Biomolecular Imaging of Cellular Uptake of Nanoparticles using Multimodal Nonlinear Optical Microscopy

Probing gold nanoparticles (AuNPs) in living systems is essential to reveal the interaction between AuNPs and biological tissues. Moreover, by integrating ...

University of Exeter

Second harmonic generation (SHG) and two-photon fluorescence (TPF) microscopy is used to image the intercellular and pericellular matrix in normal and degenerate equine articular cartilage. The polarization sensitivity of SHG can be used directly to determine fiber orientation in the superficial 10 to 20 microm of tissue, and images of the ratio of intensities taken with two orthogonal polarization states reveal small scale variations in the collagen fiber organization that have not previously been reported. The signal from greater depths is influenced by the birefringence and biattenuance of the overlying tissue. An assessment of these effects is developed, based on the analysis of changes in TPF polarization with depth, and the approach is validated in tendon where composition is independent of depth. The analysis places an upper bound on the biattenuance of tendon of 2.65 x 10(-4). Normal cartilage reveals a consistent pattern of variation in fibril orientation with depth. In lesions, the pattern is severely disrupted and there are changes in the pericellular matrix, even at the periphery where the tissue appears microscopically normal. Quantification of polarization sensitivity changes with depth in cartilage will require detailed numerical models, but in the meantime, multiphoton microscopy provides sensitive indications of matrix changes in cartilage degeneration.

Collagen fiber arrangement in normal and diseased cartilage studied by polarization sensitive nonlinear microscopy.

A combination of two-photon fluorescence (TPF), second harmonic generation (SHG) and coherent anti-Stokes Raman scattering (CARS) imaging has been used to investigate the elastin fibre network in healthy equine articular cartilage from the metacarpophalangeal joint. The elastin fibres were identified using their intrinsic two-photon fluorescence and immuno-staining was used to confirm the identity of these fibres. SHG was used to reveal the collagen matrix and the collagen fibre orientations were determined from their SHG polarization sensitivity, while CARS was used to clearly delineate the cell boundaries. Extensive elastin fibre networks were found in all the joint regions investigated. The elastin was found predominantly in the superficial zone (upper 50 microm) and was aligned parallel to the articular surface. Elastin was also detected in the pericellular matrix surrounding the superficial chondrocytes; however, individual fibres could not be resolved in this region. Variations in the density and organization of the fibres were observed in different regions on the joint surface.

The elastin network: its relationship with collagen and cells in articular cartilage as visualized by multiphoton microscopy.

Multi-modal multiphoton microscopy was used to investigate tissue microstructure in the zone of calcified cartilage, focussing on the collagen fibre organisation at the tidemark and cement line. Thick, unstained and unfixed sagittal sections were prepared from the equine metacarpophalangeal joint. Second harmonic generation (SHG) provided contrast for collagen, two-photon fluorescence (TPF) for endogenous fluorophores, and coherent anti-Stokes Raman scattering (CARS) allowed the cells to be visualised. The structure of radial and calcified cartilage was found to vary with location across the joint, with the palma regions showing a more ordered parallel arrangement of collagen fibres than the cortical ridge and dorsal regions. These patterns may be associated with regional variations in joint loading. In addition, the cell lacunae had a greater diameter in the dorsal region than in the palmar region. At the cement line some collagen fibres were observed crossing between the calcified cartilage and the subchondral bone. At the tidemark the fibres were parallel and continuous between the radial and calcified cartilage. Beneath early superficial lesions the structure of the tidemark and calcified cartilage was disrupted with discontinuities and gaps in the fibrillar organisation. Cartilage microstructure varies in the deep zones between regions of different loading. The variations in collagen structure observed may be significant to the local mechanical properties of the cartilage and therefore may be important to its mechanical interactions with the subchondral bone. The calcified cartilage is altered even below early superficial lesions and therefore is important in the understanding of the aetiology of osteoarthritis.

A multi-modal multiphoton investigation of microstructure in the deep zone and calcified cartilage.

Stimulated Raman scattering (SRS) has been applied to unstained samples of articular cartilage enabling the investigation of living cells within fresh tissue. Hyperspectral SRS measurements over the CH vibrational region showed variations in protein and lipid content within the cells, pericellular matrix and interterritorial matrix. Changes in the cells and pericellular matrix were investigated as a function of depth into the cartilage. Lipid was detected in the pericellular matrix of superficial zone chondrocytes. The spectral profile of lipid droplets within the chondrocytes indicated that they contained predominantly unsaturated lipids. The mineral content has been imaged by using the PO₄³⁻ vibration at 959 cm⁻¹ and the CO₃²⁻ vibration at 1070 cm⁻¹. Both changes in cells and mineralization are known to be important factors in the progression of osteoarthritis. SRS enables these to be visualized in fresh unstained tissue and consequently should benefit osteoarthiritis research.

