Dual-pump coherent anti-Stokes-Raman scattering microscopy

Abstract

We introduce dual-pump coherent anti-Stokes-Raman scattering (dual-CARS) microscopy. This new technique permits simultaneous imaging of two species characterized by different molecular vibrations, as well as the removal of nonresonant background. This is achieved by using three synchronized laser pulses probing two different vibrations. We demonstrate the virtues of the method by imaging a mixture of nondeuterated and deuterated lipids, clearly distinguishing the individual components and their organization in the mixed arrangement. Further, dual-CARS images of lipid stores in living Caenorhabditis elegans nematodes show that the suppression of the nonresonant background results in significantly enhanced image contrast.

Monitoring of lipid storage in Caenorhabditis elegans using coherent anti-Stokes Raman scattering (CARS) microscopy

“…After initial proof-of-principle measurements on C. elegans (16), the unique capabilities of CARS microscopy are here fully used for quantitative imaging of lipids in populations of different strains in vivo, allowing holistic understanding of lipid storage mechanisms. Specifically, we examined the influence of environmental conditions and genetically regulated signaling pathways on how energy is stored or alternatively used for reproduction and cell maintenance.…”

Coherent anti‐Stokes Raman Scattering Microscopy

“…The simultaneous use of two pump lasers allows the parallel recording of resonant and nonesonant images and the derivation of an image with a much reduced non-resonant background. [40] In nearly all cases, the purely resonant contribution is detected on a strong nonresonant background signal. In this case, laser-induced fluctuations, such as pulse-to-pulse power fluctuations, timing jitter between the pump and Stokes laser pulses, or changes in alignment, limit the detectability of the small resonant contribution on top of the large and noisy nonresonant background.…”

“…Also here, the main focus has been given to the detection of CÀH stretching vibrations of fatty acids either in mice [31,104] or in the model organism C. elegans [40] (see Figure 8). An obvious problem met in experiments with larger animals or with thick tissue slices [32] is the excitation geometry.…”

“…This can either be used to probe two Raman transitions or to reduce the nonresonant background. [40] In all cases in which a single Ti:Sapphire laser is used both as pump source and for the creation of the Stokes beam in a nonlinear process, femtosecond pulses are generally being used (on the basis of efficiency arguments). Because this limits the spectral resolution in the single-frequency CARS method, various approaches using a spectral chirp on the laser pulses have been realized to achieve spectral focusing.…”

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

“…It requires the detection of a small modulation on a large background but has been demonstrated successfully for imaging [9,10]. Dual pump CARS [11] measures the nonresonant background separately, requiring an extra laser input, long integration times, and high stability. Frequency modulation (fm) CARS [12] uses the mixing between the resonant and nonresonant signal to detect the amplitude modulation.…”