Background free CARS imaging by phase sensitive heterodyne CARS

Jurna, M.; Korterik, Jeroen P.; Otto, Cornelis; Herek, Jennifer Lynn; Offerhaus, Herman L.

doi:10.1364/oe.16.015863

Cited by 64 publications

(60 citation statements)

References 15 publications

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“…The nonresonant background, however, makes it diffi cult to perform CARS imaging based on the fi ngerprint bands. Various advanced CARS imaging techniques have been developed to suppress the nonresonant contribution, including polarization-sensitive detection ( 35 ), time-resolved detection ( 121 ), heterodyne detection ( 122,123 ), and frequency modulation ( 122,124 ). However, the complexity of these techniques hinders their biological applications.…”

Section: Study Of Myelin Diseasesmentioning

confidence: 99%

Shedding new light on lipid biology with coherent anti-Stokes Raman scattering microscopy

Yue

Cheng

2010

Journal of Lipid Research

147

130

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Despite the ubiquitous roles of lipids in biology, the detection of lipids has relied on invasive techniques and population measurements. The molecular profi les of the lipid-rich structures are evaluated by measurements of total lipid extracts with liquid or gas chromatography coupled to mass spectrometry ( 8 ). This approach is inexact because it measures lipid molecules from areas other than the structures of interest. Furthermore, spatial information, which is critical to understanding the function of lipids ( 9 ), is inaccessible with the bulk measurement techniques. To visualize the lipid-rich structures, fl uorescently tagged lipid molecules, which intercalate with the lipidrich structures, are used ( 10, 11 ). Nevertheless, the use of the fl uorescent lipid molecules to label lipid-rich structures is problematic for several reasons. First, to facilitate the transport of the fl uorescent lipid molecules into cells, cell fi xation procedures are often performed ( 12 ). This procedure prevents monitoring the dynamic responses of lipid-rich structures to stimuli or the disease processes. Second, labeling effi ciency is highly dependent on the type of fl uorescent lipid molecules ( 12 ), thus posing a signifi cant problem to the quantitation of lipid-rich structures. Third, the intercalation of fl uorescent lipid molecules can lead to changes in the properties of lipidrich structures. For instance, incorporation of fl uorescent lipid molecules into the cell membrane can induce membrane phase separation and membrane microdomain formation, which can perturb critical cellular processes including signal transduction and endocytosis ( 13 ).As an alternative to fl uorescence, signals from molecular vibration provide an attractive means for label-free chemical imaging. In particular, Raman scattering has been widely used for spectroscopic study of biomolecules ( 14 ). The Raman-scattered photons exhibit frequency Abstract Despite the ubiquitous roles of lipids in biology, the detection of lipids has relied on invasive techniques, population measurements, or nonspecifi c labeling. Such diffi culties can be circumvented by a label-free imaging technique known as coherent anti-Stokes Raman (CARS) microscopy, which is capable of chemically selective, highly sensitive, and high-speed imaging of lipid-rich structures with submicron three-dimensional spatial resolution. We review the broad applications of CARS microscopy to studies of lipid biology in cell cultures, tissue biopsies, and model organisms. Recent technical advances, limitations of the technique, and perspectives are discussed. Lipids play a critical role in human health and diseases. Phospholipids, glycolipids, and sterol lipids are the major components of the cell membrane ( 1 ). Sphingolipids constitute up to 80% of the myelin sheaths, which are the membranous structures that wrap around the axons for insulation and for proper propagation of electrical impulses ( 2 ). Glycerolipids or triglycerides serve as the cytoplasmic energy depots ( 3 ). Bioactive lip...

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Section: Study Of Myelin Diseasesmentioning

confidence: 99%

Shedding new light on lipid biology with coherent anti-Stokes Raman scattering microscopy

Yue

Cheng

2010

Journal of Lipid Research

147

130

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“…Several heterodyne detected optical imaging techniques based on interferometric CARS [1][2][3], stimulated Raman scattering (SRS) [4], transient absorption [5], and photothermal effect [6] have shown great potential for label-free imaging, e.g., SRS microscopy [7,[8][9][10][11] has been used for vibrational imaging of biomass [12], pharmaceutical samples [13,14], and lipid bodies [10,15]. All these techniques require the extraction of a small AC signal at the sub-microvolt level from a noisy environment.…”

Section: Introductionmentioning

confidence: 99%

Heterodyne detected nonlinear optical imaging in a lock‐in free manner

Slipchenko

Oglesbee

Zhang

et al. 2012

Journal of Biophotonics

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We report a compact, cost‐effective tuned amplifier for frequency‐selective amplification of the modulated signal in heterodyne detected nonlinear optical microscopy. Our method improved the signal to noise ratio by an order of magnitude compared to conventional lock‐in detection, as demonstrated through stimulated Raman scattering imaging of live cells and tissues at the speed of 2 μsec/pixel. Application of the tuned amplifier to transient absorption microscopy is also demonstrated. The increased signal to noise ratio allowed epi‐detected in vivo imaging of myelin and blood in rat spinal cord with high spatial resolution. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

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“…This is the phase difference that distinguishes between the real (nonresonant) and the imaginary (resonant, Raman) part [18]. However, this is not directly the phase measured in the focal volume; even for copropagating fields this phase is disturbed by refractive index changes in the sample, curvature of the field of view, and interferometric instabilities.…”

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confidence: 99%

Vibrational Phase Contrast Microscopy by Use of Coherent Anti-Stokes Raman Scattering

et al. 2009

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In biological samples the resonant coherent anti-Stokes Raman scattering signal of less abundant constituents can be overwhelmed by the nonresonant background, preventing detection of those molecules. We demonstrate a method to obtain the phase of the oscillators in the focal volume that allows discrimination of those hidden molecules. The phase is measured with respect to the local excitation fields using a cascaded phase-preserving chain. It is measured point-by-point and takes into account refractive index changes in the sample, phase curvature over the field-of-view, and interferometric instabilities. The detection of the phase of the vibrational motion can be regarded as a vibrational extension of the linear (refractive index) phase contrast microscopy introduced by Zernike around 1933.

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Background free CARS imaging by phase sensitive heterodyne CARS

Cited by 64 publications

References 15 publications

Shedding new light on lipid biology with coherent anti-Stokes Raman scattering microscopy

Shedding new light on lipid biology with coherent anti-Stokes Raman scattering microscopy

Heterodyne detected nonlinear optical imaging in a lock‐in free manner

Vibrational Phase Contrast Microscopy by Use of Coherent Anti-Stokes Raman Scattering

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