Abstract

Coherent Anti-Stokes Raman Scattering (CARS) microscopy has recently emerged as a new technique for imaging in cell biology, offering chemical specificity without the need of staining and fluorescence-tagging. We have developed a home-built CARS microscope [1] which exploits linearly-chirped ultrafast laser pulses to achieve spectral focussing of the vibrational excitation [2]. CARS Stokes pulses are generated by a Ti:Sa laser (835nm, 100fs), which pumps an optical parametric oscillator (OPO). The intra-cavity frequency-doubled signal of the OPO provides the CARS Pump. Both pulses are equally chirped with glass blocks of calibrated group-velocity dispersion and we studied the effect of linear chirp corresponding to pulse durations from 600fs to 2.5ps [1]. As a proof-of principle, we investigated polystyrene beads (0.2-3μm diameter) in 2% agar solution by detecting the forward-collected CARS at the C-H aromatic band (3054cm¹). By varying the delay time between Pump and Stokes their instantaneous frequency difference (IFD) changes so that CARS spectra can be measured. This is shown in Fig. 1a for three different chirped pulse durations τ. The measured spectral width of the aromatic C-H vibration in polystyrene is decreasing with increasing τ, as expected since the spectral width σ_D of the vibrational excitation decreases [1]. The peak intensity of the resonant CARS response keeps essentially constant as long as σ_D is larger than the probed Raman line-width 2T₂⁻¹~27 cm⁻¹. Vice versa, CARS from the agarose gel, dominated by the spectrally non-resonant CARS response of water, is broadening in delay time and decreasing in peak intensity with increasing pulse duration τ. The ratio between resonant and non-resonant CARS is accordingly increasing with τ and saturates for τ >> T₂~ 0.4 ps, as shown in the inset of Fig.1a. A good compromise between a high ratio improving the selectivity and increasing with τ, and a high resonant CARS intensity improving the sensitivity and decreasing with τ, is τ~T₂. For durations comparable or less than T₂, we also found that the maximum CARS is not created for zero delay between Pump and Stokes pulses. In fact, since CARS is generated modulating the Pump by the induced coherent vibration, a Pump pulse arriving after the Stokes pulse (by about 20% of the pulse duration for τ ~T₂) optimizes the CARS signal and reduces the non-resonant CARS background [1]. It is therefore better to set the frequency difference between Pump and Stokes to be larger than the vibrational resonance (by about 20% of the pulse spectral width for τ ~T₂), so that the IFD matches the resonance at the optimum delay. Our microscope also features two-photon fluorescence, second harmonic generation, and differential interference contrast imaging modalities. We have demonstrated its applicability to cell imaging on a range of live unstained cells including 3T3-L1 fibroblasts, HeLa cells (see Fig. 1b), HepG2 liver cells, and in fixed tissues (mouse mammary gland and intestine). Current progress towards a differential detection modality with improved signal-to-background ratio will also be presented.

© 2009 IEEE