Two-photon absorption and transient photothermal

imaging of pigments in tissues

Tong Yea*, Dan Fub, Thomas E. Matthewsa,

Lian Honga, John D. Simona, Warren S. Warrena,c,d

aDepartment of Chemistry, cDepartment of Biomedical Engineering, dDepartment of Radiology, Duke University, Durham, NC 27708

bDepartment of Chemistry, Princeton University, Princeton, NJ 08544

ABSTRACT

As a main pigment in skin tissues, melanin plays an important role in photo-protecting skin from UV radiation. However, melanogenesis may be altered due to disease or environmental factors; for example, sun exposure may cause damage and mutation of melanocytes and induce melanoma. Imaging pigmentation changes may provide invaluable information to catch the malignant transformation in its early stage and in turn improve the prognosis of patients. We have demonstrated previously that transmission mode, two-photon, one- or two-color absorption microscopy could provide remarkable contrast in imaging melanin in skin. In this report we demonstrate significantly improved sensitivity, so that we are now able to image in epi-mode (or back reflection) in two-photon absorption. This improvement makes possible for us to characterize the different types of pigmentation on the skin in vivo at virtually any location. Another finding is that we can also image transient photothermal dynamics due to the light absorption of melanin. By carefully choosing excitation and probe wavelengths, we might be able to image melanin in different structures under different micro-environments in skin, which could provide useful photochemical and photophysical insights in understanding how pigments are involved in photoprotection and photodamage of cells. Keywords: pump-probe, two-photon absorption, microscopy, melanin, eumelanin, pheomelanin, melanoma

1. INTRODUCTION Although melanin is the most ubiquitous and biologically important natural pigment, its biological functions are still under debate. Two types of melanins are generally found in skin1: eumelanin, a black-to-dark-brown insoluble material and pheomelanin, a yellow-to-reddish-brown alkali-soluble material. Both of them are synthesized within specialized organelles (melanosomes) belonging to melanocytes2 and distributed through dendritic extensions of melanocytes to surrounding epidermal cells (keratinocytes), resulting in pigmentation of the skin. The main function of melanin pigmentation is generally believed to shield the skin from harmful ultraviolet radiation (UVR) but the different types of melanin likely do not play the same role in photoprotection. There have been considerable evidences that suggest pheomelanin and/or its precursors contribute to carcinogenesis/mutagenesis3, perhaps because pheomelanin is more vulnerable than eumelanin to UVR to generate free radicals4, 5, leading to DNA damage and mutagenicity. This implies that people with different pigmentation have varying risk factors for developing skin cancer or melanoma, as supported by epidemiological studies6. Clinical observations have shown that the production of pheomelanin in lesions is increased significantly (compared with normal skin tissues) as melanoma progresses7, 8. Thus the relative ratio between eumelanin and pheomelanin, and the change of this ratio, might have clinical value to diagnose melanoma in early stages.

Photonic Therapeutics and Diagnostics IV, edited by Nikiforos Kollias, Bernard Choi, Haishan Zeng, Reza S. Malek,Brian Jet-Fei Wong, Justus F. R. Ilgner, Kenton W. Gregory, Guillermo J. Tearney, Henry Hirschberg, Steen J. Madsen,

Proc. of SPIE Vol. 6842, 684206, (2008) · 1605-7422/08/$18 · doi: 10.1117/12.764248

