Oblique Transillumination Research Articles

Fatigue and failure mode analyses of glass infiltrated 5Y-PSZ bonded onto dentin analogues

The purpose of this study was to evaluate the fatigue survival of 5Y-PSZ zirconia infiltrated with an experimental glass and bonded onto dentin analogues. Disc-shaped specimens of a 5Y-PSZ (Katana UTML Kuraray Noritake) were cemented onto dentin analogs (NEMA G10) and divided into four groups (n = 15): Zctrl Group (control, without infiltration); Zglz Group (Glaze, compression surface); Zinf-comp Group (Experimental Glass, compression surface); Zinf-tens Group (Experimental Glass, tension surface). Surface treatments were varied. Cyclic fatigue loading, oblique transillumination, stereomicroscope examination, and scanning electron microscopy were performed. Fatigue data were analyzed (failure load and number of cycles) using survival analysis (Kaplan–Meier and Log-Rank Mantel–Cox). There was no statistically significant difference in fatigue survival between the Zglz, Zctrl, and Zinf-comp groups. The Zinf-tens group presented a significantly higher failure load when compared to the other groups and exhibited a different failure mode. The experimental glass effectively infiltrated the zirconia, enhancing structural reliability, altering the failure mode, and improving load-bearing capacity over more cycles, particularly in the group where the glass was infiltrated into the tensile surface of the zirconia. Glass infiltration into 5Y-PSZ zirconia significantly enhanced structural reliability and the ability to withstand loads over an increased number of cycles. This approach has the potential to increase the durability of zirconia restorations, reducing the need for replacements and save time and resources, promoting efficiency in clinical practice.

Portable low-cost macroscopic mapping system for all-optical cardiac electrophysiology.

All-optical cardiac electrophysiology enables the visualization and control of key parameters relevant to the detection of cardiac arrhythmias. Mapping such responses in human induced pluripotent stem-cell-derived cardiomyocytes (hiPSC-CMs) is of great interest for cardiotoxicity and personalized medicine applications. We introduce and validate a very low-cost compact mapping system for macroscopic all-optical electrophysiology in layers of hiPSC-CMs. The system uses oblique transillumination, low-cost cameras, light-emitting diodes, and off-the-shelf components (total ) to capture voltage, calcium, and mechanical waves under electrical or optical stimulation. Our results corroborate the equivalency of electrical and optogenetic stimulation of hiPSC-CMs, and similarity in conduction under pacing. Green-excitable optical sensors are combinable with blue optogenetic actuators (chanelrhodopsin2) only under very low green light ( ). Measurements in warmer culture medium yield larger spread of action potential duration and higher conduction velocities compared to Tyrode's solution at room temperature. As multiple optical sensors and actuators are combined, our results can help handle the "spectral congestion" and avoid parameter distortion. We illustrate the utility of the system for uncovering the action of cellular uncoupling agents and show extensibility to an epi-illumination mode for future imaging of thicker native or engineered tissues.

Acute loss of renal function attenuates slow leukocyte rolling and transmigration by interfering with intracellular signaling

Acute loss of renal function reduces leukocyte recruitment into inflamed tissues, and we studied the molecular basis of this using intravital microscopy of cremaster muscle and an autoperfused flow chamber system after bilateral nephrectomy or sham operation in mice. Acute loss of renal function resulted in cessation of selectin-induced slow leukocyte rolling on E-selectin/intercellular adhesion molecule 1 (ICAM-1) and P-selectin/ICAM-1. It also reduced in vivo neutrophil extravasation (assessed by reflected light oblique transillumination) without affecting chemokine-induced arrest. This elimination of selectin-mediated slow leukocyte rolling was associated with a reduced phosphorylation of spleen tyrosine kinase, Akt, phospholipase C-γ2, and p38 MAPK. However, the levels of adhesion molecules located on the neutrophil surface were not altered. Leukocytes from critically ill patients with sepsis-induced acute kidney injury showed a significantly higher rolling velocity on E-selectin/ICAM-1- and P-selectin/ICAM-1-coated surfaces compared with patients with sepsis alone or healthy volunteers. Thus, an acute loss of renal function significantly impairs neutrophil rolling and transmigration, both in vivo and in vitro. These effects are due, in part, to decreased phosphorylation of selectin-dependent intracellular signaling pathways.

Measuring surface geometry of adherent cells using oblique transillumination

We present a new technique for estimating cell surface geometry. A dish supporting adherent cells is observed using oblique transillumination and rotated in the horizontal plane using a stepping motor. The stage rotation-dependent movements of the start and end points of a shadow formed behind the illuminated cells uniquely determine the relative height differences between points along the cell surface. Thus, using custom-made apparatuses and living endothelial cells, we demonstrate that the combination of a rotating stage and oblique lighting allows for the evaluation of three-dimensional surface geometry of adherent cells. As compared to confocal microscopy and atomic force microscopy, which are commonly used for measuring cell surface geometry, this approach can be performed rapidly and is especially suitable for the observation of unstained cells over a large surface covering multiple cells at a time.

