Energy-dispersive X-ray microanalysis of air-dried microdroplets containing a macromolecular solute.

In the preparation of microdroplets of biological fluids for X-ray microanalysis, we have found that incorporation of a macromolecular solute, dextran, to a final concentration of 1.5-2.5% retards crystal formation and produces sufficiently uniform deposits on thin films to allow droplets to be analysed without prior freeze-drying. Analyses have been carried out at 20 kV in a scanning electron microscope, using energy-dispersive spectrometry. Absorption of Na X-rays by the added solute can be significant but its effect is minimized by preparing droplets as thin as possible, and by using standards of similar composition. The minimum detectable concentrations are increased because of the extra background contribution, and for a single determination are about 6 mM for Na and 2 mM for Cl and K. These concentrations can be further reduced by measuring replicates. The reproducibility of analysis is significantly improved (to less than 5% for Na and K) over the use of calibration curves by calculating the element concentrations from a known element in the sample, chlorine. Under our analytical conditions loss of Cl did not occur. This method requires that the Cl is measured separately by microcoulometry, but eliminates the need for a range of standard droplets on the grid, and determination of the unknowns is then independent of droplet volume, beam current, counting time and magnification. We have compared, with biological samples, the results from using Cl as an internal standard with those obtained using an added standard element, cobalt. The reproducibility using Cl was approximately two-times better than that obtained with Co, probably because of unavoidable volumetric errors when the Co is pipetted separately.

Ultrastructure and chemical composition of calcite urinary calculi in Chinese swamp buffalo.

Four urinary calculi, derived from Chinese swamp buffalo, were studied by using qualitative chemical analysis, X-ray diffraction, scanning electron microscopy and qualitative energy dispersive (electron probe) microanalysis. Qualitative chemical analysis showed that the predominant ions were calcium and carbonate with small amounts of magnesium and ammonium. X-ray diffraction confirmed that the calculi were primarily composed of calcium carbonate (calcite). On ultrastructural examination, three apparently distinct structural regions were identified in the calculi: outer large laminations; cavities containing variable numbers of small spheres and rods; and large spheres. There did not appear to be material that acted as a nidus and all regions, on qualitative electron probe analysis, contained primarily calcium with trace amounts of magnesium, phosphorus, potassium and chloride. It was concluded that calcite calculi in Chinese swamp buffalo are probably formed through a process of asynchronous layering and that nidus formation may not be necessary. Moreover, the ultrastructure of the calcite calculi is similar to that reported for siliceous calculi in ruminants and this suggests that similar factors may be involved in their formation.

Morphological differences between sub-populations of human lymphocytes revealed by scanning electron microscopy.

Human lymphoid cells were examined by scanning electron microscopy (SEM) to see if a correlation existed between surface morphologic features and the presence of various surface markers and receptors. When viewed by SEM thymocytes appeared as smooth-surfaced cells with few surface microvilli; peripheral blood lymphocytes (PBL) on the other hand were moderately to densely villate with no entirely smooth-surfaced cells observed. Surface morphology within PBL samples was not uniform, due mainly to variations in the shape and number of microvilli. However, 2 distinctive types of surface morphology (termed Types 1 and 2) were discernable with a small number of cells displaying features of both groups (Type 3). The majority of E-rosette forming cells (T lymphocytes) displayed Type 1 and the majority of cells bearing demonstrable surface immunoglobulin (B lymphocytes) displayed Type 2 morphology. Exposure of PBL to anti-T cell specific ALG resulted in cytolysis of cells with Type 1 morphology while cells with Type 2 morphology appeared largely unaffected. PBL with Fc and C3 receptors displayed all 3 types of morphology. It is concluded that T and B lymphocytes do have subtle but nevertheless discernable differences in surface morphology and within these 2 groups, variations in surface morphology are probably associated with changes in the physiological status of the cell.

Structure of a calcium-binding carp myogen.

The amino-acid sequence and three-dimensional structure of a calcium-binding protein prepared from carp muscle has been determined. This protein, designated carp-muscle calcium-binding protein B, is one of three closely related parvalbumins found in this tissue. The electron density map, calculated by heavyatom substitution crystallographic methods to 2.0-A resolution, reveals the orientation of most of the amino-acid side chains. The calcium coordination site consists of one glutamic- and three aspartic-acid carboxyl groups in a tetrahedral arrangement. The core of this spherical molecule is remarkably hydrophobic, with 8 of its 10 phenylalanine side chains packed in an approximate herringbone pattern. 52 of the 108 residues are in six alpha-helixes; there is no beta-pleated sheet. The acetylated amino-terminal alanine appears not to be accessible to solvent. All of the heavy-atom derivatives are bound at the sole cysteine. The properties of this protein suggest a relationship to troponin A of mammalian tissue.