Two-photon Laser Microscopy Research Articles

Comparison of Ca2+ transients and [Ca2+]i in the dendrites and boutons of non‐fast‐spiking GABAergic hippocampal interneurons using two‐photon laser microscopy and high‐ and low‐affinity dyes

Using two-photon laser microscopy, high- and low-affinity dyes and patch clamp electrophysiology, we successfully measured somatic stimulation-evoked Ca(2+) transients simultaneously in the dendrites and axonal boutons of the same non-fast-spiking GABAergic interneurons in acute slice preparations obtained from hippocampal area CA1. The advantage of the acute preparation is that both neuronal connections and anatomy are maintained. Calculated as unperturbed values, the amplitudes of Ca(2+) transients and changes in [Ca(2+)]i in response to somatic single or burst stimulation were much higher in boutons (428 nM/AP) than in dendrites (49 nM/AP), leading to the conclusion that the much greater influx of Ca(2+) observed in terminals might be due to a higher density of N-type voltage-sensitive Ca(2+) channels compared to the L-type channels present in dendrites. Whereas the decay of Ca(2+) transients recorded in dendrites was primarily mono-exponential, the decay in boutons was bi-exponential, as indicated by an initial fast phase, followed by a much slower reduction in fluorescence intensity. The extrusion of Ca(2+) was much faster in boutons than in dendrites. To avoid saturation effects and the flawed conversion of fluorescence measures of [Ca(2+)]i, we assessed the limits of [Ca(2+)] measurements (which ranged between 6 and 82% of the applied dye saturation) when high- and low-affinity dyes were applied at different concentrations. When two APs were delivered at a high frequency (>3 Hz) of stimulation, the low-affinity indicators OGB-6F (KD = 3.0 μM) and OGB-5N (KD = 20 μM) were able to accurately reflect the changes in ΔF/F produced by the consecutive APs. There was no difference in the endogenous buffer capacity (κE), which can shape Ca(2+) signals, calculated in dendrites (κE = 354) or boutons (κE = 458).

Controlled intracellular self-assembly of gadolinium nanoparticles as smart molecular MR contrast agents

Herein we developed a new “smart” Gd-based MR contrast agent (i.e., 1) which is susceptive to furin, a protease overexpressed in tumor. Under the action of furin, 1 condenses to form dimers (1-Ds) and the latter self-assemble into gadolinium nanparticles (Gd-NPs). Relaxivity of 1-D is more than 2 folds of those of 1 and magnevist at 1.5 T, and 1.4 folds of that of 1 at 3 T. Intracellular condensation of 1 in furin-overexpressed MDA-MB-468 cells was proven with direct two-photon laser microscopy (TPLM) fluorescence imaging of the cells incubated with the europium analog of 1 (i.e., 2). Intracellular Gd-NPs of 1 were uncovered and characterized for the first time. MRI of MDA-MB-468 tumors showed that 1 has enhanced MR contrast within the tumors than that of its scrambled control 1-Scr.

Molecular cloning and characterization of ARF1 and COPI coat proteins from Medicago truncatula cv. Jemalong: Dissection of their role in vesicular transport in root cells

The integrity of the Golgi apparatus in both plant and mammalian cells is dependent upon a coordinated flow of COPII (coatomer protein) coated vesicles in anterograde (forward) and of COPI coated vesicles in retrograde (backward) direction. Although a fair amount of work on vesicular trafficking has been published in Arabidopsis thaliana not much information is available related to the secretory pathway in other higher plants. In the present study we have used Medicago truncatula, a model plant for legume species as for symbiotic and pathogenic interactions, to identify Arf1 and COPI components of the early secretory pathway. Their localisation and interaction with the Golgi apparatus in the root cells has been identified by confocal and two-photon laser microscopy. EST databases of the M. truncatula were screened and putative homologues for all seven coatomer proteins and MtArf1 were identified. Two isoforms of the ζ- COPI subunit and MtArf1 genes were isolated from M. truncatula cDNA libraries and were sequenced. GFP fusions of MtArf1, dominant active (Q71L), dominant inactive (T31N) forms of MtArf1and two isoforms of MtζCOP1 were expressed in transgenic “hairy root” and their subcellular localisation were analyzed. Our results show that MtArf111::GFP, Mtζ-COP1:: GFP and Mtζ-COP2::GFP were localized on mobile Golgi structures, streaming along the ER network and were sensitive to brefeldin A, indicating their potential association with Golgi stacks. This study demonstrates an important role of Arf1 and COP1 proteins in early secretory pathway in root cells of M. truncatula.

