Shinya Inoué

Foundations of Confocal Scanned Imaging in Light Microscopy

Seldom has the introduction of a new instrument generated as instant an excitement among biologists as the laser-scanning confocal microscope. With the new microscope, one can slice incredibly clean, thin optical sections out of thick fluorescent specimens; view specimens in planes tilted to, and even running parallel to, the line of sight; penetrate deep into light-scattering tissues; gain impressive three-dimensional (3D) views at very high resolution; obtain differential interference or phase-contrast images in exact register with confocal fluorescence images; and improve the precision of microphotometry.

Polarization optical studies of the mitotic spindle

Summary1.With an improved polarization microscope, spindle fibers and fibrils, can be seen in living, normally dividing cells.2.Chromosomal and continuous fibers are demonstrated in photographs of oöcytes ofChaetopterus pergamentaceous, as well as in pollen mother cells ofLilium longiflorum.3.Supplementary observations on spermatocytes of several species of grasshopper and ofDrosophila melanogaster, as well as on pollen mother cells ofPolygonatum biflorum, Gasteria carinata and a species ofIris, confirm the findings mentioned above.4.Some of the structural changes observed in the spindle and chromosomes during division are described.5.The appearance of spindle structures in certain fixed preparations closely resembles structures observable in living cells under polarized light.

CRYSTAL PROPERTY OF THE LARVAL SEA URCHIN SPICULE

A pair of pluteus skeletal spicules arises from a pair of calcareous granules via the triradiate form. In polarized light, each spicule behaves as though carved out of a single crystal of magnesian calcite. The optic axis lies perpendicular to the plane of the triradiate and parallel to the body rod of the pluteus. However, in the scanning electron microscope, the spicule surface appeared smooth or somewhat spongy and manifested no crystal faces. Neither etching nor fracturing revealed underlying crystalline texture. Nevertheless, rhombohedral calcite crystals could be grown epitaxially onto isolated spicules immersed in a medium containing CaCl2 and NaHCO3. The optic axes of all crystals coincided with the optic axis of the spicule on which they were grown. Corresponding faces of the crystals were all aligned parallel to each other despite the complex shape of each spicule. Where the left and right spicules joined, two mutually tilted sets of crystals were observed but not crystals of intermediate orientation. Thus, the sea urchin larval spicule is built from a stack of molecularly contiguous microcrystals but its overall shape is generated by the mesenchyme cells independent of the magnesian calcite crystal habit.

Functional organization of mitotic microtubules. Physical chemistry of the in vivo equilibrium system

Equilibrium between mitotic microtubules and tubulin is analyzed, using birefringence of mitotic spindle to measure microtubule concentration in vivo. A newly designed temperature-controlled slide and miniature, thermostated hydrostatic pressure chamber permit rapid alteration of temperature and of pressure. Stress birefringence of the windows is minimized, and a system for rapid recording of compensation is incorporated, so that birefringence can be measured to 0.1 nm retardation every few seconds. Both temperature and pressure data yield thermodynamic values (delta H similar to 35 kcal/mol, delta S similar to 120 entropy units [eu], delta V similar to 400 ml/mol of subunit polymerized) consistent with the explanation that polymerization of tubulin is entropy driven and mediated by hydrophobic interactions. Kinetic data suggest pseudo-zero-order polymerization and depolymerization following rapid temperature shifts, and a pseudo-first-order depolymerization during anaphase at constant temperature. The equilibrium properties of the in vivo mitotic microtubules are compared with properties of isolated brain tubules.

Diffraction Images in the Polarizing Microscope

In the polarizing microscope set for extinction, only that image whose polarization has been altered is available to form an image. The lenses themselves introduce such an alternation by rotation of the plane of polarization of rays having oblique incidence. This paper shows that the diffraction image of a pinhole has the form sin2θ·J3(r)/r, where θ and r are polar coordinates in the image plane. The image has four bright zones separated by a dark cross and the central pattern becomes a four-leaf clover form. The diffraction image of a point source through a plate of uniaxial crystal cut perpendicular to its optic axis (z-cut) is also a four-leaf clover when the polarizers are crossed. When the polarizers are parallel, the diffraction image becomes similar to that of an astigmatic system.

