Label-free Chemical Imaging Research Articles

Stimulated Raman Scattering Microscopy with a Robust Fibre Laser Source.

Stimulated Raman Scattering microscopy allows label-free chemical imaging and has enabled exciting applications in biology, material science, and medicine. It provides a major advantage in imaging speed over spontaneous Raman scattering and has improved image contrast and spectral fidelity compared to coherent anti-Stokes Raman. Wider adoption of the technique has, however, been hindered by the need for a costly and environmentally sensitive tunable ultra-fast dual-wavelength source. We present the development of an optimized all-fibre laser system based on the optical synchronization of two picosecond power amplifiers. To circumvent the high-frequency laser noise intrinsic to amplified fibre lasers, we have further developed a high-speed noise cancellation system based on voltage-subtraction autobalanced detection. We demonstrate uncompromised imaging performance of our fibre-laser based stimulated Raman scattering microscope with shot-noise limited sensitivity and an imaging speed up to 1 frame/s.

Hyperspectral imaging with stimulated Raman scattering by chirped femtosecond lasers.

Raman microscopy is a quantitative, label-free, and noninvasive optical imaging technique for studying inhomogeneous systems. However, the feebleness of Raman scattering significantly limits the use of Raman microscopy to low time resolutions and primarily static samples. Recent developments in narrowband stimulated Raman scattering (SRS) microscopy have significantly increased the acquisition speed of Raman based label-free imaging by a few orders of magnitude, at the expense of reduced spectroscopic information. On the basis of a spectral focusing approach, we present a fast SRS hyperspectral imaging system using chirped femtosecond lasers to achieve rapid Raman spectra acquisition while retaining the full speed and image quality of narrowband SRS imaging. We demonstrate that quantitative concentration determination of cholesterol in the presence of interfering chemical species can be achieved with sensitivity down to 4 mM. For imaging purposes, hyperspectral imaging data in the C-H stretching region is obtained within a minute. We show that mammalian cell SRS hyperspectral imaging reveals the spatially inhomogeneous distribution of saturated lipids, unsaturated lipids, cholesterol, and protein. The combination of fast spectroscopy and label-free chemical imaging will enable new applications in studying biological systems and material systems.

Multicolor stimulated Raman scattering microscopy with a rapidly tunable optical parametric oscillator

Stimulated Raman scattering (SRS) microscopy allows label-free chemical imaging based on vibrational spectroscopy. Narrowband excitation with picosecond lasers creates the highest signal levels and enables imaging speeds up to video-rate, but it sacrifices chemical specificity in samples with overlapping bands compared to broadband (multiplex) excitation. We develop a rapidly tunable picosecond optical parametric oscillator with an electro-optical tunable Lyot filter, and demonstrate multicolor SRS microscopy with synchronized line-by-line wavelength tuning to avoid spectral artifacts due to sample movement. We show sensitive imaging of three different kinds of polymer beads and live HeLa cells with moving intracellular lipid droplets.

Restoration and Spectral Recovery of Mid-Infrared Chemical Images

Fourier transform infrared (FTIR) microspectroscopy is a powerful technique for label-free chemical imaging that has supplied important chemical information about heterogeneous samples for many problems across a variety of disciplines. State-of-the-art synchrotron based infrared (IR) microspectrometers can yield high-resolution images, but are truly diffraction limited for only a small spectral range. Furthermore, a fundamental trade-off exists between the number of pixels, acquisition time and the signal-to-noise ratio, limiting the applicability of the technique. The recently commissioned infrared synchrotron beamline, infrared environmental imaging (IRENI), overcomes this trade off and delivers 4096-pixel diffraction limited IR images with high signal-to-noise ratio in under a minute. The spatial oversampling for all mid-IR wavelengths makes the IRENI data ideal for spatial image restoration techniques. Here, we measured and fitted wavelength-dependent point-spread-functions (PSFs) at IRENI for a 74× objective between the sample plane and detector. Noise-free wavelength-dependent theoretical PSFs are deconvoluted from images generated from narrow bandwidths (4 cm(-1)) over the entire mid-infrared range (4000-900 cm(-1)). The stack of restored images is used to reconstruct the spectra. Restored images of metallic test samples with features that are 2.5 μm and smaller are clearly improved in comparison to the raw data images for frequencies above 2000 cm(-1). Importantly, these spatial image restoration methods also work for samples with vibrational bands in the recorded mid-IR fingerprint region (900-1800 cm(-1)). Improved signal-to-noise spectra are reconstructed from the restored images as demonstrated for a mixture of spherical polystyrene beads in a polyurethane matrix. Finally, a freshly thawed retina tissue section is used to demonstrate the success of deconvolution achievable with a heterogeneous, irregularly shaped, biologically relevant sample with distinguishing spectroscopic features across the entire mid-IR spectral range.

