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

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  Figure 1. Customized setup design for dynamic deep-tissue imaging within intact long bones in vivo The schematics depict the main components of the customized multi-photon microscope. State-of-the-art two-photon imaging is performed using low pulse energy (<2 nJ) lasers, at 80 MHz, i.e., Ti:Sa and optical parametric oscillator (OPO, (i). The Ti:Sa beam is separated by a beam splitter (BS) into two optical pathways, one being used to pump the OPO, and the other being coupled into the microscope. Thus, the microscope covers a broad excitation range, i.e., 690–1080 nm (Ti:Sa) and 1050–1350 nm (OPO). Two types of optical parametric amplifiers (OPA), delivering high pulse energy radiation, are used as three-photon excitation sources. The Ytterbia OPA, a new OPA prototype, emits at a fixed wavelength (1650 nm, bandwidth 60 nm) and variable repetition rate between 1 and 4 MHz and delivers up to 130 nJ pulse energy at the sample (iii). The state-of-the-art OPA is tunable between 1250 and 1700 nm, at 2 MHz, and delivers up to 40 nJ pulse energy at the sample (ii). Power control of laser pulses was performed using power attenuation (PA) units consisting of a half-wave plate and a polarizing cube…

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  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…

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  Figure 3. Three-photon imaging at 1650 nm enables sub-cellular resolution throughout the cortical bone and bone marrow in the intact tibia (A) 3D reconstruction (442 × 442 × 362 μm³, 1036 × 1036 × 362 voxel) of THG in the intact tibia, at 1650 nm, 2 MHz (upper panel). Representative xy and xz projections of THG in 30 μm depth in bone cortex and in 300 μm depth in bone marrow (bottom image array), tissue layers indicated in the upper panel. Scale bar = 30 μm. Representative intensity profiles of THG signal (left, corresponding to the magenta lines in the xy and xz projections) and their first derivatives (right), measured in 30 μm (black profiles) and 300 μm tissue depth (red profiles). (B) 3D reconstruction (442 × 442 × 300 μm³, 517 × 517 × 150 voxel) of THG in the intact tibia, at 1330 nm, 2 MHz (upper panel). Representative xy and xz projections of THG in 30 μm depth in the bone cortex, and in 230 μm depth in the endosteal area, as indicated in the upper panel. Scale bar = 30 μm. Similar to a THG intensity profiles (left, corresponding to the magenta lines in the xy and xz projections) and their first derivatives (right) are shown in 30 μm (black profiles) and 230 μm depth (red…

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  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 ×…

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  Figure 5. Multimodal tissue analysis reveals no signs of photodamage by in vivo time-lapse three-photon imaging at 1650 nm in mouse tibia (A) Experimental design for immunofluorescence histological analysis of marrow tissue after intravital 3 p.m. at 1650 nm, 3 MHz in the tibia of CD19:tdRFP mice. 3D imaging was performed over 2 h, every 30 s, at 40 nJ (120 mW). (B) Representative immunofluorescence overlays of B lineage cells (tdRFP, magenta), heat shock protein (HSP70, yellow) and nuclear staining (DAPI, cyan) in tibia marrow tissue irradiated by 3p.m. and not irradiated (control). Scale bar = 100 μm. (C) Photodamage quantification by immunofluorescence analysis: frequencies of HSP70+ cells, apoptotic cells (TUNEL+), macrophages (CD68+), and neutrophil granulocytes (Lys6G+) at 3p.m. irradiated marrow tissue (n = 6 mice) is similar to controls (N = 3 mice). This indicates no signs of tissue photodamage by 3p.m. (D) Experimental design of in vivo imaging to compare the effect of 3p.m. to state-of-the-art 2p.m. on the motility of marrow B lineage cells. The same marrow tissue site in the tibia of CD19:tdRFP mice, with thinned cortex, was imaged by time-lapse 2 p.m. at 1100 nm, 80 MHz,…

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  Figure 6. Heterogeneity within the plasma cell population in the tibia marrow is defined by both their cellular migration patterns and THG signal (A) Experimental design of in vivo time-lapse 3 p.m. at 1650 nm, 3 MHz in the intact tibia of CD19:tdRFP mice to study the migratory behavior of marrow plasma cells. A first acquisition period of 1 h, every 30 s is followed by acquisition over 2 h, every 120 s. (B) 3D image of intact tibia acquired by 3 p.m. at 1650 nm, 3 MHz (exponential pulse-energy z-adaptation: 1.9 to 30.4 nJ). THG is shown in green, B lineage cells (tdRFP) in magenta. The yellow rectangle indicates the repeatedly imaged volume (≈300 μm depth). Scale bar = 100 μm. (C) Representative 3D images of B lineage cells (400 × 400 × 30 μm³, 518 × 518 × 11 voxel) acquired during the first (left) and second imaging period (middle). Result of B lineage cell segmentation based on the data acquired during the second imaging period (2 h, right). B cells (cyan) and plasma cells (yellow) were defined by volume. Scale bar = 50 μm. (D) Rose plots representing cell tracks of B cells (left; n = 765 cells) and plasma cells (right; n = 64 cells). (E) Mean displacement rate distribution of marrow…

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  Figure 7. THGhi signal defines two marrow plasma cell subsets with distinct functional capacity and migratory behavior (A) Representative 3D image (400 × 400 × 100 μm³, 518 × 518 × 11 voxel) of THG signal (green) in tibia cortex and marrow acquired by in vivo 3 p.m. at 1650 nm, 3 MHz. Scale bar = 50 μm. (i) Pixel distribution of THG signal in the tibia marrow: background (brown rectangle), THGlo (cyan rectangle), and THGhi (yellow rectangle). 2D image of THG signal in cells in the bone marrow, showing THGlo signal in cell membranes (cyan arrowheads) and granular intracellular THGhi signal (rose stars). Scale bar = 30 μm. (ii) Pixel distribution of THG signal in the tibia cortex, with the same color-coding for background, THGlo, and THGhi pixels as in the bone marrow. 2D image of THG signals in the bone tissue, showing lacunae and connecting canaliculi. Scale bar = 30 μm. (B) Cell segmentation distinguishing between THGlo (cyan) and THGhi (yellow) cells (magenta rectangle in A). (C) Merged (left) and single channel (middle and right) 3D image (442 × 442 × 102 μm³, 1036 × 1036 × 52 voxel) of THGhi (green) and endoplasmic reticulum (ER, magenta) in the bone marrow of an explanted C57/Bl6…