold field emission source close to the cathode. The properties of the electron probe (brightness, angular current density, stability) are quantitatively determined. The measured brightness is the largest reported so far for UTEMs. Examples of imaging, diffraction and spectroscopy using ultrashort electron pulses are given. Finally, the potential of this instrument is illustrated by performing electron holography in the off-axis configuration using ultrashort electron pulses. Copyright © 2017 Elsevier B.V. All rights reserved.

Improved sensitivity to fluorescence for cancer detection in wide-field image-guided neurosurgery

PubMed Central

Jermyn, Michael; Gosselin, Yoann; Valdes, Pablo A.; Sibai, Mira; Kolste, Kolbein; Mercier, Jeanne; Angulo, Leticia; Roberts, David W.; Paulsen, Keith D.; Petrecca, Kevin; Daigle, Olivier; Wilson, Brian C.; Leblond, Frederic

2015-01-01

In glioma surgery, Protoporphyrin IX (PpIX) fluorescence may identify residual tumor that could be resected while minimizing damage to normal brain. We demonstrate that improved sensitivity for wide-field spectroscopic fluorescence imaging is achieved with minimal disruption to the neurosurgical workflow using an electron-multiplying charge-coupled device (EMCCD) relative to a state-of-the-art CMOS system. In phantom experiments the EMCCD system can detect at least two orders-of-magnitude lower PpIX. Ex vivo tissue imaging on a rat glioma model demonstrates improved fluorescence contrast compared with neurosurgical fluorescence microscope technology, and the fluorescence detection is confirmed with measurements from a clinically-validated spectroscopic probe. Greater PpIX sensitivity in wide-field fluorescence imaging may improve the residual tumor detection during surgery with consequent impact on survival. PMID:26713218

On the influence of lipid-induced optical anisotropy for the bioimaging of exo- or endocytosis with interference microscopic imaging.

PubMed

Marques, D; Miranda, A; Silva, A G; Munro, P R T; DE Beule, P A A

2018-05-01

Some implementations of interference microscopy imaging use digital holographic measurements of complex scattered fields to reconstruct three-dimensional refractive index maps of weakly scattering, semi-transparent objects, frequently encountered in biological investigations. Reconstruction occurs through application of the object scattering potential which assumes an isotropic refractive index throughout the object. Here, we demonstrate that this assumption can in some circumstances be invalid for biological imaging due to the presence of lipid-induced optical anisotropy. We show that the nanoscale organization of lipids in the observation of cellular endocytosis with polarized light induces a significant change in far-field scattering. We obtain this result by presenting a general solution to Maxwell's equations describing light scattering of core-shell particles near an isotropic substrate covered with an anisotropic thin film. This solution is based on an extension of the Bobbert-Vlieger solution for particle scattering near a substrate delivering an exact solution to the scattering problem in the near field as well as far field. By applying this solution to study light scattering by a lipid vesicle near a lipid bilayer, whereby the lipids are represented through a biaxial optical model, we conclude through ellipsometry concepts that effective amounts of lipid-induced optical anisotropy significantly alter far-field optical scattering in respect to an equivalent optical model that neglects the presence of optical anisotropy. © 2018 The Authors Journal of Microscopy © 2018 Royal Microscopical Society.

Computer synthesis of high resolution electron micrographs

NASA Technical Reports Server (NTRS)

Nathan, R.

1976-01-01

Specimen damage, spherical aberration, low contrast and noisy sensors combine to prevent direct atomic viewing in a conventional electron microscope. The paper describes two methods for obtaining ultra-high resolution in biological specimens under the electron microscope. The first method assumes the physical limits of the electron objective lens and uses a series of dark field images of biological crystals to obtain direct information on the phases of the Fourier diffraction maxima; this information is used in an appropriate computer to synthesize a large aperture lens for a 1-A resolution. The second method assumes there is sufficient amplitude scatter from images recorded in focus which can be utilized with a sensitive densitometer and computer contrast stretching to yield fine structure image details. Cancer virus characterization is discussed as an illustrative example. Numerous photographs supplement the text.

An automatic chip structure optical inspection system for electronic components

NASA Astrophysics Data System (ADS)

Song, Zhichao; Xue, Bindang; Liang, Jiyuan; Wang, Ke; Chen, Junzhang; Liu, Yunhe

2018-01-01

An automatic chip structure inspection system based on machine vision is presented to ensure the reliability of electronic components. It consists of four major modules, including a metallographic microscope, a Gigabit Ethernet high-resolution camera, a control system and a high performance computer. An auto-focusing technique is presented to solve the problem that the chip surface is not on the same focusing surface under the high magnification of the microscope. A panoramic high-resolution image stitching algorithm is adopted to deal with the contradiction between resolution and field of view, caused by different sizes of electronic components. In addition, we establish a database to storage and callback appropriate parameters to ensure the consistency of chip images of electronic components with the same model. We use image change detection technology to realize the detection of chip images of electronic components. The system can achieve high-resolution imaging for chips of electronic components with various sizes, and clearly imaging for the surface of chip with different horizontal and standardized imaging for ones with the same model, and can recognize chip defects.

Enhancing image contrast of carbon nanotubes on cellular background using helium ion microscope by varying helium ion fluence.