Chemically specific imaging and in-situ chemical analysis of articular cartilage with stimulated Raman scattering.

The growing world population puts ever-increasing demands on the agricultural and agrochemical industries to increase agricultural yields. This can only be achieved by investing in fundamental plant and agrochemical research and in the development of improved analytical tools to support research in these areas. There is currently a lack of analytical tools that provide noninvasive structural and chemical analysis of plant tissues at the cellular scale. Imaging techniques such as coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS) microscopy provide label-free chemically specific image contrast based on vibrational spectroscopy. Over the past decade, these techniques have been shown to offer clear advantages for a vast range of biomedical research applications. The intrinsic vibrational contrast provides label-free quantitative functional analysis, it does not suffer from photobleaching, and it allows near real-time imaging in 3D with submicrometer spatial resolution. However, due to the susceptibility of current detection schemes to optical absorption and fluorescence from pigments (such as chlorophyll), the plant science and agrochemical research communities have not been able to benefit from these techniques and their application in plant research has remained virtually unexplored. In this paper, we explore the effect of chlorophyll fluorescence and absorption in CARS and SRS microscopy. We show that with the latter it is possible to use phase-sensitive detection to separate the vibrational signal from the (electronic) absorption processes. Finally, we demonstrate the potential of SRS for a range of in planta applications by presenting in situ chemical analysis of plant cell wall components, epicuticular waxes, and the deposition of agrochemical formulations onto the leaf surface.

Label-free Chemically Specific Imaging in Planta with Stimulated Raman Scattering Microscopy.

La croissance d&eacute;mographique mondiale met les demandes croissantes sur l&#39;agriculture et industries agrochimique pour augmenter agricoles rendements. Cela n&#39;est possible en investissant dans les plantes fondamentales et de la recherche de l&#39;agrochimie et &agrave; l&#39;&eacute;laboration d&#39;outils d&#39;analyse am&eacute;lior&eacute;es pour soutenir la recherche dans ces domaines. Il y a actuellement un manque d&#39;outils d&#39;analyse qui fournissent non invasive analyse structurale et chimique des tissus de la plante &agrave; l&#39;&eacute;chelle cellulaire. Imagerie des techniques telles que les Raman anti-Stokes coh&eacute;rente (automobiles) et stimul&eacute; Raman, microscopie de diffusion (SRS) offrir un contraste exempte d&#39;&eacute;tiquette sp&eacute;cifique chimiquement image bas&eacute; sur la spectroscopie vibrationnelle. Ces dix derni&egrave;res ann&eacute;es, ces techniques ont d&eacute;montr&eacute; d&#39;offrir des avantages clairs pour une vaste gamme d&#39;applications de la recherche biom&eacute;dicale. Le contraste vibratoire intrins&egrave;que fournit une analyse fonctionnelle quantitative exempte d&#39;&eacute;tiquette, il ne souffre pas de photoblanchiment et il autorise pr&egrave;s de l&#39;imagerie en temps r&eacute;el en 3D avec une r&eacute;solution spatiale SUBMICROMETRIQUES. Toutefois, en raison de la sensibilit&eacute; des syst&egrave;mes de d&eacute;tection actuels &agrave; absorption optique et &agrave; fluorescence de pigments (tels que la chlorophylle), les communaut&eacute;s de recherche plant science et agrochimiques n&#39;ont pas pu b&eacute;n&eacute;ficier de ces techniques et leur application dans la recherche de la plante est rest&eacute;e pratiquement inexplor&eacute;e. Dans cet article, nous explorons l&#39;effet de fluorescence de la chlorophylle et l&#39;absorption en microscopie SRS et de voitures. Nous montrons qu&#39;avec ce dernier, il est possible d&#39;utiliser la d&eacute;tection sensible &agrave; la phase de s&eacute;parer le signal vibratoire des processus d&#39;absorption (&eacute;lectronique). Finalement, nous d&eacute;montrons le potentiel du SRS pour un &eacute;ventail d&#39;applications de planta en pr&eacute;sentant une analyse chimique in situ de la paroi cellulaire des composants v&eacute;g&eacute;taux, cires &eacute;picuticulaires et la d&eacute;position de formulations agrochimiques sur la surface de la feuille.

Exempt d&#39;&eacute;tiquette l&#39;imagerie chimique sp&eacute;cifique en Planta la microscopie de diffusion Raman stimul&eacute;e.

