Virtual reflected-light microscopy

Research on better methods to digitally represent microscopic specimens has increased over recent decades. Opaque specimens, such as microfossils and metallurgic specimens, are often viewed using reflected light microscopy. Existing 3D surface estimation techniques for reflected light microscopy do not model reflectance, restricting the representation to only one illumination condition and making them an imperfect recreation of the experience of using an actual microscope. This paper introduces a virtual reflected-light microscopy (VRLM) system that estimates both shape and reflectance from a set of specimen images. When coupled with anaglyph creation, the system can depict both depth information and illumination cues under any desired lighting configuration. Digital representations are compact and easily viewed in an online setting. A prototype used to construct VRLM representations is comprised only of a microscope, a digital camera, a motorized stage and software. Such a system automatically acquires VRLM representations of large batches of specimens. VRLM representations are then disseminated in an interactive online environment, which allows users to change the virtual light source direction and type. Experiments demonstrate high quality VRLM representations of 500 microfossils.     

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.     

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.     

Microtribological applications of probe microscopy

This paper introduces the new field of microtribology; it gives a general overview and then presents some typical research results. Micromachines use very lightweight sliding parts, and their wear is primarily due to surface interaction forces rather than load or weight. The ultimate goal of microtribology is to create practical zero-wear devices. Microtribological evaluations of surfaces have started using new tools, such as the scanning probe microscope. Quasi-static indentation tests, impact indentation tests, line scratch tests, and scanning scratch tests have been performed using an atomic force microscope with a diamond tip. Frictional force distributions and adhesive force distributions have also been obtained using atomic force microscopes. Water clusters adsorbed onto solid surfaces have been observed using a scanning tunnelling microscope. The configuration, adsorption, and mobility of lubricant molecules have also been evaluated using a scanning tunnelling microscope.     

Proton microscopy and microanalysis--biological applications

Several nuclear and atomic techniques are available to a scanning proton microprobe for examining the elemental distributions and microstructure within a specimen. The instrument and its techniques are discussed together with some examples of non-destructive elemental microanalysis. The handling and display of quantitative maps and their relationship to regional spectra are shown with an emphasis in this paper on biological applications.     

激光干涉光刻技术应用概述

激光干涉光刻作为一种新兴的光刻技术,具有设备结构简单、价格低廉、高效率、高分辨率和大视场曝光等特点。介绍通过激光干涉光刻产生的一维光栅结构、二维网格结构以及二维点阵结构在生物传感、太阳能电池和光子晶体等领域的应用前景。     

Linear phase imaging using differential interference contrast microscopy

We propose an extension to Nomarski differential interference contrast microscopy that enables isotropic linear phase imaging. The method combines phase shifting, two directions of shear and Fourier‐space integration using a modified spiral phase transform. We simulated the method using a phantom object with spatially varying amplitude and phase. Simulated results show good agreement between the final phase image and the object phase, and demonstrate resistance to imaging noise.     

Aberration-corrected microscopy for structural biology applications

This study explores the potential of a C _s-corrected transmission electron microscope for structural studies of biological samples, in particular isolated macromolecular complexes. A 300-kV transmission electron microscope, equipped with a C _s corrector was employed to record sets of images at different defocus and C _s settings. The experiments were designed to determine whether imaging with large defocus benefits from C _s correction. Defocus contrast in biological imaging has a stronger influence on image resolution than any other parameter. We find the results are in good agreement with theoretical framework, verifying that the typical imaging conditions required for biological investigations are not affected by C _s correction.     

High-resolution electron microscopy and its applications

A review of research on high-resolution electron microscopy (HREM) carried out at the Institute of Physics, the Chinese Academy of Sciences, is presented. Apart from the direct observation of crystal and quasicrystal defects for some alloys, oxides, minerals, etc., and the structure determination for some minute crystals, an approximate image-contrast theory named pseudo-weak-phase object approximation (PWPOA), which shows the image contrast change with crystal thickness, is described. Within the framework of PWPOA, the image contrast of lithium ions in the crystal of R-Li₂Ti₃O₇ has been observed. The usefulness of diffraction analysis techniques such as the direct method and Patterson method in HREM is discussed. Image deconvolution and resolution enhancement for weak-phase objects by use of the direct method are illustrated. In addition, preliminary results of image restoration for thick crystals are given.     

Optical interference artifacts in contact atomic force microscopy images

Atomic force microscopy images are usually affected by different kinds of artifacts due to either the microscope design and operation mode or external environmental factors. Optical interferences between the laser light reflected off the top of the cantilever and the light scattered by the surface in the same direction is one of the most frequent sources of height artifact in contact (and occasionally non-contact) images. They are present when imaging highly reflective surfaces, or even when imaging non-reflective materials deposited onto reflective ones. In this study interference patterns have been obtained with a highly polished stainless steel planchet. The influence of these artifacts in surface roughness measurements is discussed, and a semi-quantitative method based on the fast Fourier transform technique is proposed to remove the artifacts from the images. This method improves the results obtained by applying the usual flattening routines.     

Combined atomic force and scanning reflection interference contrast microscopy

A sphere attached to a cantilever is used simultaneously as an atomic force microscope (AFM) tip and as a curved reflective surface for producing scanning reflection interference contrast microscope (RICM) images of fluorescent beads dried onto a glass slide. The AFM and RICM images are acquired in direct registration which enables the identification of individually excited beads in the AFM images. The addition of a sharp, electron beam-deposited tip to the sphere gives nanometer resolution AFM images without loss of optical contrast.     

微分相衬干涉显微镜定量测量表面形貌

改进了用于定量测量样品表面形貌的微分相衬干涉显微镜系统,对系统中由Nomarski棱镜引起的相位差β的消零工作提出了一种新的方法,实验证明是有效可靠的,并且对样品表面三维形貌重构、截面轮廓比对及系统的测量精度进行了实验研究.     

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.     

Surface reflection interference microscopy:* A new method for visualizing cytoskeletal components by light microscopy

Surface reflection interference microscopy of detergent resistant residues of cultured cells stained with protein dyes can be used to obtain high resolution images of the cytoskeleton. We have compared the images obtained using different dyes and have examined the effect of four of these dyes on the visualization of different parts of the cytoskeleton in detail. The dependence of contrast of the images obtained on the illuminating numerical aperture and the wavelength of incident light was determined. Staining with Acid Yellow 36, Guinea Green B and Naphtol Blue Black produces images from the entire cytoskeleton and contrast in these images is relatively insensitive to changes in the incident wavelength. Coomassie Brilliant Blue R250 images, on the other hand, result primarily from reflection from the lower surface of the cytoskeleton and the contrast of these images is sensitive to changes in incident wavelength dropping abruptly in the region of the transmission peak of the stain. From the different spectral sensitivities of the reflection images obtained and from differential interference effects at low and high illuminating numerical apertures, we conclude that the reflection images obtained using the first three stains result from modulation of the reflection by interference effects. In contrast, in the case of Coomassie Brilliant Blue R250 the resulting image originates mainly from selective reflection of wavelength near the absorption range of the dye.     

激光共焦扫描显微镜及其应用

介绍了共焦激光显微镜的基本光路、成像原理、关键技术及应用。