Significance: Stimulated Raman scattering (SRS) and pump-probe microscopy are implementations of multiphoton microscopy that acquire high-resolution, label-free images of live samples encoded with molecular contrast. Most commercial multiphoton microscopes cannot access these techniques since they require sample illumination by two temporally synchronized ultrafast pulse trains. We present a compact and robust way of synchronizing an additional Ti:sapphire laser with a conventional single-beam multiphoton microscope to realize an instrument that can acquire images with enhanced molecular specificity.
Aim: A passive optical synchronization scheme for a pair of commercially available, unmodified modelocked Ti:sapphire lasers was developed. The suitability of this synchronization scheme for advanced biomedical microscopy was investigated.
Approach: A pair of modelocked Ti:sapphire lasers were aligned in master–slave configuration. Five percent of the master laser output was used to seed the modelocking in the slave laser cavity. The timing jitter of the master and slave pulse trains was characterized using an optical autocorrelator. The synchronized output of both lasers was coupled into a laser scanning microscope and used to acquire spectral focusing SRS and pump-probe microscopy images from biological and nonbiological samples.
Results: A timing jitter between the modelocked pulse trains of 0.74 fs was recorded. Spectral focusing SRS allowed spectral discrimination of polystyrene and polymethyl methacrylate beads. Pump-probe microscopy was used to record excited state lifetime curves from hemoglobin in intact red blood cells.
Conclusion: Our work demonstrates a simple and robust method of upgrading single-beam multiphoton microscopes with an additional ultrafast laser. The resulting dual-beam instrument can be used to acquire label-free images of sample structure and composition with high biochemical specificity.
The world’s population is increasing rapidly and higher calorific diets are becoming more common; as a consequence the demand for grain is predicted to increase by more than 50% by 2050 without a significant increase in the available agricultural land. Maximising the productivity of the existing agricultural land is key to maintaining food security and agrochemicals continue to be a key enabler for the efficiency gains required. However, agrochemicals can be susceptible to significant losses and thus often require further chemical to be applied to compensate. Sources of such losses include spray drift, poor spray retention/capture by the target and poor penetration through the plant cuticle. Adjuvants can be used to help mitigate such losses but characterising how they alter the movement of the active ingredients (AIs) can be challenging. In this contribution we demonstrate the use of coherent Raman Scattering (CRS) as a tool to enable in-situ, real-time, label free characterisation of agrochemical AI as they move through wax.
Treatment of dandruff condition usually involves use of antidandruff shampoos containing antifungal agents. Different antifungal agents show variable clinical efficacy based on their cutaneous distribution and bioavailability. Using stimulated Raman scattering (SRS), we mapped the distribution of unlabeled low-molecular weight antifungal compounds zinc pyrithione (ZnPT) and climbazole (CBZ) on the surface of intact porcine skin with cellular precision. SRS has sufficient chemical selectivity and sensitivity to detect the agents on the skin surface based on their unique chemical motifs that do not occur naturally in biological tissues. Moreover, SRS is able to correlate the distribution of the agents with the morphological features of the skin using the CH2 stretch mode, which is abundant in skin lipids. This is a significant strength of the technique since it allows the microscopic accumulation of the agents to be correlated with physiological features and their chemical environment without the use of counter stains. Our findings show that due to its lower solubility, ZnPT coats the surface of the skin with a sparse layer of crystals in the size range of 1 to 4 μm. This is consistent with the current understanding of the mode of action of ZnPT. In contrast, CBZ being more soluble and hydrophobic resulted in diffuse homogeneous distribution. It predominantly resided in microscopic lipid-rich crevasses and penetrated up to 60 μm into the infundibular spaces surrounding the hair shaft. The ability of the SRS to selectively map the distribution of agents on the skin’s surface has the potential to provide insight into the mechanisms underpinning the topical application of antifungal or skin-active agents that could lead to the rational engineering of enhanced formulations.
The tissues surrounding the seeds play an important role in the control of germination vigour and in the uptake of active ingredients (AIs) applied as seed dressings. The seeds can reduce costs through more efficient germination rates and have less environmental impact due to more efficient use of AIs. In this study, we use epi-detected stimulated Raman scattering (SRS) microscopy with a fiber laser source for analyzing modes of D2O into intact seeds and determining uptake kinetics. We demonstrate that SRS microscopy is an ideal tool for visualizing uptake of AIs and worthy of further applications in real field conditions.
