Photothermal Off-Resonance Tapping for Rapid and Gentle Atomic Force Imaging of Live Cells

Imaging living cells by atomic force microscopy (AFM) promises not only high-resolution topographical data, but additionally, mechanical contrast, both of which are not obtainable with other microscopy techniques. Such imaging is however challenging, as cells need to be measured with low interaction forces to prevent either deformation or detachment from the surface. Off-resonance modes which periodically probe the surface have been shown to be advantageous, as they provide excellent force control combined with large amplitudes, which help reduce lateral force interactions. However, the low actuation frequency in traditional off-resonance techniques limits the imaging speed significantly. Using photothermal actuation, we probe the surface by directly actuating the cantilever. Due to the much smaller mass that needs to be actuated, the achievable measurement frequency is increased by two orders of magnitude. Additionally, photothermal off-resonance tapping (PORT) retains the precise force control of conventional off-resonance modes and is therefore well suited to gentle imaging. Here, we show how photothermal off-resonance tapping can be used to study live cells by AFM. As an example of imaging mammalian cells, the initial attachment, as well as long-term detachment, of human thrombocytes is presented. The membrane disrupting effect of the antimicrobial peptide CM-15 is shown on the cell wall of Escherichia coli. Finally, the dissolution of the cell wall of Bacillus subtilis by lysozyme is shown. Taken together, these evolutionarily disparate forms of life exemplify the usefulness of PORT for live cell imaging in a multitude of biological disciplines.

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

Mechanical and Functional Study of Cell Physiology at the Nanoscale

Pascal Damian Odermatt

By live-cell imaging of biological samples dynamic cellular processes can be resolved. Fluorescence microscopy (FM) and atomic force microscopy (AFM) are both capable of imaging live cells. By combining these techniques structural as well as functional information of the same biological samples are measured. While the dynamics of specific proteins can be imaged by FM, the AFM provides the structural context. To study how intracellular processes affect the mechanics, structure and kinetics of cells at the nanoscale we combined a high-speed AFM (HS-AFM) and an inverted optical microscope (OM) into one instrument. With this system cell physiological processes at different time scales are imaged. Mammalian cell migration happening at minute time scales is studied by correlated super-resolution microscopy and HS-AFM. Bacterial cell separation happening within 10s of milliseconds is characterized as well as bacterial growth over multiple generations. Additionally to imaging, the capability of the AFM to quantitatively measure mechanical properties of tissues is deployed in combination with FM on mouse corneal tissues. Different to most other rod-shaped bacteria where division happens through constriction of the cell wall at midcell, mycobacteria keep the shape of their outer cell envelope unchanged during septation. The septal wall is formed by peptidoglycan branching off from the outer cell envelop eventually splitting the cell into two sister compartments. Nanomechanical measurements show that from the initiation of septation the stiffness at the septum increases significantly. High-temporal resolution imaging of the following bacterial separation process indicated that the sibling cells separate within tens of milliseconds. The connection of the outer cell envelop at the former septum is broken apart suggesting a mechanical break. The timing of the cell separation is regulated by the tensile stress at the septum and the ultimate tensile strength of the cell wall material. The positions where these separations occur are closely associated with morphological features on the undulating cell surface. These features are formed at the poles and migrate towards midcell as the cell grows. Interestingly, sibling cell separation is restricted to the center-most wave-trough making these features the first characteristic associated with division site placement. Experiments with mutant strains show that even asymmetric divisions occur within wave-troughs emphasizing their role in division site placement. Chronic inflammation induces an increase in the stiffness of the corneal tissue and is associated with dramatic changes in the composition of the tissue. Stem cells migrating along this tissue layer of increased stiffness experience increased activation of mechanotransduction pathways. This eventually leads to induction of a skin-like rather than corneal-like character of these regenerating stem cells. It demonstrates that the stiffness of the tissue and thus pathways involved in mechanotransduction decisively influence the stem cell fate.

EPFL2016

Intravital Immunofluorescence for Visualizing the Microcirculatory and Immune Microenvironments in the Mouse Ear Dermis

Witold Waldemar Kilarski, Amanda Waite Lund, Scott Oliver, Melody Swartz, Jeremy Teo

Visualizing the dynamic behaviors of immune cells in living tissue has dramatically increased our understanding of how cells interact with their surroundings, contributing important insights into mechanisms of leukocyte trafficking, tumor cell invasion, and T cell education by dendritic cells, among others. Despite substantial advances with various intravital imaging techniques including two-photon microscopy and the generation of multitudes of reporter mice, there is a growing need to assess cell interactions in the context of specific extracellular matrix composition and microvascular functions, and as well, simpler and more widely accessible methods are needed to image cell behaviors in the context of living tissue physiology. Here we present an antibody-based method for intravital imaging of cell interactions with the blood, lymphatic, and the extracellular matrix compartments of the living dermis while simultaneously assessing capillary permeability and lymphatic drainage function. Using the exposed dorsal ear of the anesthetized mouse and a fluorescence stereomicroscope, such events can be imaged in the context of specific extracellular matrix proteins, or matrix-bound chemokine stores. We developed and optimized the method to minimize tissue damage to the ear, rapidly immunostain for multiple extracellular or cell surface receptors of interest, minimize immunotoxicity with pre-blocking Fc gamma receptors and phototoxicity with extracellular antioxidants, and highlight the major dermal tissue structures with basement membrane markers. We demonstrate differential migration behaviors of bone marrow-derived dendritic cells, blood-circulating leukocytes, and dermal dendritic cells, with the latter entering sparse CCL21-positive areas of pre-collecting lymphatic vessels. This new method allows simultaneous imaging of cells and tissue structures, microvascular function, and extracellular microenvironment in multiple skin locations for 12 hours or more, with the flexibility of immunolabeling in addition to genetic-based fluorescent reporters.

Public Library of Science2013