Studying Genes the Ophthalmic Route by MRI, and That too in Living Subjects

It is said that the eyes are the windows to the soul, though science is yet to prove that given the elusive nature of ‘soul’. But researchers has now been able to probe genes in a traumatized brain using the eyes as a gateway.

The brain is normally ‘secured’ from the circulating blood directly, so that endogenous and exogenous toxic substances, macromolecules can not gain entry easily into (and out of) the brain. More importantly, this ‘firewall’ like barrier, called the ‘blood brain barrier’ maintains the constancy of ions inside the brain such as K+, H+, Mg++, Ca++, which is vitally important for the neurons to function normally.

The ‘blood brain barrier’ (BBB: see picture) results from the ‘relative’ impermeability of both the capillaries supplying the brain as well as that of the ‘choroid plexus’ covering the brain. Actually, the endothelial cells of the capillaries are tightly packed (tight junctions) and they are non-fenestrated too. In addition, end feet of astrocytes, a type of glial cells (cells that support and aid neurons), cover these capillaries.

But there are disease conditions in which the BBB becomes leaky. For example, in traumatic brain injury, cardiac arrest, stroke and multiple sclerosis the blood brain barrier is breached, to different extents. In Alzheimer’s disease too, there is thinning of the capillaries as the disease progresses. As expected, the supporting glial cells, particularly the astrocytes, jump into action to seal the leaks. They proliferate, resulting in ‘gliosis’. Gliosis is also found in a tumorous condition of the glial cells called ‘glioma’.

These glial cells contain a protein in them called the glial fibrillary acidic protein (GFAP). Naturally, there is an mRNA for it that ‘translates’ its formation in the cytoplasm. Scientists target this mRNA molecule because tagging it will track the GFAP and consequently the astrocytes in whom GFAP is expressed.

Previously scientists had to inject MR contrast agents intra-cerebro-ventricularly or by other invasive techniques to map these leaking areas. Scientists at Harvard embarked on a novel idea. They produced a short cDNA sequence ‘complementary’ to the mRNA of GFAP. This short stretch of this ‘antisense’ oligodeoxynucleotide (ODN-gfap) would latch onto the GFAP mRNA just as a lock would to its key. They then tagged it with a paramagnetic molecule that they designed, called superparamagnetic iron oxide nanoparticles or SPION, a magnetic resonance susceptibility contrast agent. The SPION-ODN ‘report’ any inhomogeneity in transverse magnetization in ‘T2 star’weighted MRI scan, due to the paramagnetic properties of iron oxide. Liu et al also used a sequence complementary to the mRNA of beta-actin as well (actin is the most abundant protein in mammalian cells and its mRNA is found in all types of cells) to act as a ‘control probe’.

They then anesthetized the mice, the animal model they selected; and caused BBB leakage by inflicting a small puncture or by performing bilateral carotid artery occlusion (BCAO) for 60 minutes. They also tried other methods (see reference). They subjected another group of mice to a sham (=false) operation (no puncture or vessel occlusion but the same operation) at the same time. BBB leakage was checked by T1 weighted Gadolinium-DTPA contrast MRI scan. Gd-DTPA was injected into the jugular veins of the mice. Leakage would show up as enhanced areas on T1 weighted scan (normally Gd-DTPA does not cross the BBB). Due to repair process to seal the leak, glial cells would be recruited and gliosis would result.

The telltale signature of gliosis (and BBB breach) may be found in postmortem tissue samples of the brain. Previously, the GFAP antigen was detected by immunohistochemical methods. But the Harvard team was looking for a non invasive method to detect GFAP. They instilled ‘SPION-ODN gfap’ reporter into the conjunctival sac of the mice by means of eyedrops. They then measured the ‘T2 star’ values in MRI scan and transformed the values to ‘R2 star’ maps (R2 star = 1/T2 star). Areas of leakage showed up as elevated (hyperintense) signals in R2 star maps. It corroborated well with Gd-DTPA scans and also on post mortem examination. SPION-beta actin, the control probe, got bound to the endothelial cells of the vasculature as expected.

