Image comparisons of snow and ice crystals photographed by light (video) microscopy and low-temperature scanning electron microscopy

Light (video) microscopy and low-temperature scanning electron microscopy (SEM) were used to examine and record images of identical precipitated and metamorphosed snow crystals as well as glacial ice grains. Collection procedures enabled numerous samples from distant locations to be shipped to a laboratory for storage and/or observation. The frozen samples could be imaged with a video microscope in the laboratory at ambient temperatures or with the low-temperature SEM. Stereo images obtained by video microscopy or low-temperature SEM greatly increased the ease of structural interpretations. The preparation procedures that were used for low-temperature SEM did not result in sublimation or melting. However, this technique did provide far greater resolution and depth of focus over that of the video microscope. The advantage of resolution was especially evident when examining the small particles associated with rime and graupel (snow crystals encumbered with frozen water droplets), whereas the greater depth of focus provided clearer photographs of large crystals such as depth hoar, and ice. Because the SEM images contained only surface information while the video images were frequently confounded by surface and internal information, the SEM images also clarified the structural features of depth hoar crystals and ice grains. Low-temperature SEM appears to have considerable promise for future investigations of snow and ice.     

Single-lens theta microscopy — a new implementation of confocal theta microscopy

A new microscopical technique based on the principle of confocal theta microscopy (Stelzer, E.H.K., Lindek, S. & Pick, R. (1996) Konfokales Mikroskop . German Patent Office DE 43 26 473 (filed 6.8.1993, granted 6.12.1996)) is described. It uses a single objective lens in combination with a mirror unit to achieve the theta configuration that leads to axial and volume resolution improvements. In this paper we present technical details of possible microscopical set-ups, and we discuss different versions of mirror units.     

Axial resolution of confocal fluorescence microscopy

A simple analytic expression is given for the axial resolution of a confocal fluorescence microscope. The expression, which is based on the spatial frequency cut-off criterion of resolution, is valid for high aperture optics and arbitrary fluorescence wavelength.     

Measurement and restoration of the point spread function of fluorescence confocal microscopy

The point spread function of an objective lens of a fluorescence confocal microscope was directly measured by imaging fluorescent beads. We analysed how the measurement of the point spread function was influenced by the diameter of the fluorescent beads and how the restoration technique with a deconvolution algorithm improved the measuring performance. Numerical and experimental results are presented for a typical point spread function and a zero‐centred point spread function.     

Application of regularized Richardson-Lucy algorithm for deconvolution of confocal microscopy images

Although confocal microscopes have considerably smaller contribution of out-of-focus light than widefield microscopes, the confocal images can still be enhanced mathematically if the optical and data acquisition effects are accounted for. For that, several deconvolution algorithms have been proposed. As a practical solution, maximum-likelihood algorithms with regularization have been used. However, the choice of regularization parameters is often unknown although it has considerable effect on the result of deconvolution process. The aims of this work were: to find good estimates of deconvolution parameters; and to develop an open source software package that would allow testing different deconvolution algorithms and that would be easy to use in practice. Here, Richardson-Lucy algorithm has been implemented together with the total variation regularization in an open source software package IOCBio Microscope. The influence of total variation regularization on deconvolution process is determined by one parameter. We derived a formula to estimate this regularization parameter automatically from the images as the algorithm progresses. To assess the effectiveness of this algorithm, synthetic images were composed on the basis of confocal images of rat cardiomyocytes. From the analysis of deconvolved results, we have determined under which conditions our estimation of total variation regularization parameter gives good results. The estimated total variation regularization parameter can be monitored during deconvolution process and used as a stopping criterion. An inverse relation between the optimal regularization parameter and the peak signal-to-noise ratio of an image is shown. Finally, we demonstrate the use of the developed software by deconvolving images of rat cardiomyocytes with stained mitochondria and sarcolemma obtained by confocal and widefield microscopes.     

Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy

Lateral resolution that exceeds the classical diffraction limit by a factor of two is achieved by using spatially structured illumination in a wide-field fluorescence microscope. The sample is illuminated with a series of excitation light patterns, which cause normally inaccessible high-resolution information to be encoded into the observed image. The recorded images are linearly processed to extract the new information and produce a reconstruction with twice the normal resolution. Unlike confocal microscopy, the resolution improvement is achieved with no need to discard any of the emission light. The method produces images of strikingly increased clarity compared to both conventional and confocal microscopes.     

