Defocusing microscopy

Transparent objects (phase objects) are not visible in a standard brightfield optical microscope. In order to see such objects the most used technique is phase-contrast microscopy. In phase-contrast microscopy the contrast observed is proportional to the optical path difference introduced by the object. If the index of refraction is uniform, phase-contrast microscopy then yields a measure of the thickness profile of phase objects. We show that by slightly defocusing an optical microscope operating in brightfield, phase objects become visible. We modeled such an effect and show that the image contrast of a phase object is proportional to the amount of defocusing and proportional to the two-dimensional Laplacian of the optical path difference introduced by the object. For uniform index of refraction, defocusing microscopy then yields a measure of the curvature profile of phase objects. We extended our previous model for thin objects to thick objects. To check our theoretical model, we use as phase objects polystyrene spherical caps and compare their curvature radii obtained by defocusing microscopy (DM) to those obtained with atomic force microscopy (AFM). We also show that for thick curved phase objects one can reconstruct their thickness profiles from DM images. We illustrate the utility of defocusing microscopy in biological systems to study cell motility. In particular, we visualize and quantitatively measure real-time cytoskeleton curvature fluctuations of macrophages (a cell of the innate immune system). The study of such fluctuations might be important for a better understanding of the engulfment process of pathogens during phagocytosis.     

Improving DIC microscopy with polarization modulation

It is demonstrated experimentally, as well as analytically, that when the polarization of the light incident upon the first Nomarski–Wollaston prism in a differential interference contrast (DIC) light microscope is switched by 90°, image highlights are changed into shadows and vice versa. Using an inexpensive ferroelectric liquid-crystal modulator, which is easily installed in the microscope, this switching can be done at 30 frames s⁻¹, synchronized to the camera. Subtraction of alternate digitized frames generates a stream of images in which contrast is doubled, compared with conventional video-enhanced DIC, while image defects and noise tend to cancel. Subtraction of alternate images is carried out efficiently by frame buffer operations and amounts to massively parallel synchronous detection. The new method eliminates the problems inherent in obtaining a separate background image, as required by current video-enhanced DIC practice, without loss of resolution.     

Scanning interference and confocal microscopy

The form of the interference term image in scanning confocal and scanning conventional interference microscopes is identical in all respects including optical sectioning. This observation is used to obtain confocal images and surface profiles from conventional scanning interference microscope images.     

On the extinction coefficient in confocal polarization microscopy

The extinction coefficient in conventional polarized light microscopes is non-zero, even with perfect polars, solely because of the image formation process in these instruments. The imaging in confocal microscopes is different from conventional instruments and it is shown that in this case an infinite extinction coefficient results in the ideal case whereas a finite value is always to be expected with conventional instruments. Images of the same microelectronic specimen taken in both conventional and confocal polarized light microscopes are compared.     

Use of a white light supercontinuum laser for confocal interference-reflection microscopy

Shortly after its development, the white light supercontinuum laser was applied to confocal scanning microscopy as a more versatile substitute for the multiple monochromatic lasers normally used for the excitation of fluorescence. This light source is now available coupled to commercial confocal fluorescence microscopes. We have evaluated a supercontinuum laser as a source for a different purpose: confocal interferometric imaging of living cells and artificial models by interference reflection. We used light in the range 460-700 nm where this source provides a reasonably flat spectrum, and obtained images free from fringe artefacts caused by the longer coherence length of conventional lasers. We have also obtained images of cytoskeletal detail that is difficult to see with a monochromatic laser.     

The use of a digitizing frame store in microscopy

A digitizing frame store giving real time read out of the image for TV or slow scan rates is now available. This equipment has a number of applications in both optical and electron microscopy. Examples of results obtained by linkage to a low light level TV system are given showing that use of the frame store yields image information not otherwise available. Other possibilities of the system are indicated.     

Towards automatic cell identification in DIC microscopy

A general method is proposed for constructing templates of cells in differential interference contrast (DIC) microscopy. This takes account of the optics which generate DIC images, and is applicable to both transparent and semi-transparent cells of simple and complex shapes. Then, a template matching methodology is presented, which uses fast Fourier transforms to fit templates of a range of sizes and orientations to images. For illustration, this is used to automatically identify and measure individual Candida yeast cells in clusters.     

