von Andrian Lab Videos
 

 

INTRAVITAL MICROSCOPY - VISUALIZING IMMUNITY IN CONTEXT


Immunity Supplementary Movie s1.mp4

T cells and B cells.  Low magnification overview of a popliteal LN in vivo.  Fluorescently labeled T cells (green, stained with CellTracker Green) and B cells (red, stained with CellTracker Orange) were injected i.v. ~24 hours before the beginning of this recording. Lymphocytes entered the LN via high endothelial venules in the T cells area of the paracortex. While T cells mostly remain in this region, B cells become sequestered in more peripheral B cell follicles (upper right corner). There are two sources of blue signals in this recording: second harmonic UV photons are generated by collagen fibers (the filamentous, stationary structures) within the fibrous LN capsule and the reticular fiber network in the LN parenchyma; in addition, this mouse was injected subcutaneously with blue fluorescent beads (400 mm diameter), which were engulfed by phagocytic cells (mostly dendritic cells), and now delineate these cells in the cortex of the LN. Images are renditions of 11 optical sections each spaced 3 mm apart in z corresponding to 30 mm thick volumes of the LN. Scale bar = 100 mm. Image stacks were acquired at 30 second intervals and this movie is accelerated 450x over real-time

 


B cell follicle. Purified T cells (green, stained with CellTracker Green) and B220+ B cells (red, stained with CellTracker Orange) migrating within the reticular collagen network (blue, denoting second harmonic generation) of a B cell follicle (imaged between 100 µm and 130 µm below the LN capsule). Note that T cells move more vigorously than B cells even when both are within a B follicle, indicating that their differential migratory behavior reflects cell-intrinsic properties. Images are renditions of 11 optical sections spaced 3 mm apart in z, corresponding to 30 mm thick volumes of the LN. Scale bar = 25 mm. Image stacks were acquired at 30 second intervals and this movie is accelerated 450x over real-time.


Immunity Supplementary Movie s3.mp4

B cells in subcapsular sinus. Purified B cells were labeled with the blue fluorescent dye Hoechst 33342 and injected i.v. into a mouse.  Cells homed to the popliteal lymph node and migrated to a B cell follicle just beneath the subcapsular sinsus (SCS). A green fluorescent soluble marker (FITC-Dextran) was then injected into the footpad to visualize the lymph within the draining popliteal LN. Due to its high molecular mass (2 MDa), the fluorescent dextran stays confined to the SCS, which is sectioned tangentially. Some B cells can be seen flowing with the lymph. Images are sequential optical sections, spaced 0.5 mm apart, scanning through a 50 mm thick slice of tissue. Scale bar = 25 mm. Images were acquired at ~0.5 second intervals and this movie is accelerated ~7.5x over real-time.


Immunity Supplementary Movie s4.mp4

T cell area. The experimental protocol was as descibed for movie 1.  Purified T cells (green) were recorded 160-190 mm below the LN capsule. Blue fluorescent beads (400 mm diameter) were injected into the skin ~24 hours before this recording and have accumulated within phagocytic cells (presumably DC) in the T cell area. Some of the phagocytes can be seen to adhere to reticular fibers (the blue filamentous structures), while others seem more motile. Images are renditions of 11 optical sections spaced 3 mm apart in z, corresponding to 30 mm thick volumes of the LN. Scale bar = 25 mm. Image stacks were acquired at 30 second intervals and this movie is accelerated 450x over real-time.


Immunity Supplementary Movie s5.mp4

Superficial T cells area. Purified T cells (blue, stained with CellTracker Blue) migrating in the vicinity of a branched high endothelial venule connected to a network of capillaries in the superficial T cell area bordering a B cell follicle (80-110 mm below the LN capsule).  the intravascular compartment was visualized by intravenous injection of TRITC-Dextran (red). In this region T cells constitutively migrate at a lower velocity than in the deep cortex (compare movie 4), and stationary cells are more frequent. Images are renditions of 11 optical sections spaced 3 mm apart in z, corresponding to 30 mm thick volumes of the LN. Scale bar = 25 mm. Image stacks were acquired at 30 second intervals and this movie is accelerated 450x over real-time.


Immunity Supplementary Movie s6.mp4

Transgenic T cells expressing either DsRed or EGFP. In this recording transgenic lymphocytes from mice expressing either DsRed or EGFP under a T cell-specific promoter were adoptively transferred into a non-transgenic recipient. This movie illustrates the feasibility of using genetically encoded fluorochromes to visualize and distinguish migrating immune cells in vivo. Use of such fluorescent tags is often advantageous because ex vivo handling of cells can be avoided thus allowing imaging of endogenous, unmanipulated cells. The white, punctate signals stem from autofluorescent tissue components (possibly macrophages), which become particularly apparent when using excitation wavelength and emission filters for optimal viewing of fluorescent proteins. Shown are X-Y (top) and Y-Z (bottom) projections of a 624 x 624 x 90 mm volume within a popliteal LN.  Projections were from 19 optical z-sections with 5 mm vertical spacing. Scale bar = 100 mm. Image stacks were acquired at 30 second intervals and this movie is accelerated 450x over real-time.


