Photometric calibration for quantitative spectral microscopy under transmitted illumination.

Over the past decade, there have been significant developments in the mechanisms for examination of biological and material samples. These developments exploit techniques in light microscopy to elucidate specific parts of cells and tissues, as well as inorganic particles. In recent years, spectral microscopy has become more prevalent for characterization of samples. Simultaneously, sensor technology has progressed as well and modern charge-coupled devices (CCD) cameras are now capable of achieving high spatial resolution and high sensitivity measurements of signals in the optical microscope. One major impediment in obtaining absolute quantitative information of imaged samples is the lack of automated photometric calibration mechanisms for spectral microscopes. In this paper, we present a methodology for achieving photometric calibration of an automated spectral imaging system targeted towards examination of biological samples. By acquiring spatial and spectral data simultaneously, spectral imaging allows one to exploit physical connections between a particle's morphology and its characteristic response to the optical spectrum. In composite biological material, the interpretation of the spectra is a complicated problem. This is because any light source and charge-coupled device camera used for data acquisition does not have a uniform illumination spectra and quantum efficiency, respectively, across the emitted light spectra. To balance the spectral response across individual wavelengths, our method modulates the exposure duration for the charge-coupled device camera during image acquisition. We present an image similarity based method to calibrate the system. Experiments to test the effectiveness of the calibration method under the various image similarity metrics are presented along with results to show the calibrated system's ability to accurately measure spectra based on the measured transmission profiles of optical filters.

Angiogenesis in cultured and cryopreserved pancreatic islet grafts.

BACKGROUND: The ability of rat pancreatic islets to revascularize after transplantation was examined via in vitro and in vivo imaging of the microvasculature using laser scanning confocal microscopy (LSCM). METHODS: Cultured or cryoprocessed islets were transplanted at the renal subcapsular site in rats. At various time intervals after transplantation, three-dimensional imaging of the graft was performed by LSCM. In vitro studies were conducted via microvascular corrosion casting of the grafted kidney in situations where it was difficult to obtain in vivo confocal data due to surgical complications. The vascular morphology of the islet grafts was evaluated quantitatively via digital image analysis algorithms to determine the morphology of the neovascular ingrowth and the rate of revascularization. RESULTS: In cultured islet grafts, the initiation of angiogenesis was observed within 1 week, characterized by the presence of capillary sprouts, tortuous vessels, and blood vessels with blind ends. The revascularization of the graft was typically completed within 2 weeks and could be distinguished as a network of completely perfused blood vessels consisting of intertwining capillaries, with surrounding arterioles and venules. The angiogenesis process in cryopreserved islet grafts required a longer time period to initiate (approximately 2 weeks), and the revascularization was completed in 1 week after the initiation. CONCLUSIONS: These results successfully demonstrate the potential of the described in vivo and in vitro LSCM techniques to measure the angiogenesis process in pancreatic islet grafts.

Network thermodynamic model of coupled transport in a multicellular tissue--the islet of Langerhans.

Network thermodynamic modeling via bond graphs was used to describe the water and cryoprotectant additive (CPA) transport in a multicellular tissue. The model is presented as a tool to understand the osmotic behavior of the islets of Langerhans when exposed to ternary aqueous solutions containing an electrolyte and a CPA. It accounts for the effects of the location of cells within the tissue and an interstitial matrix, plus differential permeabilities to water and CPA. The interstitial matrix was assumed to be a porous medium able to store the chemical species being transported. Controlled osmotic stress experiments were conducted on isolated rat pancreas islets to measure the transient volumetric response to step-wise changes in dimethyl sulfoxide, Me2SO, concentration. The model provides a tool for predicting the transient volumetric response of peripheral and interior cells and of interstitial tissue, as well as the build up of solute concentration, during addition and removal of CPAs and freezing and thawing protocols. Inverse solution methods were applied to determine values for standard cell membrane permeability parameters Lp, omega and sigma as well as for the interstitial flow conductivities Kw and Kp'.

Regulation of the pentose phosphate pathway by an androgen receptor-mTOR-mediated mechanism and its role in prostate cancer cell growth.

Cancer cells display an increased demand for glucose. Therefore, identifying the specific aspects of glucose metabolism that are involved in the pathogenesis of cancer may uncover novel therapeutic nodes. Recently, there has been a renewed interest in the role of the pentose phosphate pathway in cancer. This metabolic pathway is advantageous for rapidly growing cells because it provides nucleotide precursors and helps regenerate the reducing agent NADPH, which can contribute to reactive oxygen species (ROS) scavenging. Correspondingly, clinical data suggest glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the pentose phosphate pathway, is upregulated in prostate cancer. We hypothesized that androgen receptor (AR) signaling, which plays an essential role in the disease, mediated prostate cancer cell growth in part by increasing flux through the pentose phosphate pathway. Here, we determined that G6PD, NADPH and ribose synthesis were all increased by AR signaling. Further, this process was necessary to modulate ROS levels. Pharmacological or molecular inhibition of G6PD abolished these effects and blocked androgen-mediated cell growth. Mechanistically, regulation of G6PD via AR in both hormone-sensitive and castration-resistant models of prostate cancer was abolished following rapamycin treatment, indicating that AR increased flux through the pentose phosphate pathway by the mammalian target of rapamycin (mTOR)-mediated upregulation of G6PD. Accordingly, in two separate mouse models of Pten deletion/elevated mTOR signaling, Pb-Cre;Pten(f/f) and K8-CreER(T2);Pten(f/f), G6PD levels correlated with prostate cancer progression in vivo. Importantly, G6PD levels remained high during progression to castration-resistant prostate cancer. Taken together, our data suggest that AR signaling can promote prostate cancer through the upregulation of G6PD and therefore, the flux of sugars through the pentose phosphate pathway. Hence, these findings support a vital role for other metabolic pathways (that is, not glycolysis) in prostate cancer cell growth and maintenance.

