Delong Zhang

Quantitative vibrational imaging by hyperspectral stimulated Raman scattering microscopy and multivariate curve resolution analysis.

Spectroscopic imaging has been an increasingly critical approach for unveiling specific molecules in biological environments. Toward this goal, we demonstrate hyperspectral stimulated Raman loss (SRL) imaging by intrapulse spectral scanning through a femtosecond pulse shaper. The hyperspectral stack of SRL images is further analyzed by a multivariate curve resolution (MCR) method to reconstruct quantitative concentration images for each individual component and retrieve the corresponding vibrational Raman spectra. Using these methods, we demonstrate quantitative mapping of dimethyl sulfoxide concentration in aqueous solutions and in fat tissue. Moreover, MCR is performed on SRL images of breast cancer cells to generate maps of principal chemical components along with their respective vibrational spectra. These results show the great capability and potential of hyperspectral SRL microscopy for quantitative imaging of complicated biomolecule mixtures through resolving overlapped Raman bands.

Fast Vibrational Imaging of Single Cells and Tissues by Stimulated Raman Scattering Microscopy

Conspectus Traditionally, molecules are analyzed in a test tube. Taking biochemistry as an example, the majority of our knowledge about cellular content comes from analysis of fixed cells or tissue homogenates using tools such as immunoblotting and liquid chromatography–mass spectrometry. These tools can indicate the presence of molecules but do not provide information on their location or interaction with each other in real time, restricting our understanding of the functions of the molecule under study. For real-time imaging of labeled molecules in live cells, fluorescence microscopy is the tool of choice. Fluorescent labels, however, are too bulky for small molecules such as fatty acids, amino acids, and cholesterol. These challenges highlight a critical need for development of chemical imaging platforms that allow in situ or in vivo analysis of molecules. Vibrational spectroscopy based on spontaneous Raman scattering is widely used for label-free analysis of chemical content in cells and tissues. However, the Raman process is a weak effect, limiting its application for fast chemical imaging of a living system. With high imaging speed and 3D spatial resolution, coherent Raman scattering microscopy is enabling a new approach for real-time vibrational imaging of single cells in a living system. In most experiments, coherent Raman processes involve two excitation fields denoted as pump at ωp and Stokes at ωs. When the beating frequency between the pump and Stokes fields (ωp – ωs) is resonant with a Raman-active molecular vibration, four major coherent Raman scattering processes occur simultaneously, namely, coherent anti-Stokes Raman scattering (CARS) at (ωp – ωs) + ωp, coherent Stokes Raman scattering (CSRS) at ωs – (ωp – ωs), stimulated Raman gain (SRG) at ωs, and stimulated Raman loss (SRL) at ωp. In SRG, the Stokes beam experiences a gain in intensity, whereas in SRL, the pump beam experiences a loss. Both SRG and SRL belong to stimulated Raman scattering (SRS), in which the energy difference between the pump and Stokes fields is transferred to the molecule for vibrational excitation. The SRS signal appears at the same wavelengths as the excitation fields and is commonly extracted through a phase-sensitive detection scheme. The detected intensity change because of a Raman transition is proportional to Im[χ(3)]IpIs, where χ(3) represents the third-order nonlinear susceptibility, Ip and Is stand for the intensity of the pump and Stokes fields. In this Account, we discuss the most recent advances in the technical development and enabling applications of SRS microscopy. Compared to CARS, the SRS contrast is free of nonresonant background. Moreover, the SRS intensity is linearly proportional to the density of target molecules in focus. For single-frequency imaging, an SRS microscope offers a speed that is ∼1000 times faster than a line-scan Raman microscope and 10 000 times faster than a point-scan Raman microscope. It is important to emphasize that SRS and spontaneous Raman scattering are complementary to each other. Spontaneous Raman spectroscopy covers the entire window of molecular vibrations, which allows extraction of subtleties via multivariate analysis. SRS offers the speed advantage by focusing on either a single Raman band or a defined spectral window of target molecules. Integrating single-frequency SRS imaging and spontaneous Raman spectroscopy on a single platform allows quantitative compositional analysis of objects inside single live cells.

Heterodyne detected nonlinear optical imaging in a lock-in free manner

We report a compact, cost-effective tuned amplifier for frequency-selective amplification of the modulated signal in heterodyne detected nonlinear optical microscopy. Our method improved the signal to noise ratio by an order of magnitude compared to conventional lock-in detection, as demonstrated through stimulated Raman scattering imaging of live cells and tissues at the speed of 2 μsec/pixel. Application of the tuned amplifier to transient absorption microscopy is also demonstrated. The increased signal to noise ratio allowed epi-detected in vivo imaging of myelin and blood in rat spinal cord with high spatial resolution.

