CHAPTER
Fluorescence Correlation Spectroscopy and Photon-Counting Histogram Analysis of Receptor–Receptor Interactions
10
Katharine Herrick-Davis and Joseph E. Mazurkiewicz Center for Neuropharmacology & Neuroscience, Albany Medical College, Albany, New York, USA
CHAPTER OUTLINE Introduction ............................................................................................................ 182 10.1 Materials........................................................................................................184 10.2 Methods .........................................................................................................185 10.2.1 Sample Preparation .................................................................... 185 10.2.1.1 Choice of Cell Type.............................................................185 10.2.1.2 Choice of Fluorescent Tag...................................................185 10.2.1.3 Labeling the Receptor.........................................................185 10.2.1.4 Plating Cells .......................................................................186 10.2.1.5 Transfecting Cells ...............................................................186 10.2.2 Instrument Setup ....................................................................... 186 10.2.2.1 Environment .......................................................................186 10.2.2.2 Imaging Setup ....................................................................186 10.2.2.3 FCS Setup ..........................................................................186 10.2.2.4 Instrument Alignment and Calibration .................................187 10.2.3 Data Acquisition......................................................................... 188 10.2.3.1 FCS Recording ...................................................................188 10.2.4 Data Analysis ............................................................................. 190 10.2.4.1 Diffusion Coefficient............................................................190 10.2.4.2 Molecular Brightness ..........................................................192 10.3 Discussion......................................................................................................194 Summary ................................................................................................................ 195 Acknowledgments ................................................................................................... 195 References ............................................................................................................. 195
Methods in Cell Biology, Volume 117 Copyright © 2013 Elsevier Inc. All rights reserved.
ISSN 0091-679X http://dx.doi.org/10.1016/B978-0-12-408143-7.00010-4
181
182
CHAPTER 10 Fluorescence Correlation Spectroscopy
Abstract Fluorescence correlation spectroscopy (FCS) performed using a laser scanning confocal microscope is a technique with single-molecule sensitivity that is becoming more accessible to cell biologists. In this chapter, we describe the use of FCS for the analysis of diffusion coefficients and receptor–receptor interactions in live cells in culture. In particular, we describe a protocol to collect fluorescence fluctuation data from fluorescence-tagged receptors as they diffuse into an out of a small laser-illuminated observation volume using a commercially available system such as the Zeiss ConfoCor 3 or LSM-780 microscope. Autocorrelation analysis of the fluctuations in fluorescence intensity provides information about the diffusion time and number of fluorescent molecules in the observation volume. A photon-counting histogram can be used to examine the relationship between fluorescence intensity and the number of fluorescent molecules to estimate the average molecular brightness of the sample. Since molecular brightness is directly proportional to the number of fluorescent molecules, it can be used to monitor receptor–receptor interactions and to decode the number of receptor monomers present in an oligomeric complex.
INTRODUCTION Fluorescence correlation spectroscopy (FCS) is a single-molecule detection technique that measures the fluorescence fluctuations of molecules diffusing through a well-defined volume. Introduced over 40 years ago by Magde, Elson, and Webb (1972), one of the initial applications of FCS was to analyze the interaction of ethidium bromide with DNA in solution to measure diffusion and chemical reaction kinetics. The application of FCS to address such questions in live cells, especially the measurement of diffusion of cell surface proteins in biological membranes, was limited by the lack of sufficiently sensitive instrumentation, stable lasers for excitation and a means to reduce the volume in which the measurements were made. Most of these concerns were addressed in the early 1990s with the adoption of the confocal microscope for FCS measurements, providing a sensitive method for monitoring protein dynamics in living cells (reviewed in Elson, 2013). The main advantage of FCS over other currently used techniques for monitoring receptor interactions is that it provides real-time information about the twodimensional dynamics of single molecules diffusing within a plasma membrane with diffraction-limited spatial and sub-microsecond temporal resolution. In addition, the most accurate FCS measurements are made in samples with very low protein expression levels, making this technique ideal for monitoring receptors at physiological expression levels. Confocal microscopy-based FCS experiments are performed by focusing a laser beam into a small diffraction-limited spot (0.3 mm) using a high numerical aperture objective to create an observation volume on the order of 0.5 1015 L (Fig. 10.1).
