Melanoma is responsible for around 10,000 deaths annually in the US. While simple excision can cure most melanomas, some progress into metastatic cancer, necessitating advanced treatment options. However, the current clinical diagnosis methods are insufficient in accurately identifying metastasis. There is a need for better biomarkers for patients at risk of developing metastases to enable timely intervention and appropriate treatment. We present a biomarker based on femtosecond pump-probe microscopy and supervised learning techniques to diagnose metastatic melanoma. Pump-probe microscopy images of primary melanomas reveal the chemical and physical structure of melanin, a naturally occurring pigment in most melanoma tumors. Leveraging supervised learning models, we classify melanin features and utilize them to guide the diagnosis of metastatic disease. Our proposed biomarker is compatible with the current clinical protocol, as it only requires a slice of the primary tumor, which is routinely excised following clinical guidelines. In our preliminary dataset of approximately 50 patients, the biomarker demonstrates encouraging sensitivity and specificity, exceeding 80%.
We discuss applications of pump-probe microscopy to the investigation of pigments in artworks. In pump-probe, a series of ultrafast laser pulses probe molecular state dynamics, which is governed by several physical mechanisms that evolve on timescales ranging from sub-picoseconds to many microseconds. The interplay of these mechanisms can lead to complex dynamics and is sensitive to a pigment’s structure, composition, and environment. We describe strategies to interpret pump-probe dynamics and to disentangle the underlying contributions from different physical mechanisms. We also report on applications to the studies of Cadmium pigments, carbon-based black pigments, and their degradation mechanism.
More patients diagnosed with stage I (local) melanoma die than any other metastatic stage, because there exists no biomarker to reliably diagnose metastatic melanoma, preventing many patients to receive appropriate treatment. We pursue an approach based on femtosecond pump-probe microscopy of melanin; a natural pigment found in most melanoma. The measured pump-probe signals of melanin are complex superpositions of multiple nonlinear processes, making interpretation challenging. We demonstrate how polarization control and data fitting are used to decompose melanin signals into their underlying nonlinear interactions. False colored images of a small set of melanoma tumors, based on specific nonlinear interactions, correlate with clinical concern. This approach of decomposing pump-probe signals is applicable to a multitude of different samples.
More people die from melanoma after a stage I diagnosis than after a stage IV diagnosis, because the tools available to clinicians do not readily identify which early-stage cancers will be aggressive. Near-infrared pump-probe microscopy detects fundamental differences in melanin structure between benign human moles and melanoma and also correlates with metastatic potential. However, the biological mechanisms of these changes have been difficult to quantify, as many different mechanisms can contribute to the pump-probe signal. We use model systems (sepia, squid, and synthetic eumelanin), cellular uptake studies, and a range of pump and probe wavelengths to demonstrate that the clinically observed effects come from alterations of the aggregated mode from “thick oligomer stacks” to “thin oligomer stacks” (due to changes in monomer composition) and (predominantly) deaggregation of the assembled melanin structure. This provides the opportunity to use pump-probe microscopy for the detection and study of melanin-associated diseases.
Optical coherence tomography (OCT) enables non-invasive, high-resolution, tomographic imaging of biological tissues by leveraging principles of low coherence interferometry; however, OCT lacks molecular specificity. Spectroscopic OCT (SOCT) overcomes this limitation by providing depth-resolved spectroscopic signatures of chromophores, but SOCT has been limited to a couple of endogenous molecules, namely hemoglobin and melanin. Stimulated Raman scattering, on the other hand, can provide highly specific molecular information of many endogenous species, but lacks the spatial and spectral multiplexing capabilities of SOCT. In this work we integrate the two methods, SRS and SOCT, to enable simultaneously multiplexed spatial and spectral imaging with sensitivity to many endogenous biochemical species that play an important role in biology and medicine. The method, termed SRS-SOCT, has the potential to achieve fast, volumetric, and highly sensitive label-free molecular imaging, which would be valuable for many applications. We demonstrate the approach by imaging excised human adipose tissue and detecting the lipids’ Raman signatures in the high-wavenumber region. Details of this method along with validations and results will be presented.
Stimulated Raman scattering (SRS) enables fast, high resolution imaging of chemical constituents important to biological structures and functional processes. While this technology has shown remarkable potential, it is currently limited to point scanning and can only probe a few Raman bands at a time. In this work we take a fundamentally different approach to detecting the small nonlinear signals based on dispersion effects that accompany the loss/gain processes in SRS. We use a modified pump-probe system (pulses with duration of ~0.5 ps and 75 fs, respectively) with interferometric detection in the Fourier-domain to demonstrate that the dispersive measurements are more robust to noise (e.g., laser noise) compared to conventional amplitude measurements, which in turn permits facile spectral and spatial multiplexing.
