ABSTRACT: Optical microscopy is an indispensable tool in biomedical sciences, but its reach in deep tissues is limited due to aberrations and scattering. This problem can be overcome by wavefront-shaping techniques, albeit at limited fields of view (FOVs). Inspired by astronomical imaging, conjugate wavefront shaping can lead to an increased field of view in microscopy, but this correction is limited to a set depth and cannot be dynamically adapted. Here, we present a conjugate wavefront-shaping scheme based on focus scanning holographic aberration probing (F-SHARP). We combine it with a compact implementation that can be readily adapted to a variety of commercial and home-built two-photon microscopes. We demonstrate the power of the method by imaging with high resolution over extended FOV (>80 µm) deeper than 400 μm inside a mouse brain through a thinned skull.
Project description:Although near-field imaging techniques reach sub-nanometer resolution on rigid samples, it remains extremely challenging to image soft interfaces, such as biological membranes, due to the deformations induced by the probe. In photonic force microscopy, optical tweezers are used to manipulate and measure the scanning probe, allowing imaging of soft materials without force-induced artifacts. However, the size of the optically trapped probe still limits the maximum resolution. Here, we show a novel and simple nanofabrication protocol to massively produce optically trappable quartz particles which mimic the sharp tips of atomic force microscopy. Imaging rigid nanostructures with our tips, we resolve features smaller than 80 nm. Scanning the membrane of living malaria-infected red blood cells reveals, with no visible artifacts, submicron features termed knobs, related to the parasite activity. The use of nanoengineered particles in photonic force microscopy opens the way to imaging soft samples at high resolution.
Project description:The uptake of menadione (2-methyl-1,4-naphthoquinone), which is toxic to yeast cells, and its expulsion as a glutathione complex were studied by scanning electrochemical microscopy. The progression of the in vitro reaction between menadione and glutathione was monitored electrochemically by cyclic voltammetry and correlated with the spectroscopic (UV-visible) behavior. By observing the scanning electrochemical microscope tip current of yeast cells suspended in a menadione-containing solution, the export of the conjugate from the cells with time could be measured. Similar experiments were performed on immobilized yeast cell aggregates stressed by a menadione solution. From the export of the menadione-glutathione conjugate detected at a 1-microm-diameter electrode situated 10 microm from the cells, a flux of about 30,000 thiodione molecules per second per cell was extracted. Numerical simulations based on an explicit finite difference method further revealed that the observation of a constant efflux of thiodione from the cells suggested the rate was limited by the uptake of menadione and that the efflux through the glutathione-conjugate pump was at least an order of magnitude faster.
Project description:We investigate the radiation patterns of sharp conical gold tapers, which were designed as adiabatic nanofocusing probes for scanning near-field optical microscopy (SNOM). Field calculations show that only the lowest order eigenmode of such a taper can reach the very apex and thus induce the generation of strongly enhanced near-field signals. Higher-order modes are coupled into the far field at finite distances from the apex. Here, we demonstrate experimentally how to distinguish and separate between the lowest and higher-order eigenmodes of such a metallic taper by filtering in the spatial frequency domain. Our approach has the potential to considerably improve the signal-to-background ratio in spectroscopic experiments at the nanoscale.
Project description:Research tasks in microgravity include monitoring the dynamics of constituents of varying size and mobility in processes such as aggregation, phase separation, or self-assembly. We use differential dynamic microscopy, a method readily implemented with equipment available on the International Space Station, to simultaneously resolve the dynamics of particles of radius 50?nm and 1??m in bidisperse aqueous suspensions. Whereas traditional dynamic light scattering fails to detect a signal from the larger particles at low concentrations, differential dynamic microscopy exhibits enhanced sensitivity in these conditions by accessing smaller wavevectors where scattering from the large particles is stronger. Interference patterns due to scattering from the large particles induce non-monotonic decay of the amplitude of the dynamic correlation function with the wavevector. We show that the position of the resulting minimum contains information on the vertical position of the particles. Together with the simple instrumental requirements, the enhanced sensitivity of differential dynamic microscopy makes it an appealing alternative to dynamic light scattering to characterize samples with complex dynamics.
