Phase Contrast Microscopy

We introduce a laser-patterned tape as a master for replica moulding microfluidics in poly(dimethylsiloxane) (PDMS). Normally, a laser is applied to scribe microchannels directly on poly(methyl methacrylate) (PMMA) substrates. This direct engraving usually offers a faster turn-around time than conventional soft lithography but generates rough surfaces which perform poorly under phase-contrast microscopy imaging. Using a laser-patterned tape as the master

Microscopy images must be acquired at the optimal focal plane for the objects of interest in a scene. Although manual focusing is a standard task for a trained observer, automatic systems often fail to properly find the focal plane under different microscope imaging modalities such as bright field microscopy or phase contrast microscopy. This article assesses several autofocus algorithms applied in the study of fluorescence-labeled tuberculosis bacteria. The goal of this work was to find the optimal algorithm in order to build an automatic real-time system for diagnosing sputum smear samples, where both accuracy and computational time are important. We analyzed 13 focusing methods, ranging from well-known algorithms to the most recently proposed functions. We took into consideration criteria that are inherent to the autofocus function, such as accuracy, computational cost, and robustness to noise and to illumination changes. We also analyzed the additional benefit provided by preprocessing techniques based on morphological operators and image projection profiling. © 2012 International Society for Advancement of Cytometry

Rhodomyrtone [6,8-dihydroxy-2,2,4,4-tetramethyl-7-(3-methyl-1-oxobutyl)-9-(2-methylpropyl)-4,9-dihydro-1H-xanthene-1,3(2H)-di-one] from Rhodomyrtus tomentosa (Aiton) Hassk. displayed significant antibacterial activities against Gram-positive bacteria including Bacillus cereus, Bacillus subtilis, Enterococcus faecalis, Staphylococcus aureus, methicillin-resistant S. aureus (MRSA), Staphylococcus epidermidis, Streptococcus gordonii, Streptococcus mutans, Streptococcus pneumoniae, Streptococcus pyogenes, and Streptococcus salivarius. Especially noteworthy was the activity against MRSA with a minimum inhibitory concentration (MIC) and a minimum bactericidal concentration (MBC) ranging from 0.39 to 0.78 μg/ml. As shown for S. pyogenes, no surviving cells were detected within 5 and 6 h after treatment with the compound at 8MBC and 4MBC concentrations, respectively. Rhodomyrtone displays no bacteriolytic activity, as determined by measurement of the optical density at 620 nm. A rhodomyrtone killing test with S. mutans using phase contrast microscopy showed that this compound caused a few morphological changes as the treated cells were slightly changed in color and bigger than the control when they were killed. Taken together, the results support the view that rhodomyrtone has a strong bactericidal activity on Gram-positive bacteria, including major pathogens.

Plasmodium berghei sporozoites delivered by mosquito bite were more infectious to outbred CD-1 mice than were sporozoites delivered by intravenous inoculation. The route of challenge also affected vaccine efficacy. In view of these findings and the fact that mosquito bites are the natural mode of sporozoite delivery, infectious mosquito bites should be considered the challenge protocol of choice for sporozoite vaccine efficacy trials.

The ocular humors are relatively low in protein, yet cell growth in the human capsular bag still occurs after extracapsular cataract extraction (ECCE) surgery. This resilient growth gives rise to posterior capsule opacification (PCO) in a significant proportion (30%) of patients. This study compared the ability of human lens cells to proliferate in serum-supplemented and protein-free medium. Sham cataract operations were performed on human donor eyes. The capsular bag was dissected free, pinned flat on a petri dish, and incubated in Eagle's minimal essential medium (EMEM) alone or in EMEM supplemented with 10% fetal calf serum. Observations were made by phase-contrast microscopy. At the endpoint, capsules were studied by fluorescence or electron microscopy. Mitotic activity was identified using Bromo-2-deoxyuridine labeling and detection techniques. When required, an intraocular lens was implanted when surgery was performed. It was found that human lens cells from a wide age spe...

Several strains of a novel heterotrophic gas vacuolate bacterium were isolated from antarctic marine waters. The results of phylogenetic analyses in which 16S ribosomal DAN sequencing was used, coupled with phenotypic tests, indicated that strain 34-P(T) (T = type strain) belongs to a new genus and species of the beta subgroup of the Proteobacteria, for which the name Polaromonas vacuolata is proposed. Although the other four strains studied probably belong to this new species, DNA-DNA hybridization tests were not conducted. The closest phylogenetic relatives of P. vacuolata are the photosynthetic nonsulfur purple bacterium Rhodoferax fermentans and the hydrogen autotroph Variovorax paradoxus.

Our previous work demonstrated that the 12-lipoxygenase metabolite of arachidonic acid, 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE] induced a nondestructive and reversible retraction of cultured endothelial cells. In the current study we tested the hypothesis that tumor cells produce 12(S)-HETE during their interactions with endothelial cells which in turn induces endothelial cell retraction. Coincubation of Lewis lung carcinoma cells or elutriated B16 amelanotic melanoma (B16a) cells but not 3T3 fibroblasts with microvascular endothelial cells (CD3) resulted in a time- and concentration-dependent retraction of the CD3 monolayers as revealed by quantitative binding assays and phase contrast microscopy. Lewis lung carcinoma cell-induced endothelial cell retraction was blocked by specific lipoxygenase inhibitors but not by cyclooxygenase inhibitors, suggesting the involvement of a lipoxygenase metabolite(s). Radioimmunoassay and high-performance liquid chromatography analysis of t...

... The “spatiotemporal” imaging method will permit direct examination of nonlinear lattice dynamics and will provide feedback in coherent control systems that use spatiotemporal femtosecond pulse shaping to manipulate propagating excitations with ultrashort light pulses directed ...

A simple and fast method is presented which detects mitotic cells from two cell lines imaged in two phase-contrast microscopy techniques. Such detection is a first step to more sophisticated image processing tasks like determination of mitotic index or mitotic cell tracking in time-lapse movies. Detection algorithm is based on template matching approach that provides a list of candidates. The list is then pruned by validation algorithm that takes into account a priori information about mitotic cells. The method has been implemented as plugin for ImageJ and has been tested for several different data sets.

Experiments were performed to demonstrate that adherence of Helicobacter pylori to gastric epithelial cells causes alterations in the cell cytoskeleton. H. pylori intimately attached to cultured human gastric epithelial cells on small cellular projections, while there was no intimate association of H. pylori with cultured human esophageal epithelial cells. Fluorescein-conjugated phalloidin staining of gastric epithelial cells showed that H. pylori adherence stimulated actin polymerization; this stimulation was not observed with esophageal cells. Also, this organism's selectivity for gastric mucosa was supported by rare binding of bacteria to esophageal epithelial cells and gastric fibroblasts.