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Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

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JoVE Journal Biologia
Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo
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1. Experiment Preparation

  1. Prepare the isotonic balanced salt solution (50 ml) containing: 140 mM NaCl, 5 mM KCl, 2.5 mM CaCl2, 2 mM MgCl2 and 10 mM HEPES (pH 7.2) for in vivo skeletal muscle imaging.
  2. Prepare 1 L of Krebs-Henseleit buffer (KHB) containing: 118 mM NaCl, 5.3 mM KCl, 1.2 mM MgSO4, 0.5 mM EDTA and 25 mM NaHCO3.
  3. Bubble the KHB with gas containing 95% O2/5% CO2 for 10-15 min prior to the addition of 2 mM CaCl2. Add metabolic substrates (e.g. 10 mM glucose and 0.5 mM pyruvate).
  4. Prepare the surgical instruments by sterilizing the clamp, scissors, and forceps in 70% ethanol and then rinsing in sterilized ddH2O.
  5. Turn on the heart perfusion system, adjust the temperature on the water bath and circulator, set the speed of peristaltic pump to 6 ml/min.
  6. Bubble the KHB with 95% O2/5% CO2 gas, and monitor the flow rate and temperature (37 °C, by a fiber optic temperature probe) of the effluent. The system needs about 20 min for gas and temperature equilibration.

2. Confocal Imaging of Skeletal Muscles In Vivo

  1. Anesthetize mouse with pentobarbital (80 mg/kg, i.p.). The animal will reach surgical anesthesia (no response to toe pinch) within 10-15 min and remain in this status for 1-1.5 hr, sufficient for the in vivo imaging of skeletal muscles.
  2. Remove hairs on one of the hindlimbs and sterilize the skin with 70% ethanol.
  3. Make an incision on the skin along the outer side of the limb to expose the gastrocnemius muscles.
  4. Pick up the epimysium gently with a sharp forceps and make an incision through it with scissors. Further dissect to remove the epimysium and expose the muscle fibers beneath it.
  5. For in vivo loading of TMRM into the skeletal muscle, include TMRM (500 nM) in the isotonic balanced salt solution to immerse muscle for 30 min and then wash out by indicator-free solution.
  6. Put the mouse on its side on the confocal microscope (Zeiss LSM 510) stage and restrain the rear limb in a position that the exposed skeletal muscle is facing against the coverslip that forms the bottom of the chamber. The coverslip is in between the muscle tissue and the inverted objective (40X oil).
  7. Press the leg down gently to make tight contact between the tissue and the coverslip. Immerse the exposed muscles in isotonic balanced salt solution.
  8. Record two dimensional (2D, xy) confocal images at a sampling rate of one second per frame. The intensity of each pixel is digitized at 8-bit depth. Usually, a serial scan contains 100 frames.
  9. Collect sequential images by first exciting at 405 nm and collecting at >505 nm then at 488 nm while collecting at >505 nm for dual wavelength excitation imaging of mt-cpYFP. Use sequential excitation at 405, 488 and 543 nm, and collect emission at 505-545, 505-545, and >560 nm, respectively, for triple wavelength excitation imaging of mt-cpYFP and TMRM.