Immediate and long-term consequences of vascular toxicity during zebrafish development.

Proper formation of the vascular system is necessary for embryogenesis, and chemical disruption of vascular development may be a key event driving developmental toxicity. In order to test the effect of environmental chemicals on this critical process, we evaluated a quantitative assay in transgenic zebrafish using angiogenesis inhibitors that target VEGFR2 (PTK787) or EGFR (AG1478). Both PTK787 and AG1478 exposure impaired intersegmental vessel (ISV) sprouting, while AG1478 also produced caudal and pectoral fin defects at concentrations below those necessary to blunt ISV morphogenesis. The functional consequences of vessel toxicity during early development included decreased body length and survival in juvenile cohorts developmentally exposed to inhibitor concentrations sufficient to completely block ISV sprouting angiogenesis. These data show that concentration-dependent disruption of the presumed targets for these inhibitors produce adverse outcomes at advanced life stages.

Viability analysis of cryopreserved rat pancreatic islets using laser scanning confocal microscopy.

We have developed a digital image analysis technique to assay the viability of frozen-thawed pancreatic islets by using laser scanning confocal microscopy (LSCM) in conjunction with double fluorescent staining [acridine orange/propidium iodide (AO/PI)]. Freshly isolated rat pancreatic islets were cultured for 18-24 h and then brought to a 2 M concentration of dimethyl sulfoxide (Me2SO) by serial addition at decreasing temperatures. Ice was nucleated in the islet suspension at a defined temperature (-10 degrees C), followed by a controlled period for equilibration and then cooling in a programmable bulk freezer at rates of 0.3, 1, 3, 10 and 30 degrees C/min to -70 degrees C. Samples were then stored in liquid nitrogen. Subsequent to rapid thawing and serial dilution with sucrose solution to remove Me2SO, AO/PI-stained individual islets were prepared for imaging on the LSCM. A series of optical sections through individual stained islets were obtained and processed to obtain high-contrast images at two different wavelengths; 488 nm and 514 nm for viable and damaged tissue, respectively. Image analysis algorithms consisted of template masking, generation of histograms of the pixel intensity profile, and gray level thresholding to obtain binary images. The total percentages of both types of tissue in the islet were computed by summing the two populations in each serial section. The spatial distributions of viable and damaged tissue were calculated from the three-dimensional (3-D) data base for both cultured (control) and cryopreserved islets. The 3-D spatial distributions of damaged and viable tissue in the islets were computed by determining the normalized distance of each viable/damaged voxel from the centroid of the islet volume to a mathematically estimated 3-D superquadric surface used to estimate the outer boundary of the islet. Further, each isolated damaged cell was identified and its volume determined. These studies indicate that cryopreserved islets exhibit shape distortion and a decrease in the numerical density of cells in comparison to unfrozen controls. Maximal survival was observed at the slower cooling rates. Accordingly, damage was found to occur throughout the 3-D islet volume in distinct spatial distributions for islets frozen at the slower and the faster cooling rates. Further, it was found that the volume of the majority of damaged cells identified was consistent with that of cells ranging in diameter from 5 to 9 micrometers.

In-vivo analysis of angiogenesis and revascularization of transplanted pancreatic islets using confocal microscopy.

A technique to measure angiogenesis and revascularization in pancreatic islets transplanted at the renal subcapsular site in the rat has been developed. In-vivo imaging of the microcirculation of transplanted pancreatic islets was conducted using a confocal scanning laser microscope (CSLM) to achieve optical sectioning through the graft in order to perform a computer reconstruction of the three-dimensional neovascular morphology. Individual islets were harvested by enzymatic digestion of excised pancreas from Fischer 344 rats. Isolated islets were cultured for 24 h, and approximately 300-350 islets were transplanted at the renal subcapsular site of the left kidney in an anaesthetized rat. Six to 14 days post-transplantation, the animal was anaesthetized and prepared for in-vivo imaging of the microvasculature on a Zeiss LSM-10. Optical contrast of the microvasculature was enhanced by the administration of fluorescein-labelled dextran into the circulating blood. The transplant site was identified and serial sections were obtained through the vascular bed at varying z-intervals. Complementary fluorescence video images were also obtained via a silicon intensifier tube camera mounted on the CSLM. At completion of the imaging procedure, the kidney was returned into the body cavity, the area was sutured and the animal was allowed to recuperate for subsequent examinations. Image processing algorithms, such as grey-level thresholding, median filtering, skeletonization and template matching, were applied to compute the vessel density and diameters and extrapolated to measure 3-D vessel lengths and the tortousity index of the neovasculature.

