FRET-based voltage probes for confocal imaging: membrane potential oscillations throughout pancreatic islets

Andrey Kuznetsov,1 Vytautas P. Bindokas,2 Jeremy D. Marks,3 and Louis H. Philipson1

Departments of 1Medicine, 2Neurobiology, Pharmacology and Physiology, and 3Pediatrics, Division of Biological Sciences, Pritzker School of Medicine, The University of Chicago, Chicago, Illinois

Submitted 5 January 2005 ; accepted in final form 3 March 2005


    ABSTRACT
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 ABSTRACT
 METHODS
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Insulin secretion is dependent on coordinated pancreatic islet physiology. In the present study, we found a way to overcome the limitations of cellular electrophysiology to optically determine cell membrane potential (Vm) throughout an islet by using a fast voltage optical dye pair. Using laser scanning confocal microscopy (LSCM), we observed fluorescence (Förster) resonance energy transfer (FRET) with the fluorescent donor N-(6-chloro-7-hydroxycoumarin-3-carbonyl)-dimyristoylphosphatidyl-ethanolamine and the acceptor bis-(1,3-diethylthiobarbiturate) trimethine oxonol in the plasma membrane of essentially every cell within an islet. The FRET signal was approximately linear from Vm –70 to +50 mV with a 2.5-fold change in amplitude. We evaluated the responses of islet cells to glucose and tetraethylammonium. Essentially, every responding cell in a mouse islet displayed similar time-dependent changes in Vm. When Vm was measured simultaneously with intracellular Ca2+, all active cells showed tight coupling of Vm to islet cell Ca2+ changes. Our findings indicate that FRET-based, voltage-sensitive dyes used in conjunction with LSCM imaging could be extremely useful in studies of excitation-secretion coupling in intact islets of Langerhans.

pancreatic {beta}-cell; optical electrophysiology; islet electrical coupling


ION CHANNEL MUTATIONS can be a cause of human diabetes and hypoglycemia (2). Cells within pancreatic islets of Langerhans respond to increases in extracellular glucose with alterations in K+ conductance, leading to oscillations in plasma membrane potential (Vm) and free intracellular Ca2+ concentration ([Ca2+]i) (13). However, how Vm oscillations are coordinated with each other and whether they occur in complete synchrony with other cells within the islet are unknown because it has been difficult to obtain simultaneous microelectrode recordings of Vm changes in individual cells within an islet. These experiments are even more difficult when simultaneous optical detection of [Ca2+]i in multiple islet cells is required. Only a few observations have been reported using two-electrode recordings in a single isolated islet in vitro (6, 16, 23), although electrical activity and Ca2+ oscillations in mouse islets appear to be synchronous in vivo (7, 8).

Cellular voltage changes include slow, prolonged shifts in Vm that may last from a few seconds to several minutes, as well as varying numbers and patterns of spikes lasting on the order of milliseconds. Optical voltage dyes are classified as fast or slow, depending on the speed of signal response to voltage changes (26). Optical recording with fast dyes has been used in various excitable cells, but these measurements suffer from small changes in signal, typically 0.1%/mV. In contrast, slow dyes report larger responses but cannot resolve action potentials. However, newer formulations have used dye pairs to enhance the magnitude and temporal responses by ~10-fold (26).

Herein we report the use of fluorescence (Förster) resonance energy transfer (FRET) between two voltage reporting dyes in combination with laser scanning confocal microscopy (LSCM) to record changes in Vm throughout optical sections of whole pancreatic islets and the combination of this FRET-based Vm reporter with fluorophores reporting [Ca2+]i. The amount of FRET is profoundly affected by distance (inverse sixth power of distance) between the donor and acceptor dye molecules and typically falls to nearly zero at >10 nm separation of the two. The voltage sensor probes (VSPs; Invitrogen), first described by González and Tsien (9, 10) and initially developed by Aurora Biosciences, were studied in our experiments. The FRET ratio pair that we used is able to report changes in Vm with relatively large signal changes (>1% ratio value per mV) with the time constant of ~200 ms. N-(6-chloro-7-hydroxycoumarin-3-carbonyl)-dimyristoylphosphatidyl-ethanolamine (CC2-DMPE; coumarin fluorophore linked to a phospholipid) functions as the FRET donor, remaining embedded in the outer leaflet of the cell membrane. The bis-(1,3-diethylthiobarbiturate) trimethine oxonol component [DiSBAC2(3), a slow, voltage-sensitive dye of the oxonol class] moves from the outer side of the membrane in polarized cells to the cytoplasmic face with depolarization, thus decreasing the amount of FRET. The DMPE-CC2 and DiSBAC2(3) pair has been used previously to measure changes in membrane potential in keratinocytes (4) and population activity in cortical neurons (14, 25).

