Construction of a cryo-super-resolution microscope to guide in situ structure analysis

Vorlaufer J. 2025. Construction of a cryo-super-resolution microscope to guide in situ structure analysis. Institute of Science and Technology Austria.

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Thesis | PhD | Published | English
Supervisor

Corresponding author has ISTA affiliation

Series Title
ISTA Thesis
Abstract
The internal structure of biomolecules and their organization in higher-order arrangements are key factors governing the working principles of biological systems. Bioimaging has successfully revealed arrangements across relevant spatial scales. For example, cryo-electron tomography has become widely used for analyzing biomolecular structures in situ due to its comprehensive structural visualization of near-natively preserved samples, and its capability of sub-nm resolution via averaging. However, the identification of molecules within crowded cellular environments is often hindered by low contrast. Fluorescence microscopy, on the other hand, routinely visualizes specifically labeled targets at single-molecule contrast against essentially zero background. Moreover, it provides comparatively high throughput and is amenable to multiplexing. Due to this complementarity, combining datasets from both modalities acquired on the same region via correlative light and electron microscopy can reveal novel types of information. The spatial scale at which information can be extracted depends on imaging resolution and correlation accuracy. Since diffraction of light limits the resolution of conventional fluorescence microscopy to few hundreds of nanometers, reaching the full potential of correlative imaging requires super-resolution approaches. Performing imaging at cryogenic temperature preserves structures in a near-native state and minimizes distortions between the fluorescence and the electron microscopy datasets. Implementations of this concept have achieved correlation on the scale of cellular organelles or bacterial domains. We have worked towards pushing correlative imaging to the single-molecule scale by improving cryo-super-resolution microscopy, and devising a refined image correlation workflow. As part of this project, I constructed a microscopy setup and adopted it for super-resolution fluorescence microscopy at room temperature and cryogenic conditions. I explored different cryo-stages and acquisition strategies. Specifically, I developed a new scheme for correcting sample drift, thus increasing mechanical stability during microscopy acquisitions.
Publishing Year
Date Published
2025-08-25
Publisher
Institute of Science and Technology Austria
Acknowledgement
The project was supported by CZI grant DAF2021-234754 and grant DOI: https://doi.org/10.37921/812628ebpcwg from the Chan Zuckerberg Initiative DAF, an advised fund of Silicon Valley Community Foundation (funder DOI: https://doi.org/10.13039/100014989), as well as internal grants from ISTA’s Equipment Investment Committee and Interdisciplinary Project Committee.
Page
107
ISSN
IST-REx-ID

Cite this

Vorlaufer J. Construction of a cryo-super-resolution microscope to guide in situ structure analysis. 2025. doi:10.15479/AT-ISTA-20206
Vorlaufer, J. (2025). Construction of a cryo-super-resolution microscope to guide in situ structure analysis. Institute of Science and Technology Austria. https://doi.org/10.15479/AT-ISTA-20206
Vorlaufer, Jakob. “Construction of a Cryo-Super-Resolution Microscope to Guide in Situ Structure Analysis.” Institute of Science and Technology Austria, 2025. https://doi.org/10.15479/AT-ISTA-20206.
J. Vorlaufer, “Construction of a cryo-super-resolution microscope to guide in situ structure analysis,” Institute of Science and Technology Austria, 2025.
Vorlaufer J. 2025. Construction of a cryo-super-resolution microscope to guide in situ structure analysis. Institute of Science and Technology Austria.
Vorlaufer, Jakob. Construction of a Cryo-Super-Resolution Microscope to Guide in Situ Structure Analysis. Institute of Science and Technology Austria, 2025, doi:10.15479/AT-ISTA-20206.
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