Atomic Force Microscopy: Feels The Atoms, Sees The Bonds

When it comes to viewing things on the atomic scale, one has to be very careful and innovative. To understand how an atomic force microscope works, we should better discuss a bit about its predecessor: the scanning tunneling microscope (STM). STM was invented by Binnig and Rohrer for which they got the Nobel Prize in Physics. Binnig and colleagues later went on to develop the first Atomic Force Microscope (AFM). Both AFM and STM are types of Scanning Probe Microscopy, which employs a probe that scans the sample.

STM consists of a sharp probe tip, which scans over the specimen as the adjoining picture shows. First, the probe tip is brought near the sample manually, and then the finer adjustment of maintaining probe sample distance (height) is done by piezoelectric control. A voltage applied between the tip and the sample causes electrons to tunnel from the tip to the sample. As we have seen in Ohm’s law, the tunneling current will depend on the applied potential difference (voltage bias); and the height of tip-sample separation and the local density of states (factors determining ‘resistance’). If we know two of the three unknowns, we can calculate the other, which is actually done by the computer by data acquisition.

We can do STM in two ways. We can keep the tip position (height) fixed as it scans the specimen topography (constant height mode). Here the voltage and height are both held constant, while the tunneling current varies. In constant current mode, the tip is always at a specific height over the specimen. That is, as the tip hovers over the rugged terrain of the sample surface and comes close to a raised spot, the tunneling current will increase. The increased current will be sensed, amplified and passed to the feedback electronic circuitry which will ‘lift’ the probe-tip by applying a voltage to the piezo crystal. Hence the electronic servomechanism maintains a constant tip sample distance in constant current mode. Piezoelectric crystals translate pressure changes into electricity (and vice versa) as we see in oven gas lighters and in mobile phone speakers.

But STM has its inherent drawback: the sample has to be a conductor or a semiconductor, in order for tunneling to occur. Hence, biological tissues, non conducting polymers can not be imaged. So, the need for atomic force microscopy arose. Here again, Gerd Binnig played a pivotal role.

Atomic Force Microscopy operates on a similar principle. First, let’s discuss how the music on gramophone record grooves is translated. The stylus (which overlies a piezo crystal) feels the groovy surface of the vinyl LP disc which revolves on a turntable. The mechanical vibration sets in a voltage in the piezo via the stylus. We get the surface topology in the form of music. Another great way of viewing how the AFM scans and interprets a sample is how a blind person ‘feels’ a surface by using a stick (see picture).

In AFM, a very sharp tip (made of silicon or silicon nitride) is scanned over a surface with similar feedback mechanisms that maintain the tip at a constant force (to get height information), or height (to obtain force information) above the sample surface. This sharp tip is mounted on a cantilever, a rod like structure whose other end is fixed and unmovable. As the probe tip raster scans (i.e. scans in a zigzag fashion as done in TV scanning) the surface of the sample, a laser light is made to fall on the back of the cantilever. The light gets reflected off from this side and is detected by one of a dual photodiode. The cantilever ‘tilts’ as it slopes ‘down a bump’, causing another reflection that hits the other of the dual photodiode. A differential amplifier then calculates the difference output between the two light intensities and this is proportional to the cantilever deflection. We can calculate the force (acting between the cantilever tip and the sample) using Hooke’s law (F=-kx), where F is force, k is constant for a particular cantilever, and x is the deflection of the cantilever. The amplifier difference output may be used to keep the tip in either at a constant force or at a constant height above the sample, through piezoelectric servo mechanism (feedback).

AFM is very versatile and allows many user specific modifications. It can be done in contact mode, where the tip remains in contact with the surface as it scans over the specimen. In tapping mode, the cantilever oscillates over the sample, touching the surface intermittently. Interatomic forces like van der Waals forces and electrostatic forces cause a deflection of the cantilever when the tip comes closer to the sample. In the non contact mode, the cantilever does not touch the sample, and is oscillated at slightly above its natural resonance frequency. Any long range force like van der Waals force will decrease the frequency of the vibrating cantilever, when the tip approaches the sample. There are many other variants of AFM and combination of AFM with other imaging modalities like optical microscopy, Raman spectroscopy and so on.

