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ref: -2011 tags: two photon cross section fluorescent protein photobleaching Drobizhev gcamp date: 11-04-2020 18:07 gmt revision:9 [8] [7] [6] [5] [4] [3] [head]

PMID-21527931 Two-photon absorption properties of fluorescent proteins

  • Significant 2-photon cross section of red fluorescent proteins (same chromophore as DsRed) in the 700 - 770nm range, accessible to Ti:sapphire lasers ...
    • This corresponds to a S 0S nS_0 \rightarrow S_n transition
    • But but, photobleaching is an order of magnitude slower when excited by the direct S 0S 1S_0 \rightarrow S_1 transition (but the fluorophores can be significantly less bright in this regime).
      • Quote: the photobleaching of DsRed slows down by an order of magnitude when the excitation wavelength is shifted to the red, from 750 to 950 nm (32).
    • See also PMID-18027924
  • Further work by same authors: Absolute Two-Photon Absorption Spectra and Two-Photon Brightness of Orange and Red Fluorescent Proteins
    • " TagRFP possesses the highest two-photon cross section, σ2 = 315 GM, and brightness, σ2φ = 130 GM, where φ is the fluorescence quantum yield. At longer wavelengths, 1000–1100 nm, tdTomato has the largest values, σ2 = 216 GM and σ2φ = 120 GM, per protein chain. Compared to the benchmark EGFP, these proteins present 3–4 times improvement in two-photon brightness."
    • "Single-photon properties of the FPs are poor predictors of which fluorescent proteins will be optimal in two-photon applications. It follows that additional mutagenesis efforts to improve two-photon cross section will benefit the field."
  • 2P cross-section in both the 700-800nm and 1000-1100 nm range corresponds to the chromophore polarizability, and is not related to 1p cross section.
  • This can be useflu for multicolor imaging: excitation of the higher S0 → Sn transition of TagRFP simultaneously with the first, S0 → S1, transition of mKalama1 makes dual-color two-photon imaging possible with a single excitation laser wavelength (13)
  • Why are red GECIs based on mApple (rGECO1) or mRuby (RCaMP)? dsRed2 or TagRFP are much better .. but maybe they don't have CP variants.
  • from https://elifesciences.org/articles/12727

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ref: -0 tags: Na Ji 2p two photon fluorescent imaging pulse splitting damage bleaching date: 03-10-2020 21:44 gmt revision:6 [5] [4] [3] [2] [1] [0] [head]

PMID-18204458 High-speed, low-photodamage nonlinear imaging using passive pulse splitters

  • Core idea: take a single pulse and spread it out to N=2 kN= 2^k pulses using reflections and delay lines.
  • Assume two optical processes, signal SI αS \propto I^{\alpha} and photobleaching/damage DI βD \propto I^{\beta} , β>α>1\beta \gt \alpha \gt 1
  • Then an NN pulse splitter requires N 11/αN^{1-1/\alpha} greater average power but reduces the damage by N 1β/α.N^{1-\beta/\alpha}.
  • At constant signal, the same NN pulse splitter requires N\sqrt{N} more power, consistent with two photon excitation (proportional to the square of the intensity: N pulses of N/N\sqrt{N}/N intensity, 1/N per pulse fluorescence, Σ1\Sigma \rightarrow 1 overall fluorescence.)
  • This allows for shorter dwell times, higher power at the sample, lower damage, slower photobleaching, and better SNR for fluorescently labeled slices.
  • Examine the list of references too, e.g. "Multiphoton multifocal microscopy exploiting a diffractive optical element" (2003)

  • In practice, a pulse picker is useful when power is limited and bleaching is not a problem (as is with GCaMP6x)

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ref: -0 tags: Lucy Flavin mononucelotide FAD FMN fluorescent protein reporter date: 10-17-2019 19:54 gmt revision:1 [0] [head]

PMID-25906065 LucY: A Versatile New Fluorescent Reporter Protein

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ref: -2016 tags: fluorescent proteins photobleaching quantum yield piston GFP date: 06-19-2019 14:33 gmt revision:0 [head]

PMID-27240257 Quantitative assessment of fluorescent proteins.

  • Cranfill PJ1,2, Sell BR1, Baird MA1, Allen JR1, Lavagnino Z2,3, de Gruiter HM4, Kremers GJ4, Davidson MW1, Ustione A2,3, Piston DW
  • Model bleaching as log(F)=αlog(P)+clog(F) = -\alpha log(P) + c or k bleach=bI αk_{bleach} = b I^{\alpha} where F is the fluorescence intensity, P is the illumination power, and b and c are constants.
    • Most fluorescent proteins have α\alpha > 1, which means superlinear photobleaching -- more power, bleaches faster.
  • Catalog the degree to which each protein tends to form aggregates by tagging to the ER and measuring ER morphology. Fairly thorough -- 10k cells each FP.

