Lecture Nine: Optics 2

Question 1

What other practical factors affect resolution and their solutions?

Answer 1

1) Numerical aperture of lens >NA
2) Resolution and Contrast are linked! >contrast
2) Digitalisation: what size pixel? resolution/2.33) Refractive index of mountant match to oil
4) Depth of focus – keep close to cover slip
5) Coverslip thickness – 170 μm or #1.5
6) Noise e.g. photons , instrumentation - >photons
7) Quality of labelling – use controls e.g. blank

Question 2

What happens when we under sample?

Answer 2

We get aliasing

Question 3

What is nyquist theorem in imaging?

Answer 3

Nyquist sampling of an image of two points separated by the Rayleigh resolution

Nyquist rate is 2.3 for images unlike voltage

Question 4

What is a good approximation of an airy disk?

Answer 4

A gussian function

Question 5

What can cause spherical abberation?

Answer 5

Mismatch between emersion medium and mounting medium

Question 6

What is the optimal coverslip thickness?

Answer 6

0.17-0.18mm this has the smallest FWHM for a constant light compared to all others. (look at graph of this)

Question 7

Should everything be sampled at the diffraction limit?

Answer 7

Depends on the question

Question 8

How is photon noise modulated?

Answer 8

Poisson distribution where mean equals the variance Reduce noise by increasing number of photons e.g. improve labelling, laser intensity, averaging

Question 9

What are the types of filter averaging to remove noise

Answer 9

Mean
Median
Gussian

Question 10

How can the image be restored from PSF?

Answer 10

decovolution