Three-Dimensional imaging of fertilization and early development

The field of biological microscopy has recently enjoyed major technical advances, exemplified by the development of field-emission low-voltage scanning electron microscopes and laser scanning confocal light microscopes. In addition, computer processing of microscopical data is revolutionizing the way morphological information is imaged. In this paper, we illustrate methods by which this new technology can be used to examine events in fertilization and early development in three dimensions. Different types of specimen preparation protocols, using both echinoderm and mammalian gametes and embryos, are evaluated for their ability to preserve accurately the threedimensional organization of these specimens for imaging by both low-voltage scanning electron microscopy and laser scanning confocal light microscopy.     

Inexpensive, high-quality optical relay for use in confocal scanning beam imaging

An inexpensive, high optical-quality relay lens made up of two eyepieces arranged in an afocal assembly for use in confocal scanning laser imaging is described. In the past we have used relays, within our confocal microscopes, made up of achromats with long focal lengths (> or = 10 cm), which take up large optical tracks and suffer from significant amounts of astigmatism and curvature of field. We quantify aberrations associated with achromat and eyepiece relays using CODE V optical design and analysis software. The eyepiece relay is found to be more compact, better corrected, and not significantly more expensive than its achromat counterpart. In addition to being used to interconnect two scanning mirrors optically as well as scanning mirrors with microscope objectives, it can form part of the optics in a confocal scanning laser MACROscope-Microscope system (Biomedical Photometrics, Inc., Waterloo, Ontario, Canada). Due to design constraints, the MACROscope-Microscope system cannot incorporate a conventional wide-field microscope into its structure such as is done in most commercial confocal microscopes. The eyepiece relay is used as a stand-alone, compact optical link between the scanning mirrors and the microscope objective. This consequently makes the MACROscope-Microscope system more compact and easier to commercialize.     

Optimizing the performance of confocal point scanning laser microscopes over the full field of view

To examine many of the imaging capabilities of confocal scanning laser microscopes rapidly and reliably over the whole field of view three simple, easily prepared specimens are required: a mirror positioned on a carefully measured shallow gradient, a film of highly fluorescent material and a rectangular grid with a readily defined centre. Using these specimens the adjustment of any combination of confocal scanning laser visualization system and light microscope can be examined throughout the field of view. The effects of misalignment of the various subcomponents of a confocal scanning laser microscope on both the axial spread function of a plane and the shading pattern over the image field are described. Finally, where the design of the confocal optics permits, the three specimens can be used to facilitate the alignment of the various components to the optimal level achievable.     

The theory of the direct-view confocal microscope

A theory is presented which describes imaging in both conventional and scanning microscopes. This theory embraces conventional microscopes with partially coherent source and scanning microscopes with partially coherent effective source and detector, including confocal microscopes. The theory is applicable to the direct-view confocal microscope of Petrán?, the design of which is discussed. This microscope combines the resolution and depth discrimination improvements of confocal microscopy with the ease of operation of the conventional microscope.     

Use of a white light supercontinuum laser for confocal interference-reflection microscopy

Shortly after its development, the white light supercontinuum laser was applied to confocal scanning microscopy as a more versatile substitute for the multiple monochromatic lasers normally used for the excitation of fluorescence. This light source is now available coupled to commercial confocal fluorescence microscopes. We have evaluated a supercontinuum laser as a source for a different purpose: confocal interferometric imaging of living cells and artificial models by interference reflection. We used light in the range 460-700 nm where this source provides a reasonably flat spectrum, and obtained images free from fringe artefacts caused by the longer coherence length of conventional lasers. We have also obtained images of cytoskeletal detail that is difficult to see with a monochromatic laser.     

Fluorescence resonance energy transfer from cyan to yellow fluorescent protein detected by acceptor photobleaching using confocal microscopy and a single laser

