Focus on Multidimensional Microscopy

The following sections are included:

• Contents

• Preface

A novel illumination system allows standard objective lenses to achieve greater resolution, sharpness, contrast, depth of field, direct 3D viewing and 3D photography. The theoretical and practical basis of the high definition 3D microscope is discussed. The method employs multiple oblique illumination, which allows investigators to examine thicker preparations in order to gain a direct understanding of the interrelationships between structures within single samples.

Simultaneous recording of multiple fluorescent labels is complicated by the fact that many fluorophores have broad and partially overlapping emission spectra. Using the intensity-modulated multiple-beam scanning (IMS) technique we demonstrate that the cross-talk between channels can be substantially reduced. As a consequence it is now possible to use fluorophore combinations such as Lucifer Yellow and Cyanine-3.18. Previously this combination was very difficult to use due to excessive cross-talk.

Recently we have described the Voronoi approach to quantitative three-dimensional image analysis based on a geometrical model. This approach allows the determination of several first-order estimates such as volume, surface and shape parameters of two-dimensionally sectioned objects in the three-dimensional space. In the current paper the Voronoi method will be reviewed in short and a more detailed study on the reliability of volume estimates obtained by the Voronoi approach will be presented. Additionally, we will report on a significant improvement of the Voronoi algorithm which considerably speeds up the evaluation of a 3D image volume. We will further describe novel approaches to quantitative image analysis based on the Voronoi diagram. The new methods include the calculation of medial lines and skeletons in the 3D Voronoi diagram. These methods will be shown to be suitable for the determination of the localization of objects and for the calculation of distances between objects in the three-dimensional space. The application of these methods to three-dimensional image data obtained from a confocal laser scanning microscope (CLSM) will be demonstrated. Notably, the Voronoi approach allows the compression, three-dimensional reconstruction and visualization as well as segmentation, skeletization and quantitative analysis of large data sets on-line.

This paper investigates confocal imaging through weakly and highly scattering media under ultrashort pulsed beam illumination. In the first part of this paper, resolution in various confocal imaging methods under the first Born approximation which implies that multiple scattering caused by the object is weak is studied theoretically. For bright-field confocal microscopy, time-resolved and time-averaged imaging methods are considered, while resolution in two-photon and single-photon confocal fluorescence microscopy is examined only for the time-averaged imaging method. It is shown that the time-resolved approach can give a three-dimensional image of a thick object with axial resolution approximately 30% higher than the limiting value under continuous wave (CW) laser illumination of the same wavelength as the pulsed beam, while the transverse resolution is slightly improved. The second part of the paper presents our recent experimental results of confocal imaging through highly scattering (random) media. Various practical effects including confocal pinhole size, numerical aperture of the objective, and annular pupils on the image quality are examined experimentally. The combination of an annular objective with a finite-sized detector may prove advantageous for improving image quality through highly-scattering media.

The following sections are included:

• Introduction

• Theory

• Experiments

• Acknowledgements

• References

Embryo sac development of maize follows a well-defined temporal and spatial pattern manifested by the polarity of the embryo sac and synchronous free nuclear divisions at the opposite poles of the embryo sac. The polarized megasporocyte gives rise to the linear dyad and tetrad of megaspores after the nuclear divisions of meiosis I and meiosis II. During megagametogenesis, the micropylar and chalazal nuclei of the embryo sac undergo synchronous division and migration at the second and third mitosis. The nearly perpendicular configuration and orientation of the spindles, phragmoplasts, and pairs of nuclei result in precise positioning of the nuclei. The different patterns of microtubular organization in the cells of the mature embryo sac reflect their structural adaptation for their future function.

During fertilization the onset of degeneration in one of the synergids in Nicotiana tabacum was reflected by the electron density of cytoplasmic organelles. Rhodamine-phalloidin staining of actin reveals that the electron-dense aggregates located in the intercellular spaces at the chalazal end of the synergid and between the synergids, egg and central cell contain abundant actin. This actin appears to precisely position and facilitate the access of the male gametes to the egg and central cell for fusion. Upon pollen-tube arrival, the male gametes are released through a terminal aperture of the pollen tube into the degenerate synergid. Sperm cells undergo morphological modification before gametic fusion including an increase in their mitochondrial electron density, dilation of the endoplasmic reticulum, and vacuolation of sperm cytoplasm. Discharge of the pollen tube results in the formation of numerous enucleated cytoplasmic bodies which are either stripped or shed from sperm cells and the pollen-tube cytoplasm.

