Two-Photon Imaging of Skeletal Muscle Calcium Dynamics

Two-photon images were acquired from tibialis anterior muscle with a laser scanning system (LSM 7 MP, Carl Zeiss) equipped with 2 types of water-immersion objective lens (×10 and ×20, with numerical apertures of 0.5 and 1.0, respectively; Carl Zeiss) and a Ti:sapphire laser (Mai Tai HP, Spectra-Physics) operating at a wavelength of 950 nm (36 (link), 37 (link)). Continuous 4000-frame Ca²⁺ imaging was repeated for each imaging field. The imaged fields were 848.54 by 848.54 μm (original scan) or 425.1 by 425.1 μm (×2.0 digital zoom). The pixel size was 1.657 or 0.83 μm (×2.0 digital zoom), and the frame duration was 968 ms. FRET imaging of tibialis anterior from the surface to a maximum depth of 250 μm (pixel size, 0.83 μm; frame duration, 3.87 seconds; depth interval, 2 μm) was recorded at an excitation wavelength of 830 nm (38 (link)). Fluorescence was separated by a 509-nm dichroic mirror with 460- to 500-nm (cyan channel: for CFP fluorescence detection) and 520- to 560-nm (yellow channel: for YFP fluorescence detection) emission filters.

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Hirata Y., Nomura K., Kato D., Tachibana Y., Niikura T., Uchiyama K., Hosooka T., Fukui T., Oe K., Kuroda R., Hara Y., Adachi T., Shibasaki K., Wake H, & Ogawa W. (2022). A Piezo1/KLF15/IL-6 axis mediates immobilization-induced muscle atrophy. The Journal of Clinical Investigation, 132(10), e154611.

Publication 2022

LSM 7 MP

Digital Fluorescence Frame Fret Immersion Lens Sapphire Scan Seconds Tibialis anterior

Corresponding Organization :

Other organizations : Kobe University, Nagoya University, University of Shizuoka, Tokyo Medical and Dental University, University of Nagasaki

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Variable analysis

independent variables

Objective lens type (10x and 20x)
Numerical aperture of objective lens (0.5 and 1.0)
Excitation wavelength (950 nm and 830 nm)

dependent variables

Two-photon images of tibialis anterior muscle
Calcium (Ca2+) imaging
FRET imaging

control variables

Laser scanning system (LSM 7 MP, Carl Zeiss)
Ti:sapphire laser (Mai Tai HP, Spectra-Physics)
Imaged fields (848.54 x 848.54 μm or 425.1 x 425.1 μm)
Pixel size (1.657 μm or 0.83 μm)
Frame duration (968 ms or 3.87 seconds)
Depth interval (2 μm) for FRET imaging
Fluorescence separation (509-nm dichroic mirror, 460-500 nm and 520-560 nm emission filters)

controls

Positive control: Not mentioned
Negative control: Not mentioned

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