One manifestation of fluorescence resonance energy transfer (FRET) is an increase in donor fluorescence after photobleaching the acceptor. Published acceptor‐photobleaching methods for FRET have mainly used wide‐field microscopy. A laser scanning confocal microscope enables faster and targeted bleaching within the field of view, thereby improving speed and accuracy. Here we demonstrate the approach with CFP and YFP, the most versatile fluorescent markers now available for FRET. CFP/YFP FRET imaging has been accomplished with a single laser (argon) available on virtually all laser‐scanning confocal microscopes. Accordingly, we also describe the conditions that we developed for dual imaging of CFP and YFP with the 458 and 514 argon lines. We detect FRET in a CFP/YFP fusion and also between signalling molecules (TNF‐Receptor‐Associated‐Factors or TRAFs) that are known to homo‐ and heterotrimerize. Importantly, we demonstrate that appropriate controls are essential to avoid false positives in FRET by acceptor photobleaching. We use two types of negative control: (a) an internal negative control (non‐bleached areas of the cell) and (b) cells with donor in the absence of the acceptor (CFP only). We find that both types of negative control can yield false FRET. Given this false FRET background, we describe a method for distinguishing true positive signals. In summary, we extensively characterize a simple approach to FRET that should be adaptable to most laser‐scanning confocal microscopes, and demonstrate its feasibility for detecting FRET between several CFP/YFP partners.     

Fluorescence in the tandem scanning microscope

Our studies have shown that the fluorescence mode can be used to good effect in both tandem scanning microscopes (TSM: direct view confocal microscopes) as well as confocal scanning laser microscopes (CSLM). Applications are presented which show that the two great advantages of TSM are real-time viewing and real colour, which allow faster use and interpretation. CSLM are complementary, not competitive, being currently more sophisticated for low-level fluorescence work. This is equally possible with available TSM, but requires further development using CCD cameras and image-processing systems.     

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.     

激光扫描共聚焦光谱成像系统

在激光扫描共聚焦显微成像技术基础上引入了光谱成像技术以便区分生物组织中的不同荧光成分。采用分光棱镜对荧光进行光谱展开,在光谱谱面处设置两个可移动缝片形成出射狭缝,两个步进电机带动安装其上的两个缝片设置系统在整个工作波长（400~700 nm）内的光谱带宽,其最小光谱带宽优于5 nm。用488 nm激光和低压汞灯实际测量了几条谱线对应的狭缝位置并和理论值做了比较,结果显示实际狭缝位置和理论值的差值均小于0.1 mm。在全光谱和50 μm出射狭缝（对应2.5 nm光谱带宽）对老鼠肾脏组织进行了共聚焦光谱成像实验,获得了老鼠肾脏组织中DAPI标定的细胞核图像和Alexa Fluor^®488标定的肾脏小球曲管图像,实现了对老鼠肾脏组织不同成分的区分。实验结果表明：提出的系统能够进行共聚焦光谱成像,扩大了共聚焦显微镜的适用范围。     

Minimizing light exposure with the programmable array microscope

The exposure of fluorophores to intense illumination in a microscope often results in photobleaching and phototoxicity, thus constituting a major limiting factor in time lapse live cell or single molecule imaging. Laser scanning confocal microscopes are particularly prone to this problem, inasmuch as they require high irradiances to compensate for the inherently low duty cycle of point scanning systems. In the attempt to maintain adequate speed and signal-to-noise ratios, the fluorophores are often driven into saturation, thereby generating a nonlinear response. One approach for reducing photodegradation in the laser scanning confocal microscope is represented by controlled light exposure microscopy, introduced by Manders and colleagues. The strategy is to reduce the illumination intensity in both background areas (devoid of information) as well as in bright foreground regions, for which an adequate signal-to-noise ratio can be achieved with lower excitation levels than those required for the less intense foreground pixels/voxels. Such a variable illumination scheme can also be exploited in widefield microscopes that employ lower irradiance but higher illumination duty cycles. We report here on the adaptation of the controlled light exposure microscopy principle to the programmable array microscope, which achieves optical sectioning by use of a spatial light modulator (SLM) in an image plane as a programmable mask for illumination and conjugate (and nonconjugate) detection. By incorporating the basic controlled light exposure microscopy concept for minimizing exposure, we have obtained a reduction in the rate of photobleaching of up to ~5-fold, while maintaining an image quality comparable to regular imaging with the programmable array microscope.     

Advances in laser sources for confocal and multiphoton microscopy

The illumination source for all high-resolution, optical sectioning, scanning microscopes is crucially important to the overall performance of the system. We examine advances that have been made in laser sources for both confocal and multiphoton microscopy where the emphasis has been on the development of potentially low-cost, easy to use sources. Growing interest in temporally and spatially resolved techniques has directed laser research towards addressing these challenges. We present the most recent developments in sources for confocal and multiphoton microscopy along with the considerations that should be made when a new source is being considered.     