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ref: -2019 tags: super-resolution microscopy fluorescent protein molecules date: 05-28-2019 16:02 gmt revision:3 [2] [1] [0] [head]

PMID-30997987 Chemistry of Photosensitive Fluorophores for Single-Molecule Localization Microscopy

  • Excellent review of all the photo-convertable, photo-switchable, and more complex (photo-oxidation or reddening) of both proteins and small molecule fluorophore.
    • E.g. PA-GFP is one of the best -- good photoactivation quantum yield, good N ~ 300
    • Other small molecules, like Alexa Fluor 647 have a photon yield > 6700, which can be increased with triplet quenchers and antioxidants.
  • Describes the chemical mechanism of the various photo switching -- review is targeted at (bio)chemists interested in getting into imaging.
  • Emphasize that critical figures of merit are photoactivation quantum yield Φ pa\Phi_{pa} and N, overall photon yield before photobleaching.
  • See also Colorado lecture

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ref: -2015 tags: conjugate light electron tomography mouse visual cortex fluorescent label UNC cryoembedding date: 03-11-2019 19:37 gmt revision:1 [0] [head]

PMID-25855189 Mapping Synapses by Conjugate Light-Electron Array Tomography

  • Use aligned interleaved immunofluorescence imaging follwed by array EM (FESEM). 70nm thick sections.
  • Of IHC, tissue must be dehydrated & embedded in a resin.
  • However, the dehydration disrupts cell membranes and ultrastructural details viewed via EM ...
  • Hence, EM microscopy uses osmium tetroxide to cross-link the lipids.
  • ... Yet that also disrupt / refolds the poteins, making IHC fail.
  • Solution is to dehydrate & embed at cryo temp, -70C, where the lipids do not dissolve. They used Lowicryl HM-20.
  • We show that cryoembedding provides markedly improved ultrastructure while still permitting multiplexed immunohistochemistry.

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ref: -0 tags: nanotube tracking extracellular space fluorescent date: 02-02-2017 22:13 gmt revision:0 [head]

PMID-27870840 Single-nanotube tracking reveals the nanoscale organization of the extracellular space in the live brain

  • Extracellular space (ECS) takes up nearly a quarter the volume of the brain (!!!)
  • Used the intrinsic fluorescence of single-walled carbon nanotubes @ 1um, 845nm excitation, with super-resolution tracking of diffusion.
    • Were coated in phospholipid-polyethylene glycol (PL-PEG), which display low cytotoxicity compared to other encapsulants.
  • 5ul, 3ug/ml injected into the ventricles of young rats; allowed to diffuse for 30 minutes post-injection.
  • No apparent response of the microglia.
  • Diffusion tracking revealed substantial dead-space domains in the ECS.
    • As compared to patch-clamp loaded SWCNTs
  • Estimate from parallel and perpendicular diffusion rates that the characteristic scale of ECS dimension is 80 to 270nm, or 150 +- 40nm.
  • The ECS nanoscale dimensions as visualized by tracking similar in dimension and tortuosity to electron microscopy.
  • Viscosity of the extracellular matrix from 1 to 50 mPa S, up to two orders of magnitude higher than the CSF.
  • Positive control through hyalurinase + several hours to digest the hyaluronic acid.
    • But no observed changes in morphology of the neurons via confocal .. interesting.
    • Enzyme digestion normalized the spatial heterogenaity of diffusion.

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ref: -0 tags: voltage sensitive dyes fluorescent protein date: 01-02-2013 05:08 gmt revision:0 [head]

PMID-20622860 Imaging brain electric signals with genetically targeted voltage-sensitive fluorescent proteins.

  • Interesting: Most fluorescent fusion proteins form intracellular aggregates during long-term expression in mammalian neurons, although this effect appears to be minimal in Aequorea victoria–derived fluorescent proteins.
  • See also {1185}

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ref: Sakai-2001.06 tags: voltage scensitive fluorescent protein flourophore VSFP1 endoscope date: 01-24-2012 06:07 gmt revision:5 [4] [3] [2] [1] [0] [head]

http://www.blackwell-synergy.com/doi/full/10.1046/j.0953-816x.2001.01617.x PMID-11454036[0]


[0] Sakai R, Repunte-Canonigo V, Raj CD, Knöpfel T, Design and characterization of a DNA-encoded, voltage-sensitive fluorescent protein.Eur J Neurosci 13:12, 2314-8 (2001 Jun)
[1] van Roessel P, Brand AH, Imaging into the future: visualizing gene expression and protein interactions with fluorescent proteins.Nat Cell Biol 4:1, E15-20 (2002 Jan)
[2] Guerrero G, Siegel MS, Roska B, Loots E, Isacoff EY, Tuning FlaSh: redesign of the dynamics, voltage range, and color of the genetically encoded optical sensor of membrane potential.Biophys J 83:6, 3607-18 (2002 Dec)
[3] Jung JC, Mehta AD, Aksay E, Stepnoski R, Schnitzer MJ, In vivo mammalian brain imaging using one- and two-photon fluorescence microendoscopy.J Neurophysiol 92:5, 3121-33 (2004 Nov)
[4] Sjulson L, Miesenböck G, Optical recording of action potentials and other discrete physiological events: a perspective from signal detection theory.Physiology (Bethesda) 22no Issue 47-55 (2007 Feb)