Downloaded from http://cshprotocols.cshlp.org/ at ALBIN O KUHN LIBRARY on January 6, 2015 - Published by Cold Spring Harbor Laboratory Press
Protocol
Two-Dimensional Imaging of Fast Intracellular Ca2+ Release Qinghai Tian, Lars Kaestner, and Peter Lipp1 Institute for Molecular Cell Biology and Research Center for Molecular Imaging and Screening, School of Medicine, Saarland University, 66421 Homburg/Saar, Germany
Asynchronous release of calcium (Ca2+)—for example, the generation of Ca2+ alternans in cardiac myocytes—is a phenomenon important in the development of cardiac arrhythmogenesis. The development of a failure to release Ca2+ at individual release sites can be regarded as a major contributor to cardiac pathologies such as hypertrophy. Although confocal linescans provide sufficient temporal resolution to investigate the physiological and pathological cardiac excitation–contraction (EC) coupling, linescans can only image 1.5% of the cross section of myocytes, which raises doubts about how representative such recordings are, especially in light of nonhomogeneous uncoupling of Ca2+ channels and ryanodine receptors. Nowadays, the speed of confocal microscopes has been greatly improved, enabling two-dimensional (2D) imaging at sufficient image rates (>100 frames/sec). To understand better the physiological and pathophysiological EC coupling of cardiomyocytes, we describe here a protocol to monitor fast intracellular Ca2+ signals using fast 2D confocal scanning.
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. RECIPE: Please see the end of this article for recipes indicated by . Additional recipes can be found online at http://cshprotocols.cshlp.org/site/recipes.
Reagents
Cardiac myocytes Contraction inhibitor (e.g., blebbistatin) (optional; see Steps 2–5) Fluo-4 AM Prepare a 1 mM stock solution in DMSO containing 20% pluronic F-127.
Tyrode solution for probing Ca2+ signaling Equipment
Confocal microscope (fast, such as the Leica TCS SP5 II or SP8 [Leica Microsystems], the Nikon A1R [Nikon] or VTeye [VisiTech]) Coverslip (coated with extracellular matrix protein) Experimental chamber (for imaging with the inverted microscope) Pulse generator (Myopacer [IonOptix] or similar) Two-lead platinum electrode 1
Correspondence: [email protected]
© 2014 Cold Spring Harbor Laboratory Press Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot077032
1324
Downloaded from http://cshprotocols.cshlp.org/ at ALBIN O KUHN LIBRARY on January 6, 2015 - Published by Cold Spring Harbor Laboratory Press
2D Imaging of Fast Ca 2+ Release
METHOD Cell Culture and Confocal Imaging
1. Prepare cardiac myocytes using previously published methodologies (Kaestner et al. 2009). 2. Load the cardiac myocytes with 1 µM Fluo-4 AM in Tyrode solution on the coverslip at room temperature for 30 min, followed by deesterification for 15 min in dye-free Tyrode. (Optional) To suppress movement artifacts during imaging, add the contraction inhibitor blebbistatin (5 µM) to the deesterification solution.
3. Place the coverslip with the myocytes in an experimental chamber, typically at room temperature, that allows imaging with an inverted microscope and oil-immersion objectives (ideally a 60× objective with a high numerical aperture). See Troubleshooting.
4. Perfuse the myocytes with Tyrode solution and pace with a two-lead platinum electrode, using a pulse generator to provide the desired pacing frequency. 5. Set the scanning speed above 120 frames/sec to record precisely the upstroke of the Ca2+ transients. (Optional) Because the contraction inhibitor blebbistatin is light sensitive, it should be used continuously during the perfusion to ensure continuity of the inhibition of contraction. See Troubleshooting.
Image Analysis
6. To analyze the Ca2+ signals, use the fluorescence self-ratio F/F0 or the converted real Ca2+ concentration, where F0 is the fluorescence intensity at the time-point t = 0 (Cheng et al. 1993). This ratio calculation should only be performed on background-corrected images.
