Homogeneous multifocal excitation for high-throughput super-resolution imaging

Super-resolution microscopies have become an established tool in biological research. However, imaging throughput remains a main bottleneck in acquiring large datasets required for quantitative biology. Here we describe multifocal flat illumination for field-independent imaging (mfFIFI). By integrating mfFIFI into an instant structured illumination microscope (iSIM), we extend the field of view (FOV) to >100 x 100 mu m(2) while maintaining high-speed, multicolor, volumetric imaging at double the diffraction-limited resolution. We further extend the effective FOV by stitching adjacent images for fast live-cell super-resolution imaging of dozens of cells. Finally, we combine our flat-fielded iSIM with ultrastructure expansion microscopy to collect three-dimensional (3D) images of hundreds of centrioles in human cells, or thousands of purified Chlamydomonas reinhardtiic entrioles, per hour at an effective resolution of similar to 35 nm. Classification and particle averaging of these large datasets enables 3D mapping of posttranslational modifications of centriolar microtubules, revealing differences in their coverage and positioning.

High-throughput and adaptive super-resolution fluorescence microscopy of organelles

Dora Mahecic

Fluorescence super-resolution microscopy has allowed unprecedented insight into the workings of biological systems below the diffraction limit of light. Over the past decade, it has overcome several challenges to deliver 3D, multi-color and faster imaging techniques, which have found many applications in deciphering the workings and structure of organelles â specialized cellular compartments whose shape and dynamics serve their specific functions. However, most super-resolution techniques are laborious and slow, and there are existing challenges in achieving sufficient throughput for collection of large datasets and capturing dynamic processes in living cells. The aim of this thesis is to address issues of limited throughput, temporal resolution and live-cell compatibility, with application to centriole architecture and mitochondrial dynamics. The design of a novel flat-fielding module for multi-focal excitation which â once integrated into an instant structured illumination microscope â allows the capture of larger fields of view with uniform illumination and hence results in increased throughput. This flat-fielded microscope allows multi-color, 3-dimensional imaging at double the diffraction-limited resolution of up to 100 mammalian cells within 1-2 minutes. Furthermore, combined with advanced sample preparation using expansion microscopy, the setup achieves an effective resolution of ~35 nm, comparable to some of the more powerful super-resolution techniques, but at 100-1000 times increased throughput. To showcase this improvement, we study the distribution of post-translational modifications within the centriole, revealing previously unobserved molecular organization. Second, with fast, live-cell imaging, we investigate the role of membrane bending energy and tension in mitochondrial division. While many molecular factors involved in this process have been identified, little is known about its physical requirements. By analyzing mitochondrial constrictions enriched in Drp1 that divide successfully or reverse, we establish that mitochondrial constrictions with higher bending energy are more likely to divide. Furthermore, by analyzing changes in mitochondrial shape and employing a novel membrane tension sensor, we observe that conditions under which mitochondrial membrane tension is increased, result in more successful divisions. These observations are supported by a model in which membrane bending energy increases the elastic energy at the constriction site, bringing it closer to the energy barrier to fission, while membrane tension acts as a fluctuation to overcome the remaining residual energy. Finally, to overcome existing trade-offs between temporal resolution and imaging duration, we develop an acquisition framework for smart and adaptive temporal sampling. By adapting the temporal resolution is in response to events of interest, they are captured at high imaging speed, while preserving the sample during a lack thereof. Using the accumulation of Drp1 to detect mitochondrial constriction sites allowed us to adapt the imaging speed to capture mitochondrial constrictions at high temporal resolution while preserving the sample when such events are lacking. Overall, this framework provides both high temporal resolution and long imaging times when needed, decreasing resulting phototoxicity, photobleaching and amount of redundant data.

EPFL2020

Diamond-Based Nanoscale Quantum Relaxometry for Sensing Free Radical Production in Cells

Mayeul Sylvain Chipaux, Hoda Shirzad

Diamond magnetometry makes use of fluorescent defects in diamonds to convert magnetic resonance signals into fluorescence. Because optical photons can be detected much more sensitively, this technique currently holds several sensitivity world records for room temperature magnetic measurements. It is orders of magnitude more sensitive than conventional magnetic resonance imaging (MRI) for detecting magnetic resonances. Here, the use of diamond magnetometry to detect free radical production in single living cells with nanometer resolution is experimentally demonstrated. This measuring system is first optimized and calibrated with chemicals at known concentrations. These measurements serve as benchmarks for future experiments. While conventional MRI typically has millimeter resolution, measurements are performed on individual cells to detect nitric oxide signaling at the nanoscale, within 10-20 nm from the internalized particles localized with a diffraction limited optical resolution. This level of detail is inaccessible to the state-of-the-art techniques. Nitric oxide is detected and the dynamics of its production and inhibition in the intra- and extracellular environment are followed.

