A confocal Raman microscope is used to study the protein distribution inside biological cells. It is shown that high quality Raman imaging of the protein distribution can be obtained using confocal nonresonant Raman imaging ( exc ϭ 647.1 nm). The results are shown for two different human cell types. Perpheral blood lymphocytes are used as an example of the fully maturated cells with a low level of nuclear transcription. Human eye lens epithelial cells are used as an example of cells with a high level of nuclear activity. The protein distribution in both cell types is completely different. The nuclear distribution of the protein largely varies in the peripheral blood lymphocyte cells, while proteins are more homogenously distributed over the nuclear space in the eye lens epithelial cells. The imaging time is ϳ20 min for a field of view of 10 ϫ 10 m 2 . The size of the sampling volume is 1.4 fL using a full width at halfmaximum criterion along the z axis and a 1/e 2 criterion in the xy plane. The results presented here indicate that Raman imaging is particularly of interest in the study of cellular processes, like phagocytosis, apoptosis, chromatin compaction, and cellular differentiation, which are accompanied by relatively large-scale redistributions of the materials.

SUMMARYA number of cell types situated along interfaces of various tissues and organs such as the peritoneum and the intestine have been shown to secrete inflammatory cytokines in a polarized fashion. Retinal pigment epithelial (RPE) cells are positioned at the interface between the vascularized choroid and the avascular retina, forming part of the blood-retina barrier. These cells are potent producers of inflammatory cytokines and are therefore considered to play an important role in the pathogenesis of ocular inflammation. Whether cytokine secretion by these cells also follows a vectorial pattern is not yet known, and was therefore the subject of this study. Monolayers of human RPE cells (primary cultures and the ARPE-19 cell line) cultured on transwell filters were stimulated to produce IL-6 and IL-8 by adding IL-1b (100 U/ml) to either the upper or the lower compartment. After stimulation, the human RPE cell lines showed polarized secretion of IL-6 and IL-8 towards the basal side, irrespective of the side of stimulation. The ARPE-19 cell line also secreted IL-6 and IL-8 in a polarized fashion towards the basal side after basal stimulation; polarized secretion was, however, not apparent after apical stimulation. The observation that human RPE cells secrete IL-6 and IL-8 in a polarized fashion towards the choroid may represent a mechanism to prevent damage to the adjacent fragile retinal tissue.

Microplasmin caused vitreolysis and posterior vitreous separation in an ex vivo porcine eye model in an apparent dose- and time-dependent fashion. In this model system, the minimal effective dose appeared to be 125 microg.

This study provides scanning electron microscopic observations on the early morphogenesis of persistent hyperplastic tunica vasculosa lentis and primary vitreous (PHTVL/PHPV) in canine fetuses at days 28 35 postcoitum (D28 and D35). From previous studies regarding PHTVL/PHPV it is known that a retrolental plaque of fibrovascular tissue is present in eyes of affected canine fetuses from the D33 stage. The contribution of vitreous cells to the formation of the plaque is supported by the results of this study. The lens capsules at the stages described were not found to contain abnormalities such as transparent (thinner) parts or rents, as have been described for postnatal cases of PHTVL/PHPV. These findings support the hypothesis that the capsular anomalies observed in postnatal patients are secondary entities.

We evaluated the gross morphology, location, and fiber cell architecture of equatorial cortical opacities in the aging human lens. Using dark-field stereomicroscopy, we photographed donor lenses in toto and as thick slices. In addition, we investigated the details of the fiber cell architecture using fluorescent staining for membranes and by scanning electron microscopy. We then combined our data with data from recent studies on lens viscoelasticity. We found that small cortical and cuneiform opacities are accompanied by changes in fiber structure and architecture mainly in the equatorial border zone between the lens nucleus and cortex. Because the lens cortex and nucleus have different viscoelastic properties in young and old lenses, we hypothesize that external forces during accommodation cause shear stress predominantly in this border zone. The location of the described changes suggests that these mechanical forces may cause fiber disorganization, small cortical opacities, and ultimately, cuneiform cataracts.