Biomolecular Studies using Scanning Probe Microscopy Biomolecular Studies using Scanning Probe Microscopy

By

Krishnashish bose

Current supervisor: Assoc Prof. Dr. Anh Tuan Phan

Previous supervisors:

Prof. Dr. Bodh Raj Mehta & Assoc Prof. Dr. Bishwajit Kundu

Contents

I shall be briefly discussing the

research that I have done during

my M.Sc in Physics at IIT Delhi.

I shall start with a brief

introduction to AFM and its

potential applications.

This presentation consists of 24

slides (mostly images) and would

be for about 20 minutes.

SPM: Introduction

Images taken by me

PeakForce QNM

Nanoindentation

High Resolution Imaging

Dynamic Imaging

Limitations

TO SEE IS TO BELIEVE

To see the processes at the molecular level happening inside the living cell is the one of the greatest challenges.

It is easier to look at a star thousands of light years away, than to look at a molecule inside our own cells.

SPM: Introduction

SPM stands for Scanning Probe Microscope. It is a broad class of

microscopes that use a very sharp tip to interact locally with the

sample.

AFM is the successor of the Scanning Tunneling Microscope (STM),

developed in the 1980s by Binning et al.

STM exploits the principle of tunnelling of electrons across a barrier

with a difference in density of states of electrons. It is the highest

resolution non-destructive microscopy technique.

AFM measures directly the tip-sample interaction forces , hence the

name Force Microscopy.

Basic design of an AFM.

(a) Design of a small sample AFM

(b) Design of a large sample AFM

The small sample AFM usually has

better signal to noise ratio for

scan sizes below 500 nm.

Picture taken from Pg 10 of Bhart Bhusan’s book : Nanotribology and Nanomechanics, 2nd edition, Springer.

Why Scanning Probe Techniques?

The only non-destructive approach to reach atomic resolution. Electron

Microscopes of similar resolution burn (radiation damage) the sample.

The only technique offering true height resolution of less than 1 nm.

Not only can you see single molecules, but also play with it to extract so

much information that we are yet to explore.

Scanning Probe Microscopy is a vast field and is still growing rapidly. It can

offer the best solutions in the world of quantum mechanics. Even a quantum

computer (which according to me is the greatest invention of the century)

would use a Magnetic Resonance Force Microscope to read qubits.

Techniques like NSOM capture photons at very low cross-sections, thus

offering sub-wavelength optical microscopy and Raman spectroscopy.

Images of Beta Amyloid Nanofibers

Images taken by me

Images of α-Synuclein and Gelsolin amyloid Nanofiber

Images taken by me

Images taken by me

Imaged in tapping mode using FESP

probe having cantilever stiffness of

1.6 N/m and a tip-end diameter of

10 nm (measured using SEM).

Notice that the Prion fibrils have

just been resolved at a separation of

10 nm (see Section Analysis curve).

In the section analysis, it shows that

the protofibrils are both of width 4

nm, but the height analysis gives 3.5

nm and 2.5 nm. This is because for

lateral widths less than the tip

diameter, the Nanoscope software

cannot give the true value.

Mica

Sapphire

Images taken by me

DMT modulus image obtained (top right) of the Lysozyme oligomers (top left).Section Analysis (below) gives a DMT modulus of 3.3 GPa

PeakForce QNM mode

a)

Images taken from Bruker manual

Reduced Stiffness In general, D = Zp + Zc + δ , where Zc and δ are the deformations of

cantilever and sample respectively, D is thedistance between the tip and the sample and Zp is the distance moved by the piezo in the z-

direction. In the contact regime, D = 0 => Zp = -(Zc + δ) .

Comparing with two springs of stiffness kc (cantilever) and ks (sample) connected in series in equilibrium, the effective stiffness (keff) is given

by

sceff kkk

111

effc

effc

kk

kk

sk=>

• If the sample is much stiffer than the cantilever, then keff ≈ kc which shows that the force curve is primarily due to the stiffness of the cantilever, and not that of the sample. • There is a restraint even on the young's modulus of the tip.

