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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