Amplitude and phase reconstruction for Low-Energy Electron Holography of individual proteins

Single-molecule imaging methods are of importance in structural biology, and specifically in the imaging of proteins, since they can elucidate conformational variability and structural changes that might be lost in imaging methods relying on averaging processes. Low-energy electron holography (LEEH) is a promising technique for imaging individual proteins with negligible radiation damage, which can be combined with a sample preparation process by native electrospray ion beam deposition (native ES-IBD) ensuring the creation of chemically pure samples suitable for holographic imaging.The central step in the analysis of data measured by LEEH is the numerical reconstruction of the object from the experimentally acquired holograms.Full information about the imaged object consists of both amplitude and phase information imprinted on the scattered wave during the interaction of the electron beam with the molecule and encoded in the hologram created by the interference of the scattered wave and the transmitted incident wave. A propagation-based algorithm with the goal of reconstructing the wave field in the plane of the object is presented and applied to holograms of individual proteins prepared by ES-IBD and measured with a low-energy electron holography microscope. The information retrieved from the reconstruction of the amplitude distribution in the object plane is discussed by analysing holograms of antibodies, demonstrating that the inherent conformational variability of these molecules can be mapped by LEEH. The influence of the sample preparation process on the surface conformations is tracked by tuning the landing energy of the proteins during deposition.To complement the amplitude data, an iterative phase retrieval algorithm is implemented to reconstruct the phase distribution in the object plane along with the amplitude distribution. The algorithm's performance and robustness is thoroughly evaluated and simulations regarding multiple scattering effects and element-dependent variations in scattering strength are carried out to provide a reference for the interpretation of phase data retrieved from experimentally acquired holograms. The iterative phase retrieval algorithm is then applied to protein data, indicating that both molecular density and charges can be related to features in the phase reconstructions, while the presence of metals does not correlate with specific phase signals at the current resolution obtainable from the experimental data.Since proteins are inherently three-dimensional, approaches towards three-dimensional reconstruction schemes are discussed, which will be the focus of future work.

Progress in quantitative single-molecule localization microscopy

Paolo Annibale, Hendrik Georges Deschout, Aleksandra Radenovic, Marco Scarselli, Arun Shivanandan

With the advent of single-molecule localization microscopy (SMLM) techniques, intracellular proteins can be imaged at unprecedented resolution with high specificity and contrast. These techniques can lead to a better understanding of cell functioning, as they allow, among other applications, counting the number of molecules of a protein specie in a single cell, studying the heterogeneity in protein spatial organization, and probing the spatial interactions between different protein species. However, the use of these techniques for accurate quantitative measurements requires corrections for multiple inherent sources of error, including: overcounting due to multiple localizations of a single fluorophore (i.e., photoblinking), undercounting caused by incomplete photoconversion, uncertainty in the localization of single molecules, sample drift during the long imaging time, and inaccurate image registration in the case of dual-color imaging. In this paper, we review recent efforts that address some of these sources of error in quantitative SMLM and give examples in the context of photoactivated localization microscopy (PALM).

Springer2014

Optical microscopy for imaging through scattering media

Xin Yang

Optical microscopy has been widely used in the life sciences and biological research for several decades. It possesses many advantages over x-ray radiography, computed tomography, ultrasound imaging and magnetic resonance imaging, in terms of being low cost, high resolution, harmless and capable of multiple contrast mechanism, etc. However, the weakness of optical microscopy is the inability to perform deep tissue imaging. Owing to the short wavelength of optical waves, it experiences much more scattering than longer waves in inhomogeneous materials such as biological tissues. Scattering scrambles the wavefront and limits the penetration depth of optical microscopy to only hundreds of microns. We explore in this thesis, novel optical approaches to image through scattering media. There are two main categories of the state of art techniques: imaging with ballistic photons, which requires separation of ballistic photons from the sneak or multiple scattered ones; and imaging with scattered photons, which relies on manipulation of the scattered photons according to the specific scattering properties. We primarily focus on the second category in this thesis. Taking advantage of the reversibility of light, phase conjugation has a long history in aberration compensation. A deterministic wave is incident on a scattering medium and results in a random scattered field. If we record the full information of the scattered field (amplitude and phase) and generate a phase conjugated replica of it, the phase conjugated wave can follow the same path and reconstruct the original deterministic wave after passing back through the scattering medium. In this work, phase conjugation is achieved using digital method, called digital phase conjugation. The scattered field is recorded through digital holography, and a spatial light modulator is applied to construct the phase conjugated wave. Second harmonic radiation imaging probes are used as the original light source, rendering the generation of a focus behind the scattering medium. The memory effect, which indicates the correlation between speckles with different incident angles, is exploited to move the focus around and obtain three-dimensional scanning microscopy behind scattering medium. Speckle scanning microscopy is the second method demonstrated in this thesis for imaging through turbid media. In contrast to digital phase conjugation, which requires access from both sides of the scatterers to measure the scattered field, in speckle scanning microscopy, excitation and collection can be achieved from the same side. The specific scattering events are not taken into account, instead, the statistic property of the scatterers plays a major role. This technique makes full use of two facts: 1. The statistically averaged autocorrelation of speckle fields approaches a delta function; 2. When a speckle pattern scans across a fluorescence object, the collected fluorescence intensity is proportional to the convolution of the speckle field and the object. Linking these two facts with delicate calculation, we can eventually minimize the influence of the speckle, and reconstruct two-dimensional images of objects behind turbid medium. Encouraged by the success of two-dimensional speckle scanning microscopy, we investigate more advanced possibilities by simulation, including three-dimensional speckle scanning and reference based speckle scanning techniques. [...]

EPFL2014

3D high-density localization microscopy using hybrid astigmatic/biplane imaging and sparse image reconstruction

Lina Carlini, Seamus John Holden, Suliana Manley, Michaël Unser

Localization microscopy achieves nanoscale spatial resolution by iterative localization of sparsely activated molecules, which generally leads to a long acquisition time. By implementing advanced algorithms to treat overlapping point spread functions (PSFs), imaging of densely activated molecules can improve the limited temporal resolution, as has been well demonstrated in two-dimensional imaging. However, three-dimensional (3D) localization of high-density data remains challenging since PSFs are far more similar along the axial dimension than the lateral dimensions. Here, we present a new, high-density 3D imaging system and algorithm. The hybrid system is implemented by combining astigmatic and biplane imaging. The proposed 3D reconstruction algorithm is extended from our state-of-the art 2D high-density localization algorithm. Using mutual coherence analysis of model PSFs, we validated that the hybrid system is more suitable than astigmatic or biplane imaging alone for 3D localization of high-density data. The efficacy of the proposed method was confirmed via simulation and real data of microtubules. Furthermore, we also successfully demonstrated fluorescent-protein-based live cell 3D localization microscopy with a temporal resolution of just 3 seconds, capturing fast dynamics of the endoplasmic recticulum. (C) 2014 Optical Society of America

Optical Soc Amer2014

Optical microscopy for imaging through scattering media

Xin Yang

EPFL2014