Recent advances in pharmaceutical nanotechnology have enabled the development of nano-particulate medicines with enhanced drug performance. Although the fate of these nano-particles can be macroscopically tracked in the body (e.g. using radio-labeling techniques), there is little information about the sub-cellular scale mechanistic processes underlying the particle-tissue interactions, or how these interactions may correlate with pharmaceutical efficacy. To rationally engineer these nano-particles and thus optimize their performance, these mechanistic interactions must be fully understood. Coherent Anti-Stokes Raman scattering (CARS) microscopy provides a label-free means for visualizing biological samples, but can suffer from a strong non-resonant background in samples that are prepared using aldehyde-based fixatives. We demonstrate how formalin fixative affects the detection of polymeric nanoparticles within kidneys following oral administration using CARS microscopy, compared with samples that were snap-frozen. These findings have implications for clinical applications of CARS for probing nanoparticle distribution in tissue biopsies.
The potential of microalgae as a source of renewable energy has received considerable interest because they can produce lipids (fatty acids and isoprenoids) that can be readily converted into biofuels. However, significant research in this area is required to increase yields to make this a viable renewable source of energy. An analytical tool that could provide quantitative in situ spectroscopic analysis of lipids synthesis in individual microalgae would significantly enhance our capability to understand the synthesis process at the cellular level and lead to the development of strategies for increasing yield. Stimulated Raman scattering (SRS) microscopy has great potential in this area however, the pump-probe signal from two-color two-photon absorption of pigments (chlorophyll and carotenoids) overwhelm the SRS signal and prevent its application. Clearly, the development of a background suppression technique is of significant value for this important research area.
To overcome the limitation of SRS in pigmented specimens, we establish a frequency-modulated stimulated Raman scattering (FM-SRS) microscopy that eliminates the non-Raman background by rapidly toggling on-and-off the targeted Raman resonance. Moreover, we perform the background-free imaging and analysis of intracellular lipid droplets and extracellular hydrocarbons in a green microalga with FM-SRS microscopy. We believe that FM-SRS microscopy demonstrates the potential for many applications in pigmented cells and provides the opportunity for improved selective visualization of the chemical composition of algae and plants
Fungi have been found to be an underlying cause of 70% of all plant and animal extinctions caused by infectious
diseases. Fungal infections are a growing problem affecting global health, food production and ecosystems. Lipid
metabolism is a promising target for antifungal drugs and since effective treatment of fungal infections requires a better
understanding of the effects of antifungal agents at the cellular level, new techniques are needed to investigate this
problem.
Recent advances in nonlinear microscopy allow chemically-specific contrast to be obtained non-invasively from intrinsic
chemical bonds within live samples using advanced spectroscopy techniques probing Raman-active resonances. We
present preliminary data using Stimulated Raman Scattering (SRS) microscopy as a means to visualise lipid droplets
within individual living fungi by probing Raman resonances of the CH stretching region between 2825cm-1 and
3030cm-1.
Microscopic chemical mapping of living plant tissues without the use of extrinsic labels would represent a major
advance in analytical capability for many areas of biological research; Coherent Raman Scattering (CRS) microscopy
offers label-free chemical imaging based on vibrational spectroscopy and is an obvious solution. However, due to the
high levels of optical absorption and fluorescent emission in plant tissues the technique is severely limited for in-vivo
plant imaging. This paper reports preliminary results regarding the technical issues associated with performing label-free
imaging in plant tissues with CRS and discusses how they may be mitigated in future applications.
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 µm 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 2.65×10−4. 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.
We report on the use of phase-resolved DOCT to measure oscillatory flow in a rigid glass capillary. Experimental
measurements are obtained in which a sinusoidal pressure gradient generates a sinusoidal varying flow of a scattering
liquid (Intralipid-20%) in a 660 micron i.d. glass tube. Dynamic flow profiles are measured and compared with the
analytical theory due to Womersley. Reasonable agreement is found for one half of the flow profile (-ve velocities) but
the agreement is less good for the +ve velocities. Possible reasons for the discrepancy are discussed. The study indicates
the usefulness of DOCT for studying fluid flow dynamics.
Surface-enhanced Raman scattering (SERS) has received a great deal interest as an analytical tool due to its potential
for obtaining Raman signals from single molecules. Many methods for preparing SERS-active substrate have been
reported. These range from nano-particle based methods, which lack reproducibility, to highly reproducible nano-arrays
requiring time consuming and costly preparation. We show that highly reproducible SERS can be achieved by applying
a metallic coating to the brightly coloured regions of the graphium weiskei butterfly wing. Electron microscopy reveals
the wing exhibit nanostructures with comparable dimensions to the roughness scale of SERS substrates. SERS
measurements performed on wings coated with 60 nm of silver display enhancement factors of approximately 107 with
no apparent background contribution from the wing. To demonstrate effectiveness and reproducibility the substrate is
coated with a monoclonal antibody.
