Re-evaluation of differential phase contrast (DPC) in a scanning laser microscope using a split detector as an alternative to differential interference contrast (DIC) optics

In this paper, differential phase imaging (DPC) with transmitted light is implemented by adding a suitable detection system to a standard commercially available scanning confocal microscope. DPC, a long‐established method in scanning optical microscopy, depends on detecting the intensity difference between opposite halves or quadrants of a split photodiode detector placed in an aperture plane. Here, DPC is compared with scanned differential interference contrast (DIC) using a variety of biological specimens and objective lenses of high numerical aperture. While DPC and DIC images are generally similar, DPC seems to have a greater depth of field. DPC has several advantages over DIC. These include low cost (no polarizing or strain‐free optics are required), absence of a double scanning spot, electronically variable direction of shading and the ability to image specimens in plastic dishes where birefringence prevents the use of DIC. DPC is also here found to need 20 times less laser power at the specimen than DIC.     

Laser scanning gradient index optics microscope: submicrometre scale spectroscopy and imaging at cryogenic temperatures

A laser scanning far-field optical microscope for low-temperature imaging and spectroscopy based on gradient index optics is presented. A rod-shaped gradient index microlens is used as a zero-working-distance solid immersion objective lens. The obtained lateral resolution is 310 nm of the FWHM at a wavelength of 545 nm. A laser scanning mechanism located outside an optical cryostat enables one to achieve large scanning ranges independent of temperature. The use of the microscope for submicrometre-scale spectroscopy and low-temperature photochemistry performed on molecular J aggregates in thin polymer films is presented.     

Image sharpness and contrast transfer in coherent confocal microscopy

Confocal microscopes provide clear, thin optical sections with little disturbance from regions of the specimen that are not in focus. In addition, they appear to provide somewhat greater lateral and axial image resolution than with non-confocal microscope optics. To address the question of resolution and contrast transfer of light microscopes, a new test slide that enables the direct measurement of the contrast transfer characteristics (CTC) of microscope optics at the highest numerical aperature has been developed. With this new test slide, the performance of a confocal scanning laser microscope operating in the confocal reflection mode and the non-confocal transmission mode was examined. The CTC curves show that the confocal instrument maintains exceptionally high contrast (up to twice that with non-confocal optics) as the dimension of the object approaches the diffraction limit of resolution; at these dimensions, image detail is lost with non-confocal microscopes owing to a progressive loss of image contrast. Furthermore, we have calculated theoretical CTC curves by modelling the confocal and non-confocal imaging modes using discrete Fourier analysis. The close agreement between the theoretical and experimental CTC curves supports the earlier prediction that the coherent confocal and the incoherent non-confocal imaging mode have the same limit of resolution (defined here as the inverse of the spatial frequency at which the contrast transfer converges to zero). The apparently greater image resolution of the coherent confocal optics is a consequence of the improved contrast transfer at spacings which are close to the resolution limit.     

Laser scanning gradient index optics microscope: submicrometre scale spectroscopy and imaging at cryogenic temperatures

A laser scanning far-field optical microscope for low-temperature imaging and spectroscopy based on gradient index optics is presented. A rod-shaped gradient index microlens is used as a zero-working-distance solid immersion objective lens. The obtained lateral resolution is 310 nm of the FWHM at a wavelength of 545 nm. A laser scanning mechanism located outside an optical cryostat enables one to achieve large scanning ranges independent of temperature. The use of the microscope for submicrometre-scale spectroscopy and low-temperature photochemistry performed on molecular J aggregates in thin polymer films is presented.     

Three-dimensional transport imaging for the spatially resolved determination of carrier diffusion length in bulk materials

A contact-free optical technique is developed to enable a spatially resolved measurement of minority carrier diffusion length and the associated mobility-lifetime (μτ) product in bulk semiconductor materials. A scanning electron microscope is used in combination with an internal optical microscope and imaging charge-coupled device (CCD) to image the bulk luminescence from minority carrier recombination associated with one-dimensional excess carrier generation. Using a Green's function to model steady-state minority carrier diffusion in a three-dimensional half space, non-linear least squares analysis is then applied to extract values of carrier diffusion length and surface recombination velocity. The approach enables measurement of spatial variations in the μτ product with a high degree of spatial resolution.     

