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Extended abstract of a paper presented at Microscopy and Microanalysis 2013 in Indianapolis, Indiana, USA, August 4 – August 8, 2013.

A gas discharge strontium vapor laser has been shown to operate with up to 90% of its light emitted at 6.45 μm. We have investigated the use of this laser as a potential stand-alone, tabletop alternative to the FEL for ablation of soft tissue. This custom-made laser currently delivers up to 2.4 watts of average power at 13 kHz pulse repetition rate (range 5-20 kHz). Despite a poor spatial beam profile the laser has been shown to ablate both water and soft tissue. However, current pulse energies (< 185 μJ) are insufficient for single pulse ablation even when focused to the smallest possible spot size (130 μm). Instead, the high pulse repetition rate causes the ablation to occur in a quasi CW manner. The dynamics of ablation studied by pump-probe (Schlieren) imaging and macroscopic white light imaging showed micro-explosions but at a rate well below the pulse repetition frequency. Histological analysis of ablation craters in bovine muscle exhibited significant collateral thermal damage, consistent with the high pulse frequency, thermal superposition and heat diffusion. Efforts to increase the pulse energy in order to achieve the threshold for pulse-to-pulse ablation are ongoing and will be discussed.

The development of digital microscopy and computational power is providing new opportunities for analyzing the motility of the vesicles (proteins) within living cells. In this paper, an automatic method is developed to segment and track vesicles in large amount of fluorescence images, in order to compute a number of quantitative parameters such as displacement, residence time, binding, or immobile fraction. We present a method that permits the automatic tracking of subcellular structures in long sequences of fluorescence images (up to 100 frames). The method we propose has been tested on 92 data sets totaling 8225 frames.

Visual sensation in vertebrates is triggered when light strikes retinal photoreceptor cells causing photoisomerization of the rhodopsin chromophore 11-cis-retinal to all-trans-retinal. The regeneration of preillumination conditions of the photoreceptor cells requires formation of 11-cis-retinal in the adjacent retinal pigment epithelium (RPE). Using the intrinsic fluorescence of all-trans-retinyl esters, noninvasive two-photon microscopy revealed previously uncharacterized structures (6.9 ± 1.1 μm in length and 0.8 ± 0.2 μm in diameter) distinct from other cellular organelles, termed the retinyl ester storage particles (RESTs), or retinosomes. These structures form autonomous all-trans-retinyl ester-rich intracellular compartments distinct from other organelles and colocalize with adipose differentiation-related protein. As demonstrated by in vivo experiments using wild-type mice, the RESTs participate in 11-cis-retinal formation. RESTs accumulate in Rpe65−/− mice incapable of carrying out the enzymatic isomerization, and correspondingly, are absent in the eyes of Lrat−/− mice deficient in retinyl ester synthesis. These results indicate that RESTs located close to the RPE plasma membrane are essential components in 11-cis-retinal production.

Scanning near-field optical microscopy (SNOM) makes it routinely possible to overcome the fundamental diffraction limit of standard (far-field) microscopy. Recently, aperture-based infrared SNOM performed in the spectroscopic mode, using the Vanderbilt University free electron laser, started delivering spatially-resolved information on the distribution of chemical species and on other laterally-fluctuating properties. The practical examples presented here show the great potential of this new technique both in materials science and in life sciences. (C) 2004 Elsevier B.V. All rights reserved.

Pancreatic beta cells secrete insulin in response to changes in the extracellular glucose. However, prolonged exposure to elevated glucose exerts toxic effects on beta cells and results in beta cell dysfunction and ultimately beta cell death (glucose toxicity). To investigate the mechanism of how increased extracellular glucose is toxic to beta cells, we used two model systems where glucose metabolism was increased in beta cell lines by enhancing glucokinase (GK) activity and exposing cells to physiologically relevant increases in extracellular glucose (3.3-20 mm). Exposure of cells with enhanced GK activity to 20 mm glucose accelerated glycolysis, but reduced cellular NAD(P)H and ATP, caused accumulation of intracellular reactive oxygen species (ROS) and oxidative damage to mitochondria and DNA, and promoted apoptotic cell death. These changes required both enhanced GK activity and exposure to elevated extracellular glucose. A ROS scavenger partially prevented the toxic effects of increased glucose metabolism. These results indicate that increased glucose metabolism in beta cells generates oxidative stress and impairs cell function and survival; this may be a mechanism of glucose toxicity in beta cells. The level of beta cell GK may also be critical in this process.

Fluorescence anisotropy measurements can elucidate the microenvironment of a membrane protein in terms of its rotational diffusion, interactions, and proximity to other proteins. However, use of this approach requires a fluorescent probe that is rigidly attached to the protein of interest. Here we describe the use of one such probe, a green fluorescent protein (GFP) expressed and rigidly held within the amino acid sequence of a major histocompatibility complex (MHC) class I molecule, H2L(d). We contrast the anisotropy of this GFP-tagged MHC molecule, H2L(d)GFPout, with that of an H2L(d) that was GFP-tagged at its C-terminus, H2L(d)GFPin. Both molecules fold properly, reach the cell surface, and are recognized by specific antibodies and T-cell receptors. We found that polarized fluorescence images of H2L(d)GFPout in plasma membrane blebs show intensity variations that depend on the relative orientation of the polarizers and the membrane normal, thus demonstrating that the GFP is oriented with respect to the membrane. These variations were not seen for H2L(d)GFPin. Before transport to the membrane surface, MHC class I associates with the transporter associated with antigen processing complex in the endoplasmic reticulum. The intensity-dependent steady-state anisotropy in the ER of H2L(d)GFPout was consistent with FRET homotransfer, which indicates that a significant fraction of these molecules were clustered. After MCMV-peptide loading, which supplies antigenic peptide to the MHC class I releasing it from the antigen processing complex, the anisotropy of H2L(d)GFPout was independent of intensity, suggesting that the MHC proteins were no longer clustered. These results demonstrate the feasibility and usefulness of a GFP moiety rigidly attached to the protein of interest as a probe for molecular motion and proximity in cell membranes.