PubMed

Dykas, M M; Poddar, K; Yoong, S L; Viswanathan, V; Mathew, S; Patra, A; Saha, S; Pastorin, G; Venkatesan, T

2018-01-01

Carbon nanotubes (CNTs) have become an important nano entity for biomedical applications. Conventional methods of their imaging, often cannot be applied in biological samples due to an inadequate spatial resolution or poor contrast between the CNTs and the biological sample. Here we report a unique and effective detection method, which uses differences in conductivities of carbon nanotubes and HeLa cells. The technique involves the use of a helium ion microscope to image the sample with the surface charging artefacts created by the He + and neutralised by electron flood gun. This enables us to obtain a few nanometre resolution images of CNTs in HeLa Cells with high contrast, which was achieved by tailoring the He + fluence. Charging artefacts can be efficiently removed for conductive CNTs by a low amount of electrons, the fluence of which is not adequate to discharge the cell surface, resulting in high image contrast. Thus, this technique enables rapid detection of any conducting nano structures on insulating cellular background even in large fields of view and fine spatial resolution. The technique demonstrated has wider applications for researchers seeking enhanced contrast and high-resolution imaging of any conducting entity in a biological matrix - a commonly encountered issue of importance in drug delivery, tissue engineering and toxicological studies. © 2017 The Authors Journal of Microscopy © 2017 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

Field-testing of a cost-effective mobile-phone based microscope for screening of Schistosoma haematobium infection (Conference Presentation)

NASA Astrophysics Data System (ADS)

Ceylan Koydemir, Hatice; Bogoch, Isaac I.; Tseng, Derek; Ephraim, Richard K. D.; Duah, Evans; Tee, Joseph; Andrews, Jason R.; Ozcan, Aydogan

2016-03-01

Schistosomiasis is a parasitic and neglected tropical disease, and affects <200-million people across the world, with school-aged children disproportionately affected. Here we present field-testing results of a handheld and cost effective smartphone-based microscope in rural Ghana, Africa, for point-of-care diagnosis of S. haematobium infection. In this mobile-phone microscope, a custom-designed 3D printed opto-mechanical attachment (~150g) is placed in contact with the smartphone camera-lens, creating an imaging-system with a half-pitch resolution of ~0.87µm. This unit includes an external lens (also taken from a mobile-phone camera), a sample tray, a z-stage to adjust the focus, two light-emitting-diodes (LEDs) and two diffusers for uniform illumination of the sample. In our field-testing, 60 urine samples, collected from children, were used, where the prevalence of the infection was 72.9%. After concentration of the sample with centrifugation, the sediment was placed on a glass-slide and S. haematobium eggs were first identified/quantified using conventional benchtop microscopy by an expert diagnostician, and then a second expert, blinded to these results, determined the presence/absence of eggs using our mobile-phone microscope. Compared to conventional microscopy, our mobile-phone microscope had a diagnostic sensitivity of 72.1%, specificity of 100%, positive-predictive-value of 100%, and a negative-predictive-value of 57.1%. Furthermore, our mobile-phone platform demonstrated a sensitivity of 65.7% and 100% for low-intensity infections (≤50 eggs/10 mL urine) and high-intensity infections (<50 eggs/10 mL urine), respectively. We believe that this cost-effective and field-portable mobile-phone microscope may play an important role in the diagnosis of schistosomiasis and various other global health challenges.

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.

Ultrafast Graphene Photonics and Optoelectronics

DTIC Science & Technology

2017-04-14

SUBJECT TERMS Graphene, Ultrafast Optical Processin, Terahertz Electronics ; 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT SAR 18...Rep, (2016)) Fig. 4. (a) Images of scanning electron microscope for 1D and 2D gratings. (b) Ratio of the real part of the transmitted field

Intraoperative Fluorescence Cerebral Angiography by Laser Surgical Microscopy: Comparison With Xenon Microscopy and Simultaneous Observation of Cerebral Blood Flow and Surrounding Structures.

PubMed

Ito, Yuhei; Suzuki, Kyouichi; Ichikawa, Tsuyoshi; Watanabe, Yoichi; Sato, Taku; Sakuma, Jun; Saito, Kiyoshi

2018-06-12

Laser surgical microscopes should enable uniform illumination of the operative field, and require less luminous energy compared with existing xenon surgical microscopes. To examine the utility of laser illumination in fluorescence cerebral angiography. Fluorescein sodium (fluorescein) was used as a fluorescent dye. We first compared the clarity of cerebral blood flow images collected by fluorescence angiography between the laser illumination and xenon illumination methods. We then assessed use of the laser illuminator for simultaneous observation of blood flow and surrounding structures during fluorescence angiography. Furthermore, the study was designed to evaluate usefulness of the thus determined excitation light in clinical cases. Fluorescence angiography using blue light laser for excitation provided higher clarity and contrast blood flow images compared with using blue light generated from a xenon lamp. Further, illumination with excitation light consisting of a combination of 3 types of laser (higher level of blue light, no green light, and lower level of red light) enabled both blood flow and surrounding structures to be observed through the microscope directly by the surgeon. Laser-illuminated fluorescence angiography provides high clarity and contrast images of cerebral blood flow. Further, a laser providing strong blue light and weak red light for excitation light enables simultaneous visual observation of fluorescent blood flow and surrounding structures by the surgeon using a surgical microscope. Overall, these data suggest that laser surgical microscopes are useful for both ordinary operative manipulations and fluorescence angiography.

Tip-enhanced near-field optical microscope with side-on and ATR-mode sample excitation for super-resolution Raman imaging of surfaces

NASA Astrophysics Data System (ADS)

Heilman, A. L.; Gordon, M. J.

2016-06-01

A tip-enhanced near-field optical microscope with side-on and attenuated total reflectance (ATR) excitation and collection is described and used to demonstrate sub-diffraction-limited (super-resolution) optical and chemical characterization of surfaces. ATR illumination is combined with an Au optical antenna tip to show that (i) the tip can quantitatively transduce the optical near-field (evanescent waves) above the surface by scattering photons into the far-field, (ii) the ATR geometry enables excitation and characterization of surface plasmon polaritons (SPPs), whose associated optical fields are shown to enhance Raman scattering from a thin layer of copper phthalocyanine (CuPc), and (iii) SPPs can be used to plasmonically excite the tip for super-resolution chemical imaging of patterned CuPc via tip-enhanced Raman spectroscopy (TERS). ATR-illumination TERS is also quantitatively compared with the more conventional side-on illumination scheme. In both cases, spatial resolution was better than 40 nm and tip on/tip off Raman enhancement factors were >6500. Furthermore, ATR illumination was shown to provide similar Raman signal levels at lower "effective" pump powers due to additional optical energy delivered by SPPs to the active region in the tip-surface gap.

