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One of the largest single sources of epilepsy in the world is produced as a neurological sequela in survivors of cerebral malaria. Nevertheless, the pathophysiological mechanisms of such epileptogenesis remain unknown and no adjunctive therapy during cerebral malaria has been shown to reduce the rate of subsequent epilepsy. There is no existing animal model of postmalarial epilepsy. In this technical report we demonstrate the first such animal models. These models were created from multiple mouse and parasite strain combinations, so that the epilepsy observed retained universality with respect to genetic background. We also discovered spontaneous sudden unexpected death in epilepsy (SUDEP) in two of our strain combinations. These models offer a platform to enable new preclinical research into mechanisms and prevention of epilepsy and SUDEP.

The insulin-secreting β-cells are contained within islets of Langerhans, which are highly vascularized. Blood cell flow rates through islets are glucose-dependent, even though there are no changes in blood cell flow within in the surrounding exocrine pancreas. This suggests a specific mechanism of glucose-regulated blood flow in the islet. Pancreatic islets respond to elevated glucose with synchronous pulses of electrical activity and insulin secretion across all β-cells in the islet. Connexin 36 (Cx36) gap junctions between islet β-cells mediate this synchronization, which is lost in Cx36 knockout mice (Cx36−/−). This leads to glucose intolerance in these mice, despite normal plasma insulin levels and insulin sensitivity. Thus, we sought to investigate whether the glucose-dependent changes in intraislet blood cell flow are also dependent on coordinated pulsatile electrical activity. We visualized and quantified blood cell flow using high-speed in vivo fluorescence imaging of labeled red blood cells and plasma. With the use of a live animal glucose clamp, blood cell flow was measured during either hypoglycemia (∼50 mg/dl) or hyperglycemia (∼300 mg/dl). In contrast to the large glucose-dependent islet blood velocity changes observed in wild-type mice, only minimal differences are observed in both Cx36+/− and Cx36−/− mice. This observation supports a novel model where intraislet blood cell flow is regulated by the coordinated electrical activity in the islet β-cells. Because Cx36 expression and function is reduced in type 2 diabetes, the resulting defect in intraislet blood cell flow regulation may also play a significant role in diabetic pathology.

With the advent of in vivo laser scanning fluorescence microscopy techniques, time-series and three-dimensional volumes of living tissue and vessels at micron scales can be acquired to firmly analyze vessel architecture and blood flow. Analysis of a large number of image stacks to extract architecture and track blood flow manually is cumbersome and prone to observer bias. Thus, an automated framework to accomplish these analytical tasks is imperative. The first initiative toward such a framework is to compensate for motion artifacts manifest in these microscopy images. Motion artifacts in in vivo microscopy images are caused by respiratory motion, heart beats, and other motions from the specimen. Consequently, the amount of motion present in these images can be large and hinders further analysis of these images. In this article, an algorithmic framework for the correction of time-series images is presented. The automated algorithm is comprised of a rigid and a nonrigid registration step based on shape contexts. The framework performs considerably well on time-series image sequences of the islets of Langerhans and provides for the pivotal step of motion correction in the further automatic analysis of microscopy images.

Introduction: A significant portion of the morbidity and mortality following aneurysmal subarachnoid hemorrhage (SAH) is due to delayed ischemic neurological deficits (DIND). Although large vessel vasospasm has been implicated as a cause of DIND, the presence of such spasm is not always correlated with DIND. In the present experiment we examined the cortical microvasculature in awake mice after SAH in an experimental model typically associated with large vessel vasospasm. Methods: Twenty adult mice underwent cisterna magna (CM) injection of 60ul syngenic donor blood or artificial cerebrospinal fluid (aCSF). The mice were perfused at 72 hours, and Circle of Willis (COW) vessel diameters were measured. In a separate experiment, polished and reinforced thinned skull cranial windows were created in 10 mice that then underwent CM blood or aCSF injection. Cortical microvessels were measured in living, awake mice in the postoperative vasospasm period using two photon laser scanning microscopy. Results: A pairwise comparison of COW vessels revealed a statistically significant difference in vessel diameter between the experimental and control groups at each of the sites measured (ACA, P=0.0012; MCA, P=0.0259; Pcom, P=0.0053). In the second experiment, there were no significant differences in cortical microvessel diameter between the experimental and control groups. Conclusions: This study represents the first report of in vivo imaging of cortical microvessels during large vessel spasm following SAH. In a CM injection SAH model, we observed significant large vessel spasm but no cortical microvessel spasm. These results may have important implications for understanding the mechanisms of DIND.

