Multi-plane super-resolution microscopy

Azat Sharipov
EPFL, 2017
Thèse EPFL

Résumé

Understanding cell functions is the major goal of molecular biology, which intends to elucidate the interactions between biomolecules at a subcellular level. One of the widely used techniques in molecular biology is fluorescence microscopy, which offers high specificity and sensitivity at the submicrometer spatial scale but is limited by diffraction to about 200nm lateral resolution, which is insufficient for the observation of many molecular processes. During the last two decades several super-resolution techniques overcoming the diffraction limit have been developed. However, imaging samples in three dimensions (3D) at high speed remains a challenging and not yet resolved task. This thesis focuses on enhancing super-resolution imaging towards fast, live-cell and 3D imaging. Super-resolution optical fluctuation imaging (SOFI) is a technique based on the stochastic fluctuations of photoswitchable fluorescent markers. It possesses several unique features such as background reduction, capability of increased pixel grid generation, i.e. spatial oversampling, as well as tolerance and robustness to a wide range of photoswitching conditions. In this thesis SOFI was extended to perform 3D analysis. As a result, the resolution in all three spatial dimensions can be improved and the depth sampling increased. We present a novel design of a 3D fluorescence microscope capable of acquiring images of eight depth planes simultaneously. This design incorporates an image-splitting prism, a single optical element allowing to achieve in-depth image separation. The optical performance of the 3D microscope was described and experimentally verified. The simultaneous depth plane acquisition allows to fully exploit the 3D capabilities of SOFI while generating additional virtual depth planes. An algorithm for the extraction of switching kinetics of fluorescent markers is presented. Using appropriate imaging conditions, we demonstrate the applications of 3D SOFI on several examples of fixed and living cells. We also present the potential of the 3D microscope for phase retrieval in transparent samples.

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https://infoscience.epfl.ch/record/225626?ln=fr

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Azat Sharipov
EPFL, 2017
Thèse EPFL

Résumé

Official source

https://infoscience.epfl.ch/record/225626?ln=fr

À propos de ce résultat

Concepts associés (17)

Concepts associés

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Publications associées (42)

Publications associées

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Dielectric-sphere-based microsystem for optical super-resolution imaging

Gergely Huszka

Well-established imaging techniques proved that features below the diffraction limit can be observed optically using so-called super-resolution microscopies, which overcome Abbe's resolution limit. In traditional far-field microscopy, the introduction of fluorescent samples and engineered light paths was key for this breakthrough. In parallel, near-field techniques with similar performance were developed, but they suffered from a limited field-of-view. The merge of the two approaches was already demonstrated ~15 years ago, when micrometer-sized dielectric objects positioned on a sample were found to be able to image the sample with super-resolution. By observing the sample through the micro-object with a classical optical microscope, the latter could capture a virtual image showing sub-diffraction details. Although this way the near-field information transfer into the far-field by the micro-object was proven and found to be key for enabling super-resolution imaging, the limited field-of-view, as determined by the size of the micro-object, remained an issue. In this dissertation, a novel method is presented that provides a microscopy technique capable of achieving super-resolution without field-of-view restrictions. Based on previous studies, dielectric microspheres were chosen for this imaging technique. First, the working principle of these microspheres was explored by investigating both the illumination and the reflected light path. These findings provided a better understanding on the phenomena working behind microsphere-assisted imaging and allowed to create an engineering toolbox that can be used to design microsphere-based optical systems. This was followed by an investigation on microfabrication techniques, in order to create a microchip that can serve as a bridge between a single microsphere and the macro-sized-components of a classical optical microscope. The resulting chip was later embedded in a custom fixation system that allowed scanning of this microsphere over the sample, while keeping its position fixed compared to the microscope objective. The microscope mounted camera recorded pictures during the scan, which were used to generate a large field-of-view super-resolution image by stitching. After initial successes, the setup was improved in terms of robustness and application range. The new version allowed field-of-view in the millimeter range, while it could be operated in both oil- and water-immersion. Parallel imaging with an array of microspheres was also implemented, which further enhanced the imaging speed. The algorithmic background (including an automated scanning and image reconstruction protocol) of this microscopy method was developed in-house. Its validation showed superior performance compared to existing software. Future developments (e.g. employment of 3D-printing for mass-production, imaging in vivo biological samples, metrology applications) are envisioned. The findings presented here may pave the road for an easy-to-use, generalized super-resolution imaging system.

EPFL2018

Chargement

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

Chargement

Super-resolution fluorescence microscopy is a promising tool with the potential to strengthen our understanding of living processes. Based on the ability to switch fluorophores on and off in an either deterministic or stochastic manner, the fundamental limit of diffraction can be overcome. The first concepts have been invented 20 years ago, but first discoveries in biology have been reported only recently. As with every new technology, its successful application is challenging. In particular, super-resolution microscopy sets much higher demands on the sample preparation and labeling quality than researchers are used to. In addition, the super-resolution microscopes often require tedious alignment procedures and/or rely on complicated data analysis software. Not surprisingly, commercially available systems are rather expensive and not yet widely spread. This thesis focused on the development and characterization of novel concepts for super-resolution microscopy based on stochastic fluorescence fluctuations in order to extend and/or simplify existing techniques. In particular, we developed a framework for the image-based simultaneous estimation of three-dimensional position and orientation of fluorescent emitters. Unlike many localization-based super-resolution techniques, our approach accounts for the dipole characteristics of the fluorescence emission whilst staying compatible with standard widefield fluorescence microscopes. In a comparative study of two well-established techniques for super-resolution microscopy, namely super-resolution optical fluctuation imaging (SOFI) and stochastic optical reconstruction microscopy (STORM), we determined their characteristics and demonstrated a complementary performance, which suggested a beneficial impact upon combination of both techniques. This has led to a further development of the original SOFI analysis. The resulting balanced SOFI analyzes molecular statistics and linearizes the response with respect to brightness and blinking heterogeneities in the sample, which significantly improves the image contrast and thereby facilitates the access to higher resolutions. We experimentally demonstrated nearly five-fold resolution improvements as compared to diffraction-limited images of fluorescently labeled cells. The super-resolved molecular parameter maps obtained with balanced SOFI, such as the blinking lifetimes, fluctuation amplitudes and label densities, are sensitive to static differences and/or dynamic changes in the chemical microenvironment of the fluorophores and can thus report functional information that has not been exploited before, for instance, the local pH or the concentration of reducing and oxidizing agents. Finally, by using cross-cumulation between multiple depth and/or color channels, we demonstrated whole-cell three-dimensional super-resolution microscopy with a normal widefield illumination and without mechanical scanning as well as spectral unmixing of multiple fluorophores with overlapping emission spectra.

EPFL2013

Chargement

Nanoscopy in nonlinear scanning fluorescence imaging systems

Grégoire Pierre Jean Laporte

EPFL2016

EPFL2013

Dielectric-sphere-based microsystem for optical super-resolution imaging

Gergely Huszka

EPFL2018