But Leo Gross and colleagues at IBM Research Zurich wanted another, so they went on to develop another variant of AFM. They used a tuning fork like probe. One end of the probe was near, while the other end was away from the sample. When the fork was vibrated, the prong next to the sample experienced a minute shift in frequency due to forces acting upon it. This frequency drift when compared to the other prong, gives the molecular picture.

To get the finer detail of a molecule, one had to use a sharp AFM probe tip. They linked a molecule of carbon monoxide to the tip in such a way so that the lone oxygen atom became the de facto tip. It gave the structure of pentacene in unprecedented detail. You can clearly see even the bonds between carbon atoms, just as we read in our textbooks. However, the experiment must be vibration free, thermal noise free (at a very low temperature), and should be done in vacuum.

References: Scanning tunneling microscope (Wikipedia), Atomic Force Microscope (Wikipedia),
Single molecule's stunning image (BBC)

Last modified: never
Reference: hyper-links, unless specifically mentioned

Of Twinkling Nanostars and the Possible Application of Stroboscopes in Biological Imaging

Imagine a strong crowd, as you see in a Manchester United versus Liverpool football match and you wished to concentrate on a particular person. How would you do it? Make him wear a fluorescent shirt and dye his hair (don’t do it in the middle of the crowd, I can’t guarantee your safety).

Purdue University researchers have been successful in focusing at the cell of interest among a background of equally noisy and boisterous biomolecules and other metabolically active cells. Currently, researchers use immunological techniques to create an antibody to a molecule and then visualize the ‘molecule of interest’ by tagging the antibody to a radioisotope or a fluorescent dye; and flow cytometry can sort out different types of cells.

The Purdue University team used gold coated nanoparticles with an iron oxide core that was impregnated in the cell they wished to see. They then subjected the specimen to a periodically changing magnetic field. The superparamagnetic cores (superparamagnetic nanoparticles have no net magnetization, but an external magnetic field can magnetize them) responded by rotating as the magnetic field rotated around them. The rotation could be seen in the ‘near infra-red’ light spectrum, as the incident light bounced off (scattered) the specially designed arms of the gold nanostar as it revolved. The rate (rpm) of this gyromagnetic (gyros means to rotate) twinkling could be externally controlled by varying the rate of the externally applied field. You now could identify the cell by its characteristic ‘twinkling’ (lighthouse type) effect.

I am tempted to go beyond what’s been achieved so far. Here I go. I guess you are all familiar what happens to the rotating ceiling fan blades when you turn on a fluorescent lamp. Don’t you see a momentary snapshot of the three blades (some have 4)? That’s what where stroboscope comes in. It consists of a Xenon lamp (ordinary fluorescent lamps could do, but incandescent lamps won’t work as the glowing filament takes time to extinguish) flashing at a controllable rate. The electronic circuitry may be had here.

Suppose that the fan is revolving at 1200 RPM and it is not changing. Set your stroboscope to flash at this rate. You’ll ‘see’ that the fan blades are absolutely not moving, which is certainly not true! But be there any mechanical defect in the fan, it will stand out as the centrifugal force widens it (provided that the fault is more or less tangential to the axis of rotation). Here also we are looking at our object of interest, aren’t we?

Now lets look what implication it might have in biological imaging. We now know that the gamma subunit of mitochondrial F type ATP Synthase ‘actually’ rotates when it is synthesizing ATP (reverse rotation occurs when ATP is hydrolyzed). There are other locomotive units within the cell as well. They comprise of actin and myosin based molecular motors. Could we study them using an externally adjustable stroboscope? The optical (electromagnetic) signals so obtained may then be similarly broken down into simpler trigonometric (sine and cosine) functions by Fourier analysis (Fourier transform) as was done in the ‘twinkling nanostars’ experiment. At least, we expect to get rid of some 'noise' and some good still photos. But if we wanted better resolution and used higher frequency (electromagnetic) for it, some extraneous error will be introduced. It's a trade-off!