A series of E,E-N-phosphorylalkylene-3,5-bis(arylidene)piperid-4-ones 7a-k was prepared via the condensation of aromatic aldehydes with omega-aminophosphonates 5a-c and 6a,b bearing piperidone or a protected piperidone moiety, respectively. The synthetic routes to the starting aminophosphonates 5a-c and 6a,b varied depending on the number of methylene groups in the alkylene chain and comprised the Kabachnik-Fields reaction (n=1), the aza-Michael reaction (n=2) or alkylation of 4-piperidone hydrochloride with diethyl omega-bromoalkylphosphonates under phase transfer catalysis conditions (n=3,4). Phosphoryl substituted 3,5-bis(arylidene)piperid-4-ones 7b,c,e,f,h,i,k bearing both nitro groups and fluorine atoms in the para-position of the arene rings possess cytotoxicity toward human carcinoma cell lines CaOv3, Scov3, PC3 and A549 in the low micromolar range while their analogues having para-dimethylamino groups had IC(50) values greater than 50 microM. In contrast, only Me(2)N-substituted phosphonates 7g,j (n=3 and 4) and the salts of Me(2)N-substituted phosphonic acids 10c,f (n=2 and 3) display fluorescence.

Infrared (IR) spectroscopy of biological cells is a growing area of research, with many papers focusing on differences between the spectra of cancerous and noncancerous cells. Much of this research has been performed using a monolayer of dehydrated cells. We posit that the use of monolayers can introduce artefacts that lead to an apparent but inaccurate measurement of differences between cancerous and noncancerous cells. Additionally, the use of dried cells complicates the extraction of biochemical information from the IR spectra. We demonstrate that using suspensions of viable cells in aqueous suspension reduces measurement artefacts and facilitates determining the concentration of the major biochemical components via a linear least-squares fit of the component spectra to the spectrum of the cells.

One-photon and polarized two-photon fluorescence excitation spectra and the dispersed fluorescence spectrum of jet-cooled 2,3-dimethylindole are reported. The two-photon intensity ratios for linear versus circular polarized excitation show that the origin is primarily of 1Lb character and that lines with predominantly 1La character appear at 342, 356, 371, 383, 396, and 406 cm−1 above the 1Lb origin. These lines exhibit what appears to be methyl rotor tunneling spitting. The ab initio computed dispersed fluorescence spectrum for the 1Lb state agrees well with the observed dispersed fluorescence reported here; that computed for 1La does not. Large splitting of certain vibrational modes due to mixing with methyl motions is responsible for marked differences in the dispersed fluorescence compared to that of indole and 3-methylindole.

A site-selective study of the 3La phosphorescence from indole in solid argon at 20 K has revealed a special site from which the emission is exceptionally sharp (5 cm−1 fwhm). These spectra are compared with well-resolved (5 cm−1 fwhm) fluorescence from the 1Lb origin of indole under cold jet conditions. The results are compared with frequencies and Franck–Condon factors generated from ab initio electronic structure calculations on the ground and excited states. On the basis of the data and calculations, new assignments are made. ©1999 John Wiley & Sons, Inc. Int J Quant Chem 72: 347–356, 1999