The eye drop was absorbed by the lymphatics draining the palpebral (eyelid) and bulbar conjunctiva. The lymphatics then transferred the reporter probe into the veins which finally found their way into the brain. Since the BBB was breached, it finally came out of the circulation into the brain parenchyma. As the probe is detecting mRNA which is ‘transcribed’ from the DNA of the cell, it may be said that they are, in a sense, detecting the genes for GFAP.

Thus we may hope to detect gliosis, a pathology that occurs in a variety of diseases already mentioned, non invasively, the ophthalmic way.

Last modified: never
Reference: Liu, C., You, Z., Ren, J., Kim, Y., Eikermann-Haerter, K., & Liu, P. (2007). Noninvasive delivery of gene targeting probes to live brains for transcription MRI The FASEB Journal, 22 (4), 1193-1203 DOI: 10.1096/fj.07-9557com

fMRI, BOLD and the Beautiful

When we want to examine the brain of a person noninvasively by Computed Tomography (CT) or MRI, we get a ‘snapshot’ of the anatomy (or pathology, if any) of the subject’s brain. We are however clueless as to its functional aspect. fMRI or Functional Magnetic Resonant Imaging allows us to do just that. The difference is not unlike a ‘still picture’ versus a ‘video of a moving train’. PET scans, previously described, also can asses the functional state of the brain.

Whenever we do a task, think, dream, memorize, speak or see things, the brain is not activated as a whole; but only certain portions of it are activated. Activation, here, means increased metabolic activity of neurons in certain areas of the brain. Naturally, these ‘metabolically active’ neurons would demand more energy which would power them. The blood supply to these areas increases as a result of this metabolically driven vasodilation. The arteries then bring in glucose and oxygen with them, with Oxygen being transported in the form of Oxyhemoglobin (oxygenated hemoglobin or HbO2). Neurons on the other hand use up the oxygen contained in the blood, thereby reducing it to de-oxyhemoglobin or simply Hb. However, the alteration in tissue perfusion exceeds the extraction of oxygen by the neurons, so the concentration of deoxyhemoglobin within ‘the areas’ decreases. This causes molecular inhomogeneities in the magnetic field.

Oxyhemoglobin is diamagnetic, meaning that they align perpendicularly to magnetic field lines. On the other hand, deoxyhemoglobin is paramagnetic, i.e. it aligns parallely and proportinately with the intensity of the magnetic field. This causes the inhomogeneity within the magnetic field (magnetic susceptibility) in the tissue sampled. This inhomogeneity is exploited in fMRI in terms of decay of transverse magnetization, T2*, with longer T2* values in HbO2 blood and shorter values in Hb (paramagnetic) blood.Since this stems from the oxygen content in blood, fMRI is also known as the BOLD ((blood oxygenation level dependent) effect.

The machine is essentially the same as the MRI machine with echo planar imaging technology that permits faster imaging due to faster gradient switching, improved algorithm and faster CPU processing power. The patient/subject is placed inside the magnetic chamber and MRI signals are acquired, Fourier transformed and corrected for artifacts. Finally the computer reconstructs a 3D fMRI image out of this.

As is obvious, we can learn about the motor areas of a patient by asking him to grasp an object or giving him any motor task and noticing which area(s) of the brain lights up. A neurosurgeon can then be cautious about not hurting these areas. Similarly, the mapping will help spare motor and other vital areas like auditory, visual and language areas from damage in radiotherapy procedures, in addition to neurosurgery. It can also detect occult Alzheimer’s disease and cognitive deficits including those of the autism spectrum and dyslexia (reading disorder).

fMRI can also be employed to ‘read peoples’ minds’, thoughts, intentions including lie detection. Watch the video below which explains how an fMRI scan is done and interpreted.


Thus the legal and forensic implications are obvious. However, in fMRI, correlation doesn't always mean causation. Whatever it may be, it seems that fMRI is very much here to stay, both in the clinics as well as in cognitive neuroscience research. It may also be combined with tractography, MRI or other diagnostic radiologic modalities.