Immunity Supplementary Movie s7.mp4

DC entering LN. Purified CD11c+ splenic DC (red) were fluorescently labeled with CellTracker Orange and injected into the footpad of a mouse together with 5 ng LPS. The coinjected LPS induced DC maturation and migration via afferent lymph vessels to the SCS in the draining popliteal LN. Numerous DC can be seen below the LN capsule (blue) and migrating into and within the LN parenchyma. Areas that are free of DC are B follicles. Images are renditions of 8 optical sections with 4 mm vertical spacing corresponding to 28 mm thick volumes of the LN. Scale bar = 100 mm. Image stacks were acquired at 15 second intervals and this movie is accelerated 225x over real-time.


Immunity Supplementary Movie s8.mp4

T cell area overview. Splenic DC (red, stained with CellTracker Orange) were induced to migrate from the skin to the draining popliteal LN as described in movie 7. Naive CD8+ T cells (green, stained with CellTracker Green) were injected i.v. and allowed to home to the LN as described in movie 4. Both populations were visualized in the T cell area of the deep paracortex (200-230 mm below the LN capsule). Note the clustering of T cells in regions with high density of DC, which are themselves distributed inhomogeneously. Images are renditions of 6 optical sections spaced 5 mm apart in z corresponding to 30 mm thick volumes of the LN. Scale bar = 100 mm. Image stacks were acquired at 15 second intervals and this movie is accelerated 225x over real-time.


Immunity Supplementary Movie s9.mp4

CD4 T cell-DC interactions. Purified splenic DC (red, stained with CellTracker Orange) were pulsed with the Ovalbumin-derived peptide OVA323-339 and induced to migrate to a popliteal LN as described in movie 7. DO11.10 CD4 T cells (green, stained with CellTracker Green), which express a transgenic TCR specific for OVA323-339 in I-Ad were injected i.v. Both fluorescent cell types were viewed in the LN paracortex (120-170 mm below the LN capsule) <how many> hours thereafter. While many T cells are seen to undergo brief, sequential encounters with several Ag-pulsed DC, others form stable conjugates. One DC can be seen to interact with several T cells simultaneously while migrating through a meshwork of interstitial collagen fibers (blue). Images are renditions of 11 optical sections spaced 5 mm apart in z corresponding to 50 mm thick volumes of the LN. Scale bar = 15 mm. Image stacks were acquired at 30 second intervals and this movie is accelerated 450x over real-time.


Immunity Supplementary Movie s10.mp4

Central memory CD8 T cells in BM microvessels. The scene shows CFSE-labeled central memory CD8 T cells in the microvasculature in skull BM of an anesthetized mouse. T cells were injected into the feeding carotid artery, and their passage through BM microvessels in the skull was recorded by video-triggered stroboscopic ellumination. This movie is shown in real-time (30 frames per s). Cells are seen to roll in venules and sinusoids. Some of the rolling cells adhere firmly and may eventually emigrate into the surrounding BM cavities.


Immunity Supplementary Movie s11.mp4

Rhodamine 6G and FITC-dextran distribution in skull BM. This movie demonstrates accumulation of rhodamine 6G (red) and FITC-dextran (green) in skull BM upon i.v. injection.  The image is obtained by top-to-bottom optical sectioning through a tissue volume of 307 mm x 307 mm x 140 mm. The image stack contains 141 z-sections at 1 mm vertical spacing.  The low molecular mass of rhodamine 6G allows this dye to extravasate and to stain nuclei and mitochondria in both intra- and extravascular hematopoietic cells within the BM cavity. However, rhodamine 6G does not penetrate the surrounding solid bone, which therefore appears black. High molecular mass FITC-dextran (2 MDa) stays confined to the intravascular compartment and delineates the lumen of perfused BM microvessels.


Immunity Supplementary Movie s12.mp4

Clusters of murine multiple myeloma cells in BM. A three-dimensional rotation (60°) showing the distribution of GFP-expressing murine multiple myeloma cells (green) in the extravascular space in murine skull. This scene was taken 3 weeks after i.v. injection of the tumor cells. Several clusters of tumor cells can be seen to lodge in the vicinity of BM microvessels whose lumen was contrasted by i.v. injection of 2MDa TRITC-dextran (red).


For details on experimental methods & techniques see:
Sumen, C., Mempel, T.R., Mazo, I.B. and von Andrian, U.H. Intravital microscopy - visualizing immunity in context. Immunity 21(3): 315-329, 2004.
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