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Jason Kirk. "Beyond the Hype - Is 2-Photon Microscopy Right for You?" Microscopy Today 11, no. 2 (2003): 26–29. http://dx.doi.org/10.1017/s1551929500052469.
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Confocal microscopes have come a long way in the past decade. Not only are they more stable and easier to use than ever before, but their cost has dropped enough that multi-user facilities are finding competition from individual labs using the new breed of "personal" confocals. In fact it has, in some cases, become the de facto standard for fluorescence imaging regardless of whether the user actually has requirements for it or not.But, researchers always have an ear out for something better. Enter 2-photon microscopy (2PLSM). The “bigger & badder” cousin of the confocal microscope has become a new weapon in the arsenal of a microscopy industry that caters to researchers who can't wait to fill their labs with the latest and greatest imaging systems. Advertised by the industry and researchers alike as a technique that seems to solve most of the problems that plague confocal, “2-photon” has become the new buzzword in the vocabulary of researchers eager to enhance their fluorescence applications.
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Cooper, M. S. "Imaging cellular dynamics using scanning laser confocal microscopy." Proceedings, annual meeting, Electron Microscopy Society of America 50, no. 1 (1992): 12–13. http://dx.doi.org/10.1017/s0424820100120461.
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In recent years, the ability to image morphological dynamics and physiological changes in living cells and tissues has been greatly advanced by the advent of scanning laser confocal microscopy. Confocal microscopes employ optical systems in which both the condenser and objective lenses are focused onto a single volume element of the specimen. In practice, galvanometer-driven mirrors or acousto-optical deflectors are used to scan a laser beam over the specimen in a raster-like fashion through an epifluorescence microscope. The incident laser beam, as well as the collected fluorescent light, are passed through pinhole or slit apertures in image planes that are conjugate to the plane of the specimen. This method of illumination and detection prevents fluorescent light which is generated above and below the plane-of-focus from impinging on the imaging system's photodetector, thus rejecting much of the fluorescent light which normally blurs the image of a three-dimensional fluorescent specimen.
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Corle, Timothy R. "Confocal Scanning Optical Microscopy and Related Imaging Systems." Optical Engineering 36, no. 6 (1997): 1821. http://dx.doi.org/10.1117/1.601601.
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Chen, Yuncong, Yang Bai, Zhong Han, Weijiang He, and Zijian Guo. "Photoluminescence imaging of Zn2+in living systems." Chemical Society Reviews 44, no. 14 (2015): 4517–46. http://dx.doi.org/10.1039/c5cs00005j.
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Borg, Thomas K., James A. Stewart, and Michael A. Sutton. "Imaging the Cardiovascular System: Seeing Is Believing." Microscopy and Microanalysis 11, no. 3 (2005): 189–99. http://dx.doi.org/10.1017/s1431927605050439.
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From the basic light microscope through high-end imaging systems such as multiphoton confocal microscopy and electron microscopes, microscopy has been and will continue to be an essential tool in developing an understanding of cardiovascular development, function, and disease. In this review we briefly touch on a number of studies that illustrate the importance of these forms of microscopy in studying cardiovascular biology. We also briefly review a number of imaging modalities such as computed tomography, (CT) Magnetic resonance imaging (MRI), ultrasound, and positron emission tomography (PET) that, although they do not fall under the realm of microscopy, are imaging modalities that greatly complement microscopy. Finally we examine the role of proper imaging system calibration and the potential importance of calibration in understanding biological tissues, such as the cardiovascular system, that continually undergo deformation in response to strain.
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Piston, David W. "Two-photon excitation fluorescence microscopy in living systems." Proceedings, annual meeting, Electron Microscopy Society of America 51 (August 1, 1993): 154–55. http://dx.doi.org/10.1017/s0424820100146618.
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Two-photon excitation fluorescence microscopy provides attractive advantages over confocal microscopy for three-dimensionally resolved fluorescence imaging. Two-photon excitation arises from the simultaneous absorption of two photons in a single quantitized event whose probability is proportional to the square of the instantaneous intensity. For example, two red photons can cause the transition to an excited electronic state normally reached by absorption in the ultraviolet. In our fluorescence experiments, the final excited state is the same singlet state that is populated during a conventional fluorescence experiment. Thus, the fluorophore exhibits the same emission properties (e.g. wavelength shifts, environmental sensitivity) used in typical biological microscopy studies. In practice, two-photon excitation is made possible by the very high local instantaneous intensity provided by a combination of diffraction-limited focusing of a single laser beam in the microscope and the temporal concentration of 100 femtosecond pulses generated by a mode-locked laser. Resultant peak excitation intensities are 106 times greater than the CW intensities used in confocal microscopy, but the pulse duty cycle of 10−5 maintains the average input power on the order of 10 mW, only slightly greater than the power normally used in confocal microscopy.
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Castellano-Muñoz, Manuel, Anthony Wei Peng, Felipe T. Salles, and Anthony J. Ricci. "Swept Field Laser Confocal Microscopy for Enhanced Spatial and Temporal Resolution in Live-Cell Imaging." Microscopy and Microanalysis 18, no. 4 (2012): 753–60. http://dx.doi.org/10.1017/s1431927612000542.
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AbstractConfocal 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.
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Lee, Dongwoo, Jihye Kim, Eunjoo Song, et al. "Micromirror-Embedded Coverslip Assembly for Bidirectional Microscopic Imaging." Micromachines 11, no. 6 (2020): 582. http://dx.doi.org/10.3390/mi11060582.
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3D imaging of a biological sample provides information about cellular and subcellular structures that are important in cell biology and related diseases. However, most 3D imaging systems, such as confocal and tomographic microscopy systems, are complex and expensive. Here, we developed a quasi-3D imaging tool that is compatible with most conventional microscopes by integrating micromirrors and microchannel structures on coverslips to provide bidirectional imaging. Microfabricated micromirrors had a precisely 45° reflection angle and optically clean reflective surfaces with high reflectance over 95%. The micromirrors were embedded on coverslips that could be assembled as a microchannel structure. We demonstrated that this simple disposable device allows a conventional microscope to perform bidirectional imaging with simple control of a focal plane. Images of microbeads and cells under bright-field and fluorescent microscopy show that the device can provide a quick analysis of 3D information, such as 3D positions and subcellular structures.
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Zucker, Robert M., Paul Rigby, Ian Clements, Wendy Salmon, and Michael Chua. "Reliability of confocal microscopy spectral imaging systems: Use of multispectral beads." Cytometry Part A 71A, no. 3 (2007): 174–89. http://dx.doi.org/10.1002/cyto.a.20371.
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Aguilar, Alberto, Adeline Boyreau, and Pierre Bon. "Label-free super-resolution imaging below 90-nm using photon-reassignment." Open Research Europe 1 (March 24, 2021): 3. http://dx.doi.org/10.12688/openreseurope.13066.1.