7. After the calculation, convert F/F0 into real Ca2+ concentrations using the Ca2+ dissociation constant (Kd) values determined in vivo (Thomas et al. 2000). To analyze further the intracellular Ca 2+ release sites, a new emerging method known as “pixel-wise fitting” (Fig. 1) can be used to facilitate the extraction of quantitative information, including pixel-level amplitudes, upstroke velocities, and decay constants. With such data, the intracellular Ca 2+ release sites can be characterized in better detail than previously possible (Tian et al. 2012).
TROUBLESHOOTING Problem (Step 3): After dye loading, the myocytes show a striped fluorescence pattern or fluorescence
signals that are too weak, even with strong illumination. Solution: This is often caused by dye overloading. The overloaded Ca2+ dye has entered mitochondria
and/or the sarcoplasmic reticulum. Decreasing the dye concentration during ester loading (e.g., to 0.7 µM) or shortening the dye loading time (e.g., to 20 min) can diminish this problem. Problem (Step 5): The myocytes show immediate nonhomogeneous release of Ca2+ after illumination. Solution: As the cells are very sensitive to strong laser illumination that can damage intracellular
organelles very quickly, the maximum laser illumination energy should be carefully adjusted. DISCUSSION
Confocal 2D time-course scanning of Ca2+ release in cardiomyocytes can reveal much more spatial information on cardiac EC coupling than the classical technique of conducting a confocal linescan. However, fast scanning speed pushes the pixel/point dwell-time to integration times of a few tens of nanoseconds. Less exposure time for each single pixel/point will massively reduce the signal-to-noise ratio of the Ca2+ signal. Recent improvements to both hardware and software can help to overcome the noise by using the latest developments in detector technology that give high quantum efficiency or Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot077032
1325
Downloaded from http://cshprotocols.cshlp.org/ at ALBIN O KUHN LIBRARY on January 6, 2015 - Published by Cold Spring Harbor Laboratory Press
A
0 msec 6.8 msec 13.6 msec 20.4 msec 27.2 msec 34 msec
[a.u.] 3000 2000 1000
C Global
B Pixel
Raw [Ca2+] (nM)
Reconstructed 200 100
Residual (a.u.)
200 msec
200 0
D
1000 0
Intensity (a.u.)
2000
Pixel-wise fitting x t (nM)
Amplitude
E
1000 500 0 (msec) 20
CICR duration
F
10 0 (msec) 350
Decay t_avg
G
300 250 200
Fourier power
H
6 1.78 μm
4 2 0 0.3
0.6
0.9
1.2
1.5
1/distance (μm–1)
FIGURE 1. Pixel-wise fitting of a fast two-dimensional (2D) confocal time-series. (A) Raw images of 2D confocal data during the upstroke phase of an electrically evoked Ca2+ transient in a single rat ventricular myocyte loaded with Fluo4. Scale bar, 10 µm. (B) Upper panel: Plot of the single-pixel fluorescence over time (blue dots) and the single-pixel pixel-wise fitting data (red line). Lower panel: Plot of the residual over time for the single-pixel data. (C ) The same plots as in B but for globally averaged fluorescence data. (D) Surface representation of the pseudolinescan data along the yellow dashed line of the cell image for raw data (left panel) and after pixel-wise fitting (right panel). The arrows mark the time point of the electrical stimulation of the myocyte. (E–G) Spatial distribution of various important characteristics of cardiac excitation–contraction coupling (ECC): (E) amplitude; (F ) the calcium-induced calcium release (CICR) duration (sum of delay, refers to the fastest 5% of the transients and upstroke time); and (G) the decay time-constant (average of the two decay time constants). (H ) For the area highlighted by the black rectangle in F, the power spectrum of the fluorescence along the longitudinal axis of the box is calculated. The arrow marks the characteristic power peak at the sarcomeric spatial frequency. Abbreviation: a.u., arbitrary units. (Figure reprinted, with permission, from Tian et al. 2012.)
Downloaded from http://cshprotocols.cshlp.org/ at ALBIN O KUHN LIBRARY on January 6, 2015 - Published by Cold Spring Harbor Laboratory Press
2D Imaging of Fast Ca 2+ Release
through recent enhancements to image-processing techniques (e.g., pixel-wise fitting [Tian et al. 2012]).