Hardenbergh et al combined Tractography techniques with fMRI, using a technique capable of rendering multiple color-coded functional activation volumes and fiber tract bundles. Many pharmacologically active drugs have effect on memory impairment, which can be seen in ‘telltale’ fMRI scans. Sperling et al studied the effects of lorazepam (a benzodiazepine) and scopolamine (an anticholinergic drug once used as ‘truth serum’ by the CIA) on healthy volunteers and found that they did impair memory and their functional coordinates could be reproducively mapped on fMRI scans (see figure on the left). I still shudder at the thought of what happened during my PG exam when I took a benzodiazepine.

Last modified: Mar 09, 2014
Reference: Integrated 3D Visualization of fMRI and DTI tractography
Gore, J. (2003). Principles and practice of functional MRI of the human brain Journal of Clinical Investigation, 112 (1), 4-9 DOI: 10.1172/JCI200319010

The World of Tractography Where The White Matter Tracts Appear Colored

The Central Nervous System (CNS) communicates with the exterior (sensory e.g. gets visual, tactile information etc. on the one hand; and motor, performs limb movement, posture regulation etc. on the other) via the peripheral (somatic) nervous system. It also connects with the interior (our viscera or organs) via the Autonomic Nervous System. That is, it does its job in a bidirectional way: by the motor or the actuator arm, and sensory or the receptor arm. For such ‘actions’ to occur, cables of nerve fibers are laid within our body. Wouldn't it be nice if we could visualize these cables, their dispositional anatomy or any pathology that could afflict them?

Schematic of a 'peripheral' myelinated axon 

The brain and spinal cord together constitute the CNS. We also know that there are about a hundred billion neurons in the CNS. Each neuron has a cell body (soma), an axon wrapped by myelin, and many dendrites. (See figure). It is the axon that carries the information in the form of action potential. These cables (bunch of axons, called tracts) are not laid haphazardly. Nature tries to conserve space, length, energy and so on and thus the axons form into tracts in a topologically efficient way. They run up and down (also front-back and sideways) the cord to the brain, the organ that we will now concentrate upon.

Your electrical wiring to your ceiling fan would include a switch and the fan itself. The wire (cable) from the switch would ascend vertically up the wall, make a 90 degree angle, and then reach the fan horizontally up in the ceiling. Likewise, in our brain, which is made up of two hemispheres, would connect. Three broad fiber types are seen: from one hemisphere to the other (commisural fibers), restricted to one hemisphere (front to back or antero-posteriorly are association fibers) and finally vertically (up down orientation go the projection fibers).

Exploring the tracts can now be done in live animals including humans. Improvement in MRI technology has enabled us to see the tracts (tractography). Improved gradient coils, faster processors and superior software have shortened the scanning time, thereby reducing 'blur' due to organ movement (e.g. diaphragm) and patient movement. This procedure called Echo Planar Imaging (EPI) has given birth to Functional Magnetic Resonance Imaging (fMRI), Diffusion Weighted Imaging (DWI), tractography and many other diagnostic and research procedures.

Consider the neuron shown in the above picture. Water molecules in the axon (yellow) are constantly in Brownian (random) motion due to thermal energy within. Hence they tend to diffuse constantly to come to equilibrium. In most of the cerebrospinal fluid spaces these microscopic motions are equal in all directions. This is called isotropic diffusion. But in myelinated neurons, as in the white matter fiber tracts, water motion is constrained due to the fatty nature of the myelin sheath (in blue) which hinders water flow across it. This anisotropic diffusion allows the flow of more water molecules along (parallel) the direction of the nerve fiber. The apparent diffusion coefficient (ADC) is thus more along the nerve fiber. Diffusion Weighted Imaging (DWI) can capture this microscopic water flow and delineate anatomically the orientation of nerve fiber tracts.

Pyogenic abscesses hinder diffusion by virtue of their increased viscosity, a rheological property. In the early stages of acute cerebral infarction there is reduced diffusion too, giving rise to high signal intensity. However, in most pathologies of the brain the ADC is increased. Diffusion Tensor Imaging (DTI), a diffusion MRI technology, tracks fiber orientation by assigning values in ellipsoid voxels (VOlume piXEL). Ellipsoid because unlike isotropic diffusion where molecules diffuse equally in all directions, the anisotropy in the white matter tracts does not permit them to move with equal ease in all directions, and hence the pattern is that of an ellipsoid and not spherical. By connecting the long axes of all the ellipsoids, the trajectory/orientation is deduced.

