Correction of a Depth-Dependent Lateral Distortion in 3D Super-Resolution Imaging

Three-dimensional (3D) localization-based super-resolution microscopy (SR) requires correction of aberrations to accurately represent 3D structure. Here we show how a depth-dependent lateral shift in the apparent position of a fluorescent point source, which we term 'wobble', results in warped 3D SR images and provide a software tool to correct this distortion. This system-specific, lateral shift is typically > 80 nm across an axial range of similar to 1 mu m. A theoretical analysis based on phase retrieval data from our microscope suggests that the wobble is caused by non-rotationally symmetric phase and amplitude aberrations in the microscope's pupil function. We then apply our correction to the bacterial cytoskeletal protein FtsZ in live bacteria and demonstrate that the corrected data more accurately represent the true shape of this vertically-oriented ring-like structure. We also include this correction method in a registration procedure for dual-color, 3D SR data and show that it improves target registration error (TRE) at the axial limits over an imaging depth of 1 mu m, yielding TRE values of < 20 nm. This work highlights the importance of correcting aberrations in 3D SR to achieve high fidelity between the measurements and the sample.

Time-lapse high-resolution microscopy to study the morphogenesis of microorganisms

Mélanie Thérèse Marie Hannebelle

Mycobacterium tuberculosis, the etiological agent for the tuberculosis disease, is a bacterial pathogen thought to infect about a quarter of the global human population. It is the first cause of death among infectious diseases, and is most prevalent in low-income populations. Much remains unknown about how these bacteria grow, divide, and manage to persist in the host even during month-long antibiotic treatments. Bacteria are typically at the edge of the spatial resolution that conventional optical microscopy techniques can offer, and so new tools are required to study bacterial substructures or surface morphologies with nanometer resolution. We developed a platform for automated optical microscopy and atomic force microscopy (AFM) in order to study the morphogenesis of mycobacteria. What is the growth dynamics of mycobacteria? Opposite models coexist in the literature for the pole growth dynamics of mycobacteria: the unipolar model and the bipolar model. We show that the growth dynamics of mycobacteria follows a new end take off NETO model. There is a surprising similarity between the NETO growth dynamics of mycobacteria and of fission yeast, which hints at shared mechanisms for pole growth in evolutionarily distant pole growing organisms. How do pole-growing organisms maintain their shape through insertion of new cell wall material at the tip? To explore further pole growth morphogenesis, we developed a method for imaging with AFM the pole of a growing rod-shaped microorganism. We observed the formation of a stiff fibril mesh on the pole of fission yeast cells, and showed that this mesh is stretched as the cell grows and insert new cell wall material at the tip. How do mycobacteria divide into two sibling cells? Another essential aspect of morphogenesis of rod-shaped cell is the division of a mother cell into two sibling cells. Using AFM, we measured the relative contributions of mechanical forces and molecular mechanisms driving the division process in mycobacteria. We showed that there is a concentration of mechanical stress at the future division site, using a combination of COMSOL finite element modeling and AFM measurements while controlling the cell turgor pressure. The accumulation of mechanical stress, in combination of enzymatic activity, ultimately leads to mechanical fracture and separation of the sibling bacteria. Conventional optical microscopy techniques tend to have low resolution and high speed, while conventional AFMs have comparatively high resolution and low speed. We designed and built a 3D-printed structured illumination (SIM) module, the openSIM, as an add-on for fluorescence microscopes. It provides structured illumination to obtain SIM images with higher resolution. With the aim of improving the imaging speed of AFM, we implemented photothermal actuation in a tip-scanning AFM head, and quantified the design constraints for high-speed photothermal off-resonance tapping imaging. AFM is most often used to scan and acquire high-resolution images of a sample. Its nanometer precision make it a unique tool for other applications. We combined an AFM head with a volcano-shaped probe for electrogram measurements, and demonstrated simultaneous recording of extracellular field potentials and contraction of the cell. This new tool opens the way for future studies exploring the intriguing links between mechanobiology and electrophysiology, with single-cell resolution.