Precision of light intensity measurement in biological optical microscopy

Standardization and calibration of optical microscopy systems have become an important issue owing to the increasing role of biological imaging in high‐content screening technology. The proper interpretation of data from high‐content screening imaging experiments requires detailed information about the capabilities of the systems, including their available dynamic range, sensitivity and noise. Currently available techniques for calibration and standardization of digital microscopes commonly used in cell biology laboratories provide an estimation of stability and measurement precision (noise) of an imaging system at a single level of signal intensity. In addition, only the total noise level, not its characteristics (spectrum), is measured. We propose a novel technique for estimation of temporal variability of signal and noise in microscopic imaging. The method requires registration of a time series of images of any stationary biological specimen. The subsequent analysis involves a multi‐step process, which separates monotonic, periodic and random components of every pixel intensity change in time. The technique allows simultaneous determination of dark, photonic and multiplicative components of noise present in biological measurements. Consequently, a respective confidence interval (noise level) is obtained for each level of signal. The technique is validated using test sets of biological images with known signal and noise characteristics. The method is also applied to assess uncertainty of measurement obtained with two CCD cameras in a wide‐field microscope.     

Optical far-field microscopy of single molecules with 3.4 nm lateral resolution

Optical far‐field imaging of single molecules in a frozen solution at 1.2 K with a lateral resolution of 3.4 nm is reported. The mechanical stability of the fluorescence microscope, especially of the low‐temperature insert, allows for the localization of fluorescing molecules with a reproducibility of better than 5 nm within observation times up to 10 min. For observation times of 9 h the reproducibility of the lateral position is limited to about 20 nm due to mechanical drift. Lateral position and orientation of 314 single molecules, present within the confocal detection volume of ~10 µm³, are obtained. The possibility to correct for mechanical drift by monitoring the position of a spatial reference in the sample is demonstrated.     

Development and testing of hyperbaric atomic force microscopy (AFM) and fluorescence microscopy for biological applications

A commercially available atomic force microscopy and fluorescence microscope were installed and tested inside a custom-designed hyperbaric chamber to provide the capability to study the effects of hyperbaric gases on biological preparations, including cellular mechanism of oxidative stress. In this report, we list details of installing and testing atomic force microscopy and fluorescence microscopy inside a hyperbaric chamber. The pressure vessel was designed to accommodate a variety of imaging equipment and ensures full functionality at ambient and hyperbaric conditions (≤85 psi). Electrical, gas and fluid lines were installed to enable remote operation of instrumentation under hyperbaric conditions, and to maintain viable biological samples with gas-equilibrated superfusate and/or drugs. Systems were installed for vibration isolation and temperature regulation to maintain atomic force microscopy performance during compression and decompression. Results of atomic force microscopy testing demonstrate sub-nanometre resolution at hyperbaric pressure in dry scans and fluid scans, in both contact mode and tapping mode. Noise levels were less when measurements were taken under hyperbaric pressure with air, helium (He) and nitrogen (N(2) ). Atomic force microscopy and fluorescence microscopy measurements were made on a variety of living cell cultures exposed to hyperbaric gases (He, N(2) , O(2) , air). In summary, atomic force microscopy and fluorescence microscopy were installed and tested for use at hyperbaric pressures and enables the study of cellular and molecular effects of hyperbaric gases and pressure per se in biological preparations.     

Video microscopy of dynamic plant cell organelles: principles of the technique and practical application

Video microscopy, including video-enhanced contrast, ultraviolet and video-intensified fluorescence microscopy, was applied to the visualization and analysis of organelles and cytoskeletal elements at the border of resolution of the light microscope. We describe the principles of video microscopy and the necessary technical equipment, and discuss the advantages and limitations with the example of three selected plant cells. In characean internodal cells we observed the motility and disappearance of Golgi secretory vesicles during wound wall formation by video-enhanced contrast microscopy. In Byblis gland hairs we investigated the movement of different organelles along bundles of actin microfilaments by ultraviolet microscopy, and in onion inner epidermal cells we visualized the arrangement of actin microfilaments during different stages of plasmolysis with video-intensified fluorescence microscopy.     

Streak time-lapse video microscopy: analysis of protoplasmic motility and cell division in Tradescantia stamen hair cells

We have developed a video time-lapse analogue of a streak camera using a digital image processing technique, and have used it to analyse dynamic processes in plant cells. The same image area is selected from a succession of frames in a video tape recording. The successive images are stored in an image memory, rearranged, and displayed in a single frame according to the time course. Each image area is rectangular, with selectable length and width: the closest analogy to a streak camera is obtained when one of its sides is a single pixel. The composite image displays sequential changes in the distribution of subcellular components in the selected area. The method was shown to be useful for monitoring details of cytoplasmic streaming and organelle movement, observing the time course of chemical fixation, and also for studying kinetochore movement, shortening of chromatids and cell plate formation during mitosis and cytokinesis. Cessation and resumption of cytoplasmic movement during the cell division cycle were also clearly localized outside the nuclear region in apical cells of Tradescantia stamen hairs.     