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Background: Achieving resolutions below 100 nm is key for many fields, including biology and nanomaterial characterization. Although nearfield and electron microscopy are the gold standards for studying the nanoscale, optical microscopy has seen its resolution drastically improve in the last decades. So-called super-resolution microscopy is generally based on fluorescence photophysics and requires modification of the sample at least by adding fluorescent tags, an inevitably invasive step. Therefore, it remains very challenging and rewarding to achieve optical resolutions beyond the diffraction limit in label-free samples. Methods: Here, we present a breakthrough to unlock label-free 3D super-resolution imaging of any object including living biological samples. It is based on optical photon-reassignment in confocal reflectance imaging mode. Results: We demonstrate that we surpass the resolution of all fluorescence-based confocal systems by a factor ~1.5. We have obtained images with a 3D (x,y,z) optical resolution of (86x86x248) nm3 using a visible wavelength (445 nm) and a regular microscope objective (NA=1.3). The results are presented on nanoparticles as well as on (living) biological samples. Conclusions: This cost-effective approach double the resolution of reflectance confocal microscope with minimal modifications. It is therefore compatible with any microscope and sample, works in real-time, and does not require any signal processing.
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Lich, Ben. "Site Specific Three-dimensional Structural Analysis in Tissues and Cells Using Automated DualBeam Slice &View." Microscopy Today 15, no. 2 (2007): 26–31. http://dx.doi.org/10.1017/s1551929500050987.
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DualBeam instruments that combine the imaging capability of scanning electron microscopy (SEM) with the cutting and deposition capability of a focused ion beam (FIB) provide biologists with a powerful tool for investigating three-dimensional structure with nanoscale (1 nm-100 nm) resolution. Ever since Van Leeuwenhoek used the first microscope to describe bacteria more than 300 years ago, microscopy has played a central role in scientists' efforts to understand biological systems. Light microscopy is generally limited to a useful resolution of about a micrometer. More recently the use of confocal and electron microscopy has enabled investigations at higher resolution. Used with fluorescent markers, confocal microscopy can detect and localize molecular scale features, but its imaging resolution is still limited. SEM is capable of nanometer resolution, but is limited to the near surface region of the sample.
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Keene, Douglas R., Sara F. Tufa, Gregory P. Lunstrum, Paul Holden, and William A. Horton. "Confocal/TEM Overlay Microscopy: A Simple Method for Correlating Confocal and Electron Microscopy of Cells Expressing GFP/YFP Fusion Proteins." Microscopy and Microanalysis 14, no. 4 (2008): 342–48. http://dx.doi.org/10.1017/s1431927608080306.
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Genetic manipulation allows simultaneous expression of green fluorescent protein (GFP) and its derivatives with a wide variety of cellular proteins in a variety of living systems. Epifluorescent and confocal laser scanning microscopy (confocal) localization of GFP constructs within living tissue and cell cultures has become routine, but correlation of light microscopy and high resolution transmission electron microscopy (TEM) on components within identical cells has been problematic. In this study, we describe an approach that specifically localizes the position of GFP/yellow fluorescent protein (YFP) constructs within the same cultured cell imaged in the confocal and transmission electron microscopes. We present a simplified method for delivering cell cultures expressing fluorescent fusion proteins into LR White embedding media, which allows excellent GFP/YFP detection and also high-resolution imaging in the TEM. Confocal images from 0.5-μm-thick sections are overlaid atop TEM images of the same cells collected from the next serial ultrathin section. The overlay is achieved in Adobe Photoshop by making the confocal image somewhat transparent, then carefully aligning features within the confocal image over the same features visible in the TEM image. The method requires no specialized specimen preparation equipment; specimens are taken from live cultures to embedding within 8 h, and confocal transmission overlay microscopy can be completed within a few hours.
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Haupt, B. J., A. E. Pelling, and M. A. Horton. "Integrated Confocal and Scanning Probe Microscopy for Biomedical Research." Scientific World JOURNAL 6 (2006): 1609–18. http://dx.doi.org/10.1100/tsw.2006.269.
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Atomic force microscopy (AFM) continues to be developed, not only in design, but also in application. The new focus of using AFM is changing from pure material to biomedical studies. More frequently, it is being used in combination with other optical imaging methods, such as confocal laser scanning microscopy (CLSM) and fluorescent imaging, to provide a more comprehensive understanding of biological systems. To date, AFM has been used increasingly as a precise micromanipulator, probing and altering the mechanobiological characteristics of living cells and tissues, in order to examine specific, receptor-ligand interactions, material properties, and cell behavior. In this review, we discuss the development of this new hybrid AFM, current research, and potential applications in diagnosis and the detection of disease.
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Ueno, Yutaka, Kento Matsuda, Kaoru Katoh, Akinori Kuzuya, Akira Kakugo, and Akihiko Konagaya. "Modeling a Microtubule Filaments Mesh Structure from Confocal Microscopy Imaging." Micromachines 11, no. 9 (2020): 844. http://dx.doi.org/10.3390/mi11090844.
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This study introduces a modeling method for a supermolecular structure of microtubules for the development of a force generation material using motor proteins. 3D imaging by confocal laser scanning microscopy (CLSM) was used to obtain 3D volume density data. The density data were then interpreted by a set of cylinders with the general-purpose 3D modeling software Blender, and a 3D network structure of microtubules was constructed. Although motor proteins were not visualized experimentally, they were introduced into the model to simulate pulling of the microtubules toward each other to yield shrinking of the network, resulting in contraction of the artificial muscle. From the successful force generation simulation of the obtained model structure of artificial muscle, the modeling method introduced here could be useful in various studies for potential improvements of this contractile molecular system.
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Piston, David W. "Two-Photon Excitation Microscopy in Cellular Biophysics." Proceedings, annual meeting, Electron Microscopy Society of America 54 (August 11, 1996): 276–77. http://dx.doi.org/10.1017/s0424820100163848.