Beam characteristics for various sizes of annular aperture on scanning electron microscope.

Using an analogy between light optics and electron optics, we have calculated beam characteristics such as the beam profile and the optical transfer function for several sizes of annular and circular apertures on a scanning electron microscope (SEM). It has been found that an annular aperture improves the image quality with regard to several kinds of image resolution and the depth of focus at the price of good low-frequency (nu) contrast. In contrast with conventional circular-aperture SEM images, a combination of a low-nu-pass filtered, circular-aperture SEM image with a high-nu-pass filtered, annular-aperture SEM image has the potential to enhance the image quality in terms of both the image resolution and the depth of focus.     

Three‐dimensional visualization of rat retina by X‐ray differential phase contrast tomographic microscopy

The retina is one of the most tiny and sophisticated tissues of the body. Three dimensional (3D) visualization of the whole retina is valuable both in clinical and research arenas. The tissue has been predominantly assessed by time‐consuming histopathology and optical coherence tomography (OCT) in research and clinical arenas. However, none of the two methods can provide 3D imaging of the retina. The purpose of this study is to give a volumetric visualization of rat retina at submicron resolution, using an emerging imaging technique‐phase‐contrast X‐ray CT. A Sprague‐Dawley (SD) rat eye specimen was scanned with X‐ray differential phase contrast tomographic microscopy (DPC‐microCT) equipped at the Swiss Light Source synchrotron. After scanning, the specimen was subjected to routine histology procedures and severed as a reference. The morphological characteristics and signal features of the retina in the DPC‐microCT images were evaluated. The total retina and its sublayers thicknesses were measured on the DPC‐microCT images and compared with those obtained from the histological sections. The retina structures revealed by DPC‐microCT were highly consistent with the histological section. In this study, we achieved nondestructive 3D visualization of SD rat retina. In addition to detailed anatomical structures, the objective parameters provided by DPC‐microCT make it a useful tool for retinal research and disease diagnosis in the early stage.     

Contrast in confocal scanning microscopy with a finite detector

The optical properties of a general scanning microscope are determined within the framework of Fourier imaging theory. For a simple model optical system, with Gaussian lens and detector apertures, the contrast transfer function can be expressed in terms of elementary functions. The theory predicts that spatial resolution and depth discrimination vary continuously with detector aperture and that defocus phase contrast is present in transmission images obtained with a symmetric objective, collector lens confocal microscope.     

An in-vacuum x-ray diffraction microscope for use in the 0.7-2.9 keV range

A dedicated in-vacuum coherent x-ray diffraction microscope was installed at the 2-ID-B beamline of the Advanced Photon Source for use with 0.7-2.9 keV x-rays. The instrument can accommodate three common implementations of diffractive imaging; plane wave illumination; defocused-probe (Fresnel diffractive imaging) and scanning (ptychography) using either a pinhole, focused or defocused probe. The microscope design includes active feedback to limit motion of the optics with respect to the sample. Upper bounds on the relative optics-to-sample displacement have been measured to be 5.8 nm(v) and 4.4 nm(h) rms/h using capacitance micrometry and 27 nm/h using x-ray point projection imaging. The stability of the measurement platform and in-vacuum operation allows for long exposure times, high signal-to-noise and large dynamic range two-dimensional intensity measurements to be acquired. Finally, we illustrate the microscope's stability with a recent experimental result.     

Real‐time three‐dimensional imaging of cell division by differential interference contrast microscopy

Differential interference contrast (DIC) microscopy can provide information about subcellular components and organelles inside living cells. Applicability to date, however, has been limited to 2D imaging. Unfortunately, understanding of cellular dynamics is difficult to extract from these single optical sections. We demonstrate here that 3D differential interference contrast microscopy has sub‐diffraction limit resolution both laterally and vertically, and can be used for following Madin Darby canine kidney cell division process in real time. This is made possible by optimization of the microscope optics and by incorporating computer‐controlled vertical scanning of the microscope stage.     