Two-photon microscopy analysis of leukocyte trafficking and motility

During the last several years, live tissue imaging, in particular using two-photon laser microscopy, has advanced our understanding of leukocyte trafficking mechanisms. Studies using this technique are revealing distinct molecular requirements for leukocyte migration in different tissue environments. Also emerging from the studies are the ingenious infrastructures for leukocyte trafficking, which are produced by stromal cells. This review summarizes the recent imaging studies that provided novel mechanistic insights into in vivo leukocyte migration essential for immunosurveillance.Electronic supplementary materialThe online version of this article (doi:10.1007/s00281-010-0210-3) contains supplementary material, which is available to authorized users.

Abstract 2384: Role of bone marrow derived progenitor cells in intracranial tumor neovascularization

Abstract Background: Glial tumors are the most common and lethal adult brain tumor and the histopathological hallmark of their malignant transformation is vascular proliferation. Hence novel strategies to target tumor vascularity are continuously being explored in hopes to improve patient outcomes. More recent interest is focused on the role of bone marrow derived precursors cells (BMDCs) in tumor neovascularization. Whether BMDC differentiate into endothelial cells (EC) or perivascular support cells (PVC) is controversial and whether it is tumor type or tumor stage dependent remains unknown. Finally, impact of ionizing radiation (IR) on recruitment, migration and differentiation of BMDCs in brain tumor vasculature has not been examined. Methods: Animal Models: Bone marrow (BM) of NOD/SCID mice were stably reconstituted with BM derived from green-fluorescent protein (GFP) transgenic mice. Subsequently, brain tumor xenografts in an intracranial window chamber model (ICW) were generated using U87 glioma cells engineered to stably express mCherry fluorochrome. In-vivo imaging: Two-photon laser microscopy (2PLM) was used to obtain longitudinal real-time in-vivo images of the tumor cells, tumor vasculature and allow tracing of the circulating GFP+BMDCs in the ICW model. Mice were imaged daily for 20-30 days following cell implantation or following completion of RTx. Radiation Therapy (RTx): In order to examine the effects of IR, U87-mCherry tumors were treated with hemicranial irradiation at two regimens, 3×2Gy or 3×5Gy. U87-mCherry+RTx arm was compared to RTx alone or U87-mCherry alone. Brain Tumor Analysis: Mice were sacrificed in accordance with animal care protocol, using perfusion fixation and brains collected for correlative immunohistochemical (IHC) analysis. Results: Within 24 hours following tumor cell implantation GFP+ BMDCs can be seen circulating intravascularily and lining the vessel wall. 7d post implantation there is a decrease in GFP+BMDCs within vessels whilst cells migrating, differentiating and integrating outside the vessel lumen increases. IHC and 2PLM analysis identified three distinct GFP+ cells evolving over time: 1) circulating cells in vascular lumen in decreasing quantity, 2) supporting cells intimately encasing vessel walls that are smooth muscle cells or PVCs and 3) infiltrating cells within tumor microenvironment that are macrophages and micrglia. Control animals demonstrated a temporary recruitment of GFP+BMDCs to the injection site with no differentiation into cellular processes. Conclusion: Our results are the first to examine the real-time dynamic contribution of BMDC to glioma vasculature in-vivo. We demonstrate that BMDC differentiate to form macrophages and vascular support structures in the tumor microenvironment, with limited contribution to ECs. Ongoing work focuses on understanding alterations in response to IR and molecular regulators of BMDC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2384.

Abstract 4324: Establishing a high-resolution in vivo imaging technique for examining the dynamic contribution of bone marrow derived progenitor cells to intracranial tumor vasculature

