Single-wavelength reflected confocal and multiphoton microscopy for tissue imaging.
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.
Chen
Wei-Liang
WL
National Taiwan University, Department of Physics, No. 1 Section 4 Roosevelt Road, Taipei 10617, Taiwan.
Chou
Chen-Kuan
CK
Lin
Ming-Gu
MG
Chen
Yang-Fang
YF
Jee
Shiou-Hwa
SH
Tan
Hsin-Yuan
HY
Tsai
Tsung-Hua
TH
Kim
Ki-Hean
KH
Kim
Daekeun
D
So
Peter T C
PT
Lin
Sung-Jan
SJ
Dong
Chen-Yuan
CY
D017076:Computer-Aided Design; D004867:Equipment Design; D019544:Equipment Failure Analysis; D018613:Microscopy, Confocal / Q000295:instrumentation*; D036641:Microscopy, Fluorescence, Multiphoton / Q000295:instrumentation*; D019047:Phantoms, Imaging; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013382:Subtraction Technique / Q000295:instrumentation*