3. Confocal Imaging of Perfused Mouse Heart

  1. Immediately after the in vivo imaging of skeletal muscles, inject the mouse with 200 units of heparin (i.p.). Ten minutes later, euthanize the mouse by thoracotomy and quickly remove the heart with lungs and thymus attached to it.
  2. Quickly remove the lung in ice-cold buffer. Identify the lobes of the thymus and gently peel back to expose the ascending aorta.
  3. Remove the thymus and isolate the aorta by carefully removing any surrounding tissue.
  4. Make a cut at the upper end of ascending aorta before the first branch of aortic arch.
  5. Hold the wall of the aorta gently with two micro suturing forceps to expose the lumen and carefully place the aorta onto the cannula (PE50 tube). The aorta is held in place with a micro vessel clamp while sutures are quickly tied around the aorta.
  6. Remove the clamp, carefully check the cannula with forceps to make sure the tip of the cannula is above aortic root. Additional ties are added as necessary to hold the heart in place.
  7. Turn on the peristaltic pump and perfuse the heart at 1 ml/min. The heart will resume beating upon perfusion.
  8. Adjust the position of the perfusion system and put the heart on the microscope stage. The stage has an adaptor that allows heating of the chamber that holds the heart.
  9. Add 1 ml of KHB perfusion solution in the chamber to partially submerge the heart. Monitor temperature of the chamber at 37 °C. Remove effluent from the chamber by using a peristaltic pump.
  10. Increase the speed of peristaltic pump gradually to provide sufficient flow (approximately 2 ml/min) to the heart.
  11. After 10 min of stabilization, perfuse the heart with 10 μM blebbistatin and 100-500 nM TMRM. Heart beat will slow down after 10 min.
  12. Apply gentle pressure on the heart to ensure tight contact of the heart with the coverslip at the bottom of the chamber and to further suppress heartbeat.
  13. Follow the same procedure for confocal imaging of intact myocardium as described in step 2.8 above. Carefully adjust the focal plane to reveal the clearest image possible.

4. Image Processing and Data Analysis

  1. Use tools provided by the "Physiological Analysis" module of the confocal software to analyze single mitochondrial flashes and membrane potential changes. This module is often included in the image acquiring software of the confocal system and allows determination of certain areas in the image as well as output of fluorescence intensity with time labels.
  2. Open the database and then the serial 2D image file to be analyzed.
  3. Click the "Region of Interest (ROI) mean" to switch to the "mean of ROIs" mode. Click the "Region of Interest (ROI) mean" to switch to the "mean of ROIs" mode.
  4. Turn off the display of other channels except the channel of cpYFP 488 nm for selecting the flashes.
  5. Zoom in the image and manually move slide bar to play the serial 2D images.
  6. Identify single mitochondrial superoxide flashes by locating the site where fluorescent signal increases transiently. Use the appropriate ROI tool to mark the flashes. The trace showing time-dependent fluorescence change of each ROI will show up beside the image.
  7. After selecting all the flashes, turn on the display of other channels. Select an ROI on the image outside of the cell for background signal subtraction. Output the average fluorescence of each ROI together with the time labels.
  8. Record the number of flashes in each of the serial scanning image file together with the scan time and area of the cell. Use Excel to calculate flash frequency as number of flashes per 100 sec/1,000 μm2 cell area.
  9. Use a custom-developed program written in Interactive Data Language (IDL, ResearchSystems) to calculate the parameters of each flash (amplitude, time to peak and decay time). IDL software is commercially available and the custom-developed program for flash parameter analysis can be obtained from the author upon request.

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Learning Objectives

According to this protocol, in vivo imaging of single mitochondrial events can be done in skeletal muscles of anesthetized mice followed by in situ imaging in perfused heart (Figure 1). The optimal setting of the imaging conditions will ensure clear images of the intact muscle tissues and with single mitochondrion resolution (Figure 2). TMRM is often used to verify the location of mt-cpYFP and should show a complete overlapping pattern with the mt-cpYFP signal (Figure 2). TMRM is a commercially available indicator for mitochondrial membrane potential measurement in intact cells23. Its spectra are distinguishable from that of cpYFP. Further, by using the sequential excitation method, the emission signals of TMRM and mt-cpYFP will not interfere with each other17. Representative images shown in Figure 3 indicate that single mitochondrial superoxide flash accompanied by membrane depolarization can be identified in the serial 2D scanning images, with a transient fluorescence increase over the background signals in both skeletal muscle tissues and the myocardium (Figure 3). Besides high resolution, adequate fluorescence intensity is also required. This can be achieved by modulating the laser intensity and the gain in the collecting channels. In general, the basal fluorescence signal from the cell is set at one third to one fourth of the maximal intensity of the channel. Since the expression level of mt-cpYFP and the loading of TMRM can vary among animals, fine-tuning of the imaging conditions should be done for each experiment. Both physiological and pathological perturbations, such as metabolic substrates17,19, electrical stimulation (Figure 4)20, ischemic-reperfusion17, have been used to show that superoxide flash activity responds to changes in cellular metabolic status.

