Lecture 9: 24/10

Question 1

Provide the orders of magnitude for cell polymers and eukaryotic cells

Answer 1

add size, etc.

Question 2

What are the 4 key components of AFM?

Answer 2

• Calibrated cantilever
• Known probe geometry
• Controlled cantilever Z
• Laser and quadrant photodiode

Question 3

Describe the laser and quadrant photodiode?

Answer 3

Allows for very small displacements on the sample surface to be resolved into large linear displacements on the photodiode.

The 4 independent photodetectors allow us to resolve where the laser is spatially located, due to the long laser path.

Question 4

What makes AFM extremely versatile?

Answer 4

Generalized and non-specific in nature

Question 5

What can AFM be used to measure?

Answer 5

• Intramolecular forces (resistive vs attractive forces) in sample
• Topography of sample (stiffness and height of the surface influences this, you can separate stiffness and height measurements but processing the information in different ways)
• Resistance to indentation (i.e., stiffness, young’s modulus)
• Ligand-receptor binding (binding strength, adhesive forces, etc.)

Question 6

What are examples of two AFM modes?

Answer 6

• Contact
• Tapping (small taps with no deformation)

Question 7

What are two commonly used AFM tips?

Answer 7

• Sharp point (useful for topography and very specific, small measurements)
• Sphere (useful for larger measurements, sphere is used for its symmetry and predictability for indentation)

Question 8

What are the two AFM tip modelling approaches based on tip geometry?

Answer 8

Sharp: Sneddon Model
Sphere: Hertz Model

Question 9

Why do we measure the “apparent” Young’s modulus?

Answer 9

Young’s modulus is only valid for constant load, proportional load, and time-invariant materials

Since cells are viscoelastic (time dependence) and exhibit non-linear mechanics, only an “apparent” young’s modulus can be measured

Question 10

What happens to the modulus when probing speed is changed?

Answer 10

Too fast = high apparent modulus
Too slow = lower apparent modulus

Question 11

How do the models used impact the apparent young’s modulus?

Answer 11

Hertz model: accurate for initial contact, under-reports moduli
Brush model: accurate for large compression, over-reports moduli

Bad model leads to bad data (as it can highly impact the young’s modulus)

Question 12

How do the apparent young’s moduli of benign vs malignant cells compare?

Answer 12

Benign (MCF10a): stiffer at high indentations, strain stiffens more
Malignant (MDA): softer at high indentations

Same at low indentations in cytoplasm (similar at low indentations for nuclear and nucleolar)

Question 13

How can AFM be used as a microscope?

Answer 13

Offers a high spatial resolution microscopy picture of the topography, in addition to mechanical features

Question 14

How can adhesive forces be analyzed through AFM?

Answer 14

The release of the AFM tip is in jumps, which can tells us about the distribution of the binding forces and the height of the jump can tell us about the type of bond

It can characterize the individual rupture events

Question 15

Describe the motion of keratocytes?

Answer 15

• Contract orthogonally to the direction of motion
• Actin polymerization pushes leading edge forward, acto-myosin contraction drives cell body forward (can rip apart if myosin is inhibited)

Question 16

How do cellular forces arise from contractility?

Answer 16

The stresses are only at the leading edge (wings) and the contractility axis is orthogonal to the movement

Force can be measured by allowing cell to push against the sensor/tip (through wall, bead, etc.)

Question 17

How else can force be measured other than AFM?

Answer 17

Any force measurement system where we can get a resolvable deformation or displacement and understand the stiffness

• Applying fluid shear with pipette tip
• Bending microneedles visualized with microscopy