Stimulated Emission Depletion Microscopy (STED) for imaging at high resolution in the Biosciences

To find out how proteins are organised within cells and tissues, and how they interact with each other, we need to be able to see inside the cell with a high level of detail. Unfortunately, because of the way light microscopes work, the amount of detail we can normally see is limited. In this proposal, we want to set up a new type of microscope, that overcomes some of the shortcomings of traditional light microscopes, and will allow us to see much more detail, turning a blurry image into a much sharper one. Normally, we can only resolve two objects in a cell if they are more than 250 nanometers apart. This new microscope will allow us to resolve two objects in a cell that are more than ~30 to 40 nm apart, increasing the amount of detail we can see by about 10 fold. This will enable us to find the answers to important questions about how the cytoskeleton is organised in different types of cell, how it adopts a specialised organisation in primary cilia (small antennae like structures on the cell surface), how viruses replicate inside cells, how membrane proteins are organised on the surfaces of cells, how proteins are organised in heart muscle cells, and so on. This new microscope will bring a step change to our imaging capabilities, and the new level of detail that we'll be able to see will underpin a wide range of biological and health related research.

This application is requesting the purchase of a new STED (Stimulated Emission Depletion) Microscope, to provide a wide range of users with super-resolution capability, in a cost effective and simple way. This microscope will provide a resolution of ~40nm in X and Y, which is about 5 fold better than obtainable with standard microscopes. The STED itself is provided as a microscope attachment, which is place on the camera port of a widefield microscope. The improvement in resolution is achieved optically, and is immediate. It does not require any image processing. This new microscope will provide much improved resolution for a range of projects, from imaging the organisation of the cytoskeleton in structures as diverse as intercalated discs (Jayasinghe) and the Z-disc (Peckham) in the heart, to primary cilia (Johnson). It will enable a detailed look into virus replication (Stonehouse) as well as channel organisation (Beech), and the organisation of proteins in diverse cellular compartments such as the Golgi (Ponnambalam). The improved imaging capability at low cost, in a simple to operate system, will bring a step change to our imaging capability at the University of Leeds.

The ability to image cellular structures and protein organisation to very high resolution will provide new information that will be of benefit to a wide range of researchers across many different fields, given the diversity of projects we aim to address. The basic knowledge obtained will be taken forward to help us understand the healthy human, and what happens in disease. This will provide new information that helps us understand a range of basic biologies, that will have wide impact.