Microscope Cover Glass Research Articles

Vertical cell assembly of colloidal crystal films with controllable thickness

Formation of colloidal crystal films (CCFs) is the first step in the fabrication of inverse colloidal crystals, a class of three-dimensionally ordered, macroporous materials. A new vertical cell assembly method is described that yields CCFs with thicknesses up to 100 µm. The cell consists of two microscope cover glasses separated by a thin polymeric spacer. The lower edge of the cell is placed in a colloidal dispersion. Particles are transported to the top of the cell by capillary force and self-assemble into a CCF as the solvent evaporates. This novel vertical cell assembly method is well suited for fabrication of large area CCFs with controllable thickness that could be further processed into ICC filters for size-based separations of molecules and particles.

Direct visualisation and kinetic analysis of normal and nemaline myopathy actin polymerisation using total internal reflection microscopy

Actin filaments were formed by elongation of pre-formed nuclei (short crosslinked actin-HMM complexes) that were attached to a microscope cover glass. By using TIRF illumination we could see actin filaments at high contrast despite the presence of 150 nM TRITC-phalloidin in the solution. Actin filaments showed rapid bending and translational movements due to Brownian motion but the presence of the methylcellulose polymer network constrained lateral movement away from the surface. Both the length and the number of filaments increased with time. Some filaments did not change length at all and some filaments joined up end-to-end (annealing). We did not see any decrease in filament length or filament breakage. For quantitative analysis of polymerisation time course we measured the contour length of all the filaments in a frame at a series of time points and also tracked the length of individual filaments over time. Elongation rate was the same measured by both methods (0.23 microm/min at 0.1 microM actin) and was up to 10 times faster than previously published measurements. The annealed filament population reached 30% of the total after 40 min. Polymerisation rate increased linearly with actin concentration. K(on) was 2.07 microm min(-1) microM(-1) (equivalent to 34.5 monomers s(-1) microM(-1)) and critical concentration was less than 20 nM. This technique was used to study polymerisation of a mutant actin (D286G) from a transgenic mouse model. D286G actin elongated at a 40% lower rate than non-transgenic actin.

Viscous Drag Torque on a Rotating Nanofabricated Cylinder Near an Infinite Plane Boundary

Microrheological and single molecule biological measurements often involve the use of a microscopic probe particle near a surface. On such systems a precise understanding of the hydrodynamic interactions between the particle and the surface is required. Recently nanofabricated nearly cylindrical quartz particles have been used as ideal trapping particles for an angular optical trap. Here the rotational viscous drag torque imposed on a nanofabricated quartz cylinder near a surface is measured with the angular optical trap. The deviation of torque from the Stokes drag relation in solution is measured as a function of distance from the plane surface of a microscope cover glass. The surface effect is found to be significant for distances on the order of the characteristic dimension of the cylinder. These findings will allow for more accurate and quantitative torsional measurements of angular trapping systems.

Differential detection for measurements of Faraday rotation by means of ac magnetic fields

We demonstrate that by using a combination of a Wollaston prism and two photodiodes the accuracy in the measurements of Faraday rotation with ac magnetic fields can be greatly improved. Our experiments were performed on microscope cover glass plates with thicknesses between 0.13 and 0.16 mm. We show that our setup is capable of distinguishing between the Faraday rotation signals of glass plates having a difference in thickness of a few micrometers, corresponding to Faraday rotations of hundreds of microdegrees per Tesla only.

