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Monitoring Membrane Potential with Second-Harmonic Generation Stacy A. Wilson, Andrew Millard, Aaron Lewis and Leslie M. Loew Cold Spring Harb Protoc; doi: 10.1101/pdb.prot081786 Email Alerting Service Subject Categories
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Protocol
Monitoring Membrane Potential with Second-Harmonic Generation Stacy A. Wilson, Andrew Millard, Aaron Lewis, and Leslie M. Loew
This protocol describes the nonlinear optical phenomenon known as second-harmonic generation (SHG) and discusses its special attributes for imaging membrane-potential changes in single cells and multicellular preparations. Undifferentiated N1E-115 mouse neuroblastoma cells are used as a model cellular system for membrane electrophysiology. Styryl and naphthylstyryl dyes, also known as hemicyanines, are a class of electrochromic membrane-staining probes that have been used to monitor membrane potential by fluorescence; they also produce SHG images of cell membranes with SHG intensities that are sensitive to voltage. These experiments allow for the precise characterization of the voltage sensitivity of SHG and identification of the optimal wavelength for the incident laser fundamental light. This protocol presents the steps for the culture, staining, patching, and imaging of cells. The details of the imaging system and the measurements obtained are discussed, as are the prospects of this technology for imaging membrane potential changes in neuronal preparations.
MATERIALS It is essential that you consult the appropriate Material Safety Data Sheets and your institution’s Environmental Health and Safety Office for proper handling of equipment and hazardous materials used in this protocol. RECIPES: Please see the end of this protocol for recipes indicated by . Additional recipes can be found online at http://cshprotocols.cshlp.org/site/recipes.
Reagents
Carboxyethyl-γ-cyclodextrin (20 mM in dH2O; Cyclodextrin Technologies Development) Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum and 1% antibiotic– antimycotic (Invitrogen) Earle’s balanced salt solution (EBSS, Sigma-Aldrich) Ethanol (100%) External patch clamp buffer Internal patch pipette buffer Mouse neuroblastoma cells (N1E-115) Styryl and/or naphthylstyryl dyes (Invitrogen) Small samples of noncommercially available dyes may be obtained from the authors’ laboratory on request. Voltage-sensitive dyes are prepared using procedures adapted from Hassner et al. (1984) and Wuskell et al. (2006).
Adapted from Imaging: A Laboratory Manual (ed. Yuste). CSHL Press, Cold Spring Harbor, NY, USA, 2011. © 2014 Cold Spring Harbor Laboratory Press Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot081786
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S.A. Wilson et al.
Equipment
Argon gas Borosilicate glass capillary tubes (1.5-mm outer diameter, 0.86-mm inner diameter; Sutter Instruments) Clampex software (Axon Instruments) Culture dishes (50-mm, glass bottom; Matek Corporation) Electrophysiology rig, including a patch-clamp amplifier (Axon Instruments) Imaging software FluoView v 4.3 (Olympus’s imaging software designed for use with the FluoView scanning confocal imaging system) ImageJ (developed by Wayne Rasband; http://rsb.info.nih.gov/ij/) Imaging system (FluoView scanning confocal imaging system; see Discussion) Incubator for cell culture set at 37˚C and 5% CO2 Micropipette puller (P2000, Sutter Instruments) Rotary vacuum evaporator (Savant Instruments) METHOD Growing Cells
1. In preparation for patching and subsequent imaging, grow N1E-115 mouse neuroblastoma cells in DMEM with 10% fetal bovine serum and 1% antibiotic–antimycotic, maintaining them at 37˚C with 5% CO2 in 50-mm glass-bottomed culture dishes. Prepare hydrophobic or hydrophilic dyes by following Steps 2–5 or Steps 6–8, respectively, and then proceed to Step 9.
Preparation of Hydrophobic Dyes Aqueous solutions of the more hydrophobic dyes are complexed with cyclodextrin to facilitate and accelerate staining (Bullen and Loew 2001). The complexed form of the dye is used because it provides a more efficient method of staining than methods that use surfactants (Lojewska and Loew 1987) such as Pluronic F-127 (Molecular Probes).
2. Prepare a 4 mM stock solution of the hydrophobic dye in 100% ethanol. 3. Dilute the dye by a factor of 20 with 20 mM carboxyethyl-γ-cyclodextrin. 4. Dry small aliquots (0.5 mL) of this mixture in a rotary vacuum evaporator and store them at 4˚C.
