Integration of optical clearing and optical sectioning microscopy for three-dimensional imaging of natural biomaterial scaffolds in thin sections.
The intrinsic turbidity of scaffolds formed by natural biomaterials such as collagen fibers prevents high-resolution light microscopy in depth. In this research, we have developed a new method of using light microscopy for penetrative three-dimensional (3-D) visualization of scaffolds formed by collagen, chitosan, or cellulose. First, we applied an optical-clearing solution, FocusClear, to permeate and reduce the turbidity of the scaffolds. The improved photon penetration allowed fluorophores for efficient excitation and emission in the FocusClear solution. Confocal microscopy was applied to achieve cellular-level resolution up to 350 microm for both the fibroblast/collagen and the osteoblast/chitosan constructs and micrometer-level resolution up to 40 microm for the cellulose membrane. The depth of imaging of the cellulose membrane was further improved to 80 microm using two-photon microscopy. Significantly, these voxel-based confocal/two-photon micrographs allowed postrecording image processing via Amira projection algorithms for 3-D visualization and analysis of the scanned region. Although this optical method remains limited in viewing block scaffolds in thin sections, our approach provides a noninvasive way to microscopically examine the scaffold structure, which would be a valuable tool to studying biomaterials and their interactions with the molecule/cell of interest within the scaffold in an integrated fashion.
Tseng
S-Ja
SJ
National Tsing Hua University, Department of Chemical Engineering, 101 Section 2 Kuang Fu Road, Hsinchu 30013, Taiwan.
Lee
Ying-Hui
YH
Chen
Zhi-Hao
ZH
Lin
Hui-Hao
HH
Lin
Chih-Yung
CY
Tang
Shiue-Cheng
SC
D005109:Extracellular Matrix / Q000648:ultrastructure*; D016326:Extracellular Matrix Proteins / Q000648:ultrastructure*; D007089:Image Enhancement / Q000379:methods*; D021621:Imaging, Three-Dimensional / Q000379:methods*; D018613:Microscopy, Confocal / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods*; D013048:Specimen Handling / Q000379:methods; D018511:Systems Integration
D016326:Extracellular Matrix Proteins