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However, differentiating between eumelanin and pheomelanin quantitatively, and obtaining their distribution in skin tissue in vivo, has been a long standing challenge in pigment cell research. The current most accepted methods to quantify two types of melanin are based on HPLC quantification of the degradation products from melanin1, 9. Extracted melanins have been extensively studied using a wide variety of techniques, such as HPLC10, electron microscopy11, spectrophotometric methods12, 13 but these methods require very complicated and lengthy pre-processing of samples. They are apparently invasive and cannot provide in situ microscopic information of tissues. Optical spectroscopic methods provide possible ways to detect the melanin pigment noninvasively in vivo; for example, absorption, reflectance, Raman and fluorescence have been widely studied and shown potential in melanin detection and quantification14-16, but they only explore the surface and give far worse resolution than would histopathology. In addition, weak two-photon fluorescence (TPF) from melanin as well as two-photon absorption (TPA) has been suggested as a nonlinear contrast in skin imaging17, 18. They can provide a good melanin contrast in relatively deep tissue to do optical sectioning of melanoma lesions with resolution comparable to pathological histology, but not enough information can be provided to differentiate the two types of melanins. In a previous study, we found striking differences between eumelanin and pheomelanin in cuvette samples by using our two-color two-photon absorption measurement setup19, 20. We showed that eumelanin and pheomelanin exhibited quite different excited state dynamics; their transient absorption signals had opposite phases and recovery of ground states showed quite different rates. Results on melanosome samples initially showed similar and very large effects, but further investigation suggested that photothermal damage of melanosome samples might contribute. In this report, we present recent results characterizing differences between eumelanin and pheomelnin by using two-photon absorption imaging method. We have improved our sensitivity sufficiently that we can do imaging in epi-mode (or back reflection) in two-photon absorption microscopy, which makes it possible to image pigmentation on the skin in vivo at virtually any location. Eumelanin-dominant and pheomelanin-dominant hair samples display similar ultrafast dynamics of excited states with a few ps of lifetime in both samples, but the frequency domain measurement confirmed existence of a long-lived absorption (or loss) process with sample dependent lifetimes. This long-lived process appears to be at least partially due to a transient thermal effect, and may vary dramatically due to the light absorption, deexcitation and heat diffusion properties of different tissues.

2. METHODS AND MATERIALS

1.1 Sample preparation Melanoma lesion sample. Superficial spread human melanoma lesions were chemically induced on human skin grafting mouse model. The mice were sacrificed by CO2 inhalation and skin grafts were excised before the experiments. The grafts were fixed in 10% neutral-buffered formalin (Fisher Scientific) for 6 to 12 hours at room

Figure 1. The sample slide used in the imaging studies contained three samples: a red hair, a black hair and a capillary filled with R6G dye solution. They were fixed on a slide and aligned very close to each other so that a single scanning frame could cover all three.

Black Hair Red Hair

R6G Glass

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temperature and embedded in paraffin for storage. The sections were sliced with a microtone in the direction parallel to the surface of the skin with a thickness less than 1mm.

Hair samples. Indonesian, black human hair was purchased from R. Parrino Hair Goods (Northport, NY). Red Human hair sample was obtained from a 20 year old female locally (Durham, NC), and the hair had never been permed or dyed. The chemical degradation analysis of melanosomes from those hair samples showed a dominant eumelanin content with undetectable pheomelanin in black hair melanosome while the red-hair melanosomes contained a mixed type melanin, predominantly pheomelanin, with low content of eumelanin.

R6G solution sample. 30 mM Rhodamine 6G (R6G, Exciton) was dissolved in methanol and sealed in a capillary tube.

For convenience of imaging experiments, hair samples and R6G capillary were placed adjacent to each other on a slide and fixed by mounting media (Vectorshield hard set) as shown in Figure 1. So the three samples could be imaged in a single scanning frame without switching around between samples.

Figure 2. Schematic diagram of the laser scanning two-photon absorption microscope with simultaneous epi-mode and transmission image acquisition.

1.2 Two color two-photon /excited state absorption microscopy The method of measuring two-color TPA/ESA resembles the traditional pump-probe scheme, which is widely used in ultrafast transient absorption spectroscopy. The apparatus of two-color TPA/ESA measurement was described previously in details19. Briefly the pump pulses were split from the output of a broadband tunable mode-locked Ti:sapphire laser (Spectra Physics, Tsunami, 80MHz, 100fs), and was intensity modulated sinusoidally with an acousto-optical modulator (AOM) at 2 MHz. The modulation frequency was variable and provided by a signal generator (Stanford Research Systems). For the imaging experiments discussed in this report, the pump wavelength was tuned to 810 nm, and this particular wavelength was required to pump an optical parametric oscillator (Spectra Physic s, Opal, 80MHz, 120fs), the output of which was tunable from 1100 nm to 1500 nm. The Opal was tuned to 1480 nm, and the second harmonic generation of this beam provided 740 nm pulses that were used as the probe. The pump and probe beams were collinearly combined by a dichroic mirror and sent to a home-built laser scanning microscope for imaging. Both beams were ultimately focused onto specimens by the microscope objective.

