Thomas E. Matthews

Pump-Probe Imaging Differentiates Melanoma from Melanocytic Nevi

Multiphoton imaging reveals chemical changes in melanoma compared to benign nevi and could enhance current clinical diagnostic protocols. A Wolf in Sheep’s Clothing It is frequently difficult to distinguish whether something is dangerous or harmless. In the case of melanoma diagnosis, a misdiagnosed lesion could have deadly consequences. Rightly, doctors err on the side of caution; however, false-positive diagnoses result in unnecessary surgeries and biopsies, as well as emotional distress for the patient. Matthews et al. have developed a new imaging technique that can distinguish melanoma from benign lesions, which in concert with current techniques could improve patient diagnosis and decrease the need for unnecessary tests. The pigment melanin is the primary determinant of skin color. There are two dominant types of melanin in melanocytic lesions: eumelanin and pheomelanin. Eumelanin, which is brown/black, is the most common biological form of melanin, whereas pheomelanin is largely responsible for red hair and freckles. The authors use a multiphoton imaging technique, pump-probe spectroscopy, to determine the ratio of these different melanins in melanocytic lesions in the context of lesion architectural and cytological features. Eumelanin was found at higher levels in melanoma compared with both dysplastic and benign nevi. When combined with pathological examination, imaging-based determination of the melanin ratio decreased the number of false-positive diagnoses compared with pathological examination alone. Moreover, this imaging technique could be used on hematoxylin and eosin–stained slides, which are currently used by pathologists for melanoma diagnosis, and may even be able to be adapted for noninvasive diagnostics. In conjunction with traditional diagnostic methods, melanocytic imaging should greatly improve doctor’s ability to sort the wolves from the sheep. Melanoma diagnosis is clinically challenging: the accuracy of visual inspection by dermatologists is highly variable and heavily weighted toward false positives. Even the current gold standard of biopsy results in varying diagnoses among pathologists. We have developed a multiphoton technique (based on pump-probe spectroscopy) that directly determines the microscopic distribution of eumelanin and pheomelanin in pigmented lesions of human skin. Our initial results showed a marked difference in the chemical variety of melanin between nonmalignant nevi and melanoma, as well as a number of substantial architectural differences. We examined slices from 42 pigmented lesions and found that melanomas had an increased eumelanin content compared to nonmalignant nevi. When used as a diagnostic criterion, the ratio of eumelanin to pheomelanin captured all investigated melanomas but excluded three-quarters of dysplastic nevi and all benign dermal nevi. Additional evaluation of architectural and cytological features revealed by multiphoton imaging, including the maturation of melanocytes, presence of pigmented melanocytes in the dermis, number and location of melanocytic nests, and confluency of pigmented cells in the epidermis, further increased specificity, allowing rejection of more than half of the remaining false-positive results. We then adapted this multiphoton imaging technique to hematoxylin and eosin (H&E)–stained slides. By adding melanin chemical contrast to H&E-stained slides, pathologists will gain complementary information to increase the ease and accuracy of melanoma diagnosis.

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.

High-resolution in vivo imaging of blood vessels without labeling

We demonstrate that both oxyhemoglobin and deoxyhemoglobin have sequential two-color, two-photon absorption properties that can serve as endogenous contrasts in microvasculature imaging. Using a sensitive modulation transfer technique, we are able to image hemoglobin in red blood cells with micrometer resolution, both in vitro and in vivo. We show that excellent contrast from hemoglobin without any labeling can be obtained in tissue.

In vivo and ex vivo epi-mode pump-probe imaging of melanin and microvasculature

We performed epi-mode pump-probe imaging of melanin in excised human pigmented lesions and both hemoglobin and melanin in live xenograft mouse melanoma models to depths greater than 100 µm. Eumelanin and pheomelanin images, which have been previously demonstrated to differentiate melanoma from benign lesions, were acquired at the dermal-epidermal junction with cellular resolution and modest optical powers (down to 15 mW). We imaged dermal microvasculature with the same wavelengths, allowing simultaneous acquisition of melanin, hemoglobin and multiphoton autofluorescence images. Molecular pump-probe imaging of melanocytes, skin structure and microvessels allows comprehensive, non-invasive characterization of pigmented lesions.