Multiphoton laser scanning microscopy offers advantages in depth of penetration into intact samples over other optical sectioning techniques. To achieve these advantages it is necessary to detect the emitted light without spatial filtering. In this nondescanned (nonconfocal) approach, ambient room light can easily contaminate the signal, forcing experiments to be performed in absolute darkness. For multiphoton microscope systems employing mode-locked lasers, signal processing can be used to reduce such problems by taking advantage of the pulsed characteristics of such lasers. Specifically, by recovering fluorescence generated at the mode-locked frequency, interference from stray light and other ambient noise sources can be significantly reduced. This technology can be adapted to existing microscopes by inserting demodulation circuitry between the detector and data collection system. The improvement in signal-to-noise ratio afforded by this approach yields a more robust microscope system and opens the possibility of moving multiphoton microscopy from the research lab to more demanding settings, such as the clinic.

LiF films have been grown on silicon substrate, irradiated with soft x-rays to create fluorescent regular micrometric-spaced arrays based on colour centres, and studied by Scanning Near-field Optical Microscope (SNOM). Strong variations in the local reflectivity have been observed in the infrared region between 6.1 and 9.2 mum and tentatively ascribed to a modulated variation of the refractive index of the coloured zone with respect to that of the uncoloured LiF matrix. (C) 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Publisher Summary This chapter describes a resonance energy transfer method based on bioluminescence. Fluorescence resonance energy transfer (FRET) is a well-established phenomenon that is useful in cellular microscopy. When two fluorophores (the “donor” and the “acceptor”) with overlapping emission/absorption spectra are within ∼50Ǻ of one another and their transition dipoles are appropriately oriented, the donor fluorophore is able to transfer its excited state energy to the acceptor fluorophore. Protein–protein interactions are known to play an important role in a variety of biochemical systems. To date, thousands of protein–protein interactions are identified by using the conventional two-hybrid system, but this method is limited in that the interaction must occur in the yeast nucleus. This means interactions that strictly depend on cell type-specific processing and/or compartmentalization cannot be detected. Therefore, a number of new methods are developed that rely on reconstitution of biochemical function in vivo , such as fluorescence resonance energy transfer (FRET), protein mass spectrometry, or evanescent wave spectroscopy.

Silicon and silicon nitride surfaces have been successfully terminated with carboxylic acid monolayers and investigated by atomic force microscopy (AFM) and scanning near-field optical microscopy (SNOM). On clean Si surface, AFM showed topographical variations of 0.3-0.4 nm while for the clean Si3N4 surface the corrugation was around 3-4 nm. After material deposition, the corrugation increased in both samples with a value in topography of 1-2 nm for Si and 5-6 nm for Si3N4. The space distribution of specific chemical species was obtained by taking SNOM reflectivity at several infrared wavelengths corresponding to stretch absorption bands of the material. The SNOM images showed a constant contribution in the local reflectance, suggesting that the two surfaces were uniformly covered. (C) 2003 Elsevier B.V. All rights reserved.

Extended abstract of a paper presented at Microscopy and Microanalysis 2012 in Phoenix, Arizona, USA, July 29 – August 2, 2012.

Glucose homeostasis is maintained by small clusters of hormone secreting cells in the pancreas: the pancreatic islets. Insulin secreting β-cells make up ∼ 80% of the mouse islet and secrete insulin in a tightly regulated manner. Understanding the mechanisms that regulate insulin secretion is a key factor in developing drugs and therapies for type-2 diabetes and metabolic syndrome.The pancreatic β-cells synthesize dopamine from the circulating precursor L-dopa. We have shown that during glucose stimulated insulin secretion, co-secreted dopamine acts as an autocrine negative regulator of insulin secretion. It does so by activating the dopamine receptor D3 (DRD3) – a member of the G-protein coupled receptor family. DRD3 are present on β-cell plasma membrane, and upon dopamine binding, they attenuate intracellular Ca2+ dynamics. In fact, the frequency of the [Ca2+]i oscillations triggered by 8 mM glucose is diminished by dopamine; these effects are blocked when a selective DRD3 antagonist is added [Ustione and Piston, Mol. Endo. 2012].We investigate the molecular mechanism downstream of DRD3 activation that leads to the changes in intracellular Ca2+ dynamics and insulin secretion. We propose that dopamine activation of DRD3 directly affects β-cell calcium influx via Gβγ complex interaction with the L-type Ca2+ channel (CaV1.2). We test this hypothesis using FRET and two photon excitation FCCS experiments on βTC-3 cells with fluorescent protein labeled versions of the Gβγ complex, and of the CaV1.2 subunit α1C. The goal is to detect the dopamine triggered interaction between the Gβγ complex and the CaV1.2. We can modulate the FRET signal by using the photo-switchable version of the red fluorescent protein rsTagRFP, therefore we can measure FRET from the small percentage of interacting proteins, even in the presence of significant background signal arising from the majority of labeled but noninteracting proteins.

