Focus on Multidimensional Microscopy

The following sections are included:

• Contents

• Preface

Short optical pulses are used in a new technique for the measurement of fluorescence lifetimes. It has the potentiality to provide high time resolution - since it relies on the laser pulse duration, rather than on electronic gating techniques - and permits, in combination with bilateral confocal microscopy and the use of a (cooled) CCD, sensitive signal detection over a large dynamic range. Combined with confocal microscopy it enables the spatial determination of the fluorescence lifetimes. The principle of the technique is discussed within a theoretical framework taking into account various secondary effects.

In this chapter, we describe the determination of icosahedral virus structures using electron cryomicroscopy. We provide detailed descriptions of each of the steps leading to the three-dimensional reconstruction, including the subsequent resolution assessment and visualization of the structure. To aid in understanding this process a variety of illustrative examples are provided.

Two-dimensional image processing is not appropriate where confocal microscopes are used to acquire three-dimensional datasets. We present here the merits of processing confocal images with a combination of three-dimensional median filtering, using a cruciform structuring element, and one-dimensional transforms to improve image quality in the depth dimension.

The experimental station of X-ray microscopy, microtomography and fluorescence microprobe at the VEPP-3 storage ring is described. Some potential applications and capabilities of the X-ray imaging technique in environmental studies are presented.

Three-dimensional (3D) imaging properties in 4Pi confocal microscopy are presented in terms of 3D transfer functions. Bright-field and fluorescence imaging methods can be described using the 3D coherent transfer function and the 3D optical transfer function, respectively. The effect of apodization of a high-aperture objective on 3D 4Pi imaging can be understood from the 3D transfer functions. The further investigation on this topic is discussed.

An accurate tomographic reconstruction requires proper transformation of the recorded image intensities to projected specimen mass densities. For thick biological specimens, images are not only aberrated due to the objective lens aberrations, but also due to the large proportion of inelastic multiple scattering. Using a combination of energy filtering and through focus series restoration, these aberrations can be removed. The corrected images show enhanced contrast and resolution.

Microsomal glutathione transferase is a membrane-bound detoxification enzyme that catalyzes the conjugation of glutathione to electrophilic compounds. Large, well-ordered two-dimensional crystals of the protein were obtained after dialysis. Electron crystallography was used to determine a projection structure at 4 Å resolution. The protein forms a trimmer with an inner core of six parallel α-helices surrounded by elongated domains.

The uptake pathway of DNA during the cationic liposome-induced transfection procedure is investigated by fluorescence and confocal microscopy. The cationic lipid, dioleoyl trimethyl ammonium propane (DOTAP), is mixed with either dioleoyl-phosphatidylethanolamine (DOPE) or dioleoyl-phosphatidylcholine (DOPC), at 1:1 ratio, to form cationic liposomes. These cationic liposomes form DNA: cationic liposome complexes with the plasmid pSV-ß-galactosidase, which is used to transfect CHO cells. Freeze fracture electron microscopy shows that the morphology of DNA:cationic liposome complexes is an aggregate of DNA and cationic liposomes, with most of the visible, bilayer-ensheathed DNA strands attached to the outside of cationic liposomes. Granule formation from fluorescently labelled complexes was observed either in transfection medium, as in the case of complexes containing DOPE, or on the cell surfaces, as in the case of those containing DOPC. The transfection efficiency increases with the density of granules found on cell surfaces. Serum inhibits granule formation also inhibits transfection. Confocal microscopy of cells treated with doubly labelled DNA and lipid complexes reveals that internalized granules contain both DNA and lipid, but those on cell surfaces are mostly DNA aggregates, presumably remaining after cationic liposomes in the complexes fuse with plasma membranes. Labelled lipids begin to transfer to cell membranes immediately after the complexes are added to cells, but the transfer is significant only after an hour of treatment. Inhibition of endocytosis by low temperature or by 20 μg/ml of cytochalasin-B reduces granule formation on cell surfaces, and suppresses transfection. The results indicate that, for CHO cells, the major uptake pathway of DNA in the cationic liposome-induced transfection process is endocytosis. In the case of PC-containing cationic liposome, the formation of DNA:cationic liposome granules on cell surfaces is a cytoskeletal directed membrane event akin to the capping phenomenon, and the formation of granules may trigger endocytosis and consequent transfection. This process is more effective than the spontaneous granulation of PE-containing cationic liposomes in terms of effecting transfection. Thus granule-related endocytosis is a more important factor than membrane fusion in cationic liposome-induced gene transfer into CHO cells.

