Correlative 3D microscopy of single cells using super-resolution and scanning ion-conductance microscopy

High-resolution live-cell imaging is necessary to study complex biological phenomena. Modern fluorescence microscopy methods are increasingly combined with complementary, label-free techniques to put the fluorescence information into the cellular context. The most common high-resolution imaging approaches used in combination with fluorescence imaging are electron microscopy and atomic-force microscopy (AFM), originally developed for solid-state material characterization. AFM routinely resolves atomic steps, however on soft biological samples, the forces between the tip and the sample deform the fragile membrane, thereby distorting the otherwise high axial resolution of the technique. Here we present scanning ion-conductance microscopy (SICM) as an alternative approach for topographical imaging of soft biological samples, preserving high axial resolution on cells. SICM is complemented with live-cell compatible super-resolution optical fluctuation imaging (SOFI). To demonstrate the capabilities of our method we show correlative 3D cellular maps with SOFI implementation in both 2D and 3D with self-blinking dyes for two-color high-order SOFI imaging. Finally, we employ correlative SICM/SOFI microscopy for visualizing actin dynamics in live COS-7 cells with subdiffraction-resolution.

Biophysical applications of correlative scanning probe and super-resolution microscopy

Vytautas Navikas

Imaging live cells in their native environment is crucial for the understanding of complex biological phenomena. Modern optical microscopy methods such as fluorescence super-resolution microscopy are increasingly combined with complementary, label-free techniques to put the high-resolution fluorescence information into cellular context. Common high-resolution imaging approaches used in combination with fluorescence imaging such as electron microscopy and atomic force microscopy (AFM) were originally developed for solid-state material characterization and thus are not straightforwardly applicable to image live cells for sustained periods of time. In this thesis, I am focusing on advancing state-of-the-art correlative scanning ion-conductance microscopy (SICM) combined with novel super-resolution imaging techniques. The work presented in this thesis demonstrates various technological advancements for both imaging modalities as well as experimental proof-of-principle studies. The first part of the work focuses on a novel experimental combination of SICM and super-resolution optical fluctuation imaging for single-cell imaging (SOFI). To demonstrate the capabilities of our method we show correlative 3D cellular maps with SOFI implementation in both 2D and 3D with self-blinking dyes for two-color high-order SOFI imaging. We employ correlative SICM/SOFI microscopy for visualizing actin dynamics in live COS-7 cells with subdiffraction resolution. In order to increase the imaging depth for thick samples, a novel remote focusing modality based on an adaptive optics device was developed. Here, we use a deformable mirror to acquire multiple image planes of the labelled cells and we process the data using a single-molecule localization routine and/or computed spatiotemporal correlations, demonstrating subdiffraction resolution within a whole imaging volume. Second, we demonstrate the advantages of a new SICM probe manufacturing method allowing the batch production of glass nanopipettes used for SICM imaging and sensing. Finally, we show a new application of SICM tailored for single-molecule characterization based on electrical signatures while performing controlled translocations of surface-immobilized DNA molecules. The method is developed to be compatible with single-molecule fluorescence imaging and was applied for the detection of various analytes in a correlative manner. We believe that a synergy between fundamentally different imaging modalities is going to become necessary in an age of computational microscopy to ensure an unbiased data interpretation in the rapidly progressing field of biological research.

EPFL2021

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.

EPFL2013

Synthesis of carbon nanotubes and characterization of nanotubes as atomic force microscopy tips

Yanki Keles

This thesis describes the synthesis of carbon nanotubes (CNTs) by chemical vapor deposition (CVD) and the evaluation of them as tips of atomic force microscope (AFM) probes. Both arc-discharge and CVD grown nanotubes were investigated as AFM tips. Two methods were used to produce nanotube tipped AFM probes: (i) nanotubes were directly grown on the apex of AFM probes by CVD and (ii) pre-grown nanotubes were mechanically attached onto the apex of AFM probes. The first part of the thesis describes experiments that sought to control the growth of carbon nanotubes by chemical vapor deposition. The effects of various parameters e.g. catalyst, the carbon source, the addition of hydrogen and the growth temperature, on carbon nanotube growth were investigated. Ethylene was found to lead to better quality nanotubes than the acetylene. Grown nanotubes' quality was found to improve upon flow of hydrogen during the synthesis. Nanotubes synthesized on 3 nm iron film at 840°C using ethylene and hydrogen led to utilizable carbon nanotube tipped AFM probes. The second part of the thesis focuses on the mechanical production of nanotube tipped AFM probes in high resolution electron microscopes using nanomanipulators. Scanning electron microscopy (SEM) was found to be more convenient than transmission electron microscopy (TEM) to carry out the nanomanipulation process. Both arc-discharge and CVD grown nanotubes were successfully attached onto the AFM probes using a custom made nanomanipulator. Nanotubes were glued onto the probes by focused electron beam (FEB) induced amorphous carbon deposits. FEB irradiation deposited amorphous carbon performed more reliable attachment on CVD nanotubes than the arc-discharge nanotubes. The last part of the thesis describes tapping mode AFM measurements done using nanotube tipped AFM probes. Different nanotube tipped probes were investigated as AFM tips for scanning colloidal gold particles with different diameters and gratings from Digital Instruments™ AFMs. High resolution AFM images were obtained by arc-discharge nanotube tips. CVD grown nanotubes were shown to be more resilient to tip crashes than the arc-discharge grown nanotubes. However, all the AFM images acquired from the scans by CVD nanotubes had the same kind of ringing artifact, in good agreement with a previously reported data about the artifacts of the CVD nanotube tipped AFM probes. Open ends of the CVD nanotubes, lack of crystallinity and the defects in their structure are suggested to be the reason for these artifacts.

EPFL2006