Pump-Probe Imaging Differentiates Melanoma from Melanocytic Nevi

Abstract

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. Evaluating 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.

Pump-probe nonlinear phase dispersion spectroscopy

“…The experimental results and the theoretical simulations at τ = 0 for Φ' (solid red curves in Figs. [3][4] are in excellent agreement, where both show the same concavity (up or down) and slight asymmetry in the parabolic shape for each sample. These results can be understood by using the approximation provided in section 3 (and appendix): Specifically, the difference in concavity between the two samples results from the fact that the OKE response of CS 2 peaks at a later time compared to water, which causes the second derivatives to have opposite signs at τ = 0.…”

“…Specifically, the output of a mode-locked Ti:Sapphire laser (Tsunami, Spectra Physics, 80 MHz), with a centered wavelength of 808 nm, is split into two beams: The first beam pumps an optical parametric oscillator (Mira OPO, Coherent) that is tuned to 720 nm and serves as the pump beam. These wavelengths are chosen since they are routinely used for biological imaging with pump-probe microscopy [1][2][3][4]. The second beam from the Ti:Sapph laser is further split into two using a Michelson interferometer to provide a probe field and a reference field, separated by a constant time T = 1.25 mm/c = 4.16 ps, where c is the speed of light in vacuum.…”

“…For example, pump-probe microscopy measures the transient, excited state dynamic properties of pigmented samples resulting from interactions such as excited state absorption, ground state depletion, and stimulated emission; consequently the acquired signals exhibit unique structures that enable differentiation of many molecular species. This method has had a significant impact on both biomedical applications [1,2], particularly quantitative melanoma detection [3,4], and art conservation [5]. On the other hand, various techniques have been developed to provide contrast from nonpigmented samples by using nonlinear phase changes (e.g., self/cross phase modulation), which provide greater detail of a sample's morphology [6][7][8][9], but for the most part, lack molecular specificity.…”

“…Interferometric methods, on the other hand, are well suited for both of these tasks. Nonlinear interferometric vibrational imaging (NIVI), for example, resolves the complex, wavelengthdependent field of an anti-Stokes wave in order to remove the non-resonant background in coherent anti-Stokes Raman scattering (CARS), and to allow linear estimation of the third order optical susceptibility, χ (3) (Ω), where Ω is the vibrational frequency [11,12]. Wilson et al also proposed an interferometric method that utilizes a synthetic temporal aperture to ascertain information regarding impulsive stimulated Raman scattering by looking at the temporal phase [13] or the spectral phase of chirped pulses [14].…”

Label-Free Imaging of Melanoma with Confocal Photothermal Microscopy: Differentiation between Malignant and Benign Tissue

“…Melanin carries information about the metabolism and location of melanocytes and melanogenesis; therefore, the distribution of melanin could act as a marker for MM [13,14]). Two dominant types of melanin (eumelanin and pheomelanin) have a large absorption cross section of visible light without efficient fluorescence emission [15]), resulting in difficulty of imaging of the distribution of MM.…”