Abstract Introduction: Recent data suggests that tumor vascularization can occur by differentiation of bone marrow derived progenitor cells (BMDCs) into new vessels. However the precise mechanisms by which this process occurs remains controversial and to date very few studies have examined the contribution of BMDC to brain tumor vascularity. One of the challenges in studying vascular biology, and more so intracranial vascular biology, is the ability to study the temporal evolution and dynamic progression of vessel formation in response to oncogenic signals and stressors. We present a strategy that couples two-photon excitation laser microscopy (2PLM) with glioma xenograft models generated in an intracranial window chamber (ICW), in order to obtain high resolution (e.g. single cell) longitudinal real-time imaging of BMDC and examine their homing, differentiation and integration into intracranial tumor vasculature. Methodology: Animal Models: Bone Marrow (BM) of NOD/SCID mice were stably reconstituted with BM harvested from green-fluorescent protein (GFP) transgenic mice. Briefly, NOD/SCID mice received total body irradiation at 3Gy followed by tail vein injection of harvested GFP+BM. Brain tumor xenografts in an ICW were generated in these chimera mice using U87 glioma cells engineered to stably express mCherry fluorochrome. Intracranial glioma xenograft in ICW: Intracranial window chambers were generated using a 2.7mm trephine drill to remove the skull overlying the right frontal cortex, leaving the dura intact. 4×10E5 U87-mCherry cells were injected at a 3mm cortical depth in the centre of the window. The window was covered with a coverslip sealed in place with dental acrylic. In vivo Imaging - 2PLM: Mice were anesthetized and imaged in an inverted position. Mice were imaged over a longitudinal period, d1-35, post cell transplantation. Results: We have successfully imaged 40 mice using intracranial glioma xenograft in ICW model coupled with 2PLM. We obtained longitudinal imaging up to 5 weeks following glioma cell implantation with no evidence of intracranial infection, inflammation or damage to the window model. High-resolution images of tumor cells, tumor vasculature and single cell resolution of the GFP+BM cells can be obtained. We can visualize tumor mass upto a 4mm depth from the cortical surface as confirmed by histological sections. The dynamic flow of GFP+BM within tumor vasculature can be visualized and quantified in 3D. Longitudinal differentiation of BMDCs can be traced by following their migration and integration into the glioma tumor microenvironment over time. Conclusion: We introduce a non-invasive and reproducible strategy for examining brain tumor microenvironment, its vascular progression and more precisely understanding at a single-cell level the contribution of BMDC brain tumor vascularity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4324.

3D imaging of the response to CDC25 inhibition in multicellular spheroids

Multicellular tumor spheroids closely mimic the 3D organization of avascular microregions within tumors and thereby represent a valuable model for the evaluation of anticancer drugs. In this study, we performed a 3D analysis of the response to the CDC25 phosphatase inhibitor IRC-083864 in HCT116 spheroids. Continuous exposure to IRC-083864 strongly inhibits the growth of spheroids and is shown to correlate with a decrease in Ki-67 positive cells. The cytotoxicity induced by IRC-083864 was examined by two-photon laser microscopy imaging and 3D reconstruction. Visualization in 3D allowed us to demonstrate that IRC-083864 treatment results in the inhibition of mitosis and induces cell death specifically localized in the outer proliferative cell layers of the spheroid structure. These results emphasize the importance of 3D models and of in toto analysis for the evaluation of anticancer drugs cytotoxicity.

Maternal separation decreases the stability of mushroom spines in adult mice somatosensory cortex

Maternal-separation (MS) is an important model to study the effects of maternal care on infant neuronal development. It has been previously shown that MS contributes to not only structural changes of neurons in the infralimbic cortex but also to significant behavioral changes in adulthood. However, the underlying mechanism of the MS effect on neuronal circuits is not clearly understood. In this study, we studied the effects of MS on the function related to somatosensory cortex (SSC) and spine remodeling in the SSC. We found that MS mice showed hypersensitivity to somatosensory stimulation at post-natal 4, 8 and 12 weeks. MS enhanced the turnover of mushroom-type spines, leading to a decrease of the number of spines in the SSC in young and adult mice observed by using in vivo two-photon laser microscopy imaging. We conclude that MS during development affects the stability of dendritic mushroom spines in the SSC, which possibly produces impairment of the sensory behavior in adult mice.

Two-Photon Imaging of Cellular Activities in Oxygen Sensing Tissues

The cellular oxygen sensing system of the body ensures appropriate adaptation of cellular functions toward hypoxia by regulating gene expression and ion channel activity. Two-photon laser microscopy is an ideal tool to study and prove the relevance of the molecular mechanisms within oxygen sensing pathways on the cellular and complex tissue or organ level. Images of hypoxia inducible factor 1 (HIF-1) subunit nuclear mobility and protein-protein interaction in living cells, of hypoxia-induced changes in membrane potential and intracellular calcium of live ex vivo carotid bodies as well as of rat kidney proximal tubulus function in vivo, will be shown.