Last modified: never
Reference: hyper-links, unless specifically mentioned
Principles of Biochemistry, Lehninger, 4th ed
http://en.wikipedia.org/wiki/ATP_synthase
Wei, Q., Song, H., Leonov, A., Hale, J., Oh, D., Ong, Q., Ritchie, K., & Wei, A. (2009). Gyromagnetic Imaging: Dynamic Optical Contrast Using Gold Nanostars with Magnetic Cores Journal of the American Chemical Society, 131 (28), 9728-9734 DOI: 10.1021/ja901562j

On The Pursuit Of Arterial Plaques

In patients of disorders of lipid metabolism as in metabolic syndrome, diabetes mellitus and hypercholesterolemia, cholesterol laden plaques develop in the lumen of arteries. These plaques hinder the flow of blood leading to peripheral arterial disease (PAD). The narrowing of blood vessel that occur gives rise to symptoms of intermittent claudication and rest pain. Intermittent claudication is characterized by pain of the lower extremities as the person walks a few steps. This may progress to rest pain which is continually present, irrespective of whether the person walks or not. These plaques may also restrict blood flow to the heart causing myocardial ischemia.

What happens when these plaques rupture or loosen and detach? Fatal myocardial infarction (MI) and stroke (Cerebrovascular accident) may result when the heart or the brain arteries are involved respectively. We know that dyslipidemia of diabetes predispose us greatly to atherogenous plaque formation. LDL (low density lipoprotein), particularly oxidized LDL is a grave offender in this regard. Now imaging modalities are at hand which will let us visualize these atherosclerotic events, in real time.

Inflammation by oxidized LDL and their ilks increase the production of TNF-alpha (Tumor Necrosis Factor) and IL-1 (InterLeukin-1). They in turn increase the expression of vascular cell adhesion molecule-1 (VCAM) and P selectin. These intracellular adhesion molecules kind of attract leukocytes, which bind loosely to the plaque. These are the ominous plaques seething to rupture and loosen.

Micro Particles of Iron Oxide (MPIO) targeted with anti-VCAM-1 monoclonal antibody (mAb) are now being used to probe and identify these lesions. While the monoclonal antibody will latch onto the antigen (VCAM) as a key fits onto a lock; the microparticles of iron will act as a marker when seen in NMR (Nuclear Magnetic Resonance) imaging. Also called MRI or magnetic resonance imaging, this technique detects the density and spins of protons (H+ or hydrogen nuclei). In areas where contrast is less, contrast agents are employed to get a clear picture. Now, a team from National Taiwan University has developed this technique which employs dextran-coated iron oxide nanoparticles tagged with anti-VCAM-1 to get a glimpse of whats going on inside arterial lumen. A combination of anti-VCAM-1 mAb and anti-P-selectin mAb (VCAM-MPIO-P-selectin) is also being developed.

Although we already have angiography, intravascular ultrasound, and optical coherence tomography to detect these plaques, the newer techniques will certainly throw more light on this insidious killer process within.

Last modified: never
Reference: hyper-links, unless specifically mentioned

HistoMag: Nanomaterials in Medical Physics

Long ago when I used to be a medical student, my Anatomy teacher said that the breasts were an ornament to a lady and it gave motherhood to a woman. The words still reverberate in my ears. This aesthetic organ is frequently targeted by cancer. Put in other way, cancer of the breast is the second most common cancer worldwide (lung cancer tops the list) among both the sexes, and the most common type of cancer in women.

Diagnosing cancerous cells have been a nightmare for pathologists. We can not define a normal cell properly and differentiating them with cancerous cells can be pretty tough. Apart from their detection by radiological investigations which include X-rays, CT scan, MRI and other imaging modalities; we depend heavily on tissue biopsy samples collected from the patient's suspected specimen. We then stain the specimen using various dyes and examine them under the microscope.

For example, in the above picture, a section of breast tissue has been stained with immunoperoxidase. Such staining employs targeting and latching onto a tissue antigen of interest by using an external antibody (the immuno part), and then making these tissue antigens visible by the formation of a colored product by catalytic reactions brought about by peroxidase.

Now scientists have gone one step further. In some cancers of the breast there is an overexpression of a protein (antigen) called HER2/neu (Erb B2). This is a transmembrane protein, meaning that this molecule has an extracellular domain, a membrane spanning portion and an intracellular part. The peculiar nomenclature derives from the fact that this molecule is in fact a receptor for human epidermal growth factor and was found in rat neuroblastoma cell lines. Surprisingly, HER does not seem to have a physiological ligand, making the receptor look like a lady who does not have a mate! The protein when over-expressed, may form complexes among themselves, thereby transactivating its inherent tyrosine kinase activity. Unleashing the tyrosine kinase activity results in a cascade of activity that fosters tumorigenesis (picture on the left).