Figure 1
Figure 1. Schematic illustration of the confocal imaging of skeletal muscles and Langendorff perfused heart. The transgenic mouse expressing mt-cpYFP is anesthetized and the skeletal muscles on one of the hindlimbs are exposed for confocal imaging. The heart is then perfused in the Langendorff mode and confocal image is conducted. Image processing and data analysis used the confocal software and custom-developed program.

Figure 2
Figure 2. Confocal imaging of skeletal muscle fibers in vivo and myocardium in perfused heart. A. Representative images showing the skeletal muscles of nontransgenic (Ntg) and mt-cpYFP transgenic (mt-cpYFP) mouse loaded with mitochondrial membrane potential indicator, TMRM. B. Representative images showing the myocardium of Ntg and mt-cpYFP mice in Langendorff perfused heart. Ex: Excitation wavelength. mt-cpYFP is excited at 405 and 488 nm with emissions collected at 505-530 nm (blue) and 505-530 nm (green), respectively. TMRM is excited at 543 nm with emission collected at >560 nm (red). Scale bars = 50 μm.

Figure 3
Figure 3. Single mitochondrial superoxide flashes detected in skeletal muscles in vivo and in perfused heart. A. Representative images (upper panel) and traces of time-dependent fluorescence change (mt-cpYFP at 405 and 488 nm excitation and TMRM at 543 nm excitation, lower panel) showing a single mitochondrial superoxide flash (highlighted by orange arrows in the enlarged portion of the images) in skeletal muscle fibers. Note the increased mt-cpYFP signal (at 488 nm excitation) is accompanied by decreased TMRM signal. B. Representative images (upper panel) and traces of time-dependent fluorescence change (lower panel) during a single mitochondrial superoxide flash in perfused myocardium. Scale bars = 50 μm. Click here to view larger figure.

Figure 4
Figure 4. Increased superoxide flash frequency in perfused heart by electrical stimulation. The heart was electrically stimulated (Pacing, 4 V and 2 Hz) for 2 min. Data are mean ± SEM, n = 8 cells. *: P < 0.05 versus Resting. Click here to view larger figure.

List of Materials

Print Materials List
REAGENTS
Blebbistatin Toronto Research Chemicals B592500
CaCl2 Acros Organics AC34961-5000
EDTA Fisher Scientific BP120-500
D-Glucose Sigma-Aldrich G8270-1
HEPES Sigma-Aldrich H7006-500
KCl Sigma-Aldrich P9541-1
MgCl2•6H2O Fisher Scientific BP214-500
MgSO4•7H2O Sigma-Aldrich M1880-1
NaCl Fisher Scientific BP358-212
NaH2PO4 Sigma-Aldrich S8282-500
NaHCO3 Sigma-Aldrich S6014-1
Pyruvate Sigma-Aldrich P2256-25
TMRM Invitrogen T-668
[header]
EQUIPMENT
Confocal Line Scanning Microscope (LSM 510 Meta, Zeiss), software version 4.2 SP1 including "Physiological Analysis" module.