A new approach for improving the birefringence analysis of dental enamel mineral content using polarizing microscopy

The main problem in interpreting birefringence of dental enamel under polarizing microscopy is the lack of physical constants able to allow the Wiener equation to be applied directly to the composition of such tissue. The present study introduces a new approach to circumvent this constraint. Because the nonmineral phase of enamel is heterogeneous, its refractive index can be computed in terms of its components (namely, water, which is partially replaced by the immersion medium, and organic matter), thereby providing a more acceptable refractive index to be used in the Wiener equation. Furthermore, the enamel mineral volume is ordinarily calculated on the basis of the density 3.15 g cm^?3. The density 2.99 g cm^?3 has been, however, reported to be more accurate for enamel hydroxyapatite, so enamel mineral volumes from selected published data were converted using such a density. The birefringence of mature enamel computed by the Wiener equation, taking into account the above refinements, matched, for the first time, published experimental birefringence values. The theoretical water and organic contents were also consistent with published experimental data. Thus, a direct application of the Wiener equation to the enamel composition has now been achieved. It is speculated that quantitative data on the mineral, the water and the organic contents of mature dental enamel can be derived from interpretation of birefringence in two immersion media (obtained before and after extraction of the organic matter) with this new approach.     

Laser interference microscopy of amphibian erythrocytes: impact of cell volume and refractive index

This study examined the action of anisosmotic media on the volume of nucleated erythrocytes isolated from Rana temporaria. Elevation of medium osmolarity from 100 to 345 mOsm resulted in attenuation of mean cell volume by more than 3-fold, estimated by hematocrit measurement. By contrast to this 'classic' erythrocyte volume evaluation technique, we did not observe any significant cell volume modulation by examining the 3D reconstruction of erythrocyte interference images obtained by laser interference microscopy. Comparative analysis of mean cell volume, phase height and cell area appraised by laser interference microscopy showed that the lack of visible alterations of phase image geometry was caused by sharp elevation of the average refractive index of the cytoplasm in shrunken cells. Thus, our results show for the first time that laser interference microscopy in combination with a direct method for cell volume measurement may be employed for estimation of the refractory index of intracellular milieu and for assessment of changes of physical chemical properties of the cytoplasm evoked by diverse stimuli including osmotic stress.     

Time-resolved, three-dimensional quantitative microscopy of a droplet spreading on solid substrates

A polarized microscope was used to study the spreading of mercury droplets on thin silver films. Using the differential interference contrast (DIC) method and semi‐quantitative measurements of the optical path difference (OPD), the three‐dimensional shape of the liquid droplet that wets the solid surface was constructed with an angle resolution of 1°. The evolution of the droplet shape was determined with a time resolution of 0.04 s. The quantitative results are compared with other wetting‐reaction systems. In particular, it is demonstrated that the droplet has a spherical‐cup shape during the entire wetting‐reaction process.     

Curvature measurement with reflected-light microscopy and its application to fly facet lenses

Two methods for microscopically measuring the curvature of strongly curved surfaces are compared: one using a Michelson interferometer-type microscope and one using a novel reflectometrical method implemented in an epi-illumination microscope. The curvature values obtained with the two methods were very similar, but the latter proved to be by far the simplest. Curvature measurements on the front surface of the facet lenses of various dipteran flies revealed that facet lens diameter and radius of curvature are linearly related over a wide range of facet lens sizes.     

Development of a new bimodal imaging methodology: a combination of fluorescence microscopy and high-resolution secondary ion mass spectrometry

In this paper, we present a new experimental methodology to combine mass spectrometry (NanoSIMS) with fluorescence microscopy to provide subcellular information on the location of small molecules in cultured cells. We demonstrate this by comparing the distribution of 5-bromo-2-deoxyuridine in the same cells given by both NanoSIMS analysis and by fluorescence immunohistochemistry. Fiducial markers in the substrates ensured that the images formed by SIMS mapping of bromine ions could be co-registered exactly with images from fluorescence microscopy. The NanoSIMS was shown to faithfully reproduce the information from fluorescence microscopy, but at a much higher spatial resolution. We then show preliminary SIMS images on the distribution of ATN-224, a therapeutic copper chelator for which there is no fluorescent marker, co-registered with conventional Lysotracker and Hoechst stains on the same cells.     

Measurement of cellular elastic properties by acoustic microscopy

The acoustic microscope is used to investigate the elastic properties of living biological cells. A quantitative model is developed relating acoustic microscope image contrast to cellular elastic properties. Cytoplasmic acoustic attentuation is measured by focusing the acoustic microscope on the surface of the underlying substrate. Cytoplasmic acoustic impedance is measured by focusing the acoustic microscope on the top surface of the cell. The model allows the acoustic microscope to give quantitative information about cellular elasticity on a subcellular scale.     