Ultrathin fluorescent layers for monitoring the axial resolution in confocal and two-photon fluorescence microscopy

Monomolecular films of polymerized dimethyl-bis[pentacosadiinoic-oxyethyl] ammonium bromide (EDIPAB) provide one- and two-photon excited fluorescence that is sufficiently high to quantify the axial resolution of 3-D fluorescence microscopes. When scanned along the optical axis, the fluorescence of these layers is bright enough to allow online observation of the axial response of these microscopes, thus facilitating alignment and fluorescence throughput control. The layers can be used for directly measuring and monitoring the axial response of 4Pi-confocal microscopes, as well as for their initial alignment and phase adjustment. The proposed technique has the potential to supersede the conventional technique of calculating the derivative of the axial edges of a thick fluorescent layer. Coverslips with EDIPAB-layers can be used as substrates for the cultivation of cells.     

Characterization of uniform ultrathin layer for z-response measurements in three-dimensional section fluorescence microscopy

Layer‐by‐layer technique is used to adsorb a uniform ultrathin layer of fluorescently labelled polyelectrolytes on a glass cover slip. Due to their thickness, uniformity and fluorescence properties, these ultrathin layers may serve as a simple and applicable standard to directly measure the z‐response of different scanning optical microscopes. In this work we use ultrathin layers to measure the z‐response of confocal, two‐photon excitation and 4Pi laser scanning microscopes. Moreover, due to their uniformity over a wide region, i.e. cover slip surface, it is possible to quantify the z‐response of the system over a full field of view area. This property, coupled with a bright fluorescence signal, enables the use of polyelectrolyte layers for representation on sectioned imaging property charts: a very powerful method to characterize image formation properties and capabilities (z‐response, off‐axis aberration, spherical aberration, etc.) of a three‐dimensional scanning system. The sectioned imaging property charts method needs a through‐focus dataset taken from such ultrathin layers. Using a comparatively low illumination no significant bleaching occurs during the excitation process, so it is possible to achieve long‐term monitoring of the z‐response of the system. All the above mentioned properties make such ultrathin layers a suitable candidate for calibration and a powerful tool for real‐time evaluation of the optical sectioning capabilities of different three‐dimensional scanning systems especially when coupled to sectioned imaging property charts.     

Image division technique in pre-acquisition analysis of information content for automated microscopy

This article presents a method that allows for reliable automated image acquisition of specimens with high information content in light microscopy with emphasis on fluorescence microscopy applications. Automated microscopy typically relies on autofocusing used for the analysis of information content behaviour along the z-axis within each field of view. However, in the case of a field of view containing more objects that do not lie precisely in one z-plane, traditional autofocusing methods fail due to their principle of operation. We avoid this issue by reducing the original problem to a set of simple and performable tasks: we divide the field of view into a small number of tiles and process each of them individually. The obtained results enable discovering z-planes with rich information content that remain hidden during global analysis of the whole field of view. Our approach therefore outperforms other acquisition methods including the manual one. A large part of the contribution is oriented towards practical application.     

An efficient algorithm for measurement and correction of chromatic aberrations in fluorescence microscopy

Even the best optical microscopes available on the market exhibit chromatic aberrations to some extent. In some types of study, chromatic aberrations of current optics cannot be neglected and a software correction is highly desirable. This paper describes a novel method of chromatic aberration measurement and software correction using sub-resolution bead imaging and computer image analysis. The method is quick, precise and enables the determination of both longitudinal and lateral chromatic aberrations. Correction function can be computed in about half an hour, including image acquisition. Using this approach, chromatic aberrations can be reduced to 10–20 nm laterally and 10–60 nm axially depending on the type of optical set-up. The method is especially suitable for fluorescence microscopy, where a limited number of wavelengths are observed.     

Characterization of mixed self-assembled monolayers for immobilization of streptavidin using chemical force microscopy

Mixed self-assembled monolayers (SAMs) to immobilize streptavidin on a gold surface were investigated by measuring the pull-off force between an AFM tip and a biotin-modified surface using CFM. Biotin-LC-NHS was modified on SAMs prepared from a mixed solution of cystamine and MEOH. Increased pull-off forces between the AFM tip and the surface were observed with an increased cystamine mole fraction in the solution. Streptavidin was immobilized onto biotin-LC-NHS modified mixed SAMs and analyzed by tapping AFM. Also, the formation of mixed SAMs containing MUOH and MBDA was confirmed using CFM. The measured pull-off forces on the only MBDA surface were larger than those on the surface with MUOH. These results can be applied to determine an optimal mixing ratio of MUOH and MBDA SAMs that reduces non-specific streptavidin binding onto a surface.     