In the present study, we used the VSPs in conjunction with LSCM to scan, for the first time, simultaneous voltage changes from multiple cells within an islet. This method revealed characteristic synchronous electrical activity within islets. By combining this method with confocal imaging of changes in [Ca2+]i in the peripheral islet cells, we also simultaneously assessed the relationship between [Ca2+]i and Vm oscillations in multiple individual islet cells in response to glucose and tetraethylammonium (TEA). The use of noninvasive confocal imaging techniques with VSPs dramatically expands the ability to study excitation-secretion coupling and cell-to-cell communication within relatively intact tissue specimens, such as pancreatic islets.


    METHODS
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 METHODS
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Specimen preparation. Islets of Langerhans were isolated from C57BL/6 mice pancreata using the collagenase digestion technique (24). Isolated islets were placed on 25-mm glass coverslips in RPMI 1640-based complete medium and cultivated in a tissue culture thermostat (Water-Jacketed Incubator 3250; Forma Scientific) at 37°C in a 5% CO2 atmosphere. Islets attached to the coverslips after 48 h of cultivation were used in experiments. Loading of islets with VSPs was achieved by incubating them in a 37°C 5% CO2 thermostat for 40 min in a Krebs-Ringer bicarbonate solution (KRB) (20) containing 3 µM CC2-DMPE and 3 µM DiSBAC2(3), 0.1% Pluronic F-127, and 2 or 16 mM glucose (VSPs were obtained from Invitrogen, Carlsbad, CA). The staining solution was protected from light and was used only once immediately after preparation. After being loaded, the islets were rinsed with an aliquot of the dye-free KRB and transferred to a required experimental solution. In some experiments, before being loaded with the VSPs, islets were preloaded with 5 µM Ca2+ indicator fluo-4 AM (Molecular Probes, Eugene, OR) in complete medium for 45 min in the thermostat. Dye loading for islets was compared with that in primary cultures of isolated chick cardiomyocytes and hippocampal neurons.

Confocal microscopy. Experiments were performed using the Leica TCS SP2 AOBS spectral laser scanning confocal microscopy system (Leica Microsystems, Mannheim, Germany). Coverslips containing islets were placed into an open perfusion chamber (Medical Systems International, Greenvale, NY) on a stage of an inverted Leica DMIRE2 microscope and maintained at 37°C. Structural data acquisition was performed in the sequential line mode for the best spatiotemporal reliability. CC2-DMPE was excited with a 405-nm diode laser, and DiSBAC2(3) was excited with the 476-nm line of an argon laser. The resulting fluorescence was recorded in two channels set up to detect emitted light correspondingly in the ranges 430–490 nm and 545–625 nm. For FRET experiments only, the donor dye CC2-DMPE was excited using the 405-nm diode laser. Donor and acceptor dye fluorescence signals were recorded in line mode simultaneously in two independent channels set to detect light in the ranges 430–490 nm and 545–625 nm, respectively.

To simultaneously record [Ca2+]i, fluo-4 was excited with the 488-nm line of the argon laser, and a 25-nm-wide band (505–530 nm) of fluorescence was acquired with a third detector. Data were collected using a x63 magnification, 1.40 numerical aperture (NA) oil-immersion UV or a x20 0.70 NA air objective using 1-kHz unidirectional or 2-kHz bidirectional scan rates.

