This document discusses the applications of atomic force microscopy (AFM) in various fields. It begins with a brief history of AFM and description of its basic components and working principle. Then it discusses some key abilities and applications of AFM like high-resolution imaging of biomolecules, studying drug-DNA interactions, imaging ion channels, nano-scale drug delivery, observing dynamic cellular responses to drugs. AFM allows direct visualization and analysis of samples without labeling or coating and can be used under physiological conditions to study structure-function relationships at a nanoscale level.

1. Master’s Seminar On
Presented By
Chandan Patil
I MVSc
V-1661/16
Department Of Veterinary Pharmacology & Toxicology
DUVASU ,Mathura

2. Types of microscopy

3. History
The precursor to the AFM, the Scanning
Tunneling Microscope (STM), was
developed by Gerd Benning and Heinrich
Rohrer in the early 1980s at IBM Research
–Zurich, a development that earned them
the Nobel Prize for Physics in 1986.
Binnig invented the Atomic-Force
Microscope and the first experimental
implementation was made by
Binnig,Quate and Gerber in 1986.

4. Salient Features
Very-high-resolution type of Scanning Probe Microscopy (SPM), with demonstrated resolution on the
order of fractions of a nanometer.
The information is gathered by "feeling" or "touching" the surface with a mechanical probe.
AFM permits the three dimensional(3D) imaging of membranes and biomolecules with molecular and
sub-molecular resolution.
AFM sample preparation is fairly simple and quick.
AFM does not require any stains, contrast agents, or conductive coatings that can cause partial
obstruction of the actual sample
Ability to image a variety of materials (i.e., nonconductive, magnetic, biological) under a variety of
environmental conditions (i.e., ambient air, various gases, different humidity levels and temperatures)
AFM is a nondestructive method that allows the sample to be reused for additional analyses over
time

5. Salient Features continued…
The precise nanometer control of the position and the picoNewton
control of the force of the cantilever allows the physical manipulation
of biomolecules, the dissection of biological structures the delivery of
ligands, drugs or other materials to specific locations,and the precise
measurement of interacting forces at specific sites;
The AFM apparatus can be modified by adding complementary
methodologies such as the measurement of ionic conductance, total
internal reflection fluorescence (TIRF), fluorescence resonance energy
transfer (TIRF) and fluorescence imaging, microfluidics,
physicochemical measurements that allow the measurement of
structure and function of biological tissues among other modalities.

6. Abilities of AFM
Force
measurment
Imaging
Manipulation

7. Basic components of Atomic Force
Microscopy
• Piezoelectric scanner
• Flexible Cantilever
with sharp probe
• Laser source
• Photodiode detector
• Feedback electronics

8. Principle Of AFM
• The AFM consists of a cantilever with a sharp tip
(probe) at its end that is used to scan the specimen
surface. When the tip is brought into proximity of a
sample surface, forces between the tip and the
sample lead to a deflection of the cantilever. This
cantilever deflection is monitored by bouncing the
laser beam on the back on the cantilever to a set of
photodetectors which convert light signals into
electrical signals.

10. APPLICATIONS

11. Imaging of DNA
• Watson & Crick model was based on the X-RAY
Crystallography & Electron Microscopy
• In AFM there is no necessity to crystallize the
DNA prior to imaging, and the possibility to
study protein– DNA interactions and
processes as they occur in normal cell

12. Drug DNA Complex
• This is of considerable interest since nucleic
acid ligands are commonly used as anticancer
drugs and in the treatment of genetic diseases
• AFM was used to study drug binding mode,
affinity, and exclusion number by comparing
the length of DNA fragments that have and
have not been exposed to the drug.

13. • When exposed to ethidium, a well characterized
inter-calator, the DNA strand was shown through
AFM to have increased in length from 3300 nm to
5250 nm (see fig. 3).