Diallyl disulphide depletes glutathione in Candida albicans: oxidative stress‐mediated cell death studied by two‐photon microscopy

Using two-photon scanning laser microscopy, we investigated the effect of an Allium sativum (garlic) constituent, diallyl disulphide (DADS), on key physiological functions of the opportunistic pathogen Candida albicans. A short 30 min exposure to 0.5 mM DADS followed by removal induced 70% cell death (50% necrotic, 20% apoptotic) within 2 h, increasing to 75% after 4 h. The early intracellular events associated with DADS-induced cell death were monitored with two-photon fluorescence microscopy to track mitochondrial membrane potential (Deltapsi(m)), reactive oxygen species (ROS) and NADH or reduced glutathione (GSH) under aerobic conditions. DADS treatment decreased intracellular GSH and elevated intracellular ROS levels. Additionally, DADS induced a marked decrease of Deltapsi(m) and lowered respiration in cell suspensions and isolated mitochondria. In vitro kinetic experiments in cell-free extracts suggest that glutathione-S-transferase (GST) is one of the intracellular targets of DADS. Additional targets were also identified, including inhibition of a site or sites between complexes II-IV in the electron transport chain, as well as the mitochondrial ATP-synthase. The results indicate that DADS is an effective antifungal agent able to trigger cell death in Candida, most probably by eliciting oxidative stress as a consequence of thiol depletion and impaired mitochondrial function.

Local Retinal Circuits of Melanopsin-Containing Ganglion Cells Identified by Transsynaptic Viral Tracing

Intrinsically photosensitive melanopsin-containing retinal ganglion cells (ipRGCs) control important physiological processes, including the circadian rhythm, the pupillary reflex, and the suppression of locomotor behavior (reviewed in [1]). ipRGCs are also activated by classical photoreceptors, the rods and cones, through local retinal circuits [2, 3]. ipRGCs can be transsynaptically labeled through the pupillary-reflex circuit with the derivatives of the Bartha strain of the alphaherpesvirus pseudorabies virus(PRV) [4, 5] that express GFP [6-12]. Bartha-strain derivatives spread only in the retrograde direction [13]. There is evidence that infected cells function normally for a while during GFP expression [7]. Here we combine transsynaptic PRV labeling, two-photon laser microscopy, and electrophysiological techniques to trace the local circuit of different ipRGC subtypes in the mouse retina and record light-evoked activity from the transsynaptically labeled ganglion cells. First, we show that ipRGCs are connected by monostratified amacrine cells that provide strong inhibition from classical-photoreceptor-driven circuits. Second, we show evidence that dopaminergic interplexiform cells are synaptically connected to ipRGCs. The latter finding provides a circuitry link between light-dark adaptation and ipRGC function.

Cell signaling and trafficking of human melanocortin receptors in real time using two-photon fluorescence and confocal laser microscopy: differentiation of agonists and antagonists.

Melanocortin hormones and neurotransmitters regulate a vast array of physiologic processes by interacting with five G-protein-coupled melanocortin receptor types. In the present study, we have systematically studied the regulation of individual human melanocortin receptor wild subtypes using a synthetic rhodamine-labeled human melanotropin agonist and antagonist, arrestins fused to green fluorescent protein in conjunction with two-photon fluorescence laser scanning microscopy and confocal microscopy. Stimulation of the melanocortin receptors by its cognate agonist triggered rapid arrestin recruitment and receptor internalization for all four human melanocortin receptors examined. Antagonists-bound melanocortin receptors, on the other hand, did not recruit beta-arrestins, and remained in the cell membrane even after long-term (30 min) treatment. Agonist-mediated internalization of all melanocortin receptor subtypes was sensitive to inhibitors of clathrin-dependent endocytosis, but not to caveolae inhibitors. In summary, agonist-mediated internalization of all subtypes of melanocortin receptors are dependent upon beta-arrestin-mediated clathrin-coated pits, whereas, beta-arrestin-2 conjugated green fluorescence protein (beta-arrestin-2-GFP) recruitment is not dependent on protein kinase A activation. Real time two-photon fluorescence laser scanning microscopy is a most powerful tool to study the dynamic processes in living cells and tissues, without inflicting significant and often lethal damage to the specimen.

