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

2. Point spread function in scanning microscopy?

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

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

5. 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

6. What really is the diffraction limit?

7. Spherical aberration
Positive No aberration Negative z

8. Cover glass and spherical aberration

9. Cover glass and spherical aberration

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

12. Chromatic aberration

13. Pulsed lasers can’t have line spectra!

14. Group delay dispersion (GDD)

15. Prism compressor / prechirper

16. Scattering and absorption
Increase power?

17. The mysterious glow from the deep