The Use of Atomic Force Microscopy in Biological Imaging Robert Meservy, Undergraduate of Physics, Utah State University, 2006.

The Use of Atomic The Use of Atomic Force Microscopy in Force Microscopy in Biological ImagingBiological Imaging

Robert Meservy, Undergraduate of Physics, Utah State Robert Meservy, Undergraduate of Physics, Utah State University, 2006 University, 2006

Atomic Force MicroscopyAtomic Force Microscopy

Atomic Force Microscopes or “AFMs” are Atomic Force Microscopes or “AFMs” are a family of microscopes that are capable a family of microscopes that are capable of seeing objects on the submicron scale.of seeing objects on the submicron scale.

They use a small tip or cantilever to They use a small tip or cantilever to either touch or scan the surface and either touch or scan the surface and measure the forces. A computer gathers measure the forces. A computer gathers this information and translates it into an this information and translates it into an image that can be seen on a computer image that can be seen on a computer screen.screen.

Simple Schematic of a Simple Schematic of a Typical AFM SetupTypical AFM Setup

What Makes the AFM so What Makes the AFM so great?great?

It is very versatile- It can image objects in the following It is very versatile- It can image objects in the following ways:ways: ““contact” mode - In this mode the tip touches the surface and contact” mode - In this mode the tip touches the surface and

is dragged across it. In this way the physical characteristics of is dragged across it. In this way the physical characteristics of the surface can be easily measured. Such as friction the surface can be easily measured. Such as friction (deflection), adhesion, elasticity, and height etc.(deflection), adhesion, elasticity, and height etc.

““scanning” mode - In this case the tip is moved just above the scanning” mode - In this case the tip is moved just above the surface. The atomic forces acting between the tip and sample surface. The atomic forces acting between the tip and sample are measured and thus information can be obtained about the are measured and thus information can be obtained about the different types of atoms/ molecules that make up the sample.different types of atoms/ molecules that make up the sample.

““Tapping” mode – In this method the tip is vibrated so that it Tapping” mode – In this method the tip is vibrated so that it repeatedly makes contact with the surface. In this way the repeatedly makes contact with the surface. In this way the elasticity and hardness of the object can be measured and elasticity and hardness of the object can be measured and objects in solution can be imaged without fear of displacement.objects in solution can be imaged without fear of displacement.

AFMs and BiologyAFMs and Biology

AFMs have proven particularly useful in AFMs have proven particularly useful in the field of biology. The fact that the the field of biology. The fact that the samples need no special preparation samples need no special preparation means that living cells can be easily means that living cells can be easily imaged with no damage to them. Thus imaged with no damage to them. Thus living processes can be studied with as living processes can be studied with as little interference as possible. little interference as possible.

This has enabled us to better understand This has enabled us to better understand things such as protein folding, cell bio-things such as protein folding, cell bio-processes, DNA and protein structure, processes, DNA and protein structure, enzyme catalysis, and protein crystal enzyme catalysis, and protein crystal growth.growth.

Biology AdvantagesBiology Advantages In biological imaging we’re not so concerned about the atomic In biological imaging we’re not so concerned about the atomic

scale. We might want to see a protein molecule but such scale. We might want to see a protein molecule but such things are made up of thousands of atoms. This means that things are made up of thousands of atoms. This means that the AFM can provide all the resolution we could possibly the AFM can provide all the resolution we could possibly need.need.

Until the invention of the AFM the best resolution was Until the invention of the AFM the best resolution was obtained using Scanning Electron Microscopes and Tunneling obtained using Scanning Electron Microscopes and Tunneling Electron Microscopes. To obtain a reasonable resolution with Electron Microscopes. To obtain a reasonable resolution with these methods however required that the sample be killed these methods however required that the sample be killed and very carefully prepared. And imaging often required a and very carefully prepared. And imaging often required a vacuum, which precluded using live samples. If it were vacuum, which precluded using live samples. If it were somehow kept alive the electron radiation was sure to kill it.somehow kept alive the electron radiation was sure to kill it.

