Super-resolution microscopy live cell imaging and image analysis

Tomas Lukes
Czech Technical University in Prague, 2017
Thèse hors EPFL

Résumé

Novel fundamental research results provided new techniques going beyond the diffraction limit. These recent advances known as super-resolution microscopy have been awarded by the Nobel Prize as they promise new discoveries in biology and live sciences. All these techniques rely on complex signal and image processing. The applicability in biology, and particularly for live cell imaging, remains challenging and needs further investigation. Focusing on image processing and analysis, the thesis is devoted to a significant enhancement of structured illumination microscopy (SIM) and super-resolution optical fluctuation imaging (SOFI)methods towards fast live cell and quantitative imaging. The thesis presents a novel image reconstruction method for both 2D and 3D SIM data, compatible with weak signals, and robust towards unwanted image artifacts. This image reconstruction is efficient under low light conditions, reduces phototoxicity and facilitates live cell observations. We demonstrate the performance of our new method by imaging long super-resolution video sequences of live U2-OS cells and improving cell particle tracking. We develop an adapted 3D deconvolution algorithm for SOFI, which suppresses noise and makes 3D SOFI live cell imaging feasible due to reduction of the number of required input images. We introduce a novel linearization procedure for SOFI maximizing the resolution gain and show that SOFI and PALM can both be applied on the same dataset revealing more insights about the sample. This PALM and SOFI concept provides an enlarged quantitative imaging framework, allowing unprecedented functional exploration of the sample through the estimation of molecular parameters. For quantifying the outcome of our super-resolutionmethods, the thesis presents a novel methodology for objective image quality assessment measuring spatial resolution and signal to noise ratio in real samples. We demonstrate our enhanced SOFI framework by high throughput 3D imaging of live HeLa cells acquiring the whole super-resolution 3D image in 0.95 s, by investigating focal adhesions in live MEF cells, by fast optical readout of fluorescently labelled DNA strands and by unraveling the nanoscale organization of CD4 proteins on a plasma membrane of T-cells. Within the thesis, unique open-source software packages SIMToolbox and SOFI simulation tool were developed to facilitate implementation of super-resolution microscopy methods.

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https://infoscience.epfl.ch/record/231317?ln=fr

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Tomas Lukes
Czech Technical University in Prague, 2017
Thèse hors EPFL

Résumé

Official source

https://infoscience.epfl.ch/record/231317?ln=fr

À propos de ce résultat

Concepts associés (27)

Concepts associés

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Publications associées (39)

Publications associées

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Imaging tissues and cells beyond the diffraction limit with structured illumination microscopy and Bayesian image reconstruction

Tomas Lukes, Jakub Pospisil

Background: Structured illumination microscopy (SIM) is a family of methods in optical fluorescence microscopy that can achieve both optical sectioning and super-resolution effects. SIM is a valuable method for high-resolution imaging of fixed cells or tissues labeled with conventional fluorophores, as well as for imaging the dynamics of live cells expressing fluorescent protein constructs. In SIM, one acquires a set of images with shifting illumination patterns. This set of images is subsequently treated with image analysis algorithms to produce an image with reduced out-of-focus light (optical sectioning) and/or with improved resolution (super-resolution). Findings: Five complete, freely available SIM datasets are presented including raw and analyzed data. We report methods for image acquisition and analysis using open-source software along with examples of the resulting images when processed with different methods. We processed the data using established optical sectioning SIM and super-resolution SIM methods and with newer Bayesian restoration approaches that we are developing. Conclusions: Various methods for SIM data acquisition and processing are actively being developed, but complete raw data from SIM experiments are not typically published. Publically available, high-quality raw data with examples of processed results will aid researchers when developing new methods in SIM. Biologists will also find interest in the high-resolution images of animal tissues and cells we acquired. All of the data were processed with SIMToolbox, an open-source and freely available software solution for SIM.