In muscle, insulin enhances influx of glucose and its conversion to glucose 6-phosphate (G6P) by hexokinase (HK). While effects of insulin on glucose transport have been demonstrated, its effect on the activity of HK of cells has not. In L6 myotubes treated for 24h with insulin there was increased expression of the HK isoform, HKII, and increased glucose phosphorylation without a concomitant increase in glucose transport, indirectly suggesting that phosphorylation of glucose was a target of insulin action [Osawa, Printz, Whitesell and Granner (1995) Diabetes 44, 1426—1432]. In the present work the same treatment led to a 2-fold rise in G6P, suggesting that transport and/or HK were important targets of insulin action. We used a method to identify the site of rate control involving the specificity of phosphorylation towards 2-deoxy-[1-14C]glucose and d-[2-3H]glucose. Glucose transport does not greatly discriminate between these two tracers while HK shows increased specificity for glucose. Specificity of the glucose phosphorylation of the cells increased with addition of insulin and when extracellular glucose was raised. Specificity was reduced at low glucose concentrations or when the inhibitor of transport, cytochalasin B, was added. We conclude that transport and HK share nearly equal control over glucose phosphorylation in these cells. A computer program was used to test models for compatibility with the different types of experiments. The predicted intracellular glucose and transport rates associated with phosphorylation activity were lower than their measured values for the whole cell. In the most likely model, 15±4% of the glucose transporters serve a proportionate volume of the cytoplasm. Insulin activation of glucose phosphorylation might then result from stimulation of these transporters together with HK recruitment or relief from inhibition by G6P.

Glucokinase (GK) activity is essential for the physiological regulation of insulin secretion by glucose. Because the enzyme exerts nearly total control over glucose metabolism in the beta-cell, even small changes in GK activity exert effects on glucose-stimulated insulin secretion and, consequently, the blood glucose concentration. Using quantitative imaging of multicolor fluorescent proteins fused to GK, we found that the association of GK with insulin granules is regulated by glucose in the beta-cell. Glucose stimulation increased the rate of fluorescence recovery after photobleaching of GK to insulin granules, indicating that GK is released into the cytoplasm after glucose stimulation. Changes in fluorescence resonance energy transfer between two different fluorescent protein variants inserted on opposing ends of GK were observed after glucose stimulation and correlated with increased enzyme activity. Furthermore, glucose-stimulated changes in GK regulation were blocked by two inhibitors of insulin secretion. Insulin treatment restored GK regulation in inhibited cells and stimulated GK translocation and activation by itself. Together, these data support a model for post-translational regulation of GK whereby insulin regulates both the association of GK with secretory granules and the activity of the enzyme within the pancreatic beta-cell.

Multiphoton laser scanning microscopy offers numerous advantages, but sensitivity can be seriously affected by contamination from ambient room light. Typically, this forces experiments to be performed in an absolutely dark room. Since mode-locked lasers are used to generate detectable signals, signal-processing can be used to avoid such problems by taking advantage of the pulsed characteristics of such lasers. Demodulation of the fluorescence signal generated at the mode-locked frequency can result in significant reduction of interference from ambient noise sources. Such demodulation can be readily adapted to existing microscopes by inserting appropriate processor circuitry between the detector and data collection system, yielding a more robust microscope.

The corepressor CtBP (carboxyl-terminal binding protein) is involved in transcriptional pathways important for development, cell cycle regulation, and transformation. We demonstrate that CtBP binding to cellular and viral transcriptional repressors is regulated by the nicotinamide adenine dinucleotides NAD + and NADH, with NADH being two to three orders of magnitude more effective. Levels of free nuclear nicotinamide adenine dinucleotides, determined using two-photon microscopy, correspond to the levels required for half-maximal CtBP binding and are considerably lower than those previously reported. Agents capable of increasing NADH levels stimulate CtBP binding to its partners in vivo and potentiate CtBP-mediated repression. We propose that this ability to detect changes in nuclear NAD + /NADH ratio allows CtBP to serve as a redox sensor for transcription.

Complete lack of transcription factor PDX-1 leads to pancreatic agenesis, whereas heterozygosity for PDX-1 mutations has been recently noted in some individuals with maturity-onset diabetes of the young (MODY) and in some individuals with type 2 diabetes. To determine how alterations in PDX-1 affect islet function, we examined insulin secretion and islet physiology in mice with one PDX-1 allele inactivated. PDX-1(+/-) mice had a normal fasting blood glucose and pancreatic insulin content but had impaired glucose tolerance and secreted less insulin during glucose tolerance testing. The expression of PDX-1 and glucose transporter 2 in islets from PDX-1(+/-) mice was reduced to 68 and 55%, respectively, whereas glucokinase expression was not significantly altered. NAD(P)H generation in response to glucose was reduced by 30% in PDX-1(+/-) mice. The in situ perfused pancreas of PDX-1(+/-) mice secreted about 45% less insulin when stimulated with 16.7 mm glucose. The K(m) for insulin release was similar in wild type and PDX-1(+/-) mice. Insulin secretion in response to 20 mm arginine was unchanged; the response to 10 nm glucagon-like peptide-1 was slightly increased. However, insulin secretory responses to 10 mm 2-ketoisocaproate and 20 mm KCl were significantly reduced (by 61 and 66%, respectively). These results indicate that a modest reduction in PDX-1 impairs several events in glucose-stimulated insulin secretion (such as NAD(P)H generation, mitochondrial function, and/or mobilization of intracellular Ca(2+)) and that PDX-1 is important for normal function of adult pancreatic islets.

Extended abstract of a paper presented at Microscopy and Microanalysis 2011 in Nashville, Tennessee, USA, August 7–August 11, 2011.