Dichroic Mirror

Avalanche Photodiode

1480nm 740nm Ti:Sapphire Oscillator 690-1080 nm

Lock-in Amplifier 1

Photodiode

Optical Delay

SH

Modulator 810nm

Pump

Probe

Motorized Specimen stage

Scan Lens

Trinocular/Tube Lens

Polarizing Beamsplitter Or Dichroic Mirror

Objective

Condenser

Scan Mirror

Lock-in Amplifier 2

Motion Control

Computer/DAQ

Signal Generator

OPO 1100-1500nm

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The two-color two-photon absorption/excited state absorption microscope used in our imaging experiment was built on a home-made two-photon fluorescence laser scanning microscope, which followed the design described in the literature.21 The laser scanning microscope in the upright configuration was assembled on an XT95 rail (Thorlabs), on which successively were attached the XY-axis scanning mirrors (Cambridge Technologies), scan lens (Olymous), trinocular(Olympus), focusing unit (Nikon) and a condenser (home-built). A polarizing cube beam splitter (Newport) replaced the dichroic mirror between the tube lens and the objective used in the two-photon fluorescence microscope. This design made possible to image simultaneously in both the transmission mode and the epi-mode. In transmission-mode imaging, the light transmitted through the sample was first collimated and then focused onto an amplified large area photodiode (Thorlabs, PDA55); an edgepass filter was placed before the photodiode to block out the pump beam. A radio-frequency lock-in amplifier (Stanford Research Systems, SR844) was used to measure the signals at the modulation frequency. In epi-mode imaging, the polarized beam splitter allowed both the pump and the probe beams to fully pass through while the backscattered laser light from specimen with the polarization perpendicular to the pump beam was reflected by the beam splitter and focused onto an avalanche photodiode (Hamamatsu); an edge filter in the front to block out the pump beam. The voltage signal from the avalanche photodiode was sent to another radio-frequency lock-in amplifier (Stanford Research Systems, SR844) to extract the signals at the modulation frequency. The analog outputs from both of lock-in amplifiers were digitized by a data acquisition board (DAQ) (National Instrument, PXI-6115) in a PXI chassis (National Instrument, PXI-1042). Since each dual-phase lock-in amplifier had two analog outputs (Channel X and Channel Y), the 4 A/D channels of the DAQ board were fully occupied. The image acquisition software was written on the platform of Labwindows/CVI (National Instruments) and the data was analyzed in Matlab (Mathworks) environment.

3. RESULTS AND DISCUSSION

3.1 Two-photon absorption imaging in epi-mode

The capability of imaging in epi-mode is crucial for in vivo applications where measuring transmission of laser beam through bulk tissues is impossible. In transmission imaging TPA/ESA signals are extracted from the transmitted probe

(A) Transmission mode (B) Epi-mode

Fig. 3 A melanoma lesion was imaged both in (A)the transmission mode and (B)the epi-mode. TPA/ESA signals from melanin gave the almost identical patterns in both imaging modes. Imaging parameters: Objective,20x; Frame size: 256x256 µm; the pump wavelength, 810 nm; the probe wavelength, 740 nm; the average powers measured after the objective were, 1.3 mW for the pump, and 1.0 mW for the probe.