The expression of the NADPH oxidase subunit p22phox is regulated by a redox-sensitive pathway in endothelial cells

Endothelial dysfunction is characterized by increased levels of reactive oxygen species (ROS) and a prothrombotic state. The mechanisms linking thrombosis to ROS production in the endothelium are not well understood. We investigated the role of thrombin in regulating NADPH oxidase-dependent ROS production and expression of its subunit p22phox in the endothelial cell line EaHy926. Thrombin elicited a biphasic increase in ROS generation peaking within 15 min, but also at 3 h. The delayed response was accompanied by increased p22phox mRNA and protein expression. Two-photon confocal laser microscopy showed colocalization between p22phox and ROS production. Antioxidant treatment with vitamin C or diphenyleneiodonium abrogated thrombin-induced ROS production and p22phox expression, whereas H 2O 2 elevated ROS production and p22phox levels. Both responses were dependent on p38 MAP kinase and phosphatidylinositol-3-kinase (PI3 kinase)/Akt. Finally, p22phox was required for thrombin- or H 2O 2-stimulated proliferation. These data show that thrombin rapidly increases ROS production in endothelial cells, resulting, via activation of p38 MAP kinase and PI3 kinase/Akt, in upregulation of p22phox accompanied by a delayed increase in ROS generation and enhanced proliferation. These findings suggest a positive feedback mechanism whereby ROS, possibly generated by the NADPH oxidase, lead to elevated levels of p22phox and, thus, sustained ROS generation as is observed in endothelial dysfunction.

Deciphering the Oxygen Sensing Pathway by Microscopy

This chapter focuses on discovering oxygen sensing pathways by microscopy. Under normoxic conditions, O 2 sensing leads to an optimizing of cell function as exemplified by an enhanced phosphoenol pyruvate carboxykinase (PCK) expression in the periportal liver zone with high PO 2 levels and of enhanced glukokinase (GK) expression in the perivenous liver zone with low PO 2 levels. Under arterial hypoxia tissue, PO 2 frequency distribution is left shifted, leading to a hypoxic response of O 2 sensing for adaptation of cell function. In this chapter, three optical methods used to identify, locate, and image several putative members of the oxygen sensing pathway and their reaction to hypoxia are discussed. The three optical methods are based on light absorption photometry, the optical probe technique, and two-photon confocal laser microscopy. The described microscopic techniques are unique tools to reveal nature, regulation, and localization of critical members of the oxygen sensing pathway. Three-dimensional imaging of intracellular oxygen sensing and concepts related to two photon confocal laser-scanning microscopy and intracellular localization of prolyl hydroxylases are described.

T cell-dendritic cell interaction in vivo: random encounters favor development of long-lasting ties.

Understanding the complexity of the functional communication between cells composing the immune system is central to improving our capacity to manipulate it and conceive better strategies to combat microbial pathogens. So far, these studies have been based on immunohistochemistry of fixed tissues and in vitro attempts to reproduce functional connections between cells. The application of two-photon laser microscopy to the observation of viable immune cells in their natural environment where foreign antigens are carried to trigger an immune response opens a new era for these studies. They reveal exceptional properties of the locomotion of T cells that facilitate encounters with dendritic cells and the receipt of information that promotes T cell survival, death, or initiation of immune responses. These studies also complement in vitro observations addressing the importance of time of stimulation in determining T cell fates.

T Cell-Dendritic Cell Interaction in Vivo: Random Encounters Favor Development of Long-Lasting Ties

Understanding the complexity of the functional communication between cells composing the immune system is central to improving our capacity to manipulate it and conceive better strategies to combat microbial pathogens. So far, these studies have been based on immunohistochemistry of fixed tissues and in vitro attempts to reproduce functional connections between cells. The application of two-photon laser microscopy to the observation of viable immune cells in their natural environment where foreign antigens are carried to trigger an immune response opens a new era for these studies. They reveal exceptional properties of the locomotion of T cells that facilitate encounters with dendritic cells and the receipt of information that promotes T cell survival, death, or initiation of immune responses. These studies also complement in vitro observations addressing the importance of time of stimulation in determining T cell fates.

Synthesis and visualization of a membrane-permeable MRI contrast agent.

The study of in vivo developmental events has undergone significant advances with the advent of biological molecular imaging techniques such as computer enhanced light microscopy imaging, positron emission tomography (PET), micro-CT, and magnetic resonance imaging (MRI). MRI has proven to be a particularly powerful tool in clinical and biological settings. Images can be acquired of opaque living animals, with the benefit of tracking events of extended periods of time on the same specimen. Contrast agents are routinely used to enhance regions, tissues, and cells that are magnetically similar but histologically distinct. A principal barrier to the development of MR contrast agents for investigating developmental biological questions is the ability to deliver the agent across cellular membranes. As part of our research, we are investigating a number of small molecules that facilitate transport of charged and uncharged species across cell membranes. Here we describe the synthesis and testing of a Gd(III)-based MR contrast agent conjugated to polyarginine that is able to permeate cell membranes. We confirmed cellular uptake of the agent using two-photon laser microscopy to visualize a Eu(III) derivative of the contrast agent in cell culture, and verified this uptake by T(1) analysis of the Gd(III) agent in cells.