A low cost image and data acquisition system for use with the Vickers M85 scanning microdensitometer

A simple and inexpensive interface has been constructed between the Vickers M85 microdensitometer and a BBC model B microcomputer. The interface incorporates three sensitivity ranges and enables the production of pseudocolour images of the specimen using the two-dimensional scanning mode of the M85. The operator can select a 160times256 pixel image with eight colours or a 320times256 display using only four colours. Each colour represents a defined range of transmittance which is software controlled. The image histogram can be displayed and the interval between colours redefined so as to enable contrast stretching. Intervals between colours can be either linear or logarithmic and the images thus obtained can be stored on disc or videotape, or a hard copy can be obtained using a screen dump routine. Two-dimensional absorption images can thus be obtained at any single wavelength from 400 to 700 nm at normal magnifications of the light microscope. In addition, the system can be used to acquire, store and process data from one-dimensional scans to obtain quantitative information about variations in optical density within the specimen, so considerably increasing the usefulness of the instrument. Although obviously limited in its capabilities, the system produces images of very high quality and one-dimensional data of high sensitivity. The interface can be constructed for less than £40. A small modification to one of the M85 circuit boards is necessary to obtain maximum resolution.     

Development of x-ray laminography under an x-ray microscopic condition

An x-ray laminography system under an x-ray microscopic condition was developed to obtain a three-dimensional structure of laterally-extended planar objects which were difficult to observe by x-ray tomography. An x-ray laminography technique was introduced to an x-ray transmission microscope with zone plate optics. Three prototype sample holders were evaluated for x-ray imaging laminography. Layered copper grid sheets were imaged as a laminated sample. Diatomite powder on a silicon nitride membrane was measured to confirm the applicability of this method to non-planar micro-specimens placed on the membrane. The three-dimensional information of diatom shells on the membrane was obtained at a spatial resolution of sub-micron. Images of biological cells on the membrane were also obtained by using a Zernike phase contrast technique.     

A simple,combined diffractometer and scanning microdensitometer

A simple device for densitometering electron micrographs or other transparencies by one-dimensional scanning is described. Micrographs of crystalline materials are oriented by means of their optical diffraction patterns displayed with a slightly modified microscope.     

Near-field scanning optical microscopy and near-field induced photocurrent investigations of buried heterostructure multiquantum well lasers

Buried heterostructure multiquantum well laser devices are investigated utilizing a near-field scanning optical microscope to characterize and correlate the surface topography, optical output and electronic properties of the device. Near-field photocurrent imaging has been used to accurately measure the unbiased buried heterostructure multiquantum well device in cross-section, successfully revealing the distribution of pn-junctions and their associated fields. Moreover, this has been accurately correlated with the physical structure of the device determined by simultaneous shear-force imaging of the surface. Topographic structure is manifested as a result of strain relaxation (∼10⁻¹⁰ m) of the cleaved cross-section. These imaging modes are similarly correlated with the optical output of the operational device mapped with 50 nm lateral resolution. The collection-mode measurements detected electroluminescence external to the active region, highlighting the existence of carrier recombination away from the multiquantum well device region. The combination and correlation of different near-field scanning optical microscope imaging modes proved powerful in the analysis of the buried heterostructure multiquantum well device, and was shown to assist in the identification of current leakage pathways within the structure.     

Three‐dimensional optical transfer functions in the aberration‐corrected scanning transmission electron microscope

In the scanning transmission electron microscope, hardware aberration correctors can now correct for the positive spherical aberration of round electron lenses. These correctors make use of nonround optics such as hexapoles or octupoles, leading to the limiting aberrations often being of a nonround type. Here we explore the effect of a number of potential limiting aberrations on the imaging performance of the scanning transmission electron microscope through their resulting optical transfer functions. In particular, the response of the optical transfer function to changes in defocus are examined, given that this is the final aberration to be tuned just before image acquisition. The resulting three‐dimensional optical transfer functions also allow an assessment of the performance of a system for focal‐series experiments or optical sectioning applications.     