(1)

Stiffness and Young's Modulus

s

s

t

t

tot EEE

22 11

4

31

The stiffness of the sample is related to its Young's modulus by ks = 1.5 aEtot ;

where a is the tip-sample contact radius, Etot is the reduced Young's modulus for

perfectly elastic solids, given by

s

s

tot EE

21

4

31 If the tip deformation is neglected, then

21

2s

sEa

effc

effcss kk

kk

aE

2

1 2

Therefore, ks =

which gives,

(2)

(3)

(4)

(5)

H.J.Butt, B.Cappella, M.Kappl; “Force measurements with the atomic force microscope: Technique, interpretation and applications”. Elsevier Surface Science Reports 59 (2005).

The Hertz Model

totE

RFa 3

The first paper on Nanoindentation was published in 1882 by the great

physicist Prof. Heinrich Rudolf Hertz.

Hertz theory can only be applied when the adhesion force is much smaller

than the maximum load.

In this model, following relations have been established:

2/3REF tot

a = Tip-Sample contact radiusR = Effective tip radius of curvature =

F = Force exerted by tip on sampleEtot = Reduced Young's modulusσt = Poisson's ratio of tipδ = Indentation = d (in figure)

1

11

sampletip RR

(6) (7)

(8)

The DMT model

RWFE

Ra

tot

23

This model was forwarded in 1974 by Derjaguin, Muller and Toporov.

This model considers adhesion just outside the area of contact of the

tip and sample.

In this model, following relations have been established:

RWREF tot 22/3

a = Tip-Sample contact radiusR = Effective tip radius of curvatureδ = Indentation

F = Force exerted by tip on sampleEtot = Reduced Young's modulusW = Work of adhesion per unit area

(9) (10)

My results and conclusion

The DMT modulus of Prion & Gelsolin amyloids were found for the

first time.

The DMT modulus of Lysozyme fibrils agreed well with the reported

value.

The DMT modulus of amyloids in oligomeric stage and pre-fibrillary

stage were found for the first time.

Amyloids responsible for more probable diseases show high elastic

modulus.

As the amyloids keep maturing, their elastic modulus decreases.

Significance of Elastic Modulus of Amyloid Nanofibers

The elastic modulus is a fundamental property of matter that owes its

origin to a molecule's restraint to deformation.

It gives clue about the molecular packing density and structural rigidity.

Using this, it is possible to characterize different materials based only on

their elastic modulus.

A 'normal' protein and an amyloid forming protein could be distinguished

just by their elastic modulus values. This can help in early detection of

diseases marked by amyloid formation.

It is also possible to monitor the effect of external agents on the

mechanical strength of amyloids, which would help in finding better cure

for diseases caused by amyloids.

STM images of DNA

Source: Biochemical and Biophysical Research Communications 303 (2003) 154

Source: J. Vac. Sci. Technol. B 9, 1306 (1991)

Microtubule imaged with AFM in liquid. Single protofilaments are visible (Schaap et al. 2004). Microtubule decorated with kinesin motors in presence of AMP-PNP. The motors are visible as blobs on the microtubule. (Schaap et al. 2007).

Single actin filaments scanned in liquid with monomer (4 nm) resolution. (Schmitz et al. 2010).

An actin filament simultaneously imaged with total internal reflection fluorescence microscopy and AFM. (Schaap 2006).

Dynamic Images captured by AFM

Single kinesin motor moving along the microtubule at 1 µM ATP. Both heads of the motor are clearly visible (Schaap et al. 2011)

Two single kinesin motors moving along a single protofilament (Schaap et al. 2011).

Dynamics of DNA loosely bound to a mica surface (Sebastian Hanke 2011).

Hurdles for reaching ultimate resolutionThe height & lateral resolution of the best AFMs in the world is 0.1 nm & 0.5 nm, but on non-deformable samples.

The tip-sample contact area limits the lateral resolution.

Low stiffness cantilevers are prone to noise. It can be taken care of by increasing AFM stability.

Fabrication of sharp tips is a great technological challenge. The sharpest tip available has an end-radius of 1 nm.

Tip shape is also a big issue, depending on your sample topography.

AFM stability

Vibration Isolation

Acoustic Isolation

Small dedicated room

Constant temperature