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Tools & Techniques

AFM Principles and


Application in Biosciences
Mr. R. Rajasekaran

Abstract considerable progress has been made, but The Principles of Atomic
applications in the field of biology are
In recent year, Atomic Force exploring biological structures under Force Microscopy
Microscope (AFM) has provided a conditions in which living organisms exist.
Atomic force microscopy (AFM) is a member
range of now opportunities for viewing, AFM is a kind of scanning probe microscope
of a family of new microscopic techniques
Manipulating and analyzing where imaging of the sample is realized by
that are referred to as scanning-probe
biomolecules in the environments. And interaction of the probe with the sample
microscopes (SPMs). The concept on which
will hopefully allow application to be surface and no imaging beam (light or
all SPMs are based is the generation of images
developed for AFM in Medicine and electron) is involved in the process. The tip of
of surfaces by measuring the physical
biotechnology. the probe is mounted on the end of a flexible
interaction between a sharp tip and the sample
cantilever (1).
Keywords:Atomic Force Microscope rather than by using an incident beam (light or
(AFM), Biomolecules, Interaction tip, Sample preparation and recording conditions, electrons) as in classical microscopy. The
Piconewton (10-12N). has revolutionized the way in which main parts of an atomic force microscope are
microscopists explore biological structures. the sample stage, the CANTILEVER ( Box 2)
Introduction This surface imaging technique involves and the optical detection system, which
scanning a sharp tip over the surface of a comprises a laser diode and a hotodetector.
The invention of ATOMIC FORCE sample, while sensing the interaction force The sample is moved relative to the cantilever
MICROSCOPY (AFM) in the mid-1980s6, between the tip and the sample of biological in three dimensions using PIEZOELECTRIC
followed by continuous progress in specimens at sub-nanometre resolution under CERAMICS (Materials that expand or
instrumentation, (AFM). A relatively new physiological conditions. contract when subjected to a potential
form of microscopy in which a sharp tip is difference.) The force interacting between the
scanned over the surface of a sample, while In fact, an atomic force microscope is more
tip and the sample is monitored with
sensing the interaction force between the tip than just a microscope as it can also measure
piconewton (10-12N) sensitivity, by attaching
and the sample. Because AFM does not rely minute forces within or between biological
the tip to a soft cantilever, which acts as a
on an incident beam, as in electron or light molecules a method known as Force
spring, and measuring the bending (or
microscopy, the specimen can be directly spectroscopy (Box 1) in a way that was not
deflection) of the cantilever (Fig-1). The
observed at high Resolution in aqueous previously possible the last decade of using
cantilever deflection is usually detected by a
solution. AFM and related Scanning Probe Microscopy
laser beam focused on the free end of the
techniques in biology showed that their
cantilever and is reflected into a photodiode
The newly developed atomic force popularity and power continue growing.
(2). AFM cantilevers and tips are usually
microscope is a valuable tool for studying Numerous reviews both comprehensive and
made of silicon or silicon nitride using
physical and biological structures provides a specialized cited in the reference list and
MICROFABRICATION techniques ( Box .3)
unique window to the microworld of cells, articles devoted to biological applications of
subcellular structures, and biomolecules. The Atomic Force Microscopy prove this fact (2). Box - 2
AFM can image the three-dimensional
structure of biological specimens in a Box - 1
Cantilver
physiological environment.
Force spectroscopy AFM tips are mounted on cantilever beams or
This enables real-time biochemical and
triangles, which are typically made of silicon
physiological processes to be monitored at a A form of AFM in which the force acting on
or silicon nitride, that behave like springs.
resolution similar to that obtained for the the tip is measured with piconewton
Using Hooke's law, the magnitude of the
electron microscope. Atomic force (10-12 N) sensitivity as the tip is pushed
tipsample force is proportional to the
microscopy since the initial reports of towards the sample. Then retracted from it.
deflection of the cantilever.

38 | Advanced Biotech | June 2008


Tools & Techniques

the constituents of the sample to each other or


to the substrate. Cooling can also stiffen the
sample. Nevertheless, all these methods have
significant influence on the properties of
biomolecules. The sharper tip now is
available commercially and really does help a
lot.

