Lecture 2 - Methods

Question 1

Methods in cell biology

Answer 1

Light microscopy:

• non fluorescent
• fluorescent: tags, fluorophores, antibodies, specific applications, resolution

Electron microscopy:

-TEM Correlative microscopy

Question 2

Diseases arise from

Answer 2

abnormal cellular function

Question 3

What does the normal protein do?

Answer 3

Location within the cell - location suggests function and specifity

Time of expression - switching on/off suggests function

Partners - other proteins that work together/influence on another, eg. protein A cannot function without protein B, this also suggests function

Regulation - when and how proteins are regulated, e.g. phosphorylation switches proteins on and off suggests function

Question 4

What does the abnormal protein do?

Answer 4

Delocalisation of the protein from normal site of activity

Degradation/timing of expression

Partners - cannot interact with its associated proteins

Dys/unregulation

Question 5

Identification and cure

Answer 5

Can we retarget the protein?

Can we make sure the protein is there?

Can we force interactions?

Can we mimic its regulations?

Question 6

Light microscopy

Answer 6

Light is electromagnetic radiation

Radiation has 3 properties: energy, frequency, wavelength lambda is the distance between tops of waves, defines the length of the wave

Wavelength 380-760nm is detected by human eye and is visible light

Different wavelengths are seen as different colours - white light is a combination

Question 7

Light microscopy

Answer 7

When white light passes through a living cell, the phase of the wave is changed depending on the density of the part of the specimen

These phase changes are undetectable by the human eye under normal (brightfield) microscopy

Phase Contrast and Differential Interference Contrast (DIC) microscopy: very dense regions appear darker than the background. DIC highlights the 3d nature of the specimen

Darkfield microscopy illuminates the sample from the side so only scattered rays hit the lens

Question 8

Data Handling Practice - types of microscopy

Answer 8

A, Brightfield

B, Phase contrast

C, DIC

D, Darkfield

Question 9

Fluorescence

Answer 9

Must be excited to glow

More sensitive than non‐fluo dyes

Very specific

Possibility to co‐stain

Question 10

Principle of fluorescent microscopy

Answer 10

Fluorescent molecules absorb photons of light at one wavelength and emit at a lower energy one – If illuminated at the correct wavelength and viewed through a filter corresponding to the emission, will “glow” against a dark background

Fluorescent molecules can be chemical compounds (dyes, aka fluorophores)

proteins (eg. GFP)

or others (eg. nano‐dots).

Question 11

Green Fluorescent Protein GFP

Answer 11

Can use standard molecular biology to tag your protein with GFP or derivative (either N‐ or C‐ terminal), and express in cells of interest

Question 12

Data Handling Practice

Answer 12

Correct answer is C, S2 is required and sufficient for nuclear localization

Question 13

Bimolecular fluorescence complementation (BiFC)

Answer 13

Used to determine if 2 proteins interact

• Each protein is tagged with half the fluorophore.
• If the two proteins come close and interact, the 2 halves of the fluorophore are reconstituted.
• Each half emits no light, but the reconstituted fluorophore does
• Hence, if the 2 proteins interact, light is emitted.

Question 14

Fluorescence Recovery After Photobleaching (FRAP)

Answer 14

Used to measure protein dynamics in cells

• Photobleaching = fading = fluorescence is permanently lost
• A strong, focussed laser beam irreversibly bleaches the fluorophore it is exposed to
• Recovery of fluorescence has to come from elsewhere in the cell
• Provides a measure of diffusion coefficients (in the cytosol) or the dissociation of a protein from its location

Question 15

Data Handling Practice

Answer 15

Correct answer: 1

Question 16

Immunocytochemistry

Answer 16

• Sometimes a protein cannot be tagged
• Specificity can be achieved using antibodies (indirect immunocytochemistry)
• Can combine multiple primary and secondary antibodies to co‐localise molecules of interest
• Can use live (if inject into cells), but generally used in fixed tissue

Question 17

Resolution of light microscopy

Answer 17

• The limit of resolution is the limiting separation at which 2 objects can still be seen as distinct.
• The resolution (R) depends on the wavelength of light () and the ability of the objective to gather light (NA) of the lens used.
• R is a distance
• Under the best condition, with violet light ( = 400 nm) and NA=1.4, a limit resolution of just under 0.2 m can be achieved by a light microscope. A resolution of less than 0.2 m by light microscopy is simply impossible

Question 18

Data Handling Practice

Answer 18

Correct answer: R= (0.61 x 520)/1.4 = 227nm

Question 19

Spatial vs. temporal resolution

Answer 19

Light microscope

Light (photons)

Limit of resolution 200 nm ‐

Fixed or live sample

Electron microscope

Electrons

Limit of resolution 0.05 nm

Fixed

Question 20

Transmission electron microscopy TEM

Answer 20

Can use antibodies in much the same way as light microscopy to highlight specific proteins

• Instead of fluorophore, gold particles are used (gold is electron dense)

Question 21

EM tomography

Answer 21

The specimen is tilted to varying angles along an axis perpendicular to the electron beam

‐ Each image is projected in 2D

‐ All 2D images are stacked into a 3D reconstitution

Question 22

Correlative Light Electron Microscopy

Answer 22

Combines advantages of both types of microscopy (eg: dynamics + resolution)

• The slide is a grid, essential to navigate and re‐identify the cell of interest.
• Technically very challenging

Question 23

Techniques are not isolated but are complimentary

Answer 23

Answer 24

Summary

• The microscope is a cell biologist’s best friend
• Snap‐shots of cellular and sub‐cellular organisation can be studied by fixing, staining and imaging samples – allowing excellent spatial resolution
• Temporal aspects can be studied using the light microscope and molecule‐specific fluorescent markers
• Advanced techniques in both electron and light microscopy combine expert observation with computational and physical tools to yield more detail than ever before
• Complementary techniques required to answer one question/hypothesis