Journal: Journal of structural biology

Article Title: Quantitative assessment of forward and backward second harmonic three dimensional images of collagen Type I matrix remodeling in a stimulated cellular environment

doi: 10.1016/j.jsb.2012.05.004

Figure Lengend Snippet: (A and B) Photographs of the fibroblast–collagen gel systems in culture wells at 0 days and after 7 days of contraction. (C) The SHG and MPEF spectral scan data measured from the fibroblast-collagen gel systems. The detected signals show SHG originating from the fibrillar collagen Type 1 matrix which is manifested by a narrow peak arising only at the expected wavelength 425 nm which is half of the excitation wavelength 850 nm and Alexa 488 signal originating from actin filaments of fibroblasts. (D) Schematics of the in vitro fibroblast-mediated collagen gel contraction model, where fibroblasts are seeded into a 3-dimensional collagen matrix. (E and F) Multiphoton microscopy setup: Femto-second IR laser pulses tuned to 850 nm is directed through a 690 nm DCXRU dichroic mirror before being focused on the specimen through a high resolution 63X/1.2 NA HCX PL APO water immersion objective. The backscattered emissions from the sample is collected through the objective lens and directed to the non-de-scanned PMT detectors in the reflection geometry (R-NDD) using the same 690 nm DCXRU dichroic mirror for generating images. A 700 nm short pass filter is used to prevent the scattered IR laser radiation from reaching the non-de-scanned PMT detectors and a 455 nm DCXRU dichroic mirror is used to separate SHG signal from the MPEF signal originating from the Alexa 488 signals. SHG signal in forward direction is captured using a non-descanned detector in the transmission geometry (T-NDD) equipped with similar IR block filter (700 SP) and a 445/60 nm band pass filter and 1.4 NA oil condenser. (G) Representative 3D rendered forward SHG image originating from the collagen Type 1 matrix (green color) overlaid with the MPEF image of stimulated fibroblasts (red color) after 2 days of contraction. The stimulated collagen raft system reveals that the fibroblasts induce lateral orientation of collagen fibers. The fibrillar collagen appears to have criss-cross oriented fiber morphology. (H and I) Similar 3D rendered image and an xy-plane image after 7 days of contraction. The lateral orientation of fibrillar collagen and the formation of long laterally oriented collagen fibers are particularly evident after 7 days of contraction (Scale bar: 25 μm).

Article Snippet: Upon entering the microscope, the laser beam was directed to the scanning mirrors, then through a 670 nm long pass dichroic mirror (DCXRU 690, Chroma Technology, USA) and subsequently focused on the specimens through the water dipping objective lens.

Techniques: In Vitro, Microscopy, Transmission Assay, Blocking Assay