Atomic Force Microscopy

Objectives
General Applications Background and History How Does AFM Work? 3 Modes: Contact mode Non-contact mode Tapping Mode Figures of Merit Advantages of AFM Biological Applications

General Applications
Materials Investigated: Thin and thick film coatings, ceramics, composites, glasses, synthetic and biological membranes, metals, polymers, and semiconductors. Used to study phenomena of: Abrasion, adhesion, cleaning, corrosion, etching, friction, lubricating, plating, and polishing. AFM can image surface of material in atomic resolution and also measure force at the nanoNewton scale.

Background and History

1st AFM made by Gerd Binnig and Cristoph Gerber in 1985 Constructed by gluing tiny shard of diamond onto one end of tiny strip of gold foil Small hook at end of the tip pressed against sample surface Sample scanned by tracking deflection of cantilever by monitoring tunneling current to 2nd tip position above cantilever Developed in order to examine insulating surfaces

ATOMIC FORCE MICROSCOPE

Atomic Force Microscopes or AFMs are a family of microscopes that are capable of seeing objects on the submicron scale. They use a small tip or cantilever to either touch or scan the surface and measure the forces. A computer gathers this information and translates it into an image that can be seen on a computer screen.

Parts of AFM
1. Laser deflected off cantilever

2. Mirror reflects laser beam to photodetector 3. Photodetector dual element photodiode that measures differences in light intensity and converts to voltage 4. Amplifier 5. Register 6. Sample 7. Probe tip that scans sample made of Si 8. Cantilever moves as scanned over sample and deflects laser beam

Necessary Components
Indirect detection of force Vibration Isolation Flexibility of Cantilever

OPERATION OF THE AFM

Topography
Contact Mode
High resolution Damage to sample Can measure frictional forces

Non-Contact Mode
Lower resolution No damage to sample

Tapping Mode
Better resolution Minimal damage to sample
http://stm2.nrl.navy.mil/how-afm/how-afm.html#imaging%20modes

3 Modes of AFM
Contact Mode Non-Contact Mode Tapping (Intermittent contact) Mode

Contact Mode
Measures repulsion between tip and sample Force of tip against sample remains constant Feedback regulation keeps cantilever deflection constant Voltage required indicates height of sample Problems: excessive tracking forces applied by probe to sample

Non-Contact Mode
Measures attractive forces between tip and sample Tip doesnt touch sample Vander Waals forces between tip and sample detected Problems: Cant use with samples in fluid Used to analyze semiconductors Doesnt degrade or interfere with sample- better for soft samples

Tapping (Intermittent-Contact) Mode

Tip vertically oscillates between contacting sample surface and lifting of at frequency of 50,000 to 500,000 cycles/sec. Oscillation amplitude reduced as probe contacts surface due to loss of energy caused by tip contacting surface Advantages: overcomes problems associated with friction, adhesion, electrostatic forces More effective for larger scan sizes

Figures of Merit
Can measure surface features with dimensions ranging from inter-atomic spacing to 0.1mm Resolution limited by size of tip (2-3nm) Resolution of imaging 5nm lateral and .01nm vertical

WHAT MAKES THE AFM SO GREAT ?

It is very versatile- It can image objects in the following ways: contact mode - In this mode the tip touches the surface and is dragged across it. In this way the physical characteristics of the surface can be easily measured. Such as friction (deflection), adhesion, elasticity, and height etc. scanning mode - In this case the tip is moved just above the surface. The atomic forces acting between the tip and sample are measured and thus information can be obtained about the different types of atoms/ molecules that make up the sample. Tapping mode In this method the tip is vibrated so that it repeatedly makes contact with the surface. In this way the elasticity and hardness of the object can be measured and objects in solution can be imaged without fear of displacement.

AFMs and Biology

AFMs have proven particularly useful in the field of biology. The fact that the samples need no special preparation means that living cells can be easily imaged with no damage to them. Thus living processes can be studied with as little interference as possible. This has enabled us to better understand things such as protein folding, cell bioprocesses, DNA and protein structure, enzyme catalysis, and protein crystal growth.

Advantages of AFM
AFM versus STM (scanning tunneling microscope): both conductors and Insulators AFM versus SEM (scanning electron microscope): greater topographic contrast AFM versus TEM (transmission electron microscope): no expensive sample prep

Advantages Over Other Imaging Methods But cant other microscopy methods obtain images just as easily?
Yes, in fact Scanning Tunneling Microscopes are actually capable of even higher resolution.

So why use an AFM at all?

AFM imaging requires no special preparation of the sample. Imaging of live cells can be done in-situ. Physical characteristics of the sample can be measured. The AFM can actually be used as a manipulation device. I.e. Images can be cut into samples and objects can be moved or otherwise adjusted.

With living cells it can often be difficult to obtain images of their actual surfaces. For these often transparent parts to be imaged requires staining or relies on the differences between the refractive indexes of the various layers. An AFM provides a truly 3-Dimensional image of the surface including also various mechanical information, and is particularly useful in the study of the membrane surface and cytoskeleton. Such imaging can even be performed in cell culture medium on currently living cells enabling us to see the various processes as they happen. The tip can also be modified to stimulate the cells using specific interactions

Biological Applications
Used to analyze DNA, RNA, protein-nucleic acid complexes, chromosomes, cell membranes, proteins and peptides, molecular crystals, polymers, biomaterials, ligand-receptor binding Little sample prep required Nanometer resolved images of nucleic acids Imaging of cells Quantification of molecular interactions in biological systems Quantification of electrical surface charge

Who Needs It
Vacuum, Air, Aqueous Medium - Mimic Biological Environment Sub-nanometer resolution Manipulate Surface with Molecular Precision Real Time Direct Structure-Function Studies

Sample Preparation
AFM Does require minimum of sample preparation: No clean room handling No thin film metal coating Works in liquids, gases, and vacuum Works at elevated or sub ambient temperatures Dimensions are not critical
Spring 2009 AFM Lab

Instrument Setting
Sample: Center it in the middle of the sample plate and immobilize it using dual side sticky pads Laser: Laser beam has to bounce on the tip of the cantilever. Photodiode: Reflected beam signal has to be shared equally between the 4 cells System adjustment: Servo Gain (PI values), Force, Raster speed
Spring 2009 AFM Lab

Applications
Study Unfolding Of Proteins Imagining Of Biomolecular Force Measurements In Real Solvent Environments Antibody-Antigen Binding Studies Ligand-Receptor Binding Studies Binding Forces Of Complimentary DNA Strands Study Surface Frictional Forces Ion Channel Localization

VIDEOS