Scientists are now tagging magnetic nanoparticles with antibody to HER2/neu. Trastuzumab (herceptin), as the antibody is known, is a humanized monoclonal antibody (only one type/clone of molecule). Thus magnetic nanoparticles attaches to HER proteins (via trastuzumab); the density (intensity) of which can then be probed by applying the specimen to a magnetic field and extrapolating the the distortion these nanoparticles induce in them. Researchers at University College London (UCL) in the UK have developed HistoMag system which uses this technology. A drive coil (picture shown) produces a magnetic field which is made to vary with time, and a magnetometer using SQUID (superconducting quantum-interference device) as the pick up coil. The tissue section was sandwiched in the middle. Apart from detection of malignant cells it is also capable of predicting what population of women are likely to benefit from Herceptin (Trastuzumab) therapy. (In passing, it may be said that the same magnetometer device is being developed further so that it may, one day be able to pick-up your thoughts non invasively.) Many different approaches are cropping up which use nanotechnology, optical imaging and other modalities for the imaging of tissue sections. Quentin Pankhurst, professor of physics at UCL, the man behind HistoMag, has previously developed and commercialized SentiMag, a device which detects sentinel lymph nodes (lymph nodes first to enlarged in cancer).

In another development, researchers at the University of Debrecen, Debrecen, Hungary and Max Planck Institute for Biophysical Chemistry, Göttingen, Germany, have used paramagnetic microspheres coated with ligands (Herceptin) to attach to HER2. They then used confocal laser microscopy and digital image processing to 'see' the trans-activation (of ErbB2 (HER-2)). In confocal laser microscopy, a laser light is thrown into the sample through a special dichroic mirror, which allows light of one (long) wavelength to pass and that of other (short) wavelengths to reflect. When a laser of blue color (say) strikes the sample, which has been treated with a fluorescent dye, light of another wavelength say green is emitted. The dichromatic mirror now filters the blue light, while letting the green light pass, which is then amplified by photomultiplier tube and visualized. They found this to be an efficient tool in assessing Erb (HER2) activation, signal propagation and heterodimer formation.

In vivo neoplasms may be imaged by administering the patient a dose of these nanomagnets by mouth or by injection, and then imaging the patient. A modification of the device will be necessary.

Friedländer, E., Arndt-Jovin, D.J., Nagy, P., Jovin, T.M., Szöllősi, J., Vereb, G. (2005). Signal transduction of erbB receptors in trastuzumab (Herceptin) sensitive and resistant cell lines: Local stimulation using magnetic microspheres as assessed by quantitative digital microscopy. Cytometry Part A, 67A(2), 161-171. DOI: 10.1002/cyto.a.20173
Last modified: never
Reference: hyper-links, unless otherwise mentioned

Duroquinone: A Parallelly Processing Chemical Computer

Dr Anirban Bandyopadhyay of the National Institute for Materials Science, Tsukuba, Japan, have developed a tiny chemical nano-brain, that could one day be guided by remote control. These machines could make surgery on human bodies easier and help revolutionize the computing power of future computers.

Scientists have built nanobots (nanoscale robots; nano means a billionth of a metre) previously but these bots could not be controlled by outside means. Dr. Bandyopadhyay has now devised a nanobot, a chemical one and not mechanical or electronic one, that can be controlled from outside.

This promising nanomachine, just 2 billionths of a meter across, consists of a molecule called duroquinone. A single nanomachine comprises of 17 duroquinone molecules; with one molecule at the center and remaining 16 surrounding it. All these molecules are connected by hydrogen bonding. As is shown in the figure, each duroquinone molecule has four spoke like arms jutting out from it, which can be independently rotated to represent four different states. Thus they can be made to represent four different 'logic states', bits: 0,1,2,3. While ordinary computers work on binary logic (0,1), computers using this technology would have four billion possible combinations with this chemical brain.

The molecule at the center, to which the rest are connected, can be controlled by a scanning tunneling microscope (STM). This machine is not only capable of 'manipulating/directing' their (nanobots') orientations, it is also capable of 'reading' the states they are in. They act rather like both a transducer and a receiver. By tweaking the central molecule, one could switch the nanobot's configurations. Comparable switches in electronic circuits include CD4066, a quad bilateral switch, electrical relays, transistors and others. But here, we are controlling a chemical device by using STMs. In future, we may be able to operate the duroquinone machinery by using the conformational properties of proteins, by optical devices like lasers and may be other electromagnetic devices too.