Colors are added to it with respect to the three principal axes (x, y and z) and the result is a stunning tractography! (see left)



The YouTube video below describes the colorful realms of tractography and how they are used.

When fMRI and DTI are combined together, a whole new world emerges. But, I prefer to keep it on hold till I discuss Functional Magnetic Resonance Imaging.

NB: The picture of the neuron/axon shown is not representative of CNS neurons. Oligodendrocytes that form myelin in the CNS does not encircle so many times as the Schwann cells in peripheral neurons do. It has been shown here only for the sake of clarity.

Last modified: Mar 10, 2014
Reference: Diffusion Tensor Tractography: Exploring the Cost-Benefit Ratio of Incorporating CSF Suppression into Fiber Tracing Algorithms

William P. Dillon. Neuroimaging in Neurologic Disorders. In: Harrison's Principles of Internal Medicine, 17th Ed., Volume 2, McGraw Hill; 2008. p. 2491-2497.

P. Mukherjee,, J.I. Berman,, S.W. Chung,, C.P. Hess, & R.G. Henry (2008). Diffusion Tensor MR Imaging and Fiber Tractography: Theoretic Underpinnings AM J Neuroradiol DOI: 10.3174/ajnr.A1051

Relaxation in the Nuclear Microcosm

All of us want to give themselves a hard earned ‘rest’ after a “hard day’s night”, don’t we? So do the protons, perturbed by the destabilizing magnetic component of the radio-frequency pulse [which previously ‘happily’ aligned themselves to the externally applied magnetic field; one way (parallel) or the other (antiparallel)] applied at the Larmor frequency. It is like slapping an individual in a “merry go round” each time he came near a person who is paid just for slapping that person. But, when we call ‘spin’, we do not mean ‘spin’ the way we see them in a classical world. [We’ve given various names to the ‘quarks’: up, down, strange, bottom etc. depending on ‘something’ called ‘flavor’; and red, green and blue depending on ‘something’ called ‘color’.However, spin, flavor, color etc.‘in the quantum world’ have ‘no relevance’ to what we usually attribute to them in our everyday life. Things are a bit crazy in the quantum world, but I will take recourse to some ‘classical world’ analogies to make the description lucid.]

Thus, the already aligned nuclei (parallel or anti-parallel to the applied steady external magnetic field B0), has now been perturbed owing to the ‘knocking’ by the ‘magnetic component’ (B1) of the electromagnetic RF pulse. The nuclei gain energy and sway away from the perpendicular to the horizontal (90 degree) depending on how long the RF pulse is applied. So, now the nuclei behave like ‘punch-drunk’( like a person who’s been reeling due to a strong blow to the head!). Remember, that this new angular momentum is also a vector quantity having magnitude and direction. It can be resolved in terms of a horizontal component (Mxy) and a vertical component Mz. Anyway, the proton does recover from this situation, after some time, once the external RF field has stopped. Typically, Mxy component decays faster than the recovery of Mz.

The excited proton recovers in two ways and both forms occur simultaneously: (1) The excited nuclei which now have been ‘forced’ to lie horizontally (90 degree), ‘re-align’ themselves back to their ‘original position’ as they were before the RF pulse (perpendicularly towards the field of externally applied field B0); and (2) the energized protons dissipate their energies to the surrounding nuclei (horizontally) at their level. The first example, obviously, is called the (spin-lattice, or longitudinal) relaxation; while the second one, transverse relaxation (T2). There is little energy loss due to RF emission.

T1 relaxation, also known as, longitudinal relaxation or spin-lattice relaxation can be best understood if you see the following Youtube video. [The spiral trajectory, in this case, reminds me of the laser experiment I did to satisfy my lesser friends. Analogically, the trajectory would be such, if the power supply were switched off.] In T1 relaxation, the proton loses energy to the surrounding lattice, by interacting with nuclei in the lattice which are in vibrational, translational and rotational motion. Clearly, the surrounding nuclei (lattice) having the same (or nearly same) Larmor frequency will efficiently absorb energy of the excited proton, resulting in a tiny rise of temperature.