5. Just before use, reconstitute an aliquot of the dye with EBSS to make a 100 µM aqueous dye solution. This solution can be applied directly to the cells (see Step 12).
Preparation of Dyes That Are Soluble in Water
6. Prepare a 4 mM stock solution of the dye in 100% ethanol. Store it at 4˚C.
7. Just before use, take small aliquots of the dye stock solution and dry them under argon gas until all of the ethanol has evaporated. 8. Dissolve the dried dye in EBSS to make a 100 µM dye solution. This solution can be applied directly to the cells (see Step 12). Long-term storage of dilute aqueous dye solutions is not recommended.
Electrophysiology Preparation
9. Prepare patch pipettes on a micropipette puller from 1.5-mm outer diameter, 0.86-mm inner diameter borosilicate glass. The pipettes should have a resistance of 5 MΩ when filled with internal patch pipette buffer.
644
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Monitoring Membrane Potential with SHG
10. Set up the electrophysiology rig. The authors control a patch-clamp amplifier by a computer running Clampex. The software provides diagnostics such as seal resistance during patching and synchronization of voltage-clamping operations with nonlinear imaging.
11. Remove the DMEM from the cells, gently rinse with 2 mL of external patch clamp buffer, and then add 3 mL of this buffer. Staining, Patching, and Imaging Cells
12. Stain the cells by adding aqueous dye solution (from Steps 5 or 8) to the cells in the glass-bottom dish (Step 1) and swirling it immediately to promote prompt mixing and to avoid prolonged exposure of the cells to locally high dye concentrations. An overall dye concentration of 3–4 µM is typically used for staining.
13. Using the air objective and bright-field imaging, select a cell for patching to a gigaohm seal (Penner 1995), and then switch to the water-immersion objective. After forming the whole cell patch, carefully switch the microscope configuration from bright-field to nonlinear imaging. If this presents difficulty, the configuration of the microscope can be switched after gigaohm seal formation but before breaking in to establish the more delicate whole cell patch.
14. Perform imaging analysis. The authors use FluoView v.4.3. The vertical sync trigger from FluoView (pin 5 of the FluoView output cable) is used to trigger Clampex as appropriate. Combined second-harmonic generation (SHG) and two-photon excitation of fluorescence (2PF) images are typically recorded in a time series and analyzed within FluoView or using ImageJ software. For further details and examples, see Discussion.
DISCUSSION
The work of Lawrence Cohen and colleagues in the mid-1970s led to the establishment of optical methods as a way to measure the electrical activity of cells in situations in which traditional microelectrode methods are not possible or are too limiting (Cohen et al. 1974). The authors’ laboratory soon joined the effort to develop potentiometric dyes by applying rational design methods based on molecular orbital calculations of the dye chromophores and characterization of their binding and orientations in membranes (Loew et al. 1978, 1979a). Several important general-purpose hemicyanine dyes have emerged from this effort, including di-5-ASP (Loew et al. 1979b), di-4-ANEPPS (Fluhler et al. 1985; Loew et al. 1992), and di-8-ANEPPS (Bedlack et al. 1992; Loew 1994). The one-photon excitation of fluorescence (1PF) properties of the dyes is characterized using a hemispherical lipid bilayer apparatus (Loew et al. 1979a; Loew and Simpson 1981). The fluorescence signals from the naphthylstyryl ANEP dyes have been particularly effective in studies aimed at mapping the activity of excitable cells in complex preparations (Wu et al. 1998; Obaid et al. 1999; Antic et al. 2000; Zochowski et al. 2000; Loew 2001, 2010a). An important and advantageous attribute of the hemicyanine dyes is their strong fluorescence when bound to membranes and the almost insignificant fluorescence from dye in the bathing aqueous medium. This feature not only increases the sensitivity of membranepotential measurements, but also has led to a completely different application for these dyes as indicators of synaptic vesicle release and recycling; the “FM” series of dyes are styryl dyes that have been optimized for such measurements (Betz and Bewick 1992; Betz et al. 1992). In the mid-1980s, investigators realized that the large charge redistribution that occurs on absorption of a photon by the ANEP chromophores, which makes these dyes electrochromic, should also make them promising materials for SHG (Huang et al. 1988). SHG is a nonlinear optical process that can take place at the focus of an ultrafast near-infrared laser. As in the case of 2PF, the probability of SHG is proportional to the square of the incident light intensity, so that threedimensional (3D) optical sectioning is a natural benefit of scanning microscopy with either of these nonlinear optical modalities. The physics behind these phenomena are, however, quite distinct. Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot081786
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S.A. Wilson et al.
