Francisco E. Robles

Dual window method for processing spectroscopic optical coherence tomography signals with simultaneously high spectral and temporal resolution.

Current methods for analysis of spectroscopic optical coherence tomography (SOCT) signals suffer from an inherent tradeoff between time (depth) and frequency (wavelength) resolution. Here, we present a dual window (DW) method for reconstructing time frequency distributions (TFDs) that applies two orthogonal Gaussian windows that independently determine the spectral and temporal resolution. The effectiveness of the method is demonstrated in simulations and in processing of measured OCT signals that contain fields which vary in time and frequency. The DW method yields TFDs that maintain high spectral and temporal resolution and are free from the artifacts and limitations commonly observed with other processing methods.

Invited Review Article: Pump-probe microscopy

Multiphoton microscopy has rapidly gained popularity in biomedical imaging and materials science because of its ability to provide three-dimensional images at high spatial and temporal resolution even in optically scattering environments. Currently the majority of commercial and home-built devices are based on two-photon fluorescence and harmonic generation contrast. These two contrast mechanisms are relatively easy to measure but can access only a limited range of endogenous targets. Recent developments in fast laser pulse generation, pulse shaping, and detection technology have made accessible a wide range of optical contrasts that utilize multiple pulses of different colors. Molecular excitation with multiple pulses offers a large number of adjustable parameters. For example, in two-pulse pump-probe microscopy, one can vary the wavelength of each excitation pulse, the detection wavelength, the timing between the excitation pulses, and the detection gating window after excitation. Such a large parameter space can provide much greater molecular specificity than existing single-color techniques and allow for structural and functional imaging without the need for exogenous dyes and labels, which might interfere with the system under study. In this review, we provide a tutorial overview, covering principles of pump-probe microscopy and experimental setup, challenges associated with signal detection and data processing, and an overview of applications.

Measuring morphological features using light-scattering spectroscopy and Fourier-domain low-coherence interferometry

We present measurements of morphological features in a thick turbid sample using light-scattering spectroscopy (LSS) and Fourier-domain low-coherence interferometry (fLCI) by processing with the dual-window (DW) method. A parallel frequency domain optical coherence tomography (OCT) system with a white-light source is used to image a two-layer phantom containing polystyrene beads of diameters 4.00 and 6.98 mum on the top and bottom layers, respectively. The DW method decomposes each OCT A-scan into a time-frequency distribution with simultaneously high spectral and spatial resolution. The spectral information from localized regions in the sample is used to determine scatterer structure. The results show that the two scatterer populations can be differentiated using LSS and fLCI.

Phasor analysis for nonlinear pump-probe microscopy

Pump-probe microscopy provides molecular information by probing transient, excited state dynamic properties of pigmented samples. Analysis of the transient response is typically conducted using principal component analysis or multi-exponential fitting, however these methods are not always practical or feasible. Here, we show an adaptation of phasor analysis to provide an intuitive, robust, and efficient method for analyzing and displaying pump-probe images, thereby alleviating some of the challenges associated with differentiating multiple pigments. A theoretical treatment is given to understand how the complex transient signals map onto the phasor plot. Analyses of cutaneous and ocular pigmented tissue samples, as well as historical pigments in art demonstrate the utility of this approach.

Nonlinear phase dispersion spectroscopy

Nonlinear phase dispersion spectroscopy is introduced as a means to retrieve wideband, high spectral resolution profiles of the wavelength-dependent real part of the refractive index. The method is based on detecting dispersion effects imparted to a light field with low coherence transmitted through a thin sample and detected interferometrically in the spectral domain. The same sampled signal is also processed to yield quantitative phase maps and spectral information regarding the total attenuation coefficient using spectral-domain phase microscopy and spectroscopic optical coherence tomography (SOCT), respectively. Proof-of-concept experiments using fluorescent and nonfluorescent polystyrene beads and another using a red blood cell demonstrate the ability of the method to quantify various absorptive/dispersive features. The increased sensitivity of this method, novel to our knowledge, is compared to intensity-based spectroscopy (e.g., SOCT), and potential applications are discussed.

Detecting precancerous lesions in the hamster cheek pouch using spectroscopic white-light optical coherence tomography to assess nuclear morphology via spectral oscillations.

We have developed a novel dual-window approach for spectroscopic optical coherence tomography (OCT) measurements and applied it to probe nuclear morphology in tissue samples drawn from the hamster cheek pouch carcinogenesis model. The dual-window approach enables high spectral and depth resolution simultaneously, allowing detection of spectral oscillations, which we isolate to determine the structure of cell nuclei in the basal layer of the epithelium. The measurements were executed with our parallel frequency domain OCT system, which uses light from a thermal source, providing high bandwidth and access to the visible portion of the spectrum. The structural measurements show a highly statistically significant difference between untreated (normal) and treated (hyperplastic/dysplastic) tissues, indicating the potential utility of this approach as a diagnostic method.

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.

Detection of early colorectal cancer development in the azoxymethane rat carcinogenesis model with Fourier domain low coherence interferometry

Fourier domain low coherence interferometry (fLCI) is an emerging optical technique used to quantitatively assess cell nuclear morphology in tissue as a means of detecting early cancer development. In this work, we use the azoxymethane rat carcinogenesis model, a well characterized and established model for colon cancer research, to demonstrate the ability of fLCI to distinguish between normal and preneoplastic ex-vivo colon tissue. The results show highly statistically significant differences between the measured cell nuclear diameters of normal and azoxymethane-treated tissues, thus providing strong evidence that fLCI may be a powerful tool for non-invasive, quantitative detection of early changes associated with colorectal cancer development.

Separating the scattering and absorption coefficients using the real and imaginary parts of the refractive index with low-coherence interferometry.

We present an analytical method that yields the real and imaginary parts of the refractive index (RI) from low-coherence interferometry measurements, leading to the separation of the scattering and absorption coefficients of turbid samples. The imaginary RI is measured using time-frequency analysis, with the real part obtained by analyzing the nonlinear phase induced by a sample. A derivation relating the real part of the RI to the nonlinear phase term of the signal is presented, along with measurements from scattering and nonscattering samples that exhibit absorption due to hemoglobin.

Multispectral nanoparticle contrast agents for true-color spectroscopic optical coherence tomography

We have recently developed a novel dual window scheme for processing spectroscopic OCT images to provide spatially resolved true color imaging of chromophores in scattering samples. Here we apply this method to measure the extinction spectra of plasmonic nanoparticles at various concentrations for potential in vivo applications. We experimentally demonstrate sub-nanomolar sensitivity in the measurement of nanoparticle concentrations, and show that colorimetric imaging with multiple species of nanoparticles produces enhanced contrast for spectroscopic OCT in both tissue phantom and cell studies.