Introduction
Laser-illuminated region Confocal observation volume ( 7.5), or GFP for instrument calibration. HPLC-grade water for dilution of dye and for placing on the 40 water immersion objective Isotonic solution For washing cells and performing FCS experiments, use phosphate-buffered saline, Krebs’ ringer solution, or phenol red-free minimal essential media (MEM, CellGro). Add 10 mM HEPES to the solution to keep pH constant during FCS recording. Avoid substances with fluorescence such as phenol. Ethanol and lens cleaning solution Clean the bottom of the coverslip or MatTek dish immediately prior to the FCS experiment. Use ethanol on a Kimwipe to clean glass and wipe dry, and then use a lens cleaning solution and wipe dry.
10.2 METHODS 10.2.1 Sample preparation 10.2.1.1 Choice of cell type HEK293 and CHO cells are cell types that work nicely for FCS experiments. Primary cell cultures, such as neurons or epithelial cells, endogenously expressing the receptor of interest may be used. However, this requires a suitable fluorescent tag capable of labeling native receptors in an exact 1:1 stoichiometry. Membrane stability is important as movement of the membrane within the observation volume during an FCS recording will directly impact the diffusion time and molecular brightness.
10.2.1.2 Choice of fluorescent tag The ideal fluorescent tag has a large quantum yield (brightness), has a good photostability, and is monomeric. The higher the quantum yield, the better the signal to noise ratio. Photostability is important since photobleaching will decrease fluorescence intensity, producing an artificially fast diffusion time as bleaching will mimic the disappearance of the fluorescence signal from the observation volume. GFP variants, such as eGFP and eYFP, and the newer variants are suitable for FCS. Others, such as CFP, mCherry, and dsRed, have low quantum yield and have the potential to form aggregates (dsRed).
10.2.1.3 Labeling the receptor Plasmids containing GFP variants are useful for attaching fluorescent labels to the C-terminus of the receptor of interest. This is a commonly employed method to ensure an exact 1:1 labeling of the receptor with the fluorescent probe, which is critical
185
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CHAPTER 10 Fluorescence Correlation Spectroscopy
for molecular brightness analysis of receptor–receptor interactions. Fluorescent ligands have been used to label receptors for monitoring diffusion and binding kinetics (Briddon et al., 2010) but would require very slow dissociation kinetics in order to be useful for molecular brightness analysis. Alternatively, a monoclonal, monovalent Fab, recognizing the native conformation of the receptor, could be used provided each Fab has exactly one fluorescent tag.
10.2.1.4 Plating cells Seed cells at 5 105 cells per 25 mm coverslip or MatTek dish (coated with appropriate substrate) 12–18 h prior to transfection or following electroporation (40% confluence). Note that the type of substrate will affect cell attachment and overall cell shape, which has important implications for making FCS recordings from the plasma membrane.
10.2.1.5 Transfecting cells Lipofectamine, calcium phosphate, and electroporation are common transfection methods. End the transfection in phenol red-free culture medium, as phenol has autofluorescence. Wait 24 h prior to the start of the FCS experiment to allow time for the receptors to reach the plasma membrane. Fluorescent receptors in the ER/Golgi or in vesicles can enter the observation volume complicating interpretation of plasma membrane FCS results. If the endogenous ligand for the chosen receptor is present in serum, use media with dialyzed serum to minimize receptor internalization.
10.2.2 Instrument setup 10.2.2.1 Environment The room should be maintained at constant temperature during FCS recording (68– 70 F). Detector sensitivity can decrease at high room temperatures, and changes in temperature surrounding the sample can affect diffusion time.
10.2.2.2 Imaging setup Turn on the instrument, allow 30 min for the laser to stabilize, and establish the proper settings for the fluorescent probe. Choose the appropriate laser line, dichroic mirror, and set the appropriate emission range to capture as much of the emission spectrum as possible. Select the fastest scan speed using 12 bits with 514 514 resolution, high detector gain (1100), pinhole of 1 airy unit, and low laser intensity (1%) to minimize photobleaching.
10.2.2.3 FCS setup Choose the appropriate laser line, dichroic mirror or main bean splitter and set the appropriate emission range to capture as much of the emission spectrum as possible. Use a pinhole diameter of 1 airy unit and the lowest laser setting that gives good signal to noise with minimal photobleaching. Note that laser intensity will depend on the
10.2 Methods
sensitivity of the instrument (on the Zeiss ConfoCor 3, we use 1%, and on the LSM780, we use 0.1%) and must be determined experimentally. Select a correlation bin time of 0.2 ms, PCH bin time of 10 ms, and data acquisition time as 10 s for 10 consecutive repetitions (runs). For proper signal statistics, the acquisition time should be 1000-fold over the diffusion time of the protein being studied. For membrane receptors, with diffusion times on the order of 10–100 ms, a total acquisition time of 100 s is sufficient. It is essential to use the same settings for all FCS recordings.