Whereas 2PF involves the near-simultaneous absorption of two photons to excite a fluorophore, followed by relaxation and noncoherent emission, SHG is a near-instantaneous process in which two photons are converted into a single photon of precisely twice the energy. The SHG light propagates coherently in the forward direction. The intensity of the SHG signal depends on the molecular hyperpolarizability of the array of molecules that experience the intense laser field. The molecular hyperpolarizability, in turn, can be resonance-enhanced when the incident laser wavelength is close to twice the wavelength of an absorption band of the molecules; this resonance enhancement also depends on the sensitivity of this absorption band to electric fields (i.e., their degree of electrochromism). The full theory of SHG and its application to biological systems is beyond the scope of this chapter, but has been thoroughly reviewed elsewhere (Moreaux et al. 2000; Loew et al. 2002, 2010b; Campagnola and Loew 2003; Millard et al. 2003a; Pons et al. 2003; Jiang et al. 2007). One key condition for SHG, in addition to a large molecular hyperpolarizability, is that the molecules producing an SHG signal be organized in a noncentrosymmetric array—a condition that is nicely met when the dye molecules stain one side of a cell membrane. The relationship between SHG and electrochromism also prompted investigation of whether SHG from membranes stained with ANEP dyes could be sensitive to membrane potential (Bouevitch et al. 1993; Ben-Oren et al. 1996; Campagnola et al. 1999). Many of these earlier studies provided indications that the SHG sensitivity to membrane potential could be much larger than the typical best sensitivity of 10% fluorescence change per 100 mV. However, it has only been recently that SHG has been measured in stained cells that are simultaneously voltage-clamped via whole-cell patch clamp (Millard et al. 2003b, 2004, 2005a,b; Dombeck et al. 2004, 2005; Araya et al. 2006; Nuriya et al. 2006; Teisseyre et al. 2007). The voltage sensitivity of this technique can be significantly greater than the voltage sensitivity of fluorescence for many of the dyes that have been tested. Furthermore, the ability of SHG to image only the plasma membrane and not intracellular organelles is especially useful for experiments in which dye is applied internally to an individual cell. Thus, second-harmonic imaging of membrane potential has great promise for spatiotemporal mapping of electrical activity in neurons. Imaging Setup
We have adapted a FluoView scanning confocal imaging system (Olympus) with an inverted Axiovert 100TV microscope (Carl Zeiss) for nonlinear optical imaging, as shown in Figure 1. A Mira 900 Ti: sapphire (Ti:S) ultrafast laser (Coherent) is pumped by a 10-W Verdi doubled solid-state laser (Coherent) and purged with nitrogen gas to make wavelengths >930 nm accessible. A second excitation source, a FemtoPower fiber laser (Fianium) operating at a fixed wavelength of 1064 nm, provides a robust, turnkey source of long-wavelength excitation. ER4-protected gold-coating mirrors (Newport) are used to direct the beam through the various optical components, through the FluoView scan head, and finally into the microscope. The Ti:S beam is first passed through a Faraday isolator (Electro-Optics Technology, Inc.) to prevent back-propagating reflections from knocking the Mira out of mode-lock. A 700-nm (CVI Laser) or 880-nm (Chroma) long-pass filter then removes residual pump light from the Ti:sapphire or the fiber laser, respectively. We use a halfwave plate and a Glan Thompson polarizer to modulate the beam intensity and then half- and quarterwave plates (CVI Laser) to produce circularly polarized light at the sample; this effectively eliminates distorting contrast patterns produced by direct excitation with the linearly polarized laser light. The scanning beam enters the microscope from below, passing through a planoconcave BK7 lens (f = –150 mm) (Newport) to colocalize the bright-field focus and the focus for nonlinear excitation. For nonlinear (simultaneous SHG and 2PF) imaging mode, an infinity-corrected 40 × 0.8 NA water-immersion IR-Achroplan objective (Zeiss) focuses the ultrafast beam into the sample. 2PF is collected by the water-immersion objective, reflected by a dichroic mirror, and passed through filters to select emission wavelengths of interest. For imaging of bluer styryl dyes, a 770-nm long-pass dichroic mirror (Chroma; D in Fig. 1) is used with a 750-nm short-pass filter (CVI Laser) in combination with either a 540-nm band-pass filter (Chroma) or a 675-nm band-pass filter (Chroma). This setup covers wavelength ranges to either side of the emission spectrum maximum wavelength (i.e., 615 nm for di646
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Monitoring Membrane Potential with SHG
Olympus FluoView scan-head Diverging lens Dichroic mirror D
Coherent Mira Ti:sapphire laser
Fianium FemtoPower fiber laser
Quarterwave plate
F2PF R3896 PMT
Halfwave plate Faraday isolator
0.8 NA 40x
Pockels cell
Filter
0.55 NA FSHG Mirror M
Dichroic mirror
H7421-40 Photon-counting head
FIGURE 1. Schematic of our nonlinear imaging system. Excitation light leaves the scan head and is directed through a diverging lens into the microscope. The light passes through the dichroic (D) to enter the objective. 2PF is collected back through the objective, and the dichroic reflects it through filters (F2PF) into the photomultiplier tube. The condenser collects all transmitted light, directing it to the mirror (M), which selectively reflects SHG through filters (FSHG) into the photon-counting head.