Experimental and numerical investigation into the joule heating effect for electrokinetically driven microfluidic chips utilizing total internal reflection fluorescence microscopy

This paper presents a detection scheme for analyzing the temperature distribution nearby the channel wall in a microfluidic chip utilizing a temperature-dependent fluorescence dye. An advanced optical microscope system—total internal reflection fluorescence microscope (TIRFM) is used for measuring the temperature distribution on the channel wall at the point of electroosmotic flow in an electrokinetically driven microfluidic chip. In order to meet the short working distance of the objective type TIRFM scheme, microscope cover glass slits are used to fabricate the microfluidic chips. The short fluorescence excitation depth from a TIRFM system makes the intensity information obtained using TIRFM is not sensitive to the channel depth variation which ususally biases the measured results while using a conventional Epi-fluorescence microscope (EPI-FM). Therefore, a TIRFM can precisely describe the temperature profile of the distance within 100 nm of the channel wall where consists of the Stern layer and the diffusion layer for an electrokinetic microfluidic system. Results indicate the proposed TIRFM provides higher measurement sensitivity over the EPI-FM. Significant temperature gradient along the channel depth is experimentally observed. In addition, the measured wall temperature distributions can be the boundary conditions for numerical investigation into the joule heating effect. The proposed method gives a precise temperature profile of microfluidic channels and shows the substantial impact on developing a numerical simulation model for precisely predicting the joule heating effect in microfluidic chips.

Myostatin Directly Regulates Skeletal Muscle Fibrosis

Skeletal muscle fibrosis is a major pathological hallmark of chronic myopathies in which myofibers are replaced by progressive deposition of collagen and other extracellular matrix proteins produced by muscle fibroblasts. Recent studies have shown that in the absence of the endogenous muscle growth regulator myostatin, regeneration of muscle is enhanced, and muscle fibrosis is correspondingly reduced. We now demonstrate that myostatin not only regulates the growth of myocytes but also directly regulates muscle fibroblasts. Our results show that myostatin stimulates the proliferation of muscle fibroblasts and the production of extracellular matrix proteins both in vitro and in vivo. Further, muscle fibroblasts express myostatin and its putative receptor activin receptor IIB. Proliferation of muscle fibroblasts, induced by myostatin, involves the activation of Smad, p38 MAPK and Akt pathways. These results expand our understanding of the function of myostatin in muscle tissue and provide a potential target for anti-fibrotic therapies.

A desktop Faraday rotation instrument in the ultraviolet

A simple versatile desktop instrument for the measurement of Faraday rotation in the ultraviolet range has been designed and constructed. A high pressure short arc mercury lamp is used for the light source. By using interference filter for mercury, the desired wavelength of the available lines for mercury (e.g., 365, 405, and 436 nm) is selected. Our instrument measures hysteresis loops in magnetic fields up to 900 kAm within a few minutes. With a light beam intensity monitor, the detrimental effect from fluctuations in the light source has been virtually eliminated. The instrument can operate at photocurrents from the picoampere regime and above with a Faraday rotation sensitivity of around 1 millideg. By incorporating a higher order Taylor expansion approach, we improve the linearity of the Faraday rotation to transmitted light relationship by two orders of magnitude. The electronics is custom designed analog type, rendering relaxed dynamic requirements for the analog-to-digital converter. The design is fully protected from ambient light which makes operation with the equipment in darkness superfluous; neither does it need any optical table. The data acquisition and operation of the instrument are fully supported by a LABVIEW program. Measurements on a magnetite thin film and on microscope cover glass are given as examples for the performance and sensitivity of the equipment.

Effect of salt precursor on laser-assisted copper deposition

Continuous copper structures were precipitated on microscope cover glasses by the laser-induced chemical liquid phase deposition (LCLD) method. The deposition of copper was realized from both CuSO4- and CuCl2-based electrolyte solutions. The surface morphology and the chemical composition of the deposits were analyzed with a scanning electron microscope and energy dispersive X-ray spectrometer (EDX). The dependence of the characteristics (width and resistance) of the copper deposits on the laser power, temperature and the salt precursor was examined. The observed strong influence of the solution temperature and the salt precursor on the deposits characteristics was explained by the specific chemical features of the Cu(II)→Cu(0) reduction process.

