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Imaging the proteome at the nanoscale

Periodic Reporting for period 1 - IMAGEOMICS (Imaging the proteome at the nanoscale)

Reporting period: 2021-07-01 to 2022-06-30

The key principle of biological imaging is specific labeling. The protein of interest is revealed by tagging with fluorophores, either by genetic encoding or by specific affinity labeling. This procedure has the great disadvantage that each protein needs to be tagged individually. This has so far stopped imaging from becoming an “omics” approach, for large numbers of targets. We proposed to change this concept in our project, by inventing an imageomics approach. We plan to rely on small affinity probes that bind amino acid sequences (peptides) that are present in multiple proteins. We will apply these probes to biological samples in a combinatorial fashion, and we will image them at the nanoscale, with a resolution that is sufficient to image single proteins. Each protein will be identified by the binding of a unique combination of affinity probes, corresponding to its sequence. The implementation of this concept would lead to a radical new way of imaging proteins, with major implications in basic science and in medicine.
Our proposal is to rely on nanobodies, small camelid antibody fragments, which are generated against different peptides. After selecting the best specific nanobodies, they are labeled with bright fluorophores, and are imaged in a super-resolution imaging setup that enables the combinatorial analysis of all nanobodies, enabling us to decipher the identities of the different proteins.
In our four main work packages, we proposed the following:
1. To generate the nanobody candidates and to validate them through microfluidics. This work is ongoing, with many nanobodies already generated. A microfluidics-based procedure for nanobody testing has been generated, and is being now implemented to test the different nanobodies.
2. To obtain the best possible sample preparation and imaging conditions. First, this implies generating optimal expansion microscopy (ExM) conditions. We have performed this, obtaining resolutions of close to 1 nm in expanded gels. Second, this also implies obtaining excellent setups for gel imaging. This is well under way, in the form of a specialized STED setup with limited bleaching.
3. To generate IMAGEOMICS imaging protocols. The work started recently in this WP, but has already led to protocols for the optimal fluorescence labeling of nanobodies.
4. To organize and put together a semi-automated imaging setup to be used for IMAGEOMICS. The work has not yet started in this WP, according to our initial plans.
We propose here to develop a technology that will provide nanoscale proteomic images for biomedical research. We will eventually replace conventional assays implemented with antibodies, as Western Blot, immunostaining or immunohistochemistry, with a more efficient and wide-ranging method. This should have large economic implications.
The highly experimental character of our technology, and the long studies necessary for its approval for human diagnostic purposes, will slow its entry into the diagnostic market. ELISA and similar methods will still remain the tools of choice for several years. Nevertheless, IMAGEOMICS has the potential to provide superior results to such studies, especially as we expect a higher sensitivity for our technology, compared with antibody-based detection, due to the higher epitope recognition provided by small probes. This would also enable the collection of smaller biopsies, reducing patient discomfort and the risk of biopsy-induced metastasis.
The impact of this project in the basic sciences is probably even higher than that on diagnostic testing. This will be a foundational project, which will open a new world of information on cells and tissues. This will result in a substantial impetus on understanding the protein composition of different samples, from health and disease. The resulting new knowledge will offer countless projects for young scientists (MSc, PhD, Post-doc), in multiple disciplines. Biologists and medical investigators will describe the new biomedical insight obtained. Informatics experts will develop new data analysis methods, which will be able to deal with the massive amount of information provided by IMAGEOMICS, and will describe the most significant features out of thousands of protein measurements in multiple samples. Physicists will continue to develop and improve the optics setups necessary for this approach, while engineers and bio-engineers will generate better and faster setups.
Importantly, not only the ultimate result of the project is important, but also the intermediate results. Many of our intermediate results will also be important for the imaging community. For example, in the last few months we tackled the problem that fluorescence microscopy still fails to image the morphology of single proteins or small molecular complexes, either purified or in a cellular context. We found a solution to this problem, in the form of one-nanometer expansion (ONE) microscopy. We combined the 10-fold axial expansion of the specimen (1000-fold by volume) with a fluorescence fluctuation analysis to achieve resolutions down to 1 nm or better. We have successfully applied ONE microscopy to image cultured cells, tissues, viral particles, molecular complexes and single proteins. At the cellular level, using immunostaining, our technology revealed detailed nanoscale arrangements of synaptic proteins, including a quasi-regular organisation of PSD95 clusters. At the single molecule level, upon main chain fluorescent labelling, we could visualise the shape of individual membrane and soluble proteins. Moreover, conformational changes undergone by the ~17 kDa protein calmodulin upon Ca2+ binding were readily observable. We could also image and classify molecular aggregates in cerebrospinal fluid samples from Parkinson’s Disease (PD) patients, which represents a promising new development towards an improved PD diagnosis. ONE microscopy is compatible with conventional microscopes and can be performed with the software we provide here as a free, open-source package. This technology bridges the gap between high-resolution structural biology techniques and light microscopy, and provides a new avenue for discoveries in biology and medicine. ONE microscopy is a very important stepping stone for the final implementation of IMAGEOMICS.
ONE microscopy images of single proteins
ONE microscopy, combined with nanobody usage, offers hope for Parkinson's Disease diagnostics
A new microscopy approach, ONE microscopy, provides very high resolution images.
ONE microscopy images of synapses