β-cells in the pancreatic islet respond to elevated plasma glucose by secreting insulin to maintain glucose homeostasis. In addition to glucose stimulation, insulin secretion is modulated by numerous G-protein coupled receptors (GPCRs). The GPCR ligands Kisspeptin-10 (KP) and glucagon-like peptide-1 (GLP-1) potentiate insulin secretion through Gq and Gs-coupled receptors, respectively. Despite many studies, the signaling mechanisms by which KP and GLP-1 potentiate insulin release are not thoroughly understood. We investigated the downstream signaling pathways of these ligands and their affects on cellular redox potential, intracellular calcium activity ([Ca(2+)]i), and insulin secretion from β-cells within intact murine islets. In contrast to previous studies performed on single β-cells, neither KP nor GLP-1 affect [Ca(2+)]i upon stimulation with glucose. KP significantly increases the cellular redox potential, while no effect is observed with GLP-1, suggesting that KP and GLP-1 potentiate insulin secretion through different mechanisms. Co-treatment with KP and the Gβγ-subunit inhibitor gallein inhibits insulin secretion similar to that observed with gallein alone, while co-treatment with gallein and GLP-1 does not differ from GLP-1 alone. In contrast, co-treatment with the Gβγ activator mSIRK and either KP or GLP-1 stimulates insulin release similar to mSIRK alone. Neither gallein nor mSIRK alter [Ca(2+)]i activity in the presence of KP or GLP-1. These data suggest that KP likely alters insulin secretion through a Gβγ-dependent process that stimulates glucose metabolism without altering Ca(2+) activity, while GLP-1 does so, at least partly, through a Gα-dependent pathway that is independent of both metabolism and Ca(2+).

Live cell fluorescence microscopy offers a powerful way to examine structure and dynamics of proteins and protein complexes, and has been used to elucidate many of the spatial and temporal relationships between molecules within the cell. While most fluorescence microscopy relies solely on signal brightness, the photophysical properties of fluorescence provide many other parameters that can be utilized as well. These parameters include spectral shape, lifetime, and polarization. Fluorescence polarization, also referred to as anisotropy, has long been used in cuvette spectroscopy of proteins and lipids. While the basic infrastructure needed to make anisotropy measurements in the microscope has been developed, this approach has not yet found widespread use. This is somewhat surprising, considering that the polarizations of widely utilized fluorescence proteins are intrinsically high because of the fixed orientation of chromophore in the β-barrel structure (1). In this issue of the Biophysical Journal, a group at Rockefeller University describes the elegant utilization of fluorescence polarization to resolve structure of individual components of the nuclear pore complex (NPC) in live cells (2). This novel, to my knowledge, approach permits the researchers to distinguish between ordered and disordered domains within the NPC components. As the authors rightfully conclude, this information provides an important bridge between high-resolution structural information available from x-ray crystallography or cryo-electromagnetic studies and more-traditional cell biological studies that offer limited spatial resolution. This study also provides a comprehensive theoretical and experimental framework for anisotropy imaging experiments that should open this approach up for the study of many other intracellular protein complexes. Three experimental insights by Mattheyses et al. (2) made this study possible. First, the authors utilized GFP, which provides not only outstanding specificity of labeling, but also highly polarized fluorescence. Because the GFP chromophore is rigid within the overall protein structure, which is slowly rotating (10–20-ns rotational correlation time) with respect to the fluorescence lifetime of ∼3 ns, this results in highly polarized fluorescence that is easy to measure. Second, the authors used no extra amino acids between the GFP and the NPC component protein of interest. This led to a sufficiently rigid structure in which the GFP orientation reproduces the labeled protein orientation as well as possible. Third, they used the known symmetry of the NPC to obtain a full 360° orientation map at each NPC. Similar approaches have been used to map the plasma membrane as in, for instance, red blood cell ghosts (3), but this is the first such application to a single protein complex. Because many protein complexes have been shown to possess significant symmetry, this approach may prove quite general. As for any powerful technique, there are potential experimental pitfalls that must be carefully considered. For these detailed anisotropy imaging experiments, two important potential artifacts come from the possibility of resonance energy transfer between labels (homo-Forster resonance energy transfer, or homoFRET), and the use of a high numerical aperture objective. Both of these issues are addressed by the authors both theoretically and experimentally in exemplary fashion. First, because homoFRET can occur between two labels in different orientations, it can appear as a “rotation” and dramatically change the measured polarization (4). This is of particular concern at higher concentrations of labels, which might be expected within a single NPC. Fortunately, in this study, control experiments performed with different labeling concentrations showed that homoFRET was not an issue. Secondly, the sensitivity of any fluorescence microscopy experiment depends on the number of photons collected, and typically high numerical aperture objectives are used to facilitate maximal light collection. However, the use of polarized light with high NA lenses requires extra corrections, which were performed and validated as part of this study. One of the key difficulties in studying protein complexes inside the cell has been the lack of appropriate techniques for live cell imaging. Scanning near-field optical microscopy (SNOM) and total internal reflection fluorescence microscopy (TIRF) have been used to examine NPC in vitro, but because they are inherently surface measurement techniques, neither approach can be used to assay the structure or dynamics of the NPC inside the cell. Both single particle tracking (SPT) and fluorescence correlation spectroscopy (FCS) have been used to monitor molecules passing through the NPC pore, and these approaches can offer dynamic information at high spatial and temporal resolution, but they yield no information related directly to the structure of the NPC. Anisotropy microscopy with GFP labels, on the other hand, is somewhat analogous to the probe methods used to explore protein structure, such as luminescence resonance energy transfer (LRET) (5), tryptophan quenching of fluorescence (6), and site-directed spin-labeling electron paramagnetic resonance (EPR) (7). Most importantly, anisotropy imaging can be accomplished in live cells, yielding a new and complementary in vivo link among structure, dynamics, and the actual function of protein complexes in their native environment.

