Standard Optical Microscope Research Articles

Confocal Raman Microscopy. Thomas Dieing, Olaf Hollricher, and Jan Toporski (Eds.)

Confocal Raman Microscopy. Thomas Dieing, Olaf Hollricher, and Jan Toporski (Eds.). Springer, New York, 2011, 289 pages. ISBN 978-3642125218Raman microscopy is the combination of Raman spectroscopy with optical light microscopy and was first presented in the mid-1970s. It combines the advantages of vibrational Raman spectroscopy as a noninvasive technique providing a wealth of chemical information on the properties of molecules and solids with the imaging capabilities and small sample volume requirements of an optical microscope. “Seeing is believing”: Raman microscopy can visualize the inherent chemical inhomogeneity of a sample without the need of external labels, information that cannot be obtained from simply looking at an unstained sample in the standard optical microscope. During the last decade, confocal Raman microscopy has gone through a rapid development with respect to instrumentation and software as well as the diversity of applications.

Mapping of native inorganic elements and injected nanoparticles in a biological organ with laser-induced plasma

Emission spectroscopy of laser-induced plasma from a thin section of mouse kidney successfully detected inorganic elements, Na, Ca, Cu, and Gd, naturally contained in the organ or artificially injected in the form of Gd-based nanoparticle. A two-dimensional scan of the sample allowed the laser beam to explore its surface with a resolution of 100 μm, resulting in a quantitative elemental mapping of the organ with sub-mM sensitivity. The compatibility of the setup with standard optical microscopy emphasizes the potential to provide multiple images of a same biological tissue with different types of response which can be elemental, molecular, or cellular.

Measurement of single cell refractive index, dry mass, volume, and density using a transillumination microscope.

Phase contrast microscopy has become ubiquitous in the field of biology, particularly in qualitative investigations of cellular morphology. However, the use of quantitative phase retrieval methods and their connection to cellular refractive index and dry mass density remain under utilized. This is due in part to the restriction of phase and cellular mass determination to custom built instruments, involved mathematical analysis, and prohibitive sample perturbations. We introduce tomographic bright field imaging, an accessible optical imaging technique enabling the three dimensional measurement of cellular refractive index and dry mass density using a standard transillumination optical microscope. The validity of the technique is demonstrated on polystyrene spheres. The technique is then applied to the measurement of the refractive index, dry mass, volume, and density of red blood cells. This optical technique enables a simple and robust means to perform quantitative investigations of engineered and biological specimens in three dimensions using standard optical microscopes.

External and internal features of garnet revealed by the multifocus imaging technique

This publication illustrates the imaging of both external and internal features of a single-garnet crystal using the multifocus imaging technique. Based on image stacks acquired using a standard optical microscope, it demonstrates that this easily implemented technique yields information on single grains and may in some cases provide more information than that obtainable from a conventional thin section.

A Fiber-Optic-Based Imaging System for Nondestructive Assessment of Cell-Seeded Tissue-Engineered Scaffolds

A major limitation in tissue engineering is the lack of nondestructive methods that assess the development of tissue scaffolds undergoing preconditioning in bioreactors. Due to significant optical scattering in most scaffolding materials, current microscope-based imaging methods cannot "see" through thick and optically opaque tissue constructs. To address this deficiency, we developed a fiber-optic-based imaging method that is capable of nondestructive imaging of fluorescently labeled cells through a thick and optically opaque scaffold, contained in a bioreactor. This imaging modality is based on the local excitation of fluorescent cells, the acquisition of fluorescence through the scaffold, and fluorescence mapping based on the position of the excitation light. To evaluate the capability and accuracy of the imaging system, human endothelial cells (ECs), stably expressing green fluorescent protein (GFP), were imaged through a fibrous scaffold. Without sacrificing the scaffolds, we nondestructively visualized the distribution of GFP-labeled cells through a ~500 μm thick scaffold with cell-level resolution and distinct localization. These results were similar to control images obtained using an optical microscope with direct line-of-sight access. Through a detailed quantitative analysis, we demonstrated that this method achieved a resolution on the order of 20-30 μm, with 10% or less deviation from standard optical microscopy. Furthermore, we demonstrated that the penetration depth of the imaging method exceeded that of confocal laser scanning microscopy by more than a factor of 2. Our imaging method also possesses a working distance (up to 8 cm) much longer than that of a standard confocal microscopy system, which can significantly facilitate bioreactor integration. This method will enable the nondestructive monitoring of ECs seeded on the lumen of a tissue-engineered vascular graft during preconditioning in vitro, as well as for other tissue-engineered constructs in the future.

