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ref: -0 tags: VARNUM GEVI genetically encoded voltage indicators FRET Ace date: 03-18-2020 17:12 gmt revision:5 [4] [3] [2] [1] [0] [head]

PMID-30420685 Fast in-vivo voltage imaging using a red fluorescent indicator

  • Kannan M, Vasan G, Huang C, Haziza S, Li JZ, Inan H, Schnitzer MJ, Pieribone VA.
  • Other genetically encoded voltage indicators (GEVI):
    • PMID-22958819 ArcLight (Peribone also last author) ; sign of ΔF/F\Delta F / F negative, but large, 35%! Slow tho? improvement in speed
    • ASAP3 ΔF/F\Delta F / F large, τ=3ms.\tau = 3 ms.
    • PMID-26586188 Ace-mNeon FRET based, Acetabularia opsin, fast kinetics + brightness of mNeonGreen.
    • Archon1 -- fast and sensitive, found (like VARNUM) using a robotic directed evolution or direct search strategy.
  • VARNAM is based on Acetabularia (Ace) + mRuby3, also FRET based, found via high-throughput voltage screen.
  • Archaerhodopsin require 1-12 W/mm^2 of illumination, vs. 50 mw/mm^2 for GFP based probes. Lots of light!
  • Systematic optimization of voltage sensor function: both the linker region (288 mutants), which affects FRET efficiency, as well as the opsin fluorophore region (768 mutants), which affects the wavelength of absorption / emission.
  • Some intracellular clumping (which will negatively affect sensitivity), but mostly localized to the membrane.
  • Sensitivity is still imperfect -- 4% in-vivo cortical neurons, though it’s fast enough to resolve 100 Hz spiking.
  • Can resolve post-synaptic EPSCs, but < 1 % ΔF/F\Delta F/F .
  • Tested all-optical ephys using VARNAM + blueshifted channelrhodopsin, CheRiff, both sparsely, and in PV targeted transgenetic model. Both work, but this is a technique paper; no real results.
  • Tested TEMPO fiber-optic recording in freely behaving mice (ish) -- induced ketamine waves, 0.5-4Hz.
  • And odor-induced activity in flies, using split-Gal4 expression tools. So many experiments.

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ref: -0 tags: DNA paint FRET tag superresolution imaging oligos date: 02-20-2020 16:28 gmt revision:1 [0] [head]

Accelerated FRET-PAINT Microscopy

  • Well isn't that smart -- they use a FRET donor, which is free to associate and dissociate form a host DNA strand, and a more-permanently attached DNA acceptor, which blinks due to FRET, for superresolution imaging.
  • As FRET acceptors aren't subject to bleaching (or, perhaps, much less subject to bleaching), this eliminates that problem...
  • However, the light levels used ~1kW / cm^2, does damage the short DNA oligos, which interferes with reversible association.
  • Interestingly, CF488 donor showed very little photobleaching; DNA damage was instead the limiting problem.
    • Are dyes that bleach more slowly better at exporting their singlet oxygen (?) or aberrant excited states (?) to neighboring molecules?

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ref: Erickson-2003.07 tags: GFP FRET math CFP DsRed math date: 02-08-2008 16:04 gmt revision:1 [0] [head]

PMID-12829514 DsRed as a Potential FRET Partner with CFP and GFP

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ref: notes-0 tags: two-photon laser imaging fluorescence lifetime imaging FRET GFP RFP date: 01-21-2008 17:23 gmt revision:0 [head]

images/538_1.pdf