Results show that it is possible to assess a broadband dispersion spectrum (currently limited to ~400 cm-1) with a single laser shot or spectrometer acquisition (20-50 µs). For molecular imaging with broadband spectral information, we achieve spatial pixel rates of 2.5 kHz, and will discuss how this can be further improved to 20-50 kHz. We also combine SRS with optical coherence tomography (OCT) (molecular and structural information are rendered from the same data), which enables axial multiplexing by coherence gating and paves the way for volumetric biochemical imaging. The approach is tested on a thin water-and-oil phantom, a thick scattering polystyrene bead phantom, and thick freshly excised human adipose tissue. Finally, we will outline other opportunities for spatial multiplexing using wide-field holography and spectroscopic-OCT, which would massively parallelize the spatial and spectral information. The combination of dispersion-based SRS and phase imaging has the potential to enable faster wide-area and volumetric molecular imaging. Such methods would be valuable in a clinical setting for many applications.
KEYWORDS: Digital signal processing, Multiphoton microscopy, Microscopy, Field programmable gate arrays, Nonlinear optics, Tissue optics, Signal processing, Time resolved spectroscopy, Linear filtering, Luminescence, Filtering (signal processing)
The use of multiphoton interactions in biological tissue for imaging contrast requires highly sensitive optical measurements. These often involve signal processing and filtering steps between the photodetector and the data acquisition device, such as photon counting and lock-in amplification. These steps can be implemented as real-time digital signal processing (DSP) elements on field-programmable gate array (FPGA) devices, an approach that affords much greater flexibility than commercial photon counting or lock-in devices. We will present progress toward developing two new FPGA-based DSP devices for multiphoton and time-resolved microscopy applications. The first is a high-speed multiharmonic lock-in amplifier for transient absorption microscopy, which is being developed for real-time analysis of the intensity-dependence of melanin, with applications in vivo and ex vivo (noninvasive histopathology of melanoma and pigmented lesions). The second device is a kHz lock-in amplifier running on a low cost ($50--$200) development platform. It is our hope that these FPGA-based DSP devices will enable new, high-speed, low-cost applications in multiphoton and time-resolved microscopy.
KEYWORDS: Data modeling, Absorption, Signal detection, Rhodamine, Modulation, Microscopy, Field programmable gate arrays, In vivo imaging, Digital signal processing, Tissue optics
The transient absorption response of melanin is a promising optically-accessible biomarker for distinguishing malignant melanoma from benign pigmented lesions, as demonstrated by earlier experiments on thin sections from biopsied tissue. The technique has also been demonstrated in vivo, but the higher optical intensity required for detecting these signals from backscattered light introduces higher-order nonlinearities in the transient response of melanin. These components that are higher than linear with respect to the pump or the probe introduce intensity-dependent changes to the overall response that complicate data analysis. However, our data also suggest these nonlinearities might be advantageous to in vivo imaging, in that different types of melanins have different nonlinear responses. Therefore, methods to separate linear from nonlinear components in transient absorption measurements might provide additional information to aid in the diagnosis of melanoma.
We will discuss numerical methods for analyzing the various nonlinear contributions to pump-probe signals, with the ultimate objective of real time analysis using digital signal processing techniques. To that end, we have replaced the lock-in amplifier in our pump-probe microscope with a high-speed data acquisition board, and reprogrammed the coprocessor field-programmable gate array (FPGA) to perform lock-in detection. The FPGA lock-in offers better performance than the commercial instrument, in terms of both signal to noise ratio and speed. In addition, the flexibility of the digital signal processing approach enables demodulation of more complicated waveforms, such as spread-spectrum sequences, which has the potential to accelerate microscopy methods that rely on slow relaxation phenomena, such as photo-thermal and phosphorescence lifetime imaging.
We demonstrate that ultrafast pump-probe microscopy provides unique dynamics for natural iron oxide and iron hydroxide earth pigments, despite their chemical similarity. First, we conducted a pump-probe spectroscopy study on heat-treated hematite (the pure red iron oxide mineral) and found the pump-probe dynamics to be temperature dependent. Second, we investigated pottery fired under known conditions and observed firing dependent pump-probe dynamics. Finally, we imaged a New World potshard from the North Carolina Museum of Art. Our results indicate that pump-probe microscopy could be a useful tool in elucidating pottery manufacture.
New labeling, imaging, or analysis tools could provide new retrospective insights when applied to archived, paraffin-embedded
samples. Deep-tissue multiphoton microscopy of paraffin-embedded specimens is achieved using optical
clearing with mineral oil. We tested a variety of murine tissue specimens including skin, lung, spleen, kidney, and heart,
acquiring multiphoton autofluorescence and second-harmonic generation, and pump-probe images This technique
introduces the capability for non-destructive 3-dimensional microscopic imaging of existing archived pathology
specimens, enabling retrospective studies.
Phasor analysis has become a powerful tool for examining signals in fluorescence life-time microscopy (FLIM), where
the analysis provides a fast, robust and intuitive means of separating different fluorescent species and mixtures thereof.
In this work we adapt this analysis for pump-probe microscopy, a method that provides molecular contrast of pigmented
samples by probing their excited state dynamic properties. The bipolar nature of the pump-probe signals presents
important differences in the resulting phasors compared to FLIM—here, we discuss these differences and describe the
behavior of bipolar signals in phasor analysis. Results show that this method is indeed able to separate multiple
molecular species of interests and allows facile assessment of pigment chemistry and its distribution in samples.