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beam that carries the coupled modulation frequency from the pump beam. In epi-mode imaging two-photon absorption signals rely on the backscattered or back-reflected probe light after the focal spot at which TPA/ESA signals are generated. An obvious challenge is to separate the weak backscattered probe light carrying signals from the much stronger light reflected from the tissue surfaces or the backscattered probe light before the focal spot. In our microscope, a polarizing beam splitter was used to solve the problem partially. The polarizing beam splitter was used to pass the entering pump and probe laser beam and most of the returning reflection from the tissue surfaces while the back-scattered light with the polarization perpendicular to the probe beam was reflected to the detector, as shown in Figure 2. An avalanche photodiode was used to increase the sensitivity of detection. With those upgrades epi-mode images were successfully acquired from pigmented skin lesions. As shown in Figure 3, the epi-mode and the transmission images of a skin lesion are almost identical to each other though the epi-mode images have smaller signal to noise ratios (SNRs). 3.2 Imaging study of excited state dynamics of hair samples Hair is a good tissue model for studying pigment imaging. Comparing with skin tissues, hair pigments are more condensed and uniformly distributed; pigment contents are well-characterized and available in the lab; the hair and skin melanocytes share a common embryonic origin—the neural crest22 and very likely make the same melanin pigments; the studies have also shown that the contents of eumelanin and pheomelanin in human hair are well-correlated to skin pigment contents23. The slide described in Figure 1 was used in our imaging study. In the typical two-photon absorption experiment, the pump wavelength was 810 nm and the probe wavelength was 740 nm. Average powers deposited onto specimens were 2.7 mW for the pump and 0.95 mW for the probe, respectively. At those levels of the laser power, no damage of samples was observed visually. A series of images were acquired in both transmission-mode and epi-mode simultaneously when the delay between the pump and the probe pulses were set at

Channel X -2 ps 0 ps 1 ps 2 ps 4 ps 10 ps

Channel Y -2 ps 0 ps 1 ps 2 ps 4 ps 10 ps

Figure 4. The two-photon absorption images were acquired from the slide with a black hair, a red hair and a R6G sample. The upper row of images was acquired from the Channel X of the lock-in amplifier. The lower row was from the Channel Y. From the left to the right are the images collected at different interpulse delays between the pump and the probe, i.e. -2, 0, 1, 2, 4, and 10 ps. In the experiment, the pump wavelength was 810nm with 2.7mW average power deposit onto the slide; the probe was 740nm with 0.95mW. The objective was a 10X (NA, 0.25) from Olympus. The image size was 512µm x 512µm. The white squares are the ROIs within the areas of different samples.

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12 different positions from -4 to 12 ps. The total number of images collected in this single experiment was 48 as each of lock-in amplifiers had two output channels orthogonal in phase. The transmission and epi-mode images were very similar to each other. Figure 4 only shows the selected images collected in transmission mode at delays of -2, 0, 1, 2, 4 and 10 ps. The upper row is from the Channel X of the lock-in amplifier while the lower one is from the Channel Y. The initial phase of the lock-in amplifier was “auto-set” to “zero” when the Channel X displayed the maximum of signal in hair samples when varying the optical delay. This meant that the phase of the Channel X was set to the same phase with two-photon absorption signals in hair sample. Therefore the X channel should mainly reflect the two-photon absorption dynamics. From those images, several interesting observations can be made. The first is that Channel X image intensities of hair samples show a slow decay following a fast rise with the optical delays. For comparison, the R6G image is only glimpsed at the frame when the pump and the probe pulses are perfectly overlapped. These results are as expected and consistent with what we have seen before in cuvette melanin samples and R6G solution19. We also note that the Channel X hair images show completely opposite phase comparing with the R6G sample. Thus, since the R6G signal arises from instantaneous two-photon absorption signal, the signals from the hair samples likely arise from bleaching. Finally, In Channel Y the hair samples show almost constant signals at all delays, but the R6G sample shows no signal. To better quantify the images, we selected three regions of interest (ROIs, shown in the top left image in Figure 4) corresponding to the areas of three different samples on the images, and averaged the signals pixel by pixel within ROIs. After this analysis is done on all images collected at all delays in the transmission mode, the average pixel intensities of the three different samples at each channel can be plotted against the delays (Figure 5; note that for hair samples the same scale is used for both channels, but for R6G sample different scales are used for better visualization of the data points). Figure 5(c) confirms that R6G has very short-lived signals and no signal shown in the Channel Y. The two hair samples present approximately the same behavior on the excited state recovery as shown in Channel X although the black hair sample show a slightly faster rise than the red hair sample. Neither sample has completely returned to the ground state within 12 ps. However the striking difference is observed when comparing the signals from the Channel X with those from the Channel Y. The ratio between the maximum signal in Channel X and the signal in Channel Y is about 2:1 for the black hair, but is about 10:1 for the red hair. Additionally in the Channel X there are considerable amount of background sitting before the “zero” delay for both hair samples.