In Vivo Imaging and Quantitative Analysis of Leukocyte Directional Migration and Polarization in Inflamed Tissue

Directional migration of transmigrated leukocytes to the site of injury is a central event in the inflammatory response. Here, we present an in vivo chemotaxis assay enabling the visualization and quantitative analysis of subtype-specific directional motility and polarization of leukocytes in their natural 3D microenvironment. Our technique comprises the combination of i) semi-automated in situ microinjection of chemoattractants or bacteria as local chemotactic stimulus, ii) in vivo near-infrared reflected-light oblique transillumination (RLOT) microscopy for the visualization of leukocyte motility and morphology, and iii) in vivo fluorescence microscopy for the visualization of different leukocyte subpopulations or fluorescence-labeled bacteria. Leukocyte motility parameters are quantified off-line in digitized video sequences using computer-assisted single cell tracking. Here, we show that perivenular microinjection of chemoattractants [macrophage inflammatory protein-1α (MIP-1α/Ccl3), platelet-activating factor (PAF)] or E. coli into the murine cremaster muscle induces target-oriented intravascular adhesion and transmigration as well as polarization and directional interstitial migration of leukocytes towards the locally administered stimuli. Moreover, we describe a crucial role of Rho kinase for the regulation of directional motility and polarization of transmigrated leukocytes in vivo. Finally, combining in vivo RLOT and fluorescence microscopy in Cx3CR1gfp/gfp mice (mice exhibiting green fluorescent protein-labeled monocytes), we are able to demonstrate differences in the migratory behavior of monocytes and neutrophils.Taken together, we propose a novel approach for investigating the mechanisms and spatiotemporal dynamics of subtype-specific motility and polarization of leukocytes during their directional interstitial migration in vivo.

Matrix metalloproteinase-9 promotes neutrophil and T cell recruitment and migration in the postischemic liver

Matrix metalloproteinases-2 and -9 (MMP-2/9) are critically involved in degradation of extracellular matrix, and their inhibition is discussed as a promising strategy against hepatic ischemia-reperfusion (I/R) injury. Here, we analyzed the role of MMP-2 and -9 for leukocyte migration and tissue injury in sham-operated mice and in mice after I/R, treated with a MMP-2/9 inhibitor or vehicle. Using zymography, we show that the MMP-2/9 inhibitor abolished I/R-induced MMP-9 activation, whereas MMP-2 activity was not detectable in all groups. As demonstrated by intravital microscopy, MMP-9 inhibition attenuated postischemic rolling and adherence of total leukocytes in hepatic postsinusoidal venules, CD4+ T cell accumulation in sinusoids, and neutrophil transmigration. These effects were associated with reduction of plasma tumor necrosis factor alpha (TNF-alpha) levels and endothelial expression of CD62P. Motility of interstitially migrating leukocytes was assessed by near-infrared reflected light oblique transillumination microscopy in the postischemic cremaster muscle. Upon MMP-9 blockade, leukocyte migration velocity and curve-line and straight-line migration distances were reduced significantly as compared with the vehicle-treated I/R group. Postischemic sinusoidal perfusion failure, hepatocellular apoptosis, and alanine aminotransferase activity were only slightly reduced after MMP-9 inhibition, whereas aspartate aminotransferase activity and mortality were significantly lower. In conclusion, MMP-9 is involved in the early recruitment cascades of neutrophils and CD4+ T cells, promotes neutrophil and T cell transmigration during hepatic I/R, and is required for motility of interstitially migrating leukocytes. MMP-9 blockade is associated with an attenuation of TNF-alpha release and endothelial CD62P expression, weakly protects from early microvascular/hepatocellular I/R damage, but improves postischemic survival.

Chemokine receptors Ccr1, Ccr2, and Ccr5 mediate neutrophil migration to postischemic tissue

Leukocyte infiltration of reperfused tissue is a key event in the pathogenesis of ischemia-reperfusion. However, the role of chemokine receptors Ccr1, Ccr2, and Ccr5 for each single step of the postischemic recruitment process of leukocytes has not yet been characterized. Leukocyte rolling, firm adherence, transendothelial, and extravascular migration were analyzed in the cremaster muscle of anaesthetized C57BL/6 mice using near-infrared reflected light oblique transillumination microscopy. Prior to 30 min of ischemia as well as at 5, 30, 60, 90, and 120 min after onset of reperfusion, migration parameters were determined in wild-type, Ccr1-/-, Ccr2-/-, and Ccr5-/- mice. Sham-operated wild-type mice without ischemia were used as controls. No differences were detected in numbers of rolling leukocytes among groups. In contrast, the number of firmly adherent leukocytes was increased significantly in wild-type mice as compared with sham-operated mice throughout the entire reperfusion phase. Already after 5 min of reperfusion, this increase was reduced significantly in Ccr1-/- and Ccr5-/- mice, whereas only in Ccr2-/- mice, was adherence attenuated significantly at 120 min after onset of reperfusion. Furthermore, after 120 min of reperfusion, the number of transmigrated leukocytes (>80% Ly-6G+ neutrophils) was elevated in wild-type mice as compared with sham-operated animals. This elevation was significantly lower in Ccr1-/-, Ccr2-/-, and Ccr5-/- mice. Leukocyte extravascular migration distances were comparable among groups. In conclusion, these in vivo data demonstrate that Ccr1, Ccr2, and Ccr5 mediate the postischemic recruitment of neutrophils through effects on intravascular adherence and subsequent transmigration.