10.2.2.4 Instrument alignment and calibration Align the light beam path and pinhole, and determine the dimensions of the observation volume. a. Clean the lens of the 40 objective and adjust the collar to match coverslip thickness (0.17 mm for no.1.5 and 0.15 mm for no.1.0 coverslip thickness). Place a drop of HPLC-grade (ultrapure, low fluorescence) water on the objective lens. b. Prepare a 20 nM dilution of calibration dye, such as rhodamine 6G, fluorescein (pH > 7.5), or GFP. Place a 100 ml drop on a coverslip or MatTek dish. Be aware that sample evaporation will concentrate the sample and introduce error into the calibration process. c. Place the holder with dye solution on the microscope stage and raise the objective until the water just contacts the surface of the coverslip. Adjust the focus upward until the focal plane of the laser is in the middle of the dye solution. d. Using the imaging and FCS settings established in the preceding text (for the fluorescent probe used to label your receptor of interest, not the calibration dye), capture an image of the dye solution. In the imaging setup menu, set the zoom to 3 and capture another image. Locate the “region of interest” marker and position the marker in the center of the screen. Perform pinhole adjustments for the X and Y planes. e. Begin an FCS recording and adjust the laser setting to give a count rate of approximately 50 kHz. Begin an FCS recording for 10 consecutive 10 s intervals. Repeat. Fit the data to a 3D model for Brownian diffusion and perform an autocorrelation analysis. The autocorrelation curve represents the time-dependent decay in the fluorescence signal. The midpoint of the curve provides a measure of tD (the average dwell time of the fluorescent particles in the observation volume). Use tD to calculate the radius of the observation volume (o0) as in Eq. (10.1): o0 ¼
pffiffiffiffiffiffiffiffiffiffiffi 4DtD
(10.1)
where D is the known diffusion coefficient of the calibration dye (rhodamine 6G, or fluorescein, 400 mm2/s; GFP in solution, 87 mm2/s). The theoretical value of o0 is calculated as 0.61 l/NA where l is the laser
187
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CHAPTER 10 Fluorescence Correlation Spectroscopy
wavelength and NA is the numerical aperture of the objective. The number of molecules in the observation volume (N) is calculated as in Eq. (10.2): N¼
1 g G ð 0Þ
(10.2)
where G(0) is the amplitude of the autocorrelation curve (y-intercept) and g is the point spread function describing the shape of the observation volume (for calibration dye, use g ¼ 0.35 for a 3D Gaussian confocal observation volume). Commercially available software designed for FCS analysis will automatically display G(0), the number of molecules, the count rate, and the diffusion time in a table. Use the number of molecules (N) to calculate the confocal volume (V) as in Eq. (10.3): V¼
N CNa
(10.3)
where C is the concentration of calibration dye (20 nM) and Na is Avogadro’s number. Finally, calculate the structural parameter (s, the ratio of the axial length to radial width of the observation volume) as in Eq. (10.4): s¼
V po20
(10.4)
Successful instrument alignment and calibration will produce a structural parameter between 3 and 8.
