Additional file 5: Figure S4. of The chronically inflamed central nervous system provides niches for long-lived plasma cells

BAFF-positive B cells and plasma cells in the inflamed CNS. Mice were immunized and boosted (day 28) with rhMOG. Analysis of spinal cord was performed during peak after boost. The fluorescence signal of DAPI (blue), BAFF (red), kappa/lambda (κ/λ, upper panel green) and B220 (lower panel green) is shown. A plasma cell is indicated with an arrow. Three mice of two independent experiments were analyzed. Scale bars represent 50 μm. (TIFF 20356 kb

Additional file 1: Figure S1. of The chronically inflamed central nervous system provides niches for long-lived plasma cells

Non-proliferating CD138+ cells in the brain of patients with other inflammatory neurological diseases (OND). DAPI (blue), CD138 (green) and Ki67 (red) were stained in the CNS of patient biopsies with other neurological diseases (OND, n = 4) as indicated on the left. Representative images are shown. White arrows indicate Ki67+ CD138+ cells. Scale bars represent 50 μm. (TIFF 14779 kb

Additional file 4: of The chronically inflamed central nervous system provides niches for long-lived plasma cells

Supplementary methods. (PDF 133 kb

High-Resolution Intravital Microscopy

<div><p>Cellular communication constitutes a fundamental mechanism of life, for instance by permitting transfer of information through synapses in the nervous system and by leading to activation of cells during the course of immune responses. Monitoring cell-cell interactions within living adult organisms is crucial in order to draw conclusions on their behavior with respect to the fate of cells, tissues and organs. Until now, there is no technology available that enables dynamic imaging deep within the tissue of living adult organisms at sub-cellular resolution, i.e. detection at the level of few protein molecules. Here we present a novel approach called multi-beam striped-illumination which applies for the first time the principle and advantages of structured-illumination, spatial modulation of the excitation pattern, to laser-scanning-microscopy. We use this approach in two-photon-microscopy - the most adequate optical deep-tissue imaging-technique. As compared to standard two-photon-microscopy, it achieves significant contrast enhancement and up to 3-fold improved axial resolution (optical sectioning) while photobleaching, photodamage and acquisition speed are similar. Its imaging depth is comparable to multifocal two-photon-microscopy and only slightly less than in standard single-beam two-photon-microscopy. Precisely, our studies within mouse lymph nodes demonstrated 216% improved axial and 23% improved lateral resolutions at a depth of 80 µm below the surface. Thus, we are for the first time able to visualize the dynamic interactions between B cells and immune complex deposits on follicular dendritic cells within germinal centers (GCs) of live mice. These interactions play a decisive role in the process of clonal selection, leading to affinity maturation of the humoral immune response. This novel high-resolution intravital microscopy method has a huge potential for numerous applications in neurosciences, immunology, cancer research and developmental biology. Moreover, our striped-illumination approach is able to improve the resolution of any laser-scanning-microscope, including confocal microscopes, by simply choosing an appropriate detector.</p> </div

Depth-dependent SNR and maximal penetration depth in lymph node and spleen.

<p>(<b>a</b>) Second harmonic generation (SHG) images recorded in 80 µm depth at the same region of a lymph node with the MB-CCD-, SB-PMT- and MB-SI-TPLSM setups, respectively. The SHG signal mainly originates from fibrillar collagen. λ_exc = 900 nm, scale bar = 20 µm. (<b>b</b>) Depth-dependent signal-to-noise ratio recorded with the SB-PMT-, MB-CCD- and MB-SI-TPLSM at the same region of the spleen of an EGFP chimeric mouse and corresponding yz-projections of 3D fluorescence images in the spleen. (<b>c</b>) Depth-dependent signal-to-noise ratio recorded with the SB-PMT-, MB-CCD- and MB-SI-TPLSM at the same region in a lymph node of a B1–8^+/+ Jk^−/− EGFP mouse and corresponding yz-projection of the 3D fluorescence images. λ_exc = 920 nm.</p

Comparison of multi-beam striped-illumination TPLSM to standard single- and multi-beam TPLSM.