Probing skin pigmentation changes with transient absorption imaging of eumelanin and pheomelanin

As some of the most ubiquitous and biologically important natural pigments, melanins play essential roles in the photoprotection of skin. Changes in melanin production could potentially be useful for clinical diagnosis of the progression stage of melanoma. Previously we demonstrated a new method for imaging melanin distribution in tissue with two-color transient absorption microscopy. Here we extend this study to longer wavelengths and show that we are able to image melanin in fixed thin skin slices with higher signal-to-noise ratios (SNRs) and demonstrate epimode imaging. We show that both photothermal effects and long-lived excited states can contribute to the long-lived signal. Eumelanin and pheomelanin exhibit markedly different long-lived excited state absorption. This difference should enable us to map out their respective distribution in tissue samples with subcellular resolution. This technique could provide valuable information in diagnosing the malignant transformation of melanocytes.

Label-free in vivo optical imaging of microvasculature and oxygenation level

The ability to perform high-resolution imaging of microvasculature and its oxygenation is very important in studying early tumor development. Toward this goal, we improved upon our excited state absorption (ESA)-based imaging technique to allow us to not only image hemoglobin directly but also differentiate between oxy- and deoxyhemoglobin in tissue. We demonstrate the separation of arterioles from venules in a live nude mouse ear using our imaging technique.

Probing near-infrared photorelaxation pathways in eumelanins and pheomelanins.

Ultraviolet-visible spectroscopy readily discerns the two types of melanin pigments (eumelanin and pheomelanin), although fundamental details regarding the optical properties and pigment heterogeneity are more difficult to disentangle via analysis of the broad featureless absorption spectrum alone. We employed nonlinear transient absorption spectroscopy to study different melanin pigments at near-infrared wavelengths. Excited-state absorption, ground-state depletion, and stimulated emission signal contributions were distinguished for natural and synthetic eumelanins and pheomelanins. A starker contrast among the pigments is observed in the nonlinear excitation regime because they all exhibit distinct transient absorptive amplitudes, phase shifts, and nonexponential population dynamics spanning the femtosecond-nanosecond range. In this manner, different pigments within the pheomelanin subclass were distinguished in synthetic and human hair samples. These results highlight the potential of nonlinear spectroscopies to deliver an in situ analysis of natural melanins in tissue that are otherwise difficult to extract and purify.

Optical Spectroscopy of Biological Cells

Optical spectroscopy has seen expanding use for the study of biological cells in recent years. An overview of relevant spectroscopic techniques is presented, and applications to biological cells are reviewed.

Two-color excited-state absorption imaging of melanins

We have demonstrated a new method for imaging melanin with two-color 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 place in the medium. We can easily measure 10-6 absorption changes caused by either instantaneous two-photon absorption or relatively long lived excited state absorption with a RF lock-in amplifier. Eumelanin and pheomelanin exhibit similar excited state dynamics. However, their difference in excited state absorption and ground state absorption leads to change in the phase of the transient absorption signal. Scanning microscopic imaging is performed with B16 cells, melanoma tissue to demonstrate the 3D high resolution imaging capability. Different melanosome samples are also imaged to illustrate the differences between eumelanin and pheomelanin signals. These differences could enable us to image their respective distribution in tissue samples and provide us with valuable information in diagnosing malignant transformation of melanocytes.

Estimation of molar absorptivities and pigment sizes for eumelanin and pheomelanin using femtosecond transient absorption spectroscopy

Fundamental optical and structural properties of melanins are not well understood due to their poor solubility characteristics and the chemical disorder present during biomolecular synthesis. We apply nonlinear transient absorption spectroscopy to quantify molar absorptivities for eumelanin and pheomelanin and thereby get an estimate for their average pigment sizes. We determine that pheomelanin exhibits a larger molar absorptivity at near IR wavelengths (750 nm), which may be extended to shorter wavelengths. Using the molar absorptivities, we estimate that melanin pigments contain approximately 46 and 28 monomer units for eumelanin and pheomelanin, respectively. This is considerably larger than the oligomeric species that have been recently proposed to account for the absorption spectrum of eumelanin and illustrates that larger pigments comprise a significant fraction of the pigment distribution.