Lack of Casq2 causes spontaneous Ca2+ releases from the sarcoplasmic reticulum (SR) and catecholaminergic-polymorphic ventricular tachycardia. We tested the hypothesis that lack of Casq2 alters elementary Ca2+ release events (Ca2+ sparks) by comparing spark properties of ventricular myocytes isolated from wild-type (WT) and Casq2 null (Casq2-/-) mice. Sparks were recorded in line-scan mode and analyzed with SparkMaster. Spark mass was calculated as amplitude×1.206×FWHM3. SR Ca2+ load was measured by rapid application of caffeine. In intact Casq2-/- myocytes stimulated with 100nM isoproterenol, spark amplitude and spark width (FWHM) increased compared to WT (0.77±0.019 vs. 0.39±0.02 Δ(F/Fo), and 2.4±0.03 vs. 1.3±0.05μm, 1095 and 105 sparks respectively), resulting in larger spark mass (20±1.4 vs. 2.2±0.4 Δ(F/Fo).μm3). Time-to-peak and spark duration (FDHM) were 2.5-fold longer and spark frequency was 4-fold higher in Casq2-/- myocytes (2.4±0.2 vs. 0.5±0.08 sparks×100μm-1×s-1). Spark-mediated leak (spark mass×spark frequency) was much larger (47.6 vs. 1.2 Δ(F/Fo) μm3). In saponin-permeabilized myocytes, spark-mediated leak and spark frequency were also higher in Casq2-/- myocytes (249.2 vs. 171.3 Δ(F/Fo)μm3, and 9.5±1 vs. 4.2±1 sparks×100μm-1×s-1, 587 and 333 sparks for Casq-/- and WT respectively), but the differences between Casq-/- and WT were less pronounced compared to intact myocytes. This may be a consequence of the increased spark-mediated SR Ca2+ leak resulting in significantly decreased SR Ca2+ load in permeabilized Casq2-/- cells (−25% at baseline and −31% with 50μM cAMP, n= 10-16 myocytes per group).Conclusions: Lack of Casq2 in cardiac myocytes increases the spark frequency and the spark-mediated leak. This is still observed in permeabilized cells despite decreased SR Ca2+ load, suggesting that this is due to a primary Casq2 effect on RyR2 SR Ca2+ release channels. This may be in part responsible for the increased arrhythmia susceptibility in Casq2-/- mice.

Two-photon excitation microscopy was used to image and quantify NAD(P)H autofluorescence from intact pancreatic islets under glucose stimulation. At maximal glucose stimulation, the rise in whole-cell NAD(P)H levels was estimated to be approximately 30 microM. However, because glucose-stimulated insulin secretion involves both glycolytic and Kreb's cycle metabolism, islets were cultured on extracellular matrix that promotes cell spreading and allows spatial resolution of the NAD(P)H signals from the cytoplasm and mitochondria. The metabolic responses in these two compartments are shown to be differentially stimulated by various nutrient applications. The glucose-stimulated increase of NAD(P)H fluorescence within the cytoplasmic domain is estimated to be approximately 7 microM. Likewise, the NAD(P)H increase of the mitochondrial domain is approximately 60 microM and is delayed with respect to the change in cytoplasmic NAD(P)H by approximately 20 sec. The large mitochondrial change in glucose-stimulated NAD(P)H thus dominates the total signal but may depend on the smaller but more rapid cytoplasmic increase.

Extended abstract of a paper presented at Microscopy and Microanalysis 2009 in Richmond, Virginia, USA, July 26 – July 30, 2009

We describe a method for assaying protein interactions that offers some attractive advantages over previous assays. This method, called bioluminescence resonance energy transfer (BRET), uses a bioluminescent luciferase that is genetically fused to one candidate protein, and a green fluorescent protein mutant fused to another protein of interest. Interactions between the two fusion proteins can bring the luciferase and green fluorescent protein close enough for resonance energy transfer to occur, thus changing the color of the bioluminescent emission. By using proteins encoded by circadian (daily) clock genes from cyanobacteria, we use the BRET technique to demonstrate that the clock protein KaiB interacts to form homodimers. BRET should be particularly useful for testing protein interactions within native cells, especially with integral membrane proteins or proteins targeted to specific organelles.

The relationship between glucokinase (GK) and glucose-stimulated metabolism, and the potential for metabolic coupling between beta cells, was examined in isolated mouse islets by using a recombinant adenovirus that expresses Cre recombinase (AdenoCre) to inactivate a conditional GK gene allele (gklox). Analysis of AdenoCre-treated islets indicated that the gklox allele in approximately 30% of islet cells was converted to a nonexpressing variant (gkdel). This resulted in a heterogeneous population of beta cells where GK was absent in some cells. Quantitative two-photon excitation imaging of NAD(P)H autofluorescence was then used to measure glucose-stimulated metabolic responses of individual islet beta cells from gklox/lox mice. In AdenoCre-infected islets, approximately one-third of the beta cells showed markedly lower NAD(P)H responses. These cells also exhibited glucose dose responses consistent with the loss of GK. Glucose dose responses of the low-responding cells were not sigmoidal and reached a maximum at approximately 5 mM glucose. In contrast, the normal response cells showed a sigmoidal response with an KcatS0.5 of approximately 8 mM. These data provide direct evidence that GK is essential for glucose-stimulated metabolic responses in beta cells within intact islets and that intercellular coupling within the islet plays little or no role in glucose-stimulated metabolic responses.

Publisher Summary The chapter presents a discussion on quantitative imaging of metabolism by two-photon excitation microscopy. The chapter describes the use of the naturally occurring reduced nicotinamide adenine dinucleotide (phosphate) [NAD(P)H] as a monitor of cellular metabolism. To image these ultraviolet (UV)-absorbing fluorophores in living cells, two-photon excitation microscopy that minimizes the photodamage associated with NAD(P)H imaging is utilized. The chapter describes the use of NAD(P)H as a metabolic indicator and discusses the two-photon excitation microscopy methods, which is used to image its activity. The chapter details the instruments that are used for these experiments, with emphasis on the important design criteria for this demanding application. Finally, the chapter presents the application of two-photon excitation imaging of NAD(P)H to assay glucose-stimulated metabolism in both pancreatic and muscle cells. Fluorescence from naturally occurring NAD(P)H can be used as an indicator of cellular respiration and therefore as an intrinsic probe to study cellular metabolism. The chapter discusses the background and concepts of two-photon excitation microscopy, applications to quantitative metabolic imaging, quantitative metabolic imaging of β cells in intact pancreatic islets, quantitative imaging of muscle metabolism and several related concepts.

