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ref: -2019 tags: three photon imaging visual cortex THG chirp NOPA mice GCaMP6 MIT date: 03-01-2019 18:46 gmt revision:2 [1] [0] [head]

PMID-30635577 Functional imaging of visual cortical layers and subplate in awake mice with optimized three photon microscopy

  • Murat Yildirim, Hiroki Sugihara, Peter T.C. So & Mriganka Sur'
  • Used a fs Ti:Saphirre 16W pump into a non-colinear optical parametric amplifier (both from Spectra-Physics) to generate the 1300nm light.
  • Used pulse compensation to get the pulse width at the output of the objective to 40 fS.
    • Three-photon cross section is inverse quadratic in pulse width:
    • NP 3δ(τR) 2(NA 22hcλ) 3 N \sim \frac{P^3 \delta}{(\tau R)^2} (\frac{NA^2}{2hc\lambda})^3
    • P is power, δ\delta is 3p cross-section, τ\tau is pulse width, R repetition rate, NA is the numerical aperture (sixth power of NA!!!), h c and λ\lambda Planks constant, speed of light, and wavelength respectively.
  • Optimized excitation per depth by monitoring damage levels. varied from 0.5nJ to 5 nJ.
  • Imaged up to 1.5mm deep! All the way to the white matter / subplate.
  • Allegedly used a custom scan and tube lens to minimize aberrations in the excitation path (hence improve 3p excitation)
  • Layer 5 neurons are more broadly tuned for orientation than other layers. But the data is not dramatic.
  • Used straightforward metrics for tuning, using a positive and negative bump gaussian fit, then vector averaging to get global orientation selectivity.
  • Interesting that the variance between layers seems higher than between mice.