To observe biological structures in their


native state by atomic force microscopy
(AFM), samples must be firmly attached to a
solid support to resist the lateral forces
exerted by the scanning tip. Several
immobilization strategies have been
Fig: 1. Atomic force microscopy. established for microbial specimens. For
isolated membrane proteins, the most
Box - 3 In tapping mode atomic force microscopy frequently used procedure is based on
(TMAFM), an oscillating tip is scanned over physical adsorption on a flat support, such as
mica, glass or silicon oxide, in the presence of
Microfabrication the surface and the amplitude and phase of the
cantilever are monitored near its resonance the appropriate electrolytes. For whole cells,
A range of techniques that are derived from frequency. As the tip touches the sample however, immobilization by means of simple
the techniques used in Microelectronics to surface only at the very end of its downward adsorption procedures is generally
make integrated circuits and which are used to movement, lateral forces during imaging are inappropriate because the contact area
make AFM tips and cantilevers. greatly reduced, which is advantageous for between the cells and the support is very
imaging 'soft' biological samples. small, often leading to the cell being detached
The different operating by the scanning tip. To solve this problem, air-
This is recorded as the tip is pushed towards drying and chemical fixation can be used to
modes of Atomic Force the sample and retracted from it. Using promote cell attachment, but these treatments
Microscopy appropriate corrections, a force versus can cause significant denaturation of the
separation distance curve is obtained. Such a s p e c i m e n . A l t e r n a t i v e l y, g e n t l e r
Atomic force microscopes can be operated in curve can be exploited to gain insights into a immobilization procedures have been
various modes in the constant-force imaging variety of surface properties and molecular developed in which the cells are mechanically
mode, images are created by bringing the tip interactions and to manipulate single trapped either in an agar gel or in a porous
and sample into contact and scanning the tip molecules. Importantly, force distance curves membrane (4), In the latter method, a
across the surface while the sample height is can be recorded at multiple locations of the concentrated cell suspension is gently sucked
adjusted using a feedback loop to keep the (x, y) plane to yield spatially resolved maps of through an isopore polycarbonate membrane
bending or DEFLECTION (The vertical properties and interactions. with a pore size that is slightly smaller than
bending of the AFM cantilever resulting from that of the cell. This simple approach can be
the tipsample interaction force) of the atomic Advanced methodologies used to image single, spherical, bacterial,
force microscope cantilever constant. This yeast and fungal cells under aqueous
yields a topographic image that gives
for sample preparation
conditions, while minimizing denaturation of
calibrated height information about the Little sample preparation is required for the surface molecules.
sample. bioimaging with the AFM. In most cases it is
In many cases, small cantilever deflections as simple as spotting a few microliters of Biological Applications of
occur because the feedback loop is not solution on mica or glass. Of course
contaminations that cover surface features
AFM
perfect, and the resulting error signal can be
used to generate a deflection image, In have to be avoided or removed. First the One of the advantages of AFM is that it can
addition to being used as a microscope, an substrate-adsorbate should be rinsed with a image the non-conducting surfaces. So it was
atomic force microscope can measure large excess of buffer. The following immediately extended to the biological
biomolecular forces with piconewton procedures are dialysis, centrifugation and systems, such as analyzing the crystals of
(10-12N) sensitivity (3). In this mode known homogenization. In order to get good contrast amino acids and organic monolayers.
as force spectroscopy the cantilever and to reduce mechanical damage of the soft Applications of AFM in the biosciences
deflection which is useful for revealing biological materials, the samples can be include DNA and RNA analysis; Protein-
surface details of corrugated samples. stabilized by adding covalent cross-linking nucleic acid complexes; Chromosomes;
agents or certain cations that are able to link

