Two-color, two-photon, and excited-state absorption microscopy.

We develop a new approach in imaging nonfluorescent species with two-color two-photon and excited state absorption microscopy. If one of two synchronized mode-locked pulse trains at different colors is intensity modulated, the modulation transfers to the other pulse train when nonlinear absorption takes places in the medium. We can easily measure 10(-6) absorption changes caused by either two-photon absorption or excited-state absorption with a RF lock-in amplifier. Sepia melanin is studied in detail as a model system. Spectroscopy studies on the instantaneous two-photon absorption (TPA) and the relatively long-lived excited-state absorption (ESA) of melanin are carried out in solution, and imaging capability is demonstrated in B16 cells. It is found that sepia melanin exhibits two distinct excited states with different lifetimes (one at 3 ps, one lasting hundreds of nanoseconds) when pumped at 775 nm. Its characteristic TPA/ESA enables us to image its distribution in cell samples with high resolution comparable to two-photon fluorescence microscopy (TPFM). This new technique could potentially provide valuable information in diagnosing melanoma.

[1]  Tong Ye,et al.  Imaging melanin by two-photon absorption microscopy , 2006, SPIE BiOS.

[2]  R. Webb,et al.  In vivo confocal scanning laser microscopy of human skin: melanin provides strong contrast. , 1995, The Journal of investigative dermatology.

[3]  Bruce J Tromberg,et al.  Effect of pulse duration on two-photon excited fluorescence and second harmonic generation in nonlinear optical microscopy. , 2006, Journal of biomedical optics.

[4]  Andreas Volkmer,et al.  Femtosecond Two‐photon Excited Fluorescence of Melanin* , 1999, Photochemistry and photobiology.

[5]  B. Tromberg,et al.  Imaging cells and extracellular matrix in vivo by using second-harmonic generation and two-photon excited fluorescence , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[6]  Klaus Hoffmann,et al.  Fluorescence Studies of Melanin by Stepwise Two-Photon Femtosecond Laser Excitation , 2000, Journal of Fluorescence.

[7]  Melissa C Skala,et al.  Multiphoton microscopy of endogenous fluorescence differentiates normal, precancerous, and cancerous squamous epithelial tissues. , 2005, Cancer research.

[8]  Xingde Li,et al.  Fiber-optic scanning two-photon fluorescence endoscope. , 2006, Optics letters.

[9]  E. W. Stryland,et al.  Sensitive Measurement of Optical Nonlinearities Using a Single Beam Special 30th Anniversary Feature , 1990 .

[10]  M. Fischer,et al.  Two-photon absorption and self-phase modulation measurements with shaped femtosecond laser pulses. , 2005 .

[11]  V. Centonze,et al.  Multiphoton excitation provides optical sections from deeper within scattering specimens than confocal imaging. , 1998, Biophysical journal.

[12]  B R Masters,et al.  Two-photon excitation fluorescence microscopy. , 2000, Annual review of biomedical engineering.

[13]  E. W. Stryland,et al.  Measurement of nondegenerate nonlinearities using a two-color Z scan. , 1992, Optics letters.

[14]  P. So,et al.  Implementation of intensity-modulated laser diodes in time-resolved, pump-probe fluorescence microscopy. , 2001, Applied Optics.

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

[16]  Chen-Yuan Dong,et al.  Discrimination of basal cell carcinoma from normal dermal stroma by quantitative multiphoton imaging. , 2006, Optics letters.

[17]  Michael D. Cahalan,et al.  Two-photon tissue imaging: seeing the immune system in a fresh light , 2002, Nature Reviews Immunology.

[18]  E. Neher,et al.  Highly nonlinear photodamage in two-photon fluorescence microscopy. , 2001, Biophysical journal.

[19]  J. Simon,et al.  Comparison of the ultrafast absorption dynamics of eumelanin and pheomelanin , 2003 .

[20]  W. Webb,et al.  Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[21]  P. So,et al.  Two-photon 3-d mapping of tissue endogenous fluorescence Species based on fluorescence excitation and emission Spectra , 2002, Microscopy and Microanalysis.

[22]  W. Webb,et al.  Multiphoton fluorescence excitation: new spectral windows for biological nonlinear microscopy. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[23]  F. Bestvater,et al.  Two‐photon fluorescence absorption and emission spectra of dyes relevant for cell imaging , 2002, Journal of microscopy.

[24]  Peifang Tian,et al.  Ultrafast measurement of two-photon absorption by loss modulation. , 2002, Optics letters.

[25]  Enrico Gratton,et al.  Mitigating thermal mechanical damage potential during two-photon dermal imaging. , 2004, Journal of biomedical optics.

[26]  Peter T. C. So,et al.  Two-photon 3D mapping of tissue endogenous fluorescence species based on fluoresence excitation spectra , 2001, SPIE BiOS.

[27]  Joseph R. Lakowicz,et al.  Two-Color Two-Photon Excitation of Indole , 1997 .

[28]  W. Denk,et al.  Deep tissue two-photon microscopy , 2005, Nature Methods.

[29]  R. Naskrȩcki,et al.  Cross phase modulation artifact in liquid phase transient absorption spectroscopy , 2000 .

[30]  M. Bass,et al.  Dependence of two-photon absorption excited fluorescence in dye solutions on the angle between the linear polarizations of two intersecting beams , 2004 .

[31]  G. Yurtsever,et al.  Two-photon absorption and self-phase modulation measurements with shaped femtosecond laser pulses , 2005, (CLEO). Conference on Lasers and Electro-Optics, 2005..

[32]  Ulrike Tauer,et al.  Advantages and Risks of Multiphoton Microscopy in Physiology , 2002, Experimental physiology.

[33]  R. Cubeddu,et al.  Time-resolved fluorescence imaging in biology and medicine , 2002 .

[34]  W. Webb,et al.  Water-Soluble Quantum Dots for Multiphoton Fluorescence Imaging in Vivo , 2003, Science.

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

[36]  Haishan Zeng,et al.  Cutaneous melanin exhibiting fluorescence emission under near-infrared light excitation. , 2006, Journal of biomedical optics.

[37]  Bruce J. Tromberg,et al.  Increasing efficiency of two-photon excited fluorescence and second harmonic generation using ultrashort pulses , 2006, SPIE BiOS.

[38]  Haishan Zeng,et al.  Raman spectroscopy of in vivo cutaneous melanin. , 2004, Journal of biomedical optics.

[39]  M. Egger,et al.  Applications of multi-photon microscopy in cell physiology. , 2004, Frontiers in bioscience : a journal and virtual library.