10.2.3 Data acquisition 10.2.3.1 FCS recording Always perform a pinhole adjustment using a sample of the fluorescent probe chosen to label the receptor of interest before beginning an FCS recording session. Begin and end each session with control samples and perform FCS recordings on the isotonic viewing solution and in the middle of untransfected cells to establish autofluorescence levels. a. Prepare a coverslip of control cells transfected with plasmid containing the chosen fluorescent probe (e.g., monomeric GFP or YFP expressed in the cytosol) by washing three times using an isotonic solution such as PBS, Krebs’ ringer, or phenol red-free MEM. Place the coverslip in a viewing chamber and add 1 ml of room temperature isotonic solution buffered with 10 mM HEPES. Place a drop of HPLC-grade water on the 40 objective, place sample on the microscope stage, and raise the objective until the waterdrop touches the coverslip. Working quickly, illuminate the sample and focus upward until the cells are visible, and then turn off. It is important to minimize illumination and viewing of the cells to minimize photobleaching. b. Collect an image of the cells and display on the computer screen. Choose a cell of low to medium brightness (avoid bright cells) and adjust the zoom setting to 3. Working quickly, use fast continuous scanning to position the cell in the
10.2 Methods
center of the screen. For FCS measurements in the cytosol, place the region of interest marker near the center of the cell, just to one side of the nucleus where the cell is likely to be tallest, and fill the observation volume in the axial direction. Perform a pinhole adjustment in X and Y and note the settings. c. To establish the proper laser power setting, begin an FCS recording with low intensity (0.1–1%). Note the extent of photobleaching that occurs during the recording. Repeat the process on different cells testing different laser powers and monitor the extent of photobleaching. Select the lowest laser power setting that gives minimal photobleaching (no leftward shift in the autocorrelation curve) but still gives a good signal from the fluorescent control sample and low background fluorescence. Use this laser setting for all subsequent experiments. d. Perform an FCS recording (for 10 10 s intervals) in the cytosol of five different cells, saving the data files after each run for subsequent analysis at a later time. If the software allows, monitor the counts per molecule during the recording to get an idea of the molecular brightness of the control sample. e. If the receptor of interest is a plasma membrane receptor, prepare a coverslip with control cells expressing a known plasma membrane monomeric or dimeric control such as CD-86 or CD-28, respectively. Quickly capture an image of the cells (Fig. 10.2). Choose a cell of low to medium brightness (count rate of 50–250 kHz), and adjust the zoom setting to 3. Working quickly, use fast continuous scanning to position the cell in the center of the screen and focus on the upper plasma membrane. Mark a region on the upper plasma membrane (over the nucleus) and begin an FCS recording. During the first 10 s interval there will be a dramatic photobleaching of the nonmobile fraction of receptors that will appear as a rapid decline in the count rate. During the subsequent 10 s intervals, it will be necessary to adjust the focal plane of the sample
FIGURE 10.2 Confocal image of YFP-tagged receptors in the plasma membrane of HEK293 cells. (A) Betaadrenergic receptors; (B) CD-28 receptors; (C) upper plasma membrane of two transfected cells with þ marking the region where an FCS recording was made. Scale bar ¼ 10 mm.
189
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CHAPTER 10 Fluorescence Correlation Spectroscopy
up and down (along the z-axis) to get the plasma membrane positioned in the center of the observation volume. Monitor the counts per molecule while making small adjustments, in 0.1 mm steps, along the z-axis. Find the position in z corresponding to the maximal counts per molecule, and then make a 10 by 10 s FCS recording from that position. Save the data file and move to a different region of the coverslip, select a new cell, and begin again. Each time, adjust the sample focus along the z-axis to find the optimal positioning of the membrane in the observation volume. Make recordings from at least 5 to 10 different cells. Repeat the process for the tagged receptors of interest and end the session with another control sample. Repeat the process with freshly prepared cells on two additional test days.
10.2.4 Data analysis Commercially available systems can be purchased with autocorrelation analysis software. If using a homebuilt system, software such as Origin or MATLAB can be used.
10.2.4.1 Diffusion coefficient As the fluorescence-tagged receptors pass through the laser-illuminated observation volume, the fluctuations in fluorescence intensity are recorded in real time by the photon-counting detector, and a fluorescence intensity trace for the observation period is generated (Fig. 10.3A). During the first two 10 s intervals of the 100 s FCS recording time, photobleaching of the immobile fraction of plasma membrane receptors occurs. For G-protein-coupled receptors, this typically represents 40–50% of the receptor pool. Data analysis is performed on the mobile fraction of receptors monitored in the third through tenth 10 s FCS recording intervals (runs 3–10). Autocorrelation analysis of the fluorescence signal is performed as in Eq. (10.5): GðtÞ ¼
hdF ðtÞdF ðt þ tÞi hFðtÞi2
(10.5)
where G(t) is the htime averagei of the change in fluorescence fluctuation intensity (dF) at some time point (t) and at a time interval later (t þ t) divided by the square of the average fluorescence intensity. Autocorrelation analysis of the fluorescence intensity trace is performed using a nonlinear least-squares fitting routine that graphically represents the autocorrelation function G(t) on the ordinate and diffusion time on the abscissa (Fig. 10.3B). The rate at which the fluorescence-tagged receptor diffuses within the plasma membrane is reported as the average dwell time (tD) within the observation volume and is calculated from the midpoint of the autocorrelation decay curve. For autocorrelation analysis, select a 2D model for plasma membrane receptors or a 3D model for cytosolic receptors. Most autocorrelation curves will have a minimum of two components, a very fast component (
