A broadly accessible facility microscope to probe nanoscale cellular dynamics by combined live cell super-resolution microscopy and photomanipulation

Microscopy is a cornerstone of modern biological research because it enables visualisation of the dynamics of life at the subcellular level. Microscopy has led to breakthroughs in every area of biology, but it has traditionally been constrained by the diffraction limit, a 250 nm resolution barrier that was until recently thought insurmountable. Over the past few decades, super-resolution microscopy techniques have been developed that allow us to visualise cells and their inner workings far beyond the diffraction limit. Super-resolution microscopes allow observation of cellular organization and dynamics at the nanometre scale, and have revealed biologically critical, previously invisible, cellular organization in bacteria, plants and animals.

At the University of Warwick, we have world class researchers across the institution investigating areas pivotal for the future of our economy and health, such as antibiotic resistance, food security and aging. As cells are highly dynamic, the ability to perform high speed super-resolution measurements directly in live cells is an essential tool in cutting edge cell biology research. This critical capability is currently lacking at the University of Warwick.

We have defined several complementary live cell super-resolution microscopy capabilities which would enhance the research capabilities of a large number of groups at University of Warwick:
- The ability to image at a resolution of 100 nm or better, because many cellular components, such as cytoskeletal elements or cellular organelles, are organized on this scale.
- The ability to image at high speed to capture dynamic processes, as the movements of molecules and organelles in the cell can be extremely fast.
- The ability to image cells with low laser illumination - most cells are highly photosensitive. Excessive light dose in microscopy experiments damages cells - just like sunburn in humans - and confounds experiments due to activation of unwanted cellular responses.
- The ability to selectively photomanipulate fluorescent proteins in a controlled region. The cell is a dense, crowded environment which hampers the ability to track specific proteins or cellular structures. Photomanipulation techniques enable us to isolate specific proteins within the dense cellular environment by either highlighting them or photobleaching their surroundings. The movement of highlighted proteins within dense regions can then be revealed and followed over time.

We request funds to purchase a Zeiss Lattice Structured Illumination Microscope (SIM^2) system with all these capabilities. We will establish this instrument at the University of Warwick School of Life Sciences Imaging Facility. We will provide user-friendly access to a wide local user base, as well as providing national access to the system.

The proposed microscope will address our current capabilities gap in live cell super-resolution microscopy and give us nationally unique capabilities for joint lattice-SIM imaging and photomanipulation. The system will also enhance UK bioscience research capabilities as we will provide and promote national access to this instrument.

At the University of Warwick our researchers work across the BBSRC portfolio including strategic priority areas such as combatting antibiotic research, healthy aging across the life course, food nutrition and health, sustainably enhancing agricultural production, synthetic biology and systems approaches to the biosciences. To advance our research in these key areas we require microscope systems capable of resolving dynamic cellular structures in live cells at nanoscale resolution.

We are requesting funds to acquire a Zeiss Lattice SIM^2 microscope with photomanipulation capabilities capable of:
- 100nm lateral resolution in standard mode, potentially 60nm lateral resolution with SIM^2 deconvolution
- High temporal resolution - maximal 255fps
- Low phototoxicity to allow imaging over prolonged periods of dynamics processes without damaging cells or altering cellular processes
- The ability to perform photomanipulation - spatially controlled photoactivation or photobleaching of fluorescent proteins - alongside 100nm or better resolution microscopy

This system will be integrated into a successful imaging facility environment which will deliver access to local and external users. This will be managed by a Research Co-I who is an experienced imaging facility manager and will dedicate 20% of his time to this project. He will be supported by application PI, an established expert in super-resolution microscopy, together with multiple application Co-Is with substantial experience in SIM microscopy and its application to biological imaging.

This system will substantially enhance our imaging capabilities at University of Warwick. Furthermore, we believe such a system - lattice SIM^2+photomanipulation is not currently available for the UK bioimaging community, so the system - to which we will provide national access - will also enhance UK bioimaging capabilities.