On illumination schemes for wide-field CARS microscopy

Abstract

New system for a wide-field CARS microscopy is demonstrated, including two schemes of non-phase-matching illumination. Several advantages including high Stokes pulse energy, pulse-to-pulse stability and inherent synchronization between pump and Stokes pulses were brought by use of methane-filled Raman converter. Spatial resolution of the system with axially symmetric illumination, 0.5 microm, was found to correspond to diffraction limit of the imaging objective. Selective sensitivity to lipid-rich myelin sheaths in the nerve tissue has been demonstrated and confirmed by comparison with histological samples stained with myelin-specific dye. Single-shot imaging capability of the system has been demonstrated with a speckling-free illumination on a monolayer of 3 microm polystyrene beads.

Video-rate wide-field coherent anti-Stokes Raman scattering microscopy with collinear nonphase-matching illumination

“…Instead, within the experimental errors, the same CARS intensities are observed, which is in contrast to earlier wide-field CARS microscopy experiments. [11][12][13][14][15] This supports the notion that CARS signal generation in this collinear illumination approach in a nonphase-matching geometry mainly stems from laser refraction and scattering in the sample. It is noteworthy that when we changed the position of the common focus of the pump and Stokes beam from above the sample, to below the sample also no change in signal intensity was observed (Fig.…”

“…10 The latter problem prompted the development of alternative CARS excitation geometries based on wide-field excitation. [11][12][13][14][15] This technique allows for simultaneous imaging of a large field of view. Single-shot imaging capability using nanosecond pulses has been demonstrated, which makes way for investigations of fast biological processes and chemical reactions.…”

“…14,15 While both approaches are promising, their implementation is technically demanding, since high-power, low-repetition-rate laser systems such as Q-switched Nd:YAG lasers [11][12][13] or regenerative Ti:sapphire amplifiers 14,15 and special optical elements such as ultradark-field condensers 12 or Cassegrain objectives 15 are necessary. To homogeneously illuminate the sample, both approaches apply homogenizers such as multimode fibers [11][12][13] or microlens arrays, 15 which inevitably produce speckle noise in the sample plane. Dynamic averaging of the speckle noise by random waggling of the homogenizers will give a smooth image, but also limit the image acquisition speed.…”

“…Dynamic averaging of the speckle noise by random waggling of the homogenizers will give a smooth image, but also limit the image acquisition speed. 15 We present a simple video-rate wide-field CARS microscopy experiment using a nonphase-matching wide-field illumination scheme. Picosecond oscillator laser systems are used for excitation.…”

Raman Imaging of Lignocellulosic Feedstock

“…The main methods for Raman imaging are scanning (mapping) methods (Point-by-Point and Line scanning) and Wide-field source illumination approaches [114][115][116][117].…”