Chemical Imaging with Frequency Modulation Coherent Anti-Stokes Raman Scattering Microscopy at the Vibrational Fingerprint Region

Abstract

We present a new coherent anti-Stokes Raman scattering (CARS) method that can perform background-free microscopy and microspectroscopy at the vibrational fingerprint region. Chirped broad-band pulses from a single Ti:sapphire laser generate CARS signals over 800-1700 cm -1 with a spectral resolution of 20 cm -1 . Fast modulation of the time delay between the pump and Stokes pulses coupled with lock-in signal detection not only removes the nonresonant background but also produces Raman-like CARS signals. Chemical imaging and microspectroscopy are demonstrated with various samples such as edible oils, lipid membranes, skin tissue, and plant cell walls. Systematic studies of the signal generation mechanism and several fundamental aspects are discussed.

Visualization of structural changes in cellulosic substrates during enzymatic hydrolysis using multimodal nonlinear microscopy

“…Coherent Raman scattering techniques, coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering, can probe molecular vibrations, allowing chemically sensitive imaging, for example to image carbohydrates and lignin in plant cell walls. Lignin is generally imaged by probing aromatic ring stretching vibration at 1600 cm -1 (Chen et al 2010;Ding et al 2012;Pohling et al 2014;Saar et al 2010;Zeng et al 2010), while polysaccharides can be imaged either by probing C-C and C-O stretching vibrations at *1100 cm -1 (Chen et al 2010;Saar et al 2010) or C-H stretching vibrations at *2900 cm -1 (Ding et al 2012;Pohling et al 2014;Zimmerley et al 2010). If full Raman spectrum is recorded, lignin and different types of polysaccharides can be distinguished using spectral fitting (Pohling et al 2014).…”

From molecular structure to tissue architecture: collagen organization probed by SHG microscopy

“…Aside from lipids other relevant molecules yield good CARS image contrast, e.g. DNA or proteins [49,50]. Another equally powerful NLO imaging technique providing morpho-functional information on biological tissues is represented by second-harmonic generation (SHG) microscopy [3,51].…”

Dual-soliton Stokes-based background-free coherent anti-Stokes Raman scattering spectroscopy and microscopy

“…The NR background can further distort and even overwhelm the resonant signal of interest, especially in the fingerprint spectral region where molecules present their unique vibrational signature with lower cross section than the frequently used C-H stretch [2]. Several methods have been explored to overcome this problem including phase-and polarization control CARS [4], time-resolved CARS [5], Fourier transform CARS [6], heterodyne interferometric CARS [7] and frequency modulation CARS (FM-CARS) [8,9]. In particular FM-CARS technique has demonstrated its efficient suppression of NR CARS background.…”

“…In particular FM-CARS technique has demonstrated its efficient suppression of NR CARS background. However, the reported implementations require hardware modification to provide a second pump laser [8] or modulation of the time delay in the spectral focusing scheme, yielding a complex and costly setup [9]. In addition to the NR background, the complexity of the excitation sources also prevents a broader use of CARS microscopy.…”

“…This temporal interval can be naturally applied to a time-sweep chirped CARS scheme, like the spectral focusing technique [17], which establishes an ingenious time-frequency transform to allow the subsequent shifted excitation relying on an appropriate frequency distance, by scanning the time delay between the pump and the dual-soliton pulses. Shifted excitation with two excitation wavelengths shifted by an amount comparable to the bandwidth of the measured Raman bands (typically 10 cm -1 in the fingerprint region) has been demonstrated as an effective way to eliminate the large backgrounds in the FM-CARS method [9]. As an example, Fig.…”