Project description:The total number of data points required for image generation in Raman microscopy was greatly reduced using sparse sampling strategies, in which the preceding set of measurements informed the next most information-rich sampling location. Using this approach, chemical images of pharmaceutical materials were obtained with >99% accuracy from 15.8% sampling, representing an ∼6-fold reduction in measurement time relative to full field of view rastering with comparable image quality. This supervised learning approach to dynamic sampling (SLADS) has the distinct advantage of being directly compatible with standard confocal Raman instrumentation. Furthermore, SLADS is not limited to Raman imaging, potentially providing time-savings in image reconstruction whenever the single-pixel measurement time is the limiting factor in image generation.
Project description:This paper discusses several methods for manufacturing ultra-sharp probes, with applications geared toward, but not limited to, scanning microscopy (STM, AFM) and intra-cellular recordings of neural signals. We present recipes for making tungsten, platinum/iridium alloy, and nanotube fibril tips. Electrical isolation methods using Parylene-C or PMMA are described.
Project description:We describe a wavefront sensor strategy for the implementation of adaptive optics (AO) in microscope applications involving thick, scattering media. The strategy is based on the exploitation of multiple scattering to provide oblique back illumination of the wavefront-sensor focal plane, enabling a simple and direct measurement of the flux-density tilt angles caused by aberrations at this plane. Advantages of the sensor are that it provides a large measurement field of view (FOV) while requiring no guide star, making it particularly adapted to a type of AO called conjugate AO, which provides a large correction FOV in cases when sample-induced aberrations arise from a single dominant plane (e.g., the sample surface). We apply conjugate AO here to widefield (i.e., nonscanning) fluorescence microscopy for the first time and demonstrate dynamic wavefront correction in a closed-loop implementation.
Project description:Myelination processes are closely related to higher brain functions such as learning and memory. While their longitudinal observation has been crucial to understanding myelin-related physiology and various brain disorders, skull opening or thinning has been required to secure clear optical access. Here we present a high-speed reflection matrix microscope using a light source with a wavelength of 1.3 μm to reduce tissue scattering and aberration. Furthermore, we develop a computational conjugate adaptive optics algorithm designed for the recorded reflection matrix to optimally compensate for the skull aberrations. These developments allow us to realize label-free longitudinal imaging of cortical myelin through an intact mouse skull. The myelination processes of the same mice were observed from 3 to 10 postnatal weeks to the depth of cortical layer 4 with a spatial resolution of 0.79 μm. Our system will expedite the investigations on the role of myelination in learning, memory, and brain disorders.
Project description:In conventional confocal/multiphoton fluorescence microscopy, images are typically acquired under ideal settings and after extensive optimization of parameters for a given structure or feature, often resulting in information loss from other image attributes. To overcome the problem of selective data display, we developed a new method that extends the imaging dynamic range in optical microscopy and improves the signal-to-noise ratio. Here we demonstrate how real-time and sequential high dynamic range microscopy facilitates automated three-dimensional neural segmentation. We address reconstruction and segmentation performance on samples with different size, anatomy and complexity. Finally, in vivo real-time high dynamic range imaging is also demonstrated, making the technique particularly relevant for longitudinal imaging in the presence of physiological motion and/or for quantification of in vivo fast tracer kinetics during functional imaging.
Project description:Multiphoton microscopes are hampered by limited dynamic range, preventing weak sample features from being detected in the presence of strong features, or preventing the capture of unpredictable bursts in sample strength. We present a digital electronic add-on technique that vastly improves the dynamic range of a multiphoton microscope while limiting potential photodamage. The add-on provides real-time negative feedback to regulate the laser power delivered to the sample, and a log representation of the sample strength to accommodate ultrahigh dynamic range without loss of information. No microscope hardware modifications are required, making the technique readily compatible with commercial instruments. Benefits are shown in both structural and in-vivo functional mouse brain imaging applications.