A chemiluminescence sensor for the determination of hydrogen peroxide

A chemiluminescence one-shot sensor for hydrogen peroxide is described. It is prepared by immobilization of cobalt chloride and sodium lauryl sulphate in hydroxyethyl cellulose matrix cast on a microscope cover glass. Luminol, sodium phosphate and the sample are mixed before use and applied on the membrane by a micropipette. The calibration graph is linear in the range 20–1600 μg/L, and the detection limit of the method (3 σ) is 9 μg/L. A relative standard deviation of 4.5% was obtained for 100 μg/L H 2O 2 ( n = 11). The sensor has been applied successfully to the determination of hydrogen peroxide in rainwater.

Combining fluidic reservoirs and optical tweezers to control beads/living cells contacts

We have developed a complete system based on holographic optical tweezers to realize multiple-point interactions between beads and cells with control of the stimulation places, timing and durations. We introduce microstereolithography as a 3D micromanufacturing approach to the rapid prototyping of three-dimensional fluidic microchambers of complex shapes, comprising wells, channels and walls, that are afterwards placed inside the sample and used to inject beads locally and keep them separated from cells in our assays. A custom reservoir designed to keep beads and cells separated in liquid samples has been realized and successfully tested. This allows us to deposit beads locally on the microscope cover glass placed under the reservoir outlet. Limited dispersion of beads under the outlet has been confirmed, and the ability of the polymeric structures to confine beads in a restricted area has been demonstrated. Examples of manipulations consisting at first in extracting several beads from the reservoir, making them travel to the target cell, and finally depositing on its outer membrane with respect to the shape of the target cell, are finally given.

Surface Crystallization of Indomethacin Below T g

To study the surface crystallization of indomethacin (IMC) below T (g) and its effects on the kinetics of overall crystallization. Crystal growth rates in liquid layers formed between microscope cover glasses were measured with the top cover glass in place and removed. Polymorphs were identified by powder X-ray diffraction, Raman microscopy, and melting-point determination by hot-stage microscopy. Surface crystals were identified by scratching the sample surface, by cutting the sample to expose its interior, and by analyzing the intensity of X-ray diffraction. Amorphous IMC particles of different sizes were stored at 40 degrees C (T (g)-2 degrees C) and analyzed at different times by differential scanning calorimetry to obtain the kinetics of crystallization. Crystal growth of IMC below T (g) at the free surface was approximately two orders of magnitude faster than that in the bulk, resulting in a surface layer of crystals around a slower-crystallizing interior. Surface crystallization yielded mainly the gamma polymorph. Amorphous IMC powders showed rapid initial crystallization at 40 degrees C, but the crystallization abruptly slowed down at "saturation levels" below 100%; the larger the particles, the lower the "saturation level." The faster surface crystallization of IMC than the bulk crystallization leads to unusual crystallization kinetics wherein a rapid initial increase of crystallinity is followed by an abrupt slowdown of crystallization. Surface crystallization should be distinguished from bulk crystallization in modeling and controlling the crystallization of amorphous solids.

Leukemia cells interaction with plasma-polymerized acrylic acid coatings

The acrylic acid (ppAc) films were deposited on a glass substrate by plasma polymerization. The interaction of leukemia cells with the ppAc films was studied. It was found that the leukemia cells showed a higher growth on cell culture polystyrene and microscope cover glass substrates, whereas a low growth (about 60%) on the ppAc films. The presence of functional groups such as hydroxyl and carbonyl on the ppAc films can affect the cell culture.

Picoscope, a new label-free biosensor

A new label-free biosensing spectral correlation method based on measurements of a correlation signal of two interferometers in an original optical scheme has been developed. One of the interferometers used as a biochip is a simple microscope cover glass without any additional metal or dielectric film. A biosensor named “Picoscope” has been developed for real-time simultaneous detection of several biological agents by measuring picometer-range changes of the thickness of different biorecognition spots on the biochip surface. The application area of the Picoscope can be as wide as that of traditional optical microscope. However, the Picoscope can provide much more comprehensive information with outstanding resolution in depth, for example, for measuring molecular binding kinetics, monitoring of assembling and detaching in molecular structures, etc. The Picoscope technology will significantly increase the power of research instruments for bio-, nano- and pico-technologies.