Arrangement of DNA in Living Sperm: A Biophysical Analysis

The submicroscopic arrangement of DNA molecules in living sperm is analyzed by new, highly sensitive polarization optical techniques. It is concluded that the molecules are arranged as a coil of a coil in sperm chromosomes, which in turn appear to be arranged in single file with a definite sequence.

Imaging of unresolved objects, superresolution, and precision of distance measurement with video microscopy

Publisher Summary This chapter discusses the utility of video microscopy to visualize, resolve, and measure widths and distances to precisions that are conventionally considered to be below the limit of the resolution of the light microscope. In an image-forming system, resolution is generally expressed as a measure of the ability to separate the images of two neighboring object points. In the case of a light microscope, producing diffraction-limited images, the resolution limit is defined as the minimum distance between two self-luminous or incoherently illuminated objects or structures whose diffraction images can visually be distinguished as coming from two points. When the diffraction images of the two points overlap to an extent that they can no longer be distinguished from that of an individual object, the two are said not to be resolved or that the distance is less than the limit of resolution. For microscopy, the contrast boosting ability of video allows the use of the best corrected objective lenses not only for bright field and fluorescence but also for polarized light and differential interference contrast (DIC) microscopy.

Fluorescence polarization of green fluorescence protein

We report here the striking anisotropy of fluorescence exhibited by crystals of native green fluorescence protein (GFP). The crystals were generated by water dialysis of highly purified GFP obtained from the jellyfish Aequorea. We find that the fluorescence becomes six times brighter when the excitation, or emission, beam is polarized parallel (compared with perpendicular) to the crystal long axis. Thus, the major dipoles of the fluorophores must be oriented very nearly parallel to the crystal long axis. Observed in a polarizing microscope between parallel polars instead of either a polarizer or analyzer alone, the fluorescence polarization ratio rises to an unexpectedly high value of about 30:1, nearly the product of the fluorescence excitation and emission ratios, suggesting a sensitive method for measuring fluorophore orientations, even of a single fluorophore molecule. We have derived equations that accurately describe the relative fluorescence intensities of crystals oriented in various directions, with the polarizer and analyzer arranged in different configurations. The equations yield relative absorption and fluorescence coefficients for the four transition dipoles involved. Finally, we propose a model in which the elongated crystal is made of GFP molecules that are tilted 60° to align the fluorophores parallel to the crystal long axis. The unit layer in the model may well correspond to the arrangement of functional GFP molecules, to which resonant energy is efficiently transmitted from Ca2+-activated aequorin, in the jellyfish photophores.

Contractile proteins in Drosophila development

In summary, we have used a multidisciplinary approach to the analysis of actomyosin-based motility during Drosophila embryogenesis. We have documented the movements of early embryogenesis with modern, video methods. We have characterized the cytoplasmic myosin polypeptide, made specific polyclonal antisera to the molecule, studied its distribution during early embryogenesis, cloned and partially characterized the gene that encodes it, and have recently completed the nucleotide sequence of a nearly full length cDNA that encodes the entire protein-coding region. We have initiated studies on myosin function in living embryos both by direct microinjection of antibodies and through classical genetics. To better understand how myosin function is regulated, we have begun analysis of its light chains. Finally, to investigate the molecular mechanism by which its function is integrated into a labile cytoskeleton, whose architecture is constantly changing, we have also investigated Drosophila spectrins. Together, these studies are designed to shed light on the dynamics of biologic form at the cellular level, with current focus on such complex processes as cytokinesis and morphogenesis.

Orientation-independent differential interference contrast microscopy

We describe a new technique for differential interference contrast (DIC) microscopy, which digitally generates phase gradient images independently of gradient orientation. To prove the principle we investigated specimens recorded at different orientations on a microscope equipped with a precision rotating stage and using regular DIC optics. The digitally generated images successfully displayed and measured phase gradients, independently of gradient orientation. One could also generate images showing distribution of optical path differences or enhanced, regular DIC images with any shear direction. Using special DIC prisms, one can switch the bias and shear directions rapidly without mechanically rotating the specimen or the prisms and orientation-independent DIC images are obtained in a fraction of a second.