Validation and application of a novel set of “self-blinking” dyes for use in super-resolution (PALM) microscopy. This project is initiated by Fadi Jradi in collaboration with T3, the Advanced Imaging Center and others.
Nitric Oxide chemical sensor
In vitro validation of several iterations of fluorescent dye sensors for the gaseous cellular messenger nitric oxide (NO). Yoshi Aso is attempting to use these in Drosophila to elucidate the roles of NO in learning and memory. This project was initiated by Fadi Jradi.
Localized Reactive Oxygen generating tags
Fluorescent dyes were designed to produce damaging reactive oxygen species (ROS) upon light-activation (Chad Binns, Luke Lavis). When coupled to protein-specific HaloTag, this reagent is used to target the ROS damage to a particular cell or protein and can be used to ablate a cell or induce turnover of a specific protein. T3 has assisted in validating these molecules in vitro. Janelia investigators are continuing their use to ablate specific neurons in zebrafish.
Enzymatic In vivo cell surface protein labeling
T3 has spearheaded the expression and purification of an enzyme (phosphopantetheinyl transferase) and conjugation and purification of JF549-Coenzyme A. The goal is to use these reagents to resurrect a method to enzymatically label specific membrane proteins in the brain, and to assess protein localization and dynamics in vivo. Ultimately, the tools may be used to assess the functional contribution of each protein to neural computation.
Single-chain Variable Fragment (scFv) and nanobody cell surface protein labeling
T3 is optimizing scFv scFv and nanobody reagents to specifically label neuronal membrane proteins as an alternative and complementary strategy to the enzymatic method outlined above. This is also a gateway project to develop additional applications of these small affinity reagent scFv technologies within T3.
Validation and use-optimization of second and third generation Janelia Fluor dyes”
The Janelia Fluor dyes continue to be improved by the Lavis lab with expansion of the fluorescence color palette along with improvements in brightness and stability. T3 has ongoing projects in determining the optimal use of these fluorescent dyes.
Use of Akaluc as a freely behaving drosophila reporter gene
T3 is optimizing delivery of enzyme substrate and in engineering of the Akaluc enzyme in various split forms as a tool for assessing molecular events in whole organisms.