Luminal atherosclerosis imaging was demonstrated by multimodal femtosecond CARS
microscopy (MM-CARS). Using a myocardial infarction-prone rabbit model of
atherosclerosis, this study demonstrated the utility of multimodal CARS imaging in
determining atherosclerotic plaque burden through two types of image analysis procedures.
Firstly, multimodal CARS images were evaluated using a signal-intensity parameter based
on intensity changes derived from the multi-channel data (e.g. TPEF, SHG and CARS) to
classify plaque burden within the vessel. Secondly, the SHG images that mainly correspond
to collagen fibrils were evaluated using a texture analysis model based on the first-order
statistical (FOS) parameters of the image histogram. Correlation between FOS parameters of
collagen images with atherosclerosis plaque burden was established. A preliminary study
of using spectroscopic CARS in identifying the different lipid components within the plaque
was also discussed.
We demonstrate the simultaneous collection and separation of femtosecond-laser-based forward-collected coherent anti-Stokes Raman scattering (F-CARS) and two-photon-excitation-induced fluorescence lifetime images (FLIM) using time-correlated single photon counting (TCSPC). We achieve this in a nondescanned geometry using a single multimode fiber without significant loss of light, field of view, and most importantly, TCSPC timing fidelity. In addition to showing the ability to separate CARS images from FLIM images using time gating, we also demonstrate composite multimodal epicollected FLIM imaging with fiber-collected F-CARS imaging in live cells.
Label-free imaging of bulk arterial tissue is demonstrated using a multimodal nonlinear optical microscope based on a photonic crystal fiber and a single femtosecond oscillator operating at 800 nm. Colocalized imaging of extracellular elastin fibers, fibrillar collagen, and lipid-rich structures within aortic tissue obtained from atherosclerosis-prone myocardial infarction-prone Watanabe heritable hyperlipidemic (WHHLMI) rabbits is demonstrated through two-photon excited fluorescence, second harmonic generation, and coherent anti-Stokes Raman scattering, respectively. These images are shown to differentiate healthy arterial wall, early atherosclerotic lesions, and advanced plaques. Clear pathological changes are observed in the extracellular matrix of the arterial wall and correlated with progression of atherosclerotic disease as represented by the age of the WHHLMI rabbits.
Traditional CARS microscopy using picosecond (ps) lasers has been applied to a wide variety of applications;
however, the lasers required are expensive and require an environmentally stable lab. In our work, we demonstrate
CARS microscopy using a single femtosecond (fs) laser combined with a photonic crystal fiber (PCF) and optimal
chirping to achieve similar performance to the ps case with important added advantages: fs-CARS utilizes
versatile source that allows CARS to be combined with other multiphoton techniques (e.g. SHG, TPF) for
multimodal imaging without changing laser sources. This provides an attractive entry point for many researchers
to the field. Furthermore, optimal chirping in fs-CARS also opens the door to the combination and extension
of other techniques used in ps CARS microscopy such as multiplex and FM imaging. The key advantage with
chirped fs pulses is that time delay corresponds to spectral scanning and allows for rapid modulation of the
resonant CARS signal. The combination of a fs oscillator with a PCF leads to a natural extension of the
technology towards an all-fiber source for multimodal multiphoton microscopy. An all-fiber system should be
more robust against environmental fluctuations while being more compact than free-space systems. We have
constructed and demonstrated a proof of concept all-fiber based source that can be used for simultaneous CARS,
TPF and SHG imaging. This system is capable of imaging tissue samples and live cell cultures with 4 μs/pixel
dwell time at low average powers.
In this study we compare the single-photon autofluorescence and multi-photon emission spectra obtained from the
luminal surface of healthy segments of artery with segments where there are early atherosclerotic lesions. Arterial tissue
was harvested from atherosclerosis-prone WHHL-MI rabbits (Watanabe heritable hyperlipidemic rabbit-myocardial
infarction), an animal model which mimics spontaneous myocardial infarction in humans. Single photon fluorescence
emission spectra of samples were acquired using a simple spectrofluorometer set-up with 400 nm excitation. Samples
were also investigated using a home built multi-photon microscope based on a Ti:sapphire femto-second oscillator. The
excitation wavelength was set at 800 nm with a ~100 femto-second pulse width. Epi-multi-photon spectroscopic signals
were collected through a fibre-optics coupled spectrometer. While the single-photon fluorescence spectra of
atherosclerotic lesions show minimal spectroscopic difference from those of healthy arterial tissue, the multi-photon
spectra collected from atherosclerotic lesions show marked changes in the relative intensity of two-photon excited
fluorescence (TPEF) and second-harmonic generation (SHG) signals when compared with those from healthy arterial
tissue. The observed sharp increase of the relative SHG signal intensity in a plaque is in agreement with the known
pathology of early lesions which have increased collagen content.
We demonstrate high performance coherent anti-Stokes Raman scattering (CARS) microscopy using a single
femtosecond Ti:Sapphire laser source combined with a photonic crystal fiber (PCF). By adjusting the chirp of
the femtosecond pump and Stokes laser pulses, we achieve high quality multimodal imaging (simultaneous CARS,
two-photon fluorescence, and second harmonic generation) of live cells and tissues. The tuneable Ti:sapphire
output provides the pump beam directly, while part of this is converted to the red-shifted Stokes pulse using a
PCF having two close-lying zero dispersion wavelengths. This PCF gives good power and stability over Stokes
shifts ranging from below 2300 cm-1 to over 4000 cm-1. This tuning range can be accessed by simply controlling
the time delay between the input pulses. This allows fast, continuous computer-controlled tuning of the Stokes
shift over a broad range, without involving any adjustment of either the femtosecond laser or the PCF. The
simultaneous optimization of CARS, two-photon fluorescence and second harmonic generation is achieved by
controlling the degree of chirp and involves a trade-off between spectral resolution of the CARS process and
signal strength. This is illustrated by showing applications of the multimodal CARS imaging and optimization
technique to biomedical problems involving both live cells and tissues.
Nonlinear optical (NLO) microscopy provides a minimally invasive optical method for
fast molecular imaging at subcellular resolution with 3D sectioning capability in thick,
highly scattering biological tissues. In the current study, we demonstrate the imaging
of arterial tissue using a nonlinear optical microscope based on photonic crystal fiber
and a single femto-second oscillator operating at 800nm. This NLO microscope system
is capable of simultaneous imaging extracellular elastin/collagen structures and lipid
distribution within aortic tissue obtained from coronary atherosclerosis-prone WHHLMI
rabbits (Watanabe heritable hyperlipidemic rabbit-myocardial infarction) Clear
pathological differences in arterial lumen surface were observed between healthy
arterial tissue and atherosclerotic lesions through NLO imaging.
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