Nanometer targeting of microtubules to focal adhesions

Although cell movement is driven by actin, polarization and directional locomotion require an intact microtubule cytoskeleton that influences polarization by modulating substrate adhesion via specific targeting interactions with adhesion complexes. The fidelity of adhesion site targeting is precise; using total internal reflection fluorescence microscopy (TIRFM), we now show microtubule ends (visualized by incorporation of GFP tubulin) are within 50 nm of the substrate when polymerizing toward the cell periphery, but not when shrinking from it. Multiple microtubules sometimes followed similar tracks, suggesting guidance along a common cytoskeletal element. Use of TIRFM with GFP- or DsRed-zyxin in combination with either GFP-tubulin or GFP–CLIP-170 further revealed that the polymerizing microtubule plus ends that tracked close to the dorsal surface consistently targeted substrate adhesion complexes. This supports a central role for the microtubule tip complex in the guidance of microtubules into adhesion foci, and provides evidence for an intimate cross-talk between microtubule tips and substrate adhesions in the range of molecular dimensions

SUPER MULTI-VIEW NEAR-EYE DISPLAY WITH LED ARRAY AND WAVEGUIDE ILLUMINATION MODULE

A near-eye display includes an array of light sources, a reflective spatial light modulator (SLM) synchronized with the array of light sources and configured to modulate and reflect incident light beams to generate images, display optics configured to project the images generated by the reflective SLM to a user’s eye, and a waveguide between the display optics and the reflective SLM, where the waveguide is configured to guide light beams emitted by the array of light sources and direct the light beams towards the reflective SLM to illuminate the reflective SL

MesoTIRF: a novel axial super-resolution illuminator for membrane imaging over a 4.4 mm x 3.0 mm field of view

In Total Internal Reflection Fluorescence (TIRF) microscopy, a specimen is illuminated by the evanescent field produced by a beam undergoing total internal reflection, whose characteristic depth is orders of magnitude below the axial diffraction limit. The axial super-resolution and much improved contrast of TIRF gives a substantially improved signal-to-background ratio (SBR) than that which can be achieved with widefield illumination, and it is used extensively for imaging of the cell membrane [1]– [3] . Commercial TIRF objectives allow for a simple adaption to existing microscope systems. To attain a super-critical angle at the specimen plane, the illumination must enter the back focal plane of these objectives off-axis, requiring a high numerical aperture. As such, the magnification of these objectives is generally a minimum of 60x, reducing the lateral imaging field to less than 100 µm in diameter. Sub-cellular axial resolution is therefore restricted to a tiny population of cells and statistically significant data sampling may be difficult to achieve. To address this, we have developed a TIRF illuminator for the Mesolens, a custom giant objective lens with a 4x/0.47NA specification. The Mesolens provides an imaging field of 4.4 mm x 3.0 mm, and in combination with our new TIRF illuminator which we call MesoTIRF we have performed imaging of large cell populations with sub-micron resolution in three-dimensions. With MesoTIRF we demonstrate more than a 5-fold improvement in SBR and significantly reduced photobleaching rate compared to widefield epifluorescence illumination. We will present details of the MesoTIRF system together with current and emerging applications in cell imaging

Modular multimodal platform for classical and high throughput light sheet microscopy

Light-sheet fluorescence microscopy (LSFM) has become an important tool for biological and biomedical research. Although several illumination and detection strategies have been developed, the sample mounting still represents a cumbersome procedure as this is highly dependent on the type of sample and often this might be time consuming. This prevents the use of LSFM in other promising applications in which a fast and straightforward sample-mounting procedure and imaging are essential. These include the high-throughput research fields, e.g. in drug screenings and toxicology studies. Here we present a new imaging paradigm for LSFM, which exploits modularity to offer multimodal imaging and straightforward sample mounting strategy, enhancing the flexibility and throughput of the system. We describe its implementation in which the sample can be imaged either as in any classical configuration, as it flows through the light-sheet using a fluidic approach, or a combination of both. We also evaluate its ability to image a variety of samples, from zebrafish embryos and larvae to 3D complex cell cultures.The authors acknowledge financial support from the Spanish Ministerio de Economía y Competitividad (MINECO) through the “Severo Ochoa” program for Centres of Excellence in R&D (CEX2019-000910-S [MCIN/ AEI/10.13039/501100011033]), Fundació Privada Cellex, Fundació Mir-Puig, and Generalitat de Catalunya through CERCA program; MINECO/FEDER Ramón y Cajal program (RYC-2015-17935); Laserlab- Europe EU-H2020 GA no. 871124; European Union’s Horizon 2020 Framework Programme (H2020 Marie Skłodowska-Curie Innovative Training Networks ImageInLife N. 721537). We thank Verena Ruprecht (CRG- Center of Genomic Regulation, Barcelona), Paz Herráez (Universidad de León), Ester Antón-Galindo and Noelia Fernández-Castillo (Universitat de Barcelona), Marymar Becerra (Universidad Nacional Autónoma de México), Georges Lutfalla, Mai Nguyen Chi and Tamara Sipka (Université de Montpellier), Catarina Brito (ITQB/IBEQ, Lisbon), Antonia Weberling and Magdalena Zernicka-Goetz (University of Cambridge), and Corinne Lorenzo (ITAV – CNRS, Toulouse) for the samples provided. We also thank Maria Marsal and Jordi Andilla for many fruitful discussions.Postprint (published version