Moganite in the Chalcedony Varieties of Continental Cherts (Miocene, Madrid Basin, Spain)

ABSTRACT Varieties of chalcedony (calcedonite, quartzine, and lutecite) and other optical micro- and cryptocrystalline textures of quartz and opal from Miocene silcretes of the Madrid Basin were studied using a Thermo Fisher DXR Raman microscope. The microscope has a point-and-shoot Raman capability of 1 µm spatial resolution and was coupled with a standard optical microscope. Our results show that all the varieties of chalcedony can have a composite Raman spectrum of both quartz and moganite. The spectra are independent of the chalcedony origin by ageing, direct replacement, and cementation processes. Moganite was absent only in some calcedonite cements. The presence of moganite is independent of the surface sedimentary setting of the host rocks in which the silcretes are formed.

High Temperature Deformation of Superalloy Inconel 718

Experimental results on hot deformation and dynamic structural processes of nickel based alloy Inconel 718 are reviewed. The focus is the analysis of dynamic precipitation processes which operate during hot deformation of these materials at elevated temperatures. Hot compression tests were performed on the solution treated precipitation hardenable nickel based superalloy Inconel 718 at 720-1150°C with a constant true strain rates of 10-4 and 4x10-4s-1. True stress - true strain curves and microstructure analysis of the deformed nickel based superalloy is presented. The properties and dynamic behaviour are explained through observation of the microstructure using standard optical, scanning and transmission electron microscopy. Structural observations of solution treated Inconel 718 deformed at high temperatures, reveal non uniform deformation effects. The distribution of niobium-rich carbides were affected by localized flow within the strain range investigated at relatively low deformation temperatures 720 - 850°C.

Spiked Gold Beads as Substrates for Single‐Particle SERS

We introduce a new family of spiked particles resulting from the growth of high aspect ratio gold nanorods. Upon spike growth, elongated beads are obtained with sizes above 300 nm. Interestingly, and in contrast to smooth particles of the same size, these spiked-particles are not only able to sustain localized surface plasmon resonances and consequently enhance Raman signals, but are also big enough to be recognized by standard confocal optical microscopy. These spiked beads have been engineered into thin films to test their surface-enhanced Raman scattering (SERS) enhancing efficiency as a function of the particle density. Such films provide a high level of portability and easiness of use for "in-field" optical ultrasensitive analysis.

Fourier analysis of blurred images for the measurement of the in-plane dynamics of MEMS

The goal of this paper is to use FFT imaging techniques to measure in-plane resonances of MEMS devices from blurred microphotographs where the presence of resonance is not visually discernable. A method is presented for measuring the high-frequency (in the 10s–100s of kHz range) response characteristics of MEMS devices using only standard optical microscope cameras (15–30 Hz frame rate) and applying Fourier analysis of camera images of periodic patterns on the oscillating devices. In the frequency domain, in-plane blurring acts as a low pass filter, attenuating all frequency components, but preferentially attenuating the higher order harmonics. A theoretical formula for the blur-induced attenuation of the harmonics of Fourier series components is derived and it is shown that it follows a Bessel curve. The theoretical predictions were verified experimentally using a series of camera microphotographs of three different variations of an electro-thermally driven pad suspended on springs. The predicted attenuations of harmonics were observed and verified. The analysis of the measured attenuation was able to (1) determine in-plane resonant frequencies, (2) measure submicron motions and (3) characterize the nonlinear dynamics (modeled by the Duffing equation). The amplitude uncertainty of the FFT method for detecting in-plane resonant peaks at 75 kHz and 3.5 V was found to be ±0.027 µm using a single image and ±0.011 µm using an average of 10 images.

