Differential interference contrast microscopy as a polarimetric instrument

Differential interference contrast (DIC) microscopy is shown to be equivalent to an incomplete Stokes polarimeter capable of probing optical properties of materials on microscopic-length scales. The Mueller matrix for a DIC microscope is calculated for various types of samples, and the polarimetric properties for DIC component parts of a spaceflight microscope are spectrally measured. As a practical application, a measurement of the index mismatch between colloidal particles and a nearly index-matched fluid bath was performed.     

Simulation of high-resolution x-ray zone plates

A full-wave approach to quantitative characterization of x-ray zone plate lenses is proposed. Distributed focusing efficiency eta(z) of a multifocus optical element is defined as the energy flux through the Airy disk of a reference perfect lens with variable focal length z. Maxima of this function characterize diffraction efficiencies and spatial resolution of the zone plate foci. The parabolic wave equation is used to take into account diffraction effects inside the optical element. Rough and fuzzy interface models are introduced to describe realistic zone profiles. Numerical simulation reveals the limited capability of zone width reduction to improve the zone plate imaging performance. The possibilities of second-order focus enhancement by optimization of the zone plate thickness, line-to-space ratio, and zone tilt are studied numerically.     

Orientation-independent differential interference contrast microscopy

We describe a new technique for differential interference contrast (DIC) microscopy, which digitally generates phase gradient images independently of gradient orientation. To prove the principle we investigated specimens recorded at different orientations on a microscope equipped with a precision rotating stage and using regular DIC optics. The digitally generated images successfully displayed and measured phase gradients, independently of gradient orientation. One could also generate images showing distribution of optical path differences or enhanced, regular DIC images with any shear direction. Using special DIC prisms, one can switch the bias and shear directions rapidly without mechanically rotating the specimen or the prisms and orientation-independent DIC images are obtained in a fraction of a second.     

Differential interference contrast microscope with differential detection for optimizing image contrast

A laser scanning differential interference contrast microscope using a differential detection method is proposed. This microscope permits observers to control the differential image contrast with a simple operation. Utilizing this function, the observers are capable of obtaining the optimized differential image whose contrast is most favorable for observation. A simple theoretical analysis and experimental considerations are described.     

Stabilizing method for reflection interference contrast microscopy

Reflection interference contrast microscopy (RICM) is a technique for measuring the shape and position of microscopic objects in solution; it has many biological and biophysical applications. Use of RICM for long-time acquisitions requires minimizing defocusing effects that are due to thermal and mechanical drift. We present a simple stabilizing method that accomplishes this using an image-analysis-based linear focus function to establish feedback control of the focal position. While implementing this routine, we used RICM for independent measurement of the apparent fluctuation in the vertical position of an immobilized bead: the measured height had a standard deviation of 0.12 nm during a 45 min acquisition while under feedback control, demonstrating the high stability achievable with our approach.     

Fabrication of x-ray zone plates by surface-plasma chemical vapor deposition

A new cost-efficient sputter-slice technology for hard x-ray (10-30 keV) Fresnel zone plates fabrication, imposing no limitation to aspect ratio, is proposed. By means of a plasma chemical process, SiO(2)/Si(1-x)Ge(x)O(2) glassy film multilayer structures are deposited on a lateral surface of a silica rod, outermost layers being as thin as 100 nm. It has been shown by numerical simulation that for x=0.2 germanium fraction, 100-300 microm zone plate thickness and the number of zones of about 1000, first order diffraction efficiency as high as 20%-30% at the energy of approximately 20 keV can be achieved.     

Automated analysis of differential interference contrast microscopy images of the foveal cone mosaic

An algorithm is presented for processing and analysis of differential interference contrast (DIC) microscopy images of the fovea to study the cone mosaic. The algorithm automatically locates the cones and their boundaries in such images and is assessed by comparison with results from manual analysis. Additional algorithms are presented that analyze the cone positions to extract information on cone neighbor relationships as well as the short-range order and domain structure of the mosaic. The methods are applied to DIC images of the human fovea.     

Digital in-line soft x-ray holography with element contrast

Digital in-line soft x-ray holography (DIXH) was used to image immobilized polystyrene and iron oxide particles and to distinguish them based on their different x-ray absorption cross sections in the vicinity of the carbon K-absorption edge. The element-specific information from the resonant DIXH images was correlated with high-resolution scanning electron microscopy (SEM) pictures. We also present DIXH images of a cell nucleus and compare the contrast obtained for nuclear components with the appearance in optical microscopy.     

Quantitative phase and refractive index analysis of optical fibers using differential interference contrast microscopy

A systematic and straightforward image processing method to extract quantitative phase and refractive index data from weak phase objects is presented, obtained using differential interference contrast (DIC) microscopy. The method is demonstrated on DIC images of optical fibers where a directional integration routine is applied to the DIC images to extract phase and refractive index information using the data obtained across the whole DIC image. By applying the inverse Abel transform to the resultant phase images, an accurate refractive index profile is obtained. The method presented here is compared to the refracted near-field technique, typically used to obtain the refractive index profile of optical fibers, and shows excellent agreement. It is concluded that through careful image processing procedures, DIC microscopy can be successfully implemented to obtain quantitative phase and refractive index information of optical fibers.     