Direct visualization of nanopatterns by single-molecule imaging

The structures of patterned surface regions were clearly visualized by superimposing a series of single-molecule images from a total internal reflection fluorescence microscope, clearly demonstrating that a single-molecule imaging method can be used to directly visualize nanostructures. The pattern was fabricated on a microscope cover glass that formed a sandwich with a regular cover glass. The incorporated solution of fluorescent probe dyes was examined by single-molecule techniques. The effect of the surface pattern on the diffusion of the probe has also been examined.

Establishment by an Original Single‐cell Cloning Method and Characterization of an Immortal Mouse Melanoblast Cell Line (NCCmelb4)

We devised a unique new single-cell cloning method which uses microscope cover glasses and established a melanoblast cell line derived from mouse neural crest cells. A microscope cover glass was nicked and broken into small pieces and put on a dish. Culture medium and a suspension of 20-30 cells/ml were dropped in the dish. After 1-3 d, a piece of glass to which only one cell was adhered was picked up and transferred to another dish containing culture medium. The greatest advantage of this method is that the derivation of a colony from a single cell can be directly confirmed by microscopy and there is no risk of migratory cells being contaminated by other colonies. Using this single-cell cloning method, in this study we established a cell line derived from a neural crest cell line (NCC-S4.1) and designated it as NCCmelb4. When the culture medium was supplemented with stem cell factor (SCF) alone, NCCmelb4 cells were KIT-positive and tyrosinase-negative melanocyte precursors; they remained at an immature and undifferentiated stage. When the medium was supplemented with phorbol 12-o-tetradecanoyl-13-acetate (TPA) + cholera toxin (CT), the cell morphology changed and became L-3,4-dihydroxyphenylalanine (DOPA)-positive. This observation indicates that the NCCmelb4 cells are capable of further differentiation with suitable stimulation. NCCmelb4 cells derived from the mouse neural crest has characteristics of melanocyte precursors (melanoblasts), and is a cell line which can be utilized to study differentiation-inducing factors and growth factors without the effects of feeder cells.

Planar bilayer lipid membranes supported on mesoporous aerogels, xerogels, and Vycor® glass: an epifluorescence microscopy study

We report the deposition and characterization of fluid synthetic biomembranes on various mesoporous materials. Planar phospholipid bilayers consisting of 97mol% l-α-phosphatidylcholine (egg PC) and 3mol% fluorescently labeled lipid N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn -glycero-3-phosphoethanolamine (NBD-PE) were formed by fusion of ∼30nm diameter unilamellar vesicles on four different silica-based substrates: aerogels, xerogels, Vycor® glass, and microscope cover glass. Epifluorescence microscopy was used to examine the micrometer-scale homogeneity of the resulting planar membranes. Fluorescence recovery after photobleaching analysis was used to determine the lateral mobility retained in the membranes. Diffusion coefficients of 0.6±0.2, 2±1, 1.7±1.1, and 2.5±0.4μm2s−1 were obtained for membranes supported on aerogels, xerogels, Vycor®, and glass, respectively. In the same order, the average immobile lipid fractions, defined by the normalized magnitudes of recovery, were ∼75%, 81%, 94%, and 100%. Both lateral mobility and immobile fraction exhibited ranges of standard variations, especially pronounced for xerogels and Vycor®, suggesting that the long-range translational dynamics of lipids in the supported membranes are sensitive to local defects and geometry of the underlying surface. Micrometer-scale clefts on the aerogel and xerogel surfaces limited the lipid lateral exchange, as shown by the reduced diffusivity and recovery.

