Video image processing greatly enhances contrast, quality, and speed in polarization-based microscopy

Video cameras with contrast and black level controls can yield polarized light and differential interference contrast microscope images with unprecedented image quality, resolution, and recording speed. The theoretical basis and practical aspects of video polarization and differential interference contrast microscopy are discussed and several applications in cell biology are illustrated. These include: birefringence of cortical structures and beating cilia in Stentor, birefringence of rotating flagella on a single bacterium, growth and morphogenesis of echinoderm skeletal spicules in culture, ciliary and electrical activity in a balancing organ of a nudibranch snail, and acrosomal reaction in activated sperm.     

Multiple objects tracking in fluorescence microscopy

Many processes in cell biology are connected to the movement of compact entities: intracellular vesicles and even single molecules. The tracking of individual objects is important for understanding cellular dynamics. Here we describe the tracking algorithms which have been developed in the non-biological fields and successfully applied to object detection and tracking in biological applications. The characteristics features of the different algorithms are compared.     

Fibronectin molecule visualized in electron microscopy: a long, thin, flexible strand

We have determined the structure of plasma fibronectin by electron microscopy of shadowed specimens. the 440,000 molecular weight, dimeric molecule appears to be a long, thin, highly flexible strand. The contour length of the most extended molecules is 160 nm, but a distribution of lengths down to 120 nm was observed, indicating flexibility in extension as well as in bending. The average diameter of the strand is 2 nm and there are no large globular domains. the large fragments produced by limited digestion with plasmin are not globular domains but are segments of the strand, whose length corresponds to the molecular weight of the polypeptide chain. We conclude that each polypeptide chain of the dimeric molecule spans half the length of the strand, with their carboxyl termini joined at the center of the strand and their amino termini at the ends. This model is supported by images of fibronectin-fibrinogen complexes, in which the fibrinogen is always attached to an end of the fibronectin strand.     

Electron microscopy of thin protein crystal sections.

In continuation of an earlier publication (Hoppe et al., 1968), further experiments are described here on the preparation of thin film sections of embedded protein crystals for investigation by electron microscopy and electron diffraction. Several embedding media were compared, the best being Aquon. Periodicities were observed in electron micrographs as well as in electron diffraction patterns. In diffraction experiments the best resolution observed was approximately 10 to 11 Å.     

Orientation-dependent recombination hotspot activity in bacteriophage lambda.

Promoters of genetic exchange by the Escherichia coli Rec system, Chi elements, have been analyzed in λ phages carrying bacterial EcoRI restriction fragments. Some fragments confer Chi⁺ phenotype in one orientation and Chi^? in the opposite orientation. The inactivity of Chi in one orientation explains why all active Chi elements in λ manifest a certain recombinational bias of the same sense.When these studies were undertaken, we rather expected to find two classes of Chi, one class which stimulated recombinant formation stronger to its left and one class stimulating recombinant formation more strongly to its right. The failure to find the second class is now understandable by supposing that the orientation of Chi which would have permitted it to act rightward is the orientation in which Chi has no activity at all. Several models are proposed for the orientation dependence of Chi activity.     

Single long DNA molecule analysis using fluorescence microscopy.

Single long DNA molecule (T4 DNA) in agarose gel was visualized with a fluorescence microscope. We confirmed alternating current electric fields is effective for stretching of single DNA molecule in agarose gel. This stretching phenomenon was observed with wide range of agarose gel concentration from 0.5%(W/V) to 1.5%. From this observation, the presence of agarose gel fiber is essential for this stretching phenomenon. The stretching process of several DNA molecules in gel shows discontinuity, which is never observed in polymer systems. It would be based on topological restriction from gel fibers.     

Tropomyosin positions in regulated thin filaments revealed by cryoelectron microscopy.

Past attempts to detect tropomyosin in electron micrograph images of frozen-hydrated troponin-regulated thin filaments under relaxing conditions have not been successful. This raised the possibility that tropomyosin may be disordered on filaments in the off-state, a possibility at odds with the steric blocking model of muscle regulation. By using cryoelectron microscopy and helical image reconstruction we have now resolved the location of tropomyosin in both relaxing and activating conditions. In the off-state, tropomyosin adopts a position on the outer domain of actin with a binding site virtually identical to that determined previously by negative staining, although at a radius of 3.8 nm, slightly higher than found in stained filaments. Molecular fitting to the atomic model of F-actin shows that tropomyosin is localized over sites on actin subdomain 1 required for myosin binding. Restricting access to these sites would inhibit the myosin-cross-bridge cycle, and hence contraction. Under high Ca(2+) activating conditions, tropomyosin moved azimuthally, away from its blocking position to the same site on the inner domain of actin previously determined by negative staining, also at 3.8 nm radius. These results provide strong support for operation of the steric mechanism of muscle regulation under near-native solution conditions and also validate the use of negative staining in investigations of muscle thin filament structure.     