Simultaneous electrophysiological and optical recordings. Electrophysiological recordings were performed in dissociated islet cells using a L/M-EPC7 patch-clamp amplifier (List Medical Electronics, Darmstadt, Germany) controlled using Clampex 8 acquisition software (Axon Instruments, Union City, CA) via a DigiData 1200A interface (Axon Instruments). Cells were loaded with the VSPs and placed in the Leica confocal system for FRET recordings as described above. KRB supplemented with 16 mM glucose was used as the bath solution. In a typical experiment, a single cell exhibiting satisfactory voltage dye membrane loading was approached with a borosilicate recording pipette (glass type 8250, 1.5/0.9 mm diameter, pipette resistance 4–6 M{Omega}; Garner Glass, Claremont, CA) filled with the intracellular solution (in mM: 130 KCl, 5 NaCl, 2 CaCl2, 2 MgCl2, 10 EGTA, 5 MgATP, and 10 HEPES, pH 7.3). Pipette movements were controlled using the Water Robot Micromanipulator WR-88 (Narishige Scientific Instrument Lab, Tokyo, Japan). After the formation of a gigaohm seal was observed, a short negative pressure pulse was used to establish a whole cell configuration and the cell was held at –60 mV until the beginning of recordings. Electrophysiological and optical recordings were triggered simultaneously. For the electrophysiological component, the following voltage-step protocol was used. The cell was held at –60 mV, and seven steps with 20-mV increments were applied, starting from –70 mV and increasing to +50 mV at the final step. Each voltage step was 5 s long and was separated from the next one by a 5-s intermediate step at –60 mV. Electrical signals were recorded at a 10-kHz rate. The optical component was recorded as described above with the acquisition rate of 5 Hz.

Data analysis. The images were analyzed using Leica confocal software (Leica Microsystems), MetaMorph software (Universal Imaging, Downingtown, PA) and NIH ImageJ software (Wayne Rasband, Research Services Branch, National Institute of Mental Health, Bethesda, MD). The electrophysiological data were processed and analyzed using Clampfit software (Axon Instruments). Data were typically processed with a Kalman filter (0.5 gain factor) or three-dimensional (3-D) hybrid median filters (NIH ImageJ software) to minimize noise. Pixel-by-pixel ratios were calculated using MetaMorph or ImageJ software with donor/acceptor [CC2-DMPE/DiSBAC2(3)] (F460/F580). 3-D reconstructions were created with ImageJ, MetaMorph, or Voxx2 software (5). Microsoft Excel, Word, and PowerPoint software were used for preparation of the figures.


    RESULTS
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We initially explored loading of islets empirically with the individual dyes until the conditions discussed in METHODS were established. Underloading or overloading resulted in a poor FRET signal or in no response to depolarization. Both the dye ratio and the use of simultaneous loading, as opposed to sequential loading, were critical for this application.

With optimal loading established, we used LSCM to scan islet cells in three fluorescent and differential interference contrast (DIC) optical channels to assess the spatial distribution of the VSPs and the degree of FRET (Fig. 1). In individual dissociated {beta}-cells (Fig. 1A), CC2-DMPE was accumulated mostly in plasmalemma, while DiSBAC2(3) was distributed throughout the entire cell and the FRET signal was clearly observed in the cell membrane region. In small islets or islet fragments (Fig. 1B), the intracellular VSP distribution was quite similar and the FRET signal could be detected readily in the cell membranes of essentially all cells in the islet, creating a lattice-like effect. While the degree of labeling of interior cells is not uniform, useful signals can be extracted from most cells of the islet even at relatively fast acquisition times for islet responses. In the islet cells, where glucose affects the Vm, the DiSBAC2(3) distribution between the plasma membrane and the cytoplasm was dependent on glucose concentration, while in presumably more stably hyperpolarized cardiomyocytes and neurons, both VSPs were localized primarily to the plasmalemma (data not shown).