Tip-enhanced near-field optical microscope with side-on and ATR-mode sample excitation for super-resolution Raman imaging of surfaces

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

Heilman, A. L.; Gordon, M. J.

A tip-enhanced near-field optical microscope with side-on and attenuated total reflectance (ATR) excitation and collection is described and used to demonstrate sub-diffraction-limited (super-resolution) optical and chemical characterization of surfaces. ATR illumination is combined with an Au optical antenna tip to show that (i) the tip can quantitatively transduce the optical near-field (evanescent waves) above the surface by scattering photons into the far-field, (ii) the ATR geometry enables excitation and characterization of surface plasmon polaritons (SPPs), whose associated optical fields are shown to enhance Raman scattering from a thin layer of copper phthalocyanine (CuPc), and (iii) SPPs can be used tomore » plasmonically excite the tip for super-resolution chemical imaging of patterned CuPc via tip-enhanced Raman spectroscopy (TERS). ATR-illumination TERS is also quantitatively compared with the more conventional side-on illumination scheme. In both cases, spatial resolution was better than 40 nm and tip on/tip off Raman enhancement factors were >6500. Furthermore, ATR illumination was shown to provide similar Raman signal levels at lower “effective” pump powers due to additional optical energy delivered by SPPs to the active region in the tip-surface gap.« less

Non-invasive Differentiation of Kidney Stone Types using X-ray Dark-Field Radiography

PubMed Central

Scherer, Kai; Braig, Eva; Willer, Konstantin; Willner, Marian; Fingerle, Alexander A.; Chabior, Michael; Herzen, Julia; Eiber, Matthias; Haller, Bernhard; Straub, Michael; Schneider, Heike; Rummeny, Ernst J.; Noël, Peter B.; Pfeiffer, Franz

2015-01-01

Treatment of renal calculi is highly dependent on the chemical composition of the stone in question, which is difficult to determine using standard imaging techniques. The objective of this study is to evaluate the potential of scatter-sensitive X-ray dark-field radiography to differentiate between the most common types of kidney stones in clinical practice. Here, we examine the absorption-to-scattering ratio of 118 extracted kidney stones with a laboratory Talbot-Lau Interferometer. Depending on their chemical composition, microscopic growth structure and morphology the various types of kidney stones show strongly varying, partially opposite contrasts in absorption and dark-field imaging. By assessing the microscopic calculi morphology with high resolution micro-computed tomography measurements, we illustrate the dependence of dark-field signal strength on the respective stone type. Finally, we utilize X-ray dark-field radiography as a non-invasive, highly sensitive (100%) and specific (97%) tool for the differentiation of calcium oxalate, uric acid and mixed types of stones, while additionally improving the detectability of radio-lucent calculi. We prove clinical feasibility of the here proposed method by accurately classifying renal stones, embedded within a fresh pig kidney, using dose-compatible measurements and a quick and simple visual inspection. PMID:25873414

Hard x-ray phase contrastmicroscopy - techniques and applications

NASA Astrophysics Data System (ADS)

Holzner, Christian

In 1918, Einstein provided the first description of the nature of the refractive index for X-rays, showing that phase contrast effects are significant. A century later, most x-ray microscopy and nearly all medical imaging remains based on absorption contrast, even though phase contrast offers orders of magnitude improvements in contrast and reduced radiation exposure at multi-keV x-ray energies. The work presented is concerned with developing practical and quantitative methods of phase contrast for x-ray microscopy. A theoretical framework for imaging in phase contrast is put forward; this is used to obtain quantitative images in a scanning microscope using a segmented detector, and to correct for artifacts in a commercial phase contrast x-ray nano-tomography system. The principle of reciprocity between scanning and full-field microscopes is then used to arrive at a novel solution: Zernike contrast in a scanning microscope. These approaches are compared on a theoretical and experimental basis in direct connection with applications using multi-keV x-ray microscopes at the Advanced Photon Source at Argonne National Laboratory. Phase contrast provides the best means to image mass and ultrastructure of light elements that mainly constitute biological matter, while stimulated x-ray fluorescence provides high sensitivity for studies of the distribution of heavier trace elements, such as metals. These approaches are combined in a complementary way to yield quantitative maps of elemental concentration from 2D images, with elements placed in their ultrastructural context. The combination of x-ray fluorescence and phase contrast poses an ideal match for routine, high resolution tomographic imaging of biological samples in the future. The presented techniques and demonstration experiments will help pave the way for this development.

Quantitative phase imaging by wide field lensless digital holographic microscope

NASA Astrophysics Data System (ADS)

Adinda-Ougba, A.; Koukourakis, N.; Essaidi, A.; Ger­hardt, N. C.; Hofmann, M. R.

2015-05-01

Wide field, lensless microscopes have been developed for telemedicine and for resource limited setting [1]. They are based on in-line digital holography which is capable to provide amplitude and phase information resulting from numerical reconstruction. The phase information enables achieving axial resolution in the nanometer range. Hence, such microscopes provide a powerful tool to determine three-dimensional topologies of microstructures. In this contribution, a compact, low-cost, wide field, lensless microscope is presented, which is capable of providing topological profiles of microstructures in transparent material. Our setup consist only of two main components: a CMOSsensor chip and a laser diode without any need of a pinhole. We use this very simple setup to record holograms of microobjects. A wide field of view of ~24 mm², and a lateral resolution of ~2 μm are achieved. Moreover, amplitude and phase information are obtained from the numerical reconstruction of the holograms using a phase retrieval algorithm together with the angular spectrum propagation method. Topographic information of highly transparent micro-objects is obtained from the phase data. We evaluate our system by recording holograms of lines with different depths written by a focused laser beam. A reliable characterization of laser written microstructures is crucial for their functionality. Our results show that this system is valuable for determination of topological profiles of microstructures in transparent material.