Quantitative Chemical Imaging with Multiplex Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy is a newly developed label-free chemical imaging technique that overcomes the speed limitation of confocal Raman microscopy while avoiding the nonresonant background problem of coherent anti-Stokes Raman scattering (CARS) microscopy. Previous demonstrations have been limited to single Raman band measurements. We present a novel modulation multiplexing approach that allows real-time detection of multiple species using the fast Fourier transform. We demonstrate the quantitative determination of chemical concentrations in a ternary mixture. Furthermore, two imaging applications are pursued: (1) quantitative determination of oil content as well as pigment and protein concentration in microalgae cultures; and (2) 3D high-resolution imaging of blood, lipids, and protein distribution in ex vivo mouse skin tissue. We believe that quantitative multiplex SRS uniquely combines the advantage of fast label-free imaging with the fingerprinting capability of Raman spectroscopy and enables numerous applications in lipid biology as well as biomedical imaging.

Label-free chemical imaging in vivo: three-dimensional non-invasive microscopic observation of amphioxus notochord through stimulated Raman scattering (SRS)

Notochord is a rod-shaped axial supporting structure unique only to chordates. In this study, we use cephalochordate amphioxus (Branchiostroma belcheri, a living basal chordate) and zebrafish (Danio rerio), a vertebrate, as model animals and employ stimulated Raman microscopy (SRS), a newly developed label-free technique, to investigate notochord structure and chemical composition in live animals. Coherent anti-Stokes Raman scattering (CARS) images have also been acquired for comparison. Both CARS and SRS images can construct the detailed three-dimensional structure of the notochord with resolution better than 1 μm. Label-free live imaging allows us to obtain the whole animal's intact internal morphology, which is difficult to obtain through other mechanical or optical sectioning methods. Intrinsically, chemical sensitive SRS images, with a simple data processing procedure, show that the amphioxus notochord is protein-rich. Our result agrees well with that drawn from the labeling and cryo-sectioning observation, verifying that the coherent Raman scattering techniques are useful to examine the structure and chemical composition of live animals without labels in a fast, simple and accurate way.

Imaging Drug Delivery to Skin with Stimulated Raman Scattering Microscopy

Efficient drug delivery to the skin is essential for the treatment of major dermatologic diseases, such as eczema, psoriasis and acne. However, many compounds penetrate the skin barrier poorly and require optimized formulations to ensure their bioavailability. Here, stimulated Raman scattering (SRS) microscopy, a recently developed, label-free chemical imaging tool, is used to acquire high resolution images of multiple chemical components of a topical formulation as it penetrates into mammalian skin. This technique uniquely provides label-free, nondestructive, three-dimensional images with high spatiotemporal resolution. It reveals novel features of (trans)dermal drug delivery in the tissue environment: different rates of drug penetration via hair follicles as compared to the intercellular pathway across the stratum corneum are directly observed, and the precipitation of drug crystals on the skin surface is visualized after the percutaneous penetration of the cosolvent excipient in the formulation. The high speed three-dimensional imaging capability of SRS thus reveals features that cannot be seen with other techniques, providing both kinetic information and mechanistic insight into the (trans)dermal drug delivery process.

