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Asthma is a chronic inflammatory airway disease driven by various infiltrating immune cell types into the lung. Optical microscopy has been used to study immune infiltrates in asthmatic lungs. Confocal laser scanning microscopy (CLSM) identifies the phenotypes and locations of individual immune cells in lung tissue sections by employing high-magnification objectives and multiplex immunofluorescence staining. In contrast, light-sheet fluorescence microscopy (LSFM) can visualize the macroscopic and mesoscopic architecture of wholemount lung tissues in three dimensions (3D) by adopting an optical tissue-clearing method. Despite each microscopy method producing image data with unique resolution from a tissue sample, CLSM and LSFM have not been applied together because of different tissue-preparation procedures. Here, we introduce a new approach combining LSFMand CLSM into a sequential imaging pipeline. We built a new optical tissue clearing workflow in which the immersion clearing agent can be switched from an organic solvent to an aqueous sugar solution for sequential 3D LSFMand CLSMof mouse lungs. This sequential combination microscopy offered quantitative 3D spatial analyses of the distribution of immune infiltrates in the samemouse asthmatic lung tissue at the organ, tissue, and cell levels. These results show that ourmethod facilitates multiresolution 3D fluorescencemicroscopy as a new imaging approach providing comprehensive spatial information for a better understanding of inflammatory lung diseases. [ABSTRACT FROM AUTHOR]

We have developed a method to visualize fluorescent protein-labeled [beta]-cells in the intact pancreas through combined reflection and confocal imaging. This method provides a 3-D view of the [beta]-cells in situ. Imaging of the pancreas from mouse insulin I promoter (MIP)-green (GFP) and red fluorescent protein (RFP) transgenic mice shows that islets, [beta]-cell clusters, and single [beta]-cells are not evenly distributed but are aligned along the large blood vessels. We also observe the solitary [beta]-cells in both fetal and adult mice and along the pancreatic and common bile ducts. We have imaged the developing endocrine cells in the embryos using neurogenin-3 (Ngn3)-GFP mice crossed with MIP-RFP mice. The dual-color-coded pancreas from embryos (E15.5) shows a large number of green Ngn3-expressing proendocrine cells with a smaller number of red [beta]-cells. The imaging technique that we have developed, coupled with the transgenic mice in which [beta]-cells and [beta]-cell progenitors are labeled with different fluorescent proteins, will be useful for studying pancreatic development and function in normal and disease states. development of pancreas; islet formation

Insulin secretion is dependent on coordinated pancreatic islet physiology. In the present study, we found a way to overcome the limitations of cellular electrophysiology to optically determine cell membrane potential ([V.sub.m]) throughout an islet by using a fast voltage optical dye pair. Using laser scanning confocal microscopy (LSCM), we observed fluorescence (Forster) resonance energy transfer (FRET) with the fluorescent donor N-(6-chloro-7-hydroxycoumarin-3-carbonyl)-dimyristoyl phosphatidyl-ethanolamine and the acceptor bis-(1,3-diethylthiobarbiturate) trimethine oxonol in the plasma membrane of essentially every cell within an islet. The FRET signal was approximately linear from [V.sub.m] -70 to +50 mV with a 2.5-fold change in amplitude. We evaluated the responses of islet cells to glucose and tetraethylammonium. Essentially, every responding cell in a mouse islet displayed similar time-dependent changes in [V.sub.m]. When [V.sub.m] was measured simultaneously with intracellular [Ca.sup.2+], all active cells showed tight coupling of [V.sub.m] to islet cell [Ca.sup.2+] changes. Our findings indicate that FRET-based, voltage-sensitive dyes used in conjunction with LSCM imaging could be extremely useful in studies of excitation-secretion coupling in intact islets of Langerhans. pancreatic [beta]-cell; optical electrophysiology; islet electrical coupling

