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Keywords:

  • CARS;
  • coherent anti-Stokes Raman;
  • live cell imaging;
  • atomic force microscopy

Abstract

We have constructed a novel, versatile Coherent anti-Stokes Raman scattering (CARS) microscope for biological imaging. The system employs a Nd:YVO4 laser as Stokes (6 ps, 1064 nm), and a tunable optical parametric oscillators (OPOs) (5 ps, 700–1000 nm) as pump excitation, resulting in an observable range of ∼600–4000 cm−1. A second OPO can be used as a replacement pump for low wavenumber excitation (down to ∼100 cm−1). Pump and Stokes excitations are focused onto a single-mode fibre for ease of alignment, which is connected to the input of a confocal inverted optical microscope. The epi-detected CARS signal is filtered using a spectrometer, and the photons counted with an avalanche photodiode. This detection is preferable to using bandpass filters and photomultipliers, as it allows all wavenumbers to be imaged and ensures rejection of unwanted fluorescent and scattered light. Our microscope comprises a heater stage and perfusion cell for imaging live cells, and allows complementary biological imaging techniques (differential interference contrast (DIC), second harmonic generation, sum frequency generation, one- and two-photon fluorescence, fluorescence lifetime imaging). In addition, our system features an atomic force microscope (AFM) which enables CARS imaging at 10-nm resolution. The details and advantages of the modular imaging system are described more fully, and the latest results are presented. Copyright © 2009 John Wiley & Sons, Ltd.