T2 relaxation (transverse or spin-spin relaxation) on the other hand, does not involve exchange of energy with the lattice.
The magnetic moments of the protons merely changes phase. Here, the nuclei exchange “quantum states” (kind of, what Einstein called ‘spooky action at a distance’): an excited nucleus (proton) will transfer its energy and relax, while the neighboring nucleus in the lower energy state that absorbs it becomes excited. This loss of phase coherence of spins can be clearly seen in this beautiful video.

It can be understood easily that T1 and T2 values would depend on the surrounding molecular environment (tissues, for example). Hence, the values differ in different tissues. Again, since Mxy decays faster, as described, it may be understood why T1 is greater than T2 (usually, T1=5T2). Both T1 and T2 contribute toward contrast in tissues. T1 relaxation time is the time needed for 63% of protons to return to their previous equilibrium state. Likewise, T2 relaxation time is the time needed for 63% of protons to become dephased owing to their interaction with nearby protons. The contrast, naturally depends on the water content of the tissues. Grey matter has about 10% more water than white matter and this creates a contrast. We can also create contrast by varying TR and TE times.

TR (Repetition Time) refers to the time gap at which consecutive RF pulses are applied; while TE (Echo Time) refers to the time delay between the applied RF pulse and its reception (echo). T1 weighted images (T1W) are produced by keeping TR and TE relatively short, while T2 weighted images (T2W) are produced by keeping TR and TE relatively long. Water molecules being relatively light spins much faster than the Larmor frequency, making energy transfer rather tough (exchanging of packets of energy becomes more efficient as the relative angular velocity narrows). Consequently, water has a long T1 time. Proteins and nucleic acids being rather heavy, spin slowly. They also have problem with energy exchanging, and thus have a long T1. Cholesterol, a medium sized molecule, precesses near the Larmor frequency, efficiently absorbing the energy and giving a small T1 value.Thus (fat) liquid cholesterol in craniopharyngiomas, a benign tumor, appears bright on T1W images (T1 being small, the rate at which RF energy is released is fast. Hence, the signal intensity in NMR is high).

Subacute hemorrhage also has shorter T1, due to the presence of paramagnetic iron in methemoglobin present in the tissue, hence high signal intensity. Cerebrospinal fluid (CSF), edema (collection of fluid in tissue space or ECF) having more water content have both long T1 & T2 relaxation time. They give low signal intensity in T1 (dark) but higher signal intensity (bright) in T2W images. T2W images are superior to their T1 counterparts in case of infarction, edema, demyelination etc. Contrast agents like the heavy metal Gadolinium, a paramagnetic substance, has been used to reduce both T1 and T2 times by introducing inhomogeneity in the magnetic field. Gadolinium is complexed (chelated) with a substance called DTPA to prevent toxic build-up inside body tissues. This gives high signal in T1W but a low signal in T2W. It (the complex) does not cross the blood brain barrier (BBB); but disruption in the BBB or parts of the brain where it is deficient (circumventricular organs), take-up the substance and affects relaxation properties.

Below is an MRI showing changes in Subacute Sclerosing Panencephalitis, a complication of measles. Note: Panels A and C are T1-weighted images; B and D are T2-weighted images. The hypointense (darker) signal on the T1-weighted image (arrow in A) and a hyperintense (bright) signal on the T2-weighted image (arrow in B) can be clearly seen.

Given all these, it can be said that relaxation parameters of nuclei have enabled us in visualizing biological tissues nonivasively, identifying chemicals spectroscopically and a lot more as we shall see later.

• References: Magnetic Resonance Imaging: David D. Stark, William. G. Bradley, Jr.