-2000

-1000

00

1000

2000

3000

4000-4 -2 0 2 4 6 8 10 12

Delay (psec)

Ave

rage

Pix

el ln

tens

ity (a

.u.)

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Channel Y

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00

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002468

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erag

e P

ixel

lnte

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(a.u

.)

Channel X

Channel Y

(a) Black hair

(b) Red hair (c) R6G

Figure 5. The average pixel intensities from both the Channel X (red) and the Channel Y (blue) of the lock-in amplifier are plotted versus the time delay between the pump and the probe pulses.

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IOU

60

U

Consider the simplest kinetic model for recovery of an excited state that has a single lifetime τ. The number density of excitations N evolving with time t can be described as,

(1)

where G(t) is the generation rate. For a sinusoidally modulated pump at frequency ω, the solution for N(t) in Eq. (1) has to have the form:

(2)

Using Eq. (2) the solution for Eq. (1) is,

(3)

For small signals, the relative transmission change due to the pump excitation ∆Τ/Τ is proportional to N(ω), so

(4) In Eq. (4), the real part is the in-phase term that has the same phase with the pump while the imaginary part is a quadrature or out-of-phase signal that has a 90 degree phase difference to that of the pump. So the phase (φ) of the pump-probe signal follows,

(5) In most ultrafast spectroscopy studies, people are interested in lifetimes that are shorter than 1 ns; for the lifetime of 1 ns, with a modulation frequency of 1 MHz, we can calculate that φ is 1.1x10-4 degree, which is too small to be noticed. However, if the lifetime of the signal is increased to 1 µs, still with 1 MHz modulation frequency, φ is 80 degree and will have a large effect in the phase sensitive detection like what the lock-in amplifier does. So the constant signal appearing in Channel Y in our experiments is expected from some very long-lived processes.

4 MHz 2MHz 1.5 MHz 1 MHz 0.5 MHz

Figure 7. The phase images were calculated by the formula, arctan(Channel Y/Channel X) while the images from both channel were acquired at the different modulation frequencies as the interpulse delay set to -2 ps. But absolute values of the calculated phases were used to retain its physical meaning. The unit of the phase is in degree.

τ)()()( tNtG

dttdN

−=

2

2

2 )(1)(

)(1)()(

ωτωτω

ωττωω

++

+=

GiGN

ωτφ =)tan(

tieNtN ωω −= )()(

2

2

2 )(1)(

)(1)()(

ωτωτω

ωττωω

++

+∝

∆ GiGTT

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0 1 2 3 4 5 6 7 8 9 10 11 12 13 140

1

2

3

4

5

6 Red Hair Black Hair

tan(

φ)ω (radians/second)

Figure 8. The tangent of the average phases versus the modulation frequencies. The linear fitting reveals that a long-lived component for the black hair sample and the red hair sample is about 0.293± 0.008 µs and 0.148±0.007 µs respectively.

