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Optics Express

Optics Express

  • Editor: Andrew M. Weiner
  • Vol. 21, Iss. 6 — Mar. 25, 2013
  • pp: 7096–7106

Simultaneous hyperspectral differential-CARS, TPF and SHG microscopy with a single 5 fs Ti:Sa laser

Iestyn Pope, Wolfgang Langbein, Peter Watson, and Paola Borri  »View Author Affiliations

Optics Express, Vol. 21, Issue 6, pp. 7096-7106 (2013)

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We have developed a multimodal multiphoton laser-scanning microscope for cell imaging featuring simultaneous acquisition of differential Coherent Antistokes Raman Scattering (D-CARS), two-photon fluorescence (TPF) and second harmonic generation (SHG) using a single 5 fs Ti:Sa broadband (660–970 nm) laser. The spectral and temporal pulse requirements of these modalities were optimized independently by splitting the laser spectrum into three parts: TPF/SHG excitation (> 900 nm), CARS Pump excitation (< 730 nm), and CARS Stokes excitation (730–900 nm). In particular, by applying an equal linear chirp to pump and Stokes pulses using glass dispersion we achieved a CARS spectral resolution of 10 cm−1, and acquired CARS images over the 1200–3800 cm−1 vibrational range selected by the time delay between pump and Stokes. A prism pulse compressor in the TPF/SHG excitation was used to achieve Fourier limited 30 fs pulses at the sample for optimum TPF and SHG. D-CARS was implemented with few passive optical elements and enabled simultaneous excitation and detection of two vibrational frequencies with a separation adjustable from 20 cm−1 to 150 cm−1 for selective chemical contrast and background suppression. The excitation/detection set-up using beam-scanning was built around a commercial inverted microscope stand providing conventional bright-field, differential interference contrast and epi-fluorescence for user-friendly characterization of biological samples. Examples of CARS hyperspectral images and simultaneous acquisition of D-CARS, TPF and SHG images in both forward and epi-direction are shown on HeLa cells, stem-cell derived human adipocytes and mouse tissues.

© 2013 OSA

OCIS Codes
(170.3880) Medical optics and biotechnology : Medical and biological imaging
(180.6900) Microscopy : Three-dimensional microscopy
(180.4315) Microscopy : Nonlinear microscopy
(180.5655) Microscopy : Raman microscopy

ToC Category:

Original Manuscript: December 14, 2012
Revised Manuscript: February 1, 2013
Manuscript Accepted: February 14, 2013
Published: March 13, 2013

Virtual Issues
Vol. 8, Iss. 4 Virtual Journal for Biomedical Optics

Iestyn Pope, Wolfgang Langbein, Peter Watson, and Paola Borri, "Simultaneous hyperspectral differential-CARS, TPF and SHG microscopy with a single 5 fs Ti:Sa laser," Opt. Express 21, 7096-7106 (2013)

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