Distribution of troponin components in the thin filament studied by immunoelectron microscopy.

1. The localization of specific antiboidies against troponin components, i.e., troponin T(TN-T), troponin I(TN-I, and troponin C(TN-C), was studied by the use of an electrom microscope. 2. Every antibody was distributed along the thin filament with a period of 38 nm. 3. Staining with anti-TN-I or anti-TN-C formed narrow striations. The location of the first striation was 26 nm from the free end of the thin filament. 4. The width of individual striations formed by anti-TN-T was 14--20nm The H-band-side end of each striation coincided with the location of anti-TN-I or anti-TN-C.     

Fibrous long spacing collagen ultrastructure elucidated by atomic force microscopy.

Fibrous long spacing collagen (FLS) fibrils are collagen fibrils in which the periodicity is clearly greater than the 67-nm periodicity of native collagen. FLS fibrils were formed in vitro by the addition of alpha1-acid glycoprotein to an acidified solution of monomeric collagen and were imaged with atomic force microscopy. The fibrils formed were typically approximately 150 nm in diameter and had a distinct banding pattern with a 250-nm periodicity. At higher resolution, the mature FLS fibrils showed ultrastructure, both on the bands and in the interband region, which appears as protofibrils aligned along the main fibril axis. The alignment of protofibrils produced grooves along the main fibril, which were 2 nm deep and 20 nm in width. Examination of the tips of FLS fibrils suggests that they grow via the merging of protofibrils to the tip, followed by the entanglement and, ultimately, the tight packing of protofibrils. A comparison is made with native collagen in terms of structure and mechanism of assembly.     

New possibilities of studying microbial objects by laser interference microscopy

A novel method, laser interference microscopy, has been developed for studying the morphofunctional state of bacterial cells and the structure of bacterial communities. The following potentialities of the method are shown: rapid determination of the cell structure and subcellular structures (nucleus zone, vacuoles, lamellar structures) and the physiological state of the cell, as well as the study of the structure of bacterial communities (biofilm). The method does not require any additional preparation of cells before the investigation (fixation, staining, treatment with contrasting substances), which reduces the possible appearance of artifacts to a minimum and enables one to use laser interference microscopy for in vivo investigations.     

Preliminary characterization of thin biofilms by optical microscopy

A simple non‐invasive technique has been used that employs conventional optical microscopy and a glass flow cell to observe biofilms formed on opaque thin substrata. The technique allows the roughness of the biofilm and the substratum to be evaluated, and the biofilm thickness to be easily measured. The biofilm density may be quantified through colour gradients. In addition, some details of biofilm growth processes like the formation of water channels and pores, and interactions between planktonic and sessile cells can be visualized. Results related to the development of thin biofilms and their response to the environment under different conditions are reported. Pure and mixed microbial cultures and different solid substrata were assessed.     

Visualization of nucleosomes in thin sections by stereo electron microscopy

Nucleosomes (approximately diameter) were clearly visualized in thin sections (approximately 0.1 micrometer thick) of isolated chicken erythrocytes. The cells were lysed and fixed in low ionic strength buffers that maintained the chromatin as dispersed filaments and prevented the reformation of supranucleosomal structures. Stereo electron micrographs at high magnification demonstrate the stability of nucleosome structure in the dispersed chromatin state during fixation, dehydration, and embedding.     

The effect of odour priming on long latency visual evoked potentials of matching and mismatching objects.

This study reports a cross-modal, olfactory/visual event related potential (ERP) using odours as olfactory primes. The results show a difference in the ERP waveform for the N400 waveform when a visual image does not match the priming odour. An N400 peak was produced for both the matched and mismatched conditions but the peaks were significantly more negative for the mismatched condition. By the use of non-food odours this study extends an earlier finding by Grigor, who, using the same ERP paradigm, obtained similar results for food odours and photographs of food.     

Study of some neurohistologic objects by the method of scanning microscopy]

With the aid of a scanning measuring microscope (R. E. Bykov et al., 1972) different biological microobjects were measured in particular the nuclei of neurons and gliocytes at differnt levels of the spinal cord under normal and experimental conditions. The nuclei of the rabbit hypothalamus were stimulated by graded electrical current in order to induce neurogenic visceral dystrophies. Investigation of the square surfaces of cross sections of the neuron and gliocyte nuclie in different terms permitted establishment of progressing diminishing of the size of the square surface of the neuron nuclei against the background of a relatively steady state of steady state of wquare surfaces of the cross section of the nuclei of the adjacent glial cells. The obtained data are of interest not only for studying transneuronal changes in disturbed trophical function of the nervous system, but may be taken into consideration in clinical practice when elaborationg prerequistites for purposeful neurovegetative blocade in neurogenic dystrophies of the central genesis.