• NMR spectroscopy
• Magnetic Resonance Imaging
• Principles of NMR
• William P. Dillon. Neuroimaging in Neurologic Disorders. In: Harrison's Principles of Internal Medicine, 17th Ed., Volume 2, McGraw Hill; 2008. p. 2491-2497.
• Ian L. Pykett, Ph.D., Jeffrey H. Newhouse, M.D., Ferdinando S. Buonanno, M.D., Thomas J. Brady, M.D., Mark R. Goldman, M.D., J. Philip Kistler, M.D., & Gerald M. Pohost, M.D. (1982). Principles of Nuclear Magnetic Resonance Imaging Radiology
• Time constants

Last Modified: Aug 19, 2010

Understanding the Basic Principles of Nuclear Magnetic Resonance Imaging

Nuclear Magnetic Resonance Imaging (NMRI), better known as Magnetic Resonance Imaging (MRI) in medical parlance, is an invaluable tool in the study of the neurological system, soft tissue and musculo-skeletal system disorders. The word “Nuclear” was intentionally dropped later, as the procedure could then be wrongly interpreted by patients in relation to “ionizing radiation”, which certainly is not the case. However, the term Nuclear Magnetic Resonance (NMR) continues to be used in other (non-medical) fields of science, such as analytical chemistry, physics, biochemistry, petroleum industry, analysis of biological samples etc. In either case, the procedure and the basic principles remain the same. Paul Lauterbur was one of the pioneering inventors of this seemingly tough technological field.

Matter is made up of atoms, which in turn, are composed of negatively charged electrons orbiting around the nucleus (look at the animation of a Helium atom on the left), consisting of positively charged protons and charge-less neutrons (with the exception of Hydrogen 1H nucleus, which contains a single proton and no neutron). These subatomic particles (electron, proton etc) somehow, can not be understood in terms of shape or color; instead they are denoted by their charge, mass or spin (angular momentum). An even number of them will cancel each other’s spin [just like two revolving spheres, in touch with each other would, in a ‘classical world’ (if one rotated clockwise, the other would rotate anticlockwise, canceling any resultant spin)].

Hence, a net resultant spin would result in the nucleus only if it contained an odd (unpaired) number of protons, an odd number of neutron or both. [The concept that certain nuclear species had angular momentum was first suggested by Wolfgang Pauli, while explaining the fine structures in the Atomic spectra. In the presence of an external magnetic field, the spectral lines got split, depending on the strength of the field (Zeeman Effect).]

Since nucleons bear a net charge (owing to the protons contained), the spinning nuclei will generate a magnetic field (since moving charges generate magnetic field). Each of these charged spinning ‘spheres’, hence, may be thought of as a tiny bar magnet having a magnetic dipole (that is a north-south orientation). [Electrons, similarly, have their own angular momentum though, responsible for molecular structure which nature uses, but they are not used by humans (Milestones in Spin podcast)] When we talk about “MRI” in humans, we mean proton nuclear magnetic resonance; i.e. NMR that detects the presence of hydrogen (proton) nuclei.

Our bodies have a plentiful of Hydrogen atoms: from the water within us, in cells and in extracellular fuid, (and to a lesser extent to the adipose tissue (fat)). These charge-carrying ‘unpaired’ protons (Hydrogen nuclei) rotate around their axes, but since all are spinning in a random fashion (as there’s no coordinator of any sorts); their net spin is zero, or in other words, their net magnetic moment is zero (as shown on the left).

Understanding spins aren't easy either. But, Prof. Stephen Hawking made it quite simpler for us using the real classical world analogy of ‘playing cards’ in his famous book A Brief History of Time (follow the link to learn more about ‘spin’). Having said that, the unpaired, positively charged protons having half integer (1/2) spins, behave like magnetic dipoles; it may now be understood easily that the spinning protons (nuclei) would align themselves to an externally applied magnetic field.

Thus, in a static magnetic field, the randomly oriented ‘tiny bar magnets’ align themselves up according to the applied magnetic field. These spinning protons (nuclei) also precess (make an angle) with the applied magnetic field (Bo), much like a spinning top does when its angular momentum diminishes. An animation of a proton precessing around a field is shown on the right.