Poloxamer 188 enhances functional recovery of lethally heat-shocked fibroblasts.

Damage to the cell membrane has been implicated as the primary event in the pathogenesis of heat shock, generally resulting in loss of cellular homeostasis and cell death. Thus a promising mode of therapy would involve the restoration of cell membrane integrity. Surfactant molecules, specifically triblock polymers such as Poloxamer 188 (P-188), possess the ability to self-aggregate into membrane-like structures in aqueous solutions and have been shown to restore membrane integrity. The objective of this study was to develop functional and morphological assays to determine whether treatment with P-188 after heat shock enhances the recovery of thermally damaged cells. Human foreskin fibroblasts were placed in sterile vials and heated by immersion in a calibrated water bath for various lengths of time at predefined temperatures. Cell recovery after heat shock was assessed using a functional assay based on the ability of the cells to contract fibroblast populated collagen lattices (FPCLs). Subsequent to heating, collagen lattices were prepared with control (no heat, no P-188) and heat shocked cells (with and without P-188). Our results indicate that treatment with low concentrations of P-188 after heat shock was effective in ameliorating both the morphological integrity and the contractile function of thermally damaged cells. Further, we observed that P-188 was most effective in improving the contractile ability of cells heat shocked at 45 degrees C; however, it had no influence on the contractility of cells exposed to higher temperatures. Our results suggest that there exists a threshold of thermal stress (45 degrees C for 20-60 min) beyond which treatment with low concentrations of P-188 (0.5 mg/ml) is ineffective in minimizing cell damage. Moreover, the results of our morphological assays indicate that cells treated with P-188 after heat shock maintain their cytoskeletal organization, whereas untreated cells exhibit filamentous actin depolymerization.

Three-dimensional distribution of damaged cells in cryopreserved pancreatic islets as determined by laser scanning confocal microscopy.

The technique of serial optical sectioning by confocal microscopy, in conjunction with off-line digital image analysis, was used to quantify the radial distribution of damaged cells in rat pancreatic islets following cryopreservation. The process consists of imaging frozen-thawed islets of Langerhans using laser scanning confocal microscopy (LSCM). The three-dimensional (3-D) distribution and analysis of the two populations of viable and damaged cells was visualized via acridine orange/propidium iodide (AO/PI) fluorescent staining. In preparation for cryopreservation, isolated and cultured rat pancreatic islets were brought to a 2 M concentration of dimethyl sulphoxide (DMSO) by serial addition at decreasing temperatures. Ice was nucleated in the islet suspension at -10 degrees C, and individual specimens were frozen to -70 degrees C at cooling rates of 1, 3, 10 and 30 degrees C/min in a programmable bulk freezer and subsequently stored in liquid nitrogen. After rapid thawing and serial dilution to remove DMSO, individual islets were prepared with AO/PI stains for imaging on the LSCM. Serial sections of the islets, 2-7 microns in thickness, were obtained and processed to obtain high-contrast images. Analysis algorithms consisted of template masking, grey-level thresholding, median filtering and 3-D blob colouring. The radial distribution of damaged cells in the islets was determined by isolating the cell and computing its distance from the centroid of the 3-D islet volume. An increase in the number of blobs corresponding to single and/or aggregates of damaged cells was observed progressively with distance from the centre towards the periphery of the islet. This pattern of freeze-induced killing of cells within the islet was found to occur consistently in the numerous individual specimens processed.

Analysis of volumetric changes in rat pancreatic islets under osmotic stress using laser scanning confocal microscopy.

Analysis of the volumetric changes in rat pancreatic islets undergoing shrinkage and/or swelling due to osmotic stress is essential for understanding the mechanism of mass transport between cells and their environment and for optimizing cryopreservation protocols. Addition and removal of cryoprotective additives is an integral component of all cryopreservation processes. We have used laser scanning confocal microscopy (LSCM) of acridine orange/propidium iodide (AO/PI) stained Islets of Langerhans to analyze the effects of osmotic stress induced by exposure to varying concentrations of the cryoprotectant dimethyl sulfoxide (DMSO), on the islet volume at two temperatures 23 degrees C and 15 degrees C. Experiments were conducted by mounting a single islet onto a unique freeze-thaw-perfusion stage on which the system temperature and the chemical composition of the solutions can be precisely controlled. The bathing medium of the islet was rapidly changed from isotonic saline to the desired DMSO osmolality to produce a defined osmotic stress, and the islet was imaged simultaneously using 488 nm argon laser. Three to seven serial sections were obtained through each islet at increments varying between 15 microns and 20 microns. The three-dimensional (3-D) image was segmented into islet and non-islet regions using a combination of median filtering, gray level thresholding and region labeling, and the islet volume was computed by counting voxels. Further, a special analysis algorithm was applied to identify shape changes both locally and globally throughout the islet volume.