A high-pressure optical chamber, PiezoGRIN, that facilitates label-free 3D high-resolution live-cell multiphoton microscopy in thick tissue samples is presented. A set of two Gradient Index (GRIN) rod lenses is integrated into the chamber as an optical guide and allows for the adjustment of the focal plane through the sample providing a field of view volume of 450 × 450 × 500 µm (x, y, z). An optical lateral resolution of 0.8 µm is achieved by using two-photon excitation with 150 fs pulses of a 810 nm titanium-sapphire laser at hydrostatic pressures up to 200 MPa. With the PiezoGRIN setup, it is possible to follow pressure-induced changes in subcellular structure of unstained vital mouse skeletal muscle tissue up to 200 µm below the tissue surface.
Keywords: high pressure; multiphoton microscopy; second harmonic generation; skeletal muscle.