Microscope densitometer system for point measurement of autoradiograms.

A photometric system for measuring optical densities generated by tissue autoradiograms has been adapted to an ordinary light microscope. The optics of the microscope are used to direct the light transmitted through the autoradiogram to ocular-mounted photoconductive cells coupled to a bridge amplifier. Readout is on a digital panel meter. The integrated area analyzed varies between 33.16 to 0.02 0.02 mm2 depending on the objective magnification. The system is linear over a range of optical densities from 0.5 to 0.05.     

Confocal scanning optical microscopy and three-dimensional imaging

Summary— Confocal scanning optical microscopy has significant advantages over conventional fluorescence microscopy: it rejects the out-of-locus light and provides a greater resolution than the wide-field microscope. In laser scanning optical microscopy, the specimen is scanned by a diffraction-limited spot of laser light and the fluorescence emission (or the reflected light) is focused onto a photodetector. The imaged point is then digitized, stored into the memory of a computer and displayed at the appropriate spatial position on a graphic device as a part of a two-dimensional image. Thus, confocal scanning optical microscopy allows accurate non-invasive optical sectioning and further three-dimensional reconstruction of biological specimens. Here we review the recent technological aspects of the principles and uses of the confocal microscope, and we introduce the different methods of three-dimensional imaging.     

Optical sectioning microscopy

Confocal scanning microscopy, a form of optical sectioning microscopy, has radically transformed optical imaging in biology. These devices provide a powerful means to eliminate from images the background caused by out-of-focus light and scatter. Confocal techniques can also improve the resolution of a light microscope image beyond what is achievable with widefield fluorescence microscopy. The quality of the images obtained, however, depends on the user's familiarity with the optical and fluorescence concepts that underlie this approach. We describe the core concepts of confocal microscopes and important variables that adversely affect confocal images. We also discuss data-processing methods for confocal microscopy and computational optical sectioning techniques that can perform optical sectioning without a confocal microscope.     

The optical near-field and cell biology

A new form of scanning light microscopy is described in which the lens is replaced by a point of light that is smaller than the wavelength. Resolution is obtained that is defined not by the wavelength but by the size of the spot of light. This is the case so long as the point of light is within the dimension of a wavelength from the surface that is to imaged or within the optical near-field. This new form of light microscopy is called near-field scanning optical microscopy (NSOM). Resolutions are being obtained with NSOM that are similar to scanning electron microscopy but without the destructive effects of a vacuum or of an electron beam. In addition such a microscope is readily interfaced with fluorescent and non-fluorescent contrast enhancing stains that are commonly used in cell biology. The possibility of a near-field/far-field microscope is discussed with overlapping resolutions from a few hundred of a conventional microscope to the tens of thousand that can be obtained with NSOM.     

Photometric methods in quantitation of light microscope radioautography

Photometric evaluation of autoradiographic grain densities can be performed according to various optical principles. In all instances the amount of light recorded should be a measure of the radioactive substance amount in the specimens. It is shown that grain densities suitable for light microscopic autoradiography are indeed linearly related to radiation exposure. Dependent on the photometric system used, there is a larger or smaller section of linear correlation of the photometric response with grain density. The advantages of incident light bright-field illumination for silver grain counting are discussed. Quantitation of substance amounts in autoradiographs depends on the use of radioactive standard sources. Two different approaches of quantitation are discussed. In 14C-autoradiography which can be applied for cell-kinetic studies, standard plates are used consisting of 14C-polymethylmethacrylate. Allowance has to be made for the different condition of radiation geometry of the cells and the standards. In 125I-autoradiography, 125I-labeled red cells are used as standards. This technique allows for quantitating the number of antibodies bound to individual cell surfaces.     

Polarization and Filter Properties Investigation of Metal Gratings and Rings

We demonstrated the near-field optical transmission properties of nanogratings with spoke and rings structures through a near-field scanning optical microscope, and the far-field optical transmission properties with different polarization angles are investigated with an optical microscope. Our experimental results verified the polarization properties of the nanograting structures and further demonstrated the experimental results are supported by the finite difference time domain theoretical simulation. The optical microscope imaging of the spoke and ring structures also show that the grating structures can disperse visible light of different wavelengths.     

Quantitative studies on the polarization optical properties of living cells. I. Microphotometric birefringence detection system

A method of polarization optical analysis is described in which phase retardation attributable to birefringence of a minute area in a microscopic object is determined. The optical system consists of a polarizing microscope with "rectified" strain-free lenses, a photoelectric detector to determine the intensity of the light passing through a minute window located at the image plane of the specimen, and a stage that moves the specimen at appropriate velocities for scanning. The error resulting from any flare of light emerging from outside of the area to be measured is minimized by limiting the illuminated area. The specimen can be observed during the measurement of light intensity by illuminating the whole microscope field at a wavelength different from that of the light used for the measurement. The retardation of the specimen is determined by comparing the specimen and background intensities as functions of the azimuth of a Brace-Koherl compensator. Alternatively, retardation is obtained directly from the light intensity at a fixed compensator angle, using the theory of polarization optics. The basal noise level for the present apparatus is approximately 0.03 nm when measuring birefringence of a 4-micron2 area in 0.1 s, using a X 40, NA 0.65 objective. The noise decreases in inverse proportion to the square root of the area times the duration of measurement.     