To verify this, we set the delay to -2 ps where no fast decay component should be seen in the Channel X. Then we imaged the same sample slide at a series of different modulation frequencies, i.e. 0.5, 1, 1.5, 2 and 4 MHz. The phase images could be calculated according to the definition of the phase in lock-in amplifier, but we neglected the negative sign because the physical meaning of the phase in Eq. (6) allows only positive values; the negative sign of the lock-in signals just indicated that the signals were from the process causing “loss”. The calculated phase images are shown in Figure 7. The tangent of the average phases in the ROIs (black squares in Figure 7) for two hair samples are plotted versus the angular frequency of modulation in Figure 8 (the data at 4 MHz is not included because the phase is too close to 90 degree). Linear fitting to the data points should provide us the estimated lifetime for the long-lived process. According to the fitting results, the lifetime of the long-lived process is 0.293± 0.008 µs for the black hair and 0.148±0.007 µs for the red hair sample. These results confirm that a long-lived process does exist in the hair sample. One possible contribution is a thermal effect, which may cause the scattering properties of tissues to change shortly after absorption of the light. The lifetime may characterize the heat diffusion rate of the samples. Alternatively, the long-lived process may come from the formation of transient intermediate species when pigment molecules are excited.

4. CONCLUSIONS We have demonstrated that recent improvements on our two-photon absorption microscope allow us to image pigments in tissues in epi-mode, which is crucial for in vivo applications. We have also found a long lived process in hair melanins, the lifetime of which differs between the red hair and black samples. This result raises interesting questions, such as the origin of this process, how tissue properties affect it, and how it can be quantitatively linked to pigment contents. To answer those questions, further quantitative studies will need to be done in the future.

5. ACKNOWLEDGMENTS This work is supported by the Center for Molecular and Bimolecular Imaging at Duke University.

6. REFERENCES 1. S. Ito, and K. Wakamatsu, "Quantitative analysis of eumelanin and pheomelanin in humans, mice, and other animals: A comparative review," Pigment Cell Research 16, 523-531 (2003).