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Fig. 1. Voltage sensor probe (VSP) dyes loading of islet cells. A: single mouse {beta}-cell was stained with the VSPs and visualized in four optical channels. Shown are N-(6-chloro-7-hydroxycoumarin-3-carbonyl)-dimyristoylphosphatidyl-ethanol-amine (CC2-DMPE) (1), bis-(1,3-diethylthiobarbiturate) trimethine oxonol [DiSBAC3(2)] (2); fluorescence (Förster) resonance energy transfer (FRET)-derived fluorescence of DiSBAC3(2) (3), and a differential interference contrast (DIC) image (4). B: mouse islet fragment visualized as in A. DMPE-CC2, DiSBAC3(2), and FRET can be detected in the membranes of external as well as internal islet cells. Scale bars, 5 µm.

 
We then measured the responses of individual dissociated islet cells while obtaining control of the Vm using the whole cell patch-clamp technique. As shown in Fig. 2, the FRET response in a representative cell was linear across the entire range of membrane potentials tested. This result shows the broad membrane response of the VSPs and validates its use to estimate Vm changes in whole islets. The range of responses varies for different islets and individual cells, so that a calibration, once performed, is useful only for that experiment. In a series of similar experiments in which similar hardware settings and loading conditions pertain, similar ratio values can be obtained in line with other fluorescence-based reporters (e.g., fura-2 or cameleons).



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Fig. 2. Correlation between the cell membrane potential and the FRET signal. Measurements of cell membrane potential (Vm) and FRET for a typical single dissociated mouse {beta}-cell are shown (see METHODS for details). Overall, 12 similar recordings were performed.

 
The ability to visualize the entire islet is best appreciated in Fig. 3, which shows a combined view of the critical signals, CC2-DMPE and FRET, in a montage (Fig. 3A) and in an orthogonal reconstruction of a different islet (Fig. 3B). The deep penetration of the dyes and internal islet membrane structures can be appreciated.



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Fig. 3. VSP dyes stain cells throughout the entire islet of Langerhans. A: montage of a series of optical sections of a mouse islet. The images were acquired 5 µm apart along the z-axis of the islet. B: 3-dimensional reconstruction of a different mouse islet Z series with orthogonal cross sections revealing the internal cellular structures. Scale bars, 10 µm.

 
This analysis can be further used to determine membrane potential oscillations in the individual islet cells (Fig. 4). An islet was loaded with the two dyes in low glucose (C1 and C2 are the two channels; see Fig. 4 legend) and then subjected to a step change from low to high glucose (from 2 to 16 mM). Note the difference in the localization of the C2 signal [oxonol dye, DiSBAC2(3)] in this islet loaded in low glucose compared with the cells in Fig. 1, which were loaded in high glucose, reflecting the lower Vm in the cell in low glucose. The right side of Fig. 4 shows the changes in VSP fluorescence (F1, F2, and FRET) in three separate, nonadjacent cells. The high degree of electrical synchronicity in this mouse islet is clearly demonstrated. The donor dye signal and acceptor dye signal are reciprocal, reflecting the occurrence of FRET in the cell membrane. All of the cells in this figure behaved identically to the three shown. To our knowledge, this study is the first optical demonstration of electrical synchronicity in an intact pancreatic islet.



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Fig. 4. Membrane potential oscillations in individual cells of a mouse islet. The islet was loaded with the VSP dyes and continuously perifused with 16 mM glucose (see METHODS). Time series of fluorescent images were recorded at 3-Hz rate for the shown optical section in two optical channels to visualize the donor dye DMPE-CC2 (Channel 1, C1) and the FRET-derived acceptor dye DiSBAC3(2) signals (Channel 2, C2). Intensities of fluorescence of the shown regions (arrows in C1) of the membranes of the three different nonadjacent cells were measured, and the values were used for FRET ratio calculations. Scale bar, 5 µm.