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。     

Calibration and standardization of the emission light path of confocal microscopes

Recently, there has been a large expansion in the usage of optical microscopes for obtaining quantitative information from biological samples in order to determine fundamental biological information such as molecular kinetics and interaction, and heterogeneity within cell populations. Consequently, we built a highly stable, uniform, isotropically emitting and convenient‐to‐use light source, and designed image analysis procedures for calibrating the emission light path of optical microscopes. We used the source and procedures to analyse the quantitative imaging properties of a widely used model of laser scanning confocal microscope. Results showed that the overall performance was as high as could be expected given the inherent limitations of the optical components and photomultiplier tubes. We observed that the photon detection efficiency did not vary with photomultiplier tube gain and that the highest dynamic range was achieved with relatively low gain and 12‐bit digitization. Practical applications of the light source for checking the transmission of optical components in the emission light path are presented.     

显微观测技术的新进展及其应用

依据显微观测技术的发展过程 ,介绍了普通的光学显微镜和 2 0世纪流行的电子显微镜 ,详细阐述了以扫描隧道显微镜和激光扫描共焦显微镜为代表的新型显微镜系列的发展 ,以及各类显微镜的基本工作原理和应用情况     

Quality assessment of atomic force microscopy probes by scanning electron microscopy: correlation of tip structure with rendered images

While image quality from instruments such as electron microscopes, light microscopes, and confocal laser scanning microscopes is mostly influenced by the alignment of optical train components, the atomic force microscope differs in that image quality is highly dependent upon a consumable component, the scanning probe. Although many types of scanning probes are commercially available, specific configurations and styles are generally recommended for specific applications. For instance, in our area of interest, tapping mode imaging of biological constituents in fluid, double ended, oxide-sharpened pyramidal silicon nitride probes are most often employed. These cantilevers contain four differently sized probes; thick- and thin-legged 100 microm long and thick- and thin-legged 200 microm long, with only one probe used per cantilever. In a recent investigation [Taatjes et al. (1997) Cell Biol. Int. 21:715-726], we used the scanning electron microscope to modify the oxide-sharpened pyramidal probe by creating an electron beam deposited tip with a higher aspect ratio than unmodified tips. Placing the probes in the scanning electron microscope for modification prompted us to begin to examine the probes for defects both before and after use with the atomic force microscope. The most frequently encountered defect was a mis-centered probe, or a probe hanging off the end of the cantilever. If we had difficulty imaging with a probe, we would examine the probe in the scanning electron microscope to determine if any defects were present, or if the tip had become contaminated during scanning. Moreover, we observed that electron beam deposited tips were blunted by the act of scanning a hard specimen, such as colloidal gold with the atomic force microscope. We also present a mathematical geometric model for deducing the interaction between an electron beam deposited tip and either a spherical or elliptical specimen. Examination of probes in the scanning electron microscope may assist in interpreting images generated by the atomic force microscope.     

Imaging properties of high aperture multiphoton fluorescence scanning optical microscopes

A theory for multiphoton fluorescence imaging in high aperture scanning optical microscopes employing finite sized detectors is presented. The effect of polarisation of the fluorescent emission on the imaging properties of such microscopes is investigated. The lateral and axial resolutions are calculated for one-, two- and three-photon excitation of p-quaterphenyl for high and low aperture optical systems. Significant improvement in lateral resolution is found to be achieved by employing a confocal pinhole. This improvement increases with the order of the multiphoton process. Simultaneously, it is found that, when the size of the pinhole is reduced to achieve the best possible resolution, the signal-to-noise ratio is not degraded by more than 30%. The degree of optical sectioning achieved is found to improve dramatically with the use of confocal detection. For two- and three-photon excitation axial full width half-maximum improvement of 30% is predicted.     

