Coherent anti-Stokes Raman Microscopy Research Articles

Modeling nonlinear optical microscopy in scattering media, part II. Radiation from focal volume to far-field: tutorial.

The development and application of nonlinear optical (NLO) microscopy methods in biomedical research has experienced rapid growth over the past three decades. Despite the compelling power of these methods, optical scattering limits their practical use in biological tissues. This tutorial offers a model-based approach illustrating how analytical methods from classical electromagnetism can be employed to comprehensively model NLO microscopy in scattering media. In Part I, we quantitatively model focused beam propagation in non-scattering and scattering media from the lens to focal volume. In Part II, we model signal generation, radiation, and far-field detection. Moreover, we detail modeling approaches for major optical microscopy modalities including classical fluorescence, multi-photon fluorescence, second harmonic generation, and coherent anti-Stokes Raman microscopy.

Modeling nonlinear optical microscopy inscattering media, part I. Propagation from lens tofocal volume: tutorial.

The development and application of nonlinear optical (NLO) microscopy methods in biomedical research have experienced rapid growth over the past three decades. Despite the compelling power of these methods, optical scattering limits their practical use in biological tissues. This tutorial offers a model-based approach illustrating how analytical methods from classical electromagnetism can be employed to comprehensively model NLO microscopy in scattering media. In Part I, we quantitatively model focused beam propagation in non-scattering and scattering media from the lens to focal volume. In Part II, we model signal generation, radiation, and far-field detection. Moreover, we detail modeling approaches for major optical microscopy modalities including classical fluorescence, multi-photon fluorescence, second harmonic generation, and coherent anti-Stokes Raman microscopy.

Spatial frequency modulated imaging in coherent anti-Stokes Raman microscopy.

For sparse samples or in the presence of ambient light, the signal-to-noise ratio (SNR) performance of single-point-scanning coherent anti-Stokes Raman scattering (CARS) images is not optimized. As an improvement, we propose replacing the conventional CARS focus-point illumination with a periodically structured focus line while continuing to collect the transmitted CARS intensity on a single detector. The object information along the illuminated line is obtained by numerically processing the CARS signal recorded for various periods of the structured focus line. We demonstrate experimentally the feasibility of this spatial frequency modulated imaging (SPIFI) in CARS (SPIFI-CARS) and SHG (SPIFI-SHG) and identify situations where its SNR is better than that of the single-point-scanning approach.

A New Method to Characterize the Porous Carbon Materials

Compared with the traditional materials, carbon materials exhibit excellent physical, electrical, mechanical and chemical properties. Porous carbon is one of the structured carbon materials, with controlled large surface area, high surface activity, low density, superior chemicals, corrosion resistance and uniform pore features on micrometer and nanometer scales. It is explored as advanced material in lithium-ion batteries, Super capacitor, fuel cell, energy storage and conversion devices, and adsorption materials, because of its excellent property. The characterization methods were the traditional instruments, such as transmission electron microscopy, scanning electron microscopy, Raman spectrum, X-ray diffraction, X-ray photoelectron spectroscopy and infrared spectroscopy. This article demonstrated new method to character the porous carbon with coherent anti-stokes Raman microscopy.

Quantitative chemical imaging with background-free multiplex coherent anti-Stokes Raman scattering by dual-soliton Stokes pulses.

Coherent anti-Stokes Raman microscopy (CARS) is a quantitative, chemically specific, and label-free optical imaging technique for studying inhomogeneous systems. However, the complicating influence of the nonresonant response on the CARS signal severely limits its sensitivity and specificity and especially limits the extent to which CARS microscopy has been used as a fully quantitative imaging technique. On the basis of spectral focusing mechanism, we establish a dual-soliton Stokes based CARS microspectroscopy and microscopy scheme capable of quantifying the spatial information of densities and chemical composition within inhomogeneous samples, using a single fiber laser. Dual-soliton Stokes scheme not only removes the nonresonant background but also allows robust acquisition of multiple characteristic vibrational frequencies. This all-fiber based laser source can cover the entire fingerprint (800-2200 cm-1) region with a spectral resolution of 15 cm-1. We demonstrate that quantitative degree determination of lipid-chain unsaturation in the fatty acids mixture can be achieved by the characterization of C = C stretching and CH2 deformation vibrations. For microscopy purposes, we show that the spatially inhomogeneous distribution of lipid droplets can be further quantitatively visualized using this quantified degree of lipid unsaturation in the acyl chain for contrast in the hyperspectral CARS images. The combination of compact excitation source and background-free capability to facilitate extraction of quantitative composition information with multiplex spectral peaks will enable wider applications of quantitative chemical imaging in studying biological and material systems.