Two-photon polarized fluorescence excitation and vibronically resolved one-photon dispersed fluorescence spectra of the long-wavelength absorbing van der Waals complexes of indole with water, methanol, and formamide were examined for the purpose of firmly assigning the nature of the lowest singlet excited electronic state. The two-photon spectra for all of these complexes have circular/linear polarization ratios of absorptivity (Ω-values) that show that excitation is to the 1Lb state. Analysis of the Franck–Condon (FC) patterns of the dispersed fluorescence for these indole-polar solvent complexes show that emission is also from the 1Lb state in each case. In the emission spectra, the intensity ratios of the origin and ν26 lines are about 2:1, which is the value expected for 1Lb emission. The ν26 vibration is the most intense nonorigin vibronic line, as expected for 1Lb emission. Finally, there is little or no intensity from the ν8, ν9, and ν10 vibrations, which would be strongly active with emission from...

To examine the interplay between tumor cells and the microenvironment during early breast cancer metastasis, we developed a technique for ex vivo imaging of murine tissue explants using two-photon microscopy. Cancer cells in the liver and the lung were compared by imaging both organs at specific time points after the injection of the same polyomavirus middle T-initiated murine mammary tumor cell line. Extravasation was greatly reduced in the lung compared with the liver, with 56% of tumor cells in the liver having extravasated by 24 hours, compared with only 22% of tumor cells in the lung that have extravasated. In the liver, imaged cells continually transitioned from an intravascular location to an extravascular site, whereas in the lung, extravasation rates slowed after 6 hours. Within the liver microenvironment, the average size of the imaged micrometastatic lesions increased 4-fold between days 5 and 12. Histologic analysis of these lesions determined that by day 12, the micrometastases were heterogeneous, consisting of both tumor cells and von Willebrand factor–positive endothelial cells. Further analysis with intravenously administered lectin indicated that vessels within the micrometastatic tumor foci were patent by day 12. These data present the use of two-photon microscopy to directly compare extravasation times in metastatic sites using the same tumor cell line and highlight the differences in early events and metastatic patterns between two important secondary sites of breast cancer progression with implications for future therapy. Mol Cancer Res; 8(10); 1319–27. ©2010 AACR.

Two-photon photodynamic therapy has the advantages of being highly localized in its effects and allows for deeper tissue penetration, when compared to one-photon photodynamic therapy. N-alkylated 3,5-bis(arylidene)-4-piperidones, with a donor-pi-acceptor-pi-donor structure, have the potential to be useful two-photon sensitizers. We have measured two-photon cross sections (using femtosecond excitation), fluorescence quantum yields, fluorescence lifetimes, and xray crystal structures for a number of these compounds. Most two-photon cross sections are comparable to or larger than that of Rhodamine B. However, the fluorescence quantum yields are low (all less than 10%) and the fluorescence lifetimes are less than 1 ns (with one exception), suggesting that there may be a significant energy transfer to the triplet state. This would encourage singlet oxygen formation and increase cellular toxicity. Results of dark cyto-toxicity studies with several human cancer cell lines are presented. White light photo-toxicity results are also presented, and suggest that increasing the number of double bonds, from one to two, in the piperidone wings increases the photo-toxicity with little corresponding change in the dark cyto-toxicity.

We have conducted an extensive comparison of cellular biochemical composition obtained from infrared and Raman spectra of intact cells with measurements using standard extraction and chemical analysis (including NMR), and flow cytometric assay on fixed cells. Measurements were conducted on a rat fibroblast carcinogenesis model consisting of normal and tumorigenic cells assayed as exponentially growing and plateau-phase cultures. Estimates of protein, DNA, RNA, lipids, and glycogen amounts were obtained from a previous publication in which vibrational spectra were fit to a set of basis spectra representing protein, DNA, RNA, lipids, and glycogen. The Raman spectral estimates of absolute cellular composition were quite similar to the independent biochemical and flow cytometric assays. The infrared spectra gave similar results for protein, lipid, and glycogen but underestimated the DNA content while overestimating the RNA level. When ratios of biochemical concentrations in exponential and plateau-phase cultures were examined, the Raman spectroscopic results were the same, within errors, as the independent methods, in all cases. Several changes in relative biochemical composition due to tumorigenic and proliferative status previously reported using vibrational spectroscopy were confirmed by the independent methods. These results demonstrate that vibrational spectroscopy can provide reliable estimates of the biochemical composition of mammalian cells.