4-ANEPPS); by restricting the emission range to one side of the spectrum, the voltage sensitivity can reinforce the sensitivity associated with the corresponding wing of the excitation spectrum (Fluhler et al. 1985). For more recently synthesized longer wavelength dyes, an 880-nm dichroic is used with an 850nm short-pass filter and either a 640-nm band pass or a 750-nm long-pass filter, allowing access to dyes with redder wavelength maxima. The filtered 2PF is directly detected by a photomultiplier tube (Hamamatsu R3896) that is connected to one of the FluoView channel inputs via a PMT amplifier board (Olympus). Second-harmonic light is produced in the forward direction and is collected using a 0.55NA condenser (Zeiss), then reflected from a broadband dielectric mirror (Thorlabs; M in Fig. 1) to focus through filters that are appropriate for the second-harmonic wavelength (see Table 1) onto a GaAsP photon-counting head (Hamamatsu H7421-40) connected to the other FluoView channel input. Example of Application
To determine the voltage sensitivity of SHG from styryl dyes, we typically select single cells having no physical contact with other cells. Once a cell is patched and stained, we take a series of 27 images with the clamp voltage switched back and forth between 0 mV and a test voltage V after every three image frames. A total intensity value for each image is obtained by summing the intensity values of the pixels associated with the cell membrane; that is, a total intensity value I(ti) is obtained for frame i. Fifteen total intensity values, I0(ti), correspond to the 0-mV reference voltage, and 12 values, I = (ti), correspond to the test voltage V. In addition to scatter, the I0(ti) and IV(ti) drift a little over time, because of a small but continuous incorporation of dye into the membrane during the course of the experiment. Thus, a normalization process that successfully corrects for the drift has been developed as TABLE 1. Filters used for SHG detection with various excitation wavelengths Wavelengths
Filters, FSHG
830–870 nm 890–910 nm 930–970 nm 1064 nm
Chroma Technology D425/40 and CVI Laser SPF-450, passing 425 ± 10 nm Chroma Technology D460/50 and Oriel Instruments 57530, passing 450 ± 10 nm Chroma Technology D470/40 and 475 ± 50 nm band pass, passing 470 ± 15 nm Semrock FF01-530/11, passing 530 ± 11 nm
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follows: We fit a second-order polynomial A0(ti) to the 15 I0(ti) values, and then the normalized total intensity values N(ti) are just I(ti)/A0(ti) for each i. The average relative signal change C for the test voltage is kNV(ti)l − kN0(ti)l. Each image series yields one value for C, which is considered a single experimental measurement, although each measurement obtained in this way actually represents data collected over an entire cell membrane and averaged over 15 0-mV images and 12 test voltage images. To illustrate the voltage sensitivity visually, an SHG image series can be processed to produce a montage such as that shown in Figure 2. Although “on-the-fly” averaging is not generally used while obtaining image series, each image in Figure 2 is a Kalman average of three acquisitions to yield a clearer visual presentation. Modulation of SHG intensity by membrane potential is apparent. The 81 acquisitions of the original image series, each corresponding to an 2.5-sec acquisition time, took place without any major degradation in SHG intensity and with a stable response to the step changes in membrane potential. Such stability appears to be dependent on excitation wavelength, with greater degradation of both SHG and 2PF intensity at wavelengths
Monitoring Membrane Potential with Second-Harmonic Generation Stacy A. Wilson, Andrew Millard, Aaron Lewis and Leslie M. Loew Cold Spring Harb Protoc; doi: 10.1101/pdb.prot081786 Email Alerting Service Subject Categories
Receive free email alerts when new articles cite this article - click here. Browse articles on similar topics from Cold Spring Harbor Protocols. Cell Imaging (462 articles) Confocal Microscopy (88 articles) Labeling for Imaging (300 articles)
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Protocol
Monitoring Membrane Potential with Second-Harmonic Generation Stacy A. Wilson, Andrew Millard, Aaron Lewis, and Leslie M. Loew
This protocol describes the nonlinear optical phenomenon known as second-harmonic generation (SHG) and discusses its special attributes for imaging membrane-potential changes in single cells and multicellular preparations. Undifferentiated N1E-115 mouse neuroblastoma cells are used as a model cellular system for membrane electrophysiology. Styryl and naphthylstyryl dyes, also known as hemicyanines, are a class of electrochromic membrane-staining probes that have been used to monitor membrane potential by fluorescence; they also produce SHG images of cell membranes with SHG intensities that are sensitive to voltage. These experiments allow for the precise characterization of the voltage sensitivity of SHG and identification of the optimal wavelength for the incident laser fundamental light. This protocol presents the steps for the culture, staining, patching, and imaging of cells. The details of the imaging system and the measurements obtained are discussed, as are the prospects of this technology for imaging membrane potential changes in neuronal preparations.