Regio- and stereo-specific nitrile hydrolysis by the nitrile hydratase from Rhodococcus AJ270

Abstract Acid phosphatase activity was measured in individual cells by determining their optical densities through a scanning confocal laser microscope. The naphthol AS-TR (3-hydroxy-2-naphtoic acid 4'-chloro-2'-methylanilide) phosphate-hexazotized para-rosanilin method was used to visualise the acid phosphatase content in the light microscope. Evidence was obtained that the amount of enzyme varied in exponential growth phase cells as the fission age increased. By comparing the acid phosphatase activity with the rate of food vacuole formation, it appeared that the amount of enzyme inside the cells decreased in early clonal life, whereas the rate of food uptake increased. It was assumed that the reduction of acid phosphatase content could lead to a more extended life of vacuoles and to a decreased membrane recycling rate. In turn, the reduced supply of membrane available for food vacuole formation could partly be responsible for the decrease of the food uptake rate observed after the initial increase.     

Confocal microscopy via reciprocal optical fibre image detection

We describe a compact form of confocal scanning microscope using a semiconductor laser. Confocal operation is ensured by the use of a single mode optical fibre for both launching the light into the microscope and collecting the signal from the object. The collected light is allowed to re-enter the laser and the image is detected as a modulation on the signal from the laser power monitor diode. Images are compared with those obtained from traditional point detectors. The alignment tolerances of the reciprocal scheme are found to be greatly reduced over conventional confocal systems.     

The HOME microscope workstation. A new tool for cervical cancer screening.

The Highly Optimized Microscope Environment (HOME) is a computerized microscope design for assisting pathologists and cytotechnologists in routine clinical tasks. The prototype system consists of an IBM PC-compatible computer and a light microscope in which a built-in high-resolution computer display image is super-imposed on the optical image of the specimen. Also, an encoding stage and objective turret encoder are used to provide continuous monitoring of the stage coordinates and microscope magnification to the computer. This allows any position on the stage to be uniquely defined. Software, written in C language and running under the MS-DOS/MS-Windows environment, is controlled by means of a mouse-driven cursor. A specific application has been developed for cervical cancer screening, taking into account the needs and constraints of microscopists performing this task. Informatics tools offered by the HOME system provide them with precise flagging and relocation of objects on the slide, control of the scanning pathway, and ability to write and print the report directly through the microscope. The computer files generated by microscopic examination are stored and contain information available for quality control assessment and laboratory management.     

Plasmon resonant particles for biological detection

Several recent advances in the optical observation, fabrication, and bioconjugation of nanometer-sized gold or silver colloids have produced a robust new class of label. These plasmon resonant particle (PRP) conjugates have several important advantages: they are ultra-bright, so the light scattered from the individual particles can be viewed using a simple optical microscope system with a white light illumination source; they do not photo-bleach; PRPs can be prepared that preferentially scatter light of a chosen color; and it is possible to prepare bioconjugated PRPs that are stable in solution. These properties, and the automation of PRP identification, discrimination, and counting, have enabled the development of ultrasensitive, multicolor, and multiplex applications in the life science field.     

激光共聚焦显微镜与光学显微镜之比较

激光扫描共聚焦显微镜在活细胞的动态检测、光学切片和三维结构重建等方面较光学显微镜有质的飞跃。本文对激光扫描共聚焦显微镜和光学显微镜进行了比较和讨论,并简单介绍多光子激光扫描显微镜。     

虻复眼感杆光导的模式与适应状态的关系

利用逆向照明方法,不经角膜中和,通过光学显佩镜快速观察和记录了暗适应,短时间明适应和长时间明适应状态下,虻(Atylotus miser Szilady)复眼感杆光导的模式。同时与角膜中和的实验结果作了比较。本文还讨论了憎黄虻复眼角膜透镜的作用以及该复眼对视觉信憎黄息抽样方式与适应状态的关系。     

Dynamic light scattering microscopy. A novel optical technique to image submicroscopic motions. I: theory

The theoretical basis of an optical microscope technique to image dynamically scattered light fluctuation decay rates (dynamic light scattering microscopy) is developed. It is shown that relative motions between scattering centers even smaller than the optical resolution of the microscope are sufficient to produce significant phase variations resulting in interference intensity fluctuations in the image plane. The timescale and time dependence for the temporal autocorrelation function of these intensity fluctuations is derived. The spatial correlation distance, which reports the average distance between constructive and destructive interference in the image plane, is calculated and compared with the pixel size, and the distance dependence of the spatial correlation function is derived. The accompanying article in this issue describes an experimental implementation of dynamic light scattering microscopy.     