Fluorescence Correlation Spectroscopy and Photon-Counting Histogram Analysis of Receptor–Receptor Interactions
10
Katharine Herrick-Davis and Joseph E. Mazurkiewicz Center for Neuropharmacology & Neuroscience, Albany Medical College, Albany, New York, USA
CHAPTER OUTLINE Introduction ............................................................................................................ 182 10.1 Materials........................................................................................................184 10.2 Methods .........................................................................................................185 10.2.1 Sample Preparation .................................................................... 185 10.2.1.1 Choice of Cell Type.............................................................185 10.2.1.2 Choice of Fluorescent Tag...................................................185 10.2.1.3 Labeling the Receptor.........................................................185 10.2.1.4 Plating Cells .......................................................................186 10.2.1.5 Transfecting Cells ...............................................................186 10.2.2 Instrument Setup ....................................................................... 186 10.2.2.1 Environment .......................................................................186 10.2.2.2 Imaging Setup ....................................................................186 10.2.2.3 FCS Setup ..........................................................................186 10.2.2.4 Instrument Alignment and Calibration .................................187 10.2.3 Data Acquisition......................................................................... 188 10.2.3.1 FCS Recording ...................................................................188 10.2.4 Data Analysis ............................................................................. 190 10.2.4.1 Diffusion Coefficient............................................................190 10.2.4.2 Molecular Brightness ..........................................................192 10.3 Discussion......................................................................................................194 Summary ................................................................................................................ 195 Acknowledgments ................................................................................................... 195 References ............................................................................................................. 195
Methods in Cell Biology, Volume 117 Copyright © 2013 Elsevier Inc. All rights reserved.
ISSN 0091-679X http://dx.doi.org/10.1016/B978-0-12-408143-7.00010-4
181
182
CHAPTER 10 Fluorescence Correlation Spectroscopy
Abstract Fluorescence correlation spectroscopy (FCS) performed using a laser scanning confocal microscope is a technique with single-molecule sensitivity that is becoming more accessible to cell biologists. In this chapter, we describe the use of FCS for the analysis of diffusion coefficients and receptor–receptor interactions in live cells in culture. In particular, we describe a protocol to collect fluorescence fluctuation data from fluorescence-tagged receptors as they diffuse into an out of a small laser-illuminated observation volume using a commercially available system such as the Zeiss ConfoCor 3 or LSM-780 microscope. Autocorrelation analysis of the fluctuations in fluorescence intensity provides information about the diffusion time and number of fluorescent molecules in the observation volume. A photon-counting histogram can be used to examine the relationship between fluorescence intensity and the number of fluorescent molecules to estimate the average molecular brightness of the sample. Since molecular brightness is directly proportional to the number of fluorescent molecules, it can be used to monitor receptor–receptor interactions and to decode the number of receptor monomers present in an oligomeric complex.
INTRODUCTION Fluorescence correlation spectroscopy (FCS) is a single-molecule detection technique that measures the fluorescence fluctuations of molecules diffusing through a well-defined volume. Introduced over 40 years ago by Magde, Elson, and Webb (1972), one of the initial applications of FCS was to analyze the interaction of ethidium bromide with DNA in solution to measure diffusion and chemical reaction kinetics. The application of FCS to address such questions in live cells, especially the measurement of diffusion of cell surface proteins in biological membranes, was limited by the lack of sufficiently sensitive instrumentation, stable lasers for excitation and a means to reduce the volume in which the measurements were made. Most of these concerns were addressed in the early 1990s with the adoption of the confocal microscope for FCS measurements, providing a sensitive method for monitoring protein dynamics in living cells (reviewed in Elson, 2013). The main advantage of FCS over other currently used techniques for monitoring receptor interactions is that it provides real-time information about the twodimensional dynamics of single molecules diffusing within a plasma membrane with diffraction-limited spatial and sub-microsecond temporal resolution. In addition, the most accurate FCS measurements are made in samples with very low protein expression levels, making this technique ideal for monitoring receptors at physiological expression levels. Confocal microscopy-based FCS experiments are performed by focusing a laser beam into a small diffraction-limited spot (0.3 mm) using a high numerical aperture objective to create an observation volume on the order of 0.5 1015 L (Fig. 10.1).
