Coherent anti-Stokes Raman scattering (CARS) generates a strong label-free signal in the long wavenumber C─H stretching region. Lipid-rich myelinated tissues, such as brain and spinal cord, would appear to be ideal subjects for imaging with CARS laser-scanning microscopy. However, the highly ordered, biochemically complex, and highly scattering nature of such tissues complicate the use of the technique. A CARS microscopy approach is presented that overcomes the challenges of imaging myelinated tissue to achieve chemically and orientationally sensitive high-resolution images.
The pathology of multiple sclerosis involves the gray and white matter regions of the brain and spinal cord often
characterized by various combinations of demyelination, inflammatory infiltration, axonal degeneration, and later gliosis
in chronic lesions. While acute and chronic white matter lesions are well characterized and easily identified, evidence
indicates that the CNS of MS patients may be globally altered, with subtle abnormalities found in grossly normal
appearing white matter (NAWM) with histochemical stains and magnetic resonance imaging only indicating a general
alteration in tissue composition at best. Thus, the prototypical acute inflammatory lesion may merely represent the most
obvious manifestation of a chronic widespread involvement of the CNS, which is difficult to examine reliably. The
current study deals with the microstructure and biochemistry of demyelination, remyelination and axonal loss in various
regions in post-mortem human MS brain, especially NAWM areas around more typical acute and chronic lesions. The
myelin sheath, neuroglia and perivascular spaces were investigated through changes in the intrinsic molecular vibrational
signatures of lipid biochemistry using a novel, label-free Coherent anti-Stokes Raman Scattering (CARS) microscope.
The biochemistry of myelin lipids can be probed by detecting subtle changes to phospholipids and the intra-molecular
disorder of their fatty acid acyl chains, various oxidation products and general protein contributions. NAWM regions
surrounding pathological MS lesions were shown to reveal abnormalities despite morphological classifications indicating
otherwise. CARS data were correlated with immunohistochemical stains and lipophilic dyes. Spectral data were
analyzed using a unique non-linear algorithm, which allows quantification and classification through gated parameters
and displayed through bivariate histograms. Our CARS microscopy system provides high-resolution, detailed
morphological and unique biochemical information regarding CNS pathology in human MS examples and may be
applicable to a broad range of other white matter centric neurological disorders.
Today’s commercially available microscopes offer a wide array of options to accommodate common imaging experiments. Occasionally, an experimental goal will require an unusual light source, filter, or even irregular sample that is not compatible with existing equipment. In these situations the ability to modify an existing microscopy platform with custom accessories can greatly extend its utility and allow for experiments not possible with stock equipment. Light source conditioning/manipulation such as polarization, beam diameter or even custom source filtering can easily be added with bulk components. Custom and after-market detectors can be added to external ports using optical construction hardware and adapters. This paper will present various examples of modifications carried out on commercial microscopes to address both atypical imaging modalities and research needs. Violet and near-ultraviolet source adaptation, custom detection filtering, and laser beam conditioning and control modifications will be demonstrated. The availability of basic ‘building block’ parts will be discussed with respect to user safety, construction strategies, and ease of use.
The pathology of multiple sclerosis (MS) involves both the gray and white matter regions of the brain and spinal cord. It is characterized by various combinations of demyelination, inflammatory infiltration, axonal degeneration, and later
gliosis in chronic lesions. While acute and chronic white matter plaques are well characterized and easily identified,
evidence indicates that the CNS of MS patients may be globally altered, with subtle abnormalities found in grossly
normal appearing white matter (NAWM) and in diffusely abnormal white matter (DAWM) where histochemical stains
and advanced magnetic resonance imaging indicate altered tissue composition. Thus, the prototypical acute
inflammatory lesion may merely represent the most obvious manifestation of a chronic widespread involvement of the
CNS, which is difficult to examine reliably. The current study deals with the microstructure and biochemistry of demyelination, remyelination and axonal loss in various regions of post-mortem human MS brain, including NAWM, areas of remyelination and more typical acute and chronic lesions. The myelin sheath, neuroglia and perivascular spaces were investigated using a novel Coherent Anti-Stokes Raman Scattering (CARS) microscope with simultaneous Two-Photon Excited Fluorescence (TPEF) imaging. The active CH stretching region between ~ 2800 and 3000 cm-1 was probed to provide chemically specific, high resolution, label-free imaging pertaining to the progression of the disease. CARS data were correlated with TPEF and conventional histochemical and immunohistochemical stains.
Our novel CARS microscopy system provides detailed morphological and biochemical information regarding CNS
pathology in MS and that may be applicable to a broad range of other human brain and spinal cord disorders.
The current trend in laser sources for Coherent Anti-Stokes Raman Scattering (CARS) microscopy consists of picosecond optical parametric oscillators (OPO)s and femtosecond-pumped fiber supercontinuum sources. While both
methods are proven CARS performers, restricted wavelength tuning range and low power limit the Raman lines and
types of samples that may be practically interrogated. To address these limitations, we present a novel, highly tunable
spectrally focused femtosecond Optical Parametric Amplifier (OPA) and microscope system optimized for CARS
microscopy. The laser source consists of an amplified ytterbium fiber laser driving a pair of OPAs producing two outputs that produce tunable femtosecond pulses from 650 to 1300nm. Each OPA may be tuned independently of the other over its entire range, allowing the addressing of any arbitrary wavenumber from 0 to 7700 cm-1. Additionally, the complete freedom of tuning allows one beam to be set at the optimal wavelength for a complementary technique, such as twophoton fluorescence or second harmonic, while the second beam is then tuned to the desired wavenumber difference for CARS. The femtosecond pulses are chirped out to the picosecond regime, reducing non-resonant background and providing improved spectral resolution. Typically, OPA systems are limited to kHz repetition rates, making them impractical for imaging applications. In contrast, our OPA system is driven at 1 MHz, providing a sufficient pulse rate for high-resolution imaging at rates of 1-2 frames per second. The 1 MHz rate preserves good pulse energy while reducing average power, thus limiting sample photo damage.
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