[The magnet used for this purpose employs superconductivity. In a superconductive magnet, the electromagnet coils are immersed in liquid Helium at minus 269 degree Centigrade. At such a low temperature, the coils loose ‘resistance’ to the flow of electrons, resulting in a highly stable and a very strong magnet. (However, any minute vibration in the superconducting magnet can lead to runaway Eddy current leading to a phenomenon called 'quenching', that happened in the Large Hadron Collider at CERN, collapsing the whole setup.) Normally, 1.5 Tesla magnets are used, though nowadays 7 Tesla magnets have arrived. A 1 Tesla (1 Tesla=10,000 Gauss) magnet is 20,000 times stronger than the earth’s magnetic field)]. Also, note that we are considering magnetic moments along the axis of the external field only, as far as the sum-total alignment of individual magnetic torque contributing to a 'macroscopic' magnetization (M) is concerned. This is because the transverse components of the individual spins cancel out, as is seen in the 'cone' of the above picture. 600 persons of equal power, each pulling a rope either 30 degrees Northwest or 30 degree Northeast (in a 2 dimension), will certainly cancel out the 'east-west' vector, while the Northward vector will add-up. [It is this M that produces the induction current in the receiver coil].

The protons have two choices. Either they have to align parallel or anti-parallel to the applied magnetic field (known as spin-up and spin-down position respectively). In any case, the protons only ‘partially polarize’ since they tend to ‘make an angle’ with the applied static magnetic field. Spin down position is the higher energy state while spin-up state is the lower energy state of the spinning protons (in the case of 23Na, there can be 4 spin-states instead of 2 as in 1H). (Obviously, a swimmer swimming upstream has more energy than his antiparallel counterpart.) The protons revolve (precess) around the direction of the magnetic field (Bo) at an angle, while at the same time they rotate around their own axis. Just as what happens in the solar system. [However, the upper (-1/2) and lower energy (+1/2) spin states are almost equally populated with only a very small excess in the lower energy state at room temperature. Since, there are so many of them that we finally make some headway].

Let me clarify a bit. You've seen a spinning-top rotating around its own axis. Due to Earth’s gravitational field, the top ‘maintains’ an angle (with the perpendicular/vertical), more visible when its angular momentum (speed) decreases, as it continues spinning. The top may be seen to revolve around “the perpendicular” at an angle (=‘precess’), (in addition to its rotation around its “own axis”) during its course of revolution. [Watch the Video "Introductory NMR & MRI Video 01 Precession and Resonance" to see what precession in NMR is]. This is what precession is about.

The frequency of precession is given by the Larmor relationship:
f=w/2*pi=yBo/2*pi (2*pi=360 degree)
w=angular freq. in radians per second; since there are 2*pi radians (360 degree) in a circle; we can find f, the frequency of rotation.
y is the magnetogyric (gyromagnetic) ratio, nuclear constant characteristic of every isotope. For 1H it is 42.5 MHz/T;
Bo=static magnetic field

The above equation is important, as we shall see later. Now let’s summarize what we learned so far.
Protons (nuclei) spin randomly in an atom. They tend to align with respect to an external magnetic field. These protons make an angle with the magnetic field as it goes about the magnetic field (while it also dutifully goes around itself), some parallel, and some antiparallel.

In MRI, our objective will be to disturb this alignment of protons with a dose of radio frequency pulse, in a similar way I discussed in my radio transmitter article but in a much, much bigger way. But since the ‘target’ (proton) is moving (precessing) around the field, we better ‘punched’ the target as if we were moving at the same angular velocity (so that the relative velocity was zero). Thus, when we apply the RF frequency pulses at the Larmor frequency, perpendicular to the magnetic field; the magnetic component (B1) of this electromagnetic wave temporarily knocks the protons out of alignment (see picture). If energy is absorbed by the nucleus, then the angle of precession will change. Assuming the field strength to be 1 Tesla, the protons are revolving 42.5 million times per second; it is at this frequency we give the pulse (i.e. at the Larmor frequency).

The protons are pushed out of alignment and as the pulse ends, they ‘relax’ (more on how they ‘relax’, later) back to their undisturbed ‘equilibrium’ position. This causes emission of an RF signal (the Echo) that can be picked up by the receiver coil (the same transmitter coil that produced it, in most cases); a damped oscillating wave generated, as the ‘disturbed’ magnetic moments coming back to realign with the magnetic field. