RT @Austin_Lefebvre: Attention organelle enthusiasts!!.I am beyond thrilled to introduce Nellie: a fully automated pipeline for organelle s….

RT @AMMGest: Happy #FluorescenceFriday everyone! Our "Renaissance Dye" CRhOMe (and my co-first author paper from my PhD) is now published a….

RT @IlariaTesta4: RESOLFT was never faster! Happy to share the OPM with @snouty and @rsfp to craft several thin sheets of light at the same….

RT @AMMGest: Happy #FluorescenceFriday! Out now is the latest work from my PhD with @millerchembio showcasing two chemical-genetic targetin….

RT @AdamB0wman: We are hiring! If you are excited about building microscopes, voltage imaging, or techniques that can improve biological me….

RT @amoeba_wrangler: The uropod of the amoeba Chaos is labeled by microinjecting Lifeact-GFP2 protein. The #AIRR Single Objective Light She….

RT @amsikking: #AIRR microscopy has arrived! Use any immersion objective (including air!) to look into any sample refractive index! Built h….

RT @RetoPaul: We present projective oblique plane structured illumination microscopy (POPSIM):. POPSIM allows imag….

RT @joachimgoedhart: Quantitative Imaging of Genetically Encoded Fluorescence Lifetime Biosensors .

Big thanks to the team on this project @Austin_Lefebvre Rebecca Frank Hayward @DrLachie @Maria_Ingaramo @AndrewGYork . And importantly thanks to @calico for being an awesome place to do science!.

There’s lots to do: hardware to construct, theoretical foundations to build & fluorophores to engineer. Sample-side engineering is particularly needed: let’s improve linker rigidity, triplet quantum yield & rsFP activation rate. If you’re inspired to work on this, join the party!.

4. Are there existing commercial instruments that can measure tumbling of protein complexes? We used our @LeicaMicro SP8 confocal to measure tumbling of fluorescent beads. The software prevented us from measuring smaller, protein-scale objects, but the hardware is there!

3. Can we measure tumbling in millions of individual cells in an hour? Pump probe measurements are compatible with flow cytometers, enabling large-scale genetic screens. It would be awesome to rapidly ID all of the genes that affect formation of a complex

2. Can we measure tumbling in billions of voxels per second? Pump-probe tumbling measurements are compatible with widefield or lightsheet microscopes, so we can incorporate protein-protein interaction measurements as an extra channel of information.

1. How cheaply can we measure tumbling? Using “shelving” (photobleaching, triplet generation) of common fluorophores, all we need is a focused laser and a photodiode! We could imagine this as a cheap, handheld diagnostic or in a plate reader-like instrument

We explore how triggerable emission broadens the scope of tumbling applications: what photophysics can we use? What hardware can we use? (spoiler: lots of options for both!) We simulated 4 case studies & we took experimental data on an existing commercial instrument.

rsFPs and triplets have an important bonus feature: they have triggerable emission, i.e. a 2nd pulse of light causes them to emit. This lets us concentrate our photons when they are most informative…e.g. measure at 20 μs not 1 μs to distinguish 100 from 200 nm particles

Two papers recently reported watching protein complexes tumble with long-lived photophysical states:.1. Reversible switchable fluorescent proteins (rsFPs): @IlariaTesta4 @andreavolpato_ .2. Triplets: Robert Dickson.

Fluorescence anisotropy uses tumbling to measure size, BUT it can only measure tumbling during the ~5 ns fluorescence lifetime. Unfortunately, targets >30 kDa take >20 ns to tumble, so everything >30 kDa just looks “big,” with no information about binding state.

Tumbling (rotational diffusion) is exquisitely sensitive to particle size. We can optically monitor the rate of tumbling with a single label on our protein of interest. So, for a given protein, we can see the sizes and abundances of the complexes it forms! Animation: @DrLachie