Adaptive optics for correcting aberrations in cryogenic fluorescence microscopy

• Applicant: Professor Dr. Rainer Kaufmann
• Subject Area: Biophysics ; Optics, Quantum Optics and Physics of Atoms, Molecules and Plasmas
• Term: since 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 496128632

The state-of-the-art technology for structural cell biology is cryo electron microscopy (cryo-EM). It offers unprecedented resolution in specimens in a near-native state by utilizing the advantages of vitrification (ultra-fast freezing) and native contrast. However, as cryo EM lacks the possibility of specific labelling of certain proteins inside the cell, finding or validating the structures to be studied is often very challenging or even impossible. As a solution to this problem, cryo fluorescence microscopy (cryo-FM) has been developed to utilize highly specific fluorescent labelling for cryo imaging. The combination of both cryo microscopy techniques provides a powerful tool for cellular and structural biology. However, to overcome the big resolution gap between cryo-FM and cryo-EM, super-resolution cryo-FM or high-precision localization of rare objects is required. Recent developments demonstrate the enormous potential of these approaches, but also unfold the technical challenges of the relatively young field of cryo-FM. One of the major challenges are optical aberrations introduced by either the cryogenic specimen itself or the optics used for cryo-FM. The goal of this proposal is to implement adaptive optics in an existing experimental cryo-FM setup to correct for aberrations in the most typical applications of cryo-FM. This concerns: (i) limitations in high-precision 3D localization of rare objects or events inside cells due to distortions of the point spread function; (ii) artefacts and resolution loss in super-resolution cryo-FM due to asymmetries in the point spread function; (iii) resolution and sensitivity loss in cryo-immersion imaging due to aberrations introduced by refractive index mismatches of the immersion medium. We will develop correction routines for the individual cases and provide for each aim proof-of-concept demonstrations with typical biological specimens. The overarching goal of this proposal is to lay the groundwork for adaptive optics procedures for the three most important areas of cryo-FM imaging, which would strongly benefit from corrections of residual optical aberrations. We strongly believe that this will have a major impact on future technical developments in the field of cryo-FM and correlative cryo-imaging and therefore eventually also on the fields of cellular and structural biology by providing novel tools to address biological questions that currently remain unanswered.

• DFG Programme: Research Grants