Pump-probe microscopy is an imaging technique that delivers molecular contrast of pigmented samples. Here, we introduce pump-probe nonlinear phase dispersion spectroscopy (PP-NLDS), a method that ...leverages pump-probe microscopy and spectral-domain interferometry to ascertain information from dispersive and resonant nonlinear effects. PP-NLDS extends the information content to four dimensions (phase, amplitude, wavelength, and pump-probe time-delay) that yield unique insight into a wider range of nonlinear interactions compared to conventional methods. This results in the ability to provide highly specific molecular contrast of pigmented and non-pigmented samples. A theoretical framework is described, and experimental results and simulations illustrate the potential of this method. Implications for biomedical imaging are discussed.
We demonstrate a cross-phase modulation measurement technique based on the sensitive detection of modulation transfer in a pump-probe setup. By modulating the amplitude of the pump beam and ...spectrally analyzing the probe beam, we achieve a rapid, background-free measurement of nonlinear phase modulation using power levels acceptable in biological imaging. This measurement technique would allow the extension of widely employed phase microscopy methods to the nonlinear regime, providing intrinsic and universal nonlinear contrast for biological imaging.
Metastatic melanoma is associated with a poor prognosis, but no method reliably predicts which melanomas of a given stage will ultimately metastasize and which will not. While sentinel lymph node ...biopsy (SLNB) has emerged as the most powerful predictor of metastatic disease, the majority of people dying from metastatic melanoma still have a negative SLNB. Here we analyze pump-probe microscopy images of thin biopsy slides of primary melanomas to assess their metastatic potential. Pump-probe microscopy reveals detailed chemical information of melanin with subcellular spatial resolution. Quantification of the molecular signatures without reference standards is achieved using a geometrical representation of principal component analysis. Melanin structure is analyzed in unison with the chemical information by applying principles of mathematical morphology. Results show that melanin in metastatic primary lesions has lower chemical diversity than non-metastatic primary lesions, and contains two distinct phenotypes that are indicative of aggressive disease. Further, the mathematical morphology analysis reveals melanin in metastatic primary lesions has a distinct "dusty" quality. Finally, a statistical analysis shows that the combination of the chemical information with spatial structures predicts metastatic potential with much better sensitivity than SLNB and high specificity, suggesting pump-probe microscopy can be an important tool to help predict the metastatic potential of melanomas.
Microscopic variations in melanin composition can be mapped through linear and nonlinear optical responses. Though instrumentation to measure linear attenuation is simple and inexpensive, the ...nonlinear response provides more degrees of freedom with which to spectroscopically resolve pigments. The objective of this study is to assess differences in imaging melanin contrast by comparing hyperspectral (linear) versus pump-probe (nonlinear) microscopy of unstained histology sections of pigmented lesions. The images and analysis we have presented here show that pump-probe uncovers a greater variation in pigment composition, compared with hyperspectral microscopy, and that the two methods yield complimentary biochemical information.
An ultrafast pulse shaper, capable of both phase and amplitude shaping, is constructed using a single high-resolution liquid crystal phase mask. The shaper is calibrated with an inline spectral ...interferometry technique. Amplitude shaping is accomplished by writing to the mask a phase grating, whose period is smaller than the spectral focus, diffracting away selected frequencies in a controllable manner.
Melanins are biological pigments found throughout the animal kingdom that have many diverse functions. Pump–probe imaging can differentiate the two kinds of melanins found in human skin, eumelanin ...and pheomelanin, the distributions of which are relevant to the diagnosis of melanoma. The long-term stability of the melanin pump–probe signal is central to using this technology to analyze melanin distributions in archived tissue samples to improve diagnostic procedures. This report shows that most of the pump–probe signal from eumelanin derived from a Jurassic cephalopod is essentially identical to that of eumelanin extracted from its modern counterpart, Sepia officinalis. However, additional classes of eumelanin signals found in the fossil reveal that the pump–probe signature is sensitive to iron content, which could be a valuable tool for pathologists who cannot otherwise know the microscopic distributions of iron in melanins.
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.
•Identification and characterization of RNA silencing suppressors is described.•The basic steps of the agroinfitration and co-infiltration assays are presented.•The use and interpretation of ...equilibrium binding experiments is explained.•Gel mobility shift assays in the context of siRNA:suppressor binding are discussed.•Fluorescence polarization in the context of siRNA:suppressor binding is discussed.
Suppression is a common mechanism employed by viruses to evade the antiviral effects of the host’s RNA silencing pathway. The activity of suppression has commonly been localized to gene products in the virus, but the variety of mechanisms used in suppression by these viral proteins spans nearly the complete biochemical pathway of RNA silencing in the host. This review describes the agrofiltration assay and a slightly modified version of the agro-infiltration assay called co-infiltration, which are common methods used to observe RNA silencing and identify viral silencing suppressor proteins in plants, respectively. In addition, this review will provide an overview of two methods, electrophoretic mobility shift assay and fluorescence polarization, used to assess the binding of a suppressor protein to siRNA which has been shown to be a general mechanism to suppress RNA silencing by plant viruses.
We introduce a method to enable optical amplification of a coherent Raman spectroscopy signal, which we call radio frequency (RF) Doppler Raman spectroscopy. In this article, we consider the ...perturbation of a probe pulse in a sample due to an excited Raman vibrational coherence as a generalized Doppler shift, which connects a time-varying optical path length (the product of the propagation length and refractive index, OPL = n ℓ) with an optical frequency shift. Amplification of a Raman signal outside of the focused interaction is enabled by converting the Doppler frequency shift experienced by a laser probe pulse into a periodic timing jitter. This transit time perturbation is detected through the phase of a RF electronic signal measured at a harmonic of the probe pulse train with a method adapted from precision metrology techniques used to measure laser pulse train timing jitter. Measurement of a timing jitter allows access to much lower noise floors than other coherent Raman techniques, and by exploiting the new capability to scale the signal of a coherent Raman spectroscopic signal, this method opens the potential to detect very weak Raman signals that are currently not observable due to limits of illumination intensity imposed by laser damage to the specimen and noise.
Microscopy with nonlinear phase contrast is achieved by a simple modification to a nonlinear pump-probe microscope. The technique measures cross-phase modulation by detecting a pump-induced spectral ...shift in the probe pulse. Images with nonlinear phase contrast are acquired both in transparent and absorptive media. In paraffin-embedded biopsy sections, cross-phase modulation complements the chemically-specific pump-probe images with structural context.
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