Optimized intravital three-photon imaging of intact mouse tibia links plasma cell motility to functional states

Figure 4

Laser repetition rate optimization for fast image acquisition in deep tissue layers of intact tibia by three-photon excitation at 1650 nm in vivo (A) 3D reconstructions (400 × 400 × 500 μm³, 518 × 518 × 125 voxel) of tdTomato (red) and SHG (white) in the intact tibia of a Cdh5:tdTom mouse at 1650 nm, with 1, 2, 3, and 4 MHz. Deeper imaging was achieved at higher repetition rates, as indicated by the dashed lines. The pulse trains at each repetition rate for 1.98 μs pixel dwell time are shown in the graphs. (B) Left panel: Depth dependent SNR determined for tdTomato fluorescence at 1 MHz (orange), 2 MHz (green), 3 MHz (red), and 4 MHz (blue). The absolute detection limit is given by SNR = 1 (red line). SNR = 3 is needed for reliable 3D object segmentation (gray line), being reached in 300 μm depth at 1 MHz, 340 μm at 2 MHz, and ≈400 μm at both 3 and 4 MHz. Right panel: Depth dependent applied pulse energy at the tibia surface (gray line) and effective pulse energy in tissue (blue line), upper graph. The effective pulse energy is the product of the pulse energy at the tibia surface and of the normalized attenuation of radiation in tissue (bottom graph). (C) First xy image (400 × 400 μm², 518×518 pixel) of a time-lapse 2D stack acquired by in vivo 3p.m. in the tibia of a Prx1:tdRFP mouse at 1650 nm, 3 MHz (pulse energy 16 nJ, in 126 μm tissue depth, pulse train shown in graph). (D) First xy image of a similar time-lapse 2D stack as in (C) acquired at 4 MHz (pulse energy 14 nJ, in 120 μm depth, pulse train shown in graph). (C and D) tdRFP fluorescence (stroma compartment) is shown in magenta and THG in green. Among other tissue components, erythrocytes show a THG signal, enabling to visualization of blood flow in a label-free manner. Videos were acquired over 3 min, every second. To generate the time color-coded image (right images), we calculated the difference between every two consecutive THG images, color-coded the resulting images according to the acquisition time-point, and summed them up. In this way, only regions with changing structures, such as blood flow, are highlighted. 3 p.m. at both 3 and 4 MHz enables blood flow visualization over large fields of view in the tibia marrow. As at the same pulse energy, the average laser power at 3 MHz is lower than at 4 MHz, imaging at 3 MHz is less prone to induce tissue photodamage. Scale bar = 100 μm.