Assessing Cholesterol Storage in Live Cells and C. elegans by Stimulated Raman Scattering Imaging of Phenyl-Diyne Cholesterol

We report a cholesterol imaging method using rationally synthesized phenyl-diyne cholesterol (PhDY-Chol) and stimulated Raman scattering (SRS) microscope. The phenyl-diyne group is biologically inert and provides a Raman scattering cross section that is 88 times larger than the endogenous C = O stretching mode. SRS microscopy offers an imaging speed that is faster than spontaneous Raman microscopy by three orders of magnitude, and a detection sensitivity of 31 μM PhDY-Chol (~1,800 molecules in the excitation volume). Inside living CHO cells, PhDY-Chol mimics the behavior of cholesterol, including membrane incorporation and esterification. In a cellular model of Niemann-Pick type C disease, PhDY-Chol reflects the lysosomal accumulation of cholesterol, and shows relocation to lipid droplets after HPβCD treatment. In live C. elegans, PhDY-Chol mimics cholesterol uptake by intestinal cells and reflects cholesterol storage. Together, our work demonstrates an enabling platform for study of cholesterol storage and trafficking in living cells and vital organisms.

Label‐Free Quantitative Imaging of Cholesterol in Intact Tissues by Hyperspectral Stimulated Raman Scattering Microscopy

A finger on the pulse: Current molecular analysis of cells and tissues routinely relies on separation, enrichment, and subsequent measurements by various assays. Now, a platform of hyperspectral stimulated Raman scattering microscopy has been developed for the fast, quantitative, and label-free imaging of biomolecules in intact tissues using spectroscopic fingerprints as the contrast mechanism.

Imaging Lipid Metabolism in Live Caenorhabditis elegans Using Fingerprint Vibrations

Quantitation of lipid storage, unsaturation, and oxidation in live C. elegans has been a long-standing obstacle. The combination of hyperspectral stimulated Raman scattering imaging and multivariate analysis in the fingerprint vibration region represents a platform that allows the quantitative mapping of fat distribution, degree of fat unsaturation, lipid oxidation, and cholesterol storage in vivo in the whole worm. Our results reveal for the first time that lysosome-related organelles in intestinal cells are sites for storage of cholesterol in C. elegans.

Coherent Raman Scattering Microscopy in Biology and Medicine

Advancements in coherent Raman scattering (CRS) microscopy have enabled label-free visualization and analysis of functional, endogenous biomolecules in living systems. When compared with spontaneous Raman microscopy, a key advantage of CRS microscopy is the dramatic improvement in imaging speed, which gives rise to real-time vibrational imaging of live biological samples. Using molecular vibrational signatures, recently developed hyperspectral CRS microscopy has improved the readout of chemical information available from CRS images. In this article, we review recent achievements in CRS microscopy, focusing on the theory of the CRS signal-to-noise ratio, imaging speed, technical developments, and applications of CRS imaging in bioscience and clinical settings. In addition, we present possible future directions that the use of this technology may take.

Spectrally modulated stimulated Raman scattering imaging with an angle-to-wavelength pulse shaper

The stimulated Raman scattering signal is often accompanied by unwanted background arising from other pump-probe modalities. We demonstrate an approach to overcome this challenge based on spectral domain modulation, enabled by a compact, cost-effective angle-to-wavelength pulse shaper. The pulse shaper switches between two spectrally narrow windows, which are cut out of a broadband femtosecond pulse and selected for on- and off- Raman resonance excitation, at 2.1 MHz frequency for detection of stimulated Raman scattering signal. Such spectral modulation reduced the unwanted pump-probe signals by up to 20 times and enabled stimulated Raman scattering imaging of molecules in a pigmented environment.

Longitudinal in vivo coherent anti-Stokes Raman scattering imaging of demyelination and remyelination in injured spinal cord

In vivo imaging of white matter is important for the mechanistic understanding of demyelination and evaluation of remyelination therapies. Although white matter can be visualized by a strong coherent anti-Stokes Raman scattering (CARS) signal from axonal myelin, in vivo repetitive CARS imaging of the spinal cord remains a challenge due to complexities induced by the laminectomy surgery. We present a careful experimental design that enabled longitudinal CARS imaging of de- and remyelination at single axon level in live rats. In vivo CARS imaging of secretory phospholipase A(2) induced myelin vesiculation, macrophage uptake of myelin debris, and spontaneous remyelination by Schwann cells are sequentially monitored over a 3 week period. Longitudinal visualization of de- and remyelination at a single axon level provides a novel platform for rational design of therapies aimed at promoting myelin plasticity and repair.

Multimodal coherent anti-Stokes Raman spectroscopic imaging with a fiber optical parametric oscillator

We report on multimodal coherent anti-Stokes Raman scattering (CARS) imaging with a source composed of a femtosecond fiber laser and a photonic crystal fiber (PCF)-based optical parametric oscillator (FOPO). By switching between two PCFs with different zero dispersion wavelengths, a tunable signal beam from the FOPO covering the range from 840 to 930 nm was produced. By combining the femtosecond fiber laser and the FOPO output, simultaneous CARS imaging of a myelin sheath and two-photon excitation fluorescence imaging of a labeled axons in rat spinal cord have been demonstrated at the speed of 20 μs per pixel.