Limitations of two-photon microscopy

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Presentation transcript:

Limitations of two-photon microscopy Diffraction, aberration and the glow from the deep Petr Znamenskiy

Point spread function in scanning microscopy ?

Point spread function in scanning microscopy Assumption holds for f >> d θ d*sin θ Δx Destructive interference: d f >> d

Axial PSF z Two photon fluorescence is proportional to square of I

Decoupling axial and transverse resolution Annular illumination Bessel beam Path length changes by the same amount when moving along optical axis! rxy is unchanged (determined by NA of annulus) rz depends on thickness of annulus

What really is the diffraction limit?

Spherical aberration Positive No aberration Negative z

Cover glass and spherical aberration

Cover glass and spherical aberration

Aberration from thick sample Wavefront shaping with an SLM: Spatial Light Modulator

Chromatic aberration

Pulsed lasers can’t have line spectra!

Group delay dispersion (GDD)

Prism compressor / prechirper

Scattering and absorption Increase power?

The mysterious glow from the deep

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