Spectrally-broad coherent anti-Stokes Raman scattering hyper-microscopy utilizing a Stokes supercontinuum pumped at 800 nm

Abstract

We demonstrate spectral-focusing based coherent anti-Stokes Raman scattering (SF-CARS) hyper-microscopy capable of probing vibrational frequencies from 630 cm^-1 to 3250 cm^-1 using a single Ti:Sapphire femtosecond laser operating at 800 nm, and a commercially-available supercontinuum-generating fibre module. A broad Stokes supercontinuum with significant spectral power at wavelengths between 800 nm and 940 nm is generated by power tuning the fibre module using atypically long and/or chirped ~200 fs pump pulses, allowing convenient access to lower vibrational frequencies in the fingerprint spectral region. This work significantly reduces the instrumental and technical requirements for multimodal CARS microscopy, while expanding the spectral capabilities of an established approach to SF-CARS.

Keywords: (170.3880) Medical and biological imaging; (170.5810) Scanning microscopy; (180.4315) Nonlinear microscopy; (300.6230) Spectroscopy, coherent anti-Stokes Raman scattering.

Fig. 1

A simplified schematic of the multimodal CARS microscopy setup. A Ti:Sapphire oscillator generates 190 fs pulses at 800 nm, which are split into pump and Stokes-generating beams. The Stokes-generating beam passes through a Faraday isolator (FI) before being coupled into a FemtoWHITE CARS (NKT photonics) microstructured-fibre module that generates the Stokes supercontinuum. Blocks of high-dispersion S-NPH2 glass are used to disperse and match the chirps of the pump and Stokes pulses. The pump and Stokes beams are recombined using a long-pass filter (LPF) and routed to the laser-scanning microscope. Isotropically-generated TPEF is collected in the backwards direction, reflected by a dichroic and detected by a PMT. Forward-generated SHG and CARS signals are isolated with a short-pass filter (SPF) and collected using a customized multimode-fibre assembly and routed off-board, where they are wavelength-separated en route to separate PMTs.

Fig. 2

Spectra of the FemtoWHITE-CARS output vs. coupled pump power plotted in wavelength (left scale) and the difference frequency (right scale), Ω_R = ω_P – ω_S. Generated supercontinuum (a) from transform-limited 190 fs input pulses; (b) when the 190 fs input pulses are dispersed to 200 fs by a faraday isolator; (c) when the input pulses are further dispersed to 315 fs with a block of high-dispersion glass. The dashed horizontal lines are guides to the eye that correspond to Stokes wavelengths used to probe some relevant fingerprint and CH/OH vibrational frequencies. The dashed vertical line in (b) represents the slice of the Stokes spectrum (coupled power of 110 mW) used for the proceeding hyperspectral imaging experiments.

Fig. 3

Frequency-calibrating spectrogram and CARS spectrum of astaxanthin. (a) Spectrogram with a sampling spatial resolution of 50 μm (i.e. temporal resolution of 333 fs). The duration of the highly-chirped Stokes supercontinuum at the sample is approximately 25 ps. (b) CARS spectrum of astaxanthin obtained through point scan having 900 data points obtained in 100 s, and the corresponding Stokes spectrum plotted as a function of both wavelength and the vibrational frequency probed. The power densities of the Stokes at the 897 nm and 1001 nm peaks are estimated to be 43 µW/nm and 24 µW/nm, respectively. The pump power and the integrated Stokes power were measured to be 3.7 mW and 4.2 mW at the sample plane, respectively.

Fig. 4

A demonstration of the hyperspectral imaging capabilities of the multimodal CARS setup. (a) A 200 x 200 pixel multimodal image of benzonitrile (CARS; blue contrast; 12 frames centred at 3074 cm⁻¹), DMSO (CARS; green contrast; 13 frames centred at 2910 cm⁻¹), and cellulose fibre (SHG; red contrast). (b) CARS spectra from two 10 x 10 pixel regions of interest (ROIs) showing the CARS spectrum from benzonitrile and DMSO. (c) A 200 x 140 pixel (cropped) multimodal image of lily pollen containing carotenoids in the luminae (CARS; red contrast; 43 frames centered at 1154 cm⁻¹), and muri (TPEF; white contrast). (d) Raw CARS spectrum averaged over 10 x 10 pixel ROI in (c). The shaded spectral regions in (b) and (d) correspond to the image stacks averaged to produce the multimodal image. Each image stack was taken at a duration of 0.8 seconds with a 13-μs pixel dwell time using pump powers of 74 mW for (a) and 7.4 mW for (c) measured at the sample plane. The input power in the PCF was maintained at 110 mW (200 fs pulse duration) which generates supercontinuum with a power of 4.2 mW as measured at the sample plane.

Fig. 5

Demonstration of the efficacy of a Kramers-Kronig-based Raman-retrieval algorithm [35] in spectrally-broad SF-CARS. The Raman-like spectrum (blue) was retrieved from the raw CARS signal (black) and a nonresonant background spectrum collected in the sample coverslip. A comparison to the spontaneous Raman spectrum (red) is shown as a reference.