Eric O. Potma

Heterodyne coherent anti-Stokes Raman scattering (CARS) imaging

We have achieved rapid nonlinear vibrational imaging free of nonresonant background with heterodyne coherent anti-Stokes Raman scattering (CARS) interferometric microscopy. This technique completely separates the real and imaginary responses of nonlinear susceptibility chi(3) and yields a signal that is linear in the concentration of vibrational modes. We show that heterodyne CARS microscopy permits the detection of weak vibrational resonances that are otherwise overshadowed by the strong interference of the nonresonant background.

Coherent anti-stokes raman scattering spectral interferometry: determination of the real and imaginary components of nonlinear susceptibility chi(3) for vibrational microscopy.

We demonstrate coherent anti-Stokes Raman scattering (CARS) heterodyne spectral interferometry for retrieval of the real and imaginary components of the third-order nonlinear susceptibility (chi(3)) of molecular vibrations. Extraction of the imaginary component of chi(3) allows a straightforward reconstruction of the vibrationally resonant signal that is completely free of the electronic nonresonant background and resembles the spontaneous Raman spectrum. Heterodyne detection offers potential for signal amplification and enhanced sensitivity for CARS microscopy.

Characterization of Cholesterol Crystals in Atherosclerotic Plaques Using Stimulated Raman Scattering and Second-Harmonic Generation Microscopy

Cholesterol crystals (ChCs) have been identified as a major factor of plaque vulnerability and as a potential biomarker for atherosclerosis. Yet, due to the technical challenge of selectively detecting cholesterol in its native tissue environment, the physiochemical role of ChCs in atherosclerotic progression remains largely unknown. In this work, we demonstrate the utility of hyperspectral stimulated Raman scattering (SRS) microscopy combined with second-harmonic generation (SHG) microscopy to selectively detect ChC. We show that despite the polarization sensitivity of the ChC Raman spectrum, cholesterol monohydrate crystals can be reliably discriminated from aliphatic lipids, from structural proteins of the tissue matrix and from other condensed structures, including cholesteryl esters. We also show that ChCs exhibit a nonvanishing SHG signal, corroborating the noncentrosymmetry of the crystal lattice composed of chiral cholesterol molecules. However, combined hyperspectral SRS and SHG imaging reveals that not all SHG-active structures with solidlike morphologies can be assigned to ChCs. This study exemplifies the merit of combining SRS and SHG microscopy for an enhanced label-free chemical analysis of crystallized structures in diseased tissue.

Fiber delivered probe for efficient CARS imaging of tissues

We demonstrate a fiber-based probe for maximum collection of the coherent anti-Stokes Raman scattering (CARS) signal in biological tissues. We discuss the design challenges including capturing the backscattered forward generated CARS signal in the sample and the effects of fiber nonlinearities on the propagating pulses. Three different single mode fibers (fused silica fiber, photonic crystal fiber and double-clad photonic crystal fiber) were tested for the probe design. We investigated self-phase modulation, stimulated Raman scattering (SRS) and four-wave-mixing (FWM) generation in the fiber: nonlinear processes expected to occur in a two-beam excitation based probe. While SPM and SRS induced spectral broadening was negligible, a strong non phase-matched FWM contribution was found to be present in all the tested fibers for excitation conditions relevant to CARS microscopy of tissues. To spectrally suppress this strong contribution, the pro design incorporates separate fibers for excitation light delivery and for signal detection, in combination with dichroic optics. CARS images of the samples were recorded by collecting the back-scattered forward generated CARS signal in the sample through a multi-mode fiber. Different biological tissues were imaged ex vivo in order to assess the performance of our fiber-delivered probe for CARS imaging, a tool which we consider an important advance towards label-free, in vivo probing of superficial tissues.

Identification of cholesterol crystals in plaques of atherosclerotic mice using hyperspectral CARS imaging

The accumulation of lipids, including cholesterol, in the arterial wall plays a key role in the pathogenesis of atherosclerosis. Although several advances have been made in the detection and imaging of these lipid structures in plaque lesions, their morphology and composition have yet to be fully elucidated, particularly in different animal models of disease. To address this issue, we analyzed lipid morphology and composition in the atherosclerotic plaques of two animal models of disease, the low density lipoprotein receptor-deficient (LDLR−/−) mouse and the ApoE lipoprotein-deficient (ApoE−/−) mouse, utilizing hyperspectral coherent anti-Stokes Raman scattering (CARS) microscopy in combination with principal component analysis (PCA). Hyperspectral CARS imaging revealed lipid-rich macrophage cells and condensed needle-shaped and plate-shaped lipid crystal structures in both mice. Spectral analysis with PCA and comparison to spectra of pure cholesterol and cholesteryl ester derivatives further revealed these lipid structures to be pure cholesterol crystals, which were predominantly observed in the ApoE−/− mouse model. These results illustrate the ability of hyperspectral CARS imaging in combination with multivariate analysis to characterize atherosclerotic lipid morphology and composition with chemical specificity, and consequently, provide new insight into the formation of cholesterol crystal structures in atherosclerotic plaque lesions.

