传统光学显微镜与近场光学显微镜

近场光学显微镜是对于常规光学显微镜的革命。它不用光学透镜成像 ,而用探针的针尖在样品表面上方扫描获得样品表面的信息。分析了传统光学显微镜与近场光学显微镜成像原理的物理本质和两种显微镜系统结构的异同点。介绍了光纤探针的制作方法。重点讨论了近场探测原理、光学隧道效应及非辐射场的性质     

Comparison of holotomographic microscopy and coherence-controlled holographic microscopy

Quantitative phase imaging (QPI) is a powerful tool for label-free visualisation of living cells. Here, we compare two QPI microscopes – the Telight Q-Phase microscope and the Nanolive 3D Cell Explorer-fluo microscope. Both systems provide unbiased information about cell morphology, such as individual cell dry mass, perimeter and area. The Q-Phase microscope uses artefact-free, coherence-controlled holographic imaging technology to visualise cells in real time with minimal phototoxicity. The 3D Cell Explorer-fluo employs laser-based holotomography to reconstruct 3D images of living cells, visualising their internal structures and dynamics. Here, we analysed the strengths and limitations of both microscopes when examining two morphologically distinct cell lines – the cuboidal epithelial MDCK cells which form multicellular clusters and solitary growing Rat2 fibroblasts. We focus mainly on the ability of the devices to generate images suitable for single-cell segmentation by the built-in software, and we discuss the segmentation results and quantitative data generated from the segmented images. We show that both microscopes offer slightly different advantages, and the choice between them depends on the specific requirements and goals of the user.     

Emission microscopy and related techniques: resolution in photoelectron microscopy, low energy electron microscopy and mirror electron microscopy.

A unified treatment of the resolution of three closely related techniques is presented: emission electron microscopy (particularly photoelectron microscopy, PEM), low energy electron microscopy (LEEM), and mirror electron microscopy (MEM). The resolution calculation is based on the intensity distribution in the image plane for an object of finite size rather than for a point source. The calculations take into account the spherical and chromatic aberrations of the accelerating field and of the objective lens. Intensity distributions for a range of energies in the electron beam are obtained by adding the single-energy distributions weighted according to the energy distribution function. The diffraction error is taken into account separately. A working resolution is calculated that includes the practical requirement for a finite exposure time, and hence a finite non-zero current in the image. The expressions for the aberration coefficients are the same in PEM and LEEM. The calculated aberrations in MEM are somewhat smaller than for PEM and LEEM. The resolution of PEM is calculated to be about 50 A, assuming conventional UV excitation sources, which provide current densities at the specimen of 5 x 10(-5) A/cm2 and emission energies ranging up to 0.5 eV. A resolution of about 70 A has been demonstrated experimentally. The emission current density at the specimen is higher in LEEM and MEM because an electron gun is used in place of a UV source. For a current density of 5 x 10(-4) A/cm2 and the same electron optical parameters as for PEM, the resolution is calculated to be 27 A for LEEM and 21 A for MEM.     

Comparative light microscopy,scanning electron microscopy and transmission electron microscopy of selected organic walled microfossils

Two simple techniques are described and illustrated. The first is for the study of one specimen by both light microscopy (LM) and scanning electron microscopy (SEM). The second is for the study of one selected specimen by LM, SEM and in ultrathin section by transmission electron microscopy (TEM). Although these techniques were developed for the comparative study of Precambrian organic walled microfossils (OWMs), they could be used for a wide range of other specimens.     

Quantitative phase-amplitude microscopy I: optical microscopy

In this paper, the application of a new optical microscopy method (quantitative phase‐amplitude microscopy) to biological imaging is explored, and the issue of resolution and image quality is examined. The paper begins by presenting a theoretical analysis of the method using the optical transfer function formalism of Streibl (1985 ). The effect of coherence on the formation of the phase image is explored, and it is shown that the resolution of the method is not compromised over that of a conventional bright‐field image. It is shown that the signal‐to‐noise ratio of the phase recovery, however, does depend on the degree of coherence in the illumination. Streibl (1985) notes that partially coherent image formation is a non‐linear process because of the intermingling of amplitude and phase information. The work presented here shows that the quantitative phase‐amplitude microscopy method acts to linearize the image formation process, and that the phase and amplitude information is properly described using a transfer function analysis. The theoretical conclusions are tested experimentally using an optical microscope and the theoretical deductions are confirmed. Samples for microscopy influence both the phase and amplitude of the light wave and it is demonstrated that the new phase recovery method can separate the amplitude and phase information, something not possible using traditional phase microscopy. In the case of a coherent wave, knowledge of the phase and amplitude constitutes complete information that can be used to emulate other forms of microscopy. This capacity is demonstrated by recovering the phase of a sample and using the data to emulate a differential interference contrast image.     

Advanced cantilevers for magnetic force microscopy and high frequency magnetic force microscopy

In order to improve the spatial resolution achieved by magnetic force microscopy (MFM) technique and its derivatives, we employ here advanced MFM tips fabricated by means of focused ion beam (FIB) milling. The magnetic coating applied on these tips is a CoCr film of 10 nm thickness. The MFM measurements on hard disk test samples reveal the achieved high resolution, and the measurement on a garnet film demonstrates the low invasiveness. High-frequency MFM (HF-MFM) is a development of the MFM technique to observe the HF stray fields emerging from magnetic recording writer poles at their operating conditions. By means of HF-MFM, magnetic recording writer poles are characterized in the frequency range 100-1,000 MHz. Up to now, all HF-MFM experiments conducted were using standard MFM cantilevers. From the HF-MFM images obtained using the advanced MFM cantilevers, it is clearly seen that the spatial resolution is considerably improved over the images obtained using standard MFM tips. However, the 10 nm thick magnetic coating of the cantilevers is found to work properly only at frequencies of up to about 500 MHz.     

Expansion microscopy

Super‐resolution optical microscopy techniques have revolutionized how we see and understand biology. In recent past, a new super‐resolution optical microscopy technique called expansion microscopy (ExM) was developed. Unlike other pre‐existing super‐resolution imaging techniques, this technique achieves super‐resolution by physically expanding biological specimens via a swellable hydrogel. After the development of ExM, various techniques based on ExM but with improved performance in various aspects, have been developed. In this review, we introduce the basic principles of ExM and its variants. and compare the advantages and disadvantages of these techniques. In addition, we present the applications of ExM techniques in various fields.     

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.     

Desktop X-ray microscopy and microtomography

Recent developments in X-ray microtomography have made it possible to miniaturize a CT scanner into a versatile and cost-effective desktop system that fits into any laboratory environment. The possibilities of the technique are demonstrated for a range of applications. It is also shown how an existing scanning electron microscope with an X-ray detector can, with a specially developed attachment, be transformed into an X-ray microscope and microtomograph.     

Triumphs and conflicts in microscopy

The controversies surrounding the Abbe theory and its final acceptance are discussed. The practical problems of lens design and manufacture, the rise and fall of the ultraviolet microscope and reflecting microscope and the development of fluorescence microscopy are briefly surveyed. In particular, the conflicts which arose between Fellows of singular or opposing views—E. M. Nelson, A. E. Conrady, J. W. Gordon—are presented as illustrative of the failure to recognize the nature of the point at issue.     

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.     

Confocal-line microscopy

The confocal-line (CL) technique combines some of the characteristics of confocal-scanning microscopy with those of conventional-imaging methods. It is based on the introduction of line-shaped illumination and linear image detection, as an alternative to the current confocal-point (CP) approach. Although confocal only in one dimension, the proposed solution offers performance and features adequate to a variety of biological and non-biological applications and is also adaptable to an increased number of microscopical observations and measurements. The absence of moving components in the optical path and the use of electronic linear imagers permits flexible and fast operation that appears particularly relevant in many fields of basic and applied research. For instance, transmission, reflection and emission images can simultaneously be collected from the same area of the specimen, with slight adjustments to the optical setup. Useful extensions of CL microscopy to the field of spectral imaging are obtained with the introduction of a slit, a polychromator and an area detector, substituting for the linear imager. Prototype instrumentation has been constructed working from the cited principles and some tests have been performed on selected applications.     

Characterization of an oil-degrading Microcoleus consortium by means of confocal scanning microscopy, scanning electron microscopy and transmission electron microscopy

A consortium of microorganisms with the capacity to degrade crude oil has been characterized by means of confocal laser scanning microscopy (CLSM), transmission electron microscopy (TEM), and scanning electron microscopy (SEM). The analysis using CLSM shows that Microcoleus chthonoplastes is the dominant organism in the consortium. This cyanobacterium forms long filaments that group together in bundles inside a mucopolysaccharide sheath. Scanning electron microscopy and transmission electron microscopy have allowed us to demonstrate that this cyanobacterium forms a consortium primarily with three morphotypes of the heterotrophic microorganisms found in the Microcoleus chthonoplastes sheath. The optimal growth of Microcoleus consortium was obtained in presence of light and crude oil, and under anaerobic conditions. When grown in agar plate, only one type of colony (green and filamentous) was observed.     

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.     

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.     

Fluorescence microscopy and electron microscopy of adrenergic nerves after constriction at two points

Guinea pig and cat adrenergic nerves have been constricted at two points and the effects studied with the fluorescence method for the localization of noradrenaline and with the electron microscope. After operation there was a rapid accumulation of fluorescent material proximal to the constrictions. This was greater above the proximal than the distal lesion and persisted for several days. The accumulation above the distal constriction disappeared between 24–48 hours after operation. Any accumulation of fluorescent material distal to the constrictions could almost invariably be correlated with the presence of ganglion cells situated more peripherally, or with an inadequate constriction of the nerves. Electron microscopy showed an accumulation of vesicles with an electron-dense core, believed to carry noradrenaline, proximal to the constrictions. This corresponded closely to the accumulation of fluorescent material. The results suggested that the passage of noradrenaline in these nerves is from the nerve-cell body to the periphery. There was no unequivocal evidence of a retrograde flow of the transmitter.     

Cryo-fluorescence microscopy facilitates correlations between light and cryo-electron microscopy and reduces the rate of photobleaching

Fluorescence light microscopy (LM) has many advantages for the study of cell organization. Specimen preparation is easy and relatively inexpensive, and the use of appropriate tags gives scientists the ability to visualize specific proteins of interest. LM is, however, limited in resolution, so when one is interested in ultrastructure, one must turn to electron microscopy (EM), even though this method presents problems of its own. The biggest difficulty with cellular EM is its limited utility in localizing macromolecules of interest while retaining good structural preservation. We have built a cryo-light microscope stage that allows us to generate LM images of vitreous samples prepared for cryo-EM. Correlative LM and EM allows one to find areas of particular interest by using fluorescent proteins or vital dyes as markers within vitrified samples. Once located, the sample can be placed in the EM for further study at higher resolution. An additional benefit of the cryo-LM stage is that photobleaching is slower at cryogenic temperatures (−140°C) than at room temperature.