Intravital videomicroscopy of the chorioallantoic microcirculation: A model system for studying metastasis

The chick embryo is a useful model for studying hematogenous metastasis. Cancer cells injected into veins of the chorioallantoic membrane (CAM) circulate briefly through all tissues but form metastases predominantly in the CAM. This respiratory organ is particularly suitable for intravital microscope because of its accessibility without the need for surgery and the density and planar configuration of its vessels (which we confirmed by microcorrosion casting). Using an inverted microscope with oblique transillumination for high-resolution images and epifluorescence to identify labeled B16F1 melanoma cells, we studied successive stages of metastasis formation in the CAM in vivo. By 2 min postinjection (pi) all cancer cells had become arrested within the microvasculature. This initial arrest appeared to be due to size restriction, based on measurements of cell and vessel diameters. At 15–60 min pi, trapped cells were seen in tapering arterioles (27%), orifices from arterioles to the capillary plexus (61%), or in the plexus itself (12%). Some cells had extravasated into the underlying mesenchyme by 3 hr (pi), and at 24 hr all cancer cells had completed this process. The mean rate of migration out of capillary lumens was ∼1 μm/hr. Micrometastases grew in a planar configuration just beneath the capillary plexus, with a cell doubling time of ∼24 hr. Our technique is also applicable to other tumor types and host animals and provides a powerful tool to complement studies on the molecular basis of metastasis.

Selective plating medium for quantitative recovery of food-borne Listeria monocytogenes

A new plating medium (lithium chloride-ceftazidime agar [LCA]) was designed to quantitatively recover food-borne Listeria monocytogenes in the form of large colonies while inhibiting most other food-borne microorganisms. This medium included brain heart infusion agar as the nutritive agar base and a combination of selective agents (LiCl, glycine anhydride, and ceftazidime). Comparison of LCA and lithium chloride-phenylethanol-moxalactam agar (LPM) indicated that both were equally effective for the enumeration of the cold-tolerant pathogen in artificially and naturally contaminated foods. However, LCA was more effective than LPM in the recovery of sublethally heat-injured cells. Moreover, Listeria colonies on LCA exhibited a more distinct bluish hue than those on LPM when viewed by the Henry oblique transillumination technique.

Some observations on sperm transport through the uterotubal junction of the rat.

Entry of spermatozoa into the oviducts of mammals is restricted by the uterotubal junctions. The extent to which these junctions act as selective valves, or filters, for sperm transport has not been determined. A new technique has been developed that permits the direct visualization of sperm transport through the uterotubal junction of the rat in vitro. After mating or artificial insemination, the female tract is removed to a special "observation dish" containing oxygenated Earle's solution maintained at 37 degrees C. The oviducts are severed 1.0 - 1.5 mm above the uterotubal junctions. Under appropriate magnification and with oblique transillumination, spermatozoa may be observed emerging from the cut ends. It was noted that only motile spermatozoa emerged and that they usually appeared individually, with an interval of several minutes between each. Their egress was not directly related to contractions of the uterine cornu. Neither immotile spermatozoa nor a dye solution were observed to pass through the uterotubal junction. It is concluded that sperm motility is important, and probably essential, for sperm entry into the oviducts in the rat. Scanning electron microscopy revealed that the rat uterotubal junction forms a small mound or papilla projecting into the uterine cavity. No ciliated cells were observed in this region.

A method for the rapid isolation of Listeria monocytogenes from infected material.

Two media, one for enrichment and the other for differentiation of Listeria monocytogenes, are described and a method is proposed for the selective isolation of this bacterium from material containing a mixed bacterial flora such as faeces, vaginal swabs etc. Addition of potassium dichromate, chromium trioxide, thionin, nalidixic acid and amphotericin B to Todd‐Hewitt Broth (BBL) made a satisfactory enrichment broth in which good selective growth of L. monocytogenes was obtained without notable damage to cells. The differentiation agar was Trypticase Soy Agar (BBL) supplemented with gallocyanin, pyronin and nalidixic acid. On this medium L. monocytogenes colonies, when viewed by the Henry's oblique transillumination technique, were blue in contrast to colonies of other bacterial species which were pink or red. Trials with experimentally infected materials showed that L. monocytogenes could be recovered from faeces infected with as few as 20 L. monocytogenes cells/g. All common contaminants, with the exception of a few strains of Streptococcus faecalis, were inhibited.

Technical procedures for the study of the nucleolonema.

In a recent paper1 the authors have shown filamentous structures within the nucleolus of all cells. This structure was named the nucleolonema. In the present paper the technical procedures which prove their existence are described in detail.There are two groups: a) Silver impregnation procedures and iron-pyrogallic stain for fixed material; b) phase contrast microscopy, dark-field illumination and oblique transillumination for fresh material.The suitable objects are mentioned in every case in order to obtain the best results.