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Two-photon excitation microscopy (TPEM) provides attractive advantages over confocal microscopy for three-dimensionally resolved fluorescence imaging and photochemistry. Two-photon excitation arises from the simultaneous absorption of two photons in a single quantitized event whose probability is proportional to the square of the instantaneous intensity. For example, two red photons can cause the transition to an excited electronic state normally reached by absorption in the ultraviolet. In practice, two-photon excitation is made possible by the very high local instantaneous intensity provided by a combination of diffraction-limited focusing of a single laser beam in the microscope and the temporal concentration of 100 femtosecond pulses generated by a mode-locked laser. Resultant peak excitation intensities are 106 times greater than the CW intensities used in confocal microscopy, but the pulse duty cycle of 10 5 limits the average input power to less than 10 mW, only slightly greater than the power normally used in confocal microscopy.Three properties TPEM give this method a tremendous advantage over conventional optical sectioning microscopies for the study of thick samples: 1) The excitation is limited to the focal volume because of the intensity-squared dependence of the two-photon absorption. This inherent localization provides three-dimensional resolution and eliminates background equivalent to an ideal confocal microscope without requiring a confocal spatial filter, whose absence enhances fluorescence collection efficiency. Confinement of excitation to the focal volume also minimizes photobleaching and photo damage - the ultimate limiting factors in fluorescence microscopy of living cells and tissues. 2) The two-photon technique allows imaging of UV fluorophores with conventional visible light optics in both the scanning and imaging systems, because both the red excitation light (~700 nm) and the blue fluorescence (>400 nm) are within the visible spectrum. 3) Red or infrared light is far less damaging to most living cells and tissues than bluer light because fewer biological molecules absorb at the higher wavelengths. Longer wavelength excitation also reduces scattering of the incident light by the specimen, thus allowing more of the input power to reach the focal plane. This relative transparency of biological specimens to 700 nm light permits deeper sectioning, since both absorbance and scattering are reduced.
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Tominaga, Takashi, and Yoko Tominaga. "A new nonscanning confocal microscopy module for functional voltage-sensitive dye and Ca2+ imaging of neuronal circuit activity." Journal of Neurophysiology 110, no. 2 (2013): 553–61. http://dx.doi.org/10.1152/jn.00856.2012.
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Recent advances in fluorescent confocal microscopy and voltage-sensitive and Ca2+ dyes have vastly improved our ability to image neuronal circuits. However, existing confocal systems are not fast enough or too noisy for many live-cell functional imaging studies. Here, we describe and demonstrate the function of a novel, nonscanning confocal microscopy module. The optics, which are designed to fit the standard camera port of the Olympus BX51WI epifluorescent microscope, achieve a high signal-to-noise ratio (SNR) at high temporal resolution, making this configuration ideal for functional imaging of neuronal activities such as the voltage-sensitive dye (VSD) imaging. The optics employ fixed 100- × 100-pinhole arrays at the back focal plane (optical conjugation plane), above the tube lens of a usual upright microscope. The excitation light travels through these pinholes, and the fluorescence signal, emitted from subject, passes through corresponding pinholes before exciting the photodiodes of the imager: a 100- × 100-pixel metal-oxide semiconductor (MOS)-type pixel imager with each pixel corresponding to a single 100- × 100-μm photodiode. This design eliminated the need for a scanning device; therefore, acquisition rate of the imager (maximum rate of 10 kHz) is the only factor limiting acquisition speed. We tested the application of the system for VSD and Ca2+ imaging of evoked neuronal responses on electrical stimuli in rat hippocampal slices. The results indicate that, at least for these applications, the new microscope maintains a high SNR at image acquisition rates of ≤0.3 ms per frame.
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Masters, Barry R., and Andreas A. Thaer. "Real-time confocal microscopy of the human in vivo cornea." Proceedings, annual meeting, Electron Microscopy Society of America 51 (August 1, 1993): 166–67. http://dx.doi.org/10.1017/s0424820100146679.
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A new confocal microscope has several unique features which differentiates it from other in vivo confocal imaging systems.is described. • The light source is a halogen lamp. This source has many advantages over the mercury or xenon arc lamps used in other confocal designs. The mercury or xenon arc lamps have the problem of arc jitter which results in changing illumination intensity. Filters are inserted in the lamp housing to remove both short ultraviolet and infrared light. • The microscope uses standard microscope objectives which are readily interchangeable. Other in vivo confocal systems are limited to a built in objective which is not removable. In these studies we used a Leitz 50X, NA 1.0 water immersion objective.• The detection system consists of an intensitified video camera with video output to a Sony U-matic tape recorder. In parallel with the video recording there is a video monitor in order that the operator can observe in real-time the confocal images of the subject's eye.
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Robinson, J. M., and B. E. Batten. "Localization of cerium-based reaction products by scanning laser reflectance confocal microscopy." Journal of Histochemistry & Cytochemistry 38, no. 3 (1990): 315–18. http://dx.doi.org/10.1177/38.3.2406336.
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Scanning laser confocal microscopy was utilized to visualize sites of hydrogen peroxide release from stimulated neutrophils and lysosomal acid phosphatase in these and other cells using cerium in the detection systems. Imaging of the cerium-containing reactions was achieved by employing the reflectance mode of this instrument. Localization of these products at the light microscope level was direct and did not require other reactions to generate a visible product. This new approach to cerium cytochemistry should prove useful for many applications.
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Pedroso, M. Cristina, Michael B. Sinclair, Howland D. T. Jones, and David M. Haaland. "Hyperspectral Confocal Fluorescence Microscope: A New Look into the Cell." Microscopy Today 18, no. 5 (2010): 14–18. http://dx.doi.org/10.1017/s1551929510000854.
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Confocal microscopy is widely used in cell biology. Like other filter-based systems, traditional confocal microscopes are limited by the spectral bands established by each optical filter. As a result, emission spectra from labels and/or autofluorescence can be overlapped leading to spectral crosstalk and inability to quantify the amount of signal originating from each individual fluorescent species. The need for accurate quantification of in vivo cellular processes and in-depth knowledge of organelle development and microstructure led Monsanto to search for non-commercial microscopes that could achieve those goals. Through a cooperative research and development agreement (CRADA) established between Monsanto and Sandia Corporation in August 2006, we built a new 3D-hyperspectral confocal fluorescence imaging system, specifically designed to meet the analytical requirements of plant specimens.
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Ackerman, Larry D., and W. T. Jansen. "A film recording and image processing system for confocal microscopy." Proceedings, annual meeting, Electron Microscopy Society of America 48, no. 3 (1990): 798–99. http://dx.doi.org/10.1017/s0424820100161552.
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Scanning confocal microscopy has developed into a very useful technique for many scientific investigations. However, commercial development has been so rapid that some recent advances in computer graphics and imaging have not been incorporated into the commercial systems. One particular concern was high quality hard copy with alpha-numeric and graphic overlays. A subsystem was developed to provide this output for the BioRad MRC-500/600 confocal imaging system.A digital film recorder, an Agfa Matrix Procolor was chosen as the principal element of hardware. This compact unit can record an image at a resolution of 4096 horizontal by 3072 vertical pixels at a cost equivalent to popular analog video film recorders. The interface is a standard IEEE 488 GPIB board. It is compatible with various film emulsions such as Kodak Ecktachrome 100 as well as many of the major graphics arts and image processing programs. The second element of hardware in this system is an ATVista 4M image processing board.
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Lee, J. E., K. J. Liang, R. N. Fariss, and W. T. Wong. "Ex Vivo Dynamic Imaging of Retinal Microglia Using Time-Lapse Confocal Microscopy." Investigative Ophthalmology & Visual Science 49, no. 9 (2008): 4169–76. http://dx.doi.org/10.1167/iovs.08-2076.