The AFM offered biologists the opportunity to image objects The AFM offered biologists the opportunity to image objects without overly affecting them. The tip could be made to without overly affecting them. The tip could be made to gently follow the objects surface or even not touch it at all. gently follow the objects surface or even not touch it at all. No radiation was needed and images could be taken over No radiation was needed and images could be taken over and over again of the same sample!and over again of the same sample!

In fact using nano-lithography techniques it is now possible to In fact using nano-lithography techniques it is now possible to custom manufacture the tips used. Meaning sharper or duller custom manufacture the tips used. Meaning sharper or duller tips could be used depending on the tip of work required. It is tips could be used depending on the tip of work required. It is even possible to make small tweezers or other objects to be even possible to make small tweezers or other objects to be placed on the end of the cantilever.placed on the end of the cantilever.

Carbon Carbon nanotube tipnanotube tip

Normal tipNormal tip

SupertipSupertip

UltraleverUltralever

Functionalised Functionalised tiptip

Tip with Tip with atom attachedatom attached

Advantages Over Other Advantages Over Other Imaging MethodsImaging Methods

But can’t other microscopy methods obtain images just as easily?But can’t other microscopy methods obtain images just as easily?Yes, in fact Scanning Tunneling Microscopes are actually capable of even Yes, in fact Scanning Tunneling Microscopes are actually capable of even

higher resolution.higher resolution.So why use an AFM at all?So why use an AFM at all?• AFM imaging requires no special preparation of the sample. AFM imaging requires no special preparation of the sample. • Imaging of live cells can be done “in-situ”Imaging of live cells can be done “in-situ”• Physical characteristics of the sample can be measured.Physical characteristics of the sample can be measured.• The AFM can actually be used as a The AFM can actually be used as a manipulationmanipulation device. I.e. Images can device. I.e. Images can

be cut into samples and objects can be moved or otherwise adjusted. 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 With living cells it can often be difficult to obtain images of their actual

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

• The tip can also be modified to stimulate the cells using specific The tip can also be modified to stimulate the cells using specific interactions.interactions.

No Preparation NeededNo Preparation Needed60μm x 60μm60μm x 60μm

Contact AFM image taken in air showing the surface Contact AFM image taken in air showing the surface topography of a pore or stomata on the underside of a topography of a pore or stomata on the underside of a freshly picked ivy leaf.  Here the two guard cells can freshly picked ivy leaf.  Here the two guard cells can be seen, which control the flow of water vapor into be seen, which control the flow of water vapor into and out of the leaf.  and out of the leaf.  To prepare the sample a small piece of leaf tissue To prepare the sample a small piece of leaf tissue was simply stuck down with double-sided tape and was simply stuck down with double-sided tape and then imaged. STM or SEM imaging would have then imaged. STM or SEM imaging would have required metal coating and imaging of the sample in a required metal coating and imaging of the sample in a vacuum.vacuum.

1μm x 1 μm1μm x 1 μm

Apple cell wall:  The strands in the image are cellulose Apple cell wall:  The strands in the image are cellulose microfibrils. Sample was prepared by using a razor blade microfibrils. Sample was prepared by using a razor blade to cut off a section of skin which was then attached using to cut off a section of skin which was then attached using double-sided tape to a slide. double-sided tape to a slide. 

In-Situ imagingIn-Situ imagingBacterial flagellae; flagellae of Bacterial flagellae; flagellae of Pseudomonas Pseudomonas

putidaputida, non-contact mode. Imaged in , non-contact mode. Imaged in nutrient solutionnutrient solution

Movie made of a human chromosome Movie made of a human chromosome arrested in metaphase showing the arrested in metaphase showing the characteristic armscharacteristic arms

Images by Neil Thomson, movies by Peter Hallett retrieved from http://spm.phy.bris.ac.uk/movies/

The AFM is versatile enough to image objects in situations other than mounted on slides. Images can be taken in solution, in different atmospheres or conditions or even while still alive and undergoing biological processes. This makes the AFM especially useful in understanding the inner workings of living organisms.

Transcription of DNA by RNA polymeraseTranscription of DNA by RNA polymerase

DNA being transcribed by the DNA being transcribed by the enzyme RNA polymerase. enzyme RNA polymerase.