Articular cartilage possesses an extensive extracellular matrix consisting of a highly organised network of collagen
fibres embedded in a much finer mesh of proteoglycans and other glycoproteins. Many fundamental issues of cartilage
biomechanics, its ageing and the development of osteoarthritis concern the detailed organisation of this matrix. Here we
investigate the application of multi-photon microscopy to characterise the structure of the extracellular matrix. In
reflection mode both second harmonic Generation (SHG) and two photon fluorescence (TPF) imaging modalities reveal
differences in the pericellular and inter-territorial matrix in normal tissue and additional changes in degenerative lesions.
The SHG signal from the surface zone is dependent on the direction of polarization of the laser excitation beam but the
TPF signal is not. The former can be quantified to determine fibre orientation although the pattern is less well resolved
than in tendon, reflecting the less regular orientation of the finer fibres. Nevertheless, previously unreported subtle
variations in fibre orientation over the surface of the cartilage can be observed. In order to characterise variations with
depth we carried out polarization sensitivity experiments at depths up to 180 microns into the tissue. At greater depths
the polarization sensitivity is affected by the birefringence and dichroism of the overlying tissue and we have quantified
these effects to allow correction of the data.
We report on the feasibility of surface enhanced Raman scattering (SERS) as a highly sensitive technique for detecting peptide phosphorylation. Compared with existing techniques for quantifying peptide phosphorylation, such as high-performance liquid chromatography (HPLC), the short scanning and processing time associated with SERS makes it an attractive alternative for measurement on a near-real-time basis at sub micro-molar concentrations. Using the recently reported drop-coating deposition Raman method we compare our SERS spectra to normal Raman spectra that would otherwise be unobtainable at such low concentration.
We investigate the effect of multiple scattering upon Doppler optical coherence tomography images of model blood vessels immersed in a fluuid with similar optical properties to those of the human dermis. Furthermore, we quantify the deviation of the acquired velocity profiles from that known to exist within the glass capillary at various depths within the scattering media. A flow phantom consisting of a glass tube containing whole blood flowing under laminar conditions submerged in a variable depth of Intralipid was used to simulate a blood vessel within the cutaneous microcirculation. Doppler optical coherence tomography images and velocity profiles of the tube acquired at various depths within the Intralipid are compared to those obtained from the same tube in a non-scattering media with the same refractive index.
Blood, being a suspension of deformable red cells suspended in plasma, displays flow dynamics considerably more complicated than those of an ideal Newtonian fluid. Flow dynamics in blood capillaries of a few hundred micrometers in diameter are investigated using Doppler optical coherence tomography (DOCT) and Doppler amplitude optical coherence tomography (DAOCT), a novel extension of DOCT. Velocity profiles and concentration distributions of normal and rigidified in vitro red blood cell suspensions are shown to vary as functions of mean flow velocity, cell concentration, and cell rigidity. Deviation from the parabolic velocity profile expected for Pouseille flow is observed for both rigid and normal cells at low flow rates. Axial red cell migration both toward and away from the tube axis is observed for both rigid and normal cells as a function of flow velocity. Good agreement is found between our measurements, and theoretical expectations.
Flow dynamics in blood vessels of a few hundred microns in diameter were investigated using Doppler optical coherence tomography (DOCT) and Doppler Amplitude optical coherence tomography (DAOCT), a novel extension of DOCT. The motivation behind this work is discussed, followed by a brief explanation of the theory underlying the motion of blood cells in small conduits and blood vessels. Preliminary results are presented and compared to the predictions expected from theory. All significant findings are analysed along with their importance to microvascular research.
Phase-resolved Doppler optical coherence tomography is a recently reported technique for simultaneously imaging tissue structure and blood with high velocity resolution. The optical set-up consists of a fibre-based Michelson interferometer with a 1300nm superluminescent diode in the source arm. The output power is 0.6mW with a bandwidth of 50nm. The reference arm contains a grating-based Fourier domain rapid-scanning delay line with an electro-optic phase modulator to provide a stable reference frequency (800kHz). Ten axial scans sampled, at 400Hz, from the same location are processed to generate structural and velocity data from the reconstructed phase information derived from a Hilbert transform. The sample arm probe focuses light from the fibre into the tissue, producing a beam spot of diameter approximately 20micrometers . The probe is mounted on a linear translation stage, which generates a lateral step of 10micrometers between groups of ten axial scans. The Doppler shift in each pixel is calculated from the average phase shift over the ten sequential scans at each location. The acquisition time for a 100x100 pixel image is approximately 5s. We demonstrate the systems ability to image in-vivo changes in skin perfusion, induced by standard non-invasive physiological techniques.
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