Event-driven acquisition for content-enriched microscopy

Event-driven acquisition uses neural-network-based recognition of specific biological events to trigger switching between slow and fast super-resolution imaging, enriching the capture of interesting events with high spatiotemporal resolution. A common goal of fluorescence microscopy is to collect data on specific biological events. Yet, the event-specific content that can be collected from a sample is limited, especially for rare or stochastic processes. This is due in part to photobleaching and phototoxicity, which constrain imaging speed and duration. We developed an event-driven acquisition framework, in which neural-network-based recognition of specific biological events triggers real-time control in an instant structured illumination microscope. Our setup adapts acquisitions on-the-fly by switching between a slow imaging rate while detecting the onset of events, and a fast imaging rate during their progression. Thus, we capture mitochondrial and bacterial divisions at imaging rates that match their dynamic timescales, while extending overall imaging durations. Because event-driven acquisition allows the microscope to respond specifically to complex biological events, it acquires data enriched in relevant content.

Structure and dynamics of actin network at the leading edge of migrating cells

Sébastien Schaub

Crawling motion is characteristic of most animal cells and is principally based on actin-myosin II cytoskeletal system. The major components and reactions contributing to motility have been identified, but the overall picture of how these molecular events are orchestrated to result in the motion of the cell is still missing. We used a combination of experimental imaging, image analysis, and numerical modeling to analyze at a quantitative level how the elements of the actin-myosin machinery are coordinated in several important aspects of the crawling motion: the assembly of the characteristic pattern of actin network in the leading lamellipodium, the interaction between retrograde flow of the actin network and substrate adhesions, and the coordination of motion and assembly of actin and myosin II polymers over the entire cell during steady-state migration. First, we used enhanced phase contrast microscopy correlated with fluorescence and electron microscopy to investigate the supra-filament organization of actin in the lamellipodium of motile fish epidermal keratocytes. We showed a close correspondence between individual filament orientations and optical criss-cross network pattern by quantifying the orientational distribution of features in electron microscopy and optical microscopy images. We tested the hypothesis that the criss-cross pattern observed by optical microscopy results from variation of actin density, which is due to stochastic events of branching and capping of filaments. Based on this hypothesis, we produced simulated images of lamellipodium similar to experimental images. These simulations provided quantitative tools to estimate structural and dynamical parameters of the actin network in lamellipodia, notably the filament length, the actin concentration, the curvature of filaments and capping rate. Protrusion at the edge of the cell is coupled to the substrate adhesion, but at the same time is associated with the retrograde flow of the actin network away from the edge. We observed that focal adhesions were mostly localized at the boundary between the fast retrograde flow zone (the lamellipodium) and the slower flow zone (the lamellum). We analyzed simultaneously the actin flow and the formation of focal adhesion. Based on these results, we proposed a multi-stage model for the advance of the leading edge of migrating cell: advance of the lamellipodium is followed by the formation of the nascent adhesion complexes, which results in the reduction of the flow rate, advance of the lamellum, and then protrusion of the lamellipodium from a new base. We finally focused on the dynamics of the acto-myosin system in entire migrating cell. For this purpose, we developed a tracking program which allowed quantifying motion, contraction and assembly of cytoskeletal components. We measured the dynamics of actin, myosin II and their relative motion in migrating fish keratocytes. These results supported the dynamic network contraction model suggesting that the actin network deformed by myosin II drives the translocation of cell body. Focusing on the different aspects of the crawling motion, and balancing experimental and theoretical approaches, we obtained original results, which contributed to a better understanding of the dynamics of the lamellipodium and crawling motion in general.

EPFL2006

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

STED imaging of green fluorescent nanodiamonds containing nitrogen-vacancy-nitrogen centers

Grégoire Pierre Jean Laporte, Demetri Psaltis

We report Stimulated Emission Depletion (STED) imaging of green fluorescent nanodiamonds containing Nitrogen-Vacancy-Nitrogen (NVN) centers with a resolution of 70 nm using a commercial microscope. Nanodiamonds have been demonstrated to have the potential to be excellent cellular biomarkers thanks to their low toxicity and nonbleaching fluorescence, and are especially appealing for superresolution imaging technique like STED microscopy. However, only red fluorescent nanodiamonds containing Nitrogen-Vacancy (NV) centers have been used with STED microscopy so far. The existence of only one color nonbleaching center limits the possible observations, for instance it complicates spatial correlation studies with STED. To provide a nonbleaching probe in a different color, we characterize here the optical properties of the NVN defect for STED imaging. We demonstrate STED imaging of the green fluorescent nanodiamonds containing NVN centers, opening the door for long term two-color STED observation. Furthermore we exemplify the use of green nanodiamonds as a second color nonbleaching STED biomarker by imaging 70 nm fluorescent crystals up taken into HeLa cells. (C) 2015 Optical Society of America