Fast widefield techniques for fluorescence and phase endomicroscopy

Thesis (Ph.D.)--Boston UniversityEndomicroscopy is a recent development in biomedical optics which gives researchers and physicians microscope-resolution views of intact tissue to complement macroscopic visualization during endoscopy screening. This thesis presents HiLo endomicroscopy and oblique back-illumination endomicroscopy, fast widefield imaging techniques with fluorescence and phase contrast, respectively. Fluorescence imaging in thick tissue is often hampered by strong out-of-focus background signal. Laser scanning confocal endomicroscopy has been developed for optically-sectioned imaging free from background, but reliance on mechanical scanning fundamentally limits the frame rate and represents significant complexity and expense. HiLo is a fast, simple, widefield fluorescence imaging technique which rejects out-of-focus background signal without the need for scanning. It works by acquiring two images of the sample under uniform and structured illumination and synthesizing an optically sectioned result with real-time image processing. Oblique back-illumination microscopy (OBM) is a label-free technique which allows, for the first time, phase gradient imaging of sub-surface morphology in thick scattering tissue with a reflection geometry. OBM works by back-illuminating the sample with the oblique diffuse reflectance from light delivered via off-axis optical fibers. The use of two diametrically opposed illumination fibers allows simultaneous and independent measurement of phase gradients and absorption contrast. Video-rate single-exposure operation using wavelength multiplexing is demonstrated

Fast, Three-Dimensional Fluorescence Imaging of Living Cells

This thesis focuses on multi-plane fluorescence microscopy for fast live-cell imaging. To improve the performance of multi-plane microscopy, I developed new image analysis methods. I used these methods to measure and analyze the movements of cardiomyocytesand Dictyostelium discoideum cells.The multi-plane setup is based on a conventional wide-field microscope using a custom multiple beam-splitter in the detection path. This prism creates separate images of eight distinct focal planes in the sample. Since 3D volume is imaged without scanning, three-dimensional imaging at a very high speed becomes possible. However, as in conventional wide-field microscopy, the "missing cone" of spatial frequencies along the optical axis in the optical transfer function (OTF) prevents optical sectioning in such a microscope. This is in stark contrast to other truly three-dimensional imaging modalities like confocal and light-sheet microscopy. In order to overcome the lack of optical sectioning, I developed a new deconvolution method. Deconvolution describes methods that restore or sharpen an image based on physical assumptions and knowledge of the imaging process. Deconvolution methods have been widely used to sharpen images of microscopes and telescopes. The recently developed SUPPOSe algorithm is a deconvolution algorithm that uses a set of numerous virtual point sources. It tries to reconstruct an image by distributing these point sources in space and optimizing their positions so that the resulting image reproduces as good as possible the measured data. SUPPOSe has never been used for 3D images. Compared to other algorithms, this method has superior performance when the number of pixels is increased by interpolation. In this work, I extended the method to work also with 3D image data. The 3D-SUPPOSe program is suitable for analyzing data of our multi-plane setup. The multi-plane setup has only eight vertically aligned image planes. Furthermore, for accurate reconstruction of 3D images, I studied a method of correcting each image plane's relative brightness constituting an image, and I also developed a method of measuring the movement of point emitters in 3D space. Using these methods, I measured and analyzed the beating motion of cardiomyocytes and the chemotaxis of Dicyosteilium discoidem. Cardiomyocytes are the cells of the heart muscle and consist of repetitive sarcomeres. These cells are characterized by fast and periodic movements, and so far the dynamics of these cells was studied only with two-dimensional imaging. In this thesis, the beating motion was analyzed by tracing the spatial distribution of the so-called z-discs, one of the constituent components of cardiomyocytes. I found that the vertical distribution of $\alpha$-actinine-2 in a single z-disc changed very rapidly, which may serve as a starting point for a better understanding the motion of cardiomyocytes. \textit{Dictyostelium discoideum} is a well established single cell model organism that migrates along the gradient of a chemoattractant. One has conducted much research to understand the mechanism of chemotaxis, and many efforts have been made to understand the role of actin in the chemotactic motion. By suppressing the motor protein, myosin, a cell line was created that prevented the formation of normal actin filaments. In these myosin null cells, F-actin moves in a flow-like behaviour and induces cell movement. In this study, I imaged the actin dynamics, and I analyzed the flow using the newly created deconvolution and flow estimation methods. As a result of the analysis, the spatio-temporal correlation between pseudo-pod formation and dynamics and actin flow was investigated.2022-01-2