<p>(<b>a</b>) 3D fluorescence images of spleen slices of chimera EGFP mice reconstituted with a non-fluorescent immune system using single-beam scanning PMT-TPLSM (SB-PMT), multi-beam scanning CCD-TPLM (MB-CCD) and SI-TPLSM (MB-SI). λ_exc = 920 nm, grid unit = 16.5 µm, grid unit of the cropped image = 2.0 µm. (<b>b</b>) 3D fluorescence images of the same regions within a popliteal lymph node after immunization with NP-CGG as recorded by MB-CCD-TPLSM versus MB-SI-TPLSM or by SB-PMT-TPLSM versus MB-SI-TPLSM. Follicular dendritic cells (FDC) are stained by CD21/CD35-Fab fragment-ATTO590 (magenta), while antigen-specific B1–8 cells express EGFP (green). λ_exc = 860 nm, grid unit = 15 µm. (<b>c</b>) 3D second-harmonic generation (SHG) signal images of the same region within a non-fluorescent lymph node as recorded by MB-CCD-, SB-PMT- and MB-SI-TPLSM. The SHG signal mainly originates from collagen fibers. λ_exc = 900 nm, grid unit = 10 µm (grid unit = 8 µm for MB-CCD-, MB-SI-TPLSM and 6 µm for SB-PMT-TPLSM). (<b>d</b>) 3D fluorescence images of acute cerebellum slices of CerTN L15 mice (expresses Cerulean and Citrine over the Thy1 cassette) recorded with the same set-ups. λ_exc = 850 nm, grid unit = 20 µm. All experiments were performed with the 20×, NA = 0.95 objective lens at <i>z</i>-step = 500 nm.</p

Dynamic intravital imaging by MB-SI-TPLSM.

<p>(<b>a</b>) Rotation view of a 3D fluorescence image in a germinal center of the popliteal lymph node of a mouse after footpad immunization with chicken γ-globulin (NP-CGG). Imaging was performed 9 days after immunization. Follicular dendritic cells (FDCs) were <i>in situ</i> stained by anti-CD21/CD35-Fab fragments coupled to Alexa568 (red) 24 h before imaging. NP-specific GFP⁺ (green) and GFP⁻ B cells were transferred into C57BL/6 mice. The communication between antigen-carrying FDC and B cells is thought to be involved in the clonal selection process of high affinity B cells. By means of MB-SI-TPLSM we were able to reveal the dynamic nature of the contacts between FDC somata and B cells (white arrowhead) but also, due to improved resolution and contrast, interactions between fine FDC processes and B cells (white arrow). λ_exc = 800 nm, scale bar = 10 µm. In the same context, we used for the staining of FDCs anti-CD21/CD35 Fab fragments coupled to ATTO590 for a better simultaneous visualization of FDCs and B cells (<b>b–d</b>). Time-lapse 3D fluorescence images by MB-SI-TPLSM in 85 to 105 µm depth in the lymph node (<b>c</b>) revealed that CD21/CD35+ immune complexes are accumulating around the B cells and that this interaction is highly dynamic (<b>d, e</b>). The trajectories of the CD21/35 clusters are shown in (<b>d</b>), whereas their statistics concerning velocity and displacement rate are summarized in (<b>e</b>). The contact regions between the CD21/35 clusters and B cell are shown in yellow (<b>f</b>). λ_exc = 860 nm, scale bar = 10 µm, grid unit = 3.5 µm.</p

Lateral and axial resolutions measured with the 20× and 40× magnification objective lenses, respectively, on fluorescent microbeads or, for second harmonic generation, on collagen fibers within lymph nodes.

<p>Lateral and axial resolutions measured with the 20× and 40× magnification objective lenses, respectively, on fluorescent microbeads or, for second harmonic generation, on collagen fibers within lymph nodes.</p

Lateral and axial resolutions measured with the 20× and 40× magnification objective lenses, respectively, on fluorescent micorbeads embedded in agarose or, for second harmonic generation, on collagen fibers in a collagen gel.

<p>Lateral and axial resolutions measured with the 20× and 40× magnification objective lenses, respectively, on fluorescent micorbeads embedded in agarose or, for second harmonic generation, on collagen fibers in a collagen gel.</p

Depth-dependent spatial resolution in MB-SI-TPLSM.

<p>(<b>a</b>) Depth-dependence of both lateral and axial resolution of the fluorescence signal of 100 nm fluorescent beads (λ_em = 515 nm) recorded by SB-PMT-, MB-CCD- and MB-SI-TPLSM. λ_exc = 800 nm. (<b>b</b>) Representative lateral and axial profiles as well as xz-projections of the ePSFs acquired with the same set-ups. (<b>c</b>) Depth-dependence of both lateral and axial resolution of the fluorescence signal of 100 nm fluorescent beads (λ_em = 515 nm) in the same region of a lymph node recorded by SB-PMT-, MB-CCD- and MB-SI-TPLSM. λ_exc = 800 nm. (<b>d</b>) Representative lateral and axial profiles, as well as xz-projections of the ePSFs acquired by the same set-ups at the surface and in 100 µm depth in the lymph node. (<b>e</b>) xy-projection and corresponding yz-projection of a 3D image of anti-CD21/35-Fab-fragment-ATTO590 labeled follicular dendritic cells (FDCs) in a germinal center of the popliteal lymph node: intravital imaging by MB-SI-TPLSM in 80–95 µm depth. The arrowheads indicate clusters of labeled CD21/35. Their dimensions are depicted by typical profiles at the focal plane (x-profile) and along the optical axis of the microscope (z-profile). λ_exc = 860 nm, scale bar = 10 µm.</p