Video 1
Real-time monitoring of circulating immature leukocytes in blood vessels (video 1). Blocking CD45 slows down their movement and increases their interactions with the blood vessel walls (video 2).
Video 2
Real-time monitoring of circulating immature leukocytes in blood vessels (video 1). Blocking CD45 slows down their movement and increases their interactions with the blood vessel walls (video 2).
Video 3
Movement of cord blood human CD34+ cells on Hyaluronan in the absence (video 3) or the presence of SDF-1 gradient (video 4).
Video 4
Movement of cord blood human CD34+ cells on Hyaluronan in the absence (video 3) or the presence of SDF-1 gradient (video 4).
Video 5
Three dimensional view of α-SMA+ two cell complex adjacent to Nestin+ reticular cell
A Z-stack of two-photon images captured inside Sca-1-EGFP (green) mouse calvarium (4.22 MB, Download)
Blood vessels were labeled using Qtracker (red). Sca-1 positive small diameter BVs are found adjacently to calcified bone surface (second harmonic signal, blue). Note the disappearance of EGFP labeled BVs in the depth of the marrow, away from the endosteal surface, while still observing EGFP labeled round single hematopoietic cells. Scale bar indicates 25 µm.
A Z-stack of two-photon images captured inside nestin-EGFP (green) mouse calvarium.
Blood vessels were labeled using Qtracker (red). Nestin positive small diameter BVs are found adjacently to calcified bone surface (second harmonic signal, blue). Mice were injected with Di-Acetyl-LDL (pink) 2 hours prior to intravital images acquisition. Note the exclusive uptake of LDL by the large diameter sinusoids which are all negative for GFP. Scale bar indicates 15 µm.
A representative intravital live video of in vivo permeability measurements via perfusion and leakage of Rhodamine-Dextran (70 kDa) (red) in bone marrow vasculature of nestin-GFP (green) mouse calvarium (65.83 MB, Download)
Calcified bone and blood flow reflectance (in blue) were visualized by second harmonic signal. Rhodamine diffusion signal was collected from defined regions that were set immediately adjacent to the BV of interest (i.e. positive or negative for nestin expression). The average intensity within the regions of interest was measured over time and the slope calculated. The slope value indicated for the blood vessel permeability. Scale bar indicates 100 µm.
An intravital video showing only the red channel (in grey) of Supplementary video 3, allowing the measurements of Dextran diffusion (63.49 MB, Download)
Regions of interest for signal acquisition are defined by yellow borders. Note the higher diffusion in the region adjacent to the sinusoid relatively to the region adjacent to the nestin+ BV.
A representative intravital video showing PB circulating labeled RBCs (in yellow), flowing at high speed thru a small-diameter endosteal arteriole, and dramatically decelerating once entering a larger sinusoidal BV. Blood vessels are labeled with Rhodamine-Dextran (500 kDa) (red).
A representative intravital video of DiD-labeled HSPCs (red) that were injected into nestin-GFP (green) mice and monitored for their trafficking in different types of BVs.
Note that the monitored cell arrives at the endosteal area (blue) via a nestin+ BV then slows down, arrests and crawls, only when it reaches the downstream sinusoid. Scale bar indicates 50 µm.
A three dimensional reconstruction of Z-stack planes of intravital 2 photon images taken in the calvarium of nestin-GFP (green) mouse that was injected with Dil-labeled BM MNC (blue) and DiD-labeled BM HSPC (red). (3.03 MB, Download)
The video represents a network of blood vessels (grey) in the BM and adjacently adhering or extravasating cells. Note the occurrence of these events only in the large diameter nestin- sinusoids.
A representative intravital video of Dil-labeled BM MNC (blue) and DiD-labeled BM HSPC (red) that were injected into nestin-GFP (green) mice and monitored for their trafficking in different types of BVs.
Note two extravasation events (in the middle of the screen) of MNCs (blue) via BM sinusoids (gray) into the marrow. Scale bar indicates 25 µm.
A representative intravital video of Dil-labeled BM MNC (blue) and DiD-labeled BM HSPC (red) that were injected into nestin-GFP (green) mice and monitored for their trafficking in different types of BVs.
Note extravasation event (in the upper right side of the screen) of HSPC (red) via BM sinusoids (gray) into the marrow. Scale bar indicates 25 µm.
A Z-stack of intravital two-photon images captured inside nestin-EGFP (green) mouse calvarium with labeled blood vessels (Angiosense 750EX, red). Video represents the environment of the location of cellular trafficking and extravasation (sinusoid). Note the relative proximity to a nestin+ BV. Scale bar indicates 25 µm.