High-speed correlative live imaging microscope for biomedical applications

Diseases can affect humans, life stock and crops, posing one of the major risks and causing significant economic burdens. Cardiovascular diseases are currently the deadliest in humans (14.5 million deaths per year worldwide), followed by infectious diseases (lower respiratory infections, diarrhoea, HIV/AIDS, tuberculosis - 9M), cancers (8.8M) etc. For example, despite the fact that HIV was first isolated in 1983 and is one of the most well studied viruses, there is still no prospect of an HIV vaccine. As a result, there are nearly 38 million people worldwide currently living with human immunodeficiency virus type-1 (HIV). There are many other, although not life threatening, hugely debilitating diseases and conditions, particularly neurological (epilepsy, Alzheimer's disease and dementias) that affect 1 billion people worldwide. Understanding disease progression and infection spreading mechanisms at molecular and cellular level is key to the successful development of effective drugs, vaccines and therapies. This requires development of microscopy techniques capable of non-invasive, high resolution, real-time, multi-modal life cellular imaging in solutions that mimic physiological conditions so that cell functionality is retained. Almost all cell types have highly structured surfaces composed of lipid membrane and supporting cytoskeletal structures, which define microdomains tailored to perform specific function. Although single molecule fluorescence imaging enables researchers to study how individual proteins perform their function, the data lacks spatial information which could be linked to cell surface morphology. This is because fluorescence microscopy cannot be used for imaging of the cell topography over long periods of time since lipid fluorescent dyes are taken into cells due to the cell membrane recycling. Correlative imaging based on Scanning Electron Microscopy and Fluorescence Microscopy can produce combined images of cell surfaces and fluorescently labelled molecules only in chemically fixed and dried preparation, hence can not be used to study live processes.
Here we propose to develop a new correlative live imaging technique for biomedical research. The imaging technique will be based on a combination of high-speed Scanning Ion Conductance Microscopy (HS-SICM) that can produce label-free 3D images of living biological cell membrane surfaces with several nanometers resolution and a light sheet fluorescence microscopy that can deliver single molecule resolution and acquisition rates up to 800 planes per second. This imaging technique will enable researchers to find out at what locations at the cell membrane individual molecules perform their functions. We will validate our new microscopy technique by imaging the assembly of human immunodeficiency virus (HIV) -like particles in living T-cells; the release of individual insulin vesicles in beta cells that are responsible for the production of insulin - the hormone that controls blood sugar levels and is implicated in diabetes mellitus; Myddosome formation in living macrophage that is linked to inflammation and Alzheimer's disease. Such an instrument, once built, will find a widespread application in fundamental and applied biomedical sciences, as it would enable researchers to perform experiments that are impossible at present.