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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: optical gain media lasers cross section dye date: 06-13-2019 15:13 gmt revision:2 [1] [0] [head]

Eminently useful. Source: https://ocw.mit.edu/courses/electrical-engineering-and-computer-science/6-974-fundamentals-of-photonics-quantum-electronics-spring-2006/lecture-notes/chapter7.pdf

Laser Dye technology by Peter Hammond

  • This paper is another great resource!
  • Lists the stimulated emission cross-section for Rhodamine-6G as 4e-16 @ 550nm, consistent with the table above.
  • At a (high) concentration of 2mMol (1 g/l), 1/e penetration depth is 20um.
    • Depending on the solvent, there may be aggregation and stacking / quenching.
  • Tumbling time of Rhodamine 6G in ethanol is 20 to 300ps; fluorescence lifetime in oscillators is 10's of ps, so there is definitely polarization sensitive amplification.
  • Generally in dye lasers, the emission cross-section must be higher than the excited state absorption, σ eσ \sigma_e - \sigma^\star most important.
  • Bacteria can actually subsist on rhodamine-similar sulfonated dyes in aqueous solutions! Wow.

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ref: -2011 tags: HiLo speckle imaging confocal boston university optical sectioning date: 02-19-2019 06:18 gmt revision:2 [1] [0] [head]

PMID-21280920 Optically sectioned in vivo imaging with speckle illumination HiLo microscopy

  • Ah, brilliant! Illuminate a sample with a speckle pattern from a laser, and use this to optically section the data -- the contrast of the speckle pattern shows how in focus the sample is.
    • Hanece, the contrast indicates the in-focus vs out-of-focus ratio in a region.
  • The speckle statistics are invariant even in a scattering media, as scattering only further randomizes an already random laser phase front. (Within some limits.)
  • HiLo microscopy involves illuminating with a speckle pattern, then illuminating with standard uniform illumination, resulting in a diffraction-limited optically sectioned image. PMID-18709098
  • Algorithm is :
    • Take the speckle image and subtract the uniform image δI\delta I
    • Bandpass δI\delta I
    • Measure the standard deviation of the δI\delta I to get a weighting function C δs 2C^2_{\delta s}
    • Debias this estimate based on sensor..
    • Generate low-passed image from the weighted uniform image, LP[C δsI u] LP[C_{\delta s} I_u] , and high-pass from the difference HP=1LPHP = 1 - LP
    • Resultand image is a weighted sum of highpassed and lowpassed images.
  • Looks about as good as confocal.
  • Cited by...

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ref: -0 tags: locomotion decerebrated monkeys spinal cord section STN stimulation date: 03-01-2012 23:53 gmt revision:0 [head]

PMID-7326562 Locomotor control in macaque monkeys

  • Were not able to induce walking with in monkeys with a sectioned spinal cord
  • Were able to induce walking motion by pulsed stimulation of the STN, with varying walking speed with varying currents!
  • Detailed, informative report, more than I have time to record here today.