Stimulated Raman Scattering (SRS) microscopy is a nonlinear microscopy technique based on Raman vibrational resonances determined by the frequency difference between Pump and Stokes laser pulses. Modulation of one laser beam transfers the modulation to the other, as either a gain in Stokes (SRG) or a loss in Pump power (SRL). SRS microscopy does not exhibit the four-wave mixing nonresonant background characteristic of CARS microscopy. However, other background signals due to two-photon absorption, thermal lensing or cross-phase modulation (XPM) do reduce the detection sensitivity and can distort the hyperspectral scans. Phase sensitive lock-in detection can reduce contributions from two-photon absorption, which is out-of-phase for the SRG case. However, the background signal due to XPM, which can be in-phase with SRS, can reduce the detection sensitivity.
We present a novel polarization modulation (PM) scheme in SRS microscopy which greatly reduces the nonresonant XPM background, demonstrated here for the SRL case. Since many Raman vibrational transitions are parallel polarized, the SRS signal is maximum (minimum) when the polarizations of the pump and the Stokes beams are parallel (perpendicular). However, in both parallel and perpendicular Pump-Stokes geometries, XPM is non-zero in many media. Therefore, PM can remove the XPM background without significantly reducing the SRS signal. Our results show that the PM-SRS successfully removes the nonresonant signal due to XPM. High imaging contrast is observed, concomitant with high sensitivity at very low analyte concentrations and undistorted Raman spectra.
KEYWORDS: In vivo imaging, Microscopy, Raman spectroscopy, CARS tomography, Absorption, Resonance enhancement, Microscopes, Signal processing, Visualization, In vitro testing
Coherent anti-Stokes Raman scattering (CARS) and four-wave-mixing (FWM) microscopy are a related pair of
powerful nonlinear optical characterization tools. These techniques often yield strong signals from concentrated
samples, but because of their quadratic dependence on concentration, they are not typically employed for imaging or
identifying dilute cellular constituents. We report here that, depending on the excitation wavelengths employed, both
CARS and degenerate-FWM signals from carotenoid accumulations in alga cysts can be exceptionally large, allowing
for low-power imaging of astaxanthin (AXN) deposits in Haematococcus pluvialis microalga. By use of a broadband
laser pulse scheme for CARS and FWM, we are able to simultaneously collect strong intrinsic two-photon-excitation
fluorescence signals from cellular chlorophyll in vivo. We show that CARS signals from astaxanthin (AXN) samples in
vitro strictly follow the expected quadratic dependence on concentration, and we demonstrate the collection of wellresolved
CARS spectra in the fingerprint region with sensitivity below 2mM. We suggest that multimodal nonlinear
optical microscopy is sufficiently sensitive to AXN and chlorophyll concentrations that it will allow for non-invasive
monitoring of carotenogenesis in live H. pluvialis microalgae.
Hyperspectral coherent anti-Stokes Raman scattering (CARS) microscopy has provided an imaging tool for
extraction of 3-dimensional volumetric information, as well as chemically-sensitive spectral information. These
techniques have been used in a variety of different domains including biophysics, geology, and material science.
The measured CARS spectrum results from interference between the Raman response of the sample and a non-resonant
background. We have collected four dimensional data sets (three spatial dimensions, plus spectra)
and extracted Raman response from the CARS spectrum using a Kramers-Kronig transformation. However,
the three dimensional images formed by a CARS microscope are distorted by interference, some of which arises
because of the Gouy phase shift. This type of interference comes from the axial position of the Raman resonant
object in the laser focus. We studied how the Gouy phase manifests itself in the spectral domain by investigating
microscopic diamonds and nitrobenzene droplets in a CARS microscope. Through experimental results and
numerical calculation using finite-diference time-domain (FDTD) methods, we were able to demonstrate the
relationship between the spatial configuration of the sample and the CARS spectral response in three dimensional
space.
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.
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.
Biological cells can be considered as dielectric objects with a given refractive index distribution. Light scattering simulations provide us with an efficient tool for studying cell morphology as well as the nature of scattering and its sources. The analysis of this information is the basis for a better understanding and development of new optical methods for non-invasive biomedical diagnostics. Here we demonstrate the potential of Finite-Difference Time-Domain (FDTD) method based software tools for the simulation of light scattering from single cells in situations where other approaches simply do not work or the approximations inherited in them begin to be questionable.
The photolysis, geminate recombination and vibrational relaxation of the low affinity ferrous myoglobin nicotinate complex have been studied by femtosecond transient absorption spectroscopy. This is an interesting system due to the peculiar interaction between ligand and protein fluctuations. This ligand is bulky and affects the naturally occurring protein fluctuations in a way similar to a doorstop precluding a door from closing totally. The whole Q band absorption transient spectrum of the photoproduct has been monitored starting from 100 fs to 100 ps. The time evolution of the spectrum has clearly shown two distinct phases, a vibrational cooling process occurring within 4 ps after the photolyzing pulse and a geminate rebinding process with a time constant of 28.8 +/- 0.1 ps. The transient spectra show different cooling rates for the different excited normal modes. The geminate rebinding process appears to be complete within 100 ps and hence appears to be the fastest geminate recombination process reported to date for a hemoprotein. This is the first report on the photolysis of a ferrous heme adduct with a nitrogenous base, previously considered as photoinert.
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