La crescente popolazione mondiale mette sempre maggiori richieste sul settore agricolo e industrie agrochimiche aumentare agricola produce. Ci&ograve; pu&ograve; essere ottenuto solo investendo nella ricerca di agrochimici e pianta fondamentale e nello sviluppo di strumenti analitici migliorati per sostenere la ricerca in questi settori. Attualmente c&#39;&egrave; una mancanza di strumenti analitici che forniscono analisi chimiche e strutturali non invasiva dei tessuti vegetali a livello cellulare. Tecniche come coerente anti-Stokes Raman scattering (automobili) e stimolato Raman, microscopia di scattering (SRS) fornire contrasto immagine chimicamente specifico privo di etichetta basato sulla spettroscopia vibrazionale di Imaging. Nell&#39;ultimo decennio, queste tecniche hanno dimostrate di offrire chiari vantaggi per una vasta gamma di applicazioni di ricerca biomedica. Il contrasto vibrazionale intrinseco fornisce analisi funzionale privo di etichetta quantitativi, non soffrono di photobleaching e permette vicino in tempo reale immagini in 3D con risoluzione spaziale submicrometriche. Tuttavia, a causa della predisposizione degli schemi di rilevamento corrente di fluorescenza e di assorbimento ottico da pigmenti (come la clorofilla), comunit&agrave; di ricerca scienza e agrochimici di impianto non sono stati in grado di trarre beneficio da queste tecniche e loro applicazione nella ricerca della pianta &egrave; rimasta praticamente inesplorata. In questa carta, esploriamo l&#39;effetto della fluorescenza della clorofilla e assorbimento nella microscopia automobili e SRS. Mostriamo che con quest&#39;ultimo &egrave; possibile utilizzare il rilevamento phase-sensitive per separare il segnale vibrazionale dai processi di assorbimento (elettronico). Infine, noi dimostrare il potenziale di SRS per una gamma di applicazioni di planta presentando in situ analisi chimica dei componenti parete delle cellule vegetali, cere epicuticular e la deposizione di formulazioni agrochimiche sulla superficie della foglia.

Privo di etichetta chimicamente specifiche Imaging in Planta con microscopia di Scattering Raman stimolato.

Elastin is a major component of tissues such as lung and blood vessels, and endows them with the long-range elasticity necessary for their physiological functions. Recent research has revealed the complexity of these elastin structures and drawn attention to the existence of extensive networks of fine elastin fibres in tissues such as articular cartilage and the intervertebral disc. Nonlinear microscopy, allowing the visualization of these structures in living tissues, is informing analysis of their mechanical properties. Elastic fibres are complex in composition and structure containing, in addition to elastin, an array of microfibrillar proteins, principally fibrillin. Raman microspectrometry and X-ray scattering have provided new insights into the mechanisms of elasticity of the individual component proteins at the molecular and fibrillar levels, but more remains to be done in understanding their mechanical interactions in composite matrices. Elastic tissue is one of the most stable components of the extracellular matrix, but impaired mechanical function is associated with ageing and diseases such as atherosclerosis and diabetes. Efforts to understand these associations through studying the effects of processes such as calcium and lipid binding and glycation on the mechanical properties of elastin preparations in vitro have produced a confusing picture, and further efforts are required to determine the molecular basis of such effects.

The structure and micromechanics of elastic tissue.

Plant leaves are optically complex, which makes them difficult to image by light microscopy. Careful sample preparation is therefore required to enable researchers to maximize the information gained from advances in fluorescent protein labeling, cell dyes and innovations in microscope technologies and techniques. We have previously shown that mounting leaves in the non-toxic, non-fluorescent perfluorocarbon (PFC), perfluorodecalin (PFD) enhances the optical properties of the leaf with minimal impact on physiology. Here, we assess the use of the PFCs, PFD, and perfluoroperhydrophenanthrene (PP11) for in vivo plant leaf imaging using four advanced modes of microscopy: laser scanning confocal microscopy (LSCM), two-photon fluorescence microscopy, second harmonic generation microscopy, and stimulated Raman scattering (SRS) microscopy. For every mode of imaging tested, we observed an improved signal when leaves were mounted in PFD or in PP11, compared to mounting the samples in water. Using an image analysis technique based on autocorrelation to quantitatively assess LSCM image deterioration with depth, we show that PP11 outperformed PFD as a mounting medium by enabling the acquisition of clearer images deeper into the tissue. In addition, we show that SRS microscopy can be used to image PFCs directly in the mesophyll and thereby easily delimit the "negative space" within a leaf, which may have important implications for studies of leaf development. Direct comparison of on and off resonance SRS micrographs show that PFCs do not to form intracellular aggregates in live plants. We conclude that the application of PFCs as mounting media substantially increases advanced microscopy image quality of living mesophyll and leaf vascular bundle cells.