RECIPES Tyrode Solution for Probing Ca2+ Signaling
Component
Final concentration (mM)
NaCl KCl MgCl2 Glucose CaCl2 HEPES
135 5.4 1.0 10 2 10
Prepare fresh and adjust to pH 7.35 with NaOH.
ACKNOWLEDGMENTS
This work was funded by Federal Ministry for Education and Research (BMBF) Germany within the Collaborative Project CordiLux. REFERENCES Cheng H, Lederer WJ, Cannell MB. 1993. Calcium sparks: Elementary events underlying excitation-contraction coupling in heart muscle. Science 262: 740–744. Kaestner L, Scholz A, Hammer K, Vecerdea A, Ruppenthal S, Lipp P. 2009. Isolation and genetic manipulation of adult cardiac myocytes for confocal imaging. J Vis Exp 31: 1433. doi: 10.3791/1433.
Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot077032
Thomas D, Tovey SC, Collins TJ, Bootman MD, Berridge MJ, Lipp P. 2000. A comparison of fluorescent Ca2+ indicator properties and their use in measuring elementary and global Ca2+ signals. Cell Calcium 28: 213–223. Tian Q, Kaestner L, Lipp P. 2012. Noise-free visualization of microscopic calcium signaling by pixel-wise fitting. Circ Res 111: 17–27.
1327
Downloaded from http://cshprotocols.cshlp.org/ at ALBIN O KUHN LIBRARY on January 6, 2015 - Published by Cold Spring Harbor Laboratory Press
Two-Dimensional Imaging of Fast Intracellular Ca2+ Release Qinghai Tian, Lars Kaestner and Peter Lipp Cold Spring Harb Protoc; doi: 10.1101/pdb.prot077032 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. Calcium Imaging (91 articles) Confocal Microscopy (98 articles) Fluorescence (429 articles) Fluorescent Proteins (231 articles) Multi-Photon Microscopy (93 articles) Signal Transduction (51 articles)
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© 2014 Cold Spring Harbor Laboratory Press
Protocol
Two-Dimensional Imaging of Fast Intracellular Ca2+ Release Qinghai Tian, Lars Kaestner, and Peter Lipp1 Institute for Molecular Cell Biology and Research Center for Molecular Imaging and Screening, School of Medicine, Saarland University, 66421 Homburg/Saar, Germany
Asynchronous release of calcium (Ca2+)—for example, the generation of Ca2+ alternans in cardiac myocytes—is a phenomenon important in the development of cardiac arrhythmogenesis. The development of a failure to release Ca2+ at individual release sites can be regarded as a major contributor to cardiac pathologies such as hypertrophy. Although confocal linescans provide sufficient temporal resolution to investigate the physiological and pathological cardiac excitation–contraction (EC) coupling, linescans can only image 1.5% of the cross section of myocytes, which raises doubts about how representative such recordings are, especially in light of nonhomogeneous uncoupling of Ca2+ channels and ryanodine receptors. Nowadays, the speed of confocal microscopes has been greatly improved, enabling two-dimensional (2D) imaging at sufficient image rates (>100 frames/sec). To understand better the physiological and pathophysiological EC coupling of cardiomyocytes, we describe here a protocol to monitor fast intracellular Ca2+ signals using fast 2D confocal scanning.
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. RECIPE: Please see the end of this article for recipes indicated by . Additional recipes can be found online at http://cshprotocols.cshlp.org/site/recipes.
Reagents
Cardiac myocytes Contraction inhibitor (e.g., blebbistatin) (optional; see Steps 2–5) Fluo-4 AM Prepare a 1 mM stock solution in DMSO containing 20% pluronic F-127.