The biochemical composition of mammalian cells has been estimated by Raman spectroscopy and the results compared with other biochemical methods. The Raman spectroscopy estimates were performed by fitting measured Raman and infrared spectra of dense cell suspensions to a linear combination of basis components (RNA, DNA, protein, lipid, glycoen). The Raman spectroscopy results are compared to biochemical analyses performed by extraction and quantfication of the biochemical components. Both absolute and relative measurements of biochemical composition are compared. Both the Raman and biochemical results indicate that there are signficant differences in gross biochemical composition dependent on growth stage and tumorigneicity.

Raman spectra of cells and nuclei from cultures in the plateau (nonproliferating) and exponential (proliferating) phases of growth were measured and show that Raman spectroscopy can monitor changes due to cell proliferation. A simple fitting routine was developed using a basis set (lipid, protein, DNA, RNA) to estimate the relative amounts of biochemical components in cells and nuclei. Using relative amounts and ratios of biochemical components, reproducible differences can be detected and quantified that are not readily apparent by visual analysis of vibrational bands in the spectra. These differences, due to cell proliferation, can be assigned to specific biochemical changes. They include a decrease in the relative lipid and increases in the relative protein and RNA for both nontumorigenic exponential cells and nuclei, and an increase in the relative RNA for tumorigenic exponential cells. The lipid/RNA ratio decreases for nontumorigenic exponential cells and nuclei and tumorigenic exponential cells. The protein/lipid ratio increases for both tumorigenic and nontumorigenic exponential cells and nuclei. Finally, the lipid/DNA ratio decreases for tumorigenic exponential nuclei. This knowledge will be important for Raman detection of rapidly dividing populations of cancer cells in vivo.

Biochemical analysis of cells in different proliferative stages was performed using IR spectrscopy. Independent biochemical analysis of glycogen oncentration gave results that are consistent with the IR reults, i.e. plateau phase cells have more glycogen.

Analysis of Raman spectra from a pair of tumorigenic and non-tumorigenic cells suspended in phosphate buffered saline will be presented. This system allows examination of cellular changes associated strictly with carcinogenesis without interfering factors.

We have developed methods of measuring absorption of transient species utilizing stepped-scan Fourier transform interferometry that allows a combination of broad spectral coverage (10,000 - 15,000 cm-1 per spectrum), good spectral resolution, and up to ns temporal resolution with possibilities of extension to the ps domain. Nanosecond, psec or fsec laser systems, tunable from UV to IR can be used as the pump source to prepare the transient species. The absorption of that species is measured with broadband, incoherent light and can be simultaneously time and frequency resolved.© (1999) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

We have developed methods of measuring absorption of transient species utilizing both rapid scan and stepped-scan Fourier transform interferometery that allows a combination of broad spectral coverage (10,000 – 15,000 cm-1 per spectrum), good spectral resolution, and the ability to cover temporal ranges from minutes to nanoseconds. A wide range of pump sources are used including continuous, modulated, ns pulsed, and ultrafast lasers. The probe is provided by a broadband, incoherent source providing large spectral information in each experiment. This capability enables investigation of phenomena such as excited state absorption, charge transfer, formation of metastable species, and others. Extension of the technique to the ps realm is underway and will be discussed.