An update: improvements in imaging perfluorocarbon-mounted plant leaves with implications for studies of plant pathology, physiology, development and cell biology.

The cuticle is a ubiquitous, predominantly waxy layer on the aerial parts of higher plants that fulfils a number of essential physiological roles, including regulating evapotranspiration, light reflection, and heat tolerance, control of development, and providing an essential barrier between the organism and environmental agents such as chemicals or some pathogens. The structure and composition of the cuticle are closely associated but are typically investigated separately using a combination of structural imaging and biochemical analysis of extracted waxes. Recently, techniques that combine stain-free imaging and biochemical analysis, including Fourier transform infrared spectroscopy microscopy and coherent anti-Stokes Raman spectroscopy microscopy, have been used to investigate the cuticle, but the detection sensitivity is severely limited by the background signals from plant pigments. We present a new method for label-free, in vivo structural and biochemical analysis of plant cuticles based on stimulated Raman scattering (SRS) microscopy. As a proof of principle, we used SRS microscopy to analyze the cuticles from a variety of plants at different times in development. We demonstrate that the SRS virtually eliminates the background interference compared with coherent anti-Stokes Raman spectroscopy imaging and results in label-free, chemically specific confocal images of cuticle architecture with simultaneous characterization of cuticle composition. This innovative use of the SRS spectroscopy may find applications in agrochemical research and development or in studies of wax deposition during leaf development and, as such, represents an important step in the study of higher plant cuticles.

In vivo chemical and structural analysis of plant cuticular waxes using stimulated Raman scattering microscopy.

To investigate the relationships between the unique mechanical and structural properties of the superficial zone of articular cartilage on the microscopic scale.

The micromechanics of the superficial zone of articular cartilage.

The complex structure of the annulus fibrosus is strongly related to its mechanical properties. Recent work showed that it is possible to observe the relative movement of fibre bundles in loaded cow tail annulus; the aim of this work was to describe and quantify annulus fibrosus micromechanics in degenerated human disc, and compare it with cow tail annulus, an animal model often used in the literature. Second harmonic generation was used to image the collagen matrix in twenty strips of annulus fibrosus harvested from intervertebral disc of seven patients undergoing surgery. Samples were loaded to 6% tensile strain in 1% steps. Elastic modulus was calculated from loading curves, and micromechanical strains were calculated from the images using custom software. The same protocol was applied to twenty strips of annulus harvested from cow tail discs. Significant morphological differences were found between human and cow tail samples, the most striking being the lack of collagen fibre crimp in the former. Fibres were also observed bending and running from one lamella to the other, forming a strong flexible interface. Interdigitation of fibre bundles was also present at this interface. Quantitative results show complex patterns of inter-bundle and inter-lamellar behaviour, with inter-bundle sliding being the main strain mechanism. Elastic modulus was similar between species, and it was not affected by the degree of degeneration. This work gives an insight into the complex structure and mechanical function of the annulus fibrosus, which should be accounted for in disc numerical modelling.

Bovine and degenerated human annulus fibrosus: a microstructural and micromechanical comparison.

Needle puncture of the intervertebral disc can initiate a mechanical and biochemical cascade leading to disc degeneration. Puncture's mechanical effects have been shown near the puncture site, mechanical effects should be observed far, relative to needle size, from the puncture site, given the disc-wide damage induced by the stab. The aim of this work was to quantify these far-field effects, and to observe the local structural damage provoked by the needle. Strips of cow tail annulus fibrosus underwent two consecutive mechanical loadings to 5% tensile strain; fifteen samples were punctured in a radial direction with a randomly assigned needle between the two loadings (needle gauges between 19 and 23). Ten samples (control group) were not punctured. During loading, the tissue strains were imaged using second harmonic generation microscopy in a <600×800µm region about 4.4mm from the puncture site. After mechanical testing, the puncture site was imaged in 3D. Puncture had no significant effect on annulus elastic modulus. Imaging showed a modest change in the shearing between fibre bundles however, the linear strain between bundles, intra-bundle shear and linear strain were not significantly affected. At the puncture site, detached lumps of tissue were present. These results suggest that the mechanical effects observed in intact discs are due to the depressurization of the disc, rather than the local damage to the annulus. Needle profiles could be designed, aiming at separating fibre bundles rather than cutting through them, to avoid leaving dying tissue behind.

The effects of needle damage on annulus fibrosus micromechanics.