The enzyme (white spot) binds The enzyme (white spot) binds to the DNA (thin line) After to the DNA (thin line) After the third picture on the top the third picture on the top row, the enzyme starts to row, the enzyme starts to move along the DNA .move along the DNA .

A single starch granule, imaged A single starch granule, imaged under liquid by AFM, being under liquid by AFM, being degraded by the enzyme alpha-degraded by the enzyme alpha-amylaseamylase

As the enzyme moves along the DNA, it makes RNA (not visible) until it As the enzyme moves along the DNA, it makes RNA (not visible) until it comes to the end of the DNA and falls off in the bottom row of pictures. comes to the end of the DNA and falls off in the bottom row of pictures. The DNA continually wiggles around, as you can see from the pictures.The DNA continually wiggles around, as you can see from the pictures.

Physical Characteristics Can Be MeasuredPhysical Characteristics Can Be MeasuredAn AFM can be used in many ways as a set of tools by An AFM can be used in many ways as a set of tools by

which we can investigate the nano world. which we can investigate the nano world.

So sensitive are AFMs that they even been used to So sensitive are AFMs that they even been used to measure the spring coefficient and tensile strength of measure the spring coefficient and tensile strength of individual DNA strands!individual DNA strands!

When used in tapping mode AFM tips can also be used to When used in tapping mode AFM tips can also be used to measure differences in hardness and elasticity. And measure differences in hardness and elasticity. And while in contact mode properties such as adhesion, while in contact mode properties such as adhesion, resistance, and friction can be measured.resistance, and friction can be measured.

These synaptic vesicles are high (white) in the center in These synaptic vesicles are high (white) in the center in the height image but dark in the center in the hardness the height image but dark in the center in the hardness image, because their centers are harder than their image, because their centers are harder than their edges. The vesicles are on a hard surface and are from edges. The vesicles are on a hard surface and are from the electric organ of the electric organ of TorpedoTorpedo, a marine ray. They are , a marine ray. They are about one ten-thousandth of a millimeter ( 1 micron) in about one ten-thousandth of a millimeter ( 1 micron) in diameter.diameter.

Here the AFM can feel the difference between double helical Here the AFM can feel the difference between double helical DNA (blue) and triple helical DNA, which is higher (white).DNA (blue) and triple helical DNA, which is higher (white).

Hansma, H. G., I. Revenko, K. Kim, and D. E. Laney. 1996.

Nucleic Acids Res. 24:713-720

ManipulationManipulation

Both of these Both of these patterns were patterns were done using an done using an AFM tip to AFM tip to move the DNA move the DNA into the into the desired desired configurationconfiguration

A 30nm gold particle (a) A 30nm gold particle (a) before and (b) after before and (b) after being pushed over a being pushed over a 10nm high step along 10nm high step along the direction indicated the direction indicated by the arrow. Image by the arrow. Image sizes are both 1μm x sizes are both 1μm x 0.5μm.0.5μm.

Unfolding a ProteinUnfolding a Protein

Here an AFM tip is being Here an AFM tip is being used to unzip a protein used to unzip a protein known as Titan. The AFM is known as Titan. The AFM is brought close enough that it brought close enough that it experiences an attraction and experiences an attraction and is then slowly drawn away. If is then slowly drawn away. If it is done slowly enough the it is done slowly enough the tip does not release the end tip does not release the end of the chain and it is of the chain and it is unraveled. As it is drawn out unraveled. As it is drawn out the force on the tip changes the force on the tip changes as shown in the graph.as shown in the graph.