The researchers were inspired by the parallel processing circuitry of the glial cells in the brain.
Said Dr. Bandyopadhayay, "Doctors will inject molecular machines attached to similar control unit, the assembly will go to the target part inside our body through veins, and carry out bloodless surgery. Till now several molecular machines have been built, prior to this work, but there were no machine that could control them."

Nano Does a Mega Tango: FETs To Change The Fate Of Diabetics

The role of electronics in medicine or physiology is a very important one. Ranging from CT scan, fMRI, PET scan or electron microscopes and a variety of other medical investigations, medical science depends on electronics and its allied disciplines.

A simple illustration is as follows. Everyone knows that the estimation of blood glucose is the cornerstone in the diagnosis and therapy of diabetes mellitus and other hyperglycemic illnesses such as hyperthyroidism, hyperpituitarism and others. Even hypoglycemic ailments (conditions where the blood glucose levels are less than normal) like hypothyroidism, hypopituitarism or hyperinsulinemia, hypoadrenocorticism need the estimation or quantitation (quantification) of blood glucose, in order to clinch their diagnosis.

Glucose may be estimated in the blood in a variety of methods. One of the easiest methods rely on the simple reaction of glucose with potassium per-manganate. Glucose is a reducing monosachharide; while potassium permanganate is a strong oxidizing agent, so much so that it produces frank fire when added to glycerine not to mention of its emanation of oxygen (O2) when heated. When KMnO4 (potassium permanganate) is added to glucose, it (KMnO4) is reduced to manganese ions (Mn++). The colour changes from purple pink to colorless. This can be seen by the naked eye, giving us an estimate of the amount of total glucose that is present.

Another approach that is adopted nowadays is the enzymatic oxidation of glucose and their quantitation thereof. Here enzyme glucose oxidase (GOD) is used to oxidize glucose to D-gluconic acid and hydrogen peroxide (H2O2). In the presence of H2O2, hydrogen peroxidase (POD) oxidizes phenol, which combines with 4-amino antipyrine to form a red dye (quinone imine). The intensity of the red dye is linearly proportional (upto 500 mg% i.e. 500 mg per dl) to the glucose concentration in the specimen. The intensity of the color that is formed is measured by using a colorimeter; a device that measures the transmittivity of light (through the liquid) by employing LDR (Light Dependent Resistor) or phototransistors or similar devices.

Recently, advances in nanotechnology and nanoscience as a whole, is offering a brand new hope of determining blood glucose within the patients blood itself. That is you don't need to prick the guy. They are using a device called ISFET (Ion Sensitive Field Effect Transistor). It is variant of FET devices (a picture of a MOSFET i.e metal oxide semiconductor FET, is shown on the left). In a ordinary bipolar transistor, we forward bias the emitter-base junction by applying a current and make the collector strongly reverse biased. Thus any input current in the base emitter circuit is amplified several times when the electrons 'rushes' towards the attracting collector. A FET also works in much the same way. The difference is that the flow of charge carriers are controlled by a electric field applied in the Gate (G) Source (S) junction. This electric field is generated by a voltage: VGS. Thus while bipolar junction transistors (BJT) are current controlled, the FET devices are voltage controlled.

Recently, a team of physicists led by Raj Mohanty from Boston University has made a nanoscale glucose sensor using ISFETs and nanowires consisting of silicon. The silicon nanowires were primed with glucose oxidase on their surfaces and these nanowires bridged the source and the drain electrode of the MOS device. The conductivity of the 'wire' would change depending upon the glucose concentration in blood as it is reacted upon by the enzyme present on its surface. A voltage drop will ensue between the source and the drain electrodes, which may be read and interpreted by a voltage comparator. The researchers claimed that these could be put in vivo with virtually no risk. Humans do have the habit of carrying silicon on their bodies and that hardly do them any harm! We may be able to noninvasively monitor the blood glucose of diabetic persons, may even be able to act upon it, by incorporating a feedback circuit that would actuate an insulin pump, in times of hyperglycemia.