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Wang, Haoyuan, and Wei Xiong. "Vibrational Sum-Frequency Generation Hyperspectral Microscopy for Molecular Self-Assembled Systems." Annual Review of Physical Chemistry 72, no. 1 (2021): 279–306. http://dx.doi.org/10.1146/annurev-physchem-090519-050510.
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In this review, we discuss the recent developments and applications of vibrational sum-frequency generation (VSFG) microscopy. This hyperspectral imaging technique can resolve systems without inversion symmetry, such as surfaces, interfaces and noncentrosymmetric self-assembled materials, in the spatial, temporal, and spectral domains. We discuss two common VSFG microscopy geometries: wide-field and confocal point-scanning. We then introduce the principle of VSFG and the relationships between hyperspectral imaging with traditional spectroscopy, microscopy, and time-resolved measurements. We further highlight crucial applications of VSFG microscopy in self-assembled monolayers, cellulose in plants, collagen fibers, and lattice self-assembled biomimetic materials. In these systems, VSFG microscopy reveals relationships between physical properties that would otherwise be hidden without being spectrally, spatially, and temporally resolved. Lastly, we discuss the recent development of ultrafast transient VSFG microscopy, which can spatially measure the ultrafast vibrational dynamics of self-assembled materials. The review ends with an outlook on the technical challenges of and scientific potential for VSFG microscopy.
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Booth, Martin J. "Adaptive optics in microscopy." Philosophical Transactions of the Royal Society A: Mathematical, Physical and Engineering Sciences 365, no. 1861 (2007): 2829–43. http://dx.doi.org/10.1098/rsta.2007.0013.
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The imaging properties of optical microscopes are often compromised by aberrations that reduce image resolution and contrast. Adaptive optics technology has been employed in various systems to correct these aberrations and restore performance. This has required various departures from the traditional adaptive optics schemes that are used in astronomy. This review discusses the sources of aberrations, their effects and their correction with adaptive optics, particularly in confocal and two-photon microscopes. Different methods of wavefront sensing, indirect aberration measurement and aberration correction devices are discussed. Applications of adaptive optics in the related areas of optical data storage, optical tweezers and micro/nanofabrication are also reviewed.
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Miller, Christine E., Robert P. Thompson, Michael R. Bigelow, George Gittinger, Thomas C. Trusk, and David Sedmera. "Confocal Imaging of the Embryonic Heart: How Deep?" Microscopy and Microanalysis 11, no. 3 (2005): 216–23. http://dx.doi.org/10.1017/s1431927605050464.
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Confocal microscopy allows for optical sectioning of tissues, thus obviating the need for physical sectioning and subsequent registration to obtain a three-dimensional representation of tissue architecture. However, practicalities such as tissue opacity, light penetration, and detector sensitivity have usually limited the available depth of imaging to 200 μm. With the emergence of newer, more powerful systems, we attempted to push these limits to those dictated by the working distance of the objective. We used whole-mount immunohistochemical staining followed by clearing with benzyl alcohol-benzyl benzoate (BABB) to visualize three-dimensional myocardial architecture. Confocal imaging of entire chick embryonic hearts up to a depth of 1.5 mm with voxel dimensions of 3 μm was achieved with a 10× dry objective. For the purpose of screening for congenital heart defects, we used endocardial painting with fluorescently labeled poly-L-lysine and imaged BABB-cleared hearts with a 5× objective up to a depth of 2 mm. Two-photon imaging of whole-mount specimens stained with Hoechst nuclear dye produced clear images all the way through stage 29 hearts without significant signal attenuation. Thus, currently available systems allow confocal imaging of fixed samples to previously unattainable depths, the current limiting factors being objective working distance, antibody penetration, specimen autofluorescence, and incomplete clearing.
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Sheppard, C. J. R. "The influence of confocal microscope design on imaging performance." Proceedings, annual meeting, Electron Microscopy Society of America 51 (August 1, 1993): 144–45. http://dx.doi.org/10.1017/s0424820100146564.
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The confocal microscope is now widely used in both biomedical and industrial applications for imaging, in three dimensions, objects with appreciable depth. There are now a range of different microscopes on the market, which have adopted a variety of different designs. The aim of this paper is to explore the effects on imaging performance of design parameters including the method of scanning, the type of detector, and the size and shape of the confocal aperture.It is becoming apparent that there is no such thing as an ideal confocal microscope: all systems have limitations and the best compromise depends on what the microscope is used for and how it is used. The most important compromise at present is between image quality and speed of scanning, which is particularly apparent when imaging with very weak signals. If great speed is not of importance, then the fundamental limitation for fluorescence imaging is the detection of sufficient numbers of photons before the fluorochrome bleaches.
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Rzhevskii, Alexander. "The Recent Advances in Raman Microscopy and Imaging Techniques for Biosensors." Biosensors 9, no. 1 (2019): 25. http://dx.doi.org/10.3390/bios9010025.
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Raman microspectroscopy is now well established as one of the most powerful analytical techniques for a diverse range of applications in physical (material) and biological sciences. Consequently, the technique provides exceptional analytical opportunities to the science and technology of biosensing due to its capability to analyze both parts of a biosensor system—biologically sensitive components, and a variety of materials and systems used in physicochemical transducers. Recent technological developments in Raman spectral imaging have brought additional possibilities in two- and three-dimensional (2D and 3D) characterization of the biosensor’s constituents and their changes on a submicrometer scale in a label-free, real-time nondestructive method of detection. In this report, the essential components and features of a modern confocal Raman microscope are reviewed using the instance of Thermo Scientific DXRxi Raman imaging microscope, and examples of the potential applications of Raman microscopy and imaging for constituents of biosensors are presented.
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Arend, Johannes, Alexander Wetzel, and Bernhard Middendorf. "Fluorescence Microscopy of Superplasticizers in Cementitious Systems: Applications and Challenges." Materials 13, no. 17 (2020): 3733. http://dx.doi.org/10.3390/ma13173733.
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In addition to the desired plasticizing effect, superplasticizers used in high and ultra-high performance concretes (UHPC) influence the chemical system of the pastes and for example retardation of the cement hydration occurs. Thus, superplasticizers have to be chosen wisely for every material composition and application. To investigate the essential adsorption of these polymers to particle surfaces in-situ to overcome several practical challenges of superplasticizer research, fluorescence microscopy is useful. In order to make the superplasticizer polymers visible for this microscopic approach, they are stained with fluorescence dyes prior the experiment. In this work, the application of this method in terms of retardation and rheological properties of sample systems is presented. The hydration of tricalcium oxy silicate (C3S) in combination with different polycarboxylate ether superplasticizers is observed by fluorescence microscopy and calorimetry. Both methods can identify the retarding effect, depending on the superplasticizer’s chemical composition. On the other hand, the influence of the superplasticizers on the slump of a ground granulated blast furnace slag/cement paste is correlated to fluorescence microscopic adsorption results. The prediction of the efficiency by microscopic adsorption analysis succeeds roughly. At last, the possibility of high-resolution imaging via confocal laser scanning microscopy is presented, which enables the detection of early hydrates and their interaction with the superplasticizers.
