Mary Jane Simpson

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.

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.

Spatial Localization of Excitons and Charge Carriers in Hybrid Perovskite Thin Films.

The fundamental photophysics underlying the remarkably high-power conversion efficiency of organic-inorganic hybrid perovskite-based solar cells has been increasingly studied using complementary spectroscopic techniques. However, the spatially heterogeneous polycrystalline morphology of the photoactive layers owing to the presence of distinct crystalline grains has been generally neglected in optical measurements; therefore, the reported results are typically averaged over hundreds or even thousands of such grains. Here we apply femtosecond transient absorption microscopy to spatially and temporally probe ultrafast electronic excited-state dynamics in pristine methylammonium lead tri-iodide (CH3NH3PbI3) thin films and composite structures. We found that the electronic excited-state relaxation kinetics are extremely sensitive to the sample location probed, which was manifested by position-dependent decay time scales and transient signals. Analysis of transient absorption kinetics acquired at distinct spatial positions enabled us to identify contributions of excitons and free charge carriers.

Near-infrared excited state dynamics of melanins: the effects of iron content, photo-damage, chemical oxidation, and aggregate size.

Ultrafast pump–probe measurements can discriminate the two forms of melanin found in biological tissue (eumelanin and pheomelanin), which may be useful for diagnosing and grading melanoma. However, recent work has shown that bound iron content changes eumelanin’s pump–probe response, making it more similar to that of pheomelanin. Here we record the pump–probe response of these melanins at a wider range of wavelengths than previous work and show that with shorter pump wavelengths the response crosses over from being dominated by ground-state bleaching to being dominated by excited-state absorption. The crossover wavelength is different for each type of melanin. In our analysis, we found that the mechanism by which iron modifies eumelanin’s pump–probe response cannot be attributed to Raman resonances or differences in melanin aggregation and is more likely caused by iron acting to broaden the unit spectra of individual chromophores in the heterogeneous melanin aggregate. We analyze the dependence on optical intensity, finding that iron-loaded eumelanin undergoes irreversible changes to the pump–probe response after intense laser exposure. Simultaneously acquired fluorescence data suggest that the previously reported “activation” of eumelanin fluorescence may be caused in part by the dissociation of metal ions or the selective degradation of iron-containing melanin.

Comparing in vivo pump–probe and multiphoton fluorescence microscopy of melanoma and pigmented lesions

Abstract. We demonstrate a multimodal approach that combines a pump–probe with confocal reflectance and multiphoton autofluorescence microscopy. Pump–probe microscopy has been proven to be of great value in analyzing thin tissue sections of pigmented lesions, as it produces molecular contrast which is inaccessible by other means. However, the higher optical intensity required to overcome scattering in thick tissue leads to higher-order nonlinearities in the optical response of melanin (e.g., two-photon pump and one-photon probe) that present additional challenges for interpreting the data. We show that analysis of pigment composition in vivo must carefully account for signal terms that are nonlinear with respect to the pump and probe intensities. We find that pump–probe imaging gives useful contrast for pigmented structures over a large range of spatial scales (100  μm to 1 cm), making it a potentially useful tool for tracking the progression of pigmented lesions without the need to introduce exogenous contrast agents.

Nonlinear Microscopy of Eumelanin and Pheomelanin with Subcellular Resolution

Pump-probe microscopy non-destructively differentiates eumelanin and pheomelanin and can be used to quantify melanin distributions in thin biopsy slices. Here we have extended that work for imaging eumelanin and pheomelanin distributions on a sub-cellular scale, allowing elucidation of characteristics of different cell types. The results show that melanin heterogeneity, previously found to be characteristic of melanomas, persists on the sub-cellular scale. We have also found spectral changes associated with melanin located in melanophages, which could potentially differentiate invasive pigmented melanocytes from melanophages without immunohistochemical staining.

