Nonlinear optical microscopy for drug delivery monitoring and cancer tissue imaging

A multimodal nonlinear optical microscope that combines coherent anti‐Stokes Raman scattering (CARS), two‐photon excitation fluorescence (TPEF), second‐harmonic generation (SHG) and sum‐frequency generation (SFG) was developed and applied to image breast cancer tissue and MCF‐7 cells as well as monitoring anticancer drug delivery in live cells. TPEF imaging showed that drugs are preferentially localized in the cytoplasm and the nuclear envelope in resistant cells. Moreover, the extracellular matrix was observed by TPEF signals arising from elastin's autofluorescence and SHG signals from collagen fibrils in breast tissue sections. Additionally, CARS signals arising from proteins and (PO₂)⁻ allowed identification of tumors. Label‐free imaging with chemical contrast of significant components of cancer cells and tissue suggests the potential of multimodal nonlinear optical microscopy for early detection and diagnosis of cancer. Copyright © 2010 John Wiley & Sons, Ltd.     

相干反斯托克斯拉曼散射显微镜的成像特性

对自身不发荧光且不便于荧光标记的化学或生物学样品，集相干反斯托克斯拉曼散射与激光共焦扫描显微镜于一身的相干反斯托克斯拉曼散射显微镜是一种好的选择。因为相干反斯托克斯拉曼散射是一种非线性过程，相干反斯托克斯拉曼散射显微镜的显微成像特性与一般的共焦显微镜非常不同。首先计算了焦点附近相干反斯托克斯拉曼散射激发场的偏振分布，然后，利用格林函数方法，得到了以赫兹偶极子为源的波动方程的精确解，发现对于不同的成像配置和样品形状，像场的相干反斯托克斯拉曼散射场分布非常不同，因此传统的显微镜成像表征方式（如点扩展函数）将不再能描述相干反斯托克斯拉曼散射显微镜的成像特性。     

Label-free imaging of arterial cells and extracellular matrix using a multimodal CARS microscope

A multimodal nonlinear optical imaging system that integrates coherent anti-Stokes Raman scattering (CARS), sum-frequency generation (SFG), and two-photon excitation fluorescence (TPEF) on the same platform was developed and applied to visualize single cells and extracellular matrix in fresh carotid arteries. CARS signals arising from CH₂-rich membranes allowed visualization of endothelial cells and smooth muscle cells of the arterial wall. Additionally, CARS microscopy allowed vibrational imaging of elastin and collagen fibrils which are also rich in CH₂ bonds. The extracellular matrix organization was further confirmed by TPEF signals arising from elastin’s autofluorescence and SFG signals arising from collagen fibrils’ non-centrosymmetric structure. Label-free imaging of significant components of arterial tissues suggests the potential application of multimodal nonlinear optical microscopy to monitor onset and progression of arterial diseases.     

Chirped coherent anti-Stokes Raman scattering as a high-spectral- and spatial-resolution microscopy

Coherent anti-Stokes Raman scattering (CARS) microscopy is a promising tool for chemically selective imaging based on molecular vibrations. While CARS is currently used as a biological imaging tool, many variations are still being developed, perhaps the most important being multiplex CARS microscopy. Multiplex CARS has the advantage of comparing images based on different molecular vibrations without changing the excitation wavelengths. Here we demonstrate both high-spectral- and spatial-resolution multiplex CARS imaging of polymer films, using a simple scheme for chirped CARS with a spectral bandwidth of 300 cm(-1).     

Simple approach to one-laser, broadband coherent anti-Stokes Raman scattering microscopy

Coherent anti-Stokes Raman scattering (CARS) microscopy is emerging as a powerful method for imaging materials and biological systems, partly because of its noninvasiveness and selective chemical sensitivity. However, its full potential for species-selective imaging is limited by a restricted spectral bandwidth. Recent increases in bandwidth are promising but still are not sufficient for the level of robust component discrimination that would be needed in a chemically complex milieu found, for example, in intracellular and extracellular environments. We demonstrate a truly broadband CARS imaging instrument that we use to acquire hyperspectral images with vibrational spectra over a bandwidth of 2500 cm(-1) with a resolution of 13 cm(-1).     

Wide-field coherent anti-Stokes Raman scattering microscopy with non-phase-matching illumination

We have developed and tested a wide-field coherent anti-Stokes Raman scattering (CARS) microscopy technique, which provides the simultaneous imaging of an extended illuminated area without scanning. This method is based on the non-phase-matching illumination of a sample and imaging of a CARS signal with a CCD camera using conventional microscope optics. We have identified a set of conditions on the illumination and imaging optics, as well as on sample preparation. Imaging of test objects proved high spatial resolution and chemical selectivity of this technique.     

Detecting lateral interfaces with focus‐engineered coherent anti‐Stokes Raman scattering microscopy

Focus‐engineered coherent anti‐Stokes Raman scattering (FE‐CARS) microscopy is used to highlight the lateral interfaces between chemically distinct media. Interface highlighting is achieved by using a HG10 mode for the Stokes laser beam and a HG00 mode for the pump laser beam in the forward detection scheme. The spectral and the orientation dependence of FE‐CARS are found to be in agreement with theoretical predictions. A brief discussion on the relevance of this technique for imaging third‐order nonlinear susceptibility interfaces in thin samples of biological or chemical importance is presented. Copyright © 2008 John Wiley & Sons, Ltd.     