Oxford University Press (OUP)2019

Chargement

Resolution and Complex Deconvolution in Holographic Microscopy

Yann Jérôme Michel Pascal Cotte

Interest in high resolution imaging techniques has recently multiplied due to their importance in bio-medical research. Quantitative phase measurements by holographic microscopy is an extraordinary tool to gain new understanding of transparent biological samples. The extremely high demand on resolution well beyond the diffraction limit, however, sets new benchmarks for imaging techniques. Digital Holographic Microscopy is an interferometric method providing access to the complex wave front. Its capacity to image amplitude and quantitative phase simultaneously makes it an attractive research tool in many fields of biological research. It is on the verge of becoming an appealing alternative to classical fluorescence microscopy. For intensity-based microscopy, however, super-resolution methods are well established. In this thesis, approaches to unleash resolution for phase imaging techniques are explored. Based on truncated inverse filtering, a theory for deconvolution of complex fields is developed. It is a post-processing method that does not require any additional optics in the holographic microscopy setup. Gain in resolution arises by accessing the object's complex field – containing the information encoded in the phase – and deconvolving it with the characteristic coherent transfer function (CTF). By efficient harvest of a diffraction limited bandpass, complex deconvolution is demonstrated to exceed the coherent resolution limit. A novel method, called a complex point source, serves to characterize the holographic microscopy system. It consists of a coherently illuminated nano-metric hole, located on a conventional microscope slide, most common in bio-microscopy. A thin opaque film is directly evaporated on the slide and the circular sub-wavelength structures are drilled with a focused ion beam. Theoretical as well as practical work on transmission properties of apertures confirms the advantageous signal-to-noise yield of proposed method. More abstractly, the thus gained experimental amplitude point spread function is demonstrated to be suitable for experimental CTF reconstruction. Herefrom, experimental parameters are extracted and introduced into scalar and vectorial Debye theory, called synthetic CTF, adaptable to realistic imaging conditions. Also, it allows to study the role of noise in the context of complex field deconvolution. The resolution power of holographic microscopy systems is systematically examined with pairs of nano-metric apertures separated by sub-resolution pitches. Theory of information content is elaborated depending on different experimental configurations. Resolution extends beyond classical Abbe's limit by introducing spatially a varying phase into the illumination beam of a phase imaging system. In a last step, the novel deconvolution method is generalized to three-dimensional image processing of complex fields. By combining scattering theory and signal processing, the method is demonstrated to yield the reconstruction of the scattering object field under non-design microscope objective imaging conditions. The suggested technique is best suited for an implementation in high-resolution diffraction tomography based on sample and/or illumination rotation.

EPFL2012

Chargement

Fluorescence super-resolution microscopy has allowed unprecedented insight into the workings of biological systems below the diffraction limit of light. Over the past decade, it has overcome several challenges to deliver 3D, multi-color and faster imaging techniques, which have found many applications in deciphering the workings and structure of organelles â specialized cellular compartments whose shape and dynamics serve their specific functions. However, most super-resolution techniques are laborious and slow, and there are existing challenges in achieving sufficient throughput for collection of large datasets and capturing dynamic processes in living cells. The aim of this thesis is to address issues of limited throughput, temporal resolution and live-cell compatibility, with application to centriole architecture and mitochondrial dynamics. The design of a novel flat-fielding module for multi-focal excitation which â once integrated into an instant structured illumination microscope â allows the capture of larger fields of view with uniform illumination and hence results in increased throughput. This flat-fielded microscope allows multi-color, 3-dimensional imaging at double the diffraction-limited resolution of up to 100 mammalian cells within 1-2 minutes. Furthermore, combined with advanced sample preparation using expansion microscopy, the setup achieves an effective resolution of ~35 nm, comparable to some of the more powerful super-resolution techniques, but at 100-1000 times increased throughput. To showcase this improvement, we study the distribution of post-translational modifications within the centriole, revealing previously unobserved molecular organization. Second, with fast, live-cell imaging, we investigate the role of membrane bending energy and tension in mitochondrial division. While many molecular factors involved in this process have been identified, little is known about its physical requirements. By analyzing mitochondrial constrictions enriched in Drp1 that divide successfully or reverse, we establish that mitochondrial constrictions with higher bending energy are more likely to divide. Furthermore, by analyzing changes in mitochondrial shape and employing a novel membrane tension sensor, we observe that conditions under which mitochondrial membrane tension is increased, result in more successful divisions. These observations are supported by a model in which membrane bending energy increases the elastic energy at the constriction site, bringing it closer to the energy barrier to fission, while membrane tension acts as a fluctuation to overcome the remaining residual energy. Finally, to overcome existing trade-offs between temporal resolution and imaging duration, we develop an acquisition framework for smart and adaptive temporal sampling. By adapting the temporal resolution is in response to events of interest, they are captured at high imaging speed, while preserving the sample during a lack thereof. Using the accumulation of Drp1 to detect mitochondrial constriction sites allowed us to adapt the imaging speed to capture mitochondrial constrictions at high temporal resolution while preserving the sample when such events are lacking. Overall, this framework provides both high temporal resolution and long imaging times when needed, decreasing resulting phototoxicity, photobleaching and amount of redundant data.

EPFL2020

Chargement

EPFL2020

Resolution and Complex Deconvolution in Holographic Microscopy

Yann Jérôme Michel Pascal Cotte

EPFL2012