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Principal Investigator: Francois St-Pierre
Organization: BAYLOR COLLEGE OF MEDICINE
Fiscal Year: 2024
Award: $589,350
Funding agency: National Institute of Biomedical Imaging and Bioengineering
PROJECT SUMMARY/ABSTRACT
Fluorescent proteins are ubiquitous reagents in the biomedical sciences for reporting gene expression, protein and nucleic
acid localization, cell shape, and cellular activity. However, fluorescent proteins (FPs) become progressively dimmer —
they photobleach — with repeated or prolonged illumination. Photobleaching limits multiple types of biological experiments
where photostability is essential, such as single-molecule biophysics and timelapse imaging of cellular activity during
development, learning, and aging. Photobleaching often cannot simply be addressed by increasing the excitation light, as
high illumination power can induce membrane blebbing, nuclear fragmentation, alterations in the cell cycle, changes to the
concentration of intracellular calcium, and, ultimately, cell death. While over two decades of FP engineering has led to a
toolbox of bright FPs, less attention has been devoted to improving photostability because of the greater difficulty and lower
throughput endured when screening for photostable FPs. Moreover, few studies have attempted to improve photophysical
properties under two-photon illumination — a method of choice for deep-tissue imaging — because of technical challenges
associated with screening under this imaging modality. The overall objective of this research proposal is, therefore, to
develop and apply a color palette of bright and photostable FPs for one- and two-photon imaging in mammalian cells. Our
proposal leverages two specialized and synergistic approaches to FP discovery and engineering: (1) SPOTlight, a new all-
optical screening approach developed in Dr. St-Pierre's lab that circumvents technical hurdles and enables rapid screening
of both brightness and photostability at the single-cell level under one- and two-photon illumination; and (2) transcriptomic
and metagenomic mining for novel FPs from marine invertebrates, a technique pioneered by Dr. Shaner’s lab. SPOTlight
relies on light patterning technology to selectively illuminate individual cells labeled with fluorophores that can be
photoactivated from a dim to a bright state. The cells are therefore tagged with a unique fluorescence signature that can then
be distinguished and retrieved using Fluorescence Activated Cell Sorting (FACS). SPOTlight thus enables screening in
dense mixed cultures with single-cell resolution, thereby eclipsing the throughput of traditional well-based approaches.
Mining for novel FPs in marine invertebrate transcriptomes and metagenomes will allow us to rapidly identify and
characterize hundreds of novel FPs. From this pool of new FPs, we will select the most photostable for engineering with
the SPOTlight pipeline. We will also model their structures to guide site-directed mutagenesis. We propose to leverage
these new technologies and assays to develop FPs of different colors that are bright, monomeric, and sufficiently photostable
for long-term imaging experiments. We also propose to apply these new FPs to increase the photostability of genetically
encoded voltage indicators (GEVIs), which are fluorescent biosensors whose brightness reports changes in voltage. While
GEVIs are proposing tools for imaging neural electrical activity with exquisite temporal resolution, they require high
illumination power for detection and typically bleach in seconds or minutes. Overall, we anticipate that this project will
produce bright and photostable fluorophores and biosensors of broad utility for illuminating cellular dynamics and that our
procedures will inspire further multi-parameter engineering of imaging probes for long-term imaging.
Terms: <2-photon><Address><Adopted><Agar><Aging><Assay><Attention><Benchmarking><Best Practice Analysis><Bioassay><Biological><Biological Assay><Biomedical Research><Biophysics><Biosensor><Bleb><Blister><Bulla><Bullous Lesion><Calcium><Cell Body><Cell Communication and Signaling><Cell Cycle><Cell Death><Cell Division Cycle><Cell Function><Cell Physiology><Cell Process><Cell Shape><Cell Signaling><Cells><Cellular Function><Cellular Physiology><Cellular Process><Color><Coupling><Cytometry><Dedications><Detection><Development><Disease><Disorder><Dysfunction><Electrophysiology><Electrophysiology (science)><Embryo><Embryonic><Engineering><Equipment><FP593><Fainting><Flow Cytofluorometries><Flow Cytofluorometry><Flow Cytometry><Flow Microfluorimetry><Flow Microfluorometry><Fluorescence><Fluorescence Activated Cell Sorting Fractionation><Fluorescence-Activated Cell Sorting><Fluorescence-Activated Cell Sortings><Fluorescent Probes><Functional Metagenomics><Functional disorder><Gene Expression><Gene Proteins><Genetic Engineering><Genetic Engineering Biotechnology><Genetic Engineering Molecular Biology><Genetics-Mutagenesis><Health><High Throughput Assay><Illumination><Image><Imaging Device><Imaging Instrument><Imaging Tool><Individual><Intracellular Communication and Signaling><Jellyfish><Label><Laboratories><Laser Electromagnetic><Laser Radiation><Lasers><Learning><Light><Lighting><Mammalian Cell><Marine Invertebrates><Medicine><Membrane><Metagenomics><Methods><Microscopy><Mining><Modeling><Morphology><Msec><Mutagenesis><Mutagenesis Molecular Biology><Nerve Cells><Nerve Unit><Neural Cell><Neurocyte><Neurons><Neurophysiology / Electrophysiology><Nuclear><Optics><Pattern><Photobleaching><Photons><Photoradiation><Physiopathology><Procedures><Productivity><Property><Protein Engineering><Protein Gene Products><Proteins><Protocol><Protocols documentation><Rapid screening><Reagent><Recombinant DNA Technology><Recovery><Reporter><Reporter Genes><Reporting><Research Proposals><Resolution><Resource Sharing><Sampling><Science><Signal Transduction><Signal Transduction Systems><Signaling><Site-Directed Mutagenesis><Site-Specific Mutagenesis><Specificity><Structure><Subcellular Process><Syncope><System><Targeted DNA Modification><Targeted Modification><Techniques><Technology><Tissue imaging><Variant><Variation><Vesication><benchmark><biologic><biological sensor><biological signal transduction><biophysical foundation><biophysical principles><biophysical sciences><cell imaging><cell type><cellular imaging><chromophore><cofactor><college><collegiate><design><designing><developmental><drFP583><ds red protein><dsFP593><electrophysiological><experiment><experimental research><experimental study><experiments><flow cytophotometry><fluorophore><genetic protein engineering><genetically engineered><global gene expression><global transcription profile><high throughput screening><image-based method><imaging><imaging method><imaging modality><imaging probe><improved><interest><invention><membrane structure><metagenome><millisecond><monomer><necrocytosis><neural imaging><neuro-imaging><neuroimaging><neurological imaging><neuronal><new technology><novel><novel technologies><nucleic acid localization><optical><pathophysiology><protein design><red fluorescent protein><resolutions><screening><screenings><single molecule><temporal measurement><temporal resolution><time measurement><transcriptome><transcriptomics><two-photon><voltage>