Detailed three-dimensional visualization of resilin in the exoskeleton of arthropods using confocal laser scanning microscopy

Resilin is a rubber-like protein found in the exoskeleton of arthropods. It often contributes large proportions to the material of certain structures in movement systems. Accordingly, the knowledge of the presence and distribution of resilin is essential for the understanding of the functional morphology of these systems. Because of its specific autofluorescence, resilin can be effectively visualized using fluorescence microscopy. However, the respective excitation maximum is in the UV range, which is not covered by the lasers available in most of the modern commercial confocal laser scanning microscopes. The goal of this study was to test the potential of confocal laser scanning microscopy (CLSM) in combination with a 405 nm laser to visualize and analyse the presence and distribution of resilin in arthropod exoskeletons. The results clearly show that all resilin-dominated structures, which were visualized successfully using wide-field fluorescence microscopy (WFM) and a 'classical' UV excitation, could also be visualized efficiently with the proposed CLSM method. Furthermore, with the application of additional laser lines CLSM turned out to be very appropriate for studying differences in the material composition within arthropod exoskeletons in great detail. As CLSM has several advantages over WFM with respect to detailed morphological imaging, the application of the proposed CLSM method may reveal new information about the micromorphology and material composition of resilin-dominated exoskeleton structures leading to new insights into the functional morphology and biomechanics of arthropods.     

Quantitative interpretation of binding reactions of rapidly diffusing species using fluorescence recovery after photobleaching

Fluorescence recovery after photobleaching (FRAP) measurements offer an important tool for analyzing diffusion and binding processes. Confocal scanning laser microscopes that are used in FRAP experiments bleach regions with a radially Gaussian distributed profile. Previous attempts to derive analytical expressions in the case of processes governed by fast diffusion have overlooked the characteristics of the instruments used to perform FRAP measurements and therefore led to approximating solutions. In the present paper, bleaching laser beam characteristics are incorporated into an improved model to provide a more rigorous and accurate method. The proposed model simulates binding inside bounded regions, and it leads to FRAP curves that depend on the on and off rates that can be employed to determine the rate constants. It can be used in conjunction with experimental data acquired with confocal scanning laser microscopes to investigate the biophysical properties of proteins in living cells. The model aims to improve the accuracy when determining rate constants by taking into account a more realistic scenario of the light-matter interaction.     

Assessment of Congo red as a fluorescence marker for the exoskeleton of small crustaceans and the cuticle of polychaetes

In this study, the potential of the common dye Congo red as a fluorescence marker for chitin in the exoskeleton of small crustaceans and collagen in the polychaete cuticle was tested. The Congo red staining turned out to be rather efficient and yielded intensively fluorescing structures, which made a very detailed visualization by confocal laser scanning microscopy possible. The excellent results are comparable to those described for the utilization of other efficient fluorescence dyes and intense autofluorescence. The application of Congo red is easy, the fluorescence of this dye is very stable, and the excitation maximum of the structures stained with Congo red is in a range, which is covered by the lasers of most of the confocal laser scanning microscopes. These advantageous properties make the fluorescence staining by Congo red a method of choice for the detailed visualization of the external morphology of small crustaceans and polychaetes.     

High temporal and spatial resolution studies of bone cells using real-time confocal reflection microscopy

Chick and rat bone-derived cells were mounted in sealed coverslip-covered chambers; individual osteoclasts (but also osteoblasts) were selected and studied at 37°C using three different types of high-speed scanning confocal microscopes: (1) A Noran Tandem Scanning Microscope (TSM) was used with a low light level, cooled CCD camera for image transfer to a Noran TN8502 frame store-based image analysing computer to make time lapse movie sequences using 0.1 s exposure periods, thus losing some of the advantage of the high frame rate of the TSM. Rapid focus adjustment using computer controlled piezo drivers permitted two or more focus planes to be imaged sequentially: thus (with additional light-source shuttering) the reflection confocal image could be alternated with the phase contrast image at a different focus. Individual cells were followed for up to 5 days, suggesting no significant irradiation problem. (2) Exceptional temporal and spatial resolution is available in video rate laser confocal scanning microscopes (VRCSLMs). We used the Noran Odyssey unitary beam VRCSLM with an argon ion laser at 488 nm and acousto-optic deflection (AOD) on the line axis: this instrument is truly and adjustably confocal in the reflection mode. (3) We also used the Lasertec 1LM11 line scan instrument, with an He-Ne laser at 633 nm, and AOD for the frame scan. We discuss the technical problems and merits of the different approaches. The VRCSLMs documented rapid, real-time oscillatory motion: all the methods used show rapid net movement of organelles within bone cells. The interference reflection mode gives particularly strong contrasts in confocal instruments. Phase contrast and other interference methods used in the microscopy of living cells can be used simultaneously in the TSM.