Characterization of a direct detection device imaging camera for transmission electron microscopy

The complete characterization of a novel direct detection device (DDD) camera for transmission electron microscopy is reported, for the first time at primary electron energies of 120 and 200 keV. Unlike a standard charge coupled device (CCD) camera, this device does not require a scintillator. The DDD transfers signal up to 65 lines/mm providing the basis for a high-performance platform for a new generation of wide field-of-view high-resolution cameras. An image of a thin section of virus particles is presented to illustrate the substantially improved performance of this sensor over current indirectly coupled CCD cameras.     

An application of spatial deconvolution to a capillary-based high-pressure chamber for fluorescence microscopy imaging

Capillary‐based high‐pressure chambers for which the wall serves as both the optical window and mechanical support have been reported for fluorescence microscopy imaging. Although capillary chambers are straightforward and economical to construct, the curved capillary wall introduces image aberrations. The significance of these aberrations in imaging sub‐cellular‐dimension objects has yet to be assessed. Using a capillary chamber that is routinely pressurized to between 20 and 30 MPa, a pressure range suitable for studying a wide variety of cellular processes, we demonstrate sub‐cellular‐dimension spatial resolution in the imaging of fluorescent micro‐spheres. Objectives with a range of numerical apertures (0.5–1.3) and working distances (0.1–7.4 mm) are considered. We show that spatial (or point‐spread function, PSF) deconvolution improves image contrast in capillary‐based images by comparing deconvolution results with those obtained from slide‐mounted controls. Furthermore, similar deconvolution results between a measured PSF and a calculated, flat‐geometry PSF indicate that the capillary wall is optically flat on cellular length scales. Results here facilitate the application of contemporary techniques in fluorescence microscopy to high‐pressure imaging fields.     

Total internal reflection microscopy for live imaging of cellular uptake of sub-micron non-fluorescent particles

This paper presents a simple, high-resolution, non-fluorescent imaging technique called total internal reflection microscopy (TIRM) and demonstrates its potential application to real-time imaging of live cellular events. In addition, a novel instrument is introduced that combines the simplicity of TIRM with the specificity afforded by dual-colour total internal reflection fluorescence (TIRF) microscopy and allows sequential imaging with the two modalities. The key design considerations necessary to apply these imaging modes in a single instrument are discussed. The application of TIRM alone yielded high-resolution live images of cell adherence to a poly- l -lysine modified substrate, whereby fine cellular structures are imaged. Non-fluorescent imaging of the uptake of sub-micron–sized polymeric particles by live cells is also demonstrated. Finally, images of fluorescently labelled cells were obtained in TIRF mode, sequentially to images obtained of the same cell in TIRM mode. Visual information gained using TIRF is compared with TIRM to demonstrate that the level of cell structure information obtainable with our total internal reflection microscope is comparable with the TIRF technique.     

Techniques for reducing the interfering effects of autofluorescence in fluorescence microscopy: improved detection of sulphorhodamine B-labelled albumin in arterial tissue

Measurements of the transport of circulating sulphorhodamine B-labelled albumin into the arterial wall, made by applying digital imaging fluorescence microscopy to sections of arteries fixed in situ , are limited in sensitivity by the low levels of tracer fluorescence and high levels of autofluorescence emitted from the tissue. Three attempts to improve these ratios are described. In the first, spectra of the tracer in solution and of arterial autofluorescence were used to design novel microscope filters for rhodamine-like dyes. By exciting with the rarely used yellow lines of the mercury arc lamp and detecting a narrow band of emission with Stokes shifts as small as 15 nm, the ratio of tracer fluorescence to autofluorescence was tripled. In the second, effects of different fixatives were investigated. Using a model system, it was confirmed that Karnovsky's fixative gives good tracer immobilization but elevates autofluorescence, whereas fixative-free buffer solutions give low autofluorescence but do not retain the tracer. It was further found that simple formaldehyde-based fixatives, hitherto considered to be poor fixatives of albumin, immobilized the tracer as well as the glutaraldehyde-based fixative, whilst giving autofluorescence levels comparable to those seen with buffer alone; they therefore give excellent tracer fluorescence to autofluorescence ratios. In the third, lowering specimen temperature by 50 °C was found to increase the intensity of tracer fluorescence by 30% whilst autofluorescence was unaffected. These data may have relevance to microscopical studies using other tissues and fluorescent tracers.