Mapping Impurity of Single-Walled Carbon Nanotubes in Bulk Samples with Multiplex Coherent Anti-Stokes Raman Microscopy

Mapping of defects in bulk samples of single-walled carbon nanotubes (SWNT) is performed via multiplex coherent anti-Stokes Raman microscopy. The D and G vibrational bands are acquired simultaneously, and their relative amplitude is used as a criterion to quantify the local purity in spin-coated SWNT samples. We observe that defects induced by oxidation are related to the spatial dispersion of nanotubes in a solid distribution.

Scanning near-field optical coherent anti-Stokes Raman microscopy (SNOM-CARS) with femtosecond laser pulses in vibrational and electronic resonance

Accessing ultrafast photoinduced molecular dynamics on a femtosecond time-scale with vibrational selectivity and at the same time sub-diffraction limited spatial resolution would help to gain important information about ultrafast processes in nanostructures. While nonlinear Raman techniques have been used to obtain highly resolved images in combination with near-field microscopy, the use of femtosecond laser pulses in electronic resonance still constitutes a big challenge. Here, we present our first results on coherent anti-Stokes Raman scattering (fs-CARS) with femtosecond laser pulses detected in the near-field using scanning near-field optical microscopy (SNOM). We demonstrate that highly spatially resolved images can be obtained from poly(3-hexylthiophene) (P3HT) nano-structures where the fs-CARS process was in resonance with the P3HT absorption and with characteristic P3HT vibrational modes without destruction of the samples. Sub-diffraction limited lateral resolution is achieved. Especially the height resolution clearly surpasses that obtained with standard microCARS. These results will be the basis for future investigations of mode-selective dynamics in the near field.

Multiplex coherent anti-Stokes Raman microspectroscopy with tailored Stokes spectrum

We combined the ultrabroadband supercontinuum of a photonic crystal fiber with a pulse shaper, resulting in a highly flexible light source for multiplex coherent anti-Stokes Raman microscopy. Implemented as the Stokes pulse, it provides tailored selection of the relevant Raman transitions, resulting in a reduced photon load and partial suppression of the nonresonant background. This experiment exploits the advantages of multiplex excitation with the increased acquisition speed of single-channel detection. The molecule-specific Stokes pulses are demonstrated for chemical mapping of a polymer blend.

In Situ Analysis of Living Embryonic Stem Cells by Coherent Anti-Stokes Raman Microscopy

Embryonic stem cells (ESC), derived from preimplantation embryos, are defined by their ability to both self-renew and differentiate into all of the cells and tissues of a mature animal. Efforts to develop methods for in vitro culture of ESC for research or eventual therapeutic applications are hampered by the lack of rapid, nondestructive assays for distinguishing ESC from other (differentiated) cells within a growing culture. Coherent anti-Stokes Raman scattering (CARS) microscopy is shown here to be a sensitive and nondestructive method for identifying mouse ESC based on selective observation of specific molecular vibrations believed to be spectroscopic markers indicating the differentiated vs undifferentiated states of such cells. The nonlinear nature of CARS also permits imaging with subcellular resolution, potentially offering a means by which chemical changes accompanying the early stages of differentiation may be associated with certain intracellular compartments (e.g., nucleus, cytoplasm, membranes). A novel exposure/collection configuration is described, which yields high collection efficiency and low interference from nonresonant background components.