Terahertz-spectroscopy probes dynamics and spectral response of collective vibrational modes in condensed phase, which can yield insight into composition and topology. However, due to the long wavelengths employed (λ = 300 μm at 1THz), diffraction limited imaging is typically restricted to spatial resolutions around a millimeter. Here, we demonstrate a new form of subwavelength hyperspectral, polarization-resolved THz imaging which employs an optical pattern projected onto a 6 μm-thin silicon wafer to achieve near-field modulation of a co-incident THz pulse. By placing near-field scatterers, one can measure the interaction of object with the evanescent THz fields. Further, by measuring the temporal evolution of the THz field a sample's permittivity can be extracted with 65 μm spatial resolution due to the presence of evanescent fields. Here, we present the first application of this new approach to articular cartilage. We show that the THz permittivity in this material varies progressively from the superficial zone to the deep layer, and that this correlates with a change in orientation of the collagen fibrils that compose the extracellular matrix (ECM) of the tissue. Our approach enables direct interrogation of the sample's biophysical properties, in this case concerning the structure and permittivity of collagen fibrils and their anisotropic organisation in connective tissue.

Subwavelength hyperspectral THz studies of articular cartilage.

Type II collagen fibril diameters in cartilage are beneath the diffraction limit of optical microscopy, which makes the assessment of collagen organization very challenging. In this work we use polarization sensitive second harmonic generation (P-SHG) imaging to map collagen organization in articular cartilage, addressing in particular its behaviour under strain and changes which occur in osteoarthritis. P-SHG yields two parameters, molecular order and orientation, which provide measures of the degree of organization both at the molecular scale (below the diffraction limit) and above a few hundred nanometres (at the image pixel size). P-SHG clearly demonstrates the zonal collagen architecture and reveals differences in the structure of the fibrils around chondrocytes. P-SHG also reveals sub-micron scale fibril re-organization in cartilage strips exposed to tensile loading, with an increase in local organization in the superficial zone which weakly correlates with tensile modulus. Finally, P-SHG is used to investigate osteoarthritic cartilage from total knee replacement surgery, and reveals widespread heterogeneity across samples both microscale fibril orientations and their sub-micron organization. By addressing collagen fibril structure on scales intermediate between conventional light and electron microscopy, this study provides new insights into collagen micromechanics and mechanisms of degradation.

Collagen reorganization in cartilage under strain probed by polarization sensitive second harmonic generation microscopy.

Articular cartilage is a dense extracellular matrix-rich tissue that degrades following chronic mechanical stress, resulting in osteoarthritis (OA). The tissue has low intrinsic repair especially in aged and osteoarthritic joints. Here, we describe three pro-regenerative factors; fibroblast growth factor 2 (FGF2), connective tissue growth factor, bound to transforming growth factor-beta (CTGF-TGFβ), and hepatoma-derived growth factor (HDGF), that are rapidly released from the pericellular matrix (PCM) of articular cartilage upon mechanical injury. All three growth factors bound heparan sulfate, and were displaced by exogenous NaCl. We hypothesised that sodium, sequestered within the aggrecan-rich matrix, was freed by injurious compression, thereby enhancing the bioavailability of pericellular growth factors. Indeed, growth factor release was abrogated when cartilage aggrecan was depleted by IL-1 treatment, and in severely damaged human osteoarthritic cartilage. A flux in free matrix sodium upon mechanical compression of cartilage was visualised by Na -MRI just below the articular surface. This corresponded to a region of reduced tissue stiffness, measured by scanning acoustic microscopy and second harmonic generation microscopy, and where Smad2/3 was phosphorylated upon cyclic compression. Our results describe a novel intrinsic repair mechanism, controlled by matrix stiffness and mediated by the free sodium concentration, in which heparan sulfate-bound growth factors are released from cartilage upon injurious load. They identify aggrecan as a depot for sequestered sodium, explaining why osteoarthritic tissue loses its ability to repair. Treatments that restore matrix sodium to allow appropriate release of growth factors upon load are predicted to enable intrinsic cartilage repair in OA.

Jessica C. Mansfield

Biomedical Physics,
School of Physics and Astronomy,
Biomedical Physics, School of Physics and Astronomy

University of Exeter

Biomolecular Imaging of Cellular Uptake of Nanoparticles using Multimodal Nonlinear Optical Microscopy

Jessica C. Mansfield

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Biomedical Physics,School of Physics and Astronomy,Biomedical Physics, School of Physics and Astronomy

University of Exeter

Biomolecular Imaging of Cellular Uptake of Nanoparticles using Multimodal Nonlinear Optical Microscopy

Biomedical Physics,
School of Physics and Astronomy,
Biomedical Physics, School of Physics and Astronomy