Picosecond Spectral Cars Imaging with Principal Component Analysis

Imaging methods based on vibrational Raman contrast offer label-free chemical imaging of tissues. Video rate imaging acquisition times can be achieved by coherent Raman techniques, however, they do so only at a single vibrational frequency - limiting a more in-depth chemical analysis of the tissue. The most direct solution is to extend the measurement in the vibrational frequency domain, which has been manifested in the development of broadband coherent Raman methods. Unfortunately, the attributed acquisition time renders them to be impractical in examining large tissue areas. In this study, we demonstrate that picosecond spectral coherent anti-Stokes Raman scattering (CARS) imaging combined with principal component analysis (PCA) can be utilized to achieve fast high-resolution maps of lipids in tissues. Spectral CARS data sets were generated by imaging tissue sections at consecutive Raman shifts in the CH2 stretching vibrational range. By avoiding spectral redundancy, the acquisition time was shortened, whereas loss of spectral information was compensated by a much higher information density in the spatial domain via PCA. This resulted in sufficient spectral contrast, which was illustrated in our recent application in characterizing lipids distribution and composition of the meibomian glands in the context of dry eye disease. Spectral differences between CARS spectra obtained from specific regions were confirmed with a Raman microspectroscopic analysis. The resulting multi-component chemical maps revealed a lipid maturation process throughout the gland structure, which was not observed before and may provide important clues toward understanding meibomian gland dysfunction.

Multiplex Coherent Anti-Stokes Raman Spectroscopy Images Intact Atheromatous Lesions and Concomitantly Identifies Distinct Chemical Profiles of Atherosclerotic Lipids

Lipids are a key component of atherogenesis. However, their physiological role on the progression of atherosclerosis including plaque vulnerability has not been clearly understood, because of the lack of appropriate tools for chemical assessment. We aimed to develop a label-free chemical imaging platform based on multiplex coherent anti-Stokes Raman scattering (CARS) for the correlative study of the morphology and chemical profile of atherosclerotic lipids. Whole aortas from atherosclerotic apolipoprotein E knock-out mice were en face examined by multiplex CARS imaging and 4 distinctive morphologies of the lipids (intra/extracellular lipid droplets and needle-/plate-shaped lipid crystals) were classified. The chemical profiles of atherosclerotic lipids depending on morphologies were firstly identified from intact atheromatous tissue by multiplex CARS. We demonstrated that needle-/plate-shaped lipid crystals in advanced plaques had undergone a phase shift to the solid state with increased protein contents, implying that lipid modification had occurred beforehand. The validity of lipid-selective multiplex CARS imaging was supported by comparative results from oil red O staining and whole-mount immunohistochemistry. By spatial CARS analysis of atherosclerosis progression, we found greater accumulation of lipid crystals in both the lesser curvature of the aortic arch and the innominate artery. Furthermore, multiplex CARS measurement successfully demonstrated the effect of a drug, statin, on atherosclerotic lipids by showing the change of their chemical profiles. Multiplex CARS imaging directly provides intact morphologies of atherosclerotic lipids with correlative chemical information, thereby suggesting its potential applications in the investigation of lipid-associated disorders and the preclinical drug screening.

Stimulated Raman Scattering Microscopy for Label-Free Chemical Imaging

Coherent anti-Stokes Raman techniques are increasing the utility of Raman scattering for chemical and biological diagnostics.

Label-Free Chemical Imaging of Catalytic Solids by Coherent Anti-Stokes Raman Scattering and Synchrotron-Based Infrared Microscopy

Innenaufnahme: Die Kombination aus den beiden im Titel genannten Verfahren während der katalytischen Umsetzung von Thiophenderivaten an Zeolithkristallen ergibt räumlich und zeitlich aufgelöste chemische Informationen auch ohne Markierungen (siehe Bild). Das reagierende Thiophen befindet sich hauptsächlich im Kristallinneren, während das Produkt in den geraden Poren des Zeoliths gefunden wird.

Inside Cover: Label-Free Chemical Imaging of Catalytic Solids by Coherent Anti-Stokes Raman Scattering and Synchrotron-Based Infrared Microscopy (Angew. Chem. Int. Ed. 47/2009)

Inside Cover: Label-Free Chemical Imaging of Catalytic Solids by Coherent Anti-Stokes Raman Scattering and Synchrotron-Based Infrared Microscopy (Angew. Chem. Int. Ed. 47/2009)

Chemically specific imaging of cryptosporidium oocysts using coherent anti‐Stokes Raman scattering (CARS) microscopy

We demonstrate the application of coherent anti-Stokes Raman scattering microscopy for the rapid, label-free chemical imaging of waterborne pathogens. Chemically selective images of cryptosporidium were acquired in just a few seconds using coherent anti-Stokes Raman scattering microscopy, demonstrating its capability for the rapid detection of cryptosporidium at the single oocyst level. We discuss the applicability of such a technique in a near-real time automated water testing system.