Simple approach to one-laser, broadband coherent anti-Stokes Raman scattering microscopy

Abstract

Coherent anti-Stokes Raman scattering (CARS) microscopy is emerging as a powerful method for imaging materials and biological systems, partly because of its noninvasiveness and selective chemical sensitivity. However, its full potential for species-selective imaging is limited by a restricted spectral bandwidth. Recent increases in bandwidth are promising but still are not sufficient for the level of robust component discrimination that would be needed in a chemically complex milieu found, for example, in intracellular and extracellular environments. We demonstrate a truly broadband CARS imaging instrument that we use to acquire hyperspectral images with vibrational spectra over a bandwidth of 2500 cm(-1) with a resolution of 13 cm(-1).

Figure 1

Energy-level diagram for a single-frequency CARS process (solid vertical arrows) and for broadband CARS (solid and dashed vertical arrows).

Figure 2

Experimental conf iguration: DF, dispersionless filter; TF, tapered fiber; LP, long-pass filter; SP, short-pass filter; DBS, dichroic beam splitter; Obj, microscope objective. The inset spectra are (counterclockwise from top) pump, feed light to TF, and output of TF. The TF feed light is arbitrarily scaled and superimposed on the TF output spectrum.

Figure 3

Broadband CARS spectrum of benzonitrile. a, Raw CARS spectrum (solid curve) and nonresonant background (dashed curve). b, Ratio of the CARS spectrum to the nonresonant background (solid curve) and positions and amplitudes of the Raman lines. The CARS spectrum was obtained in 17 ms. c, Spontaneous Raman spectrum obtained under identical laser flux (23 mW but all in pump light). This spectrum was acquired in 1 s. The standard uncertainty of the spectrometer wavelength calibration was 5 cm⁻¹.

Figure 4

a, Broadband CARS micrograph of a phase-separated polymer blend including equal parts PMMA, PS, and PET. The pseudocolor image is 150 pixels square; the colors red, green, and blue correspond to PMMA, PS, and PET, respectively. b, Reference spectra from each of the individual polymer components (with an arbitrary vertical shift for clarity). The bold, colored line segments indicate spectral regions that were used for identification of spectra from each pixel in a.