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Wang, Guoliang, Xiaoya An, Xiaoping Zhou, et al. "Real-time confocal microscopy imaging of corneal cytoarchitectural changes induced by different stresses." Experimental Eye Research 210 (September 2021): 108706. http://dx.doi.org/10.1016/j.exer.2021.108706.
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Swedlow, Jason R., Paul D. Andrews, Ke Hu, David S. RoosT, and John M. Murray. "Defining the Tools: an Analysis of Laser Scanning Confocal and Wide-Field/Restoration Fluorescence Microscope Imaging." Microscopy and Microanalysis 7, S2 (2001): 1002–3. http://dx.doi.org/10.1017/s1431927600031081.
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Digital fluorescence microscopy is now a standard tool for determining the localization of cellular components in fixed and living cells. Two fundamentally different imaging technologies are available for imaging fluorescently labelled cells and tissues, in either the fixed or living state. The laser scanning microscope uses a diffraction-limited focused beam to scan the sample and develop an image point by point. in addition, a pinhole placed in a plane confocal to the specimen prevents emitted out-of focus fluorescence from reaching the photomultiplier tube (PMT) detector. By combining spot illumination and selection of infocus fluorescence signal, the laser scanning confocal microscope (LSCM) creates an image of the specimen largely free of out-of-focus blur. By contrast, a wide-field microscope (WFM) illuminates the whole specimen simultaneously and detects the signal with a spatial array of point detectors, usually a charge-coupled device camera (CCD). This approach collects an image of all points of the specimen simultaneously and includes all the out-of-focus blurred light. Subsequent restoration by iterative deconvolution generates an estimate of the specimen, largely free of out-of-focus blur. While many other fluorescence imaging modalities exist, these two methods represent the majority of the fluorescence imaging systems currently in use in biomedical research.
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Kerschmann, Russell. "High-Resolution Three-Dimensional Microscopy System." Microscopy and Microanalysis 6, S2 (2000): 1012–13. http://dx.doi.org/10.1017/s1431927600037557.
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The demand for new methods of three-dimensional imaging of biological systems grown significantly over the past decades. Systems for volumetric analysis of macroscopic structures have been addressed through the introduction of modem CT/MRI systems; and on the cellular level, confocal microscopy has evolved into a powerful research tool for the examination of both biological tissues and manufactured goods. However, there persists a need for the visualization and analysis of types of material in the cubic millimeter size range, a class of materials which has not been adequately addressed by either radiological or optical sectioning techniques. These materials include research and clinical biological tissue samples, as well as many types of manufactured materials such as textiles and paper.The main method currently in use for the examination of such materials is standard histopathology. Whether one is concerned with the diagnosis of a human tumor or the arrangement of cells in the leaf of a plant,
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Kramer, Jennifer A., Ramkumar K. Moorthy, William N. Casavan, and David C. Hitrys. "Exhaustive Photon Reassignment™: A Method Offering Enhanced Sensitivity and Quantitative Accuracy for High Resolution Fluorescence Microscopy." Microscopy and Microanalysis 3, S2 (1997): 379–80. http://dx.doi.org/10.1017/s1431927600008783.
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Confocal microscopy is a technique that allows researchers to obtain a highly resolved, high-contrast image of a focal plane in a fluorescent specimen by excluding or rejecting light emanating from out-of-focus planes. However, the basic design of confocals results in limitations for many biologists. It is often difficult to avoid photobleaching of specimens, to visualize fine or faintly-labeled structures, or to acquire high-quality images using short exposure times. The photomultiplier tubes used as the amplification detectors in these systems are restricted to a dynamic range of 8 bits (255 intensity levels), produce noise, and are not quantitatively linear detectors.Members of the Biomedical Imaging Group at the University of Massachusetts Medical School in Worcester, MA have spent the last fifteen years developing and perfecting a digital imaging system that helps scientists overcome these problems. Scanalytics is the exclusive worldwide licensee of this patented technology which allows researchers to obtain high-resolution, quantitatively accurate three-dimensional images of fluorescent specimens
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Ma, Yue, J. Liang, Y. Zheng, S. L. Erlandsen, L. E. Scriven, and H. T. Davis. "Direct Imaging of Sodium Stearate Crystals Dispersed in Waterpropylene Glycol Mixtures by Cryo-Electron Microscopy." Microscopy and Microanalysis 7, S2 (2001): 734–35. http://dx.doi.org/10.1017/s1431927600029743.
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Cryo-scanning electron microscopy (cryo-SEM) and cryo-transmission electron microscopy (cryo- TEM), in conjunctions with rheological measurements, light and confocal microscopy, x-ray scattering, and solid state NMR, are used to characterize sodium stearate (NaSt) crystals dispersed in waterpropylene glycol (PG) mixtures at macroscopic, microscopic, molecular, and atomic levels. NaSt is a surface-active, structural agent in household and personal cleaning products, including deodorant sticks and soap bars. A better structural characterization of NaSt/PG/water systems has practical importance in personal care and cosmetic industries. NaSt crystals and other soap crystal morphologies have been studied by the TEM/replica technique. However, the replicas were made of the residue after the original sample or its aqueous dilution were dried, and the original structure may have been lost during drying. Cryo-SEM was not used to study NaSt crystals because of its lower resolution and because the crystals are highly susceptible to radiation damage by electron beam.
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Chen, V. K.-H., and P. C. Cheng. "Real-time confocal imaging of Stentor coeruleus in epi-reflective mode by using a Tracor Northern tandem scanning microscope." Proceedings, annual meeting, Electron Microscopy Society of America 47 (August 6, 1989): 138–39. http://dx.doi.org/10.1017/s0424820100152665.
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Confocal microscopes with a spinning disk design are potentially useful in the study of dynamic processes in biological systems. However, the image brightness of the spinning disk type confocal microscope is generally quite low.A Tracor Northern Tandem Scanning confocal microscope (TSM) modified to accept a TV camera-coupled second generation image intensifier was used in this study. An Olympus 38mm Macro lens (f/2.8) was used in place of the original eyepiece to relay the image of the spinning disk onto the photocathod of the image intensifier. In order to fit the macro lens and the image intensifier onto the microscope, a set of adaptors were designed and fabricated. Figure 1 shows the microscope set-ups. To increase the image intensity, a 200W Mercury short arc lamp was used as the light source. An UV filter was used to remove radiations harmful for living specimen.