Applying the method of Coherent Anti‐stokes Raman microscopy for imaging of carotenoids in microalgae and cyanobacteria

Coherent anti‐stokes Raman scattering microscopy (CARS) was applied to visualize carotenoids in microalgae and cyanobacteria. Nonlinear light–matter interaction utilized in CARS microscopy inevitably induces a number of competing nonlinear processes, such as multiphoton excitation fluorescence. Microalgae and cyanobacteria being an intrinsically well‐fluorescent object generates a strong two‐photon‐excitation fluorescence (TPEF) signal which should be effectively suppressed during the CARS experiment. Using an energetically balanced duel‐wavelength excitation scheme and spectral purification of detecting signal, the TPEF was completely blocked providing a possibility to probe microalgae and cyanobacteria in a fingerprint region of the CARS spectrum. Microspectroscopy experiments were carried out with three species ‐ cyanobacteria Nostoc Commune, Nostoc sp. and Chlorella sp. Distinct bands obtained in CARS spectra of such species were assigned to carotenoids and were taken as spectral markers in the imaging experiment. CARS imaging known as a chemical selective and label‐free technique allows non‐invasion monitoring of accumulation and movement of chemical compound at the subcellular level. Obtained high‐resolution images of carotenoid distribution in algae and cyanobacteria clearly demonstrate the potential of CARS microscopy for spatially resolved analysis of the natural products stored in the microalgae and cyanobacteria cell. Copyright © 2013 John Wiley & Sons, Ltd.     

Differential two-signal picosecond-pulse coherent anti-Stokes Raman scattering imaging microscopy by using a dual-mode optical parametric oscillator

We propose and demonstrate a novel differential two-signal technique of coherent anti-Stokes Raman scattering (CARS) imaging microscopy using a picosecond (ps) optical parametric oscillator (OPO). By adjusting a Lyot filter inside the cavity, we operated the OPO oscillating in two stable modes separated by a few nanometers. The CARS images generated by the two modes are separated by a spectrograph behind the microscope setup, and their differential image is directly obtained by balanced lock-in detection. The feasibility of the technique is experimentally verified by imaging micrometer-sized polystyrene beads immersed in water.     

Image formation in CARS and SRS: effect of an inhomogeneous nonresonant background medium

We investigate the role of a spatially inhomogenous nonresonant background medium on several Raman-based imaging modalities. In particular, we consider a small resonant bead submerged in a spatially heterogeneous nonresonant χ(3) background. Using detailed 3D electrodynamic simulations, we compare coherent anti-Stokes Raman scattering (CARS), frequency-modulated CARS, amplitude-modulated stimulated Raman scattering (SRS), and frequency-modulated SRS. We find that only FM-SRS is background-free.     

Fourier-transform coherent anti-Stokes Raman scattering microscopy

We report a novel Fourier-transform-based implementation of coherent anti-Stokes Raman scattering (CARS) microscopy. The method employs a single femtosecond laser source and a Michelson interferometer to create two pulse replicas that are fed into a scanning multiphoton microscope. By varying the time delay between the pulses, we time-resolve the CARS signal, permitting easy removal of the nonresonant background while providing high resolution, spectrally resolved images of CARS modes over the laser bandwidth (approximately 1500 cm(-1)). We demonstrate the method by imaging polystyrene beads in solvent.     

Three-dimensional nonlinear microspectroscopy and imaging of soft condensed matter

We report on the realization of a sensitive microspectroscopy and imaging approach based on a three-color femtosecond coherent anti-Stokes Raman scattering (CARS) technique with high spectral, time, and spatial resolution. Independently tunable, high-repetition rate optical parametric oscillators were used to attain a dynamic range of 5 orders of magnitude for time-domain CARS signal. The attained sensitivity permitted tracing the decay of weak and structurally complex Raman active modes in soft condensed matter. Application of this approach to imaging of the biological specimen shows a great potential in quantitative characterization of live biological media with an ability to access inter- and intra-molecular interactions.     

Chemical imaging of glucose by CARS microscopy

Glucose is one of the most fundamental molecules within life and bioengineering sciences. Present understanding of its role in cellular and bioengineering processes relies primarily on invasive, large‐scale biochemical analysis, providing no spatial information on glucose pools or fluxes. This work identifies an emerging microscopy technique based on coherent anti‐Stokes Raman scattering (CARS), which fulfills the need of quantitative imaging of glucose at the single‐cell level with submicrometer resolution. No sample preparation with reporter molecules is required, ensuring that the low‐weight metabolite is studied under natural conditions. The potential of CARS microscopy is illustrated by quantitatively mapping glucose fluxes and distributions in a microfluidic bioreactor and in lipid‐bilayer vesicles, the latter as a model for glucose transmembrane transport. Furthermore, the metabolic response to a glucose pulse was monitored in living yeast cells. This study signifies a new era within CARS microscopy for its use of monitoring carbohydrates, in particular glucose which is one of the most abundant molecules in nature. Copyright © 2010 John Wiley & Sons, Ltd.     

