Super-Resolution Microscopy

Question 2

What is fluorescence?

Answer 2

The emission of light by a molecule after ut abosrbs higher-energy excitation light and relaxes to a lower energy level

Question 3

What causes the emission wavelength to be longer?

Answer 3

Energy loss during vibrational relaxation before photon emission

Question 4

Define excitation and emission

Answer 4

Excitation - Absorption of ligth energy, promotinf an electron to a higher orbital
Emission - Photon release when teh electorn returns to fround state

Question 5

What role does the confocal pinhole play?

Answer 5

Blocks out-of-focus light, improving resolution and contrast

Question 6

What is the trade-off in conocal microscopy?

Answer 6

Improved resolution but slower scanning speed

Question 7

What are the key unit conversions?

Answer 7

1 µm = 10⁻⁶ m
1 nm = 10⁻⁹ m
1 Å = 10⁻¹⁰ m

Question 8

What limits optical microscope resolution?

Answer 8

Diffraction - light spreads as a wave, blurring nearby points

Question 9

What is the Rayleigh criterion formula?

Answer 9

d = 0.61 / λ NA

Question 10

If λ = 532 nm and NA = 1.5, what is d?

Answer 10

220 nm

Question 11

What is the tyrpical resolution limit of light microscopy?

Answer 11

200nm laterally (xy) and 500 nm axially (z)

Question 12

What is the goeal of super-resolution microscopy?

Answer 12

To image structures smaller than the diffraction limit (<200nm)

Question 13

Which super-resolution method achieves the highest precision?

Answer 13

SMLM (Single Molecule Localisation Microscopy)

Question 14

What is the key idea behind SMLM?

Answer 14

Only a few fluorophores emit at once -> each localised precisely -> build a composite image

Question 15

How is localisation precision improved beyond diffracton?

Answer 15

By fitting each molecule’s emission to a Guassian function to find its centroid position

Question 16

The typical localisation accuracy of SMLM?

Answer 16

10 - 20 nm

Question 17

What type of fluorophores are used in STORM?

Answer 17

Photoswithcable dyes that blink between on/off states

Question 18

What enables PALM to localise proteins?

Answer 18

Photoactivation of genetically encoded fluorescent proteins (e.g., Dronpa, PA-GFP)

Question 19

List the main steps in SMLM imaging

Answer 19

1 - Stochastically switch on a subset of fluorophores
2 - Record emission and localise each to nanometre precision
3 - Switch those off, activate a new subset
4 - Repeat thousands of times -> reconstruct high-resolution image

Question 20

Why can’t all flurohores be active at once?

Answer 20

Their PSFs would overlap, making localisation impossible

Question 21

What limits SMLM?

Answer 21

• Requires high signal-to-noise
• Long acquisition and computational reconstruction
• Best suited to thin or surface samples

Question 22

Why combine TIRF with SMLM?

Answer 22

TIRF limits excitation depth -> less background -> sharper localisation for surface molecules

Question 23

What is the principle of TIRF microscopy?

Answer 23

When light hits an interface beyond the critical angle, it totally reflects and creats an evanescent field that escites nearby fluorophores

Question 24

Equation for the critical angle?

Answer 24

sin θ_c = n_2 / n_1
For glass (1.52) to water (1.33): θc = 61

Question 25

What is the depth if the evanescent field?

Answer 25

Approximately 100 -200 nm

Question 26

What does the evanescent field do?

Answer 26

Excites only fluorophores close to the glass surface, minimzing background fluorescence

Question 27

How does fluorescence intensity change with distance from the surface?

Answer 27

I(z) = I_0 e^-z/d

Question 28

What are key benefits of using TIRF?

Answer 28

- High signal-to-noise ratio - Reduced photobleaching - Ideal for studying membranes or surface proteins - Compatible with multi-colour and live-cell imaging

Question 29

What happens when TIRF is combined with SMLM?

Answer 29

Produces super-resolved images of membrane-associated proteins

Question 30

What is Light Sheet + SMLM used for?

Answer 30

3D super-resolution imaging with reduced photodamage

Question 31

Which technique achieves the best lateral resolution?

Answer 31

SMLM (10 - 20 nm)

Question 32

Which technique gives the best acial sectioning near surfaces?

Answer 32

TIRF microscopy