Techniques for quantitative analyses of calcareous marine phytoplankton

This paper discusses the techniques used to sample and analyse living marine calcareous phytoplankton. The various methods are described and tested within several research projects aimed at the determination of coccolithophore cell densities in seawater. In addition, the potential advantages and drawbacks associated with the application of light and scanning electron microscopic techniques to the quantitative analysis of coccolithophores are discussed. Several tests have been carried out in order to quantify potential errors related to: (1) homogeneity of material distribution on filter membranes; (2) use of different microscopes (scanning electron microscope versus light microscope); (3) use of different filter membranes (cellulose mixed-ester membranes versus polycarbonate membranes); and (4) Utermöhl settling versus filtration method. These tests revealed that major errors in cell density calculations could result from the uneven distribution of coccolithophore specimens on a filter membrane. The error resulting from the use of a light microscope arises from its low resolution, which restricts the identification of species, especially of small coccospheres. The use of different filter membranes does not show a statistically significant difference in cell density calculations, although polycarbonate membranes can be examined much more efficiently with the scanning electron microscopy than cellulose mixed-ester membranes. The Utermöhl method, however, gives lower cell densities consistently (several times) than the filtration method.     

Intensity of ultraviolet fluorescence of the cells depending on their size]

Using the light microscope, a correlation between ultra-violet fluorescence intensity of the cell and the cell area was established. This correlation does not depend on the large aperture of the microscope. A possible cause of this correlation is supposed to be either the proportionality between the mass of cytoplasmic protein and the squared cell radius, or a high optical density of cell elements.     

HOME: highly optimized microscope environment.

The Highly Optimized Microscope Environment (HOME) is a computerized microscope designed to assist pathologists and cytotechnicians in clinical routine tasks. The prototype system consists of a IBM-PC compatible computer and a light microscope in which a built-in high-resolution computer display image is superimposed on the optical image of the specimen. Also, a manually operated encoding stage and objective turret encoder are used to provide continuous monitoring of the stage coordinates and microscope magnification to the computer. This allows any position on a slide to be uniquely defined and makes it possible to measure interactively lengths and areas larger than the size of the microscope field. Software, written in the C language and operating under the MS-DOS/MS-Windows environment, is controlled by means of a mouse-driven cursor moving over menu light-buttons displayed on the microscope image. The HOME microscope workstation is potentially useful in a wide range of applications such as i) tagging information on particular cells and tissue structures that can thus be accurately located and relocated, ii) performing morphometric measurement, differential counting, and stereological assessment of biological specimens, and iii) training and educating laboratory personnel. Finally, HOME will offer in the near future a user-friendly interface for automatic image processing of cells and tissue entities in interactively selected specimen areas.     

How sensitive is the light microscope for observations on microorganisms in natural habitats?

Theoretical calculations based on the depth of field of standard microscope objectives and the visual acuity of normal observors show that direct microscopy of natural samples is rarely able to reveal the presence of small microorganisms at the densities found in natural systems. Over-estimation of the importance of bacterial aggregates is also likely from an uncritical use of light microscopy.     

Optical tweezers based force measurement system for quantitating binding interactions: system design and application for the study of bacterial adhesion

An optical force measurement system for quantitating forces in the pN range between micrometer-sized objects has been developed. The system was based upon optical tweezers in combination with a sensitive position detection system and constructed around an inverted microscope. A trapped particle in the focus of the high numerical aperture microscope-objective behaves like an omnidirectional mechanical spring in response to an external force. The particle's displacement from the equilibrium position is therefore a direct measure of the exerted force. A weak probe laser beam, focused directly below the trapping focus, was used for position detection of the trapped particle (a polystyrene bead). The bead and the condenser focus the light to a distinct spot in the far field, monitored by a position sensitive detector. Various calibration procedures were implemented in order to provide absolute force measurements. The system has been used to measure the binding forces between Escherichia coli bacterial adhesins and galabiose-functionalized beads.     

Monolithic Perovskite/Si Tandem Solar Cells: Pathways to Over 30% Efficiency

The article commences with a review focusing on three critical aspects of the perovskite/Si tandem technology: the evolution of efficiencies to date, comparisons of Si subcell choices, and the interconnection design strategies. Building on this review, a clear route is provided for minimizing optical losses aided by optical simulations of a recently reported high‐efficiency perovskite/Si tandem system, optimizations which result in tandem current densities of ≈20 mAcm^?2 with front‐side texture. The primary focus is on electrical modeling on the Si‐subcell, in order to understand the efficiency potential of this cell under filtered light in a tandem configuration. The possibility of increasing the Si subcell efficiency by 1% absolute is offered through joint improvements to the bulk lifetime, which exceeds 4 ms, and improves surface passivation quality to saturation current densities below 10 fA cm^?2. Polycrystalline‐Si/SiO_x passivating contacts are proposed as a promising alternative to partial‐area rear contacts, with the potential for further simplifying cell fabrication and improving device performance. A combination of optical modeling of the complete tandem structure alongside electrical modeling of the Si‐subcell, both with state‐of‐the‐art modeling tools, provides the first complete picture of the practical efficiency potential of perovskite/Si tandems.