Image_1_In Vivo Calcium Imaging of Cardiomyocytes in the Beating Mouse Heart With Multiphoton Microscopy.TIF

<p>Background: Understanding the microscopic dynamics of the beating heart has been challenging due to the technical nature of imaging with micrometer resolution while the heart moves. The development of multiphoton microscopy has made in vivo, cell-resolved measurements of calcium dynamics and vascular function possible in motionless organs such as the brain. In heart, however, studies of in vivo interactions between cells and the native microenvironment are behind other organ systems. Our goal was to develop methods for intravital imaging of cardiac structural and calcium dynamics with microscopic resolution.</p><p>Methods: Ventilated mice expressing GCaMP6f, a genetically encoded calcium indicator, received a thoracotomy to provide optical access to the heart. Vasculature was labeled with an injection of dextran-labeled dye. The heart was partially stabilized by a titanium probe with a glass window. Images were acquired at 30 frames per second with spontaneous heartbeat and continuously running, ventilated breathing. The data were reconstructed into three-dimensional volumes showing tissue structure, vasculature, and GCaMP6f signal in cardiomyocytes as a function of both the cardiac and respiratory cycle.</p><p>Results: We demonstrated the capability to simultaneously measure calcium transients, vessel size, and tissue displacement in three dimensions with micrometer resolution. Reconstruction at various combinations of cardiac and respiratory phase enabled measurement of regional and single-cell cardiomyocyte calcium transients (GCaMP6f fluorescence). GCaMP6f fluorescence transients in individual, aberrantly firing cardiomyocytes were also quantified. Comparisons of calcium dynamics (rise-time and tau) at varying positions within the ventricle wall showed no significant depth dependence.</p><p>Conclusion: This method enables studies of coupling between contraction and excitation during physiological blood perfusion and breathing at high spatiotemporal resolution. These capabilities could lead to a new understanding of normal and disease function of cardiac cells.</p

Image_2_In Vivo Calcium Imaging of Cardiomyocytes in the Beating Mouse Heart With Multiphoton Microscopy.TIF

<p>Background: Understanding the microscopic dynamics of the beating heart has been challenging due to the technical nature of imaging with micrometer resolution while the heart moves. The development of multiphoton microscopy has made in vivo, cell-resolved measurements of calcium dynamics and vascular function possible in motionless organs such as the brain. In heart, however, studies of in vivo interactions between cells and the native microenvironment are behind other organ systems. Our goal was to develop methods for intravital imaging of cardiac structural and calcium dynamics with microscopic resolution.</p><p>Methods: Ventilated mice expressing GCaMP6f, a genetically encoded calcium indicator, received a thoracotomy to provide optical access to the heart. Vasculature was labeled with an injection of dextran-labeled dye. The heart was partially stabilized by a titanium probe with a glass window. Images were acquired at 30 frames per second with spontaneous heartbeat and continuously running, ventilated breathing. The data were reconstructed into three-dimensional volumes showing tissue structure, vasculature, and GCaMP6f signal in cardiomyocytes as a function of both the cardiac and respiratory cycle.</p><p>Results: We demonstrated the capability to simultaneously measure calcium transients, vessel size, and tissue displacement in three dimensions with micrometer resolution. Reconstruction at various combinations of cardiac and respiratory phase enabled measurement of regional and single-cell cardiomyocyte calcium transients (GCaMP6f fluorescence). GCaMP6f fluorescence transients in individual, aberrantly firing cardiomyocytes were also quantified. Comparisons of calcium dynamics (rise-time and tau) at varying positions within the ventricle wall showed no significant depth dependence.</p><p>Conclusion: This method enables studies of coupling between contraction and excitation during physiological blood perfusion and breathing at high spatiotemporal resolution. These capabilities could lead to a new understanding of normal and disease function of cardiac cells.</p

Image_3_In Vivo Calcium Imaging of Cardiomyocytes in the Beating Mouse Heart With Multiphoton Microscopy.TIF