Special Section on Laser Applications in Life Sciences: Comparing in vivo pump–probe and multiphoton fluorescence microscopy of melanoma and pigmented lesions

Abstract. We demonstrate a multimodal approach that combines a pump–probe with confocal reflectance and multiphoton autofluorescence microscopy. Pump–probe microscopy has been proven to be of great value in analyzing thin tissue sections of pigmented lesions, as it produces molecular contrast which is inaccessible by other means. However, the higher optical intensity required to overcome scattering in thick tissue leads to higher-order nonlinearities in the optical response of melanin (e.g., two-photon pump and one-photon probe) that present additional challenges for interpreting the data. We show that analysis of pigment composition in vivo must carefully account for signal terms that are nonlinear with respect to the pump and probe intensities. We find that pump–probe imaging gives useful contrast for pigmented structures over a large range of spatial scales (100  μm to 1 cm), making it a potentially useful tool for tracking the progression of pigmented lesions without the need to introduce exogenous contrast agents.

Imaging Electronic Trap States in Perovskite Thin Films with Combined Fluorescence and Femtosecond Transient Absorption Microscopy.

Charge carrier trapping degrades the performance of organometallic halide perovskite solar cells. To characterize the locations of electronic trap states in a heterogeneous photoactive layer, a spatially resolved approach is essential. Here, we report a comparative study on methylammonium lead tri-iodide perovskite thin films subject to different thermal annealing times using a combined photoluminescence (PL) and femtosecond transient absorption microscopy (TAM) approach to spatially map trap states. This approach coregisters the initially populated electronic excited states with the regions that recombine radiatively. Although the TAM images are relatively homogeneous for both samples, the corresponding PL images are highly structured. The remarkable variation in the PL intensities as compared to transient absorption signal amplitude suggests spatially dependent PL quantum efficiency, indicative of trapping events. Detailed analysis enables identification of two trapping regimes: a densely packed trapping region and a sparse trapping area that appear as unique spatial features in scaled PL maps.

Separating Bulk and Surface Contributions to Electronic Excited-State Processes in Hybrid Mixed Perovskite Thin Films via Multimodal All-Optical Imaging

A comprehensive understanding of electronic excited-state phenomena underlying the impressive performance of solution-processed hybrid halide perovskite solar cells requires access to both spatially resolved electronic processes and corresponding sample morphological characteristics. Here, we demonstrate an all-optical multimodal imaging approach that enables us to obtain both electronic excited-state and morphological information on a single optical microscope platform with simultaneous high temporal and spatial resolution. Specifically, images were acquired for the same region of interest in thin films of chloride containing mixed lead halide perovskites (CH3NH3PbI3-xClx) using femtosecond transient absorption, time-integrated photoluminescence, confocal reflectance, and transmission microscopies. Comprehensive image analysis revealed the presence of surface- and bulk-dominated contributions to the various images, which describe either spatially dependent electronic excited-state properties or morphological variations across the probed region of the thin films. These results show that PL probes effectively the species near or at the film surface.

Correction to "Pump-Probe Microscopic Imaging of Jurassic-Aged Eumelanin".

We correct an error to our previous article. In Figure 5 and the text description of that data; iron concentrations were incorrectly labeled as μM, but they should have been mM. No conclusions of the paper are changed. The corrected text and figure are: Figure 5 Average spectra of iron loaded S. officinalis eumelanin obtained by varying the initial concentration of iron(III) chloride. “Figure 5 shows the pump–probe response of EDTA washed S. officinalis eumelanin loaded with different initial concentrations of iron(III) chloride. The eumelanin is saturated with iron when the initial concentration of iron is greater than 1 mM.9 Increasing the initial iron concentration causes the negative signal when the pulses are overlapped (at t = 0) to appear and the positive signal when the pump precedes the probe (t > 0) to disappear; the 0.25 mM is similar to the S. officinalis eumelanin spectrum given in Figure 1. This value is approximately in agreement with the reported concentrations found in natural S. officinalis melanin.9”