Single-wavelength reflected confocal and multiphoton microscopy for tissue imaging
Journal
Journal of Biomedical Optics
Journal Volume
14
Journal Issue
5
Date Issued
2009
Author(s)
Chen W.-L.
Chou C.-K.
Lin M.-G.
Chen Y.-F.
Jee S.-H.
Tan H.-Y.
Tsai T.-H.
Kim K.-H.
Kim D.
So P.T.C.
Abstract
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. © 2009 Society of Photo-Optical Instrumentation Engineers.
Subjects
Multiphoton fluorescence; Optical microscopy; Reflected confocal imaging; Second-harmonic generation
Other Subjects
Autofluorescence; Clinical diagnostics; Confocal imaging; Connective tissues; Femtoseconds; Image contrasts; Imaging depth; Multi-photon microscopy; Multiphoton fluorescence; Multiphotons; Near-IR; Porcine skin; Porcine skin tissues; Single wavelength; Tissue imaging; Titanium sapphire laser; Collagen; Cytology; Harmonic generation; Image quality; Laser excitation; Musculoskeletal system; Optical microscopy; Refractive index; Titanium; Tissue; Bovinae; Sus; article; computer aided design; confocal microscopy; equipment; equipment design; evaluation; image quality; image subtraction; instrumentation; multiphoton microscopy; reproducibility; sensitivity and specificity; Computer-Aided Design; Equipment Design; Equipment Failure Analysis; Microscopy, Confocal; Microscopy, Fluorescence, Multiphoton; Phantoms, Imaging; Reproducibility of Results; Sensitivity and Specificity; Subtraction Technique
Type
journal article