Anomalously Slow Diffusion of Single Molecules near a Patterned Surface

Two-dimensional hexagonal arrays of micrometer-sized spheroidal cavities were fabricated from poly(vinyl alcohol) films on microscope cover glasses. The patterned surfaces in contact with solution and another regular cover glass were used to contain single molecules. Bursts of fluorescence from a series of single molecules entering and leaving the beam focus were observed. The molecules were all rotating rapidly compared with the fastest binning time of 100 μs. The fluorescence anisotropy decayed on the nanosecond time scale. The fluorescence spectra of mixtures of dyes confirmed that the bursts separated by time intervals of several to tens of seconds are from different molecules, while those bursts spanning intervals of several to tens of milliseconds are from the same molecule continually reentering the focus. The autocorrelation function of the time-resolved fluorescence intensity suggests a translational diffusion coefficient of 1.7 × 10-8 cm2 s-1 for 6-carboxyrhodamine 6G hydrochloride molecules nea...

New direct optical biosensors for multi-analyte detection

A new label-free optical biosensing method has been developed, which can be called Spectral Correlation Method (SCM). The method is based on measurements of a correlation signal of two interferometers in an original optical scheme. The first interferometer is a scanned Fabry–Perot interferometer, in which the distance between two mirrors is periodically changed by a peizoelectric driver. The second interferometer is formed by a biochip, which is a simple glass plate with thickness of several tens or hundreds microns with different recognition spots or wells, or flow channels. In our experiments, a microscope cover glass was successfully used as a biochip to detect reactions of binding or detachment of different biological agents in real time. Two- and 96-channel SCM devices have been designed to register bio- and chemical-components in real time. The sensogram drifts for these devices expressed in terms of layer thickness were 20 and 40 pm/h, respectively.

측면 연마 광섬유와 결합된 상부 도파로의 클래딩을 이용한 편광 무의존 온도센서

본 논문에서는 측면연마 광섬유 결합기 구조에서 평면 도파로의 상부와 하부 클래딩의 열광학 효과를 이용하여 온도를 측정할 수 있는 편광 무의존 온도 센서를 제안하고 있다. 평면 도파로는 170<TEX>$\mu\textrm{m}$</TEX> 두께의 현미경용 덮게 유리를 적용함으로서 급격한 온도 변화에 대해 도파로의 변형이 발생하지 않고, 비대칭적 평면 도파로 구조로 인한 편광 의존 손실을 최소화하기 시킬 수 있는 장점을 가진다. 제작된 소자의 편광 의존 손실은 0.3dB 이하로 측정되었다. 피측정체의 온도는 센서의 결합 파장 이동으로부터 계측되어진다. 평면 도파로의 상부와 하부 클래딩의 열광학 계수에 따른 온도 센서의 온도 민감도를 측정하였고, 상ㆍ하부 클래딩의 열광학 계수 변화로 온도 민감도를 제어할 수 있음을 보였다. In this paper, we propose a polarization-insensitive temperature sensor using a thermo-optic effect of the upper and (or) lower cladding of a planar waveguide in contact with a side-polished fiber. A microscope cover glass with thickness of a 170 <TEX>${\mu}{\textrm}{m}$</TEX> is adopted as an overlay waveguide because this waveguide opposes sudden temperature change and ensures polarization-insensitive responses. The measured polarization-dependence loss is less than 0.3 dB. The temperature can be detected as a result of the shift in coupling wavelength of the sensor. We investigate the shift in coupling wavelength as a function of the temperature variation with respect to the different thermo-optic coefficients of lower and upper claddings. We also show that the temperature sensitivity of the device can be easily controlled by the thermo-optic coefficients of lower and upper claddings of the overlay waveguide.

Dispersion compensation for optical coherence tomography depth-scan signals by a numerical technique

A new numerical a posteriori dispersion compensation technique for partial coherence interferometry and optical coherence tomography depth-scan signals is presented. This technique is based on numerical correlation of the depth-scan interferometer signal with a depth-variant kernel. Examples of dispersion compensated depth-scan signals obtained from microscope cover glasses are presented.