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Chan, Tommy C. Y., Kelvin H. Wan, Kendrick C. Shih, and Vishal Jhanji. "Advances in dry eye imaging: the present and beyond." British Journal of Ophthalmology 102, no. 3 (2017): 295–301. http://dx.doi.org/10.1136/bjophthalmol-2017-310759.
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New advances in imaging allow objective measurements for dry eye as well as define new parameters that cannot be measured by clinical assessment alone. A combination of these modalities provides unprecedented information on the static and dynamic properties of the structural and functional parameters in this multifactorial disease. A literature search was conducted to include studies investigating the use of imaging techniques in dry eye disease. This review describes the application of non-invasive tear breakup time, optical coherence tomography, meibomian gland imaging, interferometry, in vivo confocal microscopy, thermography and optical quality assessment for this condition.
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Shaw, P. J. "Deconvolution in 3-D Microscopy: Applications and Limitations." Microscopy and Microanalysis 4, S2 (1998): 880–81. http://dx.doi.org/10.1017/s1431927600024521.
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Many imaging processes, including both conventional wide-field and confocal fluorescence microscopy, can be described to a good approximation as linear and spatially-invariant. Linearity means that the image of an extended specimen is simply the sum of the images of the parts of the specimen - in the limit the specimen can be regarded as a collection of points, and its image is the sum of the images of the points - i.e. weighted instances of the imaging system's point spread function (psf). In the case of spatially invariant imaging the psf is the same over the entire field of imaging. Mathematically, the image is the convolution of the specimen with the system psf. In principle, this convolution can be reversed to remove degradation introduced by the imaging process, a computational procedure often called deconvolution or, more generally, restoration.
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Hassan, A., V. Chandra, M. P. Yutkin, T. W. Patzek, and D. N. Espinoza. "Imaging and Characterization of Microporous Carbonates Using Confocal and Electron Microscopy of Epoxy Pore Casts." SPE Journal 24, no. 03 (2019): 1220–33. http://dx.doi.org/10.2118/188786-pa.
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Summary Microporous carbonates contain perhaps 50% of the oil left behind in current projects in the giant carbonate fields in the Middle East and elsewhere. Pore geometry, connectivity, and wettability of the micropore systems in these carbonates are of paramount importance in finding new improved-oil-recovery methods. In this study, we present a robust pore-imaging approach that uses confocal laser scanning microscopy (CLSM) to obtain high-resolution 3D images of etched epoxy pore casts of the highly heterogeneous carbonates. In our approach, we have increased the depth of investigation for carbonates 20-fold, from 10 µm reported by Fredrich (1999) and Shah et al. (2013) to 200 µm. In addition, high-resolution 2D images from scanning electron microscopy (SEM) have been correlated with the 3D models from CLSM to develop a multiscale imaging approach that covers a range of scales, from millimeters in three dimensions to micrometers in two dimensions. The developed approach was implemented to identify various pore types [e.g., intercrystalline microporosity (IM), intragranular microporosity (IGM), and interboundary sheet pores (SPs)] in limestone and dolomite samples.
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Angelotti, Maria Lucia, Giulia Antonelli, Carolina Conte, and Paola Romagnani. "Imaging the kidney: from light to super-resolution microscopy." Nephrology Dialysis Transplantation 36, no. 1 (2019): 19–28. http://dx.doi.org/10.1093/ndt/gfz136.
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Abstract The important achievements in kidney physiological and pathophysiological mechanisms can largely be ascribed to progress in the technology of microscopy. Much of what we know about the architecture of the kidney is based on the fundamental descriptions of anatomic microscopists using light microscopy and later by ultrastructural analysis provided by electron microscopy. These two techniques were used for the first classification systems of kidney diseases and for their constant updates. More recently, a series of novel imaging techniques added the analysis in further dimensions of time and space. Confocal microscopy allowed us to sequentially visualize optical sections along the z-axis and the availability of specific analysis software provided a three-dimensional rendering of thicker tissue specimens. Multiphoton microscopy permitted us to simultaneously investigate kidney function and structure in real time. Fluorescence-lifetime imaging microscopy allowed to study the spatial distribution of metabolites. Super-resolution microscopy increased sensitivity and resolution up to nanoscale levels. With cryo-electron microscopy, researchers could visualize the individual biomolecules at atomic levels directly in the tissues and understand their interaction at subcellular levels. Finally, matrix-assisted laser desorption/ionization imaging mass spectrometry permitted the measuring of hundreds of different molecules at the same time on tissue sections at high resolution. This review provides an overview of available kidney imaging strategies, with a focus on the possible impact of the most recent technical improvements.
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Smith, R. W. "Non-Imaging Microscopies: Flow Cytometry as a Correlative Analytical Tool in the Quantification of Cell Structure, Autofluorescence, Fluorescent Probes and Cell Populations." Microscopy and Microanalysis 5, S2 (1999): 490–91. http://dx.doi.org/10.1017/s1431927600015774.
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Non-imaging microscopy has developed somewhat independently of both traditional light microscopy and laser confocal microscopy. Flow cytometry is the chief commercial and research technology among these microscopies, and, like other nonimaging detection systems, developed around the theme of automation in clinical laboratory medicine. It is an important correlative or parallel microscopy to several image forming microscopical methods. Cell sorting is an important option as well.The basic structure of the flow cytometer certainly parallels light, laser and electron microscopes. The flow cytometer has a light source, a set of adjustable optics to focus the beam on the specimen, objective optics to collect the light and direct it to appropriate sensors, and the sensors themselves. A real image is not formed because the sensors are not in an even plane with the projection, such as provided by the retina in light microscopy or an image plane or film plate in electron microscopy, and the objective optics may not focus in the image plane.While early flow cytometers were developed primarily for the automatic counting of cells and particles, modern instruments offer particular advantages for the analysis of fluorescence, fluorescent chemicals and probes and cellular auto fluorescence.
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Conti, Giuseppina, and Yuri Uritsky. "Application of Confocal Laser Imaging Microscopy and Raman Spectroscopy to Particle Characterization in Semiconductor Industry." Microscopy and Microanalysis 7, S2 (2001): 154–55. http://dx.doi.org/10.1017/s1431927600026842.
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The analytical needs in semiconductor technology have developed according to the increasing demands imposed by size and complexity. Early optical visualization, localization and counting of ever-smaller particles extended almost immediately to their morphological and chemical characterization. This evolution was due to the shifting balance from quality control to root-cause analysis. Root-cause analysis requires a deeper analytical approach to the characterization of the observed defects. Atomic composition may suffice at times as an answer. However, this information may be ambiguous and inadequate. Therefore, deeper chemical information regarding molecular structure and crystallinity of the particle is now required.The analytical capabilities and versatilities associated with optical spectroscopy inevitably have led to their increasing applications in the semiconductor industry. in particular, the complementary Infrared and Raman spectroscopies on one hand provide huge insight into the molecular structure and crystallinity of the particles, on the other hand Raman and Photoluminescence (PL) spectroscopies are easily coupled to systems providing particle visualization and localization.
