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.
Laser-scanning optical microscopes generally do not control the polarization of the exciting laser field. We show that laser polarization and imaging mode (confocal versus two photon) exert a profound influence on the ability to detect Ca2+ changes in both cultured neurons and living myelin. With two-photon excitation, increasing ellipticity resulted in a ≈50% reduction in resting X-Rhod-1 fluorescence in homogeneous bulk solution, cell cytoplasm, and myelin. In contrast, varying the angle of a linearly polarized laser field only had appreciable effects on dyes that partitioned into myelin in an ordered manner. During injury-induced Ca2+ increases, larger ellipticities resulted in a significantly greater injury-induced signal increase in neurons, and particularly in myelin. Indeed, the traditional method of measuring Ca2+ changes using one-photon confocal mode with linearly polarized continuous wave laser illumination produced no appreciable X-Rhod-1 signal increase in ischemic myelin, compared to a robust ≈50% fluorescence increase with two-photon excitation and optimized ellipticity with the identical injury paradigm. This underscores the differences in one- versus two-photon excitation and, in particular, the under-appreciated effects of laser polarization on the behavior of certain Ca2+ reporters, which may lead to substantial underestimates of the real Ca2+ fluctuations in various cellular compartments.
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.
The rapid prototyping capability offered by 3D printing is considered advantageous for commercial applications. However, the ability to quickly produce precision custom devices is highly beneficial in the research laboratory setting as well. Biological laboratories require the manipulation and analysis of delicate living samples, thus the ability to create custom holders, support equipment, and adapters allow the extension of existing laboratory machines. Applications include camera adapters and stage sample holders for microscopes, surgical guides for tissue preparation, and small precision tools customized to unique specifications. Where high precision is needed, especially the reproduction of fine features, a printer with a high resolution is needed. However, the introduction of cheaper, lower resolution commercial printers have been shown to be more than adequate for less demanding projects. For direct manipulation of delicate samples, biocompatible raw materials are often required, complicating the printing process. This paper will examine some examples of 3D-printed objects for laboratory use, and provide an overview of the requirements for 3D printing for this application. Materials, printing resolution, production, and ease of use will all be reviewed with an eye to producing better printers and techniques for laboratory applications. Specific case studies will highlight applications for 3D-printed devices in live animal imaging for both microscopy and Magnetic Resonance Imaging.
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.
Laser-scanning non-linear optical techniques such as multi-photon fluorescence excitation microscopy (MPM), Second/
Third Harmonic Generation (SHG/THG), and Coherent Anti-Stokes Raman Scattering (CARS) are being utilized in
research laboratories worldwide. The efficiencies of these non-linear effects are dependent on the polarization state of
the excitation light relative to the orientation of the sample being imaged. In highly ordered anisotropic biological
samples this effect can become pronounced and the excitation polarization can have a dramatic impact on imaging
experiments. Therefore, controlling the polarization state of the exciting light is important; however this is challenging
when the excitation light passes through a complex optical system. In a typical laser-scanning microscope, components
such as the dichroic filters, lenses, and even mirrors can alter the polarization state of a laser beam before it reaches the
sample. We present an opto-mechanical solution to compensate for the polarization effects of an optical path, and to
precisely program the polarization state of the exciting laser light. The device and accompanying procedures allow the
delivery of precise laser polarization states at constant average power levels to a sample during an imaging experiment.