MATERIALS It is essential that you consult the appropriate Material Safety Data Sheets and your institution’s Environmental Health and Safety Office for proper handling of equipment and hazardous materials used in this protocol. RECIPES: Please see the end of this protocol for recipes indicated by . Additional recipes can be found online at http://cshprotocols.cshlp.org/site/recipes.
Reagents
Carboxyethyl-γ-cyclodextrin (20 mM in dH2O; Cyclodextrin Technologies Development) Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum and 1% antibiotic– antimycotic (Invitrogen) Earle’s balanced salt solution (EBSS, Sigma-Aldrich) Ethanol (100%) External patch clamp buffer Internal patch pipette buffer Mouse neuroblastoma cells (N1E-115) Styryl and/or naphthylstyryl dyes (Invitrogen) Small samples of noncommercially available dyes may be obtained from the authors’ laboratory on request. Voltage-sensitive dyes are prepared using procedures adapted from Hassner et al. (1984) and Wuskell et al. (2006).
Adapted from Imaging: A Laboratory Manual (ed. Yuste). CSHL Press, Cold Spring Harbor, NY, USA, 2011. © 2014 Cold Spring Harbor Laboratory Press Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot081786
643
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S.A. Wilson et al.
Equipment
Argon gas Borosilicate glass capillary tubes (1.5-mm outer diameter, 0.86-mm inner diameter; Sutter Instruments) Clampex software (Axon Instruments) Culture dishes (50-mm, glass bottom; Matek Corporation) Electrophysiology rig, including a patch-clamp amplifier (Axon Instruments) Imaging software FluoView v 4.3 (Olympus’s imaging software designed for use with the FluoView scanning confocal imaging system) ImageJ (developed by Wayne Rasband; http://rsb.info.nih.gov/ij/) Imaging system (FluoView scanning confocal imaging system; see Discussion) Incubator for cell culture set at 37˚C and 5% CO2 Micropipette puller (P2000, Sutter Instruments) Rotary vacuum evaporator (Savant Instruments) METHOD Growing Cells
1. In preparation for patching and subsequent imaging, grow N1E-115 mouse neuroblastoma cells in DMEM with 10% fetal bovine serum and 1% antibiotic–antimycotic, maintaining them at 37˚C with 5% CO2 in 50-mm glass-bottomed culture dishes. Prepare hydrophobic or hydrophilic dyes by following Steps 2–5 or Steps 6–8, respectively, and then proceed to Step 9.
Preparation of Hydrophobic Dyes Aqueous solutions of the more hydrophobic dyes are complexed with cyclodextrin to facilitate and accelerate staining (Bullen and Loew 2001). The complexed form of the dye is used because it provides a more efficient method of staining than methods that use surfactants (Lojewska and Loew 1987) such as Pluronic F-127 (Molecular Probes).
2. Prepare a 4 mM stock solution of the hydrophobic dye in 100% ethanol. 3. Dilute the dye by a factor of 20 with 20 mM carboxyethyl-γ-cyclodextrin. 4. Dry small aliquots (0.5 mL) of this mixture in a rotary vacuum evaporator and store them at 4˚C.
