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Wide-Field Detected Fourier Transform CARS Microscopy

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ABSTRACT

We present a wide-field imaging implementation of Fourier transform coherent anti-Stokes Raman scattering (wide-field detected FT-CARS) microscopy capable of acquiring high-contrast label-free but chemically specific images over the full vibrational ‘fingerprint’ region, suitable for a large field of view. Rapid resonant mechanical scanning of the illumination beam coupled with highly sensitive, camera-based detection of the CARS signal allows for fast and direct hyperspectral wide-field image acquisition, while minimizing sample damage. Intrinsic to FT-CARS microscopy, the ability to control the range of time-delays between pump and probe pulses allows for fine tuning of spectral resolution, bandwidth and imaging speed while maintaining full duty cycle. We outline the basic principles of wide-field detected FT-CARS microscopy and demonstrate how it can be used as a sensitive optical probe for chemically specific Raman imaging.

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Overview of coherent anti-Stokes Raman scattering (CARS) microscopy.(a) Pulse-timing diagram for broadband frequency-domain CARS and (b) time-domain CARS. (c) Wave-mixing energy ladder diagram describing the generation of blue-shifted CARS photons after three electric field interactions with the sample. Fourier transform CARS microscopy relying on single point (d) and wide-field image (e) detection. CARS spectra are retrieved after Fourier transformation (FT) of the obtained coherent oscillations.
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f1: Overview of coherent anti-Stokes Raman scattering (CARS) microscopy.(a) Pulse-timing diagram for broadband frequency-domain CARS and (b) time-domain CARS. (c) Wave-mixing energy ladder diagram describing the generation of blue-shifted CARS photons after three electric field interactions with the sample. Fourier transform CARS microscopy relying on single point (d) and wide-field image (e) detection. CARS spectra are retrieved after Fourier transformation (FT) of the obtained coherent oscillations.

Mentions: Taking advantage of the full potential of Raman imaging for chemical specificity ultimately demands efficient acquisition of high-quality Raman spectra over the chemically informative fingerprint region of the vibrational spectrum (500–1600 cm−1)1314. Although broadband acquisition is invariably slower compared to acquisition restricted to single vibrational features, it also opens the door to advanced chemometrics such as principle component or multivariate analysis1516. The rich information content that can be obtained from such an approach was recently demonstrated in biological tissues using broadband coherent anti-Stokes Raman scattering (b-CARS)110 In b-CARS, the combination of a broadband femtosecond pump/Stokes pulse with a narrow-band picosecond probe pulse (Fig. 1a) generates vibrational coherence over the full vibrational manifold, which is subsequently read out by a second interaction with the pump electric field. The frequency-domain approach to CARS has the advantage of producing broadband spectra in a single laser shot. At the same time, the raw acquired spectra suffer from non-resonant background, which leads to complicated dispersive lineshapes, compared to comparatively narrow Lorentzian lineshapes of a spontaneous Raman spectrum17.


Wide-Field Detected Fourier Transform CARS Microscopy
Overview of coherent anti-Stokes Raman scattering (CARS) microscopy.(a) Pulse-timing diagram for broadband frequency-domain CARS and (b) time-domain CARS. (c) Wave-mixing energy ladder diagram describing the generation of blue-shifted CARS photons after three electric field interactions with the sample. Fourier transform CARS microscopy relying on single point (d) and wide-field image (e) detection. CARS spectra are retrieved after Fourier transformation (FT) of the obtained coherent oscillations.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5121585&req=5

f1: Overview of coherent anti-Stokes Raman scattering (CARS) microscopy.(a) Pulse-timing diagram for broadband frequency-domain CARS and (b) time-domain CARS. (c) Wave-mixing energy ladder diagram describing the generation of blue-shifted CARS photons after three electric field interactions with the sample. Fourier transform CARS microscopy relying on single point (d) and wide-field image (e) detection. CARS spectra are retrieved after Fourier transformation (FT) of the obtained coherent oscillations.
Mentions: Taking advantage of the full potential of Raman imaging for chemical specificity ultimately demands efficient acquisition of high-quality Raman spectra over the chemically informative fingerprint region of the vibrational spectrum (500–1600 cm−1)1314. Although broadband acquisition is invariably slower compared to acquisition restricted to single vibrational features, it also opens the door to advanced chemometrics such as principle component or multivariate analysis1516. The rich information content that can be obtained from such an approach was recently demonstrated in biological tissues using broadband coherent anti-Stokes Raman scattering (b-CARS)110 In b-CARS, the combination of a broadband femtosecond pump/Stokes pulse with a narrow-band picosecond probe pulse (Fig. 1a) generates vibrational coherence over the full vibrational manifold, which is subsequently read out by a second interaction with the pump electric field. The frequency-domain approach to CARS has the advantage of producing broadband spectra in a single laser shot. At the same time, the raw acquired spectra suffer from non-resonant background, which leads to complicated dispersive lineshapes, compared to comparatively narrow Lorentzian lineshapes of a spontaneous Raman spectrum17.

View Article: PubMed Central - PubMed

ABSTRACT

We present a wide-field imaging implementation of Fourier transform coherent anti-Stokes Raman scattering (wide-field detected FT-CARS) microscopy capable of acquiring high-contrast label-free but chemically specific images over the full vibrational ‘fingerprint’ region, suitable for a large field of view. Rapid resonant mechanical scanning of the illumination beam coupled with highly sensitive, camera-based detection of the CARS signal allows for fast and direct hyperspectral wide-field image acquisition, while minimizing sample damage. Intrinsic to FT-CARS microscopy, the ability to control the range of time-delays between pump and probe pulses allows for fine tuning of spectral resolution, bandwidth and imaging speed while maintaining full duty cycle. We outline the basic principles of wide-field detected FT-CARS microscopy and demonstrate how it can be used as a sensitive optical probe for chemically specific Raman imaging.

No MeSH data available.


Related in: MedlinePlus