Figure 2.

Minimizing attenuation of radiation by wavelength and pulse energy optimization for in vivo deep-marrow imaging in the intact mouse tibia, through >100 μm thick cortical bone

(A) Axial xz 3D image projections acquired by three- (3p.m.) or two-photon microscopy (2p.m.) in tibia bones of Cdh5:tdTomato/Histone:GFP (Cdh5:tdTom) mice, through mechanically thinned bone cortex, <100 μm thick. tdTomato and GFP in endothelial cells (vasculature) are shown in red and blue. Second harmonics generation (SHG) and third harmonics generation (THG) are shown in white and green. 3p.m. was performed either at 1650 nm, 3 MHz using the Ytterbia OPA (left) or at 1330 nm, 2 MHz using the tunable OPA (middle). 2p.m. was performed at 1100 nm, 80 MHz using the OPO (right). All excitation schemes enable bone marrow imaging, with imaging depths >350 μm at 1650 nm, ≈300 μm at 1330 nm, and ≈200 μm at 1100 nm.

(B) Axial xz 3D image projections acquired by 3p.m. or 2p.m. in intact tibia bones of the same mouse strain, through >100 μm bone tissue. 3p.m. and 2p.m. was performed as indicated in (A), showing that bone marrow imaging through thick bone is possible only by 3p.m. Imaging depths >350 μm are achieved at 1650 nm, but only ≈230 μm (endosteal areas) at 1330 nm. 2 p.m. at 1100 nm, 80 MHz enable only signal detection in the bone cortex, not in the marrow.

(A and B) Indicated pulse energy and z-adaptation of power were chosen to prevent tissue damage.

(C) xy projections corresponding to the tissue layers indicated by dashed lines in (B), left panel (3p.m., 1650 nm). SHG and THG signals in the bone cortex (105 μm depth) are shown in the upper panels. Arrowheads indicate THG signal in single lacunae (right), with an enlarged lacuna of an osteocyte within the tibia cortex as inset. Blood vessels (tdTomato) and THG are shown in endosteal areas (230 μm depth) and in deep marrow (350 μm depth).

(D) xy projections corresponding to the tissue layers indicated by dashed lines in (B), middle panel (3p.m., 1330 nm). Similar signals as in (C) are detected in the bone cortex (67 μm depth) and in endosteal areas (180 μm, 230 μm depth), but not in deep marrow.

(A–D) Scale bar = 100 μm.

(E) Thickness of tibia cortex in the analyzed mice, either with mechanically thinned (N = 5 mice) or with intact cortex (N = 11 mice), determined relying on THG at 1330 nm and 1650 nm, and on SHG at 1100 nm. Mean values with s.d. are displayed.

(F) Effective attenuation length l_e dependence on imaging depth z in the intact mouse tibia (>100 μm thick cortex).

(G) l_e distribution in bone tissue and marrow (N = 5 mice at 1100 nm; N = 3 mice at 1330 nm; N = 8 mice at 1650 nm, mean values with s.d. are displayed). n.d. – not detected. Statistical analysis was performed using two-way ANOVA with Bonferroni post-test or t-test, significance: ∗p > 0.05, ∗∗p > 0.01, ∗∗∗p > 0.001.