During the past decade there has been considerable progress in the design of new organic materials for optical power limiting (OPL) applications. Among the more promising of the new material approaches are reverse saturable absorbers (RSAs) which derive their optical limiting capability from the photogeneration of highly absorbing transient excited states. Most of the previous research in this field has focused on excited state absorption from singlet or triplet states whose cross-sections are greater than the original S0 – S1 transition- However, other transient states, such as polaronic radical ions or bipolaronic diions, formed by photo-induced electron transfer, are attractive alternatives. We have initiated studies to determine if these transient charge states can be photo-generated efficiently, and if their excited states absorption and lifetimes are acceptable for current optical power limiting requirements.

Diabetes is a disease resulting from changes in pancreatic islets, which are insulin secreting micro-organs within the pancreas. With increased blood glucose, insulin is secreted from beta cells in the islets in a coordinated pulsatile manner. At the same time alpha cell glucagon secretion is inhibited. Mechanisms controlling these processes at the intercellular and at the inter-islet level remain unclear. We suggest that the three-dimensional organization of islet cells and the dynamics of islet blood flow have a role in regulating insulin and glucagon secretion. This is suggested by observation that the density of blood vessels within islets is much greater than in surrounding pancreatic tissue, and that most individual islet cells are adjacent to a blood vessel. As an initial test of our hypothesis, we have developed a high-speed in vivo fluorescence imaging method to track pancreatic blood flow in a living mouse. We are also developing methods necessary to analyze the large amounts of data generated. Using high speed line scan confocal microscopy the method has full frame sub-micron spatial and less than 10 ms temporal resolution. Islets are located within the pancreas by using mice with GFP-labeled beta cells. Blood plasma is labeled with a fluorescent dextran, allowing mapping of vascular dimensions and pathway. Individual blood cells are fluorescently labeled by osmotic shock loading with an Alexa dye, which allows tracking of the blood flow. We present current results for blood flow under different levels of blood glucose in clamping experiments. Our previous qualitative results have suggested that there are differences in blood flow parameters at different glucose levels. Here, a more quantitative analysis of blood flow velocity, any observed changes in vessel dimensions, and changes in blood flow coverage inside and outside islets is presented.

Two-photon photodynamic therapy has the advantages of being highly localized in its effects and allows for deeper tissue penetration, when compared to one-photon photodynamic therapy. N-alkylated 3,5-bis(arylidene)-4-piperidones, with a donor-pi-acceptor-pi-donor structure, have the potential to be useful two-photon sensitizers. We have measured two-photon cross sections (using femtosecond excitation), fluorescence quantum yields, fluorescence lifetimes, and x-ray crystal structures for a number of these compounds. Most two-photon cross sections are comparable to or larger than that of Rhodamine B. However, the fluorescence quantum yields are low (all less than 10%) and the fluorescence lifetimes are less than 1 ns, suggesting that there may be a significant energy transfer to the triplet state. This would encourage singlet oxygen formation and increase cellular toxicity. Results of dark cyto-toxicity studies with a number of human cancer cell lines are presented. White light photo-toxicity results are also presented, and suggest that increasing the number of double bonds, from one to two, in the piperidone “wings” increases the photo-toxicity with little corresponding change in the dark cyto-toxicity. Two-photon photo-toxicity studies are also underway (exposure in the range of 740 - 860 nm) as well as singlet oxygen detection studies(detection at about 1270 nm).

Both infrared and Raman spectroscopies have the potential to noninvasively estimate the biochemical composition of mammalian cells, although this cannot be unambiguously determined from analysis approaches such as peak assignment or multivariate classification methods. We have developed a fitting routine that determines biochemical composition using basis spectra for the major types of biochemicals found in mammalian cells (protein, DNA, RNA, lipid and glycogen), which is shown to be robust and reproducible. We measured both infrared and Raman spectra of viable suspensions of pairs of nontumorigenic and tumorigenic rat fibroblast cell lines. To model in vivo conditions, we compared nonproliferating, nontumorigenic cells to proliferating, tumorigenic cells. Reproducible differences in biochemical composition were found for both nontumorigenic/tumorigenic cell models, using both spectroscopic techniques. These included an increased fraction of protein and nucleic acids in the tumorigenic cells, with a corresponding decrease in lipid and glycogen fractions. Measurements of each cell type in both the proliferating and nonproliferating states showed that proliferative status was the major determinant of differences in vibrational spectra, rather than tumorigenicity per se. The smallness of the spectral changes associated with tumorgenicity may be due to the subtle nature of the oncogenic change in this system (a single mutant oncogene).