Myelination, i.e. the wrapping of axons in multiple layers of lipid-rich membrane, is a unique phenomenon in the
nervous systems of both vertebrates and invertebrates, that greatly increases the speed and efficiency of signal
transmission. In turn, disruption of axo-myelinic integrity underlies disability in numerous clinical disorders. The
dependence of myelin physiology on nanometric organization of its lamellae makes it difficult to accurately study this
structure in the living state. We expected that fluorescent probes might become highly oriented when partitioned into the
myelin sheath, and in turn, this anisotropy could be interrogated by controlling the polarization state of the exciting laser
field used for 2-photon excited fluorescence (TPEF). Live ex vivo myelinated rodent axons were labeled with a series of
lipohilic and hydrophilic fluorescenct probes, and TPEF images acquired while laser polarization was varied at the
sample over a broad range of ellipticities and orientations of the major angle [see Brideau, Micu & Stys, abstract this
meeting]. We found that most probes exhibited strong dependence on both the major angle of polarization, and perhaps
more surprisingly, on ellipticity as well. Lipophilic vs. hydrophilic probes exhibited distinctly different behavior. We
propose that polarization-dependent TPEF microscopy represents a powerful tool for probing the nanostructural
architecture of both myelin and axonal cytoskeleton in a domain far below the resolution limit of visible light
microscopy. By selecting probes with different sizes and physicochemical properties, distinct aspects of cellular
nanoarchitecture can be accurately interrogated in real-time in living tissue.
We demonstrate the operation of a novel portable, fibre delivery miniaturized multimodal microscope (exoscope) for
coherent anti-Stokes Raman scattering and two-photon excitation fluorescence imaging using a single Ti:sapphire
femtosecond pulsed laser. This microscope features a large mode area photonic crystal fibre for light delivery, as well as
biaxial scanning microelectromechanical system mirrors and custom miniaturized optics corrected for chromatic
aberration. We demonstrate imaging of polystyrene beads, two photon excitation fluorescence beads in both forward and
backward (epi) directions. This miniaturized exoscope will enable in-vivo imaging of rat spinal cord.
We discuss the design and implementation of a novel multimodal coherent anti-Stokes Raman scattering (CARS)
miniaturized microscope for imaging of injured and recovering spinal cords in a single living animal. We demonstrate
for the first time, the use of a biaxial microelectromechanical system (MEMS) mirror for scanning and diffraction
limited multiple lens miniaturized objective for exciting a CARS signal. The miniaturized microscope design includes
light delivery using a large mode area photonic crystal fiber (PCF), and multimode fiber for collection of the nonlinear
optical signal. The basic design concept, major engineering challenges, solutions, and preliminary results are presented.
We demonstrate CARS and two photon excitation fluorescence microscopy in a benchtop setup with the miniaturized
optics and MEMS scanning. The light source is based on a single femtosecond laser (pump beam) and a supercontinuum
generated in a nonlinear PCF (Stokes beam). This is coupled using free space optics onto the surface of a resonantly
driven two dimensional scanning MEMS mirror that scans the excitation light in a Lissajous pattern. The novel design of
the miniaturized microscope is expected to provide significant new information on the pathogenesis of demyelinating
diseases such as Multiple Sclerosis and Spinal Cord Injury.
As multiphoton microscopy increases in popularity users with diverse backgrounds are exploring new applications for
the technique. With the most recent 'turnkey' systems now on the market a typical user no longer has to be a laser
physicist or engineer to employ a multiphoton system in their research. However, some basic understanding of the
mechanisms of non-linear excitation will allow a user to optimize his multiphoton system for improved performance or
extend it for new applications.
We have recently demonstrated coherent anti-Stokes Raman scattering (CARS) microscopy and multiplex CARS spectroscopy of lipid-rich structures based on a single femtosecond Ti:sapphire laser. A nonlinear photonic crystal fiber (PCF) with two closely lying zero dispersion wavelengths is used to generate the Stokes pulse. Further optimization in terms of higher spectral resolution in the CARS spectra can be achieved by adding a second PCF to the pump arm to produce a spectrally compressed picosecond pulse. Theoretical predictions from modeling the propagation of the negatively chirped pump pulse in the PCF, are compared with experimental results. The effect of pulse duration, peak power and the length of the PCF in determining the bandwidth of the spectrally compressed pump pulse are considered. It is shown that for higher average output power and constant pulse duration, it is desirable to use shorter length of the PCF for attaining transform limited spectral width of the pump pulse.
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