Multispectral rapid 3D super-resolution imaging of nuclear biology

At the University of Leicester we deliver cutting-edge research on mechanisms that regulate nuclear function and genome organization and hold unique expertise in structural, chemical and single-molecule investigations to understand and manipulate their function. We have solved numerous higher-order structures of macromolecular complexes involved in physiology or disease that will allow us to test structure-function relationships within cells. The challenge of studying macromolecular complexes and the molecular organization of subcellular compartments within cells resides in their diffraction-limited sizes of few dozens of nanometers. Therefore, the study of fundamental processes of life at the necessary scale within cells relies on super-resolution microscopy (SRM) methods.

We apply for a SRM platform that offers multiple SRM modalities. These include: 1. Lattice Structured Illumination Microscopy (SIM2) reaching unprecedented resolution down to ~60 nm laterally and ~200 nm axially (8-times increase in volumetric resolution compared to conventional imaging and two times that of conventional SIM). 2. 3D-Single-Molecule Localization Microscopy (SMLM), achieving 20-30nm lateral resolution and 50-80nm volumetric resolution through 1.4 microns with a single acquisition. The instrument additionally allows for photomanipulation experiments, including single-molecule photoactivation, DNA damage, FRAP and single-molecule tracking experiments. It offers ultrafast 2-color simultaneous SRM with unprecedented mild imaging to preserve cellular integrity for live-cell observations.

We aim at equipping our multi-user, interdisciplinary advanced imaging facility (AIF) with SRM possibilities that are not currently available at UoL or our Midlands academic and industrial partners. This platform will be transformative in allowing us to answer fundamental open questions underpinning the rules of life that cannot be addressed with other methods and will bring the University to the forefront of cutting-edge SRM research.

Our objectives are to:

1. Install a SRM platform featuring lattice SIM2, 3D-SMLM with a photomanipulation module
2. Support and promote cutting-edge research on genome biology and beyond at UoL that will make us internationally competitive
3. Provide access and training to a multi-user interdisciplinary imaging facility on cutting-edge volumetric subdiffraction, subcellular and single-molecule imaging
4. Strengthen our links with industrial partners

The microscope will integrate seamlessly into the existing AIF infrastructure, in an existing room with climate control and all necessary requirements of the instrument. The facility is open to all users nationally and internationally as well as industrial partners with already established costing and online reservations booking systems in place. Dr Markaki (PI) has 12+ years of experience using SRM methods, has published numerous peer-reviewed articles on quantitative SRM imaging of the nucleus and will be instrumental in the implementation of SRM in Leicester the development of staff, faculty and students. The technical expertise of the facility manager, co-I KS, will be key to the successful daily running and training on the system. We have prepared to address big data processing and transfer particularly for supporting external users by the dedicated GPUs and connections to the UoL File Storage Drive, which will be connected to the platform upon its installation. Equipping the AIF with a SRM platform will be of huge benefit to ongoing BBSRC and UKRI-funded projects. KS and YM will run workshops and lectures, including supporting the MIBTP graduate program. The establishment of a SRM platform at the AIF will provide UoL and the broad Midlands consortia with a unique opportunity to become internationally competitive. It will further strengthen our strong links to industrial partners with novel readout methods in planned research.

The Elyra 7 lattice SIM2 +3D-SMLM+RAPP is a powerful multimodal super-resolution microscopy platform that will transform our investigations leading to fundamental discoveries in nuclear biology and subcellular compartmentalization. This instrument will offer the possibility to study diffraction-limited macromolecular complexes and subcellular structures down to the detection and live tracking of single molecules.

Three major modalities are available on the platform: 1. Lattice Structured Illumination Microscopy (SIM) where the sample is illuminated with a lattice spot pattern instead of grid lines as in the setup for conventional SIM. This inherent two-dimensionality of the lattice modulation requires only translational repositioning but no rotation, which is the case for conventional SIM, while the downstream SIM2 image reconstruction technology achieves two times the traditional SIM resolution. With lattice SIM2 subcellular components that are less than 60nm apart can be separated in the lateral and 200nm in the axial direction. Furthermore, the Dualink and Burst modes for simultaneous multispectral imaging offer unprecedented rapid and gentle acquisitions, unique for live-cell observations reducing photodamage. 2. The Single-Molecule Localization Microscopy (SMLM) modality where, individual molecules are illuminated and turned to a metastable dark state from where they stochastically blink at different time points. The rapid imaging of time series and allocation of the spatial coordinates using techniques such as PALM, dSTORM and DNA-PAINT achieves a lateral resolution of 20-30 nm. The introduction of PSF reshaping through the PRILM technology allows for spatial allocation of individual molecules over 1.4 microns with 50-80 nm resolution. 3. The RAPP photomanipulation module offers the option for photomanipulation experiments, including photoactivation such as PALM, Fluorescence Recovery After Photobleaching and UV irradiation for studies of DNA repair.