Using an AFM Using an AFM tip to cut a tip to cut a plasmidplasmid

Using an AFM tip to bend Using an AFM tip to bend carbon nanotubescarbon nanotubes

Cutting and BendingCutting and Bending

Example of an Enzyme Study Done Using Example of an Enzyme Study Done Using a Functionalised AFM Tipa Functionalised AFM Tip Here an AFM tip coated Here an AFM tip coated

with Avidin-Biotin pairs with Avidin-Biotin pairs (two types of enzyme) and (two types of enzyme) and a Biotin coated agarose a Biotin coated agarose bead are being used to bead are being used to measure the binding force measure the binding force between Avidin and between Avidin and Biotin. When the tip is Biotin. When the tip is brought close the open brought close the open sites on the Avidin bind to sites on the Avidin bind to the Biotin found on the the Biotin found on the bead. When the tip is then bead. When the tip is then withdrawn the force withdrawn the force required to remove the tip required to remove the tip from the bead surface can from the bead surface can be measured and the be measured and the binding force between the binding force between the two enzymes calculated.two enzymes calculated.

Example of Imaging Done Example of Imaging Done With an AFMWith an AFM

Below you see a human bone (a) and muscle (b) cell Below you see a human bone (a) and muscle (b) cell that have been imaged using an AFM. In the first that have been imaged using an AFM. In the first column you see an image showing the variations column you see an image showing the variations in height. In the second you are seeing the in height. In the second you are seeing the deflection or resistance of the cell as the tip was deflection or resistance of the cell as the tip was dragged across it’s surface, and in the third dragged across it’s surface, and in the third column a measurement of the cell’s elasticity column a measurement of the cell’s elasticity done by “tapping” the surface at a given rate.done by “tapping” the surface at a given rate.

Human neurons imaged in solution in tapping mode

DNA ImagesDNA Images

By adjusting the force associated with the By adjusting the force associated with the tapping mode, the image quality can be tapping mode, the image quality can be affected. Below the scan was first started with affected. Below the scan was first started with a low force; halfway through the force was a low force; halfway through the force was increased. The difference is immediately increased. The difference is immediately noticeable as the image becomes less noticeable as the image becomes less “noisy”.“noisy”.

Atomic Force image taken with an AFM tip coated Atomic Force image taken with an AFM tip coated with a certain “restrictive enzyme”. The red peaks with a certain “restrictive enzyme”. The red peaks correspond to places where the enzyme attempted correspond to places where the enzyme attempted to bind to the DNA sequence. Pinpointing the to bind to the DNA sequence. Pinpointing the locations of the desired gene.locations of the desired gene.

The AFM images of DNA here are all © A.A. Baker, University of Bristol.

Molecular Imaging Group, Oak Ridge National Laboratory

More DNA ImagesMore DNA Images

Contact image of DNA under Contact image of DNA under propanol onto Mica pre-propanol onto Mica pre-

treated with Ni ions. Note treated with Ni ions. Note how much more tightly coiled how much more tightly coiled

it is.it is.

Contact image taken Contact image taken of DNA under of DNA under propanolpropanol

Tapping image in airTapping image in airPushing DNA (a) Image of DNA before Pushing DNA (a) Image of DNA before pushing. (b) During pushing. (c) After pushing. (b) During pushing. (c) After several pushing operations.several pushing operations.

High Resolution AFM image of a bacterium and High Resolution AFM image of a bacterium and biofilmbiofilm A biofilm formed by marine-sulfate

reducing bacterium on the surface of stainless steel. Imaged in Contact mode. The bacterium was grown on 316 stainless steel surfaces which had been polished by exposure to acid steamers for 30 days. After removing the the biofilms, the AFMshowed numerous micropits on the virgin steel surface that weren’t detectable by Optical methods (Near Field Optical Microscope)

AFM image of the bacteria AFM image of the bacteria Pseudomonas putid. The left Pseudomonas putid. The left image is topographic, the right image is topographic, the right image is a phase image.image is a phase image.

In ConclusionIn ConclusionAlthough AFMs do not offer as high a resolution Although AFMs do not offer as high a resolution

as do STMs, their ability to take images in as do STMs, their ability to take images in situations that other methods cannot, and the situations that other methods cannot, and the fact that they can manipulate the very thing fact that they can manipulate the very thing that they’re imaging makes them incredibly that they’re imaging makes them incredibly useful. useful.

This is even more so in the field of biology where This is even more so in the field of biology where it is often desirable to image things while still it is often desirable to image things while still alive and with considerable resolutionalive and with considerable resolution