Digital confocal microscopy allows measurement and three-dimensional multiple spectral reconstruction of Neisseria gonorrhoeae/epithelial cell interactions in the human fallopian tube organ culture model.

Abstract

A strategy for measuring Neiseria gonorrhoeae attachment and invasion in the human Fallopian tube organ culture ( m c ) model via computerized image analysis (CIA) combined with "digital" confocal microscopy (DCM) was tested. DCM on serial image stacks of fluorescent latex beads reduced out-of-focus light propagation in theZ-axis @<0.005) and improved the shape factor of lateral three-dimensional reconstructions of the beads @<0.001). Sections of tissue infected for 44 hr with piliated, Opt+ gonococci were stained with fluorescein-labeled monoclonal a n t i -g o n d antibodies, rhodamine-labeled phalloidin, and Hoechst 33342. Serial images collected at identical focal planes for each fluorochrome were subjected to DCM. Epithelial cytoplasmic regions of interest defied by rhodamine-stained actin

Invasion of Human Fallopian Tube Epithelium byEscherichia coliExpressing Combinations of a Gonococcal Porin, Opacity‐Associated Protein, and Chimeric Lipo‐oligosaccharide

“…Contrast was adjusted to give a dark background, with the organisms appearing as bright spots. From these slides identical dilutions that gave good separation of individual organisms or organism clumps were chosen for image analysis [56]. Microscope fields were randomly selected, and digital images were acquired with a computer-controlled charge-coupled device camera (Dage MTI).…”

“…The 6 2 ϫ 10 infection was allowed to proceed for 44 h, and the tissue was fixed in 95% ethanol/5% acetic acid, was prepared for paraffin embedding, and was cut into 8-mm sections. A triple fluorescent stain protocol was used to stain bacteria, cell cytoplasm, and cell nuclei [56]. Bacteria were stained green by using a rabbit anti-E. coli primary antiserum coupled with a fluoresceinated goat antirabbit secondary antibody (Sigma).…”

“…Experimental infections of human fallopian tube organ cultures were performed, as described elsewhere (with the addition of ampicillin and chloramphenicol selection) [56,58]. In brief, viable human fallopian tube tissue was obtained from fresh surgical pathology specimens.…”

“…For each experimental group, the sizes of у2000 organism clumps were measured with computer software (Image 1; Universal Imaging). As described elsewhere [56], the software was calibrated for measurement in square micrometers with a stage micrometer, and organisms were distinguished from the dark background for measurement by using a grayscale threshold that was held constant for all groups. All computer-controlled image acquisition and analysis was carried out on a personal computer (Pentium Pro 100 MHz; Vaytek), running the Windows 95 operating system (Microsoft).…”

Aberrant DNA Polymerase α Is Excluded from the Nucleus by Defective Import and Degradation in the Nucleus

“…DNA was stained with Hoechst 33258 (blue). To detect endogenous p180 in tsFT20 cells, digital images were captured and processed with fluorescence microscopy and deconvolution analysis using a nearest neighbor method with HazeBuster software (VayTek, Inc.) (48). The stack images of 10 optical sections with a step size of 200 nm were then deconvolved in three dimensions.…”