39 | Advanced Biotech | June 2008


Tools & Techniques

Cellular membranes; Proteins and peptides; living and fixed cells such as red and white
molecular crystals; Polymers and
Application in Microbiology
blood cells, bacteria, platelets, cardiac
biomaterials; Ligand-receptor binding. Bio- myocytes, living renal epithelial cells, and The AFM has been used to viewing and
samples have been investigated on lysine- glial cells. For example, plasma membrane in analyzing the ultra structure of microbial cell
coated glass and mica substrate, and in buffer migrating epithelial cells has been imaged in surface studies and it is used to investigated
solution. By using phase imaging technique real time. The dynamic membrane the property of structure include to analyzing
one can distinguish the different components invagination process was observed in the structure of native membrane proteins at sub-
of the cell membranes. presence of calcium and when calcium levels nanometre resolution, Function-related
were reduced the process was prevented. conformational changes in single proteins,
There has been recent success imaging
30nm lipidic pore formation could also be Surface ultra structure of living cells, Cell-
individual proteins and other small molecules
resolved during calcium reduction. AFM surface dynamics, and Morphology of
with the AFM such as collogen. Employing
imaging of cells usually achieves a resolution biofilms. The physical properties and
selective affinity binding procedures has
of only 20-50 nm, not sufficient for resolving biomolecular interactions such as Stiffness of
successfully imaged smaller molecules that
membrane proteins but still suitable for cell walls, Local surface charge and
do not have a high affinity for common AFM
imaging other surface features, such as h y d r o p h o b i c i t y, E l a s t i c i t y a n d
substrates. Thiol incorporation at both the 5'
rearrangements of plasma membrane or conformational properties of single
and 3' ends of short PCR products has been
movement of submembrane filament molecules, Mechanical stability of
shown to confer a high affinity for ultra flat
bundles. The requirement for the imaging supramolecular assemblies, Unfolding
gold substrates. A similar approach was used
buffer is not restrictive, as long as the buffer pathways of membrane proteins, Molecular
to immobilize antibodies (IgG1) on treated
does not severely affect the integrity of the forces determining cell adhesion and cell
mica. In this case, the low affinity that IgG
cells. EDTA should be avoided because cells aggregation also analyzed (2, 7 & Fig-3).
molecules have for mica was overcome by
will detach from the substrate in the absence
cloning a metal-chelating peptide into the
carboxyl terminus sequence of the IgG's
of divalent cations. Cultured cells normally
adhere well to the substrate and are not
A
heavy chain. The recombinant sequence was
displaced by modest probe forces (Fig-2).
transformed into cells that expressed the
complementary light chain. The purified IgG
containing the metal-chelating peptide was
shown to bind in a regiospecific manner to
nickel-treated mica. Covalent binding of
biological structures to derivatized glass
substrates has also enabled high resolution B
imaging of some samples that are not stable
on untreated glass substrates. New
approaches in AFM have provided a solid
foundation from which research is expanding
into more complex analyses. Higher
resolution imaging of a variety of small
molecules is improving at a rapid pace.

The recent innovation, such as Digital Fig-3


Instruments BioScope system, which a. Three-dimensional AFM image of
Saccharomyces cerevisiae
combines the high resolution of AFM with the Yeast cell immobilized in a porous membrane.
Fig: 2. Protein Surface layers.
ease of use and familiarity of inverted optical b. High-resolution deflection image of the surface
microscopes, has further added to the The AFM has been used to image living cells of Phanerochaete chrysosporium Fungal
Spores.
attractiveness of AFM for biological imaging. and the underlying cytoskeleton, chromatin
Bright-field, flourescence and other optical and plasmids, ion channels, and a variety of
Application in Nucleic acid
techniques can be used to identify structures membranes. Dynamic processes such as
of interest while the AFM simultaneously crystal growth and the polymerization of research
generates nanometer-resolved images of the fibrinogen and physicochemical properties
such as elasticity and viscosity in living cells One area of significant progress is the
sample surface. (3, 5, & 9)
have been studied. Nanomanipulations, imaging of nucleic acids. The ability to
generate nanometer-resolved images of
Application in Cell Biology including dissection of DNA, plasma
unmodified nucleic acids has broad biological
membranes, and cells, and transfer of
Cell biologists have applied the AFM's unique synthetic structures have been achieved applications. Chromosome mapping,
capabilities to study the dynamic behavior of (6 & 9). transcription, translation and small molecule-
DNA interactions such as intercalating

