Energy filter with direct electron detector for electron cryo tomography

The machinery of life operates in biological cells and tissues at the molecular and atomic level. A detailed description of these molecular machines, their internal organization, and their interactions within cells is fundamental to understanding living organisms in health and disease.

At Birkbeck we study the three-dimensional structure of these machines with the aim of understanding how they work. Electron microscopy (EM) has become a major technique for observing biological machines both in isolation and in their cellular context. Electron microscopy can provide highly detailed information on the structure of biological molecules as well as overall perspectives on the organization of cells and tissues. The EM approach that allows us to determine the three-dimensional structures of cellular machinery is electron tomography. In principle, this is very similar to computed tomography in medicine, except that it works at very high magnification and shows us cells and molecules rather than bones, blood vessels and organs.

The samples are rapidly frozen using cryogenic liquids and examined at very low temperature. With this type of preparation they can be imaged in their natural, hydrated state. Advances in electron microscopy methods in the last decade have enabled us to obtain snapshots of biological molecules at different stages of their normal operation, revealing details of their mechanisms of action.

However, the unstained biological structures trapped by rapid freezing in their native environment scatter electrons very weakly and are highly sensitive to the electron beam. The resulting images have poor contrast and a high background, limiting the detectable structural features in the images. In this equipment grant, we are requesting funding for a high-resolution electron energy filter and electron detector to help us record fine structural details in tomography images of cells. The filter removes unwanted, inelastic scattering that arises when electrons pass through samples as thick as cells. Filtering out this type of scattering makes the images clearer, so that more structural details can be discerned and interpreted. This energy filter will enable us to extract more information about how the cellular machinery functions.

Specific projects that will benefit from this new equipment include: 1) studies of pathogen-infected cells (including Malaria, Chlamydia and Toxoplasma) that will reveal how these intracellular invaders sabotage host cell systems; 2) studies of bacterial secretion systems that will reveal how bacteria interact with their environment to promote their own survival and replication; 3) studies of the process by which viruses infect bacteria; 4) studies to reveal how our immune cells protect our bodies from infection or cancer; 5) studies of the architecture and microtubule cytoskeleton of neuronal cells that will reveal how they adapt their shape during brain development and how this is disrupted in human diseases; 6) studies of amyloid protein deposits in living cells that will reveal how the cell's quality control machinery handles protein misfolding that causes neurodegenerative disease.

The field of cryo-EM is increasingly important in molecular and cellular biology, owing to advances in hardware and software that improve image quality and experimental efficiency. Its scope has expanded to include three-dimensional structure determination over a wide range of samples from sections of tissues, to cell regions, isolated organelles, and individual macromolecular assemblies. In particular, new biological insights arise from structure determination of macromolecular machinery in its native cellular context. However, cellular imaging is limited by sample thickness, because inelastic scattering of electrons gives a high background that obscures structural information in the images. This background can be removed using an energy filter. Earlier generations of electron energy filters such as our current model are very slow, and alignment is difficult and unstable, causing a loss of resolution. Here we request a state-of-the-art, high-resolution post column electron energy filter and associated electron detector for our Polara microscope. Since cryo-EM operates at the limits of detection for delicate biological structures, advances in energy filters and detectors make a vast difference to feasibility, resolution and throughput of data collection, and consequently to the biological insights that can be derived from the experiments. We have recently dramatically expanded our research using cellular electron tomography and will continue to increase our activity in this area. Major projects that now require an advanced energy filter include: studies of intracellular pathogens (e.g. Malaria, Chlamydia, Toxoplasma); bacterial secretion systems in intact cells; phage-bacteria interactions; killing of infected and transformed cells at the immune synapse; neuronal migration and intracellular transport; studies of cellular amyloid deposits. Improvements in biological output resulting from improved detection lead to a much more effective use of expensive facilities.

Who will benefit from this research?
- UK Biotech and pharmaceutical drug discovery
- Patients with diseases related to malfunction of molecular machinery
- UK economy
- The wider public
- Women in science

How will they benefit from this research?

Molecular machines are fundamental to life processes and when they fail, many different types of diseases are caused. In studying their molecular mechanisms and their cellular context, our research will provide insight as to how malfunctions of these machines can be targeted to treat diseases. Examples from our current research portfolio include: 1) studies of major intracellular pathogens such as Malaria, Chlamydia and Toxoplasma that can provide new insights into the biology of these poorly understood organisms and thus possible targets for drug intervention; 2) studies of bacterial secretion systems that provide routes towards novel antibiotics; 3) studies of bacteriophages and their interactions with bacteria that could lead to new anti-bacterial tools such as phage therapy as an addition or alternative to traditional antibiotics; 4) studies of immune defense systems that target infected or cancerous cells; 5) studies of neuronal development that could bring insight into treatment of brain injury and neurodegenerative diseases; 6) studies of cellular amyloid deposits that provide molecular clues about how neurodegeneration might be reduced in conditions such as Alzheimer's disease.

Beneficiaries from this aspect of the research will include the UK biotechnology industry, as greater understanding from academic studies such as ours leads to more effective generation of improved drugs and, therefore, improved sales.
We would aim to engage these beneficiaries immediately. Science and technology will lie at the heart of global economic recovery, and we will liaise with UCL Business (UCLB), who work with Birkbeck researchers on technology development and intellectual property matters, to maximise the impact of our discoveries. In the longer term, identifying drugs for disease treatment will also benefit the millions of patients who are affected by malfunction of cellular machinery and thereby improve their quality of life. In addition, the supplier of the equipment we wish to purchase has a manufacturing unit and EM Applications lab in the UK (Gatan UK, 25 Nuffield Way, Abingdon Oxon, OX14 1RL). All together, this will ultimately have benefits for the economic competitiveness of the United Kingdom.

We will aim to make the discoveries of our research available not only to the academic community, but also to the general public. All the applicants have proven track records of public communication of science: HS, EO and CM have presented their research to a general audience as part of Birkbeck Science Week in the last few years. CM was the 2006 winner of the prestigious DeMontfort medal for science communication (SET for Britain) and has discussed her work at a Coffee for Cancer Club meeting at a local retirement community. We will work with our Departmental Web Manager to design webpages that are accessible to the general public and explain these exciting new innovations in Electron Microscopy. We will seek to participate in other public understanding of science activities, for example by inviting sixth-form students to visit the lab and experience the day-to-day life of scientists.

The EM group leaders at Birkbeck are remarkable in the field of biophysics in that they are mainly women. During the project period, we will continue to be involved in advancing gender equality in science, engineering and technology through involvement with the Athena SWAN programme at Birkbeck College. CM is a member of the Birkbeck steering committee that submitted the College's successful application for a Bronze Athena SWAN University Award and chairs the Departmental Athena SWAN committee.