Using femtosecond pump-probe microscopy to improve detection of early-stage metastatic melanoma
Optics, Quantum Optics and Physics of Atoms, Molecules and Plasmas
Final Report Abstract
We developed a systematic protocol that includes measurement procedures as well as data evaluation procedures for pump-probe microscopy that allows to rigorously decompose pump-probe images into their underlying physical interactions. Pump-probe images are a complex superposition of multiple nonlinear interactions between laser pulses and specific electronic or vibrational transitions in the sample. Each individual interaction between laser pulses and a transition appears as a single exponential decay. A measurement is typically a superposition multiple exponentials and cannot be uniquely decomposed by means of data analysis only. This makes our combination of experimental and data analysis decomposition valuable. This protocol is not only applicable to pump-probe microscopy of melanoma or melanin, but to a wide variety of samples. Pump-probe is a widely applied technique to many disciplines such as life science, material science and even cultural heritage and this protocol can be easily adapted to many different samples. We applied this protocol to melanin and identified all physical interactions that comprise a pump-probe image. We can now correlate each individual interaction between laser pulses and melanin with metastatic disease and rigorously quantify if a specific interaction is a good biomarker for metastatic melanoma. So far, we found that amplitude and lifetime of excited state absorption (a fundamental interaction between laser pulses and melanin) are a promising candidate, based on 27 tumor samples, to discriminate between patients with non-metastatic melanoma and patients that developed metastatic melanoma. The developed techniques will be applied to a larger sample set (187 patients) and used for model development (logistic regression & binary classifier). The result will be a diagnostic biomarker for metastatic melanoma, based on pump-probe microscopy of primary tumors. We developed two new pump-probe microscopes: A pump-probe microscope with two alternative modulation schemes: time delay modulation and polarization modulation. Polarization modulation allows to selectively image physical interactions. This imaging modality can be used to directly image the most relevant interaction, like excited state absorption in melanoma, and will simplify application of pump-probe microscopy outside of research labs. The second pump-probe microscope we developed is based on a single turn-key laser and a commercially available multiphoton laser scanning microscope. We significantly reduced its footprint, long-term stability and alignment complexity such that non-optics experts can use it. This microscope is available for users in a microscope user-facticity in Duke. Due to COVID we could not investigate as many melanoma biopsies as we would have liked and had to focus on microscope development and data evaluation methods instead. As a result, we developed easy to use pump-probe microscopes that can directly image the most relevant contrast. They enable us to rigorously quantify and analyze pump-probe images of melanoma tumors. Once we are finished with pathological evaluation and imaging of the full biorepository, we can develop the model for diagnosis of metastatic melanoma and test the performance characteristic (sensitivity and specificity) of this biomarker.
Publications
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Beyond intensity modulation: new approaches to pump-probe microscopy. Optics Letters, 46(6), 1474.
Jiang, Jun; Grass, David; Zhou, Yue; Warren, Warren S. & Fischer, Martin C.
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Contrast mechanisms in pump-probe microscopy of melanin. Optics Express, 30(18), 31852.
Grass, David; Beasley, Georgia M.; Fischer, Martin C.; Selim, M. Angelica; Zhou, Yue & Warren, Warren S.
