Single-wavelength reflected confocal and multiphoton microscopy for tissue imaging

• Main Authors: So, Peter T. C. (Contributor), Kim, Daekeun (Contributor), Kim, Ki Hean (Contributor), Dong, Chen-Yuan (Author), Lin, Sung-Jan (Author), Tsai, Tsung-Hua (Author), Tan, Hsin-Yuan (Author), Jee, Shiou-Hwa (Author), Chen, Yang-Fang (Author), Lin, Ming-Gu (Author), Chou, Chen-Kuan (Author), Chen, Wei-Liang (Author)
• Other Authors: Massachusetts Institute of Technology. Department of Biological Engineering (Contributor), Massachusetts Institute of Technology. Department of Mechanical Engineering (Contributor)
• Format: Article
• Language: English
• Published: Society of Photo-Optical Instrumentation Engineers, 2010-03-15T16:47:57Z.
• Subjects: Article
• Online Access: Get fulltext

Both reflected confocal and multiphoton microscopy can have clinical diagnostic applications. The successful combination of both modalities in tissue imaging enables unique image contrast to be achieved, especially if a single laser excitation wavelength is used. We apply this approach for skin and corneal imaging using the 780-nm output of a femtosecond, titanium-sapphire laser. We find that the near-IR, reflected confocal (RC) signal is useful in characterizing refractive index varying boundaries in bovine cornea and porcine skin, while the multiphoton autofluorescence (MAF) and second-harmonic generation (SHG) intensities can be used to image cytoplasm and connective tissues (collagen), respectively. In addition, quantitative analysis shows that we are able to detect MAF from greater imaging depths than with the near-IR RC signal. Furthermore, by performing RC imaging at 488, 543, and 633 nm, we find that a longer wavelength leads to better image contrast for deeper imaging of the bovine cornea and porcine skin tissue. Finally, by varying power of the 780-nm source, we find that comparable RC image quality was achieved in the 2.7 to 10.7-mW range.
National Research Program for Genomic Medicine of the National Science Council of Taiwan (NSC93-3112-B-002-033, NSC93-3112-B-002-034, and NSC-95-3112-B-002-019)