Integrated multicore fibre devices for optical trapping

The work described in this thesis details the development of a multicore fibre device that can be used to optically trap multiple cells and particles. The optical trapping of multiple cells at close proximity allows for cell-to-cell interactions to be studied. Current methods available for creating arrays of traps are free space optical systems that use diffractive optics, laser scanning techniques or the interference of multiple beams to create the multiple traps. A fully integrated, fibre optic based, multiple particles, optical trapping device could be used in non-optical research facilities such as biological laboratories to aid with their research into cellular processes. In order to create the multiple traps, the distal end of the multicore fibre needs to be modified to induce a lensing effect. The multicore fibre device presented in this thesis was lensed in a fusion splicer; this refracts the outputs from the four cores to a common point in the far field where interference fringes are formed. The initial investigation demonstrated one-dimensional interferometric optical trapping through coupling light into two of the diagonal cores of the lensed multicore fibre. This produced linear interference fringes approximately 250 ± 25 μm from the end of the fibre with a fringe spacing of 2 ± 0.3 μm. The linear interference fringes were used to optically trap polystyrene microspheres with diameters of 1.3 μm, 2 μm and 3 μm in the high intensity regions of the fringes. Coupling into all four cores using a diffractive optical element produced an array of intensity peaks across the interference pattern with high visibility fringes greater than 80 %. Each intensity peak, spaced 2.75 μm apart could trap a single particle in two dimensions. The optical trapping of multiple microspheres and Escherichia coli bacterial cells was demonstrated proving that the lensed multicore fibre has the potential to be used to trap cells in biological experiments. The active manipulation of trapped 2 μm microspheres was also demonstrated through the rotation of the input polarisation to the multicore fibre. Finally, work towards creating a “turn-key” optical trapping device was demonstrated through the fabrication of a fully integrated multicore fibre device using an ultrafast laser-inscribed fan-out to couple light into each core. Single mode operation of the device was demonstrated at 1550 nm, using a weaker lensed MCF device. The two dimensional trapping of 4.5 μm polystyrene microspheres was shown in an array of peaks spaced 11.2 μm apart at a distance of 400 ± 25 μm from the end of the fibre

Snapshot hyperspectral imaging : near-infrared image replicating imaging spectrometer and achromatisation of Wollaston prisms

Conventional hyperspectral imaging (HSI) techniques are time-sequential and rely on temporal scanning to capture hyperspectral images. This temporal constraint can limit the application of HSI to static scenes and platforms, where transient and dynamic events are not expected during data capture. The Near-Infrared Image Replicating Imaging Spectrometer (N-IRIS) sensor described in this thesis enables snapshot HSI in the short-wave infrared (SWIR), without the requirement for scanning and operates without rejection in polarised light. It operates in eight wavebands from 1.1μm to 1.7μm with a 2.0° diagonal field-of-view. N-IRIS produces spectral images directly, without the need for prior topographic or image reconstruction. Additional benefits include compactness, robustness, static operation, lower processing overheads, higher signal-to-noise ratio and higher optical throughput with respect to other HSI snapshot sensors generally. This thesis covers the IRIS design process from theoretical concepts to quantitative modelling, culminating in the N-IRIS prototype designed for SWIR imaging. This effort formed the logical step in advancing from peer efforts, which focussed upon the visible wavelengths. After acceptance testing to verify optical parameters, empirical laboratory trials were carried out. This testing focussed on discriminating between common materials within a controlled environment as proof-of-concept. Significance tests were used to provide an initial test of N-IRIS capability in distinguishing materials with respect to using a conventional SWIR broadband sensor. Motivated by the design and assembly of a cost-effective visible IRIS, an innovative solution was developed for the problem of chromatic variation in the splitting angle (CVSA) of Wollaston prisms. CVSA introduces spectral blurring of images. Analytical theory is presented and is illustrated with an example N-IRIS application where a sixfold reduction in dispersion is achieved for wavelengths in the region 400nm to 1.7μm, although the principle is applicable from ultraviolet to thermal-IR wavelengths. Experimental proof of concept is demonstrated and the spectral smearing of an achromatised N-IRIS is shown to be reduced by an order of magnitude. These achromatised prisms can provide benefits to areas beyond hyperspectral imaging, such as microscopy, laser pulse control and spectrometry