Reprint of: Atmospheric scanning electron microscope observes cells and tissues in open medium through silicon nitride film.

PubMed

Nishiyama, Hidetoshi; Suga, Mitsuo; Ogura, Toshihiko; Maruyama, Yuusuke; Koizumi, Mitsuru; Mio, Kazuhiro; Kitamura, Shinichi; Sato, Chikara

2010-11-01

Direct observation of subcellular structures and their characterization is essential for understanding their physiological functions. To observe them in open environment, we have developed an inverted scanning electron microscope with a detachable, open-culture dish, capable of 8 nm resolution, and combined with a fluorescence microscope quasi-simultaneously observing the same area from the top. For scanning electron microscopy from the bottom, a silicon nitride film window in the base of the dish maintains a vacuum between electron gun and open sample dish while allowing electrons to pass through. Electrons are backscattered from the sample and captured by a detector under the dish. Cells cultured on the open dish can be externally manipulated under optical microscopy, fixed, and observed using scanning electron microscopy. Once fine structures have been revealed by scanning electron microscopy, their component proteins may be identified by comparison with separately prepared fluorescence-labeled optical microscopic images of the candidate proteins, with their heavy-metal-labeled or stained ASEM images. Furthermore, cell nuclei in a tissue block stained with platinum-blue were successfully observed without thin-sectioning, which suggests the applicability of this inverted scanning electron microscope to cancer diagnosis. This microscope visualizes mesoscopic-scale structures, and is also applicable to non-bioscience fields including polymer chemistry. Copyright © 2010 Elsevier Inc. All rights reserved.

Automated selection of the most epithelium-rich areas in gynecologic tumor sections.

PubMed

Schipper, N W; Baak, J P; Smeulders, A W

1991-12-01

The paper describes an image analysis technique for automated selection of the epithelium-rich areas in standard paraffin tissue sections of ovarian and endometrial premalignancies and malignancies. Two staining procedures were evaluated, Feulgen (pararosanilin) and CAM 5.2, demonstrating the presence of cytokeratin 8 and 18; both were counterstained with naphthol yellow. The technique is based on the corresponding image processing method of automated estimation of the percentage of epithelium in interactively selected microscope fields. With the technique, one image is recorded with a filter to demonstrate where epithelium and stroma lie. This filter is chosen according to the type of staining: it is yellow (lambda = 552 nm) for Feulgen and blue (lambda = 470 nm) for anticytokeratin CAM 5.2. When stroma cannot be distinguished from lumina with the green filter or from epithelium with the blue filter, a second image is recorded from the same microscope field, with a blue filter (lambda = 420 nm) for Feulgen and a yellow filter (lambda = 576 nm) for anticytokeratin CAM 5.2. Discrimination between epithelium and stroma is based on the image contrast range and the packing of nuclei in the yellow image and on the automated classification of the gray value histogram peaks in the blue image. For Feulgen stain the method was evaluated on 30 ovarian tumors of the common epithelial types (8 borderline tumors and 22 carcinomas with various degrees of differentiation) and 30 endometrial carcinomas of different grades.(ABSTRACT TRUNCATED AT 250 WORDS)

3D on-chip microscopy of optically cleared tissue

NASA Astrophysics Data System (ADS)

Zhang, Yibo; Shin, Yoonjung; Sung, Kevin; Yang, Sam; Chen, Harrison; Wang, Hongda; Teng, Da; Rivenson, Yair; Kulkarni, Rajan P.; Ozcan, Aydogan

2018-02-01

Traditional pathology relies on tissue biopsy, micro-sectioning, immunohistochemistry and microscopic imaging, which are relatively expensive and labor-intensive, and therefore are less accessible in resource-limited areas. Low-cost tissue clearing techniques, such as the simplified CLARITY method (SCM), are promising to potentially reduce the cost of disease diagnosis by providing 3D imaging and phenotyping of thicker tissue samples with simpler preparation steps. However, the mainstream imaging approach for cleared tissue, fluorescence microscopy, suffers from high-cost, photobleaching and signal fading. As an alternative approach to fluorescence, here we demonstrate 3D imaging of SCMcleared tissue using on-chip holography, which is based on pixel-super-resolution and multi-height phase recovery algorithms to digitally compute the sample's amplitude and phase images at various z-slices/depths through the sample. The tissue clearing procedures and the lens-free imaging system were jointly optimized to find the best illumination wavelength, tissue thickness, staining solution pH, and the number of hologram heights to maximize the imaged tissue volume, minimize the amount of acquired data, while maintaining a high contrast-to-noise ratio for the imaged cells. After this optimization, we achieved 3D imaging of a 200-μm thick cleared mouse brain tissue over a field-of-view of <20mm2 , and the resulting 3D z-stack agrees well with the images acquired with a scanning lens-based microscope (20× 0.75NA). Moreover, the lens-free microscope achieves an order-of-magnitude better data efficiency compared to its lens-based counterparts for volumetric imaging of samples. The presented low-cost and high-throughput lens-free tissue imaging technique enabled by CLARITY can be used in various biomedical applications in low-resource-settings.

Hybrid reflecting objectives for functional multiphoton microscopy in turbid media

PubMed Central

Vučinić, Dejan; Bartol, Thomas M.; Sejnowski, Terrence J.