14. Imaging ion channels and defining
channelopathies
Amyloid ion channels and paradigm shift in amyloidogenic diseases
• In a series of AFM studies, many amyloids, including amyloid beta protein,
amylin, alpha-synuclein,ADan, ABri, and Serum Amyloid A, form ion channel-like
structures that show single ion channel activity when reconstituted in membranes
has been depicted. Lin et al.2001;Quist et al.2005

15. • Channelopathies are a category of disease that
traditionally have been thought of as being caused by
altered function of channels or of the proteins that
regulate channels, but amyloid deposition has actually
created new channels that are likely pathologic.
• For instance, amyloid beta protein (1-42)induced changes
in endothelial cells were tracked
Lin et al. 2001;Bhatia et al. 2000
• Furthermore, since the AFM operates under biological
liquids,reagents can be added and the physiologic
solutions changed during imaging. In this way, the
therapeutic (preventive) capability of an agent can be
evaluated directly and with nanometer resolution.

16. Amyloid beta channel induced cell death. Left half: Loss of synaptic arborization and neuronal death
imaged with Calcein dye and fluorescence before and after addition of AβP to the buffer solution
(compare panels A, C, E, and G (control)) with panels B, D, F, and G (after addition of nano-to-micromolar
abeta peptide). Right half: Simultaneously,AFM can be used to image the cytoskeletal structure of cells to
track effects of AβP on the cells, and to test various blocking agents for their performance in blocking
toxicity effects. For details, see Lin et al. (2001) and Bhatia et al. (2000).

17. Nano Scale Drug Delivery System
• Nano medical Research is focused on design,
Characterisation and delivery of Nano sized
drug carriers:nanoparticles,liposomes,etc,.in
the treatment of cancer.
• AFM-Directly observe very minute particles
without any cumbersome and potentially
contaminating sample preparation.

18. • By linking drug molecules to the tip, AFM can
probe the target molecules on the cell surface.
• This technique is called single-molecule force
spectroscopy (SMFS).
• By performing approach-retract cycles on the
cell surface using drug-tethered tips, force
curves can be recorded.
• The magnitude of the peak is equal to the
binding force between a target molecule and a
drug molecule.

19. The retract curve

20. Dynamic changes of cellular ultra-microstructure and
elasticity in response to drugs
• Antimicrobial peptides are a promising class of
antimicrobials that have demonstrated activity
against antibiotic-resistant bacteria, parasites,
viruses and fungi.
• AFM used to image the dynamic nanoscale
changes on single living bacterial cells in response
to CM15 (an antimicrobial peptide drug),
revealing that the cell surface changed from
smooth to corrugated after the stimulation of
CM15. - Fantner et al(2014)

21. • AFM peak force tapping mode image revealed the
effect of glyphosate and quercetin on single living
keratinocyte cells showing that cells develop a
filamentous cytoskeleton network after the
stimulation of glyphosate, whereas the filamentous
structures disappeared after the subsequent
addition of quercetin.
-Heu et al(2012)

22. • Daunorubicin (Dau) is a drug used to treat several
types of cancers, such as leukemia and
neuroblastomas.
• Dynamic changes of DNA molecules in response
to Dau as it intercalates in DNA to cause a local
unwinding of the DNA was seen by using
helix time-lapse AFM.
- Alonso-Sarduy et al(2013)

23. complement-mediated cytotoxicity (CMC)
AFM images of lymphoma cells after the
treatment of Rituximab

24. References
• Multidimensional atomic force microscopy for drug discovery:
A versatile tool for defining targets, designing therapeutics and
monitoring their efficacy by Ratnesh Lal , Morton F. Arnsdorf.
• Atomic force microscopy: A multifaceted tool to study
membrane proteins and their interactions with ligands by
Allison M. Whited, Paul S.-H. Park.
• AFM in Advanced Pharmaceutical Technology Nicolaos
Scoutaris* and Dennis Douroumis* Pharmaceutica Analytica
Acta.
• Mini Review on Feeling the forces: atomic force microscopy
in cell biology by Jose´ Luis Alonso1, Wolfgang H.
Goldmann*.
• Atomic force microscopy of macromolecular interactions by
Christopher M Yip.
• Nanoparticle Delivery of Cancer Drugs by Andrew Z. Wang,
Robert Langer, and Omid C. Farokhzad