Optical microscopy with improved resolution using two-beam interference of low-coherence light

In recent years, high-resolution microscopy using structured illumination has been practically applied for fluorescent bio-imaging. However, there is a large amount of speckle noise in reflected- and scattered-light images, because structured illumination is typically generated by laser-beam interference. Hence, this high-resolution imaging technique cannot be effectively used in industrial applications. In this study, we attempted to generate structured illumination using two-beam interference of low-coherence light for high-resolution and low-speckle imaging. First, we constructed an optical system consisting of a Michelson interferometer configured in such a manner that it achieved zero optical path-length difference and allowed the interference fringes to be manipulated. Then, we confirmed that the generated structured illumination width corresponded to the coherence length of the light source. As a final result of the resolution improvement experiment, the narrow sample pitch of 0.4 μm was successfully resolved beyond the diffraction limit of 0.74 μm with relatively less speckle noise.     

Real time uses of color for electron microscopy via a television-rate digital frame store

An on-line television-rate digital frame store device is utilized to provide color representations of a wide range of electron microscope images and image data. Various types of hardware devices in the frame store coupled with software manipulations via the host computer make rapid image acquisition, modification, measurement, and full-color display possible in real time either from micrographs or directly from an electron microscope. Lookup tables used in conjunction with grey-level image memories can be controlled from a menu display to provide a wide range of color-coding schemes and sequencing. It is also possible to use color graphics overlays and alpha numeric displays along with full-color image displays. This paper will describe many of the recent applications of color developed for electron microscopy studies of materials.     

Imaging modes for bilateral confocal scanning microscopy

The bilateral scanning approach to confocal microscopy is characterized by the direct generation of the image on a two-dimensional (2-D) detector. This detector can be a photographic plate, a CCD detector or the human eye, the human eye permitting direct visualization of the confocal image. Unlike Nipkow-type systems, laser light sources can be used for excitation. A design called a carousel has been developed, in which the bilateral confocal scan capability can be added to an existing microscope so that rapid exchange and comparison between confocal and non-confocal imaging conditions is possible. The design permits independent adjustment of confocal sectioning properties with lateral resolutions better than, or, in the worst case equivalent to, those available in conventional microscopy. The carousel can be considered as a stationary optical path in which certain imaging conditions, such as confocality, are defined and operate on part of the imaging field. The action of the bilateral scan mirror then extends this image condition over the whole field. A number of optical arrangements for the carousel are presented which realize various forms of confocal fluorescence and reflection imaging, with point, multiple point or slit confocal detection arrangements. Through the addition of active elements to the carousel direct stereoscopic, ratio, time-resolved and other types of imaging can be achieved, with direct image formation on a CCD, eye or other 2-D detectors without the need to modify the host microscope. Depending on the photon flux available, these imaging modes can run in real-time or can use a cooled CCD at (very) low light level for image integration over an extended period.     

Elimination of scanning electron microscopy image periodic distortions with digital signal-processing methods

Electromagnetic interference is one of the main distortion sources in scanning electron microscopy. Electromagnetic interference‐generated scanning electron microscopy image distortions are usually visible as edge blur (at low scan rates) or vibration (at high scan rates). Hardware solutions to this problem, e.g. electrostatic and magnetic shielding, are expensive and, in some cases, difficult to implement. The current investigations led to a significant decrease in the periodic distortions by a novel adaptation of software‐based digital signal processing to scanning electron microscopy problems, without any hardware modification.     

High-resolution cell outline segmentation and tracking from phase-contrast microscopy images

Accurate extraction of cell outlines from microscopy images is essential for analysing the dynamics of migrating cells. Phase-contrast microscopy is one of the most common and convenient imaging modalities for observing cell motility because it does not require exogenous labelling and uses only moderate light levels with generally negligible phototoxicity effects. Automatic extraction and tracking of high-resolution cell outlines from phase-contrast images, however, is difficult due to complex and non-uniform edge intensity. We present a novel image-processing method based on refined level-set segmentation for accurate extraction of cell outlines from high-resolution phase-contrast images. The algorithm is validated on synthetic images of defined noise levels and applied to real image sequences of polarizing and persistently migrating keratocyte cells. We demonstrate that the algorithm is able to reliably reveal fine features in the cell edge dynamics.