5. Just before use, reconstitute an aliquot of the dye with EBSS to make a 100 µM aqueous dye solution. This solution can be applied directly to the cells (see Step 12).
Preparation of Dyes That Are Soluble in Water
6. Prepare a 4 mM stock solution of the dye in 100% ethanol. Store it at 4˚C.
7. Just before use, take small aliquots of the dye stock solution and dry them under argon gas until all of the ethanol has evaporated. 8. Dissolve the dried dye in EBSS to make a 100 µM dye solution. This solution can be applied directly to the cells (see Step 12). Long-term storage of dilute aqueous dye solutions is not recommended.
Electrophysiology Preparation
9. Prepare patch pipettes on a micropipette puller from 1.5-mm outer diameter, 0.86-mm inner diameter borosilicate glass. The pipettes should have a resistance of 5 MΩ when filled with internal patch pipette buffer.
644
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Monitoring Membrane Potential with SHG
10. Set up the electrophysiology rig. The authors control a patch-clamp amplifier by a computer running Clampex. The software provides diagnostics such as seal resistance during patching and synchronization of voltage-clamping operations with nonlinear imaging.
11. Remove the DMEM from the cells, gently rinse with 2 mL of external patch clamp buffer, and then add 3 mL of this buffer. Staining, Patching, and Imaging Cells
12. Stain the cells by adding aqueous dye solution (from Steps 5 or 8) to the cells in the glass-bottom dish (Step 1) and swirling it immediately to promote prompt mixing and to avoid prolonged exposure of the cells to locally high dye concentrations. An overall dye concentration of 3–4 µM is typically used for staining.
13. Using the air objective and bright-field imaging, select a cell for patching to a gigaohm seal (Penner 1995), and then switch to the water-immersion objective. After forming the whole cell patch, carefully switch the microscope configuration from bright-field to nonlinear imaging. If this presents difficulty, the configuration of the microscope can be switched after gigaohm seal formation but before breaking in to establish the more delicate whole cell patch.
14. Perform imaging analysis. The authors use FluoView v.4.3. The vertical sync trigger from FluoView (pin 5 of the FluoView output cable) is used to trigger Clampex as appropriate. Combined second-harmonic generation (SHG) and two-photon excitation of fluorescence (2PF) images are typically recorded in a time series and analyzed within FluoView or using ImageJ software. For further details and examples, see Discussion.
DISCUSSION
The work of Lawrence Cohen and colleagues in the mid-1970s led to the establishment of optical methods as a way to measure the electrical activity of cells in situations in which traditional microelectrode methods are not possible or are too limiting (Cohen et al. 1974). The authors’ laboratory soon joined the effort to develop potentiometric dyes by applying rational design methods based on molecular orbital calculations of the dye chromophores and characterization of their binding and orientations in membranes (Loew et al. 1978, 1979a). Several important general-purpose hemicyanine dyes have emerged from this effort, including di-5-ASP (Loew et al. 1979b), di-4-ANEPPS (Fluhler et al. 1985; Loew et al. 1992), and di-8-ANEPPS (Bedlack et al. 1992; Loew 1994). The one-photon excitation of fluorescence (1PF) properties of the dyes is characterized using a hemispherical lipid bilayer apparatus (Loew et al. 1979a; Loew and Simpson 1981). The fluorescence signals from the naphthylstyryl ANEP dyes have been particularly effective in studies aimed at mapping the activity of excitable cells in complex preparations (Wu et al. 1998; Obaid et al. 1999; Antic et al. 2000; Zochowski et al. 2000; Loew 2001, 2010a). An important and advantageous attribute of the hemicyanine dyes is their strong fluorescence when bound to membranes and the almost insignificant fluorescence from dye in the bathing aqueous medium. This feature not only increases the sensitivity of membranepotential measurements, but also has led to a completely different application for these dyes as indicators of synaptic vesicle release and recycling; the “FM” series of dyes are styryl dyes that have been optimized for such measurements (Betz and Bewick 1992; Betz et al. 1992). In the mid-1980s, investigators realized that the large charge redistribution that occurs on absorption of a photon by the ANEP chromophores, which makes these dyes electrochromic, should also make them promising materials for SHG (Huang et al. 1988). SHG is a nonlinear optical process that can take place at the focus of an ultrafast near-infrared laser. As in the case of 2PF, the probability of SHG is proportional to the square of the incident light intensity, so that threedimensional (3D) optical sectioning is a natural benefit of scanning microscopy with either of these nonlinear optical modalities. The physics behind these phenomena are, however, quite distinct. Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot081786
645
Downloaded from http://cshprotocols.cshlp.org/ at HARVARD UNIVERSITY on June 6, 2014 - Published by Cold Spring Harbor Laboratory Press
S.A. Wilson et al.
