| Spectral Unmixing of Flavin Autofluorescence Components in Cardiac Myocytes Biophysical Journal, Volume 89, Issue 6, 1 December 2005, Pages L55-L57 D. Chorvat, J. Kirchnerova, M. Cagalinec, J. Smolka, A. Mateasik and A. Chorvatova Abstract We applied linear unmixing approach to reveal individual components of intrinsic flavin fluorescence signal recorded in living cardiac cells by spectrally resolved confocal microscopy. Responses of whole-cell autofluorescence to modulators of cell metabolism and respiration were used as a tool of separation of its components; their spectral profiles, estimated by principal component analysis, correspond to free FAD and FAD bound to different enzymes of electron transport chain. Abstract | Full Text | PDF (160 kb) |
| Microspectroscopic imaging of nodulation factor-binding sites on living Vicia sativa roots using a novel bioactive fluorescent nodulation factor Biophysical Journal, Volume 72, Issue 5, 1 May 1997, Pages 1986-1996 T.W. Gadella, G. Vereb, A.E. Hadri, H. Röhrig, J. Schmidt, M. John, J. Schell and T. Bisseling Abstract A novel bioactive fluorescent nodulation (Nod) factor, NodRlv-IV(BODIPY FL-C16), has been synthesized by attaching a BODIPY FL-C16 acyl chain to the primary amino group of chitotetraose deacetylated at the nonreducing terminus by recombinant NodB. The binding of the fluorescent Nod factor to root systems of Vicia sativa was investigated with fluorescence spectral imaging microscopy (FSPIM) and fluorescence ratio imaging microscopy (FRIM). Spatially resolved fluorescence spectra of living and labeled Vicia sativa root systems were measured by FSPIM. Strong autofluorescence, inherent to many plant systems when excited at 488 nm, was corrected for by utilizing the difference in fluorescence emission spectra of the autofluorescence and NodRlv-IV(BODIPY FL-C16). A methodology is presented to break down the in situ fluorescence emission spectra into spatially resolved autofluorescence and BODIPY FL fluorescence spectra. Furthermore, an FRIM method was developed for correcting autofluorescence in fluorescence micrographs for this system. After autofluorescence correction it was shown that NodRlv-IV(BODIPY FL-C16) was concentrated in the root hairs, but was also bound to other parts of the root surface. Abstract | PDF (2327 kb) |
| Different Metabolic Responses in α-, β-, and δ-Cells of the Islet of Langerhans Monitored by Redox Confocal Microscopy Biophysical Journal, Volume 90, Issue 7, 1 April 2006, Pages 2641-2650 Ivan Quesada, Mariana G. Todorova and Bernat Soria Abstract Blood glucose homeostasis is mainly achieved by the coordinated function of pancreatic -, -, and -cells, which secrete glucagon, insulin, and somatostatin, respectively. Each cell type responds to glucose changes with different secretion patterns. Currently, considerable information can be found about the signal transduction mechanisms that lead to glucose-mediated insulin release in the pancreatic -cell, mitochondrial activation being an essential step. Increases in glucose stimulate the mitochondrial metabolism, activating the tricarboxylic acid cycle and raising the source of redox electron carrier molecules needed for respiratory ATP synthesis. However, little is known about the glucose-induced mitochondrial response of non--cells and its role in the stimulus-secretion coupling process. This limited information is probably a result of the scarcity of these cells in the islet, the lack of identification patterns, and the technical limitations of conventional methods. In this study, we used flavin adenine dinucleotide redox confocal microscopy as a noninvasive technique to specifically monitor mitochondrial redox responses in immunoidentified -, -, and -cells in freshly isolated intact islets and in dispersed cultured cells. We have shown that glucose provokes metabolic changes in - and -cell populations in a dose-dependent manner. Conversely, no significant responses were observed in -cells, despite the sensitivity of their metabolism to drugs acting on the mitochondrial function, and their intact ability to develop Ca signals. Identical results were obtained in islets and in cultures of dispersed cells. Our findings indicate metabolic differences in glucose utilization among the -, -, and -cell populations, which might be important in the signal transduction events that lead to hormone release. Abstract | Full Text | PDF (471 kb) |
Copyright © 1998 The Biophysical Society. All rights reserved.
Biophysical Journal, Volume 74, Issue 4, 1722-1731, 1 April 1998
doi:10.1016/S0006-3495(98)77883-X
Richard Bertram*,
,
and Mark Pernarowski#
* School of Science, Pennsylvania State University, Erie, Pennsylvania 16563 USA
# Department of Mathematical Sciences, Montana State University, Bozeman, Montana 59717 USA
Address reprint requests to Dr. Richard Bertram, School of Science, Pennsylvania State University, Erie, PA 16563. Tel.: 814-898-6090; Fax: 814-898-6213.We investigate the time required for glucose to diffuse through an isolated pancreatic islet of Langerhans and reach an equilibrium. This question is relevant in the context of in vitro electrophysiological studies of the response of an islet to step changes in the bath glucose concentration. Islet cells are electrically coupled by gap junctions, so nonuniformities in islet glucose concentration may be reflected in the activity of cells on the islet periphery, where electrical recordings are made. Using a mathematical model of hindered glucose diffusion, we investigate the effects of the islet porosity and the permeability of a surrounding layer of acinar cells. A major factor in the determination of the equilibrium time is the transport of glucose into islet β-cells, which removes glucose from the interstitial spaces where diffusion occurs. This transport is incorporated by using a model of the GLUT-2 glucose transporter. We find that several minutes are required for the islet to equilibrate to a 10mM change in bath glucose, a typical protocol in islet experiments. It is therefore likely that in electrophysiological islet experiments the glucose distribution is nonuniform for several minutes after a step change in bath glucose. The delay in glucose penetration to the inner portions of the islet may be a major contributing factor to the 1–2-min delay in islet electrical activity typically observed after bath application of a stimulatory concentration of glucose.