Adult male mice were exposed to either alternating illumination or constant illumination for 70 days. Light and dark pinealocytes were compared as to distribution within the gland and ultrastructure. Quantitative studies with the electron microscope revealed a significant reduction in pinealocyte size and Golgi complex size in constant light treatment, as well as a marked but nonsignificant reduction in the concentration of lipid droplets and irregular vacuoles. Under constant light treatment the cross-sectional area of pinealocyte pericapillary terminals and the number of granulated vesicles per terminal decreased significantly. A greater number of mitochondria appeared swollen, with rarified matrix and reduced numbers of cristae, with constant light treatment. These results provide ultrastructural correlation with the known reduction of pineal weight, protein synthesis and antigonadotrophic activity that is seen with constant light treatment. The marked decrease in concentration of pinealocyte granulated vesicles in constant light treatment gives morphological support to the theory that these vesicles contain antigonadotrophic secretory material.
Examination of surface markers on lymphocytes from the normal peripheral circulation has revealed a heterogeneity of lymphocyte populations.' Many of the surface marker tests performed in a routine clinical immunology and pathology laboratory utilise specific marker particles which adhere to receptors on the cell surface, forming a rosette which is visible by light microscopy. The ultrastructural detail of the central rosette-forming cell (CRFC) and the mode of interaction between the CRFC and its marker particles have been investigated in only a limited number of situations. In addition, the necessity for examining lymphocytes at the ultrastructural level has received new impetus after Payne and Glasser2 determined that lymphocytes which contain specific organelles called parallel tubular arrays represent a distinct subpopulation of lymphocytes.We have examined many rosetted preparations at the ultrastructural level3 4 and have encountered two major technical difficulties in preparing them for transmission electron microscopy. The first involved the undesirable packing of unrosetted cells around the rosettes after centrifugation. The second involved the dissolution of the pellet during subsequent dehydration steps, often resulting in an inadequate specimen for evaluation. A survey of the literature revealed that most authors did not discuss these problems in their methodology sections.5-12 Most authors simply state that 'cell pellets were fixed, dehydrated, and embedded' for electron microscopy. We therefore present a simple technique for routinely preparing rosettes for electron microscopy which overcomes these two major technical difficulties and may be incorporated into any clinical pathology laboratory set-up. Material and methodsRosettes were prepared by mixing a suspension of white blood cells (1 x 106 cells/ml) with an equal Received for publication 12 September 1979 volume of a red blood cell suspension (1-5 x 107 cells/ml). The volume of the final rosetted preparation prepared for electron microscopy varied between 0-2 and 4 ml. An equal volume of 3% glutaraldehyde in 0*1 M phosphate buffer (pH 7-2) was then added to the rosetted cell suspension and gently mixed by drawing the suspension up and down in a glass pipette. Any pellet present was likewise resuspended. The rosetted preparations were allowed to pre-fix in the diluted glutaraldehyde for 1 hour at room temperature. The suspension was then gently centrifuged at 1000 rpm in a Sorvall GLC-1 swinging bucket clinical centrifuge for 5 minutes. The pellet was resuspended in a small aliquot of human plasma and transferred to a large (17 x 100 mm) round-bottomed plastic centrifuge tube (No. 2059 tube; Falcon, Oxnard, Calif). The suspension was again centrifuged at 1000 rpm in the swinging bucket clinical centrifuge for 5 minutes.The supernatant was removed and replaced with cold 3 % glutaraldehyde in 0 1 M phosphate buffer. The pellet was allowed to fix for 1 hour at 4°C. The fixative was then decanted and replaced with 0-1 M phosphate buffer. After ...
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