A universal fluid cell for the imaging of biological specimens in the atomic force microscope

Recently, atomic force microscope (AFM) manufacturers have begun producing instruments specifically designed to image biological specimens. In most instances, they are integrated with an inverted optical microscope, which permits concurrent optical and AFM imaging. An important component of the set-up is the imaging chamber, whose design determines the nature of the experiments that can be conducted. Many different imaging chamber designs are available, usually designed to optimize a single parameter, such as the dimensions of the substrate or the volume of fluid that can be used throughout the experiment. In this report, we present a universal fluid cell, which simultaneously optimizes all of the parameters that are important for the imaging of biological specimens in the AFM. This novel imaging chamber has been successfully tested using mammalian, plant, and microbial cells. Microsc. Res. Tech. 76:357363, 2013. (c) 2013 Wiley Periodicals, Inc.

Effects of antibacterial agents and drugs monitored by atomic force microscopy

Sandor Kasas, Giovanni Longo

Originally invented for topographic imaging, atomic force microscopy (AFM) has evolved into a multifunctional biological toolkit, enabling to measure structural and functional details of cells and molecules. Its versatility and the large scope of information it can yield make it an invaluable tool in any biologically oriented laboratory, where researchers need to perform characterizations of living samples as well as single molecules in quasi-physiological conditions and with nanoscale resolution. In the last 20years, AFM has revolutionized the characterization of microbial cells by allowing a better understanding of their cell wall and of the mechanism of action of drugs and by becoming itself a powerful diagnostic tool to study bacteria. Indeed, AFM is much more than a high-resolution microscopy technique. It can reconstruct force maps that can be used to explore the nanomechanical properties of microorganisms and probe at the same time the morphological and mechanical modifications induced by external stimuli. Furthermore it can be used to map chemical species or specific receptors with nanometric resolution directly on the membranes of living organisms. In summary, AFM offers new capabilities and a more in-depth insight in the structure and mechanics of biological specimens with an unrivaled spatial and force resolution. Its application to the study of bacteria is extremely significant since it has already delivered important information on the metabolism of these small microorganisms and, through new and exciting technical developments, will shed more light on the real-time interaction of antimicrobial agents and bacteria. For further resources related to this article, please visit the . Conflict of interest: The authors have declared no conflicts of interest for this article.

Wiley-Blackwell2014

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

Computational methods for live heart imaging with speed-constrained microscopes

Olivia Mariani

Imaging methods to capture the beating and developing heart inside embryonic animal models such as the zebrafish are a key component for the study of fundamental biological processes such as cardiac birth defects or tissue regeneration. However, live heart imaging is subject to many challenges that limit the achievable spatial and temporal resolutions. While many commonly available microscopes offer the necessary optical sectioning capabilities to produce slices or volumes at the required scale, they often lack the necessary speed to seamlessly resolve the beating heart within a still frame, the entire heartbeat, or over a complete developmental sequence. In this thesis, I aim to assemble dynamic image series of the heart by using a wide range of microscopes, including, in particular, scanning microscopes---which are widely available but slow---and microscopes with a slow framerate. First, I considered the problem of reconstructing an image sequence covering one heartbeat from still images arbitrarily triggered throughout multiple cardiac cycles, when the underlying cardiac motion is temporally asymmetrical. I proposed to sort the images such as to maximize the overall similarity between contiguous frames, which I formulated as a TSP. The performance of the method increases with the number of images. I reconstructed image sequences of the heart at a virtual frame-rate of up to 300 frames per second. Second, to resolve the ill-posedness of the sorting problem when identical object poses appear multiple times in one cycle, I proposed to illuminate alternate frames with a temporally-asymmetric pattern. Sorting issues are mitigated at the cost of a custom setup and additional light exposure, without adversely affecting the reconstructed sequence. Third, I considered the problem of imaging the beating heart on conventional scanning confocal microscopes (LSM) that have a frame scanning rate comparable to the heartbeat rate. My approach takes a set of arbitrarily triggered images that may contain scanning aberrations and assembles it into a sequence that covers one heartbeat. Reconstructing scanning-aberrated frames offers a robust alternative for LSM. The quality of recovered cardiac heartbeat dynamics is comparable to that obtained by fast microscopes. Finally, I considered the problem of building smooth time-lapse sequences in the cardiac phase versus development or imaging depth spaces, based on a set of still images captured at weakly-constrained times within the heartbeat and over the course of development or at various depths. This method determines a smooth path in cardiac phase-development or depth space enforcing neighborhood similarity via a within-stage (or within-depth) sorting followed by a cyclical least-squares phase alignment approach that I formulated as a mixed integer linear problem. Constraining the paths of time-lapses by providing key-points while enforcing local similarity successfully stabilizes the the excursion error from which recursive registration approaches suffer. The results suggest that reliably imaging the heart on a wide range of microscopes is possible, provided the heart motions are stereotypical and that minimal sampling constraints are met. The newly gained possibility of using conventional instruments may be particularly relevant for applications with emerging modalities that do not allow wide-field capture.