Longitudinal intravital imaging of the femoral bone marrow reveals plasticity within marrow vasculature

The bone marrow is a central organ of the immune system, which hosts complex interactions of bone and immune compartments critical for hematopoiesis, immunological memory, and bone regeneration. Although these processes take place over months, most existing imaging techniques allow us to follow snapshots of only a few hours, at subcellular resolution. Here, we develop a microendoscopic multi-photon imaging approach called LIMB (longitudinal intravital imaging of the bone marrow) to analyze cellular dynamics within the deep marrow. The approach consists of a biocompatible plate surgically fixated to the mouse femur containing a gradient refractive index lens. This microendoscope allows highly resolved imaging, repeatedly at the same regions within marrow tissue, over months. LIMB reveals extensive vascular plasticity during bone healing and steady-state homeostasis. To our knowledge, this vascular plasticity is unique among mammalian tissues, and we expect this insight will decisively change our understanding of essential phenomena occurring within the bone marrow.

File Name: Supplementary Movie 2 Description: Open field behavior of animals that underwent LIMB-implantation. The activity of mice at different time points shows no impairment in motility after surgery of the right hind limb. The same individual mouse is shown from above the cage in movie sequences at day 2, 6, 9, 13 and 21 post-surgery.
File Name: Supplementary Movie 3 Description: Intravital imaging and tracking of B lymphocytes for motility analysis in the femoral bone marrow at day 90 after LIMB-implantation. Intravital imaging was performed in CD19:tdRFP mice at day 90 after implantation. The mice received an intravenous injection of Qdots before imaging. Left and right: B lymphocytes are shown in green, vasculature in red. Right: Tracking the motility of the B cells revealed that some of the small (<500 µm³) RFP + cells are motile (cyan objects/ tracks), whereas all of the large (>500 µm³) RFP + cells are stationary (yellow objects/ tracks). Scale bars = 20 µm.
File Name: Supplementary Movie 4 Description: Intravital imaging and tracking of B lymphocytes for motility analysis in the calvarial bone marrow. Intravital imaging of B lymphocytes in the calvarial bone marrow of a CD19:tdRFP mouse was performed under steady state conditions. The mouse received an intravenous injection of Qdots before imaging. Left: B cells are shown in green, vasculature in red. Right: Tracking the motility of the B cells reveals that some of the small (<500 µm³) RFP + cells are motile (cyan objects/ tracks), whereas all of the large (>500 µm³) RFP + cells are stationary (yellow objects). Scale bars = 20 µm.
File Name: Supplementary Movie 5 Description: Intravital imaging and tracking of B lymphocytes for motility analysis in the femoral bone marrow at day 60 after LIMB-implantation. Intravital imaging was performed in CD19:tdRFP mice at day 60 after implantation. The mouse received an intravenous injection of Qdots before imaging. Left and right: B lymphocytes are shown in green, vasculature in red. Right: Tracking the motility of the B cells reveals that some of the small (<500 µm³) RFP + cells are motile (cyan objects/ tracks), whereas almost all of the large (>500 µm³) RFP + cells are stationary (yellow objects/ tracks). Small RFP + cells can be observed as they enter and leave the bone marrow via the bloodstream. A single large RFP + cell moves back and forth along a vessel. Scale bars = 20 µm. Grid size = 10 µm.
File Name: Supplementary Movie 6 Description: Longitudinal intravital imaging of B lymphocytes in the femoral bone marrow at day 30 after limb-implantation. LIMB was performed a CD19:tdRFP mouse at day 30 (see also Supplementary Movie 7) after implantation. The singlet GRIN lens, which gives a larger field of view of 280 µm in diameter (as compared to 150 µm with the triplet GRIN lens, compare also Supplementary Movie 3, 5) stayed stably glued within the implant during the experiment. The mouse received an intravenous injection of Qdots before each imaging session. B cells are shown in green, vasculature/Qdots in red. Vessels are well delimited and RFP-cells appear as dark shadows in the blood vessels. Video is exemplary for n = 3 experiments. Scale bar = 50 µm. Grid size = 20 µm.
File Name: Supplementary Movie 7 Description: Longitudinal intravital imaging of B lymphocytes in the femoral bone marrow at day 38 after LIMB-implantation. LIMB was again performed in the same CD19:tdRFP mouse 8 days later at day 38 after implantation (see also Supplementary Movie 6). For both time points, the singlet GRIN lens stably glued into the tubing was used, which gives a larger fieldof-view of 280 µm in diameter as compared to 150 µm with the non-fixed triplet GRIN lens (compare also Supplementary Movie 3 and 5). The mouse received an intravenous injection of Qdots before imaging. B cells are shown in green, vasculature/Qdots in red. Number of RFP + cells is slightly decreased as compared to day 30. RFPcells appear as dark shadows in the blood vessels. Video is exemplary for n = 3 experiments. Scale bar = 50 µm. Grid size = 20 µm.
File Name: Supplementary Movie 8 Description: Intravital imaging and tracking of B lymphocytes for motility analysis in the calvarial bone marrow. As an example for recording B lymphocyte migration in BM cavities of flat bones, intravital imaging was performed in the calvarium of a CD19:tdRFP mouse that received an intravenous injection of Qdots before imaging. Left: blood vessels/Qdots are shown in red, B lymphocytes in green, bone/collagen detected by second harmonic generation in blue. Right: surfaces of single cells were digitally reconstructed and tracked over time. Color-coding shows cells >500 µm³ and its tracks in yellow and cells <500 µm³ and its tracks in cyan. B lymphocytes <500 µm³ have significantly higher displacement rates than B lymphocytes >500 µm³ (see also Fig. 4). Scale bar = 100 µm. Grid size = 50 µm.
File Name: Supplementary Movie 9 Description: Intravital imaging and tracking of B lymphocytes for motility analysis in the tibial bone marrow. As an example for imaging of B lymphocyte migration in long bones which was recorded using a method other than LIMB, intravital imaging was performed after removing the cortex in the tibia of CD19:tdRFP mice. The animals had received an intravenous injection of Qdots before imaging. Left: blood vessels are shown in red, B lymphocytes in green, bone/collagen detected by second harmonics generation in blue. Right: surfaces of single cells were digitally reconstructed and tracked over time. Colorcoding shows cells >500 µm³ and its tracks in yellow and cells <500 µm³ and its tracks in cyan. Most of the tracked cells are sessile, lymphocytes showing high motility are exclusively <500 µm³. Scale bar = 100 µm. Grid size = 50 µm. File Name: Supplementary Movie 12 Description: Intravital imaging of vasculature and SHG in the bone marrow 115 days after surgery. 115 days after implantation of the LIMB-implant vasculature (red) and newly formed collagen structures (SHG, blue) are detected in the imaging volume. Non-fluorescent cells can be observed in the vessels as dark spots. Vessels were visualized by intravenous injection of Qdots before imaging. Elapsed time is shown in minutes in the lower right corner of the movie. Scale bar = 20 µm. Grid size = 20 µm.
File Name: Supplementary Movie 13 Description: Intravital imaging of vasculature in the bone marrow at day 35 after surgery and 24 hours later. The left panel depicts the 3D reconstruction of the bone marrow vasculature 35 days after implantation (green, 0 h and red, 24 hours later). Vessels were visualized by intravenous injection of Qdots before imaging.The right panel depicts the 3D reconstruction of the differential image of the vasculature acquired by LIMB, at 0 and 24 hours, i.e. 24 h -0 h. Positive values, i.e. appearance of blood vessels, are depicted in cyan and negative values, i.e. disappearance of blood vessels, are depicted in yellow.