Finding the bottom and using it: Offsets and sensitivity in the detection of low intensity values in vivo with 2-photon microscopy

Maximizing 2-photon parameters used in acquiring images for quantitative intravital microscopy, especially when high sensitivity is required, remains an open area of investigation. Here we present data on correctly setting the black level of the photomultiplier tube amplifier by adjusting the offset to allow for accurate quantitation of low intensity processes. When the black level is set too high some low intensity pixel values become zero and a nonlinear degradation in sensitivity occurs rendering otherwise quantifiable low intensity values virtually undetectable. Initial studies using a series of increasing offsets for a sequence of concentrations of fluorescent albumin in vitro revealed a loss of sensitivity for higher offsets at lower albumin concentrations. A similar decrease in sensitivity, and therefore the ability to correctly determine the glomerular permeability coefficient of albumin, occurred in vivo at higher offset. Finding the offset that yields accurate and linear data are essential for quantitative analysis when high sensitivity is required

Optical Aberrations and Objective Choice in Multicolor Confocal Microscopy

Refinements in design have simplified confocal microscopy to the extent that it has become a standard research tool in cell biology. However, as confocal microscopes have become more powerful, they have also become more demanding of their optical components. In fact, optical aberrations that cause subtle defects in image quality in wide-field microscopy can have devastating effects in confocal microscopy. Unfortunately, the exacting optical requirements of confocal microscopy are often hidden by the optical system that guarantees a sharp image, even when the microscope is performing poorly. Optics manufacturers provide a wide range of microscope objectives, each designed for specific applications. This report demonstrates how the trade-offs involved in objective design can affect confocal microscopy

Sepsis-Induced Glomerular Endothelial Dysfunction Mediates Reductions in GFR and Increases in Protein Filtration

poster abstractBackground: Sepsis is now the leading cause of acute kidney injury (AKI) known to decrease Glomerular filtration rate (GFR) and increase proteinuria. There also exists a discrepancy between renal perfusion and GFR. Methods: To evaluate the potential role of the glomerulus in the overall pathogenesis of these abnormalities, we studied surface glomeruli in 8-10 week old Munich Wistar Frmter rats using intravital 2-photon microscopy in a cecal ligation and puncture (CLP) model of sepsis to ask targeted questions and compare the metric of measured GFR to serum creatinine changes at 24 hours post CLP. Results: Male rats undergoing CLP showed an increase in serum creatinine from 0.23 +/- 0.06 mg/dl to 0.80 +/-0.17 (P0.01) and a decrease in real time GFR from 0.69 +/- 0.06 ml/min/100gm body wt to 0.34 +/-0.15 (P0.01). Hemodynamic monitoring revealed normal and hyperdynamic cardiac status within the CLP group. Quantitative analysis of 15 glomeruli in three CLP septic rats revealed a reduction in red blood cell flow rates within capillary loops from 1,771 +/- 467 to 576 +/- 327 um/sec (P0.01); an increase in WBC adherence to glomerular capillary endothelial cells from 0.42 +/-0.33 to 7.25 +/- 5.82 WBC's/standardized glomerular volume (P0.05) in CLP rats; and an increase in the glomerular sieving coefficient (GSC) of a 150kD dextran from 0.007 +/- 0.003 to 0.097 +/- 0.046 (P0.05). Rouleaux formations were seen only in septic rats. Conclusions: These data indicate glomerular endothelial-WBC interactions during sepsis, in part, explain the reduction in GFR and increased filtration of large molecular weight proteins. The results from real time GFR accurately detected the drop in renal function for this model of sepsis

TMEM27 Suppresses Tumor Development by Promoting Ret Ubiquitination, Positioning, and Degradation

The TMEM127 gene encodes a transmembrane protein of poorly known function that is mutated in pheochromocytomas, neural crest-derived tumors of adrenomedullary cells. Here, we report that, at single-nucleus resolution, TMEM127-mutant tumors share precursor cells and transcription regulatory elements with pheochromocytomas carrying mutations of the tyrosine kinase receptor RET. Additionally, TMEM127-mutant pheochromocytomas, human cells, and mouse knockout models of TMEM127 accumulate RET and increase its signaling. TMEM127 contributes to RET cellular positioning, trafficking, and lysosome-mediated degradation. Mechanistically, TMEM127 binds to RET and recruits the NEDD4 E3 ubiquitin ligase for RET ubiquitination and degradation via TMEM127 C-terminal PxxY motifs. Lastly, increased cell proliferation and tumor burden after TMEM127 loss can be reversed by selective RET inhibitors in vitro and in vivo. Our results define TMEM127 as a component of the ubiquitin system and identify aberrant RET stabilization as a likely mechanism through which TMEM127 loss-of-function mutations cause pheochromocytoma

Optical Aberrations and Objective Choice in Multicolor Confocal Microscopy

Refinements in design have simplified confocal microscopy to the extent that it has become a standard research tool in cell biology. However, as confocal microscopes have become more powerful, they have also become more demanding of their optical components. In fact, optical aberrations that cause subtle defects in image quality in wide-field microscopy can have devastating effects in confocal microscopy. Unfortunately, the exacting optical requirements of confocal microscopy are often hidden by the optical system that guarantees a sharp image, even when the microscope is performing poorly. Optics manufacturers provide a wide range of microscope objectives, each designed for specific applications. This report demonstrates how the trade-offs involved in objective design can affect confocal microscopy

Dynamic Fingering in Adhered Lipid Membranes

Artificial lipid membranes incorporating proteins have frequently been used as models for the dynamic organization of biological structures in living cells as well as in the development of biology-inspired technologies. We report here on the experimental demonstration and characterization of a pattern-forming process that occurs in a lipid bilayer membrane adhered via biotin–avidin binding to a second lipid membrane that is supported by a solid substrate. Adhesion regions are roughly circular with a diameter of about 25 μm. Using confocal fluorescence microscopy, we record time series of dynamic fingering patterns that grow in the upper lipid membrane and intermembrane biotin–avidin bonds. The fingers are micrometer-scale elongated pores that grow from the edge of an already-stabilized hole. Finger growth is saltatory on the scale of tens of seconds. We find that as the fingers grow and the density of adhesion proteins increases, the rate of finger growth decreases exponentially and the width of newly formed fingers decreases linearly. We show that these findings are consistent with a thermodynamic description of dynamic pore formation and stabilization