Diffraction Tomography from Single-Molecule Localization Microscopy: Numerical Feasibility

Single-molecule localization microscopy (SMLM) is a fluorescence microscopy technique that achieves super-resolution imaging by sequentially activating and localizing random sparse subsets of fluorophores. Each activated fluorophore emits light that then scatters through the sample, thus acting as a source of illumination from inside the sample. Hence, the sequence of SMLM frames carries information on the distribution of the refractive index of the sample. In this proof-of-concept work, we explore the possibility of exploiting this information to recover the refractive index of the imaged sample, given the localized molecules. Our results with simulated data suggest that it is possible to exploit the phase information that underlies the SMLM data.

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

Optical diffraction tomography from single-molecule localization microscopy

Thanh-An Michel Pham, Emmanuel Emilien Louis Soubies, Ferréol Arnaud Marie Soulez, Michaël Unser

Single-molecule localization microscopy (SMLM) is a powerful method for the imaging of cellular structures. This modality delivers nanoscale resolution by sequentially activating a subset of fluorescent molecules and by extracting their super-resolved positions from the microscope images. The emission patterns of individual molecules can be distorted by the refractive-index (RI) map of the sample, which reduces the accuracy of the molecule localization if not accounted for. In this work, we show that one can exploit those sample-induced aberrations to reveal the structural information of the specimen. Our work is related to the optical diffraction tomography in that we aim to recover the RI map. To that end, we propose an optimization framework in which we reconstruct the RI map and optimize the positions of the molecules in a joint fashion. The benefits of our method are twofold. On one side, we effectively recover the RI map of the sample. On the other side, we further improve the molecule localization—the primary purpose of SMLM. We validate our joint-optimization framework on simulated data. Our results lay the foundation of an exciting and novel extension of SMLM.

2021

Cryo-AFM development for high resolution DNA imaging and spectroscopy

Susana del Carmen Tobenas Borron

The thesis presents the development of a custom made atomic force microscope (AFM) conceived to image biological molecules at low temperatures in ultrahigh vacuum conditions, and its application to DNA single molecule studies. Starting from a first prototype working in contact mode, our aim was to implement non-contact AFM in order to reduce possible sample damaging due to strong tip interactions. The first chapter of the thesis discusses the technical solutions developed to improve the previous system. A complete setup for in situ exchange of samples and tips was designed and implemented. This permits a continuous operation of the microscope under the experimental conditions of UHV. Thermal instabilities and temperature differences between the tip and the sample were solved with the design of a movable shield to avoid heating by thermal radiation. The introduction of a new high voltage signal generator with variable frequency and the development of a user-friendly LabVIEW interface to communicate with this device notably improved the piezomotors driving. These piezomotors perform the micro-positioning of all the microscope parts and are driven through this interface. Finally, the previous tip-holder design was modified to incorporate a piezo actuator behind the cantilever, which provides the necessary excitation for the implementation of dynamic AFM modes. The installation of a Phase Lock Loop (PLL) circuit to lock the cantilever frequency and the replacement of old feedback electronics for a digital SPM Controller, with advanced options of frequency-shift feedback, was another requirement for the correct implementation of non-contact AFM. The second chapter deals with DNA sample preparation for high resolution imaging by AFM. Based on our studies, the advantages and drawbacks of the most commonly used techniques for DNA adsorption are discussed in detail. Our measurements in contact mode demonstrate the convenience of using hydrophobic substrates for high resolution DNA imaging. At hydrophilic surfaces, like mica, the presence of a thin water layer could be responsible of strong adhesion forces between the cantilever and the sample. In order to overcome this limitation we tested HOPG as hydrophobic substrate. HOPG, chemically modified with amphiphilic compounds, allowed us to deposit DNA without molecular damage or stress. The HOPG surface treatment was significantly improved. New protocols were developed to control the formation of dodecylamine disordered monolayers or lamella structures on the substrate. The lamella formation induces a subsequent self assembly of DNA single molecules. In the last chapter we discuss our results on DNA imaging obtained with the different operation modes implemented in our microscope: contact mode, tapping mode and frequency modulation (non-contact) AFM. We conclude that the last one provides higher signal stability and the necessary sensitivity to observe DNA details at a single-molecule level. We also present a study of frequency shift versus distance dependence (force spectroscopy experiments) performed in DNA samples. We discuss for the first time a plausible explanation to the origin of the atomic force contrast between the substrate and the DNA molecules. Force spectroscopy measurements performed along DNA molecules reveal two distinguishable types of interaction. This result suggests that the chemical composition of the minor-major groove and/or of the sugar-phosphate backbone along the molecule can be detected, opening numerous possibilities for further studies and applications of this technique.