Preparação do Laboratório

Print Setup Guide

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial dysfunction often accompanies and contributes to human disease. Majority of the approaches that have been developed to evaluate mitochondrial function and dysfunction are based on in vitro or ex vivo measurements. Results from these experiments have limited ability in determining mitochondrial function in vivo. Here, we describe a novel approach that utilizes confocal scanning microscopy for the imaging of intact tissues in live aminals, which allows the evaluation of single mitochondrial function in a real-time manner in vivo. First, we generate transgenic mice expressing the mitochondrial targeted superoxide indicator, circularly permuted yellow fluorescent protein (mt-cpYFP). Anesthetized mt-cpYFP mouse is fixed on a custom-made stage adaptor and time-lapse images are taken from the exposed skeletal muscles of the hindlimb. The mouse is subsequently sacrificed and the heart is set up for Langendorff perfusion with physiological solutions at 37 °C. The perfused heart is positioned in a special chamber on the confocal microscope stage and gentle pressure is applied to immobilize the heart and suppress heart beat induced motion artifact. Superoxide flashes are detected by real-time 2D confocal imaging at a frequency of one frame per second. The perfusion solution can be modified to contain different respiration substrates or other fluorescent indicators. The perfusion can also be adjusted to produce disease models such as ischemia and reperfusion. This technique is a unique approach for determining the function of single mitochondrion in intact tissues and in vivo.

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial dysfunction often accompanies and contributes to human disease. Majority of the approaches that have been developed to evaluate mitochondrial function and dysfunction are based on in vitro or ex vivo measurements. Results from these experiments have limited ability in determining mitochondrial function in vivo. Here, we describe a novel approach that utilizes confocal scanning microscopy for the imaging of intact tissues in live aminals, which allows the evaluation of single mitochondrial function in a real-time manner in vivo. First, we generate transgenic mice expressing the mitochondrial targeted superoxide indicator, circularly permuted yellow fluorescent protein (mt-cpYFP). Anesthetized mt-cpYFP mouse is fixed on a custom-made stage adaptor and time-lapse images are taken from the exposed skeletal muscles of the hindlimb. The mouse is subsequently sacrificed and the heart is set up for Langendorff perfusion with physiological solutions at 37 °C. The perfused heart is positioned in a special chamber on the confocal microscope stage and gentle pressure is applied to immobilize the heart and suppress heart beat induced motion artifact. Superoxide flashes are detected by real-time 2D confocal imaging at a frequency of one frame per second. The perfusion solution can be modified to contain different respiration substrates or other fluorescent indicators. The perfusion can also be adjusted to produce disease models such as ischemia and reperfusion. This technique is a unique approach for determining the function of single mitochondrion in intact tissues and in vivo.

Procedimento

Print Procedure Steps

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial dysfunction often accompanies and contributes to human disease. Majority of the approaches that have been developed to evaluate mitochondrial function and dysfunction are based on in vitro or ex vivo measurements. Results from these experiments have limited ability in determining mitochondrial function in vivo. Here, we describe a novel approach that utilizes confocal scanning microscopy for the imaging of intact tissues in live aminals, which allows the evaluation of single mitochondrial function in a real-time manner in vivo. First, we generate transgenic mice expressing the mitochondrial targeted superoxide indicator, circularly permuted yellow fluorescent protein (mt-cpYFP). Anesthetized mt-cpYFP mouse is fixed on a custom-made stage adaptor and time-lapse images are taken from the exposed skeletal muscles of the hindlimb. The mouse is subsequently sacrificed and the heart is set up for Langendorff perfusion with physiological solutions at 37 °C. The perfused heart is positioned in a special chamber on the confocal microscope stage and gentle pressure is applied to immobilize the heart and suppress heart beat induced motion artifact. Superoxide flashes are detected by real-time 2D confocal imaging at a frequency of one frame per second. The perfusion solution can be modified to contain different respiration substrates or other fluorescent indicators. The perfusion can also be adjusted to produce disease models such as ischemia and reperfusion. This technique is a unique approach for determining the function of single mitochondrion in intact tissues and in vivo.

Tags

Keywords: Confocal ImagingMitochondrial SuperoxideSingle Mitochondrial FlashesIntact HeartIn VivoTransgenic MiceMt-cpYFPLangendorff PerfusionReal-time ImagingMitochondrial FunctionMitochondrial DysfunctionIschemiaReperfusion