 
We next assessed the extent to which glucose-induced membrane potential oscillations were synchronized with previously reported oscillations in [Ca2+]i using the combination of VSPs with the Ca2+ indicator fluo-4 AM (Fig. 5). To enhance their response, islets were perfused with elevated glucose (16 mM) and TEA (20 mM), a K+ channel blocker that enhances the rate and amplitude of electrical bursting and Ca2+ oscillations in islet cells (12, 21). Like other acetoxymethyl ester derivatives, fluo-4 AM does not penetrate the islet, allowing imaging of only cells with membrane exposed to the extracellular solution. Therefore, we compared the cytoplasmic Ca2+ signal in a peripheral cell (arrow in Fig. 5B) with the plasma membrane FRET signals of cells within the islet (arrow in Fig. 5A). Every peripheral cell loaded with fluo-4 behaved identically. Similarly, every interior cell labeled with the VSPs that we analyzed also behaved identically. Note that the FRET signal ratio values are not comparable between experiments without internal calibration of some kind, so that the scale of the FRET signal shown in Fig. 5 varies from the FRET signal recordings shown in Fig. 4. In a series of similar experiments in which similar hardware settings and loading conditions pertained, similar ratio values were obtained. Again, the data show the striking connection of the cells from the periphery to the center of the islet. Thus the VSPs allowed the coupling of electrical Vm oscillations to cytoplasmic Ca2+ oscillations to be observed using confocal techniques simultaneously and in real time.



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Fig. 5. Simultaneous recording of oscillations of the Vm and intracellular Ca2+ concentration ([Ca2+]i). The islet was loaded with the VSP dyes (staining with donor dye DMPE-CC2 shown in A) and the Ca2+ indicator fluo-4 (staining of the same optical section shown in B) and perifused with the solution containing 16 mM glucose and 20 mM tetraethylammonium (TEA). Time series of the images of the shown optical section were recorded at 3 Hz in the three optical channels to visualize the donor dye, acceptor dye, and Ca2+ indicator dye. Fluorescence intensities were measured for the region of interest corresponding to the section of the membrane of a cell located in the middle of the optical section of the islet (arrow in A) and for the cytoplasmic region of a peripheral cell (arrow in B). A representative fragment of the experiment from 20 to 160 s exhibiting sustained oscillations is shown. Scale bar, 10 µm.

 

    DISCUSSION
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 METHODS
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A limitation in the understanding of islet physiology has been the lack of efficient experimental approaches to studying intercellular coordination of responses of the individual cells within an islet to various secretagogues. It is uncertain whether there is a gradient of responses such that cells in the periphery respond differently from cells in the interior. Piston and colleagues (3, 19) have used two-photon confocal microscopy to explore differential responses of individual islet cells by following the redox state of the cell determined using the endogenous fluorescence of NAD(P)H. They concluded that most cells of the islet have similar temporal metabolic responses after stimulation with high concentrations of glucose (3). Other attempts to measure responses of individual cells in intact islets have relied on simultaneous electrophysiological recordings with two microelectrodes (23) or on measurement of islet responses using Ca2+ indicators such as fluo-4 or indo-1 and dividing the acquired image of the islet into arbitrary subregions (1, 15, 18). These kinds of techniques have limitations due to either a small number of cells actually recorded or insufficient spatial resolution and are restricted by the inability of acetoxymethyl-esterified dyes such as fluo-4 AM or indo-1 AM to penetrate beyond the outer layer of cells in intact islets.

It is therefore of considerable interest to record simultaneous electrical activity throughout the islet. One approach has been the use of optical probes that change their emission on the basis of electrical changes such as the membrane potential. Previous methods, such as those based on oxonol dyes alone, were very slow and suffered from a low signal-to-noise ratio or did not return quickly to a basal state.

Recently, several new approaches using pairs of molecules, including CC2-DMPE and DiSBAC2(3), that couple by FRET have been reported (17). With cell depolarization, FRET becomes less efficient; correspondingly, the CC2-DMPE signal increases and the DiSBAC2(3) signal decreases. The FRET signal, calculated as the ratio of CC2-DMPE to DiSBAC2(3) signal values in our experiments, matches the known electrical behavior of islet cells. We have also verified the efficacy of these dyes using isolated chick cardiomyocytes and mouse hippocampal neurons in culture (Kuznetsov A, Bindokas VP, Philipson LH, and Marks JD, manuscript in preparation), supporting its application to cardiac and cortical imaging (11). These and similar dyes have been used primarily as tools to report relative changes in membrane potential using individual cells in multiwell plates (17) as, for example, in high-throughput screening assays of drugs (26) that might affect ion channels or ion transporters.

