Techniques

1. Video Enhanced Light Microscopy

Observation of living cells using video-enhanced light microscopy

Chromosome movements are recorded via time-lapse video until chemical fixation. Contrast is produced by differential interference. The final frame above is a record of the cell a moment before chemical fixation. The cell was then prepared for electron microscopy by dehydration and embeddment in plastic. For viewing, the specimen is cut into serial thin sections (0.07 - 0.25 microns).

2. Transmission Electron Microscopy

An electron micrograph showning one serial section corresponding to the cell in the above video sequence.

Transmission electron microscopy is useful to view high resolution structural determinations. Images are formed by passing a beam of electrons through the specimen.

3. 3D-Imaging: Tomography

Electron tomography is an image processing technique used for reconstructing a 3D volume from a set of 2D images collected on the electron microscope. The 3D reconstruction offers a significant improvement in clarity as compared to a 2D image where features from different depths are superimposed. Tomography has become an effective tool for structure/function determination at the cellular level. We used tomography to investigate the interaction of microtubules at the kinetochore plate.

The 2D images are collected from the electron microscope at 2 degree tilt intervals over an angular range from -60 to +60 degrees about a single axis. For higher resolution 3D volumes, a second set of projections is collected about a tilt axis orthogonal to the first. Colloidal gold particles placed on the specimen serve as fiduciary markers for alignment of the projections.

-60 0 +60

Projections from single tilt series

-60 0 +60

Projections from orthogonal, single tilt series

The computation of the 3D reconstruction is achieved using the modified back projection method.

The 3D reconstruction is analyzed by tracing consecutive slices of the volume.

4. Specimen Preparation

4A. Silver-enhancement of immuno-staining

Silver-enhancement of microtubules in a 0.65 micron thick section from a half spindle of a newt lung cell.

In this project the interaction of microtubules with chromosome arms is being studied using laser microsurgery to sever acentric fragments from the chromosome arms. Fragment ejection, or that lack of ejection, is followed for about 1 minute via DIC-video microscopy, and then the specimen is rapidly fixed using glutaraldehyde perfusion. We are examining cells that adopt an anaphase-like prometaphase configuration (about 4% of newt lung mitosis in culture) because the two half spindles are separated by a sufficient distance that interaction with microtubules from the opposite pole is improbable.

In this project it is vital to identify the microtubules that interact with the ejected fragment at the EM level of resolution. Our approach is to immuno-stain the microtubules with anti-tubulin primary and FITC-conjugated secondary antibodies, stain the chromosome with Hoechst 33258 dye, and use a through focus series with deconvolution software to obtain a low resolution 3D reconstruction of the half-spindle. The specimen is then stained with a tertiary antibody conjugated either with 1 nm gold or 1.4 nm Nanogold, and the gold particles silver-enhanced using the Danscher procedure. Stained specimens are embedded in Epon and 0.5 –1.0um thick serial sections cut. The immuno-fluorescence 3D reconstruction enables us to follow individual microtubules over their full length, which in turn provides a guide as to which microtubules should be followed via electron microscopy. Tomographic reconstructions are then computed for the fragment in appropriate sections. Silver staining is required to give the microtubules sufficient contrast to be reliably followed in thick sections.

High Magnification view of the above spindle. Sliver-stained microtubules are readily apparent.

4B. High pressure freezing/freeze-substituition
Conventional specimen preparation protocols include chemical fixation, dehydration, and plastic embedment procedures that were primarily developed more than 30 years ago. These methods are known to extract the cytoplasmic components and induce subtle, and even not so subtle, structural changes in cellular ultrastructure. Indeed, although largely ignored when interpreting kinetochore structure, chromosomes in cells fixed by conventional buffered glutaraldehyde/osmium protocols shrink up to 9% in width and length during the dehydration process. The situation is worse for pre embedment labeling immuno-EM studies, which are based exclusively on detergent extraction and fixatives that maintain antigenicity but not high resolution structure. For these reasons we have begun using specimen preparation methods based upon rapid vitreous freezing followed by freeze substitution. Although suspensions of cellular components, and a few types of whole cell preparations, can be vitreously frozen by plunging into a cryogen, most tissue culture specimens require use of a high pressure freezing apparatus to avoid structural damage from the formation of crystalline ice. Thus for most tissue culture samples, high-pressure freezing followed by freeze substitution is generally considered to be the method-of- choice for faithfully preserving cell structure at the highest possible resolution. During the freezing process internal and external cellular constituents, including their soluble components, are immobilized in their native structure and position in just a few milliseconds. During the freeze substitution step an organic solvent (methanol or acetone) is then substituted for the vitreous ice at -90oC, which minimizes the surface tension-mediated collapse and shrinkage that occurs when water is rapidly and progressively removed at room temperature. By incorporating a fixative into the freeze substitution dehydrant, the structure, location, and spatial relationships of components are preserved from distortions that occur in conventional preparations due to a fixation front that sweeps through the cell at room temperature. The superior preservation produced by high pressure freezing/freeze substitution is clear from the observations that: 1) vesicles appear circular and their surrounding membrane is smooth and continuous rather than broken rippled; 2) the cytoplasm is free from signs of extraction and coagulation; 3) ribosomes are evenly distributed and not clumped or extracted; and 4) cytoskeleton elements, particularly actin and microtubules, appear as straight linear elements or smooth arcs.: Return to Main Menu