Visualization, Modeling, and Spatial Statistics of Mitochondrial Assembly in Adult Cardiomyocytes using Serial Block-Face Scanning Electron Microscopy

Mitochondria are recognized as dynamic organelles, which constantly undergo morphological remodeling through fusion and fission processes. However, their geometric details including individual shape, size, and spatial distribution have not been quantitatively characterized in adult cardiomyocytes. Standard optical microscopy is unable to resolve neighboring mitochondria, which are intensely packed between myofilament bundles and narrow sub-sarcolemmal space. On the other hand, 3D reconstructs generated by serial thin-section transmission electron microscopy (EM) or EM tomography can only provide a limited field of view. We applied a novel advanced 3D electron microscopic technology, serial block-face scanning electron microscopy (SBFSEM), to adult mouse ventricular tissues, segmented mitochondria, and created geometric models of mitochondrial assembly. Substantially large volumes enclosing neighboring myocytes were imaged using SBFSEM. Subsequently, we applied spatial analysis tools using Spatstat R package to quantify organization of mitochondria. In cross-sectional slices generated from SBFSEM volumes, the boundaries of individual mitochondria were extracted and the centroid of each boundary was plotted. Analysis of the distribution of these centroids using a quadrat test of the intensity plot indicated borderline inhomogenity (p=0.053). The test was rerun after redrawing quadrats, now incorporating the location of the nucleus in the 3D stack. The results confirmed that mitochondria were clustered near the nucleus (p=0.017). The cell-wide inter-point interaction between mitochondrial centroids calculated by the L-function and the pair correlation function failed to support organized mitochondrial clustering. In summary, the study revealed the strength of the new integrated use of SBFSEM imaging and computational statistics to characterize and parameterize the spatial distribution of cellular organelles such as mitochondria that are dynamically remodeled under a physiological condition and more intensely in disease settings such as diabetic cardiomyopathy and heart failure.

ИДЕНТИФИКАЦИЯ ПО СЛЕДАМ НА ГИЛЬЗАХ С ИСПОЛЬЗОВАНИЕМ ТОПОГРАФИЧЕСКИХ ИЗМЕРЕНИЙ МИКРОНЕОДНОРОДНОСТЕЙ И КОРРЕЛЯЦИИ. ОБЪЕДИНЕНИЕ МИКРОСКОПИИ И СТАТИСТИЧЕСКИХ МЕТОДОВ

A comparison microscope employing the standard optical comparison method and confocal microscopy with subsequent cross correlation topography analysis were used to correctly identify cases fired from a set of ten (10) consecutively made pistol slides. The mathematical identifications were based on the breech face impression without the firing pin aperture shear marks. Fifteen (15) unknown cartridge cases were compared to test fired cartridge case sets from the consecutively manufactured slides.

Super-resolution fluorescence microscopy as a tool to study the nanoscale organization of chromosomes

Chromatin organization spans a wide range of structural complexity. Substructures at the 10-200nm scale are poorly characterized, especially in living cells, due to the limitations of electron microscopy and standard optical microscopy. Recently developed super-resolution fluorescence microscopy methods represent an exciting opportunity to access those substructures, and recent progress with these techniques has yielded insights into chromatin organization at different condensation stages. Recent studies have focused on confronting the challenges that are specific to chromatin super-resolution imaging, such as the high packing density of mitotic chromosomes and difficulties in interpreting interphase chromatin images. Building on these first results and with ongoing rapid technical advances in super-resolution fluorescence imaging there is great potential to uncover new features with unprecedented detail.