With the availability of appropriate lasers and optics, we have used these dyes with live cell LSCM at frame rates of 0.2–30 Hz. The higher frame rates are possible using subregions, and even higher ones can be used in conjunction with line-scanning modes (>1,000 Hz; data not shown). The duration of image acquisition in the time-lapse mode is essentially unlimited, provided that illumination is set low to prevent photobleaching. Recently introduced fast line scan confocal imaging technologies (e.g., the Carl Zeiss "5 live" system) will further increase full-resolution x-y capture rates to >120 Hz as well as provide faster x-y-z capabilities.

With a time constant of ~200 ms for 100-mV depolarization at 20°C (10), these dyes are well suited for pancreatic islet and {beta}-cell physiology. Moreover, the VSPs makes it possible to conduct studies of many different biological processes, from very fast ones, such as subsecond-scale individual Vm spikes in neurons (25), to much slower events, such as changes in the Vm of stimulated cells of islets of Langerhans occurring during a period of minutes or tens of minutes.

We also found that the VSPs give an essentially linear response to graded membrane potential. With the appropriate loading conditions, isolated cells and intact islets could be loaded, showing unambiguous localization of the dye to the plasma membrane compartment. In contradistinction to such widely used dyes as fura-2 AM and fluo-4 AM, which allow imaging of only peripherally located cells, CC2-DMPE and DiSBAC2(3) demonstrate excellent penetration throughout the islet, allowing measurement of Vm in multiple cells deep inside the islet. Glucose challenge resulted in membrane depolarization and repolarization cycles that temporally clearly resembled oscillations detected with microelectrode perforated patch recordings. We also increased the frequency of glucose-induced oscillations using TEA and found that the dye provided excellent fidelity, with the FRET signal continuing to report membrane oscillations.

When cells are loaded with both the Vm and [Ca2+]i indicators, the corresponding fluorescent signals can be recorded and quantified with high fidelity because of the excellent spatial separation of the cytoplasmic and plasmalemmal areas of the resulting images. We added fluo-4 AM to the islet bath during the loading conditions and found that the Ca2+ signals in the outer cells, the only ones that are capable of being loaded by acetoxymethyl ester dyes in intact islets, matched the electrical changes reported by the FRET signal extremely well.

In conclusion, this first use of the FRET pair system to report Vm changes simultaneously within multiple cells of a pancreatic islet represents a significant advance in the ability to understand integrated islet physiology. Continued advances, such as the use of faster DiSBAC derivatives [e.g., DiSBAC4(2), also available from Invitrogen] should continue to improve this approach to measuring Vm in islets. The faster dyes should provide better temporal resolution even with the currently used LSCM system, in which time resolution of ~30 ms can be achieved. It is important to apply these tools to islets of other species, models of diabetes mellitus, and other experimental models of islet dysfunction (2, 22).


    GRANTS
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 METHODS
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This work was partially supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-44840, DK-48494, DK-63493, and DK-20595 (to the Diabetes Research and Training Center, University of Chicago), National Institute of Neurological Disorders and Stroke Grant NS-38547, and the Blum-Kovler Foundation.


    ACKNOWLEDGMENTS
 
Invitrogen kindly provided the VSP dye samples. We thank James Lopez for skillful islet preparation. We also thank the University of Chicago Integrated Microscopy Facility.


    FOOTNOTES
 

Address for reprint requests and other correspondence: L. H. Philipson, Dept. of Medicine, MC1027, Univ. of Chicago, 5841 S. Maryland Ave., Chicago, IL 60637 (e-mail: l-philipson{at}uchicago.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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