Multi scale hard x-ray microscopy

The structure of crystalline materials is typically organised hierarchically on several length scales. Hard x-ray microscopy is presented as a collection of modalities that allows to zoom into a mm-sized sample to acquire 3D maps of any embedded region and at essentially all relevant length scales. For coarse mapping of grains, their orientations and average stress state diffraction based tomography methods can sample thousands of grains with a resolution of 2 µm. At the 100 nm scale, domains and dislocations and their associated strain fields can be visualised by diffraction microscopy. Similar to dark field electron microscopy, diffraction and imaging can be combined in several ways. For the ultimate resolution, a bulk version of coherent diffraction imaging is introduced. Hard x-ray microscopy is optimised for acquisition of 3D movies: directly visualising the structural changes during nucleation and growth, deformation or damage. The state of art is provided along with examples of use. I discuss how hard x-ray microscopy studies can enable the formulation and validation of improved multiscale models that account for the entire heterogeneity of materials.     

Direct observation of single native DNA molecules in a microchannel by differential interference contrast microscopy

Direct observation of single native DNA molecules in a microchannel was monitored without fluorescence-dye labeling. At a PDMS/glass microchip, the image of individual lambda-DNA molecules appear sharp and distinct in Nomarski differential interference contrast microscopy. Intercalator dyes affected the physical properties and dynamic behavior of individual DNA molecules. From the migration velocities in the microchannel it is evident that native DNA molecules migrated faster than DNA molecules labeled with the intercalator YOYO-1. This is because YOYO-1 increases the molecular weight and size of lambda-DNA and decreases the charge. The electric field strength and pH also affected the dynamics of single DNA molecules. We also observed that YOYO-labeled DNA was more stretched out compared to native DNA.     

Stroboscopic white-light interference microscopy

The principle of stroboscopic motion freezing of oscillating objects extends directly to interference microscopes that use coherence as part of the measurement principle. Analysis shows, however, that the fringe contrast loss for out-of-plane motion in stroboscopic interferometry is a wavelength-dependent phenomenon, which can alter the apparent nominal center wavelength of the white-light source. As in monochromatic systems, the key adjustable parameter is the duty cycle, equal to the product of the vibrational frequency and the pulse width. This theoretical study provides detailed graphs of expected errors as a function of the duty cycle, including fringe contrast loss, apparent wavelength shift, and measurement error.     

Surface relief zone plates fabricated with photographic emulsions

The use of photographic emulsions as a photoresist medium for fabricating relief blazed zone plates is described. Lithographic methods with masks with 256 gray levels were used in the fabrication process. An example of a zone plate working in the reflection mode at 45 degrees and having 82% diffraction efficiency is shown.     

Influence of polarization setting on gold nanorod signal at nonplasmonic wavelengths under differential interference contrast microscopy

Researchers rely on a variety of microscopic techniques for observing and tracking anisotropic nanoparticles in real time experiments. This technical note focuses on the optical behavior exhibited by gold nanorods at nonplasmonic wavelengths under differential interference contrast microscopy (DIC). Intense diffraction patterns appear at nonplasmonic wavelengths, and the behavior of these patterns can be altered by adjusting the surrounding medium or the polarizer setting. Such patterns are absent when linear and crossed polarizations are utilized. Making polarization adjustments is important in DIC microscopy, because it affects bias retardation and image contrast. The nonplasmonic diffraction bands that were observed could potentially be exploited for rotational tracking, but more importantly, researchers should exhibit care in selecting a nanorod sample and the polarization setting when working with DIC microscopy.     

Polarization-modulated differential-interference contrast microscopy with a variable retarder

A liquid-crystal variable retarder inserted into a differential-interference contrast video microscope switches image highlights into shadows and vice versa in alternate frames. Synchronous computation and display of the difference between alternate frames yield a stream of images with doubled contrast and reduced fixed-position noise because of the automatic background subtraction. The measured signal-to-noise ratio (SNR) peaks when the modulation +/- Gamma of the retarder equals the phase shift delta of the sample. A Jones calculus model of the central ray in the polarization-modulated differential-interference contrast microscope yields SNR = (sin Gamma sin delta)/((1 - cos Gamma cos delta)N), where N is the rms time-dependent photon noise. This expression fits the experiments closely for 1.8 degrees < or = Gamma < or = 115 degrees.     

Super-resolved spatial light interference microscopy

We report a scheme to achieve resolution beyond the diffraction limit in spatial light interference microscopy (SLIM). By adding a grating to the optical path, the structured illumination technique can be used to improve the resolution by a factor of 2. We show that a direct application of the structured illumination technique, however, has proved to be unsuccessful. Through two crucial modifications, namely, one to the pupil plane of the objective and the other to the demodulation procedure, faithful phase information of the object is recovered and the resolution is improved by a factor of 2.     

Optical contrast in apertureless microscopy

We report on optical image contrast for a specific apertureless near-field optical microscope. We demonstrate that the main part of the optical image's contrast results from the sample's topography. The coupling mechanism is analyzed, and we show that the microscope can be regarded as an interferometer that sensitively detects near-field components. However, in the basic configuration the reference field of the interferometer is coupled to the topography. Finally, it is demonstrated that, by controlling the phase of the reference field, one can largely decorrelate the optical image from the topography.