This RFA will accelerate the development of imaging probes that can leverage and combine the strengths of 1) light microscopy to study cell function and 2) cryo-electron tomography to study sub-cellular structure. Development of genetically-encoded tags (green fluorescent protein, or GFP, and its variants), chemical tags (HaloTag, SNAP-tag), nanobodies, and other imaging labels have revolutionized live-cell and super-resolution fluorescence microscopy. In correlative light and electron microscopy (CLEM), registration of fluorescence microscopy with electron microscopy images can be used to identify specific structures or molecules, such as proteins, organelles, or other cellular components.
Recent advances in cryo-ET technologies have the potential to image the molecular architecture of the cell with atomic resolution. The field currently lacks a robust general-purpose label that is simultaneously visible by light and electron microscopy, can be directed to any intracellular protein of interest, and will only minimally perturb the assembly state or biological function of the targeted molecule. Visibility by cryo-ET is defined as detectable or computationally recognizable in unaveraged tomograms.
The long-term goal of this opportunity is to drive technology development aimed at visualizing intracellular structure and function using cryo-ET. We specifically seek proposals that support the development of intracellular imaging probes that can withstand cryogenic preservation, provide high photon yields to allow accurate localization in cryo-CLEM, and have sufficient electron contrast or a unique shape to be detectable in cryo tomograms. This funding opportunity is explicitly aimed at the design and demonstration of a complete solution for general-purpose, intracellular cryo-CLEM imaging probes that could be broadly disseminated to the cryo-CLEM/cryo-ET field.
Examples of potential areas within the scope of this RFA include, but are not limited to:
- Complete solution for cryo-CLEM labeling that includes high-yield fluorophores and features that are detectable in electron microscopy (EM),
- Genetically encoded fluorescent and electron-dense tags with unique shapes for cryo-CLEM, ideally self assembling only when attached to protein of interest,
- Carbon nanodots or chemically synthesized probes adapted for cryo-CLEM,
- Nanobody binders with unique shapes for cryo-CLEM, and
- AI and deep learning approaches to the design of cryo-CLEM labels.
Successful designs should be:
- Applicable to targets inside of intact cells,
- Kept as small as possible,
- Minimally perturbative of the structure or function of target protein,
- Visible at a single or low copy number by cryo fluorescence microscopy,
- Detectable in an unaveraged cryo-EM tomogram (either significant metal density or have a unique shape), and
- Result in low EM background (minimal false positives).
We particularly encourage applications from:
- Women, those underrepresented or marginalized in science, and diverse groups worldwide; and
- Early career investigators, defined as principal investigators who have been in an independent faculty role for less than six years at the time of application, i.e. have started their first independent position between July 20, 2017, and July 20, 2023.
Collaboration and Open Science
CZI seeks investigators who will enthusiastically contribute to and benefit from a highly collaborative, dynamic, and interdisciplinary approach.
- Investigators and members of their project teams will participate in meetings throughout the grant period to encourage collaboration. Project teams will present progress on their design concepts or results of their proof-of-principle demonstrations, including project outcomes, lessons learned, and negative results.
- Investigators and CZI staff will work together to identify resources and technology that can drive the imaging field forward.
- Investigators will commit to rapid dissemination of all resulting data, protocols, code, reagents, and results (including negative results) prior to publication through resources such as protocols.io, GitHub, Addgene, and preprints. Please see the Policies section for additional details.