Screening for photostability in addition to color and brightness creates better fluorescent proteins.

The green fluorescent protein (GFP) from the jellyfish Aequorea victoria is a versatile reporter protein for monitoring gene expression and protein localization in a variety of systems. Applications using GFP reporters have expanded greatly due to the availability of mutants with altered spectral properties, including several blue emission variants, all of which contain the single point mutation Tyr-66 to His in the chromophore region of the protein. However, previously described “BFP” reporters have limited utility, primarily due to relatively dim fluorescence and low expression levels attained in higher eukaryotes with such variants. To improve upon these qualities, we have combined a blue emission mutant of GFP containing four point mutations (Phe-64 to Leu, Ser-65 to Thr, Tyr-66 to His, and Tyr-145 to Phe) with a synthetic gene sequence containing codons preferentially found in highly expressed human proteins. These mutations were chosen to optimize expression of properly folded fluorescent protein in mammalian cells cultured at 37 °C and to maximize signal intensity. The combination of improved fluorescence and higher expression levels yield an enhanced blue fluorescent protein that provides greater sensitivity and is suitable for dual color detection with green-emitting fluorophores.

Publisher Summary This chapter discusses the properties of green fluorescent protein (GFP) that are important for quantitative imaging. Spectral and physical properties of GFP affect the accuracy and usefulness of any quantitative measurement. Many of these properties, such as extinction coefficient, quantum yield, photobleaching rate, and pH dependence, can be measured with purified GFP in vitro. However, other important properties, especially the time course of chromophore formation and protein degradation in vivo , cannot be easily determined. In general, one chooses the brightest, most photostable GFP available, which may make complicated corrections for background and photobleaching unnecessary in less demanding applications. The chapter also discusses the properties of the fluorescence microscope that are important in quantitative imaging, such as microscope components (objective lenses, fluorescence filters, etc.), signal-to-noise ratio, detection linearity, and fluorophore saturation. Because of the large number of GFP mutants and the variety of potential biological applications, a comprehensive description of all possible quantitative imaging situations is not possible. Thus, most descriptions of methods for quantitative imaging will be limited to the use of fluorescein-like GFP mutants with laser scanning confocal microscopy.

A fluorescence-based, high-resolution imaging approach was used to visualize longitudinally the cellular events unfolding during T cell-mediated tumor destruction. The dynamic interplay of T cells, cancer cells, cancer antigen loss variants, and stromal cells—all color-coded in vivo—was analyzed in established, solid tumors that had developed behind windows implanted on the backs of mice. Events could be followed repeatedly within precisely the same tumor region—before, during and after adoptive T cell therapy—thereby enabling for the first time a longitudinal in vivo evaluation of protracted events, an analysis not possible with terminal imaging of surgically exposed tumors. T cell infiltration, stromal interactions, and vessel destruction, as well as the functional consequences thereof, including the elimination of cancer cells and cancer cell variants were studied. Minimal perivascular T cell infiltrates initiated vascular destruction inside the tumor mass eventually leading to macroscopic central tumor necrosis. Prolonged engagement of T cells with tumor antigen-crosspresenting stromal cells correlated with high IFNγ cytokine release and bystander elimination of antigen-negative cancer cells. The high-resolution, longitudinal, in vivo imaging approach described here will help to further a better mechanistic understanding of tumor eradication by T cells and other anti-cancer therapies.

The dominant motional mode for membrane proteins is uniaxial rotational diffusion about the membrane normal axis, and investigations of their rotational dynamics can yield insight into both the oligomeric state of the protein and its interactions with other proteins such as the cytoskeleton. However, results from the spectroscopic methods used to study these dynamics are dependent on the orientation of the probe relative to the axis of motion. We have employed polarized fluorescence confocal microscopy to measure the orientation of eosin-5-maleimide covalently reacted with Lys-430 of human erythrocyte band 3. Steady-state polarized fluorescence images showed distinct intensity patterns, which were fit to an orientation distribution of the eosin absorption and emission dipoles relative to the membrane normal axis. This orientation was found to be unchanged by trypsin treatment, which cleaves band 3 between the integral membrane domain and the cytoskeleton-attached domain. this result suggests that phosphorescence anisotropy changes observed after trypsin treatment are due to a rotational constraint change rather than a reorientation of eosin. By coupling time-resolved prompt fluorescence anisotropy with confocal microscopy, we calculated the expected amplitudes of the e-Dt and e-4Dt terms from the uniaxial rotational diffusion model and found that the e-4Dt term should dominate the anisotropy decay. Delayed fluorescence and phosphorescence anisotropy decays of control and trypsin-treated band 3 in ghosts, analyzed as multiple uniaxially rotating populations using the amplitudes predicted by confocal microscopy, were consistent with three motional species with uniaxial correlation times ranging from 7 microseconds to 1.4 ms.

One blastomere of the two-cell stage sea urchin embryo (Lytechinus variegatus) was labeled with an intracellular fluorescent lineage tracing stain to determine, from the lineage of that blastomere, the orientation of the first cleavage furrow with regard to the axes of bilateral symmetry in the gastrula and pluteus larva. Two methods were used to mark the blastomere: In the first, the lipophilic carbocyanine dye DiIC16was microinjected directly into the blastomere after first cleavage was completed. In the second, caged (nonfluorescent) fluorescein–dextran was microinjected into the single-celled zygote and uncaged (made fluorescent) in one of the blastomeres at the two-cell stage using two-photon excitation microscopy (TPEM). This is the first use of TPEM for embryonic lineage tracing. In both methods the dye proved to be nontoxic and fluorescence was confined to lineally related cells. The results from both methods were similar and showed that the first cleavage furrow was variable in its orientation. Results were similar using animals obtained from different geographic locations. These results differ from those of McCain and McClay (Development,1994, 120, 395–404), who reported that the median orientation was invariant in this species. The differences between the two studies are discussed. We conclude that first cleavage does not specify nor is it predictive of the bilateral axes in this species. The technique of TPEM is proffered as a powerful new tool that will enable the marking and tracing of embryonic cell lineages with less injury and more precision than current methods.