Introduction
Laser-illuminated region Confocal observation volume ( 7.5), or GFP for instrument calibration. HPLC-grade water for dilution of dye and for placing on the 40 water immersion objective Isotonic solution For washing cells and performing FCS experiments, use phosphate-buffered saline, Krebs’ ringer solution, or phenol red-free minimal essential media (MEM, CellGro). Add 10 mM HEPES to the solution to keep pH constant during FCS recording. Avoid substances with fluorescence such as phenol. Ethanol and lens cleaning solution Clean the bottom of the coverslip or MatTek dish immediately prior to the FCS experiment. Use ethanol on a Kimwipe to clean glass and wipe dry, and then use a lens cleaning solution and wipe dry.
10.2 METHODS 10.2.1 Sample preparation 10.2.1.1 Choice of cell type HEK293 and CHO cells are cell types that work nicely for FCS experiments. Primary cell cultures, such as neurons or epithelial cells, endogenously expressing the receptor of interest may be used. However, this requires a suitable fluorescent tag capable of labeling native receptors in an exact 1:1 stoichiometry. Membrane stability is important as movement of the membrane within the observation volume during an FCS recording will directly impact the diffusion time and molecular brightness.
10.2.1.2 Choice of fluorescent tag The ideal fluorescent tag has a large quantum yield (brightness), has a good photostability, and is monomeric. The higher the quantum yield, the better the signal to noise ratio. Photostability is important since photobleaching will decrease fluorescence intensity, producing an artificially fast diffusion time as bleaching will mimic the disappearance of the fluorescence signal from the observation volume. GFP variants, such as eGFP and eYFP, and the newer variants are suitable for FCS. Others, such as CFP, mCherry, and dsRed, have low quantum yield and have the potential to form aggregates (dsRed).
10.2.1.3 Labeling the receptor Plasmids containing GFP variants are useful for attaching fluorescent labels to the C-terminus of the receptor of interest. This is a commonly employed method to ensure an exact 1:1 labeling of the receptor with the fluorescent probe, which is critical
185
186
CHAPTER 10 Fluorescence Correlation Spectroscopy
for molecular brightness analysis of receptor–receptor interactions. Fluorescent ligands have been used to label receptors for monitoring diffusion and binding kinetics (Briddon et al., 2010) but would require very slow dissociation kinetics in order to be useful for molecular brightness analysis. Alternatively, a monoclonal, monovalent Fab, recognizing the native conformation of the receptor, could be used provided each Fab has exactly one fluorescent tag.
10.2.1.4 Plating cells Seed cells at 5 105 cells per 25 mm coverslip or MatTek dish (coated with appropriate substrate) 12–18 h prior to transfection or following electroporation (40% confluence). Note that the type of substrate will affect cell attachment and overall cell shape, which has important implications for making FCS recordings from the plasma membrane.
10.2.1.5 Transfecting cells Lipofectamine, calcium phosphate, and electroporation are common transfection methods. End the transfection in phenol red-free culture medium, as phenol has autofluorescence. Wait 24 h prior to the start of the FCS experiment to allow time for the receptors to reach the plasma membrane. Fluorescent receptors in the ER/Golgi or in vesicles can enter the observation volume complicating interpretation of plasma membrane FCS results. If the endogenous ligand for the chosen receptor is present in serum, use media with dialyzed serum to minimize receptor internalization.
10.2.2 Instrument setup 10.2.2.1 Environment The room should be maintained at constant temperature during FCS recording (68– 70 F). Detector sensitivity can decrease at high room temperatures, and changes in temperature surrounding the sample can affect diffusion time.
10.2.2.2 Imaging setup Turn on the instrument, allow 30 min for the laser to stabilize, and establish the proper settings for the fluorescent probe. Choose the appropriate laser line, dichroic mirror, and set the appropriate emission range to capture as much of the emission spectrum as possible. Select the fastest scan speed using 12 bits with 514 514 resolution, high detector gain (1100), pinhole of 1 airy unit, and low laser intensity (1%) to minimize photobleaching.
10.2.2.3 FCS setup Choose the appropriate laser line, dichroic mirror or main bean splitter and set the appropriate emission range to capture as much of the emission spectrum as possible. Use a pinhole diameter of 1 airy unit and the lowest laser setting that gives good signal to noise with minimal photobleaching. Note that laser intensity will depend on the
10.2 Methods
sensitivity of the instrument (on the Zeiss ConfoCor 3, we use 1%, and on the LSM780, we use 0.1%) and must be determined experimentally. Select a correlation bin time of 0.2 ms, PCH bin time of 10 ms, and data acquisition time as 10 s for 10 consecutive repetitions (runs). For proper signal statistics, the acquisition time should be 1000-fold over the diffusion time of the protein being studied. For membrane receptors, with diffusion times on the order of 10–100 ms, a total acquisition time of 100 s is sufficient. It is essential to use the same settings for all FCS recordings.