Nonlinear imaging takes advantage of the localized nature of the interaction to achieve high spatial resolution, optical
sectioning, and deeper penetration in tissue. However, nonlinear contrast (other than fluorescence or harmonic
generation) is generally difficult to measure because it is overwhelmed by the large background of detected illumination
light. Especially challenging to measure is the nonlinear refractive index - accessing this quantity would allow the
extension of widely employed phase microscopy methods to the nonlinear regime. We have developed a technique to
suppress the background in these types of measurements by using femtosecond pulse shaping to encode nonlinear
interactions in background-free regions of the frequency spectrum. Using this individual pulse shaping based technique
we have been able to measure self-phase modulation (SPM) in highly scattering environments, such as biological tissue,
with very modest power levels. Using our measurement technique we have demonstrated strong intrinsic SPM signatures
of glutamate-induced neuronal activity in hippocampal brain slices. We have also extended this measurement method to
cross-phase modulation, the two-color analogue to SPM. The two-color approach dramatically improves the
measurement sensitivity by reducing undesired background and associated noise. We will describe the nonlinear phase
contrast measurement technique and report on its application for imaging neuronal activity.
Nonlinear microscopies (most commonly, two-photon fluorescence, second harmonic generation, and coherent
anti-Stokes Raman scattering (CARS)) have had notable successes in imaging a variety of endogenous and exogenous targets
in recent years. These methods generate light at a color different from any of the exciting laser pulses, which makes the
signal relatively easy to detect. Our work has focused on developing microscopy techniques using a wider range of
nonlinear signatures (two-photon absorption of nonfluorescent species, self phase modulation) which have some specific
advantages - for example, in recent papers we have shown that we can differentiate between different types of melanin
in pigmented lesions, image hemoglobin and its oxygenation, and measure neuronal activity. In general, these signatures
do not generate light at a different color and we rely on the advantages of femtosecond laser pulse shaping methods to
amplify the signals and make them visible (for example, using heterodyne detection of the induced signal with one of the
co-propagating laser pulses). Here we extend this work to stimulated Raman and CARS geometries. In the simplest
experiments, both colors arise from filtering a single fs laser pulse, then modulating afterwards; in other cases, we
demonstrate that spectral reshaping can retain high frequency resolution in Raman and CARS geometries with
femtosecond laser pulses.
Even though multi-photon fluorescence microscopy offers higher resolution and better penetration depth than traditional
fluorescence microscopy, its use is restricted to the detection of molecules that fluoresce. Two-photon absorption (TPA)
imaging can provide contrast in non-fluorescent molecules while retaining the high resolution and sectioning capabilities
of nonlinear imaging modalities. In the long-wavelength water window, tissue TPA is dominated by the endogenous
molecules melanin and hemoglobin with an almost complete absence of endogenous two-photon fluorescence. A
complementary nonlinear contrast mechanism is self-phase modulation (SPM), which can provide intrinsic signatures
that can depend on local tissue anisotropy, chemical environment, or other structural properties. We have developed a
spectral hole refilling measurement technique for TPA and SPM measurements using shaped ultrafast laser pulses. Here
we report on a microscopy setup to simultaneously acquire 3D, high-resolution TPA and SPM images. We have
acquired data in mounted B16 melanoma cells with very modest laser power levels. We will also discuss the possible
application of this measurement technique to neuronal imaging. Since SPM is sensitive to material structure we can
expect SPM properties of neurons to change during neuronal firing. Using our hole-refilling technique we have now
demonstrated strong novel intrinsic nonlinear signatures of neuronal activation in a hippocampal brain slice. The
observed changes in nonlinear signal upon collective activation were up to factors of two, unlike other intrinsic optical
signal changes on the percent level. These results show that TPA and SPM imaging can provide important novel
functional contrast in tissue using very modest power levels suitable for in vivo applications.
Multiphoton excitation fluorescence microscopy has proven to be a powerful method for non-invasive, in vivo, thick tissue imaging with molecular specificity. However, many important endogenous biomolecules do not fluoresce (NAD) or fluoresce with low efficiency (Melanin). In this report femtosecond pulse shaping methods are used to measure two-photon absorption (TPA) directly with very high sensitivity. Combining with the laser scanning microscope, this Two-photon Absorption Microscopy (TPAM) retains the penetration and localization advantages of two-photon fluorescence microscopy and permits direct observation of important endogenous molecular markers (melanin or hemoglobin) which are invisible in multiphoton fluorescence microscopy. We have demonstrated here for the first time that TPAM can successfully and more efficiently image melanoma cells and tissues and provide a good melanin contrast in optical sectioning of the melanoma lesions which are comparable to pathological histology. Combining with the two-photon fluorescence images acquired simultaneously, the distribution patterns of the melanocytes and their intratissue behavior could be studied without cutting the lesions from patients. TPAM will undoubtedly find the applications in the clinical diagnosis and biomedical research.
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