A multiple-angle viewing stage has been built at the Forschungseinrichtung Röntgenphysik, Universität Göttingen and was implemented on the Göttingen transmission x-ray microscope (TXM) at BESSY (Schmahl et al, 1993). TXM stereo images have been taken of dry spores, diatoms and of sheaths of mineral accumulating bacteria Leptothrix ochracea. The stereo angles were in the range of 5° to 12.5°. Three-dimensional structure of those specimens is revealed at very high resolution. For these studies an objective-zoneplate with an outermost zonewidth of 40nm was used at 2.4nm wavelength. Structures of 40nm size can clearly be seen.

Taste buds are distributed randomly on the trench wall of gerbil vallate papillae and the taste pore is small (about 1mm in diameter). The average number of taste pores per bud is 85 ± 1.67 (N=4, mean ± standard error of mean) and the distribution density of the taste pore is 1.89±0.5 per 1000 μm² (N=8). Four types of taste cells, i.e., type I, II, intermediate and basal cells, were identified by their ultrastructural characteristics and each type comprised about 78%, 14%, 5 % and 1-2% of the total taste cell population, respectively. Type I, Type II and intermediate cells were elongated with apical microvilli protruding into the taste pore. Type I cells were characterized by the presence of dense granules (diameter: 100-200 nm) in their apical cytoplasm. They possessed slender nucleus and displayed well-developed Golgi apparatus in their cytoplasm. Specific features of Type II cells were the presence of a characteristic double ring vesicles in the apical cytoplasm and an abundance of smooth endoplasmic reticulum in the supranuclear cytoplasm. Intermediate cells exhibited morphological characteristics of both Type I and Type II cells. Degenerating cells containing vacuolated and/or electron-dense structures were also observed. Afferent (taste cells to nerve) and efferent (nerve to taste cell) synapses were also found in gerbil circumvallate taste buds.

For the analysis of the spatial relationship between different biological structures, multi-colour confocal microscopy has shown to be an adequate technique. To acquire more information from biological specimens a confocal microscope has been extended for the registration of three different fluorescence signals at the same time: pixel-simultaneous scanning. Major interfering effects in multi-colour confocal microscopy are: (1) cross-talk between the fluorescence signals and (2) positional shift between the colour components of an image. In the new set-up cross-talk has been reduced to a negligible level by using Intensity-modulated Multiple-beam Scanning (IMS). The effect of positional shift has been minimized by scanning pixel simultaneously. The new set-up has been tested for the fluorophore combination FITC, Texas Red and Cy5.18. Cross-talk between different fluorescence signals has been measured. Finally, the set-up has been tested on preparations of triple-stained nuclei. The results of these tests show that three-colour pixel-simultaneous intensity-modulated confocal microscopy is a useful method for the analysis of biological structures in three dimensions.

In light microscopy, it is common to consider resolution, contrast, and signal-to-noise ratio as three separate subjects. In practice however, these three variables are intimately linked through the Rose Equation which quantifies the limits placed on the detectable resolution by the intrinsic contrast of the interaction being visualized (as modified by the imaging apparatus) and the number of quanta that must be utilized in order to form the image. Specifically, the this formulation suggest that isolated features will only be recognizeable by the unaided eye if their intensity differs from the background by 5 times RMS level. By quantifying the number of quanta/pixel that must be detected in order to “unambiguously” discriminate two objects whose spacing satisfies the Rayleigh Criterion (25% contrast), the Rose Equation explains why the observed resolution in confocal fluorescent images often seems so much worse than that set by such optical considerations. While 25 quanta/pixel (q/pix) can delineate a point feature as different from a background of 1 q/pix, more than 40 times more signal is needed to defined the difference between two features which differ in intensity by only 25% with a similar accuracy. In the light microscope, small features have low contrast. As a result, signals from them can often be inadvertantly eliminated by simple image processing steps such as thresholding. This simple insight has important implications for the image processing of 3D microscopic images.

The molecular specializations associated with the neuromuscular junction (NMJ) and the myotendinous junctions (MTJ) were examined with light and electron microscopy in muscle cells isolated from the amphibian Xenopus embryos. Using conventional and confocal fluorescence microscopy coupled with specific antibodies to sarcolemma proteins, we have characterized the protein complexes associated with these two specializations in skeletal muscle. Our results suggest that each specialization is associated with a distinct set of peripheral and integral membrane proteins. To understand the development of these specializations, a non-cellular stimulus involving microbeads coated with factors residing in the extracellular matrix of skeletal muscle was devised to mimic the endogenous cellular interactions that lead to their formation. Both NMJ and MTJ can be induced by beads in cultured Xenopus muscle cells. Through the use of this procedure, the signal transduction mechanisms involved in the formation of these specializations can be conveniently explored.