Whereas 2PF involves the near-simultaneous absorption of two photons to excite a fluorophore, followed by relaxation and noncoherent emission, SHG is a near-instantaneous process in which two photons are converted into a single photon of precisely twice the energy. The SHG light propagates coherently in the forward direction. The intensity of the SHG signal depends on the molecular hyperpolarizability of the array of molecules that experience the intense laser field. The molecular hyperpolarizability, in turn, can be resonance-enhanced when the incident laser wavelength is close to twice the wavelength of an absorption band of the molecules; this resonance enhancement also depends on the sensitivity of this absorption band to electric fields (i.e., their degree of electrochromism). The full theory of SHG and its application to biological systems is beyond the scope of this chapter, but has been thoroughly reviewed elsewhere (Moreaux et al. 2000; Loew et al. 2002, 2010b; Campagnola and Loew 2003; Millard et al. 2003a; Pons et al. 2003; Jiang et al. 2007). One key condition for SHG, in addition to a large molecular hyperpolarizability, is that the molecules producing an SHG signal be organized in a noncentrosymmetric array—a condition that is nicely met when the dye molecules stain one side of a cell membrane. The relationship between SHG and electrochromism also prompted investigation of whether SHG from membranes stained with ANEP dyes could be sensitive to membrane potential (Bouevitch et al. 1993; Ben-Oren et al. 1996; Campagnola et al. 1999). Many of these earlier studies provided indications that the SHG sensitivity to membrane potential could be much larger than the typical best sensitivity of 10% fluorescence change per 100 mV. However, it has only been recently that SHG has been measured in stained cells that are simultaneously voltage-clamped via whole-cell patch clamp (Millard et al. 2003b, 2004, 2005a,b; Dombeck et al. 2004, 2005; Araya et al. 2006; Nuriya et al. 2006; Teisseyre et al. 2007). The voltage sensitivity of this technique can be significantly greater than the voltage sensitivity of fluorescence for many of the dyes that have been tested. Furthermore, the ability of SHG to image only the plasma membrane and not intracellular organelles is especially useful for experiments in which dye is applied internally to an individual cell. Thus, second-harmonic imaging of membrane potential has great promise for spatiotemporal mapping of electrical activity in neurons. Imaging Setup
We have adapted a FluoView scanning confocal imaging system (Olympus) with an inverted Axiovert 100TV microscope (Carl Zeiss) for nonlinear optical imaging, as shown in Figure 1. A Mira 900 Ti: sapphire (Ti:S) ultrafast laser (Coherent) is pumped by a 10-W Verdi doubled solid-state laser (Coherent) and purged with nitrogen gas to make wavelengths >930 nm accessible. A second excitation source, a FemtoPower fiber laser (Fianium) operating at a fixed wavelength of 1064 nm, provides a robust, turnkey source of long-wavelength excitation. ER4-protected gold-coating mirrors (Newport) are used to direct the beam through the various optical components, through the FluoView scan head, and finally into the microscope. The Ti:S beam is first passed through a Faraday isolator (Electro-Optics Technology, Inc.) to prevent back-propagating reflections from knocking the Mira out of mode-lock. A 700-nm (CVI Laser) or 880-nm (Chroma) long-pass filter then removes residual pump light from the Ti:sapphire or the fiber laser, respectively. We use a halfwave plate and a Glan Thompson polarizer to modulate the beam intensity and then half- and quarterwave plates (CVI Laser) to produce circularly polarized light at the sample; this effectively eliminates distorting contrast patterns produced by direct excitation with the linearly polarized laser light. The scanning beam enters the microscope from below, passing through a planoconcave BK7 lens (f = –150 mm) (Newport) to colocalize the bright-field focus and the focus for nonlinear excitation. For nonlinear (simultaneous SHG and 2PF) imaging mode, an infinity-corrected 40 × 0.8 NA water-immersion IR-Achroplan objective (Zeiss) focuses the ultrafast beam into the sample. 2PF is collected by the water-immersion objective, reflected by a dichroic mirror, and passed through filters to select emission wavelengths of interest. For imaging of bluer styryl dyes, a 770-nm long-pass dichroic mirror (Chroma; D in Fig. 1) is used with a 750-nm short-pass filter (CVI Laser) in combination with either a 540-nm band-pass filter (Chroma) or a 675-nm band-pass filter (Chroma). This setup covers wavelength ranges to either side of the emission spectrum maximum wavelength (i.e., 615 nm for di646
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Monitoring Membrane Potential with SHG
Olympus FluoView scan-head Diverging lens Dichroic mirror D
Coherent Mira Ti:sapphire laser
Fianium FemtoPower fiber laser
Quarterwave plate
F2PF R3896 PMT
Halfwave plate Faraday isolator
0.8 NA 40x
Pockels cell
Filter
0.55 NA FSHG Mirror M
Dichroic mirror
H7421-40 Photon-counting head
FIGURE 1. Schematic of our nonlinear imaging system. Excitation light leaves the scan head and is directed through a diverging lens into the microscope. The light passes through the dichroic (D) to enter the objective. 2PF is collected back through the objective, and the dichroic reflects it through filters (F2PF) into the photomultiplier tube. The condenser collects all transmitted light, directing it to the mirror (M), which selectively reflects SHG through filters (FSHG) into the photon-counting head.