Image contrast of dielectric specimens in transmission mode near-field scanning optical microscopy: imaging properties and tip artefacts

Near-field scanning optical microscopy (NSOM) is a scanned probe technique utilizing a subwavelength-sized light source for high-resolution imaging of surfaces. Although NSOM has the potential to exploit and extend the experimental utility of the modern light microscope, the interpretation of image contrast is not straightforward. In near-field microscopy the illumination intensity of the source (probe) is not a constant value, rather it is a function of the probe–sample electronic environment. A number of dielectric specimens have been studied by NSOM to elucidate the contrast role of specimen type, topography and crystallinity; a summary of metallic specimen observations is presented for comparative purposes. Near-field image contrast is found to be a result of lateral changes in optical density and edge scattering for specimens with little sample topography. For surfaces with considerable topography the contributions of topographic (Z) axis contrast to lateral (X,Y) changes in optical density have been characterized. Selected near-field probes have also been shown to exhibit a variety of unusual contrast artefacts. Thorough study of polarization contrast, optical edge (scattering) contrast, as well as molecular orientation in crystalline specimens, can be used to distinguish lateral contrast from topographic components. In a few cases Fourier filtering can be successfully applied to separate the topographic and lateral contrast components.     

A high stability beam-scanning confocal optical microscope for low temperature operation

We report on the design and performance of a high stability scanning confocal microscope for optical microscopy at low temperatures. By scanning the beam in a cold objective lens system, we achieve wide fields of view without compromising image quality. Photoluminescence from single nitrogen-vacancy centers in high purity diamond is used to illustrate the imaging and stability performance of the microscope.     

激光共焦高速扫描显微成像的高帧速重构算法

针对激光共焦扫描显微镜的往复式逐行扫描成像方式带来的帧图像数据分割难的问题,在分析系统扫描方式、振镜的实际运动方式与理论运动方式差异的基础上,利用相邻两帧图像相似性大的特点,提出了一套完整的高帧速重构算法。该算法通过连续帧特征区域差分的方式实现了一维信号序列的自适应分割,即实现了对一维信号序列进行动态排列及分割成二维阵列图像数据,从而重构出多帧高精度图像。实验表明,该算法的成像误差低于1.6%,适用于成像速度高达300帧/s的激光共焦扫描显微成像。     

Scanning microphotolysis: A new photobleaching technique based on fast intensity modulation of a scanned laser beam and confocal imaging

The fluorescence photobleaching method has been widely used to study molecular transport in single living cells and other microsystems while confocal microscopy has opened new avenues to high-resolution, three-dimensional imaging. A new technique, scanning microphotolysis (Scamp), combines the potential of photobleaching, beam scanning and confocal imaging. A confocal scanning laser microscope was equipped with a sufficiently powerful laser and a novel device, the ‘Scamper’. This consisted essentially of a filter changer, an acousto-optical modulator (AOM) and a computer. The computer was programmed to activate the AOM during scanning according to a freely defined image mask. As a result almost any desired pattern could be bleached (‘written’) into fluorescent samples at high definition and then imaged (‘read’) at non-bleaching conditions, employing full confocal resolution. Furthermore, molecular transport could be followed by imaging the dissipation of bleach patterns. Experiments with living cells concerning dynamic processes in cytoskeletal filaments and the lateral mobility of membrane lipids suggest a wide range of potential biological applications. Thus, Scamp offers new possibilities for the optical manipulation and analysis of both technical and biological microsystems.     

Quantitative DNA measurements in an instrument combining scanning electron microscopy and light microscopy

An instrument for combined scanning electron microscopy (SEM) and light microscopy (LM) to which a photometer unit is attached is described. A special stage in the vacuum chamber of a scanning electron microscope incorporates light microscope optics (objective and condenser) designed for transmission and epi-illumination fluorescence LM. An optical bridge connects these optics to a light microscope, without objective and condenser. The possibility of performing quantitative DNA measurements in this combined microscope (the LM/SEM) was tested using preparations of either chicken erythrocytes, human lymphocytes, or mouse liver cells. The cells were fixed, brought on a cover-glass, quantitatively stained for DNA, dehydrated, and critical point dried (CPD). After mounting the cells were coated with gold. The specimens were brought into the vacuum chamber of the combined microscope and individual cells were studied with SEM and LM. Simultaneously DNA measurements were performed by means of the photometer unit attached to the microscope. It is shown in this study that DNA measurements of cells in the combined microscope give similar results when compared to DNA measurements of embedded cells performed with a conventional fluorescence microscope. Furthermore, it is shown that although the gold layer covering the LM/SEM specimens weakens the fluorescence signal, it does not interfere with the DNA measurements.