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

Pope, Iestyn ORCID: https://orcid.org/0000-0002-4104-0389, Langbein, Wolfgang Werner ORCID: https://orcid.org/0000-0001-9786-1023, Watson, Peter Duncan ORCID: https://orcid.org/0000-0003-0250-7852 and Borri, Paola ORCID: https://orcid.org/0000-0002-7873-3314 2013. Simultaneous hyperspectral differential-CARS, TPF and SHG microscopy with a single 5 fs Ti:Sa laser. Optics Express 21 (6) , pp. 7096-7106. 10.1364/OE.21.007096

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Abstract

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.

Item Type:	Article
Date Type:	Publication
Status:	Published
Schools:	Biosciences Physics and Astronomy
Subjects:	Q Science > QC Physics R Medicine > RE Ophthalmology
Publisher:	Optical Society of America
ISSN:	1094-4087
Funders:	BBSRC, EPSRC
Date of First Compliant Deposit:	30 March 2016
Last Modified:	10 Jul 2024 19:16
URI:	https://orca.cardiff.ac.uk/id/eprint/48754

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