The ratio of nonresonant two-photon induced fluorescence excitation spectra using circularly and linearly polarized light for jet-cooled 3-methylindole complexed with a series of increasingly basic hydrogen bond acceptors (water, methanol, ethanol, diethylether, diethylamine and triethylamine) is consistent with an avoided crossing of the two lowest excited singlet states, 1La and 1Lb. The dispersed fluorescence of these from this series also reflects the crossing, providing a definitive 1La jet-cooled fluorescence spectrum. The jet-cooled 1La fluorescence spectrum is not broad and redshifted, but has vibronic structure that agrees well with ab initio predictions and is similar to that of 3La phosphorescence.

Gramicidin crystals, dimyristoylphosphatidylcholine (DMPC)/gramicidin dispersions, and DMPC/gramicidin cocrystals were examined by Raman scattering to determine lipid/gramicidin stoichiometries and lipid organization. Calibrations of the choline (716-cm-1) and tryptophan (756-cm-1) peaks indicate that the cocrystals contain two lipids for each gramicidin monomer, a result confirmed by chemical analyses of washed crystals. In dispersions with high lipid/gramicidin ratios (e.g., 25:1), the lipid is ordered but becomes increasingly disordered as the gramicidin content is increased. Paradoxically, the DMPC/gramicidin cocrystals have highly ordered lipids that possibly contain no gauche bonds at all, despite their low lipid/gramicidin ratio. In addition, the polypeptide amide I peak position near 1670 cm-1 is found to be independent of the lipid/gramicidin ratio in the complexes and may indicate a beta-helix-type secondary structure at all ratios. However, the amide I peak broadens significantly at low lipid/gramicidin ratios and broadens still further in the cocrystals, suggesting that protein-protein interactions may induce band-broadening distortions of the polypeptide structure.

Pancreatic islets are central in regulating blood glucose levels. Thus, controlling delivery of secreted hormones such as insulin, glucagon, and somatostatin likely depends upon regulating blood flow through each individual islet. The mechanism of control is expected to have one or more components that are localized to the islets, and perhaps to specific cell types within an islet. In wild type mice we have observed localized regulation of blood flow with varying glycemic conditions. Where, under hypoglycemic conditions there is a greater loss of blood flow in islets than in the exocrine pancreas, while for hyperglycemic conditions both islets and exocrine pancreas are well perfused. We hypothesize that this localized regulation of blood flow is related to coordinated electrical activity in islets that couples into vascular endothelial cells to control blood flow. To test this, we use high-speed confocal microscopy to image blood flow in the pancreas of wild type, Cx36(+/-), and Cx36(-/-) (Connexin 36 knockout in beta cells) mice. Data is collected at different z-positions through the islets and surrounding exocrine tissue, for a series of X-Y-Time scans. Vasculature is labeled with a fluorescent rhodamine dextran in the plasma, and a portion of blood cells are labeled by osmotic shock loading with a fluorescent Alexa dye. Results are presented that quantify differences between the three genotypes. Specifically, average blood flow densities and velocities for whole islets and the exocrine pancreas under glycemic conditions from 300 mg/dl (hyperglycemia) are presented. In addition, localized blood flow differences present in single islets, as a function of glycemic conditions, are analyzed. We expect in Cx36(-/-) mice, with islets that lack coordinated electrical activity, much less localized regulation of blood flow under hypoglycemic conditions.