10.2.2.4 Instrument alignment and calibration Align the light beam path and pinhole, and determine the dimensions of the observation volume. a. Clean the lens of the 40 objective and adjust the collar to match coverslip thickness (0.17 mm for no.1.5 and 0.15 mm for no.1.0 coverslip thickness). Place a drop of HPLC-grade (ultrapure, low fluorescence) water on the objective lens. b. Prepare a 20 nM dilution of calibration dye, such as rhodamine 6G, fluorescein (pH > 7.5), or GFP. Place a 100 ml drop on a coverslip or MatTek dish. Be aware that sample evaporation will concentrate the sample and introduce error into the calibration process. c. Place the holder with dye solution on the microscope stage and raise the objective until the water just contacts the surface of the coverslip. Adjust the focus upward until the focal plane of the laser is in the middle of the dye solution. d. Using the imaging and FCS settings established in the preceding text (for the fluorescent probe used to label your receptor of interest, not the calibration dye), capture an image of the dye solution. In the imaging setup menu, set the zoom to 3 and capture another image. Locate the “region of interest” marker and position the marker in the center of the screen. Perform pinhole adjustments for the X and Y planes. e. Begin an FCS recording and adjust the laser setting to give a count rate of approximately 50 kHz. Begin an FCS recording for 10 consecutive 10 s intervals. Repeat. Fit the data to a 3D model for Brownian diffusion and perform an autocorrelation analysis. The autocorrelation curve represents the time-dependent decay in the fluorescence signal. The midpoint of the curve provides a measure of tD (the average dwell time of the fluorescent particles in the observation volume). Use tD to calculate the radius of the observation volume (o0) as in Eq. (10.1): o0 ¼
pffiffiffiffiffiffiffiffiffiffiffi 4DtD
(10.1)
where D is the known diffusion coefficient of the calibration dye (rhodamine 6G, or fluorescein, 400 mm2/s; GFP in solution, 87 mm2/s). The theoretical value of o0 is calculated as 0.61 l/NA where l is the laser
187
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CHAPTER 10 Fluorescence Correlation Spectroscopy
wavelength and NA is the numerical aperture of the objective. The number of molecules in the observation volume (N) is calculated as in Eq. (10.2): N¼
1 g G ð 0Þ
(10.2)
where G(0) is the amplitude of the autocorrelation curve (y-intercept) and g is the point spread function describing the shape of the observation volume (for calibration dye, use g ¼ 0.35 for a 3D Gaussian confocal observation volume). Commercially available software designed for FCS analysis will automatically display G(0), the number of molecules, the count rate, and the diffusion time in a table. Use the number of molecules (N) to calculate the confocal volume (V) as in Eq. (10.3): V¼
N CNa
(10.3)
where C is the concentration of calibration dye (20 nM) and Na is Avogadro’s number. Finally, calculate the structural parameter (s, the ratio of the axial length to radial width of the observation volume) as in Eq. (10.4): s¼
V po20
(10.4)
Successful instrument alignment and calibration will produce a structural parameter between 3 and 8.