<p>Background: Understanding the microscopic dynamics of the beating heart has been challenging due to the technical nature of imaging with micrometer resolution while the heart moves. The development of multiphoton microscopy has made in vivo, cell-resolved measurements of calcium dynamics and vascular function possible in motionless organs such as the brain. In heart, however, studies of in vivo interactions between cells and the native microenvironment are behind other organ systems. Our goal was to develop methods for intravital imaging of cardiac structural and calcium dynamics with microscopic resolution.</p><p>Methods: Ventilated mice expressing GCaMP6f, a genetically encoded calcium indicator, received a thoracotomy to provide optical access to the heart. Vasculature was labeled with an injection of dextran-labeled dye. The heart was partially stabilized by a titanium probe with a glass window. Images were acquired at 30 frames per second with spontaneous heartbeat and continuously running, ventilated breathing. The data were reconstructed into three-dimensional volumes showing tissue structure, vasculature, and GCaMP6f signal in cardiomyocytes as a function of both the cardiac and respiratory cycle.</p><p>Results: We demonstrated the capability to simultaneously measure calcium transients, vessel size, and tissue displacement in three dimensions with micrometer resolution. Reconstruction at various combinations of cardiac and respiratory phase enabled measurement of regional and single-cell cardiomyocyte calcium transients (GCaMP6f fluorescence). GCaMP6f fluorescence transients in individual, aberrantly firing cardiomyocytes were also quantified. Comparisons of calcium dynamics (rise-time and tau) at varying positions within the ventricle wall showed no significant depth dependence.</p><p>Conclusion: This method enables studies of coupling between contraction and excitation during physiological blood perfusion and breathing at high spatiotemporal resolution. These capabilities could lead to a new understanding of normal and disease function of cardiac cells.</p

Image_4_In Vivo Calcium Imaging of Cardiomyocytes in the Beating Mouse Heart With Multiphoton Microscopy.TIF

<p>Background: Understanding the microscopic dynamics of the beating heart has been challenging due to the technical nature of imaging with micrometer resolution while the heart moves. The development of multiphoton microscopy has made in vivo, cell-resolved measurements of calcium dynamics and vascular function possible in motionless organs such as the brain. In heart, however, studies of in vivo interactions between cells and the native microenvironment are behind other organ systems. Our goal was to develop methods for intravital imaging of cardiac structural and calcium dynamics with microscopic resolution.</p><p>Methods: Ventilated mice expressing GCaMP6f, a genetically encoded calcium indicator, received a thoracotomy to provide optical access to the heart. Vasculature was labeled with an injection of dextran-labeled dye. The heart was partially stabilized by a titanium probe with a glass window. Images were acquired at 30 frames per second with spontaneous heartbeat and continuously running, ventilated breathing. The data were reconstructed into three-dimensional volumes showing tissue structure, vasculature, and GCaMP6f signal in cardiomyocytes as a function of both the cardiac and respiratory cycle.</p><p>Results: We demonstrated the capability to simultaneously measure calcium transients, vessel size, and tissue displacement in three dimensions with micrometer resolution. Reconstruction at various combinations of cardiac and respiratory phase enabled measurement of regional and single-cell cardiomyocyte calcium transients (GCaMP6f fluorescence). GCaMP6f fluorescence transients in individual, aberrantly firing cardiomyocytes were also quantified. Comparisons of calcium dynamics (rise-time and tau) at varying positions within the ventricle wall showed no significant depth dependence.</p><p>Conclusion: This method enables studies of coupling between contraction and excitation during physiological blood perfusion and breathing at high spatiotemporal resolution. These capabilities could lead to a new understanding of normal and disease function of cardiac cells.</p

Chronic imaging of stimulus-induced calcium transients after a microhemorrhage.

<p><b>A.</b> Schematic of a re-openable chronic cranial window preparation for mouse. A layer of silicone coated the skull around the craniotomy, and the glass was glued to the silicone. The window was reopened by gently detaching the silicone from the skull, enabling reinjection of OGB and sulforhodamine 101 into the cortex. <b>B.</b> Low- and <b>C.</b> high-magnification 2PEF images of the same regions of the brain before, immediately after, and one day after inducing a microhemorrhage. The hematoma is visible in the center of the second and third panels in (B). The arrow in the second panel of (C) indicates the direction to the microhemorrhage, located 40 µm away. <b>D.</b> Stimulus-induced calcium responses from the neuronal cell body and region of neuropil indicated on panel (C) by color coding.</p

Changes in somatosensory calcium responses in neuronal cell bodies and regions of neuropil after a microhemorrhage.