2010-01-01

Most multiphoton imaging of biological specimens is performed using microscope objectives optimized for high image quality under wide-field illumination. We present a class of objectives designed de novo without regard for these traditional constraints, driven exclusively by the needs of fast multiphoton imaging in turbid media: the delivery of femtosecond pulses without dispersion and the efficient collection of fluorescence. We model the performance of one such design optimized for a typical brain-imaging setup and show that it can greatly outperform objectives commonly used for this task. PMID:16880851

Wide-field microscopy using microcamera arrays

NASA Astrophysics Data System (ADS)

Marks, Daniel L.; Youn, Seo Ho; Son, Hui S.; Kim, Jungsang; Brady, David J.

2013-02-01

A microcamera is a relay lens paired with image sensors. Microcameras are grouped into arrays to relay overlapping views of a single large surface to the sensors to form a continuous synthetic image. The imaged surface may be curved or irregular as each camera may independently be dynamically focused to a different depth. Microcamera arrays are akin to microprocessors in supercomputers in that both join individual processors by an optoelectronic routing fabric to increase capacity and performance. A microcamera may image ten or more megapixels and grouped into an array of several hundred, as has already been demonstrated by the DARPA AWARE Wide-Field program with multiscale gigapixel photography. We adapt gigapixel microcamera array architectures to wide-field microscopy of irregularly shaped surfaces to greatly increase area imaging over 1000 square millimeters at resolutions of 3 microns or better in a single snapshot. The system includes a novel relay design, a sensor electronics package, and a FPGA-based networking fabric. Biomedical applications of this include screening for skin lesions, wide-field and resolution-agile microsurgical imaging, and microscopic cytometry of millions of cells performed in situ.

Optical Scatter Imaging with a digital micromirror device.

PubMed

Zheng, Jing-Yi; Pasternack, Robert M; Boustany, Nada N

2009-10-26

We had developed Optical Scatter Imaging (OSI) as a method which combines light scattering spectroscopy with microscopic imaging to probe local particle size in situ. Using a variable diameter iris as a Fourier spatial filter, the technique consisted of collecting images that encoded the intensity ratio of wide-to-narrow angle scatter at each pixel in the full field of view. In this paper, we replace the variable diameter Fourier filter with a digital micromirror device (DMD) to extend our assessment of morphology to the characterization of particle shape and orientation. We describe our setup in detail and demonstrate how to eliminate aberrations associated with the placement of the DMD in a conjugate Fourier plane of our microscopic imaging system. Using bacteria and polystyrene spheres, we show how this system can be used to assess particle aspect ratio even when imaged at low resolution. We also show the feasibility of detecting alterations in organelle aspect ratio in situ within living cells. This improved OSI system could be further developed to automate morphological quantification and sorting of non-spherical particles in situ.

Optical computing.

NASA Technical Reports Server (NTRS)

Stroke, G. W.

1972-01-01

Applications of the optical computer include an approach for increasing the sharpness of images obtained from the most powerful electron microscopes and fingerprint/credit card identification. The information-handling capability of the various optical computing processes is very great. Modern synthetic-aperture radars scan upward of 100,000 resolvable elements per second. Fields which have assumed major importance on the basis of optical computing principles are optical image deblurring, coherent side-looking synthetic-aperture radar, and correlative pattern recognition. Some examples of the most dramatic image deblurring results are shown.

Aplanatic and quasi-aplanatic diffraction gratings

DOEpatents

Hettrick, M.C.

1987-09-14

A reflection diffraction grating having a series of transverse minute grooves of progressively varying spacing along a concave surface enables use of such gratings for x-ray or longer wavelength imaging of objects. The variable groove spacing establishes aplanatism or substantially uniform magnetification across the optical aperture. The grating may be sued, for example, in x-ray microscopes or telescopes of the imaging type and in x-ray microprobed. Increased spatial resolution and field of view may be realized in x-ray imaging. 5 figs.

Cluster secondary ion mass spectrometry microscope mode mass spectrometry imaging.

PubMed

Kiss, András; Smith, Donald F; Jungmann, Julia H; Heeren, Ron M A

2013-12-30

Microscope mode imaging for secondary ion mass spectrometry is a technique with the promise of simultaneous high spatial resolution and high-speed imaging of biomolecules from complex surfaces. Technological developments such as new position-sensitive detectors, in combination with polyatomic primary ion sources, are required to exploit the full potential of microscope mode mass spectrometry imaging, i.e. to efficiently push the limits of ultra-high spatial resolution, sample throughput and sensitivity. In this work, a C60 primary source was combined with a commercial mass microscope for microscope mode secondary ion mass spectrometry imaging. The detector setup is a pixelated detector from the Medipix/Timepix family with high-voltage post-acceleration capabilities. The system's mass spectral and imaging performance is tested with various benchmark samples and thin tissue sections. The high secondary ion yield (with respect to 'traditional' monatomic primary ion sources) of the C60 primary ion source and the increased sensitivity of the high voltage detector setup improve microscope mode secondary ion mass spectrometry imaging. The analysis time and the signal-to-noise ratio are improved compared with other microscope mode imaging systems, all at high spatial resolution. We have demonstrated the unique capabilities of a C60 ion microscope with a Timepix detector for high spatial resolution microscope mode secondary ion mass spectrometry imaging. Copyright © 2013 John Wiley & Sons, Ltd.