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Zucker, R. M., and O. T. Price. "Evaluation of Confocal Microscopy System Performance: Applications for Imaging Morphology and Death in Embryos and Rerpoductive Tissue/Organs." Microscopy and Microanalysis 7, S2 (2001): 1020–21. http://dx.doi.org/10.1017/s1431927600031172.
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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 operation are working at levels of sub optimal performance. When these machines are checked, it is usually a subjective assessment of performance accomplished by primarily evaluating the system using a specific test slide provided by each user’s laboratory. We have devised test methods on the Leica TCS-SP and TCS-4D systems to ensure that these machines were working properly and delivering their correct performance. Tests were derived or perfected that measure field illumination, lens clarity, laser power, laser stability, dichroic functionality, spectral registration, axial resolution, overall machine stability, and system noise. It is anticipated this type of data will help to set performance standards for confocal microscopes and eliminate the current subjectivity in evaluating the CLSM. These tests will also serve as a guide for other investigators to insure that their machines are working correctly and providing data that is accurate and having the necessary resolution, sensitivity and precision.
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Ryan, Denise S., Rose K. Sia, Marcus Colyer, et al. "Anterior Segment Imaging in Combat Ocular Trauma." Journal of Ophthalmology 2013 (2013): 1–8. http://dx.doi.org/10.1155/2013/308259.
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Purpose. To evaluate the use of ocular imaging to enhance management and diagnosis of war-related anterior segment ocular injuries.Methods. This study was a prospective observational case series from an ongoing IRB-approved combat ocular trauma tracking study. Subjects with anterior segment ocular injury were imaged, when possible, using anterior segment optical coherence tomography (AS-OCT), confocal microscopy (CM), and slit lamp biomicroscopy.Results. Images captured from participants with combat ocular trauma on different systems provided comprehensive and alternate views of anterior segment injury to investigators.Conclusion. In combat-related trauma of the anterior segment, adjunct image acquisition enhances slit lamp examination and enables real timeIn vivoobservation of the cornea facilitating injury characterization, progression, and management.
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Lewandowski, Z., P. Stoodley, S. Altobelli, and E. Fukushima. "Hydrodynamics and kinetics in biofilm systems - recent advances and new problems." Water Science and Technology 29, no. 10-11 (1994): 223–29. http://dx.doi.org/10.2166/wst.1994.0765.
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Application of microelectrode techniques, Nuclear Magnetic Resonance Imaging, and Confocal Laser Microscopy permitted analysis of hydrodynamics, kinetics, and internal structure in biofilm systems. The commonly accepted concept of one dimensional diffusion through a three compartment model (bulk solution, biofilm, and substratum) requires revision based on recent progress in understanding the internal structures of biofilms. Biofilms seem to form three dimensional porous structures with a network of interstitial voids filled with water, forming a network of channels connected with each other and with the biofilm surface. The basic unit of this structure appears to be a bacterial cluster (sometimes called microcolony).
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POP, Oana Lelia, Loredana Florina LEOPOLD, Olivia Dumitrita RUGINA, et al. "GOLD NANOPARTICLES ENCAPSULATED IN A POLYMERIC MATRIX OF SODIUM ALGINATE." Bulletin of University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca. Food Science and Technology 73, no. 2 (2016): 134. http://dx.doi.org/10.15835/buasvmcn-fst:12340.
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Plasmonic nanoparticles can be used as building blocks for the design of multifunctional systems based on polymeric capsules. The use of functionalised particles in therapeutics and imaging and understanding their effect on the cell functions are among the current challenges in nanobiotechnology and nanomedicine. The aim of the study was to manufacture and characterize polymeric microstructures by encapsulating plasmonic gold nanoparticles in biocompatible matrix of sodium alginate. The gold nanoparticles were obtained by reduction of tetracluoroauric acid with sodium citrate. To characterize the microcapsules, UV-Vis and FTIR spectroscopy, optical and confocal microscopy experiments were performed. In vitro cytotoxicity tests on HFL-1 cells were also performed. The capsules have spherical shape and 120 μm diameter. The presence of encapsulated gold nanoparticles is also shown by confocal microscopy. In vitro tests show that the microcapsules are not cytotoxic upon 24 h of cells exposure to microcapsules concentrations ranging from 2.5 to 25 capsules per cell. The obtained microcapsules of sodium alginate loaded with plasmonic gold nanoparticles could potentially be considered as release systems for biologically relevant molecules.
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Wagner, Roger, Denis Van Loo, Fred Hossler, Kirk Czymmek, Elin Pauwels, and Luc Van Hoorebeke. "High-Resolution Imaging of Kidney Vascular Corrosion Casts with Nano-CT." Microscopy and Microanalysis 17, no. 2 (2010): 215–19. http://dx.doi.org/10.1017/s1431927610094201.
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AbstractA vascular corrosion cast of an entire mouse kidney was scanned with a modular multiresolution X-ray nanotomography system. Using an isotropic voxel pitch of 0.5 μm, capillary systems such as the vasa recta, peritubular capillaries and glomeruli were clearly resolved. This represents a considerable improvement over corrosion casts scanned with microcomputed tomography systems. The resolving power of this system was clearly demonstrated by the unique observation of a dense, subcapsular mat of capillaries enveloping the entire outer surface of the cortical region. Resolution of glomerular capillaries was comparable to similar models derived from laser scanning confocal microscopy. The high-resolution, large field of view and the three-dimensional nature of the resulting data opens new possibilities for the use of corrosion casting in research.
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Ionescu, Ana-Maria, Mihaela-Adriana Ilie, Virginia Chitu, et al. "In vivo Diagnosis of Primary Cutaneous Amyloidosis —the Role of Reflectance Confocal Microscopy." Diagnostics 9, no. 3 (2019): 66. http://dx.doi.org/10.3390/diagnostics9030066.
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Primary cutaneous amyloidosis (PCA) is a form of localized amyloidosis. It is characterized by the deposition of a fibrillar material in the superficial dermis, without affecting other systems or organs. The diagnosis can be made clinically, but usually a skin biopsy is performed in order to exclude other skin diseases with similar appearance. Reflectance confocal microscopy (RCM) is a novel imaging tool that enables in vivo characterization of various skin changes with a high, quasi-microscopic resolution. This technique might have an important role in the differential diagnosis of cutaneous amyloidosis, by the in vivo assessment of epidermal changes and dermal amyloid deposition. Moreover, it is completely non-invasive and can be safely repeated on the same skin area. However, to date, there is only one published paper presenting the confocal features of primary cutaneous amyloidosis. Hereby, we describe the in vivo RCM features of PCA lesions from a patient with diabetes and correlate them with histologic findings. This strengthens the clinical usefulness of in vivo RCM examination for the non-invasive diagnosis of cutaneous amyloidosis, especially in patients that might associate diseases with impaired wound healing.