Extended abstract of a paper presented at Microscopy and Microanalysis 2004 in Savannah, Georgia, USA, August 1–5, 2004.

To determine the application limits of green fluorescent protein (GFP) as a reporter gene or protein tag, we expressed GFP by itself and with fusion protein partners, and used three different imaging methods to identify GFP fluorescence. In conventional epifluorescence photomicroscopy, GFP expressed in cells could be distinguished as a bright green signal over a yellow-green autofluorescence background. In quantitative fluorescence microscopy, however, the GFP signal is contaminated by cellular autofluorescence. Improved separation of GFP signal from HeLa cell autofluorescence was achieved by the combination of confocal scanning laser microscopy using 488-nm excitation, a rapid cut-on dichroic mirror and a narrow-bandpass emission filter. Two-photon excitation of GFP fluorescence at the equivalent of approximately 390 nm provided better absorption than did 488-nm excitation. This resulted in increased signal/background but also generated a different autofluorescence pattern and appeared to increase GFP photobleaching. Fluorescence spectra similar to those of GFP alone were observed when GFP was expressed as a fusion protein either with glutathione-S-transferase (GST) or with glucokinase. Furthermore, purified GST.GFP fusion protein displayed an extinction coefficient and quantum yield consistent with values previously reported for GFP alone. In HeLa cells, the cytoplasmic GFP concentration must be greater than approximately 1 microM to allow quantifiable discrimination over autofluorescence. However, lower expression levels may be detectable if GFP is targeted to discrete subcellular compartments, such as the plasma membrane, organelles or nucleus.

The significance of glucagon in glucose homeostasis is becoming ever clearer, yet the mechanisms underlying its secretion from pancreatic α-cells remain poorly understood. Clinical studies show that drugs that suppress glucagon secretion can restore normoglycemia in diabetic patients, suggesting a possible alternative to insulin treatments. Two classes of models have been proposed to explain glucose inhibition of glucagon secretion (GIGS), but both share a requirement for Ca2+ triggering. In contrast, data from our lab show that changes in α-cell Ca2+ activity do not correlate with GIGS. This suggests that glucagon secretion is suppressed downstream of membrane depolarization, potentially at the level of exocytosis. The F-actin modifying Rac1-GTPase can regulate insulin secretion from β-cells by direct action on the exocytotic machinery. It has been shown that cAMP can regulate Rac1 activation in β-cells through PKA activation. Additionally, glucose metabolism activates the cAMP pathway independently of Ca2+ oscillations, though the complete role of metabolic activity in exocytosis is unknown.Since the α-cell and β-cell are closely related, we hypothesize that GIGS depends on cAMP signaling that leads to deactivation of Rac1 and down-regulation of exocytosis. To identify the role of Rac1 in regulating GIGS, we have developed EGFP-tagged dominant negative and constitutively active Rac1 viral constructs that we use to infect murine islets with RFP-labeled α-cells. This allows us to study directly the effects of Rac1-GTP and Rac1-GDP on α-cell glucagon secretion, Ca2+ activity, and glucose metabolism to determine their relative roles in Rac1-mediated GIGS. Additionally, we are characterizing the glucose dose-response of Rac1 activation in α-cells. Finally, to determine the role of cAMP signaling we are using cAMP and PKA agonists at low and high glucose to identify their effects on Rac1 activation state and glucagon secretion.

With the development of sensitive and specific fluorescent indicators, modern laser scanning microscopies enable visualization and measurement of submicron, dynamic processes inside living cells and tissues. Here we describe the working principles of new, nonlinear laser microscopies based on two-photon molecular excitation. In these techniques, a pulsed laser produces peak photon densities high enough that when focused into an appropriate medium, excitation by photon energy combinations can occur. For example, two red photons interacting simultaneously with a fluorescent molecule can excite within it a UV electronic transition, one corresponding to twice the energy of each single photon. Because the amount of two-photon excitation depends on the square of the local illumination intensity, this process exhibits a unique localization to the diffraction-limited spot of the beam focus. Elsewhere along the beam, excitation of background and photodamage is virtually nonexistent. Focal point localization of two-photon excitation lends to all visualization, measurement, and photopharmacology studies an intrinsic, three-dimensional resolution. We describe some preliminary biological applications, specifically, imaging of vital DNA stains in developing cells and embryos, imaging of cellular metabolic activity from NADH autofluorescence, spatially resolved measurements of cytoplasmic calcium ion activity, and optically induced micropharmacology using caged bioeffector molecules.

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.

