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Optical nanoscopy at 1 nm resolution: far-field fluorescence control at cryogenic temperatures

Periodic Reporting for period 3 - OptnanoATcryo (Optical nanoscopy at 1 nm resolution: far-field fluorescence control at cryogenic temperatures)

Reporting period: 2018-07-01 to 2019-12-31

Optical nanoscopy is a powerful technique used in biology to study subcellular structures and functions via specifically targeted fluorescent labels. Localization microscopy in particular offers a much better resolution (~10-50 nm) than conventional microscopy (~250 nm) while being relatively undemanding on the experimental setup and the subsequent image analysis. For these developments the Nobel Prize in Chemistry 2014 has been awarded.
The increase in resolution from traditional imaging to super-resolution has been tremendous but is still short of imaging larger biomolecules or structure of biomolecules assemblies. Cell biologists want to understand the function of proteins and sub-cellular machinery such as DNA at the molecular level. In order to do so, another increase of resolution with light microscopy is needed. Moreover, this improvement must be realized in 3D
In this project we propose such an increase, to be precise: The next revolution in imaging to 1 nm isotropic resolution in 3D. To go from the current state of the art towards this goal we must realize a big increase in the number of collected photons from single fluorescent emitters as well as in the labelling density. Only then can subcellular structures be imaged at the molecular level to study the molecular machinery of the cell. In a larger perspective, the outcome of this research will enable the combination of structural cryo-electron microscopy imaging at subnanometer resolutions with functional fluorescent imaging at the nanometer scale.
The overall objective is twofold: 1) to build and realize a localization based super-resolution microscope and image paradigm at cryogenic temperatures and 2) enable ultra-resolution close to 1 nm by data fusion algorithms on identical bio macromolecules.
We have completed the construction of a cryotank for 12h measurements with optical microscopy. It is based on the design from Joerg Enderlein in Goettingen, but with several improvements such as longer measurement times, possibility to move and position the sample with piezo motors and image from the side on the optical table for improved vibrational stability and axial drift. We have started designed a cryo-stage together with an industrial collaborator that could allow us a similar cooling capacity as the cryo tank filled with liquid nitrogen, but is much smaller/lighter and allows directly for dip/tilt xy-stage control of the sample without complicated constructions. In addition, imaging from both sides of the sample should be possible then. With another collaborator we currently build a waveguide based TIRF imaging system that is suitable for cryogenic temperatures.
We already investigated several red dyes for imaging at cryogenic temperatures and have a manuscript of this study under revision currently.
With regards to the polarization control have we completed the calibration setup for the excitation and STED light paths. We have summarized the technicalities of the setup and calibration protocol in a manuscript that is accepted. This is the basis for the polarization controlled induced sparsity. We have already first experiments on room temperature, but fixed dipole emitters.
We have developed an information optimal algorithm for data fusion in super-resolution light microscopy. From several talks at conferences about the topic we received very positive feedback. Several leading groups are interested in collaboration with us and currently we are in the final phase for a manuscript to a high impact journal. The resolution achieved by the data fusion approach is reaching towards 1 nm in 2D.
Already now we have several findings beyond the state of the art. The data fusion algorithm presents a unique approach to data fusion that has been introduced to the field of fluorescent microscopy. We believe that his approach will be the basis for developments and biological findings. The current concept by just applying algorithms from the field of cryo-EM SPA will become redundant as we can show that a clear better performance can be achieved with our approach.
On the experimental side we have implemented a polarization control system that is significantly better than prior literature as it allows controlling the (linear) polarization on the sample for 2 different wavelengths. We have shown very good modulation depth for a polarization sweep and our superior setup and calibration is to our mind the key why we have this much improved modulation depth compared to the prior art.

• We expect to have a TIRF cryo imaging platform based on a waveguide. This should allow SIM imaging at cryo temperatures with little effort.
• We expect to have a robust data fusion not only in 2D but also in 3D (GPU accelerated). We plan to demonstrate this on PAINT labeled NPC structures at room temperatures.
• We expect to have a sparsity enabling protocol at cryogenic temperatures which allows to collect >10^5 photons per emitter. In addition we expect to be able to localize the emitter in 3D with an isotropic resolution.