Imaging endoplasmic reticulum calcium with a fluorescent biosensor in transgenic mice. Am J Physiol Cell Physiol 287: C932-C938, 2004. First published May 26. 2004; 10.1152/ajpcell.00151.2004.--The use of biosynthetic fluorescent sensors is an important new approach for imaging [Ca.sup.2+] in cells. Genetically encoded indicators based on green fluorescent protein, calmodulin, and fluorescence resonance energy transfer (FRET) have been utilized to measure [Ca.sup.2+] in nonmammalian transgenic organisms and provide information about the organization and regulation of [Ca.sup.2+] signaling events in vivo. However, expression of biosynthetic FRET-based [Ca.sup.2+] indicators in transgenic mammals has proven to be problematic. Here, we report transgenic expression of an endoplasmic reticulum (ER) [Ca.sup.2+] biosensor in mouse pancreas. We targeted expression of a yellow cameleon3.3er (YC3.3er) transgene with mouse insulin I promoter. YC3.3er protein expression was limited to pancreatic [beta]-cells within islets of Langerhans and absent in the exocrine pancreas and other tissues. Animals developed and matured normally; sensor expression was unaffected by age. Glucose tolerance in transgenic mice was also unaffected, indicating the transgenic biosensor did not impair endocrine pancreas function. ER [Ca.sup.2+] responses after administration of thapsigargin, carbachol, and glucose were measured in individual [beta]-cells of intact islets using confocal microscopy and confirmed the function of the biosensor. We conclude that controlling transgene transcription with a cell-specific promoter permits transgenic expression of FRET-based [Ca.sup.2+] sensors in mammals and that this approach will facilitate real-time optical imaging of signal transduction events in living tissues. intracellular calcium; cameleon; fluorescence resonance energy transfer

An oligonucleotide-based microarray analysis of 9,500 genes and expressed sequence tags (ESTs) demonstrated that the type 1 inositol 1,4,5-trisphosphate receptor (I[P.sub.3]R) was significantly down-regulated in Bcl-[X.sub.L]-expressing as compared with control cells. This result was confirmed at the mRNA and protein levels by Northern and Western blot analyses of two independent hematopoietic cell lines and murine primary T cells. Bcl-[X.sub.L] expression resulted in a dose-dependent decrease in I[P.sub.3]R protein. I[P.sub.3]R expression is regulated as part of a mitochondrion-to-nucleus stress-responsive pathway. The uncoupling of mitochondrial oxidative phosphorylation resulted in induction of binding of the transcription factor NFATc2 to the I[P.sub.3]R promoter and transcriptional activation of I[P.sub.3]R. Expression of Bcl-[X.sub.L] led to a decreased induction of both NFATc2 DNA binding to the I[P.sub.3]R promoter and I[P.sub.3]R expression in response to the inhibition of mitochondrial oxidative phosphorylation. The Bcl-[X.sub.L]-dependent decrease in I[P.sub.3]R expression also correlated with a reduced T cell antigen receptor ligation-induced [Ca.sup.2+] flux in Bcl-[X.sub.L] transgenic murine T cells, and microsomal vesicles prepared from Bcl-[X.sub.L]-overexpressing cells exhibited lower I[P.sub.3]-mediated [Ca.sup.2+] release capacity. Furthermore, reintroducing I[P.sub.3]R into Bcl-[X.sub.L]-transfected cells partially reversed Bcl-[X.sub.L]-dependent anti-apoptotic activity. These results suggest that even under non-apoptotic conditions, expression of Bcl-2-family proteins influences a signaling network that links changes in mitochondrial metabolism to alterations in nuclear gene expression.

Isolated rat basilar smooth muscle cells were treated with hemolysate from red blood cells to identify the vasoconstrictive agent which causes cerebral vasospasm following subarachnoid hemorrhage. The results show that hemolysate triggers the release of intracellular Ca2+ stores and Ca2+ entry into cells via a voltage-independent Ca2+ channel. Furthermore, a molecule less than 1.0 kilodalton, possibly ATP, was identified as the active hemolysate component involved in the elevation of intracellular Ca2+ concentration leading to vasoconstriction in cerebral arteries.

The transforming growth factor type beta (TGF-beta) mediates calcium homeostasis and improves neuronal Bcl2 protein expression in rat hippocampal neurons, thereby protecting them from toxic insults. TGF-beta prevents neuronal death associated with Ca2+ overloading in response to the glutamatergic agonist N-methyl-D-aspartate or the Ca2+ ionophore 4-Br-A23187. Rat hippocampal neurons are also protected from death associated with excitotoxicity, trophic factor removal and oxidative injury.