4-ANEPPS); by restricting the emission range to one side of the spectrum, the voltage sensitivity can reinforce the sensitivity associated with the corresponding wing of the excitation spectrum (Fluhler et al. 1985). For more recently synthesized longer wavelength dyes, an 880-nm dichroic is used with an 850nm short-pass filter and either a 640-nm band pass or a 750-nm long-pass filter, allowing access to dyes with redder wavelength maxima. The filtered 2PF is directly detected by a photomultiplier tube (Hamamatsu R3896) that is connected to one of the FluoView channel inputs via a PMT amplifier board (Olympus). Second-harmonic light is produced in the forward direction and is collected using a 0.55NA condenser (Zeiss), then reflected from a broadband dielectric mirror (Thorlabs; M in Fig. 1) to focus through filters that are appropriate for the second-harmonic wavelength (see Table 1) onto a GaAsP photon-counting head (Hamamatsu H7421-40) connected to the other FluoView channel input. Example of Application
To determine the voltage sensitivity of SHG from styryl dyes, we typically select single cells having no physical contact with other cells. Once a cell is patched and stained, we take a series of 27 images with the clamp voltage switched back and forth between 0 mV and a test voltage V after every three image frames. A total intensity value for each image is obtained by summing the intensity values of the pixels associated with the cell membrane; that is, a total intensity value I(ti) is obtained for frame i. Fifteen total intensity values, I0(ti), correspond to the 0-mV reference voltage, and 12 values, I = (ti), correspond to the test voltage V. In addition to scatter, the I0(ti) and IV(ti) drift a little over time, because of a small but continuous incorporation of dye into the membrane during the course of the experiment. Thus, a normalization process that successfully corrects for the drift has been developed as TABLE 1. Filters used for SHG detection with various excitation wavelengths Wavelengths
Filters, FSHG
830–870 nm 890–910 nm 930–970 nm 1064 nm
Chroma Technology D425/40 and CVI Laser SPF-450, passing 425 ± 10 nm Chroma Technology D460/50 and Oriel Instruments 57530, passing 450 ± 10 nm Chroma Technology D470/40 and 475 ± 50 nm band pass, passing 470 ± 15 nm Semrock FF01-530/11, passing 530 ± 11 nm
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S.A. Wilson et al.
follows: We fit a second-order polynomial A0(ti) to the 15 I0(ti) values, and then the normalized total intensity values N(ti) are just I(ti)/A0(ti) for each i. The average relative signal change C for the test voltage is kNV(ti)l − kN0(ti)l. Each image series yields one value for C, which is considered a single experimental measurement, although each measurement obtained in this way actually represents data collected over an entire cell membrane and averaged over 15 0-mV images and 12 test voltage images. To illustrate the voltage sensitivity visually, an SHG image series can be processed to produce a montage such as that shown in Figure 2. Although “on-the-fly” averaging is not generally used while obtaining image series, each image in Figure 2 is a Kalman average of three acquisitions to yield a clearer visual presentation. Modulation of SHG intensity by membrane potential is apparent. The 81 acquisitions of the original image series, each corresponding to an 2.5-sec acquisition time, took place without any major degradation in SHG intensity and with a stable response to the step changes in membrane potential. Such stability appears to be dependent on excitation wavelength, with greater degradation of both SHG and 2PF intensity at wavelengths