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2. M. Inazu, and Y. Mishima, "Detection of Eumelanogenic and Pheomelanogenic Melanosomes in the Same Normal Human Melanocyte," Journal of Investigative Dermatology 100, S172-S175 (1993). 3. E. Wenczl, G. P. Van der Schans, L. Roza, R. M. Kolb, A. J. Timmerman, N. P. M. Smit, S. Pavel, and A. A. Schothorst, "(Pheo)melanin photosensitizes UVA-induced DNA damage in cultured human melanocytes," Journal of Investigative Dermatology 111, 678-682 (1998). 4. M. R. Chedekel, S. K. Smith, P. W. Post, A. Pokora, and D. L. Vessell, "Photodestruction of Pheomelanin - Role of Oxygen," Proceedings of the National Academy of Sciences of the United States of America 75, 5395-5399 (1978). 5. T. Ye, L. Hong, J. Garguilo, A. Pawlak, G. S. Edwards, R. J. Nemanich, T. Sarna, and J. D. Simon, "Photoionization Thresholds of Melanins Obtained from Free Electron Laser–Photoelectron Emission Microscopy, Femtosecond Transient Absorption Spectroscopy and Electron Paramagnetic Resonance Measurements of Oxygen Photoconsumption," Photochemistry and Photobiology 82, 733–737 (2006). 6. M. R. Vincensi, M. d'Ischia, A. Napolitano, E. M. Procaccini, G. Riccio, G. Monfrecola, P. Santoianni, and G. Prota, "Phaeomelanin versus eumelanin as a chemical indicator of ultraviolet sensitivity in fair-skinned subjects at high risk for melanoma: a pilot study," Melanoma Research 8, 53-58 (1998). 7. T. G. Salopek, K. Yamada, S. Ito, and K. Jimbow, "Dysplastic Melanocytic Nevi Contain High-Levels of Pheomelanin - Quantitative Comparison of Pheomelanin Eumelanin Levels between Normal Skin, Common Nevi, and Dysplastic Nevi," Pigment Cell Research 4, 172-179 (1991). 8. K. Jimbow, S. K. Lee, M. G. King, H. Hara, H. Chen, J. Dakour, and H. Marusyk, "Melanin Pigments and Melanosomal Proteins as Differentiation Markers Unique to Normal and Neoplastic Melanocytes," Journal of Investigative Dermatology 100, S259-S268 (1993). 9. S. Ito, and K. Fujita, "Microanalysis of Eumelanin and Pheomelanin in Hair and Melanomas by Chemical Degradation and Liquid-Chromatography," Analytical Biochemistry 144, 527-536 (1985). 10. B. Kongshoj, A. Thorleifsson, and H. C. Wulf, "Pheomelanin and eumelanin in human skin determined by high-performance liquid chromatography and its relation to in vivo reflectance measurements," Photodermatology, Phtotoimmunology and Photomedicine 22, 141-147 (2006). 11. N. Nagai, Y. J. Lee, N. Nagaoka, M. Gunduz, K. Nakano, T. Nojima, H. Tsujigiwa, E. Gunduz, C. H. Siar, and H. Nagatsuka, "Elemental sulphur and alkali elutable melanin detected in oral melanosis and malignant melanoma by energy-filtering transmission electron microscopy," Journal of Oral Pathology & Medicine 31, 481-487 (2002). 12. N. Kollias, "The spectroscopy of human melanin pigmentation," Journal of Investigative Dermatology 102, 268 (1994). 13. H. Ozeki, S. Ito, K. Wakamatsu, and A. J. Thody, "Spectrophotometric characterization of eumelanin and pheomelanin in hair," Pigment Cell Research 9, 265-270 (1996). 14. G. N. Stamatas, B. Z. Zmudzka, N. Kollias, and J. Z. Beer, "Non-invasive measurements of skin pigmentation in situ," Pigment Cell Research 17, 618-626 (2004). 15. M. RAJADHYAKSHA, M. GROSSMAN, D. ESTEROWITZ, and R. H. Webb, "In-vivo confocal scanning laser microscopy of human skin - melanin provides strong contrast," Journal Of Investigative Dermatology 104, 946-952 (1995). 16. Z. Huang, H. Lui, X. K. Chen, A. Alajlan, D. I. McLean, and H. Zeng, "Raman spectroscopy of in vivo cutaneous melanin," J Biomed Opt 9, 1198-1205 (2004). 17. K. Teuchner, W. Freyer, D. Leupold, A. Volkmer, D. J. S. Birch, P. Altmeyer, M. Stucker, and K. Hoffmann, "Femtosecond two-photon excited fluorescence of melanin," Photochemistry and Photobiology 70, 146-151 (1999). 18. T. Ye, G. Yurtsever, M. Fischer, J. D. Simon, and W. S. Warren, "Imaging melanin by two-photon absorption microscopy," Proceedings of SPIE-The International Society for Optical Engineering 6089, 60891X/60891-60891X/60897 (2006). 19. D. Fu, T. Ye, T. E. Matthews, G. Yurtsever, and W. S. Warren, "Two-color, two-photon, and excited-state absorption microscopy," Journal of Biomedical Optics 12, (2007). 20. D. Fu, T. Ye, T. E. Matthews, G. Yurtsever, L. Hong, J. D. Simon, and W. S. Warren, "Two-color excited-state absorption imaging of melanins," Proceedings of SPIE-The International Society for Optical Engineering 6424, 642402/642401-642402/642407 (2007).

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21. P. S. Tsai, N. Nishimura, E. J. Yoder, E. M. Dolnick, G. A. White, and D. Kleinfeld, "Principles, Design, and Construction of a Two-Photon Laser Scanning Microscope for In Vitro and In Vivo Brain Imaging," in In vivo optical imaging of brain function, (2002), pp. 113-171. 22. J. L. Rees, "Genetics of hair and skin color," Annual Review of Genetics 37, 67-90 (2003). 23. A. J. Thody, E. M. Higgins, K. Wakamatsu, S. Ito, S. A. Burchill, and J. M. Marks, "Pheomelanin as Well as Eumelanin Is Present in Human Epidermis," Journal of Investigative Dermatology 97, 340-344 (1991).

* Correspondence: current affiliation is Department of Neurobiology, University of Alabama at Birmingham, Al

35294-2182; Email: tye@nrc.uab.edu; Telephone: (205)934-5626; Fax: (205)975-7394

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