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Squirrell, J. M., D. L. Wokosin, B. D. Bavister, and J. G. White. "Imaging Subcellular Changes in Living Mammalian Embryos Using 1047 Nm Two Photon Excitation Fluorescence Microscopy." Microscopy and Microanalysis 5, S2 (1999): 1060–61. http://dx.doi.org/10.1017/s1431927600018626.
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A major challenge for fluorescence imaging of living cells is maintaining viability during and following prolonged exposure to excitation illumination, especially when imaging over hours or even days, as when studying mammalian embryonic development. The use of specific fluorescently labeled components in living embryos promises to reveal the roles of organelles and molecules in a native and reproducible context. However, to gain a thorough understanding of dynamic biological systems, events of interest must be recorded as they occur, while limiting perturbations caused by the observation technique. Therefore, establishing long-term fluorescence imaging methods that maintain viability is critical for advancing our understanding of cell and developmental biology.One promising technique for imaging living cells is two photon laser scanning microscopy (TPLSM). The lower energy per photon and the restriction of fluorophore excitation to the imaged focal plane should reduce the total photodamage to thick specimens when compared to conventional laser scanning confocal microscopy (LSCM).
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North, Alison J. "Seeing is believing? A beginners' guide to practical pitfalls in image acquisition." Journal of Cell Biology 172, no. 1 (2006): 9–18. http://dx.doi.org/10.1083/jcb.200507103.
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Imaging can be thought of as the most direct of experiments. You see something; you report what you see. If only things were truly this simple. Modern imaging technology has brought about a revolution in the kinds of questions we can approach, but this comes at the price of increasingly complex equipment. Moreover, in an attempt to market competing systems, the microscopes have often been inappropriately described as easy to use and suitable for near-beginners. Insufficient understanding of the experimental manipulations and equipment set-up leads to the introduction of errors during image acquisition. In this feature, I review some of the most common practical pitfalls faced by researchers during image acquisition, and how they can affect the interpretation of the experimental data. This article is targeted neither to the microscopy gurus who push forward the frontiers of imaging technology nor to my imaging specialist colleagues who may wince at the overly simplistic comments and lack of detail. Instead, this is for beginners who gulp with alarm when they hear the word “confocal pinhole” or sigh as they watch their cells fade and die in front of their very eyes time and time again at the microscope. Take heart, beginners, if microscopes were actually so simple then many people (including myself) would suddenly be out of a job!
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Vanek, Martin, Filip Mravec, Martin Szotkowski, et al. "Fluorescence lifetime imaging of red yeast Cystofilobasidium capitatum during growth." EuroBiotech Journal 2, no. 2 (2018): 114–20. http://dx.doi.org/10.2478/ebtj-2018-0015.
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AbstractRed yeast Cystofilobasidium capitatum autofluorescence was studied by means of confocal laser scanning microscopy (CLSM) to reveal distribution of carotenoids inside the cells. Yeasts were cultivated in 2L fermentor on glucose medium at permanent light exposure and aeration. Samples were collected at different times for CLSM, gravimetric determination of biomass and HPLC determination of pigments. To compare FLIM (Fluorescence Lifetime Imaging Microscopy) images and coupled data (obtained by CLSM) with model systems, FLIM analysis was performed on micelles of SDS:ergosterol and SDS:coenzyme Q with different content of ergosterol and coenzyme Q, respectively, and with constant addition of beta-carotene. Liposomes lecithin:ergosterol:beta-carotene were investigated too. Two different intracellular forms of carotenoids were observed during most of cultivations, with third form appeared at the beginning of stationary phase. Observed behavior is probably due to formation of some kind of carotenoid protective system in membranes of different compartments of yeast cell, especially cytoplasmic membrane.
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Stevens, John K., and Judy Trogadis. "The application of 3D volume investigation methods to serial confocal and serial EM data: Distribution of microtubules in PC12 cells." Proceedings, annual meeting, Electron Microscopy Society of America 49 (August 1991): 146–47. http://dx.doi.org/10.1017/s0424820100085034.
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CAD or Computer Assisted Design uses a computer workstation to create or design new objects. Volume Investigation (VI) uses a computer workstation to understand or analyze existing objects. CAD systems are used to produce a mathematical model of a new object, stored in the workstation's memory. This model is created interactively by the user of the workstation. In contrast, VI systems synthesize a similar mathematical-model automatically from an existing object. The model is usually created or “reconstructed” from a stack of serial cross-sections of the original object. These cross-sections may be collected non-destructively from computerized tomography scans (CT), magnetic resonance imaging scans (MRI), confocal microscopy or destructively from serial light microscopy, serial electron microscopy, or any other source of serial sections. VI workstations are in widespread use in clinical settings, but have not been used at all in scientific research.
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Quelch, Darren R., Loukia Katsouri, David J. Nutt, Christine A. Parker, and Robin J. Tyacke. "Imaging Endogenous Opioid Peptide Release with [11C]Carfentanil and [3H]Diprenorphine: Influence of Agonist-Induced Internalization." Journal of Cerebral Blood Flow & Metabolism 34, no. 10 (2014): 1604–12. http://dx.doi.org/10.1038/jcbfm.2014.117.
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Understanding the cellular processes underpinning the changes in binding observed during positron emission tomography neurotransmitter release studies may aid translation of these methodologies to other neurotransmitter systems. We compared the sensitivities of opioid receptor radioligands, carfentanil, and diprenorphine, to amphetamine-induced endogenous opioid peptide (EOP) release and methadone administration in the rat. We also investigated whether agonist-induced internalization was involved in reductions in observed binding using subcellular fractionation and confocal microscopy. After radioligand administration, significant reductions in [11C]carfentanil, but not [3H]diprenorphine, uptake were observed after methadone and amphetamine pretreatment. Subcellular fractionation and in vitro radioligand binding studies showed that amphetamine pretreatment only decreased total [11C]carfentanil binding. In vitro saturation binding studies conducted in buffers representative of the internalization pathway suggested that μ-receptors are significantly less able to bind the radioligands in endosomal compared with extracellular compartments. Finally, a significant increase in μ-receptor-early endosome co-localization in the hypothalamus was observed after amphetamine and methadone treatment using double-labeling confocal microscopy, with no changes in δ- or κ-receptor co-localization. These data indicate carfentanil may be superior to diprenorphine when imaging EOP release in vivo, and that alterations in the ability to bind internalized receptors may be a predictor of ligand sensitivity to endogenous neurotransmitter release.