Tyrode solution for probing Ca2+ signaling Equipment
Confocal microscope (fast, such as the Leica TCS SP5 II or SP8 [Leica Microsystems], the Nikon A1R [Nikon] or VTeye [VisiTech]) Coverslip (coated with extracellular matrix protein) Experimental chamber (for imaging with the inverted microscope) Pulse generator (Myopacer [IonOptix] or similar) Two-lead platinum electrode 1
Correspondence: [email protected]
© 2014 Cold Spring Harbor Laboratory Press Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot077032
1324
Downloaded from http://cshprotocols.cshlp.org/ at ALBIN O KUHN LIBRARY on January 6, 2015 - Published by Cold Spring Harbor Laboratory Press
2D Imaging of Fast Ca 2+ Release
METHOD Cell Culture and Confocal Imaging
1. Prepare cardiac myocytes using previously published methodologies (Kaestner et al. 2009). 2. Load the cardiac myocytes with 1 µM Fluo-4 AM in Tyrode solution on the coverslip at room temperature for 30 min, followed by deesterification for 15 min in dye-free Tyrode. (Optional) To suppress movement artifacts during imaging, add the contraction inhibitor blebbistatin (5 µM) to the deesterification solution.
3. Place the coverslip with the myocytes in an experimental chamber, typically at room temperature, that allows imaging with an inverted microscope and oil-immersion objectives (ideally a 60× objective with a high numerical aperture). See Troubleshooting.
4. Perfuse the myocytes with Tyrode solution and pace with a two-lead platinum electrode, using a pulse generator to provide the desired pacing frequency. 5. Set the scanning speed above 120 frames/sec to record precisely the upstroke of the Ca2+ transients. (Optional) Because the contraction inhibitor blebbistatin is light sensitive, it should be used continuously during the perfusion to ensure continuity of the inhibition of contraction. See Troubleshooting.
Image Analysis
6. To analyze the Ca2+ signals, use the fluorescence self-ratio F/F0 or the converted real Ca2+ concentration, where F0 is the fluorescence intensity at the time-point t = 0 (Cheng et al. 1993). This ratio calculation should only be performed on background-corrected images.
7. After the calculation, convert F/F0 into real Ca2+ concentrations using the Ca2+ dissociation constant (Kd) values determined in vivo (Thomas et al. 2000). To analyze further the intracellular Ca 2+ release sites, a new emerging method known as “pixel-wise fitting” (Fig. 1) can be used to facilitate the extraction of quantitative information, including pixel-level amplitudes, upstroke velocities, and decay constants. With such data, the intracellular Ca 2+ release sites can be characterized in better detail than previously possible (Tian et al. 2012).
TROUBLESHOOTING Problem (Step 3): After dye loading, the myocytes show a striped fluorescence pattern or fluorescence
signals that are too weak, even with strong illumination. Solution: This is often caused by dye overloading. The overloaded Ca2+ dye has entered mitochondria
and/or the sarcoplasmic reticulum. Decreasing the dye concentration during ester loading (e.g., to 0.7 µM) or shortening the dye loading time (e.g., to 20 min) can diminish this problem. Problem (Step 5): The myocytes show immediate nonhomogeneous release of Ca2+ after illumination. Solution: As the cells are very sensitive to strong laser illumination that can damage intracellular
organelles very quickly, the maximum laser illumination energy should be carefully adjusted. DISCUSSION
Confocal 2D time-course scanning of Ca2+ release in cardiomyocytes can reveal much more spatial information on cardiac EC coupling than the classical technique of conducting a confocal linescan. However, fast scanning speed pushes the pixel/point dwell-time to integration times of a few tens of nanoseconds. Less exposure time for each single pixel/point will massively reduce the signal-to-noise ratio of the Ca2+ signal. Recent improvements to both hardware and software can help to overcome the noise by using the latest developments in detector technology that give high quantum efficiency or Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot077032
1325
Downloaded from http://cshprotocols.cshlp.org/ at ALBIN O KUHN LIBRARY on January 6, 2015 - Published by Cold Spring Harbor Laboratory Press
A
0 msec 6.8 msec 13.6 msec 20.4 msec 27.2 msec 34 msec
[a.u.] 3000 2000 1000
C Global
B Pixel
Raw [Ca2+] (nM)
Reconstructed 200 100
Residual (a.u.)
200 msec
200 0
D
1000 0
Intensity (a.u.)