Molecular vibration imaging in the fingerprint region by use of coherent anti-Stokes Raman scattering microscopy with a collinear configuration

We have developed a new coherent anti-Stokes Raman scattering (CARS) microscopy system with a collinear configuration for use in the fingerprint region. The system consists of a picosecond laser system and a transmission-type laser scanning microscope without a pinhole in front of the detector. The observable Raman-shift region is 900-1750 cm(-1), the spectral resolution is 30cm(-1), and the spatial resolution is smaller than 1 mum in the lateral direction and 3.2 mum in the depth direction, with objectives with a numerical aperture of 0.65. CARS spectra and images of polystyrene beads are demonstrated, and CARS imaging of a viable yeast cell is attempted.     

Raman Microscopy and Associated Techniques for Label-Free Imaging of Cancer Tissue

Abstract

Raman spectroscopy can identify cancerous from healthy tissue, with a chemical analysis from the measurement of vibrational bond frequencies. However, to detect small tumors a form of Raman imaging is required. Such imaging—by acquiring a Raman spectrum at each imaging pixel—can detect tumors but is rather slow. Multiphoton versions of Raman—anti-Stokes Raman scattering (CARS) microscopy and stimulated Raman scattering (SRS) microscopy—offer similar accuracies in identifying cancerous tissue and tumor margins but with a far higher speed, which is beneficial for diagnosis of small tumors in tissue. SRS microscopy can also be used to image extrinsic molecules in living cells, such as anti-cancer drugs at typical concentrations.     

基于光强传输方程相位成像的宽场相干反斯托克斯拉曼散射显微背景抑制

相干反斯托克斯拉曼散射(CARS)显微能够对样品的特殊化学组分进行选择性成像,无需荧光标记,在生物医学领域被广泛应用.然而,传统的CARS图像往往存在非共振背景信号.本文将基于光强传输方程的单光束相位成像技术用于CARS显微成像,来抑制CARS的非共振背景信号.该方法通过记录样品在三个相邻平面上的CARS图像,然后利用光强传输方程获取CARS光场的相位分布,最后利用共振CARS信号和非共振背景信号在相位上的差异,实现了对背景噪声的抑制.该方法无需参考光,通过三次测量可完成CARS的背景噪声抑制,具有良好的应用前景.     

Molecularly sensitive optical coherence tomography

Molecular contrast in optical coherence tomography (OCT) is demonstrated by use of coherent anti-Stokes Raman scattering (CARS) for molecular sensitivity. Femtosecond laser pulses are focused into a sample by use of a low-numerical-aperture lens to generate CARS photons, and the backreflected CARS signal is interferometrically measured. With the chemical selectivity provided by CARS and the advanced imaging capabilities of OCT, this technique may be useful for molecular contrast imaging in biological tissues. CARS can be generated and interferometrically measured over at least 600 microm of the depth of field of a low-numerical-aperture objective.     

Nonlinear interferometric vibrational imaging

Coherent anti-Stokes Raman scattering (CARS) processes are "coherent," but the phase of the anti-Stokes radiation is lost by most incoherent spectroscopic CARS measurements. We propose a Raman microscopy imaging method called nonlinear interferometric vibrational imaging, which measures Raman spectra by obtaining the temporal anti-Stokes signal through nonlinear interferometry. With a more complete knowledge of the anti-Stokes signal, we show through simulations that a high-resolution Raman spectrum can be obtained of a molecule in a single pulse using broad band radiation. This could be useful for identifying the three-dimensional spatial distribution of molecular species in tissue.     

Two‐beam multiplexed CARS based on a broadband oscillator

A two‐beam multiplexed coherent anti‐Stokes Raman scattering (CARS) microscopy setup is demonstrated by using a broadband (BB) Ti:sapphire oscillator without using any specialty fibres. A well‐defined spectral structure of the source leads to a delay‐sensitive CARS measurement in two‐colour CARS and also provides an efficient means of obtaining three‐colour CARS signals combined with the dispersion compensation of the BB pulse. Our result implies that the background suppression is limited by the onset of the spurious signals caused by the different CARS process, qualitatively differing from what is typically observed in the CARS microscopy. Copyright © 2010 John Wiley & Sons, Ltd.     

Chirped-pulse adiabatic control in coherent anti-Stokes Raman scattering for imaging of biological structure and dynamics

Two novel control methods based on adiabatic passage are proposed to be implemented in coherent anti-Stokes Raman scattering (CARS) microscopy for noninvasive imaging of biological structure and dynamics. The first method provides optimal pulse-area control of the resonant vibrational transitions by using a pair of equally linear-chirped pulses. The second method, named the 'roof' method, utilizes the chirp sign variation at the central time and gives robust adiabatic excitation of the resonant vibrational mode. Both methods are robust with respect to suppression of the off-resonant transitions. The methods allow one to achieve chemical sensitivity with high resolution and can be used to obtain CARS spectra of biological molecules with efficiently suppressed background.