Autonomous T cell trafficking examined in vivo with intravital two-photon microscopy.

The recirculation of T cells between the blood and secondary lymphoid organs requires that T cells are motile and sensitive to tissue-specific signals. T cell motility has been studied in vitro, but the migratory behavior of individual T cells in vivo has remained enigmatic. Here, using intravital two-photon laser microscopy, we imaged the locomotion and trafficking of naive CD4(+) T cells in the inguinal lymph nodes of anesthetized mice. Intravital recordings deep within the lymph node showed T cells flowing rapidly in the microvasculature and captured individual homing events. Within the diffuse cortex, T cells displayed robust motility with an average velocity of approximately 11 microm x min(-1). T cells cycled between states of low and high motility roughly every 2 min, achieving peak velocities >25 microm x min(-1). An analysis of T cell migration in 3D space revealed a default trafficking program analogous to a random walk. Our results show that naive T cells do not migrate collectively, as they might under the direction of pervasive chemokine gradients. Instead, they appear to migrate as autonomous agents, each cell taking an independent trafficking path. Our results call into question the role of chemokine gradients for basal T cell trafficking within T cell areas and suggest that antigen detection may result from a stochastic process through which a random walk facilitates contact with antigen-presenting dendritic cells.

Switch to Anaerobic Glucose Metabolism with NADH Accumulation in the β-Cell Model of Mitochondrial Diabetes: CHARACTERISTICS OF βHC9 CELLS DEFICIENT IN MITOCHONDRIAL DNA TRANSCRIPTION

To elucidate the mechanism underlying diabetes caused by mitochondrial gene mutations, we created a model by applying 0.4 microg/ml ethidium bromide (EtBr) to the murine pancreatic beta cell line betaHC9; in this model, transcription of mitochondrial DNA, but not that of nuclear DNA, was suppressed in association with impairment of glucose-stimulated insulin release (Hayakawa, T., Noda, M., Yasuda, K., Yorifuji, H., Taniguchi, S., Miwa, I., Sakura, H., Terauchi, Y., Hayashi, J.-I., Sharp, G. W. G., Kanazawa, Y., Akanuma, Y., Yazaki, Y., and Kadowaki, T. (1998) J. Biol. Chem. 273, 20300-20307). To elucidate fully the metabolism-secretion coupling in these cells, we measured glucose oxidation, utilization, and lactate production. We also evaluated NADH autofluorescence in betaHC9 cells using two-photon excitation laser microscopy. In addition, we recorded the membrane potential and determined the ATP and ADP contents of the cells. The results indicated 22.2 mm glucose oxidation to be severely decreased by EtBr treatment compared with control cells (by 63% on day 4 and by 78% on day 6; both p < 0.01). By contrast, glucose utilization was only marginally decreased. Lactate production under 22.2 mm glucose was increased by 2.9- and 3.5-fold by EtBr treatment on days 4 and 6, respectively (both p < 0.01). Cellular NADH at 2.8 mm glucose was increased by 35 and 43% by EtBr on days 4 and 6 (both p < 0.01). These data suggest that reduced expression of the mitochondrial electron transport system causes NADH accumulation in beta cells, thereby halting the tricarboxylic acid cycle on one hand, and on the other hand facilitating anaerobic glucose metabolism. Glucose-induced insulin secretion was lost rapidly along with the EtBr treatment with concomitant losses of membrane potential depolarization and the [Ca(2+)](i) increase, whereas glibenclamide-induced changes persisted. This is the first report to demonstrate the connection between metabolic alteration of electron transport system and that of tricarboxylic acid cycle and its impact on insulin secretion.

Two-photon imaging of lymphocyte motility and antigen response in intact lymph node.

Lymphocyte motility is vital for trafficking within lymphoid organs and for initiating contact with antigen-presenting cells. Visualization of these processes has previously been limited to in vitro systems. We describe the use of two-photon laser microscopy to image the dynamic behavior of individual living lymphocytes deep within intact lymph nodes. In their native environment, T cells achieved peak velocities of more than 25 micrometers per minute, displaying a motility coefficient that is five to six times that of B cells. Antigenic challenge changed T cell trajectories from random walks to "swarms" and stable clusters. Real-time two-photon imaging reveals lymphocyte behaviors that are fundamental to the initiation of the immune response.