Correlative multicolor 3D SIM and STORM microscopy

Within the last decade, super-resolution methods that surpass the diffraction limit of light microscopy have provided invaluable insight into a variety of biological questions. Each of these approaches has inherent advantages and limitations, such that their combination is a powerful means to transform them into versatile tools for the life sciences. Here, we report the development of a combined SIM and STORM setup that maintains the optimal resolution of both methods and which is coupled to image registration to localize biological structures in 3D using multicolor labeling. We utilized this workflow to determine the localization of Bld12p/CrSAS-6 in purified basal bodies of Chlamydomonas reinhardtii with utmost precision, demonstrating its usefulness for accurate molecular mapping in 3D. (C) 2014 Optical Society of America

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

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

Development of novel experimental and computational methods for three-dimensional coherent and super-resolution microscopy

Adrien Charles-François Raymond Descloux

Optical microscopy is one widely used tool to study cell functions and the interaction of molecules at a sub-cellular level. Optical microscopy techniques can be broadly divided into two categories: partially coherent and incoherent. Coherent microscopy techniques are usually label-free and provide diffraction limited structural information about the sample refractive index distribution though the measurement of phase delay induced by the sample. Since they can be very conservative in the illumination power directed toward the sample, they exhibit very low photo-toxicity and are suitable for both high speed and time lapse imaging. Fluorescence microscopy is an incoherent microscopy method which uses biochemistry techniques to label cellular structures with fluorescent molecules which emit light when excited by a laser. Fluorescence microscopy provides diffraction limited high specificity imaging but is limited in time due to photo-bleaching and photo-toxicity. Super-resolution microscopy is a sub-category of fluorescence microscopy which manipulates and exploits some properties of the fluorescent molecules to achieve high specificity sub-diffraction imaging. Super-resolution comes however at the price of an increased total acquisition time, which limits the applications of super-resolution microscopy to relatively slow cellular processes. The ideal microscope however does not exist; due to the limited spatio-temporal bandwidth of far-field microscopy, there will always be unavoidable trade-offs. There is therefore a need to find new ways to ensure that the methods are reaching their optimal performances and a need to study how different methods can complement each others. I start by presenting a new method for three-dimensional quantitative phase retrieval. I derive a model for the image formation of three-dimensional bright field images and extrapolate a novel expression allowing to retrieve the phase distribution from a bright field image stack. Using a unique image-splitting multi-plane prism that allows to acquire 8 distinct focal planes in a single exposure, I demonstrate three-dimensional quantitative phase imaging at 200 Hz . Finally, I show the association of three-dimensional super-resolution SOFI with phase imaging. To improve the imaging speed and lower the illumination intensity, I combine the same prism platform with a high-speed structured illumination. Since the structured illumination presented is using a digital micro-mirror device, about 90 % of the laser light is diffracted outside of the optical path. To improve the illumination efficiency but keep its speed and flexibility, I present a new approach for achromatic high power high speed SIM, based on a Michelson interferometer. With the access to high illumination power density, I also show the first experimental combination of SIM with SOFI using a self-blinking dye. Motivated by the absence of tools to objectively judge the performance of the microscopy methods I develop, I present a novel algorithm for image resolution estimation. The method estimate the resolution by correlating the image with several filtered version of itself without any external parameters. Finally, in the context of the AD-gut consortium, I show a practical application of deep neural networks used to assists the segmentation and mapping of the microscopy image of enzymatically labeled DNA molecules.

EPFL2020