Microscopic analysis of minas frescal cheese sold in city of Londrina-PR

<p>Cheese are considered highly perishable foods, so it must be produced with raw materials of good quality, subject to an efficient quality control, besides their transport, storage and marketing appropriate to avoid  vehiculation of diseases. The presence of impurities in food may be indicative of neglect at some point in the process, moreover, the insects and mites can act as backers of microbiological contaminants such as fungi, bacteria, viruses, protozoa and helminths. This study aimed to verify the hygienic conditions of Minas frescal type cheese sold in food markets in the city of Londrina-PR, according to the nature and amount of foreign material (dirt) present. Twenty samples of cheese were washed with kerosene and filtered on filter paper, whose content was analyzed retained a standard optical microscope (increased 400 times). The results showed that 100% of the samples contained at least one type of foreign matter, demonstrating poor hygienic quality. These date showed that is important to monitor these products since they can be vehicles of diseases, endangering the health of consumers.</p><p> </p><p>DOI: <a href="http://dx.doi.org/10.14685/rebrapa.v2i1.16">http://dx.doi.org/10.14685/rebrapa.v2i1.16</a></p>

Microscopie SEEC : la microscopie optique comme outil de caractérisation nanométrique

These last decades, numerous signal enhancement techniques to push away the detection limits were born. Most of them implement the use of supports with specific properties. Indeed, supports made of thin layers, or micro- or nano-structured layers are present in topics such as unlabelling biological analysis, RAMAN spectroscopy, SNOM microscopy... Contrast enhanced supports were also developed for the optical microscopy but their performances remained so far very limited. Indeed, the optical microscopy has particular constraints due to the geometry of the incidental lightbeam. Recently, a study concerning the modelling of the polarized light beam allowed the elaboration of supports having contrast-enhancement properties. Due to their characteristics, these supports enable to visualize with a standard optical microscope, layers and objects having nanometric thicknesses (nanofilm, biofilm, biochip, ADN strength, nanoparticle, carbon nanotube, graphene sheet...).

Constituent Particle Clustering and Pitting Corrosion

Corrosion is a primary degradation mechanism that affects the durability and integrity of structures made of aluminum alloys, and it is a concern for commercial transport and military aircraft. In aluminum alloys, corrosion results from local galvanic coupling between constituent particles and the metal matrix. Due to variability in particle sizes, spatial location, and chemical composition, to name a few critical variables, corrosion is a complex stochastic process. Severe pitting is caused by particle clusters that are located near the material surface, which, in turn, serve as nucleation sites for subsequent corrosion fatigue crack growth. These evolution processes are highly dependent on the spatial statistics of particles. The localized corrosion growth rate is primarily dependent on the galvanic process perpetuated by particle-to-particle interactions and electrochemical potentials. Frequently, severe pits are millimeters in length, and these pits have a dominant impact on the structural prognosis. To accommodate large sizes, a model for three-dimensional (3-D) constituent particle microstructure is proposed. To describe the constituent particle microstructure in three dimensions, the model employs a fusion of classic stereological techniques, spatial point pattern analyses, and qualitative observations. The methodology can be carried out using standard optical microscopy and image analysis techniques.

High-resolution digital transmitting microscope with a large objective lens

Standard optical microscopes are based on diffraction imaging, and their resolution is limited to half the wavelength of the illuminating light. In contrast, scattered-light imaging depends on the sharpness of the optics and is not limited by the wavelength. This technique is used mainly for reflective dark-field observations. The combination of a scattered-light illumination system and sharp optics may improve the resolution of the transmitting microscope. It is well known that the large objective lens in a telescope improves the resolution by reducing the diffraction rings. The digital microscope, designed for industrial applications, has a large objective lens and a 16-bit high-gradation image-processing system, resulting in a sharper image. In this paper, a digital microscope is compared with a standard microscope using the same condenser lens in both cases, which produces scattered light. To amplify the scattered light, the light beam around the optical axis is masked in the condenser lens. Although the standard microscope uses an objective lens with a higher numerical aperture (NA = 0.92) than the digital microscope (NA = 0.82), the digital microscope achieved a higher resolution (over 2500 line-pairs/mm) than the standard microscope, even under dry conditions. This resolving power is greater than that predicted by classical optical theory.

