Figure 1.
Collagen fibers and striated muscle.
A) The relative SHG signal intensity ratio collagen/myosin is higher in backward SHG (green) than in forward SHG (red). B) While in striated muscle SHG visualizes myosin and thus A-bands of the sarcomeres, THG signals are from the interjacent I-bands as well as from the muscle fiber border (b) and muscle cell nuclei (n). C) Forward THG signal of a muscle fiber running parallel to the optical axis, single optical section. The fiber border is delineated by a surrounding THG signal. The small bubble at the upper fiber border may belong to a cell nucleus. No SHG signal was detected in this case. D) Thick collagen fiber producing a double signal in THG (left, cyan in overlay) which is filled by the SHG signal (center, red in overlay). Scale bars 20 µm.
Figure 2.
THG of blood vessels and blood cells in explanted, unfixed cremaster muscles recorded in forward direction.
A) Projection of optical sections. Without flow, red blood cells (RBCs) sink to the bottom of the vessel, forming a tightly packed mass. B) Visualization of typical RBC shape. C,D) Leukocytes adhering to the inner vessel wall. E–G) Blood vessel with strong THG signal from the vessel wall. Vessel diameter is between 40 and 45 µm. E shows a 3D rendering with a 2x magnification in the inset. A section perpendicular to the vessel axis is shown in F and the same section is magnified in G. The arrows point to the prominent THG signal from wall sections parallel to the optical axis, the arrowhead points to the RBCs at the vessel bottom. All scale bars are 20 µm.
Figure 3.
A) Fixed cremaster muscle, optical section about 50 µm from the surface. THG (left, cyan) revealed shapes reminiscent of migrating leukocytes deep in the tissue. DNA counterstaining (right, red) confirmed that these shapes are nucleated cells. In some cases, nuclei were lobed, a morphology typical for mature neutrophil granulocytes (Gr). Note that some cells identified by DNA stain are not visible in THG. B) Projections of THG (cyan) and SHG (red) in a time series of a freshly explanted cremaster, recorded in forward direction. While most leukocytes were stationary one moved ∼25 µm in 385 seconds (arrowheads). Image on the right shows a yz-section (view from the side, at end of the time series) illustrating that the moving cell is inside the tissue, not at the surface. The respective movie is shown in Movie S2. C) Migrating leukocyte in the freshly explanted cremaster forming and retracting pseudopodia. Single optical section. The respective movie is shown in Movie S3. Scale bars are 20 µm.
Figure 4.
Nuclei and chromatin structure.
A,B) Single optical forward THG sections from unstained, explanted cremaster. A, Section near the surface, nuclei most likely belong to macrophages. B, In deeper regions, nuclei of muscle fibers (m) can be observed, in addition to leukocytes. C,D) Nuclei in the cremaster after DNA-staining with Draq5. Chromocenters colocalize in THG channels and by DNA staining. C, Single optical section. D, Projections of five sections spaced 3 µm each. Not all DNA-stained-nuclei are visible in THG. Scale bar 20 µm for all images.
Figure 5.
Nerve fibers in the cremaster.
A) Unfixed, explanted muscle, projection of several optical sections covering 50 µm depth. Peripheral nerve (forward THG, cyan) surrounded by collagen fibers (forward SHG in red, backward SHG in green). Some blood vessels and small structures also produce THG signals. B–E) Single channel images of A near the nerve junction. Forward THG and SHG images have a much better signal to noise ratio than backward images. While forward and backward SHG signal differ substantially in some image details, backward THG (not included in A) does not contain any information in addition to the crisper forward THG image. F,G) Fixed cremaster with fluorescent DNA counterstain (red) and forward THG signal visualizing nerves. Several cell nuclei are associated with the nerve fibers. Scale bars are 20 µm, bar in F valid also for G.
Figure 6.
Deep tissue imaging in the mouse thigh muscle with backward GaAsP-detectors.
The first 100 µm consist of connective tissue, followed by a dense meshwork of collagen fibers (150 and 200 µm) and finally striated muscle fibers with interspersed collagen. For this display, at each depth a different x,y position from the original 200×200 µm stack was selected. Each focal plane was adjusted for brightness individually to compensate for intensity differences. Scale bar is 20 µm.