40 | Advanced Biotech | June 2008


Tools & Techniques

mutagens, provide exciting topics for high- measurements is to image or quantify 4. Kasas, S. & Ikai, A, 1995. A method for anchoring
resolution studies. The first highly electrical surface charge. The dynamics of round shaped cells for atomic force microscope
reproducible AFM images of DNA were imaging, Biophysics Journal, Vol. 68, pp.1678-
many biological systems depends on the
1680.
obtained only in 1991. Four major advances electrical properties of the sample surface. In
that have enabled clear resolution of nucleic 5. Shao, Z., Mou, J., Czajkowsky, D. M., Yang, J. &
addition to measuring binding forces and
Yuan, J.Y, 1996. Biological atomic force
acids are: Control of the local imaging electrostatic forces, the AFM can also probe microscopy: what is achieved and what is needed,
environment including sample modification; the micromechanical properties of biological Adv. Phys, Vol. 45, pp.186.
Tapping Mode scanning techniques; samples. Specifically, the AFM can observe 6. Jena, B. P. & Hörber, J. K. H, 2002. Methods in Cell
Improved AFM probes (such as standard the elasticity and, in fact, the viscosities of Biology, Vol. 68, 3350 (Academic Press, San
silicon nitride probes modified by electron samples ranging from live cells and Diego).
beam deposition and Oxide Sharpened membranes to bone and cartilage (10). AFM 7. Pum, D. & Sleytr, U. B, 1995. Monomolecular
NanoProbes) and Compatible substrates gives scientists a key tool to investigate the reassembly of a crystalline bacterial cell surface
layer (S-layer) on untreated and modified silicon
(such as salinized mica and carbon coated real world.
surfaces, Supramol. Sci, Vol. 2, pp.193197.
mica) (8).
8. Clausen-Schaumann, H., Seitz, M., Krautbauer, R.
Conclusion & Gaub, H. E, 2000. Force spectroscopy with
Application in Biomolecular single bio-molecules, Curr. Opin. Chem. Biol, Vol.
Now applications of AFM probing is far 4, pp.524530.
Interaction Studies beyond of these approved ones. It is found to 9. W ww.chembio.uoguelph.ca/educmat/chm729/

Along with direct imaging of biological be useful in pharmacology, biotechnology, afm/applicat.htm Hong-Qiang Li: Biological

objects Atomic Force Microscopy plays a microbiology, structural biology, molecular Applications of AFM (1997)

significant role among numerous biophysical biology, genetics and other biology related 1 0 . W w w. s p m t i p s . c o m / b i b l i o g r a p h y /
fields. biology/introduction SPM Applications in Biology
methods for investigation of specific and non-
specific molecular interactions such as DNA About the Author
replication, protein synthesis, protein- Suggested Reading
protein, enzyme-substrate, antigen-antibody, 1. T. Guha, R. Bhar, V. Ganesan, A. Sen, R. L.
receptor-ligand and drug-target association's Brahmachray, 2000. Application of AtomicForce
microscopy in seed surface studies, Current
interaction, and many others - are largely
Science journal, Vol.78, No.11, pp.1294-1295.
governed by intermolecular forces. AFM has
2. Yves F. Dufrêne, 2004. Using Nanotechniques to
the ability to measure forces in the R. Rajasekaran.
explore microbial surfaces, Nature Review
nanonewton range. This makes it possible to East Street, Sirupuliyur,
Microbiology, Vol.2, pp.451-460. Thirupalanam (PO), Thiruvaiyaru (TK).
quantify the molecular interaction in 3. Morris, V. J., Kirby, A. R. & Gunning, A. P. Atomic Thanjavur (DT), Pin- 613204. TN
biological systems such as a variety of Force Microscopy for Biologists (Imperial College Phone : 04362-261187.
Mobile: 9943511685
important ligand-receptor interactions. Press, London, 1999).
E-mail:rrs_rajesh2004@yahoo.com
Another application of AFM force

41 | Advanced Biotech | June 2008