Simultaneous multiview capture and fusion improves spatial resolution in wide-field and light-sheet microscopy

PubMed Central

Wu, Yicong; Chandris, Panagiotis; Winter, Peter W.; Kim, Edward Y.; Jaumouillé, Valentin; Kumar, Abhishek; Guo, Min; Leung, Jacqueline M.; Smith, Corey; Rey-Suarez, Ivan; Liu, Huafeng; Waterman, Clare M.; Ramamurthi, Kumaran S.; La Riviere, Patrick J.; Shroff, Hari

2016-01-01

Most fluorescence microscopes are inefficient, collecting only a small fraction of the emitted light at any instant. Besides wasting valuable signal, this inefficiency also reduces spatial resolution and causes imaging volumes to exhibit significant resolution anisotropy. We describe microscopic and computational techniques that address these problems by simultaneously capturing and subsequently fusing and deconvolving multiple specimen views. Unlike previous methods that serially capture multiple views, our approach improves spatial resolution without introducing any additional illumination dose or compromising temporal resolution relative to conventional imaging. When applying our methods to single-view wide-field or dual-view light-sheet microscopy, we achieve a twofold improvement in volumetric resolution (~235 nm × 235 nm × 340 nm) as demonstrated on a variety of samples including microtubules in Toxoplasma gondii, SpoVM in sporulating Bacillus subtilis, and multiple protein distributions and organelles in eukaryotic cells. In every case, spatial resolution is improved with no drawback by harnessing previously unused fluorescence. PMID:27761486

Digital holographic microscopy for detection of Trypanosoma cruzi parasites in fresh blood mounts

NASA Astrophysics Data System (ADS)

Romero, G. G.; Monaldi, A. C.; Alanís, E. E.

2012-03-01

An off-axis holographic microscope, in a transmission mode, calibrated to automatically detect the presence of Trypanosoma cruzi in blood is developed as an alternative diagnosis tool for Chagas disease. Movements of the microorganisms are detected by measuring the phase shift they produce on the transmitted wave front. A thin layer of blood infected by Trypanosoma cruzi parasites is examined in the holographic microscope, the images of the visual field being registered with a CCD camera. Two consecutive holograms of the same visual field are subtracted point by point and a phase contrast image of the resulting hologram is reconstructed by means of the angular spectrum propagation algorithm. This method enables the measurement of phase distributions corresponding to temporal differences between digital holograms in order to detect whether parasites are present or not. Experimental results obtained using this technique show that it is an efficient alternative that can be incorporated successfully as a part of a fully automatic system for detection and counting of this type of microorganisms.

Multimodal full-field optical coherence tomography on biological tissue: toward all optical digital pathology

NASA Astrophysics Data System (ADS)

Harms, F.; Dalimier, E.; Vermeulen, P.; Fragola, A.; Boccara, A. C.

2012-03-01

Optical Coherence Tomography (OCT) is an efficient technique for in-depth optical biopsy of biological tissues, relying on interferometric selection of ballistic photons. Full-Field Optical Coherence Tomography (FF-OCT) is an alternative approach to Fourier-domain OCT (spectral or swept-source), allowing parallel acquisition of en-face optical sections. Using medium numerical aperture objective, it is possible to reach an isotropic resolution of about 1x1x1 ìm. After stitching a grid of acquired images, FF-OCT gives access to the architecture of the tissue, for both macroscopic and microscopic structures, in a non-invasive process, which makes the technique particularly suitable for applications in pathology. Here we report a multimodal approach to FF-OCT, combining two Full-Field techniques for collecting a backscattered endogeneous OCT image and a fluorescence exogeneous image in parallel. Considering pathological diagnosis of cancer, visualization of cell nuclei is of paramount importance. OCT images, even for the highest resolution, usually fail to identify individual nuclei due to the nature of the optical contrast used. We have built a multimodal optical microscope based on the combination of FF-OCT and Structured Illumination Microscopy (SIM). We used x30 immersion objectives, with a numerical aperture of 1.05, allowing for sub-micron transverse resolution. Fluorescent staining of nuclei was obtained using specific fluorescent dyes such as acridine orange. We present multimodal images of healthy and pathological skin tissue at various scales. This instrumental development paves the way for improvements of standard pathology procedures, as a faster, non sacrificial, operator independent digital optical method compared to frozen sections.

A method for determining electrophoretic and electroosmotic mobilities using AC and DC electric field particle displacements.

PubMed

Oddy, M H; Santiago, J G

2004-01-01

We have developed a method for measuring the electrophoretic mobility of submicrometer, fluorescently labeled particles and the electroosmotic mobility of a microchannel. We derive explicit expressions for the unknown electrophoretic and the electroosmotic mobilities as a function of particle displacements resulting from alternating current (AC) and direct current (DC) applied electric fields. Images of particle displacements are captured using an epifluorescent microscope and a CCD camera. A custom image-processing code was developed to determine image streak lengths associated with AC measurements, and a custom particle tracking velocimetry (PTV) code was devised to determine DC particle displacements. Statistical analysis was applied to relate mobility estimates to measured particle displacement distributions.

Full field study of strain distribution near the crack tip in the fracture of solid propellants via large strain digital image correlation and optical microscopy

NASA Astrophysics Data System (ADS)

Gonzalez, Javier

A full field method for visualizing deformation around the crack tip in a fracture process with large strains is developed. A digital image correlation program (DIC) is used to incrementally compute strains and displacements between two consecutive images of a deformation process. Values of strain and displacements for consecutive deformations are added, this way solving convergence problems in the DIC algorithm when large deformations are investigated. The method developed is used to investigate the strain distribution within 1 mm of the crack tip in a particulate composite solid (propellant) using microscopic visualization of the deformation process.

