John H. Luft

The influence of buffer systems on fixation with osmium tetroxide

Comparison of the microscopic and ultrastructural morphology of rat liver and pancreas after fixation in osmium tetroxide buffered with six different solutions indicates that the buffer affects fixation significantly. Differences in penetration of the fixative, stainability, and cellular appearances by light microscopy and on sectioning properties, acceptance of heavy metal “stains”, and general morphology for electron microscopy were observed. The results are not easily explained on the basis of tonicity differences alone and are interpreted as evidence in favor of the concept of specific ion effects.

Effects of glutaraldehyde fixation on the structure of tight junctions. A quantitative freeze-fracture analysis

The effects of aldehyde fixation on the structure of tight junctions in rat liver and small intestine were studied by quantitations of freeze-fractured material. The length of fibrils in percentage of the total length of the junctional strands (ridges) on P-faces and the number of particles per micrometer of E-face groove were calculated in a series of experiments. After fixation in 2.5% glutaraldehyde (GA) for 1 or 3 hr, the tight junctions of liver and small intestine were comprised of strands with about 60–80% fibrils on the P-faces, whereas the E-face grooves exhibited only a very few particles (2–5/ μm). When the tissue was fixed in 0.1% GA for 24 hr similar results were obtained. However, with decreasing time of fixation (3 hr, 1 hr, 15 min, and 0 min) in 0.1% GA before glycerol cryoprotection, a significant decrease in percentage fibrils and a corresponding increase in number of particles per micrometer of E-face groove were seen. In the 15- and 0-min material, values of only about 1–3% fibrils and as much as about 60 particles per micrometer were obtained. In fresh-frozen material (nonfixed, noncryoprotected) fibrils were completely absent and all tight junction particles (approximately 65/μm) were located in chains on the E-faces. After fixation in 1.5% formaldehyde for 1 and 3 hr, only about 6 and 9% fibrils in the liver, and about 4 and 15% fibrils in the small intestine, were obtained. With respect to E-face grooves, about 31 and 25, and 44 and 42 particles per micrometer of liver and small intestine, respectively, were obtained. These results indicate that the tight junction fibrils may be polymerization products caused by the formidable crosslinking effect of GA. It is suggested that tight junctions in vivo are comprised of discrete integral proteins with stronger binding to the external side of the membrane or to opposing junctional proteins in the adjacent membrane than to the inner side of the membrane. However, GA fixation causes (1) lateral linking of the junctional proteins resulting in the formation of fibrils, and (2) binding of the fibrils to the protein-rich inner or cytoplasmic side of the membrane. Some functional consequences of this concept are considered.

Use of a nitrogen mustard derivative in fixation for electron microscopy and observations on the ultrastructure of Tetrahymena

The ultrastructure of the ciliated protozoan Tetrahymena pyriformis has been studied from thin sections of cells fixed with a mixture of glutaraldehyde and tris-(1-aziridinyl) phosphine oxide. Some improvement in fixation has been obtained compared with results employing other fixatives in common use. The fine structure of coated pits, peroxisomes, mucocysts, nuclear pores, cilia, basal bodies, and fibrillar elements are described. Of particular interest are the cilia, nascent kinetosomes in the oral area, and the fine filamentous reticulum of the oral apparatus. These and other observations are discussed in relation to previous descriptions and possible functional significance.

Fine structure of the apex of absorptive cells from rat small intestine

These cells were studied in rats, using buffered osmium or aldehyde fixatives followed by osmium. Three groups of filaments and the terminal web were visualized in the apex. One group, seen in aldehyde-fixed tissue formed the cores and rootlets of the microvilli; they showed no continuity with other filaments. Another group formed the desmosomal web, best seen in tissue stained en bloc with uranyl acetate of phosphotungstic acid (PTA). The third group appeared as short, branched profiles between the rootlets after staining with PTA. The terminal web appeared as a dense layer with gaps, extending out to the zonula adhaerens. It seemed to embed the rootlets penetrating between their filaments. Although it appeared granular with the techniques used here, it is probable that at the molecular level the terminal web is formed by elongated branching elements. By supporting the rootlets and microvilli, the terminal web endows the apex with mechanical stability and anchors it to the body of the cell.

An Index for Quantitative Measurement of Vasoconstriction in Histologic Sections of Blood Vessels

Comparisons among blood vessels on the basis of similarity of dimensions are inaccurate and complicated because of wide variations in the amount of constriction in the vessels. A given diameter, for example, may represent a dilated state of a smaller vessel or a constricted state of a larger one. This has been long recognized as a complication in the diagnosis of vascular wall hypertrophy. In a study of hypertensive vascular hypertrophy, Furuyamal applied a standardizing criterion of vasoconstriction to vessel sections. He

Fixation for biological ultrastructure. I: A viscometric analysis of the interaction between glutaraldehyde and bovine serum albumin

The increase in viscosity resulting from mixing concentrated solutions of albumin with dilute glutaraldehyde has been investigated. The viscosity changes are slow at first, very sensitive to small changes in glutaraldehyde concentration, and non‐linear with time. The cross‐linking of the albumin by glutaraldehyde seems to be retarded by the weak mechanical shear forces produced by the viscometer. The significance of these findings to the events in tissue fixation is discussed.

Fixation for biological ultrastructure. II. Cross-linking of bovine serum albumin by nanosecond pulses of ionizing radiation

Very large doses of ionizing radiation were delivered quickly to concentrated albumin solutions by pulses of 500 keV in air. The electrons penetrated the aluminium foil bottom of the test cell and into the solution. A dose of 4.6 Mrad (4.6 × 104 Gy) produced a gel in the albumin solution comparable to the long‐term effect of 0.36% glutaraldehyde in the same albumin solution. The cross‐links created by the radiation leading to gel formation are probably irreversible and quite different from those leading to gelation in the glutaraldehyde‐albumin reaction. Single large pulses of ionizing radiation may be useful for fast fixation of cells and tissues for microscopy.

Chatterbox: A vibration monitor to detect chattered sections at the ultramicrotome

The “chatterbox” is a device for amplifying the vibrations produced as thin sections are cut. With acceptable chatterfree sections a crunching noise is heard; chattered sections produce an audible squeal which is easily distinguished from crunch. With the device the microtomist can determine at the microtome when his sections are chattered, and can then alter his cutting conditions until chatter disappears.

Effects of ruthenium red and KCL on responses of guinea pig umbilical veins.

5-Hydroxytryptamine (10(-5) M), norepinephrine (10(-5) M) and acetylcholine (10(-5) M) induced maximal contractions in isolated guinea pig umbilical vein strips. Pretreatment with ruthenium red (2.7--90 mug/ml) for ten minutes reduced 5-hydroxytryptamine and norepinephrine responses in a dose-related manner but did not affect acetylcholine-induced responses. Ruthenium red alone did not produce contractions. In the presence of KC1 (125 MM) the responses to norepinephrine and acetylcholine were enhanced significantly. The sensitivity of umbilical veins to ruthenium red may be useful in determining to what extent various vasoactive agents utilize extracellular calcium to induce contractions.