Effects of coverslip on PSF
We use water-dip type objective for our two-photon microscopy. However, if there is a coverslip between the objective and the sample, the PSF(Point spread function) is degraded significantly.
This is because the these objectives are not designed for this situation.
The refractive index of water is 1.33, and coverslip is 1.52. Thus if there is a coverslip between a sample and an objective, an incident light is refracted by the coverslip and make a focus at a different point than the focal point without the coverslip. The shift (D) can be calculated as:
D(θ) = a(tanθ - tanθ')/tanθ'
Where N/N' = sinθ / sinθ' (N: refractive index of water, N': of coverslip), and a is coverslip thickness (typically 170 μm).
If optics are at the near-axis, or sinθ ~ tanθ, D does not depend on the incident angle θ. However, for a high NA objective, this is not the case. For example, for NA=0.9 water-dip objective, maximum θ is Asin 0.9/1.33~42o. In this case the shift D is 42 μm compared to 24 μm at the near axis, the focus position differs by δD~18 μm in z-axis.For NA=0.5, δD~3 μm. Thus, a coverslip degrades PSF significantly.
Therefore, in my opinion, for an in vivo application, one should underfill the backfocal plane with the laser to save laser power.
This is because the these objectives are not designed for this situation.
The refractive index of water is 1.33, and coverslip is 1.52. Thus if there is a coverslip between a sample and an objective, an incident light is refracted by the coverslip and make a focus at a different point than the focal point without the coverslip. The shift (D) can be calculated as:
D(θ) = a(tanθ - tanθ')/tanθ'
Where N/N' = sinθ / sinθ' (N: refractive index of water, N': of coverslip), and a is coverslip thickness (typically 170 μm).
If optics are at the near-axis, or sinθ ~ tanθ, D does not depend on the incident angle θ. However, for a high NA objective, this is not the case. For example, for NA=0.9 water-dip objective, maximum θ is Asin 0.9/1.33~42o. In this case the shift D is 42 μm compared to 24 μm at the near axis, the focus position differs by δD~18 μm in z-axis.For NA=0.5, δD~3 μm. Thus, a coverslip degrades PSF significantly.
Therefore, in my opinion, for an in vivo application, one should underfill the backfocal plane with the laser to save laser power.
posted by Ryohei at 1:16 PM
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