EPFL2020

Nanoscopy in nonlinear scanning fluorescence imaging systems

Grégoire Pierre Jean Laporte

In the last 30 years, superresolution in optical microscopy has been a major field of research. During this time, different techniques have been created to break the diffraction limit in order to make observations at a nanometric scale. Given that optical microscopy is non-invasive, those superresolution methods pave the way for a better understanding of biological mechanism at a molecular level. Most of those methods are based on a nonlinear interaction between the excitation light intensity and the sample response (often fluorescent signal). In the same time, nanodiamonds containing fluorescent defects have been proven to be a choice probe for superresolution nanos-copy since they exhibit a strong and stable fluorescent signal even under high light intensities exposure (often required to obtain nonlinear photoresponse). Nanodiamonds containing Nitrogen Vacancy (NV) defects that exhibit a red fluorescent signal had been previously shown to be a viable biomarker for STED superresolved image. First, we demonstrated that green fluorescent nanodia-monds containing Nitrogen-Vacancy-Nitrogen (NVN) defects can be used with a Stimulated Emission Depletion (STED) superreso-lution microscope. Then, we implemented a STED microscope in our lab and compared the properties of NVN and NV centers for STED imaging. We conclude that even if nanodiamonds with NVN defects are less intense, they can be used as a second color nonbleaching biomarker. To illustrate the potential use of green nanodiamonds as bio-compatible probe, we superresolved them internalized into a cell with STED microscopy. Second, we tried to work on one of the main limitation in STED nanoscopy: the lack of information in the axial direction within a single scan. We combined our home made STED microscope with a Double Helix phase mask that modifies the detection point spread function in order to obtain axial localization of the superresolved emitters. We achieved three dimensional localization of nanometric fluorescent emitters but we note that photobleaching was the main limitation of this approach with organic dyes. We discussed different solutions to limit the photobleaching and their feasibility. We also worked on a different superresolution technique that we named Computational Nonlinear Saturated (CNS) microscopy. We showed that with digital post treatment of the acquired data, a nonlinear photoresponse can be harnessed to any scanning microscope equipped with a camera detector to enhance the resolution. We demonstrated that increasing the excitation power and inducing fluorescence saturation, it is possible to break the diffraction limit in a conventional confocal microscope (after data post-treatment). However, with this method, we did not obtain a gain in resolution as high as with other superresolution tech-niques involving fluorescence saturation, such as saturated structured illumination microscopy. To understand the origin of this limitation, we carried out simulation to investigate the performance of CNS microscopy in noisy environments compared with wide field techniques. We propose alternative implementation and quantify the possible resolution gain with simulations. Finally, we demonstrated how a technique, initially created for optical microscopy, can be adapted to lensless endoscopic imaging...

EPFL2016

Chromatin organization, transcription factor dynamics and gene expression in higher eukaryotes with superresolution imaging and single molecule tracking

Aleksandr Benke

Gene expression is dynamic and heterogeneous across all cell types. It serves several fundamental functions such as adaptation for changing environment and developing tissue specific phenotype and functionality during development and in adult organism. Control of gene expression mostly occurs at the transcription stage. Two main factors that regulate transcription in high eukaryotes are chromatin organization and transcription factor (TF) protein dynamics. Until recently they were mostly studied by methods that are performed in vitro or/and require ensemble averaging. We have been contributing to a development of new single molecule imaging methods that allow quantifying chromatin organization and TF dynamics in vivo and at single cell/single molecule level. Here we present our methodological developments and apply them to study how chromatin organization and TF dynamics regulate transcription. On the technical side of the study we, first, developed a method of imaging DNA with enhanced resolution in living cells. Super-resolution (SR) point localization based microscopy allows visualizing sub-diffraction organization of cellular structures and their dynamics in vivo. However, until recently almost exclusively proteins were imaged in SR microscopy. We found photoswitching conditions to image with stochastic optical reconstruction microscopy (STORM) DNA directly using the site-specific DNA –binding dye Picogreen (Chapter 2). We achieved a resolution of 50-70 nm which is ~5 fold better than conventional microscopy. Due to the excellent dye preservation we were able to do time lapse SR imaging. This study was a first demonstration of using a site-specific dye for STORM SR imaging in living cells. Photoswitching principle used in SR microscopy can be applied to single molecule (SM) tracking. It provides up to 1000x increase in density of trajectories compared to classic SM tracking. Initially, photoactivatable (PA) fusion proteins were used for high density SR based tracking. However, PA proteins are relatively dim and there is a limited choice of them. We demonstrated a new approach to SR based high density tracking by using organic dyes’ photoswitching to track proteins (Chapter 4). We showed that different dyes can be photoswitched in similar live-cell compatible media on membrane and in different organelles. Together with orthogonal protein labelling schemes this allows us to perform multicolour high density tracking across different compartments. This approach gives flexibility of labelling and increased track length compared to PA-FP SR based tracking. In a separate study, using tracking we developed algorithm which can account for long-living molecules in live cell SR imaging and allows us to correct clustering artefacts (Chapter 5). [...]

EPFL2015