Biomolecular Imaging with Coherent Nonlinear Vibrational Microscopy

Chung, Chao-Yu; Potma, Eric O.

doi:10.1146/annurev-physchem-040412-110103

Cited by 105 publications

(91 citation statements)

References 140 publications

(130 reference statements)

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“…Various implementations of SFG microscopy have been realized, based on either a point-or widefield-illumination configuration. [7][8][9][10][11][12] These various forms of SFG microscopy have been used to study a growing number of spatially heterogeneous samples, including patterned molecular monolayers on planar surfaces 7 , biomolecular microcrystals [13][14] and biofibers. 9,[15][16] Among the various forms of SFG microscopy, the point-scanning configuration with collinear excitation beams is attractive because the microscope layout is similar to a standard laser-scanning nonlinear optical microscope.…”

Section: Introductionmentioning

confidence: 99%

Polarization-Sensitive Sum-Frequency Generation Microscopy of Collagen Fibers

Han

Hsu

et al. 2015

J. Phys. Chem. B

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Point-scanning sum-frequency generation (SFG) microscopy enables the generation of images of collagen I fibers in tissues by tuning into specific vibrational resonances of the polypeptide. It is shown that when collagen-rich tissues are visualized near the 2954 cm -1 stretching vibration of methylene groups, the SFG image contrast is higher compared to the contrast seen in nonresonant second-harmonic generation (SHG) imaging.Polarization and spectrally resolved analysis of the SFG signal as a function of fiber orientation in the CH-stretching range of the vibrational spectrum enabled a comparative characterization of the achiral tensor elements of collagen's second-order susceptibility. This analysis reveals that selected on-resonance tensor elements are enhanced over other elements, giving rise to a much stronger anisotropy ρ of the signal for SFG ( ρ≈15 ) compared to SHG ( ρ≈3 ). The improved anisotropy of the vibrationally resonant signal contributes to the higher contrast seen in the SFG tissue images.

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Section: Introductionmentioning

confidence: 99%

Polarization-Sensitive Sum-Frequency Generation Microscopy of Collagen Fibers

Han

Hsu

et al. 2015

J. Phys. Chem. B

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“…In SFG, the sample is illuminated by a mid-IR beam at ω 1 and a vis/NIR beam at ω 2 , producing a signal at ω 3 = ω 1 + ω 2 [4][5][6]. It can be considered a hybrid between SHG and CARS.…”

Section: Introductionmentioning

confidence: 99%

Hyperspectral imaging with laser-scanning sum-frequency generation microscopy

Hanninen¹,

Shu²,

Potma³

2017

Biomed. Opt. Express

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Vibrationally sensitive sum-frequency generation (SFG) microscopy is a chemically selective imaging technique sensitive to non-centrosymmetric molecular arrangements in biological samples. The routine use of SFG microscopy has been hampered by the difficulty of integrating the required mid-infrared excitation light into a conventional, laser-scanning nonlinear optical (NLO) microscope. In this work, we describe minor modifications to a regular laserscanning microscope to accommodate SFG microscopy as an imaging modality. We achieve vibrationally sensitive SFG imaging of biological samples with sub-µm resolution at image acquisition rates of 1 frame/s, almost two orders of magnitude faster than attained with previous point-scanning SFG microscopes. Using the fast scanning capability, we demonstrate hyperspectral SFG imaging in the CH-stretching vibrational range and point out its use in the study of molecular orientation and arrangement in biologically relevant samples. We also show multimodal imaging by combining SFG microscopy with second-harmonic generation (SHG) and coherent anti-Stokes Raman scattering (CARS) on the same imaging platfrom. This development underlines that SFG microscopy is a unique modality with a spatial resolution and image acquisition time comparable to that of other NLO imaging techniques, making point-scanning SFG microscopy a valuable member of the NLO imaging family.

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“…Coherent anti-Stokes Raman scattering (CARS) microscopy is a nonlinear, label-free imaging technique that has matured into a reliable tool for visualizing lipids, proteins and other endogenous compounds in biological tissues and cells based on their spatially-dependent third-order polarization [1][2][3]. In the CARS process, a pair of incoming beams (named "pump" and "Stokes") are exploited to coherently and resonantly excite selected vibrational levels of a population of molecules.…”

Section: Introductionmentioning

confidence: 99%

Effect of scattering on coherent anti-Stokes Raman scattering (CARS) signals

Ranasinghesagara¹,

Vito²,

Piazza³

et al. 2017

Opt. Express

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Abstract:We develop a computational framework to examine the factors responsible for scattering-induced distortions of coherent anti-Stokes Raman scattering (CARS) signals in turbid samples. We apply the Huygens-Fresnel Wave-based Electric Field Superposition (HF-WEFS) method combined with the radiating dipole approximation to compute the effects of scatteringinduced distortions of focal excitation fields on the far-field CARS signal. We analyze the effect of spherical scatterers, placed in the vicinity of the focal volume, on the CARS signal emitted by different objects (2µm diameter solid sphere, 2µm diameter myelin cylinder and 2µm diameter myelin tube). We find that distortions in the CARS signals arise not only from attenuation of the focal field but also from scattering-induced changes in the spatial phase that modifies the angular distribution of the CARS emission. Our simulations further show that CARS signal attenuation can be minimized by using a high numerical aperture condenser. Moreover, unlike the CARS intensity image, CARS images formed by taking the ratio of CARS signals obtained using xand y-polarized input fields is relatively insensitive to the effects of spherical scatterers. Our computational framework provide a mechanistic approach to characterizing scattering-induced distortions in coherent imaging of turbid media and may inspire bottom-up approaches for adaptive optical methods for image correction.

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Biomolecular Imaging with Coherent Nonlinear Vibrational Microscopy

Cited by 105 publications

References 140 publications

Polarization-Sensitive Sum-Frequency Generation Microscopy of Collagen Fibers

Polarization-Sensitive Sum-Frequency Generation Microscopy of Collagen Fibers

Hyperspectral imaging with laser-scanning sum-frequency generation microscopy

Effect of scattering on coherent anti-Stokes Raman scattering (CARS) signals

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