2000
Pixel-wise fitting x t (nM)
Amplitude
E
1000 500 0 (msec) 20
CICR duration
F
10 0 (msec) 350
Decay t_avg
G
300 250 200
Fourier power
H
6 1.78 μm
4 2 0 0.3
0.6
0.9
1.2
1.5
1/distance (μm–1)
FIGURE 1. Pixel-wise fitting of a fast two-dimensional (2D) confocal time-series. (A) Raw images of 2D confocal data during the upstroke phase of an electrically evoked Ca2+ transient in a single rat ventricular myocyte loaded with Fluo4. Scale bar, 10 µm. (B) Upper panel: Plot of the single-pixel fluorescence over time (blue dots) and the single-pixel pixel-wise fitting data (red line). Lower panel: Plot of the residual over time for the single-pixel data. (C ) The same plots as in B but for globally averaged fluorescence data. (D) Surface representation of the pseudolinescan data along the yellow dashed line of the cell image for raw data (left panel) and after pixel-wise fitting (right panel). The arrows mark the time point of the electrical stimulation of the myocyte. (E–G) Spatial distribution of various important characteristics of cardiac excitation–contraction coupling (ECC): (E) amplitude; (F ) the calcium-induced calcium release (CICR) duration (sum of delay, refers to the fastest 5% of the transients and upstroke time); and (G) the decay time-constant (average of the two decay time constants). (H ) For the area highlighted by the black rectangle in F, the power spectrum of the fluorescence along the longitudinal axis of the box is calculated. The arrow marks the characteristic power peak at the sarcomeric spatial frequency. Abbreviation: a.u., arbitrary units. (Figure reprinted, with permission, from Tian et al. 2012.)
Downloaded from http://cshprotocols.cshlp.org/ at ALBIN O KUHN LIBRARY on January 6, 2015 - Published by Cold Spring Harbor Laboratory Press
2D Imaging of Fast Ca 2+ Release
through recent enhancements to image-processing techniques (e.g., pixel-wise fitting [Tian et al. 2012]).
RECIPES Tyrode Solution for Probing Ca2+ Signaling
Component
Final concentration (mM)
NaCl KCl MgCl2 Glucose CaCl2 HEPES
135 5.4 1.0 10 2 10
Prepare fresh and adjust to pH 7.35 with NaOH.
ACKNOWLEDGMENTS
This work was funded by Federal Ministry for Education and Research (BMBF) Germany within the Collaborative Project CordiLux. REFERENCES Cheng H, Lederer WJ, Cannell MB. 1993. Calcium sparks: Elementary events underlying excitation-contraction coupling in heart muscle. Science 262: 740–744. Kaestner L, Scholz A, Hammer K, Vecerdea A, Ruppenthal S, Lipp P. 2009. Isolation and genetic manipulation of adult cardiac myocytes for confocal imaging. J Vis Exp 31: 1433. doi: 10.3791/1433.
Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot077032
Thomas D, Tovey SC, Collins TJ, Bootman MD, Berridge MJ, Lipp P. 2000. A comparison of fluorescent Ca2+ indicator properties and their use in measuring elementary and global Ca2+ signals. Cell Calcium 28: 213–223. Tian Q, Kaestner L, Lipp P. 2012. Noise-free visualization of microscopic calcium signaling by pixel-wise fitting. Circ Res 111: 17–27.
1327
Downloaded from http://cshprotocols.cshlp.org/ at ALBIN O KUHN LIBRARY on January 6, 2015 - Published by Cold Spring Harbor Laboratory Press
Two-Dimensional Imaging of Fast Intracellular Ca2+ Release Qinghai Tian, Lars Kaestner and Peter Lipp Cold Spring Harb Protoc; doi: 10.1101/pdb.prot077032 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. Calcium Imaging (91 articles) Confocal Microscopy (98 articles) Fluorescence (429 articles) Fluorescent Proteins (231 articles) Multi-Photon Microscopy (93 articles) Signal Transduction (51 articles)
To subscribe to Cold Spring Harbor Protocols go to:
http://cshprotocols.cshlp.org/subscriptions
© 2014 Cold Spring Harbor Laboratory Press
