Pulsed-interleaved MINFLUX super-resolution microscopy

Zähringer, Jonas (2023): Pulsed-interleaved MINFLUX super-resolution microscopy. Dissertation, LMU München: Fakultät für Chemie und Pharmazie

Abstract

Light microscopy has become a powerful tool to investigate structures, dynamics, and interactions in natural science such as cell biology. Especially fluorescence microscopy has seen a rapid development during the last decades. The emergence of far-field fluorescence super-resolution microscopy even overcame the fundamental diffraction limit, enabling imaging with high contrast and specificity of structures below 200 nm. With less than a tenth of the photons needed compared to previous super-resolution methods, MINFLUX is the most recent development to push the resolution limit to truly molecular dimensions. This is achieved by combining the excitation information of a structured illumination featuring a minimum with the respective emission information. While MINFLUX enables the visualization of structures and dynamics with 1 nm precision, the size of a fluorophore, only individual fluorophores are localized, thus information about their environment is missing. The method of choice to report about the environment of a fluorophore is the fluorescence lifetime. To this end, a combination of MINFLUX coupled with the fluorescence lifetime would vastly increase the information wealth of MINFLUX localizations. In this thesis, I built a pulsed-interleaved MINFLUX (pMINFLUX) that extends the nanometer precise localizations of MINFLUX with the fluorescence lifetime domain while additionally simplifying the technological complexity of pMINFLUX. I demonstrated the performance of this setup using DNA origami structures which act as nanoruler. The unprecedented combination of fluorescence lifetime and nanometer precise localizations was employed in four novel methodologies to make the investigation of structures, interactions, dynamics, and their interplay on the nanometer length scale more accessible. In combination with graphene energy transfer (GET), I extended the nanometer precise lateral localizations of MINFLUX to the third dimension, by using the fluorescence lifetime encoded axial distance information for nanometer precise 3D super-resolution microscopy. I demonstrated the resolution of GET-pMINFLUX on DNA origami structures using DNA-PAINT with axial precisions below 0.4 nm. DNA-PAINT was used to generate stochastic blinking, which is necessary for the localization method MINFLUX to resolve distances smaller than the diffraction limit. To increase the imaging speed and overcome issues with high fluorescent background of DNA-PAINT, I established local-PAINT (L-PAINT). In contrast to DNA-PAINT, L-PAINT imager strands have two binding sequences with a designed binding hierarchy such that the L-PAINT DNA-strand binds longer on one side. This allows the fluorescent dye-modified second end of the strand to locally scan for binding sites at a rapid rate. While MINFLUX is able to give insight into structural information, the interplay with the environment remains unknown as only individual fluorophores are localized. I addressed this problem by first combining Förster resonance energy transfer (FRET) with MINFLUX to simultaneously localize the donor dye and map the distance to an acceptor dye. With the multilateration of several donor dye positions I localized the acceptor dye with a full width half maximum of 0.17 nm. To overcome the limited working range of FRET of 2-10 nm, I developed a pMINFLUX lifetime multiplexing approach. pMINFLUX lifetime multiplexing uses the fluorescence lifetime to colocalize two spectrally similar dyes without photo-switching over a large field-of-view. Beyond the FRET range, pMINFLUX lifetime multiplexing enabled the co-localization of two dyes by separating their fluorescence intensities according to their fluorescence lifetimes. I demonstrated this in simulation and experiment using two independent L-PAINT pointer systems, whose dynamics were imaged with nanometer precision. Inside the FRET range, two dyes were co-localized using a newly developed combined phasor-microtime gating approach. As a result, the combination of both multiplexing approaches closed the resolution gap between single-molecule FRET and co-tracking.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: Super-resolution microscopy, DNA origami, microscopy, MINFLUX
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 540 Chemie
• Fakultäten: Fakultät für Chemie und Pharmazie
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 30. Juni 2023
• 1. Bericht­erstatter:in: Tinnefeld, Philip