A multi-user confocal superresolution microscope for cell and developmental biology

Developments in microscopy have catalyzed ever more sophisticated understanding of how cells and organisms work. Fluorescence microscopy, allows us to monitor individual proteins, protein complexes, or organelles, using antibodies or protein tagging, in either fixed or live preparations; this in turn helps us link the behavior of individual proteins or protein complexes with molecular and genetic characterization of their properties.

However, normal microscopes cannot distinguish between objects that are separated by less than half the wavelength of visible light, or around 200-300 nanometers (millionths of a millimeter). Since many components of the cell machinery are separated by distances below this limit, understanding how this machinery works requires microscopy techniques that can resolve even below this fundamental limit. This compelling need has driven the recent development of a number of "super-resolution" microscopy approaches by (among others) the 2014 Chemistry Nobel Laureates Betzig, Hell and Moerner. Each of these approaches has its own strengths and limitations.

We are applying for one type of super-resolution microscope, called a Stimulated Emission Depletion (STED) microscope. In this, laser beams use properties of fluorescent labels to generate "pixels" of light from the preparation that are smaller than the 200-300 nanometer limit - typically 50 nanometers or less, which is sometimes enough to distinguish even the opposite ends of the same protein molecule. We have chosen STED because it meets the needs of a wide range of users in cell and developmental biology: it allows super-resolution in all three dimensions, is fast enough to image live preparations in real time, and allows us to image at greater depths into preparations than other super-resolution methods.

We will set up this microscope in an environment that permits its use by as wide a range of users as possible. STED is now mature enough that we can procure a commercially available instrument that is sufficiently well configured and supported to allow use by trained non-specialists. Wide use will be facilitated by specialised technical support, a management committee, a web-based booking system, extensive documentation, user training, and a program of education for users.

We already have an active user community who need super-resolution microscopy to understand the molecular basis of fundamental cellular processes in a range of model organisms including yeast, fruitflies, and human cells. These processes represent the core biological functions of the cell, all of which can go awry in a large variety of of long term health problems that include neurological diseases and cancer. Our findings will therefore underpin our knowledge of these diseases. Examples of how a STED microscope will help advance these projects are:

1. To localize membrane structures within neurons, especially in the confined spaces of axons and synapses where signals are transmitted; and detecting defects in these structures in fruitflies carrying mutations homologous to human axon degeneration mutations.

2. To understand how neuronal networks form by studying the mechanisms that regulate the development and maintenance of synaptic contacts between identified neurons.

3. To understand the organization and localization of multifunctional cellular machines that can choreograph cell division to achieve equal segregation of cell components into two daughter cells, and that can also organise cilia, cellular outgrowths that can have both motor functions and also act as signalling antennae.

4. To understand mechanisms that achieve unequal segregation of proteins and RNAs during cell division, to ensure correct differentiation of different cell types.

5. To assess overlap and cooperativity in the functions of nuclear proteins through studies of their precise three-dimensional organization to regulate gene expression.

Understanding cellular and developmental mechanisms continues to be underpinned by advances in optical microscopy. Recent development of light microscopy able to resolve below the diffraction limit of around 200-300 nm now facilitates interrogation of cellular processes at the sub-organelle level, and below the level of protein complexes. The cellular and molecular detail revealed by these "super-resolution" techniques brings understanding of the dynamic cellular processes that include organization and function of organelles, trafficking of cellular components, cell:cell interactions including synaptic organization and function, the cell division machinery, nuclear organisation and functions during differentiation, and gene expression. Our aim is to open this level of analysis to a local community of users who work on a wide range of cell and developmental biology.

Of the different super-resolution methods available, the most suited to the wide range of users in our community is a commercially available STED (Stimulated Emission Depletion) configured to be accessible for trained non-specialists, for supported multi-user use. The features making STED widely applicable to cell biological studies are: its ability to resolve below 50 nm with super-resolution in all three dimensions; real time acquisition permitting live imaging; its confocal nature, which permits imaging through several tens of micrometres into tissues.

The instrument will be used to image fixed and live preparations of cultured cells and tissues of model organisms. It will be used to study the organisation and dynamic functions of individual proteins components of endoplasmic reticulum; neuro-synapses; the Golgi and vesicle trafficking machinery; centrosome and spindles; centrioles and cilia; organelles for RNA processing, trafficking and localization; and the nuclear machinery for gene expression.

Impact Statement

Who will benefit?

Both the successful applications of bioscience, and the expensive and disappointing series of failed advance-phase clinical trials, illustrate the vital importance of basing applications on a deep understanding of the fundamental biology. Most of our research will contribute to the underlying basic knowledge. However, as well as academic users, the Biotech industry, healthcare professionals, the agri-food sector, patients, are non-academic users who can potentially benefit from applications of our fundamental work. All of these constituencies, as well as educational or campaign/charity professionals and the wider public, can also benefit through outreach about the basic processes though which cells function.

How will they benefit from this research?

Academic users:

For wider dissemination beyond immediate users, the facility will encourage all users to publish in Open Access formats, and ask them to acknowledge BBSRC funding for the instrument, as a way of supporting institutional applications for Open Access funds. We will encourage posting of pre-publication manuscripts on pre-print servers such as BioRxiv to speed up dissemination.

To ensure that users can take advantage of a state-of-the-art instrument for cutting-edge research, training will be an important part of our mission. Our strategies for this include formal training sessions, expert lectures, and activities such as social media to build a community of users and facilitate sharing of experience and expertise. Our social media presence (e.g. Twitter accounts) will be completely in the public domain, to achieve the widest possible training impact.

Industrial/Biotech/Healthcare/AgriFood/Patients:

Research by users will underpin the knowledge base of these industries and thus foster their competitive abilities internationally. Much of the applicants' work is of direct interest to these sectors, even if only some of it has immediate applications. This includes neurodegeneration, synapse development, cell growth and cancer, and insect receptors. Better understanding of these processes allows more rational identification of druggable targets, for example kinases and ROS-generating or protective enzymes. Organoids are also likely to have a major future impact upon translational and pharmaceutical research.

Exploitation of these possibilities depends on contacts with Pharma, Biotech and Agri-Food companies; many of these are clustered around Cambridge, or have excellent Cambridge contacts, and at least two of us (Glover, Russell) have direct experience of such interactions. The STED facility will support such interactions by any of its users.

Outreach

As a facility we will use a range of possibilities to promote outreach. The Genetics Department has an open day with hands-on displays during Science Week. These can be developed to include super-resolution imaging outputs and interactive computer exercises - although it is unlikely that we would include the instrument itself in public events because of the risk of damage to both equipment and the public. We will use our departmental and laboratory and facility public websites to highlight our findings, and University press officers to communicate with the press to disseminate news rapidly. We will institute a Twitter account aimed at the general public, and at non-science professionals such as education or charity or science communication professionals. The investigators named on the application perform a range of regular outreach including schools presentations, Twitter feeds, radio/TV broadcasts, scientific talks to patient groups, and public panel discussions, and will be able to incorporate the findings of super-resolution work into their future activities.