In vivo cardiac nano-imaging: A new technology for high-precision analyses of sarcomere dynamics in the heart

Optimization of Fluorescent Labeling for In Vivo Nanoimaging of Sarcomeres in the Mouse Heart

“…It is, however, more likely that collagenase treatment increased the accessibility of ADV to myocytes, resulting in an increase in the influx of Ca 2+ into the cells, based on the mechanism described above. It is well established that, in the heart, intracellular Ca 2+ overload in myocytes results in the occurrence of arrhythmias due to depolarization of cellular membranes via influx of Na + by means of the Na + -Ca 2+ exchanger ([5] and references therein). Therefore, although the collagenase treatment ( Figure 2 ) (or a large volume / a high viral particles of ADV) increases the expression fraction of AcGFP-expressing cardiomyocytes (in other words, enhances the ease of finding of fluorescently labeled cells), Ca 2+ -overloaded myocytes are likely to affect the heart's conductance system, thereby affecting the precision of the SL analysis.…”

“…The details of the microscopic system for in vivo nanoimaging have been described in our previous studies [5, 6]. In brief, an upright microscope (BX-51WI, Olympus Co., Tokyo, Japan) combined with a Nipkow confocal scanner (CSU21, Yokogawa Electric Co., Tokyo, Japan) and an electron multiplying CCD (EMCCD) camera (iXonEM+, Andor Technology Ltd, Belfast, Northern Ireland) were used at a 512 × 512 (or 512 × 170) pixel resolution at an exposure time of 28 (or 9.8) ms. A water immersion lens, either 60× (LUMPLFLN 60XW, N/A 1.00, Olympus Co.), 40× (LUMPLFLN 40XW, N/A 0.80, Olympus Co.), or 20× (XLUMPLFLN 20XW, N/A 1.00, Olympus Co.), and also a 2× lens (XLFluor 2X/340, N/A 0.14, Olympus Co.) were used to visualize the LV surface.…”

“…Nanoimaging was performed as in our previous studies [5, 6]. Namely, the anesthetized open-chest mouse under ventilation was placed on a custom-made microscope stage (200 × 250 mm 2 ), and the animal was warmed at 38°C throughout imaging.…”

“…In 2014, Aguirre and colleagues captured images of the fluorescence-labeled T-tubules in the mouse heart, via two-photon microscopy following reconstruction of the original images [4]. However, the confocal system combined with a two-photon microscope has substantial limitations in the imaging of consistently working cardiac muscle in vivo , because the image acquisition time is extremely long compared with the physiologically relevant heartbeat frequencies of rodents (as discussed in [5]). In order to solve the shortcomings of these previous studies, we recently developed a new microscopic system for high-precision measurements of SL in mice via expression of α -actinin-AcGFP in the left ventricle (LV) [6].…”

Real-Time In Vivo Imaging of Mouse Left Ventricle Reveals Fluctuating Movements of the Intercalated Discs

“…The details of the microscopic system for in vivo nano-imaging have been described in our previous studies [25][26][27]. In brief, an upright microscope (BX-51WI, Olympus Co., Tokyo, Japan) combined with a Nipkow confocal scanner (CSU21, Yokogawa Electric Co., Tokyo, Japan) and an electron multiplying CCD camera (iXonEM+, Andor Technology Ltd., Belfast, Northern Ireland) were used at a 512 × 512 (or 512 × 170) pixel resolution at an exposure time of 28 (or 9.8) ms. A 60× water immersion lens (LUMPLFLN 60 × W, N/A 1.00, Olympus Co., Tokyo, Japan) was used to visualize the LV surface.…”