The optical system used was similar to that used in our previous work, with some modifications17 (link),18 (link). Briefly, an inverted microscope (IX-70, Olympus) was equipped with a high-sensitivity camera (iXon 3 860, iXon Ultra, Andor Technology) and an oil immersion objective (UPlanSapo 100 XO, Olympus). The vibration noise of the microscope was reduced by making the microscope stage rigid and optimizing the performance of a vibration-free table. The size of a pixel on the camera corresponded to 150 nm in the sample. The frame rate was adjusted to 500 fps for high-temporal-resolution analysis. The temperatures of the glass slides seeded with cardiomyocytes were controlled to 37.0 ± 0.2 °C by a thermostatically controlled incubator on the sample stage (INUG 2—ONICS, Tokai Hit Co.). A 1550 nm laser (FPL 1055 T, Thorlabs) was used as a heat source to enable rapid alteration of temperature in the vicinity of the cardiac myocytes being monitored. A 488 nm laser (FITEL HPU50211 (Blue), Furukawa Electric) was used as an excitation light source for fluorescence observation of AcGFP-actinin.
Inug2 onics
Manufactured by Tokai Hit
Sourced in Japan
The INUG2-ONICS is a compact and versatile laboratory equipment designed for general use in research and analytical settings. It serves as a multi-purpose instrument that can perform a variety of functions to support various experimental and testing requirements. The core function of the INUG2-ONICS is to provide a reliable and consistent platform for researchers and technicians to conduct their work efficiently.
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5 protocols using inug2 onics
1
High-speed Cardiac Myocyte Imaging
2
Neuron Imaging Temperature Control
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With the exception of fluorescence imaging experiments and experiments using chemical agents, neurons were observed in the same medium used for culturing (Supplementary Methods ). The solution temperature was adjusted during microscopy using a thermostatically controlled incubator mounted on the sample stage (INUG2-ONICS; Tokai Hit Co., Shizuoka, Japan). To stabilize the culture temperature, neurons were incubated on the microscope for 10 min prior to observations, and observations were performed within 1 h. The details are described in the Supplementary Methods .
3
Perfusion Culture of Cells in Microfluidic Device
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The device was sterilized with a 70% ethanol wash and in an autoclave (20 min at 120°C). The sterilized device was connected to a vacuum pump (DAP-15, ULVAC KIKO, Miyazaki, Japan) and placed on a collagen-coated glass substrate. To completely flush out air in the microchannel, the cell outlets were closed with plugs and the culture medium was perfused at a flow rate of 5.0 µl min−1 for 1 h with a micro-syringe pump. After the device had been filled with the medium, the cell outlet plugs were removed and the cell suspension (2 × 106 cells ml−1) was loaded into the fluidic inlet. After the cells had been loaded, the outlets were closed with the plugs. The device was placed in a 5% CO2 stage-top incubator (INUG2-ONICS, TOKAI HIT, Shizuoka, Japan) at 37°C. The cells were cultured at a flow rate of 0.0, 5.0 and 10.0 µl min−1.
4
Ratiometric Quantum Dot Thermometry
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For ratiometry, a confocal laser scanning microscope and its software (FV1000, Olympus) and 40× (numerical apeture (N.A.) 1.3) or 100× (N.A. 1.4) objective were used. Ambient temperature near the region of interest was monitored with a thermocouple (5SC-TT-K-36-36, Omega Engineering) and Arduino (ARDUINO-A000057, Switch Science) and controlled with a stage-top incubator (INUG2-ONICS, Tokai Hit). Diluted quantum dot solution was mounted on the 35 mm glass base dish and placed in the stage-top incubator set on the microscope stage. Quantum dots were excited by 405 nm laser, then its emission spectrum was dispersed in the range of 630–650 nm and 650–670 nm by a monochromator. The two emission spectra were collected by PMT and imaged with software. Total fluorescence intensity in the region of interest for 650–670 nm was divided by that for 630–650 nm and the value (fluorescence intensity ratio) was defined as the thermometric parameter.
For the acquisition of fluorescence spectrum of 50 nM quantum dots solution, 405 nm laser was applied to the sample. The emission in the range of 600–700 nm was split every 2 nm and detected by PMT. Acquired spectrum was fitted by Gaussian function and the mean value was estimated.
For the acquisition of fluorescence spectrum of 50 nM quantum dots solution, 405 nm laser was applied to the sample. The emission in the range of 600–700 nm was split every 2 nm and detected by PMT. Acquired spectrum was fitted by Gaussian function and the mean value was estimated.
5
Cardiomyocyte Heat-Induced Spontaneous Oscillations
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We used previous data [8 (link)] acquired as briefly described below. An inverted microscope (IX-70, Olympus) was equipped with a high-sensitivity camera (iXon 3 860, iXon Ultra, Andor Technology) and an oil-immersed objective lens (UPlanSapo 100 XO, Olympus). The pixel size of the camera corresponded to the cell length of 150 nm, and a frame rate of 500 fps was used for high-resolution temporal analysis. The temperature of the glass slide seeded with cardiomyocytes was maintained at (37.0±0.2)°C using a thermostat-controlled incubator (INUG 2-ONICS, Tokai Hit) on the sample stage. A 1550 nm laser (FPL 1055 T, Thorlabs) was used as a heat source to rapidly change the temperature near the cardiomyocytes to be monitored. HSOs were induced by application of heat in the vicinity of cardiomyocytes. A 488 nm laser (FITEL HPU50211, Furukawa Electric Co., Ltd.) was used as an excitation light source for observing the fluorescence of AcGFP-α-actinin.
The observation of HSOs with simultaneous measurement of changes in Fluo4 fluorescence intensity used previous data [7 (link)]. In this observation, HSOs were observed in cases where there was a change in intracellular calcium concentration (HSOs with beating) and when the intracellular calcium concentration was maintained constant in the same manner as during Cell-SPOCs observation (HSOs without beating).
The observation of HSOs with simultaneous measurement of changes in Fluo4 fluorescence intensity used previous data [7 (link)]. In this observation, HSOs were observed in cases where there was a change in intracellular calcium concentration (HSOs with beating) and when the intracellular calcium concentration was maintained constant in the same manner as during Cell-SPOCs observation (HSOs without beating).
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