10.2.3 Data acquisition 10.2.3.1 FCS recording Always perform a pinhole adjustment using a sample of the fluorescent probe chosen to label the receptor of interest before beginning an FCS recording session. Begin and end each session with control samples and perform FCS recordings on the isotonic viewing solution and in the middle of untransfected cells to establish autofluorescence levels. a. Prepare a coverslip of control cells transfected with plasmid containing the chosen fluorescent probe (e.g., monomeric GFP or YFP expressed in the cytosol) by washing three times using an isotonic solution such as PBS, Krebs’ ringer, or phenol red-free MEM. Place the coverslip in a viewing chamber and add 1 ml of room temperature isotonic solution buffered with 10 mM HEPES. Place a drop of HPLC-grade water on the 40 objective, place sample on the microscope stage, and raise the objective until the waterdrop touches the coverslip. Working quickly, illuminate the sample and focus upward until the cells are visible, and then turn off. It is important to minimize illumination and viewing of the cells to minimize photobleaching. b. Collect an image of the cells and display on the computer screen. Choose a cell of low to medium brightness (avoid bright cells) and adjust the zoom setting to 3. Working quickly, use fast continuous scanning to position the cell in the
10.2 Methods
center of the screen. For FCS measurements in the cytosol, place the region of interest marker near the center of the cell, just to one side of the nucleus where the cell is likely to be tallest, and fill the observation volume in the axial direction. Perform a pinhole adjustment in X and Y and note the settings. c. To establish the proper laser power setting, begin an FCS recording with low intensity (0.1–1%). Note the extent of photobleaching that occurs during the recording. Repeat the process on different cells testing different laser powers and monitor the extent of photobleaching. Select the lowest laser power setting that gives minimal photobleaching (no leftward shift in the autocorrelation curve) but still gives a good signal from the fluorescent control sample and low background fluorescence. Use this laser setting for all subsequent experiments. d. Perform an FCS recording (for 10 10 s intervals) in the cytosol of five different cells, saving the data files after each run for subsequent analysis at a later time. If the software allows, monitor the counts per molecule during the recording to get an idea of the molecular brightness of the control sample. e. If the receptor of interest is a plasma membrane receptor, prepare a coverslip with control cells expressing a known plasma membrane monomeric or dimeric control such as CD-86 or CD-28, respectively. Quickly capture an image of the cells (Fig. 10.2). Choose a cell of low to medium brightness (count rate of 50–250 kHz), and adjust the zoom setting to 3. Working quickly, use fast continuous scanning to position the cell in the center of the screen and focus on the upper plasma membrane. Mark a region on the upper plasma membrane (over the nucleus) and begin an FCS recording. During the first 10 s interval there will be a dramatic photobleaching of the nonmobile fraction of receptors that will appear as a rapid decline in the count rate. During the subsequent 10 s intervals, it will be necessary to adjust the focal plane of the sample
FIGURE 10.2 Confocal image of YFP-tagged receptors in the plasma membrane of HEK293 cells. (A) Betaadrenergic receptors; (B) CD-28 receptors; (C) upper plasma membrane of two transfected cells with þ marking the region where an FCS recording was made. Scale bar ¼ 10 mm.
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CHAPTER 10 Fluorescence Correlation Spectroscopy
up and down (along the z-axis) to get the plasma membrane positioned in the center of the observation volume. Monitor the counts per molecule while making small adjustments, in 0.1 mm steps, along the z-axis. Find the position in z corresponding to the maximal counts per molecule, and then make a 10 by 10 s FCS recording from that position. Save the data file and move to a different region of the coverslip, select a new cell, and begin again. Each time, adjust the sample focus along the z-axis to find the optimal positioning of the membrane in the observation volume. Make recordings from at least 5 to 10 different cells. Repeat the process for the tagged receptors of interest and end the session with another control sample. Repeat the process with freshly prepared cells on two additional test days.
10.2.4 Data analysis Commercially available systems can be purchased with autocorrelation analysis software. If using a homebuilt system, software such as Origin or MATLAB can be used.
10.2.4.1 Diffusion coefficient As the fluorescence-tagged receptors pass through the laser-illuminated observation volume, the fluctuations in fluorescence intensity are recorded in real time by the photon-counting detector, and a fluorescence intensity trace for the observation period is generated (Fig. 10.3A). During the first two 10 s intervals of the 100 s FCS recording time, photobleaching of the immobile fraction of plasma membrane receptors occurs. For G-protein-coupled receptors, this typically represents 40–50% of the receptor pool. Data analysis is performed on the mobile fraction of receptors monitored in the third through tenth 10 s FCS recording intervals (runs 3–10). Autocorrelation analysis of the fluorescence signal is performed as in Eq. (10.5): GðtÞ ¼
hdF ðtÞdF ðt þ tÞi hFðtÞi2
(10.5)
where G(t) is the htime averagei of the change in fluorescence fluctuation intensity (dF) at some time point (t) and at a time interval later (t þ t) divided by the square of the average fluorescence intensity. Autocorrelation analysis of the fluorescence intensity trace is performed using a nonlinear least-squares fitting routine that graphically represents the autocorrelation function G(t) on the ordinate and diffusion time on the abscissa (Fig. 10.3B). The rate at which the fluorescence-tagged receptor diffuses within the plasma membrane is reported as the average dwell time (tD) within the observation volume and is calculated from the midpoint of the autocorrelation decay curve. For autocorrelation analysis, select a 2D model for plasma membrane receptors or a 3D model for cytosolic receptors. Most autocorrelation curves will have a minimum of two components, a very fast component (