WILEY-V C H VERLAG GMBH2022

Developing luminescent Brownian probes for near-field investigations of the intracellular environment

Flavio Maurizio Mor

Life sciences have a constantly growing need for novel methodological approaches suitable to investigate the intracellular environment with increased temporal and spatial resolutions. Recently, optical near-field probes, such as laser-irradiated pointed metal, uncoated/metal-coated tapered optical fibers, as well as nano-emitters, such as single molecules or nanoparticles, have attracted increasing attention as key components of high-resolution microscopes. In parallel, super-resolution fluorescence microscopy, operating in far-field regime and overcoming the light diffraction limit, has markedly improved imaging resolution. Although both near- and far-field optical approaches drastically improve image resolution, they still represent numerous drawbacks, such as, technical difficulties concerning probe preparation (near-field) or potential damaging through very high light intensities (far-field). The aforementioned shortcomings of high-resolution optical microscopy can be overcome to a great extent by photonic force microscopy (PFM). PFM employs a strongly focused near-infrared (NIR) laser light to hold a dielectric or metallic particle as a local probe. Such an optical trap enables one to ‘cage’ a mesoscopic particle and track its three-dimensional (3D) thermal fluctuations in the surrounding environment, e.g. a viscous liquid. Therefore, besides offering near-field 3D imaging, PFM reports also on other important quantities concerning local mechanical properties, including force and viscosity as well as dynamical properties of the surrounding medium. This additional information is derived from the careful analysis of the jittery motion (so-called Brownian motion) of the trapped single-particle probe that collides with thermally-activated surrounding molecules. In this thesis, we first study in detail the Brownian motion of a single spherical particle that is confined within the harmonic potential of the optical trap. To this end, we optimize the NIR light path and electronic noise floor of our custom-built PFM set-up for detecting and quantifying resonances in the Brownian motion. These resonances arise from the coupling between the hydrodynamic memory and strong strength of the optical trap. Due to the high sensitivity of the short-time dynamics, the size of the particle can be measured by simultaneous fitting of the velocity autocorrelation function and power spectral density of its thermal fluctuations. In order to choose the best-suitable spherical probe for a given experiment in PFM, computational modeling based on a Matlab toolbox is performed. The generalized Lorenz-Mie theory is computed using the T -matrix method for various experimental conditions, including changes in the size and refractive index of the sphere, as well as different laser polarization states. The axial equilibrium position is examined to predict its location compared with the position of the laser focus. Optical forces acting on the sphere are investigated in 3D to highlight, in particular, the limits of the perfectly harmonic potential assumption. These limits are quantified and discussed. Subsequently, we identify different types of inorganic nano/micro-sized particles that can be trapped by the NIR laser of the PFM and used simultaneously as mechanical and near-field light sources. To this end, we take advantage of the visible light emission from single probes confined in the optical trap and excited by the NIR laser light. The emphasis is on particles revealing nonlinear optical properties. In particular, nonlinear optical field enhancement resulting from excitation of surface plasmon resonance (SPR) on trapped gold particles of 200 nm is demonstrated by measuring the emission of that second-harmonic generation (SHG). Furthermore, we show, under optical trapping conditions, SHG emission from single potassium niobate (KNbO3) nano/micro-sized particles. We also report on the multicolor upconversion luminescence (UCL), at the single particle level, for randomly shaped crystals of sodium yttrium fluoride codoped with ytterbium and erbium, NaYF4:Yb,Er (UCC), when they are held in the optical trap. In parallel, 3D Brownian fluctuations of a single trapped particle are quantified. Moreover, luminescence resonance energy transfer (LRET) between a KNbO3 particle or an UCC and molecules of an organic dye (rose bengal) adsorbed on their surface is highlighted and analyzed. Finally, randomly shaped UCCs and hexagonal nanoplates are characterized in detail at the single particle level. The hexagonal β phase in the polycrystalline and monocrystalline crystals is identified, in both randomly and hexagonally shaped particles by electron microscopy. UCL emission from the trapped crystal is quantified in terms of power and found to be dependent on the laser power density, size and shape of the particle as well as its orientation within the trap. Most importantly, the different colors in the UCL spectrum do not vary with the same trend for different orientations of the randomly or hexagonally shaped crystal upon trapping. This suggests the existence of crystalline anisotropy-dependent UCL. Thereby, we should be able to design the best-suited particle probe for LRET experiments with subwavelength resolution.