The following sections are included:

• Introduction

• Particle Number

• The Disector

• The Optical Disector or Unbiased Brick

• The Fractionator

• The Optical Fractionator

• Optimizing the Sampling Scheme

• Conclusions

• References

The optical fibre probe microscope is a compact system which allows investigation of surface topography. The effects of surface height, tilt and refractive index on the image were investigated both eoretically and experimentally.

The Special Affine Fourier Transform (SAFT) represents the most general lossless inhomogenous linear mapping in phase space, as an integral transformation of the wavefunction. In this monograph an overview of recent work relating to the area of linear transformations and optical signal processing is presented. In particular the relationship between the SAFT and the Fractional Fourier Transform (FRT) is examined. In order to fully appreciate these transformations the use of the Wigner Distribution Function (WDF) is reviewed. The Abelian subgroups of the SAFT are also discussed and several potentially useful ‘new’ transformations are introduced. Application of the resulting ideas in the areas of imaging and signal processing are illustrated.

Confocal theta microscopy is a novel method by which the three-dimensional resolution of a confocal fluorescence microscope can be substantially improved. We present the basic principle, a short theoretical description of the technique and discuss some practical aspects. In addition, we describe the instrument built at the European Molecular Biology Laboratory (EMBL). Finally, measurements that prove the feasibility and the resolution improvement achievable by this method are shown.

NuMA (Nuclear Mitotic Apparatus protein), originally described as a nuclear protein, is involved in nuclear structure, spindle assembly, and nuclear re-formation. During the past several years, we have isolated and characterized NuMA cDNAs and their corresponding proteins. Our results show that NuMA is composed of multiple isoforms and heterogeneous in size and cellular localization. Southern blot analysis suggests that the heterogeneity of NuMA may arise by alternative RNA splicing from a single gene. In this report, we have analyzed the subcellular localization of various NuMA isoforms and have mapped the NuMA gene to human chromosome 11q12-q13.3 by confocal immunofluorescence microscopy.

The following sections are included:

• Introduction

• Exact Reconstruction
  • Sufficient Condition
  • Exact Algorithms

• Approximate Reconstruction
  • Why Approximate Reconstruction
  • Derivative-Free Non-Circular Fan-Beam Algorithm
  • Generalized Feldkamp Algorithm

• Between Exact and Approximate Reconstruction
  • Exact Reconstruction to Approximate Reconstruction
  • Approximate Reconstruction to Exact Reconstruction
    • Stereo-Imaging
    • Sufficient Condition: Revisited

• Discussion and Conclusion

• Acknowledgements

• References

Low temperature scanning electron microscopy (LTSEM), which enables observations of frozen hydrated samples, permits imaging of some specimens that cannot be processed or properly preserved for ambient temperature SEM studies. LTSEM specimen preparation merely consists of cryofixation; therefore, artifacts arising from chemical fixation, solvent dehydration and critical point drying are averted. The cryotechnique is particularly useful to observe specimens that are: (1) soluble in aqueous fixatives or dehydrating solvents; (2) impermeable to chemical fixatives; (3) delicate and thereby subject to mechanical damage; (4) loosely associated or easily dislodged or; (5) physiologically altered by either the water or the time that is required for conventional chemical fixation. LTSEM also allows comparisons of freeze-fractured and freeze-etched faces of a specimen; this advantage can be used to determine the location of the water in the specimen and to distinguish aqueous areas from naturally occurring air pockets and non etchable solids. In the past, the resolution of frozen hydrated samples was limited by the conventional SEM; however use of cryotechniques on a field emission SEM enables imaging of macromolecular structures such as membrane particles, which measure less than 10 nm.

We study numerically the constant-height image in transmission mode of photon scanning tunneling microscopy (PSTM). The local field inside the probe tip is calculated, which scans over a dielectric rectangular structure. The substrate is illuminated by a s-polarized incident light with oblique angle. The influence by the dielectric functions of the tip and the lateral size of the structure on the optical signal are analyzed. We find out that the transmitted intensity is correlated with the shape of the sample and the assumption of passive probe may be a reasonable approximation when the optical constant of the tip is close to that of air.

The following sections are included:

• Author Index

• Index of Contents

• COLOUR PLATES