Quantitative Assessment of Fat Levels in Caenorhabditis elegans Using Dark Field Microscopy

PubMed Central

Fouad, Anthony D.; Pu, Shelley H.; Teng, Shelly; Mark, Julian R.; Fu, Moyu; Zhang, Kevin; Huang, Jonathan; Raizen, David M.; Fang-Yen, Christopher

2017-01-01

The roundworm Caenorhabditis elegans is widely used as a model for studying conserved pathways for fat storage, aging, and metabolism. The most broadly used methods for imaging fat in C. elegans require fixing and staining the animal. Here, we show that dark field images acquired through an ordinary light microscope can be used to estimate fat levels in worms. We define a metric based on the amount of light scattered per area, and show that this light scattering metric is strongly correlated with worm fat levels as measured by Oil Red O (ORO) staining across a wide variety of genetic backgrounds and feeding conditions. Dark field imaging requires no exogenous agents or chemical fixation, making it compatible with live worm imaging. Using our method, we track fat storage with high temporal resolution in developing larvae, and show that fat storage in the intestine increases in at least one burst during development. PMID:28404661

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.

Applications of Real Space Crystallography in Characterization of Dislocations in Geological Materials in a Scanning Electron Microscope (SEM)

NASA Astrophysics Data System (ADS)

Kaboli, S.; Burnley, P. C.

2017-12-01

Imaging and characterization of defects in crystalline materials is of significant importance in various disciplines including geoscience, materials science, and applied physics. Linear defects such as dislocations and planar defects such as twins and stacking faults, strongly influence many of the properties of crystalline materials and also reflect the conditions and degree of deformation. Dislocations have been conventionally imaged in thin foils in a transmission electron microscope (TEM). Since the development of field emission scanning electron microscopes (FE-SEM) with high gun brightness and small spot size, extensive efforts have been dedicated to the imaging and characterization of dislocations in semi-conductors using electron channeling contrast imaging (ECCI) in the SEM. The obvious advantages of using SEM over TEM include easier and non-destructive sample preparation and a large field of view enabling statistical examination of the density and distribution of dislocations and other defects. In this contribution, we extend this technique to geological materials and introduce the Real Space Crystallography methodology for imaging and complete characterization of dislocations based on bend contour contrast obtained by ECCI in FE-SEM. Bend contours map out the distortion in the crystal lattice across a deformed grain. The contrast of dislocations is maximum in the vicinity of bend contours where crystal planes diffract at small and positive deviations from the Bragg positions (as defined by Bragg's law of electron diffraction). Imaging is performed in a commercial FE-SEM equipped with a standard silicon photodiode backscattered (BSE) detector and an electron backscatter diffraction (EBSD) system for crystal orientation measurements. We demonstrate the practice of this technique in characterization of a number of geological materials in particular quartz, forsterite olivine and corundum, experimentally deformed at high pressure-temperature conditions. This new approach in microstructure characterization of deformed geologic materials in FE-SEM, without the use of etching or decoration techniques, has valuable applications to both experimentally deformed and naturally deformed specimens.

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.

Instruments for Imaging from Far to Near

NASA Technical Reports Server (NTRS)

Mungas, Greg; Boynton, John; Sepulveda, Cesar

2009-01-01

The acronym CHAMP (signifying camera, hand lens, and microscope ) denotes any of several proposed optoelectronic instruments that would be capable of color imaging at working distances that could be varied continuously through a range from infinity down to several millimeters. As in any optical instrument, the magnification, depth of field, and spatial resolution would vary with the working distance. For example, in one CHAMP version, at a working distance of 2.5 m, the instrument would function as an electronic camera with a magnification of 1/100, whereas at a working distance of 7 mm, the instrument would function as a microscope/electronic camera with a magnification of 4.4. Moreover, as described below, when operating at or near the shortest-working-distance/highest-magnification combination, a CHAMP could be made to perform one or more spectral imaging functions. CHAMPs were originally intended to be used in robotic geological exploration of the Moon and Mars. The CHAMP concept also has potential for diverse terrestrial applications that could include remotely controlled or robotic geological exploration, prospecting, field microbiology, environmental surveying, and assembly- line inspection. A CHAMP (see figure) would include two lens cells: (1) a distal cell corresponding to the objective lens assembly of a conventional telescope or microscope and (2) a proximal cell that would contain the focusing camera lens assembly and the camera electronic image-detector chip, which would be of the active-pixel-sensor (APS) type. The distal lens cell would face outward from a housing, while the proximal lens cell would lie in a clean environment inside the housing. The proximal lens cell would contain a beam splitter that would enable simultaneous use of the imaging optics (that is, proximal and distal lens assemblies) for imaging and illumination of the field of view. The APS chip would be mounted on a focal plane on a side face of the beam splitter, while light for illuminating the field of view would enter the imaging optics via the end face of the beam splitter. The proximal lens cell would be mounted on a sled that could be translated along the optical axis for focus adjustment. The position of the CHAMP would initially be chosen at the desired working distance of the distal lens from (corresponding to an approximate desired magnification of) an object to be examined. During subsequent operation, the working distance would ordinarily remain fixed at the chosen value and the position of the proximal lens cell within the instrument would be adjusted for focus as needed.

Computerized detection of leukocytes in microscopic leukorrhea images.

PubMed

Zhang, Jing; Zhong, Ya; Wang, Xiangzhou; Ni, Guangming; Du, Xiaohui; Liu, Juanxiu; Liu, Lin; Liu, Yong

