Chemically Selective Imaging of Subcellular Structure in Human Hepatocytes with Coherent Anti-Stokes Raman Scattering (CARS) Near-Field Scanning Optical Microscopy (NSOM)

Abstract

We demonstrate the combination of coherent anti-Stoke Raman scattering (CARS) with near-field scanning optical microscopy (NSOM) for chemically selective imaging via intrinsic vibrational resonances with spatial resolution below the diffraction limit. Femtosecond, near-IR pulses are used to produce CARS signals from human hepatocytes, and by tuning the CARS signal to be resonant with C−H stretching frequencies, image contrast is observed with an optical spatial resolution of ∼128 nm.

Coherent anti‐Stokes Raman Scattering Microscopy

“…Similarly, atomic force microscopy (AFM) can be combined with CARS microscopy, thereby using the field-enhancement effect that is observed when the excitation fields are focused on the cantilever tip. [93,94] In another approach to realize superresolution CARS microscopy, Schaller et al [95] combined nearfield scanning optical microscopy (NSOM) with CARS. In this application a spatial resolution of 128 nm was achieved.…”

Linear and Nonlinear Raman Microspectroscopy: From a Molecule to Single Living Cells

“…This low efficiency originates from the small scattering cross section of the spontaneous Raman process. An alternative approach to obtain vibrational images with high speed is coherent anti-Stokes Raman scattering (CARS) [5,[22][23][24][25][26][27]. In particular, multiplex CARS microspectroscopy is promising because of its capability to obtain vibrational spectra efficiently [24,[28][29][30][31].…”

Hyperspectral coherent Raman imaging – principle, theory, instrumentation, and applications to life sciences

“…For instance, pulse shaping technique was applied to CARS microscopy, in which multiple spectral components of femtosecond laser pulses were used simultaneously to accomplish the CARS process. Moreover, spatial resolution has been improved with the use of metal tips and near‐field optics …”