Phasor analysis of multiphoton spectral images distinguishes autofluorescence components of in vivo human skin

Skin contains many autofluorescent components that can be studied using spectral imaging. We employed a spectral phasor method to analyse two photon excited autofluorescence and second harmonic generation images of in vivo human skin. This method allows segmentation of images based on spectral features. Various structures in the skin could be distinguished, including Stratum Corneum, epidermal cells and dermis. The spectral phasor analysis allowed investigation of their fluorescence composition and identification of signals from NADH, keratin, FAD, melanin, collagen and elastin. Interestingly, two populations of epidermal cells could be distinguished with different melanin content.

[1]  Iris Riemann,et al.  High-resolution multiphoton tomography of human skin with subcellular spatial resolution and picosecond time resolution. , 2003, Journal of biomedical optics.

[2]  Hans C Gerritsen,et al.  Design and implementation of a sensitive high-resolution nonlinear spectral imaging microscope. , 2008, Journal of biomedical optics.

[3]  E. Gratton,et al.  The phasor approach to fluorescence lifetime imaging analysis. , 2008, Biophysical journal.

[4]  H. S. de Bruijn,et al.  In vivo monitoring of protein-bound and free NADH during ischemia by nonlinear spectral imaging microscopy , 2011, Biomedical optics express.

[5]  Guy Cox,et al.  3-dimensional imaging of collagen using second harmonic generation. , 2003, Journal of structural biology.

[6]  Karsten König,et al.  Clinical multiphoton tomography , 2008, Journal of biophotonics.

[7]  Hans C Gerritsen,et al.  Spectrally resolved multiphoton imaging of in vivo and excised mouse skin tissues. , 2007, Biophysical journal.

[8]  Fabian J Theis,et al.  Blind source separation techniques for the decomposition of multiply labeled fluorescence images. , 2009, Biophysical journal.

[9]  Chen-Yuan Dong,et al.  Multiphoton microscopy in dermatological imaging. , 2009, Journal of dermatological science.

[10]  F Flament,et al.  Clinical study on the effects of a cosmetic product on dermal extracellular matrix components using a high‐resolution multiphoton tomograph , 2010, Skin research and technology : official journal of International Society for Bioengineering and the Skin (ISBS) [and] International Society for Digital Imaging of Skin (ISDIS) [and] International Society for Skin Imaging.

[11]  Hans C. Gerritsen,et al.  Fast nonlinear spectral microscopy of in vivo human skin , 2011, Biomedical optics express.

[12]  A. Pena,et al.  Spectroscopic analysis of keratin endogenous signal for skin multiphoton microscopy. , 2005, Optics express.

[13]  Takashi Kitahara,et al.  Imaging of melanin distribution using multiphoton autofluorescence decay curves , 2010, Skin research and technology : official journal of International Society for Bioengineering and the Skin (ISBS) [and] International Society for Digital Imaging of Skin (ISDIS) [and] International Society for Skin Imaging.

[14]  Farzad Fereidouni,et al.  A modified phasor approach for analyzing time‐gated fluorescence lifetime images , 2011, Journal of microscopy.

[15]  Yuval Garini,et al.  Spectral imaging: Principles and applications , 2006, Cytometry. Part A : the journal of the International Society for Analytical Cytology.

[16]  A. Pena,et al.  Second harmonic imaging and scoring of collagen in fibrotic tissues. , 2007, Optics express.

[17]  E. Gratton,et al.  Phasor approach to fluorescence lifetime microscopy distinguishes different metabolic states of germ cells in a live tissue , 2011, Proceedings of the National Academy of Sciences.

[18]  S. Laquièze,et al.  Photoaging of the chest analyzed by capacitance imaging , 2010, Skin research and technology : official journal of International Society for Bioengineering and the Skin (ISBS) [and] International Society for Digital Imaging of Skin (ISDIS) [and] International Society for Skin Imaging.

[19]  Hans C Gerritsen,et al.  Spectral phasor analysis allows rapid and reliable unmixing of fluorescence microscopy spectral images. , 2012, Optics express.

[20]  H. S. de Bruijn,et al.  In vivo nonlinear spectral imaging in mouse skin. , 2006, Optics express.

[21]  P. So,et al.  Two-photon 3-D mapping of ex vivo human skin endogenous fluorescence species based on fluorescence emission spectra. , 2005, Journal of biomedical optics.

[22]  Paul J Campagnola,et al.  Optical diagnostics of tissue pathology by multiphoton microscopy. , 2010, Expert opinion on medical diagnostics.

[23]  W. Webb,et al.  Three‐dimensionally resolved NAD(P)H cellular metabolic redox imaging of the in situ cornea with two‐photon excitation laser scanning microscopy , 1995, Journal of microscopy.

[24]  Watt W Webb,et al.  Two-photon fluorescence spectroscopy and microscopy of NAD(P)H and flavoprotein. , 2002, Biophysical journal.

[25]  Karsten König,et al.  Spectral fluorescence lifetime detection and selective melanin imaging by multiphoton laser tomography for melanoma diagnosis , 2009, Experimental dermatology.