Glucose homeostasis is a tightly regulated process coordinated by insulin and glucagon secretion. In α- and β-cells, G-protein coupled receptor (GPCR) activation facilitates the optimization of insulin and glucagon secretion. Glucagon-like peptide-1 (GLP-1), a ligand activating the Gs pathway, increases insulin secretion, but inhibits glucagon secretion. Neuropeptide-Y (NPY) and somatostatin (SST), whose receptors are coupled to Gi, inhibit insulin secretion; however, NPY stimulates glucagon release, while SST inhibits glucagon secretion. Here, we question whether and how these GPCR ligands impact cellular metabolism and oscillations of intracellular Ca2+ activity ([Ca2+]i) to alter secretion in the pancreatic islet. We utilized two-photon excitation microscopy to measure the combined autofluorescence of NADH and NADPH (NAD(P)H, a cellular redox state indicator) upon application of NPY, SST, or GLP-1. Under untreated conditions, an increase in extracellular glucose results in elevated NAD(P)H fluorescence intensities. Treatment with NPY and SST produces a supplementary increase in NAD(P)H autofluorescence at glucose concentrations above 5 mM compared to untreated control. NAD(P)H autofluorescence is not impacted significantly (relative to untreated control) by GLP-1 treatment, which is consistent with a previous report that found that GLP-1 does not alter β-cell metabolism. At glucose concentrations greater than ∼7 mM, pancreatic islets display synchronous oscillations in [Ca2+]i, leading to the pulsatile release of insulin. The [Ca2+]i oscillation frequency increases significantly in the presence of NPY. SST treatment decreases the oscillation frequency in β-cells, but does not affect α-cells. Addition of GLP-1 does not significantly alter the [Ca2+]i oscillation frequency. Thus, GLP-1 likely modulates insulin and glucagon secretion downstream of Ca2+ signaling. NPY may inhibit insulin secretion downstream of Ca2+ signaling, but stimulates glucagon secretion upstream of Ca2+ activity. Islet hormone secretion may be inhibited by SST through alteration of pathways up- and downstream of Ca2+ signaling.

The cloning of the green fluorescent protein (GFP) from the jellyfish Aequoria victoria and its expression in heterologous systems was a significant advance for optical microscopy of living cells ([Chalfie et al., 1994][1]). Mutagenesis of jellyfish GFP has yielded proteins that fluoresce from blue

Glucagon is released into the bloodstream when glucose reaches threateningly low levels. Its secretion from α-cells, within pancreatic islets of Langerhans, stimulates hepatic glucose release and, therefore, restores proper glycemia. Once normoglycemia is reestablished, glucagon release is inhibited. Impairment of this inhibition has been observed in diabetes mellitus where greater amount of glucagon worsens the chronic hyperglycemic state. However, the mechanisms mediating this glucose suppression of glucagon secretion are poorly understood. Two models have been proposed: direct inhibition by glucose, or paracrine inhibition from non α-cells within pancreatic islets. We report here the use of transgenic mouse lines that specifically express Red Fluorescent Protein within α-cells. This strategy makes it possible to easily identify α-cells and study their intracellular calcium ([Ca2+]i) dynamics by Fluo4 imaging. Our glucagon measurements from flow-sorted α-cells indicate that glucose does not directly inhibit α-cells (+57% increase in glucagon secretion from 1 to 20mM, p

Pancreatic beta-cells secrete insulin in response to elevated blood glucose levels. Glucose stimulated insulin secretion depends on glucose metabolism that produces ATP. The resulting increase in [ATP]/[ADP] ratio closes ATP-sensitive potassium (KATP) channels, which leads to membrane depolarization and opening of voltage-dependent Ca2+ channels. This causes an elevation of intracellular free Ca2+ and insulin exocytosis. Insulin is secreted in a pulsatile manner, which is thought to be regulated in part by oscillations in glucose metabolism. Such metabolic oscillations would also lead to oscillations in the [ATP]/[ADP] ratio and hence regulate KATP channel activity.Oscillations in [ATP]/[ADP] ratio have been demonstrated using biochemical and luciferase assays, but neither approach allows measurements of such oscillations in single cells. Perceval is a recently developed fluorescent protein biosensor for [ATP]/[ADP] ratio, and it permits direct measurement of [ATP]/[ADP] ratios inside living cells. We use Perceval in combination with quantitative confocal and two-photon excitation microscopy for direct measurement of the [ATP]/[ADP] ratio in beta-cells during glucose stimulated insulin secretion. For this purpose we have developed an adenoviral vector to express Perceval specifically in the beta-cells of intact mouse islets. Dynamic changes in [ATP]/[ADP] ratio can be correlated with glucose metabolism (by simultaneous imaging of Perceval fluorescence and NAD(P)H autofluorescence) and with intracellular free Ca2+ levels (by simultaneous imaging of Perceval fluorescence and the calcium sensor, FuraRed). This data allows us to test hypotheses regarding the role of localized subcellular signaling complexes and putative microdomains of glucose metabolism, [ATP]/[ADP] ratio, and Ca2+ dynamics in the regulation of glucose stimulated insulin secretion.

Glucose homeostasis is maintained by small clusters of hormone secreting cells in the pancreas: the pancreatic islets. Insulin secreting β-cells make for 90% of each islet and secrete insulin in a tightly regulated manner.Scattered observations in the literature report that β-cells express the required machinery to synthesize and secrete dopamine. Other lines of evidence show that dopamine inhibits glucose stimulated insulin secretion (GSIS) in vitro, and the effect is mediated by the D2 isoform of the dopamine receptor. Yet, there is no evidence of dopaminergic neurons innervating pancreatic islets, and therefore, the biological relevance of such sensitivity is not clear.We test the hypothesis that pancreatic islets produce dopamine from circulating precursor L-dopa and that the resulting dopamine is released as an autocrine inhibitory signal to regulate insulin secretion. We use microfluidic devices to maintain isolated intact islets viable during imaging experiments: we monitor islet metabolic activity by imaging of NAD(P)H autofluorescence with two photon excitation and we measure intracellular [Ca2+]i oscillations by confocal microscopy. Our data from wild type and transgenic mice lacking D2 dopamine receptor support the hypothesis that dopamine is an autocrine regulator of GSIS. The results show that metabolic activity is not affected by dopamine. On the contrary, [Ca2+]i oscillation frequency is reduced by both dopamine and L-dopa, suggesting that D2 receptor activation affects GSIS downstream of glucose metabolism.This finding provides a new target for drug development in the treatment of diabetes and could help understanding the reported increased risk of developing type 2 diabetes by patients treated with antipsychotic drugs.