Measurement of Fluorescence in a Rhodamine-123 Doped Self-Assembled “Giant” Mesostructured Silica Sphere Using a Smartphone as Optical Hardware

The blue OLED emission from a mobile phone was characterised, revealing a sharp emission band centred at λ = 445 nm with a 3dB bandwidth Δλ ∼ 20 nm. It was used to excite Rhodamine 123 doped within a “giant” mesostructured silica sphere during fabrication through evaporative self-assembly of silica nanoparticles. Fluorescence was able to be detected using a standard optical microscope fitted with a green transmission pass filter and cooled CCD and with 1 ms exposure time demonstrating the potential of mobile platforms as the basis for portable diagnostics in the field.

Modeling and simulation of constituent particle clustering and distribution in rolled aluminum alloys

It is recognized that corrosion damage in aluminum alloys is the direct result of local galvanic coupling between constituent particles and the metal matrix. Heavily clustered groups of constituent particles have been shown to form the most severe corrosion pits, and the morphology of these pits has been found to exhibit a strong correlation with that of the instigating particle cluster. To predict damage evolution accurately in three dimensions, the spatial distribution of the constituent particles in a given material must be quantified. The tomographic reconstruction methods currently available are time consuming and possess characteristic length scales that are insufficient for large-scale corrosion studies. To accommodate this larger length scale, a model for three-dimensional constituent particle microstructure is proposed. This model employs a fusion of classic stereological techniques and qualitative observations to describe the constituent particle microstructure of rolled aluminum alloys in three dimensions. A method is described for generating statistically representative three-dimensional constituent particle microstructure from two-dimensional orthogonal cross sections. This novel simulation algorithm allows for detailed three-dimensional particle microstructure simulations to be carried out using standard optical microscopy and image analysis techniques.

Multicolored nanometre-resolution mapping of single protein–ligand binding complexes using far-field photostable optical nanoscopy (PHOTON)

Mapping of individual ligand molecules and their binding sites in single protein-ligand complexes at nanometer resolution in real-time would enable probing their structures and functions in vitro and in vivo. In this study, we have developed far-field photostable optical nanoscopy (PHOTON) for mapping single ligand molecules (biotin) and their binding sites in individual protein-ligand complexes (streptavidin-biotin) with 1.2 nm spatial resolution and 100 ms temporal resolution. PHOTON includes one standard far-field optical microscope with a halogen-lamp illuminator; single-molecule-nanoparticle-optical-biosensors (SMNOBS) with exceptionally high quantum-yield (QY) of Rayleigh scattering and photostability (non-photobleaching, non-photoblinking) as imaging probes; and Multispectral Imaging System (MSIS) for spectral isolation of individual SMNOBS with 1 nm wavelength resolution. Intrinsic size- and shape- dependent localized-surface-plasmon-resonance (LSPR) spectra of single SMNOBS provide multiple-spectral (color) nanoprobes for sub-diffraction imaging, offering feasibility of probing of binding structures and functions of single protein-ligand complexes at nm (potentially achieving Ångstrom) resolution in real-time.

FRET in Cell Biology: Still Shining in the Age of Super‐Resolution?

Interest in imaging of Förster resonance energy transfer (FRET) in biological systems has been steadily increasing in the last 30 years. The ability to transduce a near-field interaction into a far-field signal has provided a unique optical tool to assess biological phenomena well below the resolution of standard optical microscopy. In recent years, sub-diffraction microscopy techniques have achieved maturation and are increasingly used in biological applications. As the resolution of these methods increases they will slowly encroach on the domains where FRET is now dominant. Herein we review the major applications in biological FRET imaging and we discuss the possibilities and challenges in the super-resolution era.