<p><b>A.</b> 2PEF images of cortex (120 µm beneath brain surface; same labeling as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065663#pone-0065663-g001" target="_blank">Figure 1B</a>) before and over time after a microhemorrhage. The arrow in the second panel indicates the direction to the microhemorrhage, located 250 µm away. <b>B.</b> Images from a control experiment. <b>C.</b> Stimulus-induced calcium responses from two neuronal cell bodies and one region of neuropil (indicated by color coded regions in (A)) before and over time after a microhemorrhage. <b>D.</b> Calcium responses from a control experiment.</p

Two-photon imaging of stimulus-induced calcium transients in somatosensory cortex.

<p><b>A.</b> Animals received an electrical stimulus to the hind paw while 2PEF microscopy was used to monitor changes in neural activity with calcium-sensitive dyes that were bulk loaded into the cortex. <b>B.</b> 2PEF image frame taken about 100 µm beneath the cortical surface in a rat. Blood vessels are labeled red (TRITC), neurons are green (OGB), and astrocytes are yellow (OGB and surlforhodamine 101). <b>C.</b> Calcium transients from the neuronal cell body circled in (B). Peripheral stimulus times are indicated by arrows. <b>D.</b> Average amplitude of the calcium transient across ten stimuli for the neuronal cell body circled in (B).</p

Postmortem histology of tissue near microhemorrhages.

<p><b>A.</b> Low- and <b>B.</b> high-magnification fluorescence images for TUNEL labeling of a representative tissue section containing a microhemorrhage near the brain surface. RBCs are stained with DAB). No TUNEL-positive cells were found across 7 microhemorrhages. <b>C.</b> Low- and <b>D.</b> high-magnification fluorescence images for TUNEL labeling of a tissue section containing a larger photothrombotic ischemic lesion. TUNEL-positive cells are visible.</p

Changes in average neuronal response over one day after a microhemorrhage.

<p>Average of the post-hemorrhage stimulus-induced calcium response amplitudes, expressed as a fraction of the baseline response amplitudes, at 0.5 and 24 hours after a microhemorrhage across 7 mice (and for controls at 24 hours across 3 mice) for neuronal cell bodies (light grey), regions of neuropil (dark grey), and astrocytes (black) located within 150 µm of the RBC-filled hematoma, as indicated in the inset. Error bars represent the standard error of the mean. Dashed line indicates a response amplitude after the microhemorrhage that is equal to the baseline response. Levels of significance for differences of the average amplitude ratios from one are indicated by: * p<0.05, ** p<0.01, † p<0.001, †† p<0.0001 (Wilcoxon signed-rank test).</p

Post-hemorrhage neural response as a function of distance from the hematoma.

<p><b>A.</b> Amplitude of the stimulus-induced calcium responses for neuronal cell bodies at different distances from the hematoma core, measured within 30 minutes of lesioning, and expressed as a fraction of the baseline responses. Grey points represent measurements from individual neuronal cell bodies rats, while the red line indicates a moving median of the response amplitude. The blue line represents the fraction of neuronal cell bodies that continued to respond normally to the peripheral stimulus after the microhemorrhage (i.e. with a response amplitude ratio that was greater than the mean minus one standard deviation of the amplitude ratio for control and sham data). The red and blue shaded regions represent 95% confidence intervals about the median response amplitude and fraction of cells normally responding, respectively. Distance is defined as the three-dimensional path from the edge of the spherical RBC-filled hematoma core to the center of the neuronal cell body (inset). <b>B.</b> Box plot of the amplitude of the calcium response from neuronal cell bodies expressed as a fraction of the baseline response, for control and sham experiments. The measurements from individual cells are indicated with green (control) and orange (sham) circles. <b>C.</b> Amplitude of the stimulus-induced calcium responses for regions of neuropil at different distances from the hematoma core, within 30 minutes of lesioning, and expressed as a fraction of the baseline responses. <b>D.</b> Box plot of the amplitude of the calcium response from regions of neuropil, expressed as a fraction of the baseline response, for control and sham experiments. <b>E.</b> Amplitude of the stimulus-induced calcium responses for astrocytes at different distances from the hematoma core, within 30 minutes of lesioning, and expressed as a fraction of the baseline responses. <b>F.</b> Box plot of the amplitude of the calcium response from astrocytes, expressed as a fraction of the baseline response, for control and sham experiments. Data was taken across 15, 5 and 3 rats for hemorrhage, control and sham experiments, respectively.</p