We introduce methods for a three-dimensional structural representation of mammalian lungs. Computer guided imaging of large volumes representing respiratory units as well as stereoscopical tracings of casts describing the conductive, macroscopical part of lung are discussed. Quantization of these multiscale data sets is based on topological investigations to characterize branching patterns and self-similarities. Subsequently these properties are used for the development of a lung modeler using fractal graphics. As a result, a computer model of a complete bronchial tree could be designed to simulate functional properties.

Plant cells are confined to semi-rigid cell walls and do not undergo morphogenetic movement during development, hence plant morphogenesis is the result of orchestrated cell divisions and cell expansion in precise orientations. Many specialized structures in plants are initiated by asymmetric cell divisions. This report summarizes the recent advances in the studies of specialized structures in leaf epidermis, then gives a detailed account on the patterning of lateral root distribution in the aquatic fern Marsilea.

The increased specimen thickness made possible by the higher accelerating voltages of high- and intermediate-voltage electron microscopes provides direct 3D imaging, compared to the 2D images of cells obtained using conventional transmission electron microscopes. Stereo pair analysis, serial-section reconstruction from thick serial sections, and electron tomography are useful methods in the interpretation of this 3D information. Descriptions of these methods, and advice on selecting the appropriate method for the specimen at hand, are presented.

A numerical procedure to calculate images of highly reflecting metallic surfaces in optical scanning microscopy is described. The interaction between the focused beam and the sample is describe fairly rigorously with an approach based on Green's integral theorem and an impedance boundary condition. The procedure is applied to the study of the confocal scanning optical microscope in the profiling mode of operation.

Interactions of solid materials with liquids, vapors or other solids are of concern in materials and earth sciences, especially the diagenesis, weathering and metamorphic processes which involve the compositional, structural and morphological changes down to the nanometer scale. This article summarizes electron microscopy studies of some materials with emphases on (1) dislocations, (2) shape and orientation changes, and (3) amorphization due to interdiffusion. These, results provide new insights to several solid-state processes, such as cooperative migration and rotation of particles and diffusion induced amorphization (DIA) which may occur at varied spatial and time scales in natural occurrences.

The measurement of surface profiles using confocal microscopy or interferometric imaging is investigated by computer modelling. Effects of surface roughness and numerical aperture on the fidelity are studied.

For the observation of macroscopic and microscopic life in aquatic and marine environments we treat the animals and algae as phase objects. The combination of a collimated light beam with a spatial filter and a matched filter in the focal planes allow us to image even the most transparent objects. We illustrate the capabilities of our optical designs with three cases: the Schlieren observations of swimming tracks of planktonic animals, the observations in three-dimensions of the swimming behavior of mating copepods, and the observations of planktonic and benthic life in situ. We discuss the basics of our design and suggest further technical developments.

This paper presents an analysis of the pathways of low density lipoproteins (LDL) and high density lipoproteins (HDL) across the endothelium at the branched regions of arteries in high-cholesterol diet-and normal diet-fed rats. Rat tissues were analyzed by perfusing in situ human LDL labeled with colloidal gold or fluorescein 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) and by immunoelectron microscopy to localize LDL and HDL across the arterial walls. Accumulation of LDL-DiI in the intima was markedly increased in the high cholesterol diet-fed rats as compared to the normal diet-fed rats. In addition, more LDL-DiI accumulated in the branched regions than those in the unbranched regions of the arteries. LDL-gold conjugates were observed in the intracytoplasmic vesicles and in the subendothelial space. Some of the tracer molecules were found within the open junctions at the branched regions of the arteries. After immunogold labeling of the tissue sections, HDL was revealed within the open junctions. More gold particles were observed over the subendothelial space and the smooth muscle cells. The intensity of the labeling in the vessel wall of hypercholesterolemic rats was significantly increased as compared with that in control animals. There was no significant difference in the distribution of HDL between the branched and the unbranched regions of the arteries.

We consider the form of the optical field in the exit pupil of a high numerical aperture microscope objective when the lens is focussed onto an isotropic thin film specimen. The form of the optical field is particularly sensitive to specimen properties and is not usually circularly symmetric. We propose a number of imaging modes which take advantage of this asymmetry to provide images of greatly enhanced contrast. A method is also presented whereby full ellipsometric data may be extracted.

Recent developments in near-field fluorescence microscopy have made it possible to conduct single molecule spectroscopy in ambient environments, with a spatial resolution beyond the diffraction limit. We discuss simultaneous near-field fluorescence and shear-force imaging, with particular emphasis on single molecule spectroscopy. Experiments probing the dynamics of single molecules on both the 10^-2 to 10³ sec time scale and the picosecond to nanosecond time scale will be presented along with a discussion of the ground rules for performing nonperturbative near-field spectroscopic measurements. Finally, examples of applications to studies of single proteins and biological membranes will be presented.

The following sections are included:

• Author Index

• Index of Contents

• COLOUR PLATES