2017-09-01

Detection of leukocytes is critical for the routine leukorrhea exam, which is widely used in gynecological examinations. An elevated vaginal leukocyte count in women with bacterial vaginosis is a strong predictor of vaginal or cervical infections. In the routine leukorrhea exam, the counting of leukocytes is primarily performed by manual techniques. However, the viewing and counting of leukocytes from multiple high-power viewing fields on a glass slide under a microscope leads to subjectivity, low efficiency, and low accuracy. To date, many biological cells in stool, blood, and breast cancer have been studied to realize computerized detection; however, the detection of leukocytes in microscopic leukorrhea images has not been studied. Thus, there is an increasing need for computerized detection of leukocytes. There are two key processes in the computerized detection of leukocytes in digital image processing. One is segmentation; the other is intelligent classification. In this paper, we propose a combined ensemble to detect leukocytes in the microscopic leukorrhea image. After image segmentation and selecting likely leukocyte subimages, we obtain the leukocyte candidates. Then, for intelligent classification, we adopt two methods: feature extraction and classification by a support vector machine (SVM); applying a modified convolutional neural network (CNN) to the larger subimages. If different methods classify a candidate in the same category, the process is finished. If not, the outputs of the methods are provided to a classifier to further classify the candidate. After acquiring leukocyte candidates, we attempted three methods to perform classification. The first approach using features and SVM achieved 88% sensitivity, 97% specificity, and 92.5% accuracy. The second method using CNN achieved 95% sensitivity, 84% specificity, and 89.5% accuracy. Then, in the combination approach, we achieved 92% sensitivity, 95% specificity, and 93.5% accuracy. Finally, the images with marked and counted leukocytes were obtained. A novel computerized detection system was developed for automated detection of leukocytes in microscopic images. Different methods resulted in comparable overall qualities by enabling computerized detection of leukocytes. The proposed approach further improved the performance. This preliminary study proves the feasibility of computerized detection of leukocytes in clinical use. © 2017 American Association of Physicists in Medicine.

Nanometric depth resolution from multi-focal images in microscopy.

PubMed

Dalgarno, Heather I C; Dalgarno, Paul A; Dada, Adetunmise C; Towers, Catherine E; Gibson, Gavin J; Parton, Richard M; Davis, Ilan; Warburton, Richard J; Greenaway, Alan H

2011-07-06

We describe a method for tracking the position of small features in three dimensions from images recorded on a standard microscope with an inexpensive attachment between the microscope and the camera. The depth-measurement accuracy of this method is tested experimentally on a wide-field, inverted microscope and is shown to give approximately 8 nm depth resolution, over a specimen depth of approximately 6 µm, when using a 12-bit charge-coupled device (CCD) camera and very bright but unresolved particles. To assess low-flux limitations a theoretical model is used to derive an analytical expression for the minimum variance bound. The approximations used in the analytical treatment are tested using numerical simulations. It is concluded that approximately 14 nm depth resolution is achievable with flux levels available when tracking fluorescent sources in three dimensions in live-cell biology and that the method is suitable for three-dimensional photo-activated localization microscopy resolution. Sub-nanometre resolution could be achieved with photon-counting techniques at high flux levels.

Nanometric depth resolution from multi-focal images in microscopy

PubMed Central

Dalgarno, Heather I. C.; Dalgarno, Paul A.; Dada, Adetunmise C.; Towers, Catherine E.; Gibson, Gavin J.; Parton, Richard M.; Davis, Ilan; Warburton, Richard J.; Greenaway, Alan H.

2011-01-01

We describe a method for tracking the position of small features in three dimensions from images recorded on a standard microscope with an inexpensive attachment between the microscope and the camera. The depth-measurement accuracy of this method is tested experimentally on a wide-field, inverted microscope and is shown to give approximately 8 nm depth resolution, over a specimen depth of approximately 6 µm, when using a 12-bit charge-coupled device (CCD) camera and very bright but unresolved particles. To assess low-flux limitations a theoretical model is used to derive an analytical expression for the minimum variance bound. The approximations used in the analytical treatment are tested using numerical simulations. It is concluded that approximately 14 nm depth resolution is achievable with flux levels available when tracking fluorescent sources in three dimensions in live-cell biology and that the method is suitable for three-dimensional photo-activated localization microscopy resolution. Sub-nanometre resolution could be achieved with photon-counting techniques at high flux levels. PMID:21247948

Design and fabrication of x-ray Kirkpatrick-Baez microscope for ICF

NASA Astrophysics Data System (ADS)

Mu, Baozhong; Wang, Zhanshan; Huang, Shengling; Yi, Shengzhen; Shen, Zhengxiang

2007-12-01

A hard x-ray (8 keV, Kα line of Cu) Kirkpatrick-Baez (KB) microscope was designed for the diagnostics of inertial confinement fusion (ICF). Three main parts including optical design, fabrication of multilayers, and alignment method were discussed in this paper. According to the deduced equation of aberration in whole field, an optical system was designed, which gives attention to not only spatial resolution but also the collection efficiency. Tungsten (W) and boron carbide (B4C) were chosen as multilayer materials and the non-periodic multilayer with 40 layers was deposited. The measured reflectivity by XRD is better than 18% in the bandwidth range of about 0.3%. Super accurately alignment is another difficulty in the application of KB microscope. To meet the requirements of pointing and co-focusing, a binocular laser pointer which is flexible enough was designed. Finally, an 8keV x-ray tube was used as source in x-ray imaging experiment and images with magnification of 2× were obtained.

MEMS-based handheld confocal microscope for in-vivo skin imaging

PubMed Central

Arrasmith, Christopher L.; Dickensheets, David L.; Mahadevan-Jansen, Anita

2010-01-01

This paper describes a handheld laser scanning confocal microscope for skin microscopy. Beam scanning is accomplished with an electromagnetic MEMS bi-axial micromirror developed for pico projector applications, providing 800x600 (SVGA) resolution at 56 frames per second. The design uses commercial objective lenses with an optional hemisphere front lens, operating with a range of numerical aperture from NA=0.35 to NA=1.1 and corresponding diagonal field of view ranging from 653 μm to 216 μm. Using NA=1.1 and a laser wavelength of 830 nm we measured the axial response to be 1.14 μm full width at half maximum, with a corresponding 10%-90% lateral edge response of 0.39 μm. Image examples showing both epidermal and dermal features including capillary blood flow are provided. These images represent the highest resolution and frame rate yet achieved for tissue imaging with a MEMS bi-axial scan mirror. PMID:20389391