Four-wave mixing microscopy of nanostructures

The basics of four-wave mixing (FWM) and recent advances in FWM microscopy are reviewed with a particular emphasis on applications in the field of nanomaterials. The vast progress in nanostructure synthesis has triggered a need for advanced analytical tools suitable to interrogate nanostructures one at a time. The single-nanostructure sensitivity of optical microscopy has solidified the optical approach as a reliable technique for examining the electronic structure of materials at the nanoscale. By zooming in on the individual, optical microscopy has permitted detailed investigations of the linear optical response of nanomaterials such as semiconducting quantum dots and plasmon active nanometals. Besides studying the linear optical properties of nanostructures, optical microscopy has also been used to probe the nonlinear optical properties of nanoscale materials. FWM microscopy, a coherent third-order optical imaging technique, has shown great potential as a tool for investigating the nonlinear optical response of nanostructures. FWM microscopy not only permits the characterization of the nonlinear susceptibility of individual nanostructures, it also offers a route to explore the time-resolved dynamics of electronic and vibrational excitations on single structures. In addition, FWM produces strong signals from nanomaterials that are compatible with fast imaging applications, which holds promise for biological imaging studies based on nanoparticle labels that are not prone to photobleaching.

Multimodal CARS microscopy determination of the impact of diet on macrophage infiltration and lipid accumulation on plaque formation in ApoE-deficient mice

We characterized several cellular and structural features of early stage Type II/III atherosclerotic plaques in an established model of atherosclerosis—the ApoE-deficient mouse—by using a multimodal, coregistered imaging system that integrates three nonlinear optical microscopy (NLOM) contrast mechanisms: coherent anti-Stokes Raman scattering (CARS), second harmonic generation (SHG), and two-photon excitation fluorescence (TPEF). Specifically, the infiltration of lipid-rich macrophages and the structural organization of collagen and elastin fibers were visualized by CARS, SHG, and TPEF, respectively, in thick tissue specimens without the use of exogenous labels or dyes. Label-free CARS imaging of macrophage accumulation was confirmed by histopathology using CD68 staining. A high-fat, high-cholesterol Western diet resulted in an approximate 2-fold increase in intimal plaque area, defined by CARS signals of lipid-rich macrophages. Additionally, analysis of collagen distribution within lipid-rich plaque regions revealed nearly a 4-fold decrease in the Western diet–fed mice, suggesting NLOM sensitivity to increased matrix metalloproteinase (MMP) activity and decreased smooth muscle cell (SMC) accumulation. These imaging results provide significant insight into the structure and composition of early stage Type II/III plaque during formation and allow for quantitative measurements of the impact of diet and other factors on critical plaque and arterial wall features.

Synchronization of two passively mode-locked, picosecond lasers within 20 fs for coherent anti-Stokes Raman scattering microscopy

We report on the synchronization of two commercial picosecond Ti:sapphire lasers with unprecedented low temporal jitter between the pulse trains. Pulse jitter is reduced from a few picoseconds to 20 fs with a stability of several hours. The technology enabling the tight pulse synchronization is reviewed in this article. We demonstrate the usefulness of the synchronization scheme by applying the technique to coherent anti-Stokes Raman scattering (CARS) microscopy. It is shown that CARS images can be acquired with a significant improvement in signal-to-noise ratio. This level of performance brings the fluctuations of the CARS signal down to the fundamental photon shot-noise limit. We present detailed statistical analysis of the pulse jitter and CARS noise along with enhanced CARS vibrational images of polymer beads.

Following dimethyl sulfoxide skin optical clearing dynamics with quantitative nonlinear multimodal microscopy

Second-harmonic generation (SHG) imaging is combined with coherent anti-Stokes Raman scattering (CARS) microscopy to follow the process of optical clearing in human skin ex vivo using dimethyl sulfoxide (DMSO) as the optical clearing agent. SHG imaging revealed that DMSO introduces morphological changes to the collagen I matrix. By carefully measuring the dynamic tissue attenuation of the coherent nonlinear signal, using CARS reference signals during the clearing process, it is found that DMSO reduces the overall SHG response from dermal collagen. Evidence is provided for a role of DMSO in compromising the structure of collagen fibers, associated with a reduction of the tissues scattering properties.

CARS Microscopy for Biology and Medicine

The key requirements imposed on optical imaging techniques for the visualization of living biological specimens are noninvasiveness, chemical selectivity and high sensitivity. Coherent anti-Stokes Raman scattering (CARS) microscopy, which meets all three requirements, casts a new light on vibrational microscopy and paves the way for exciting new applications.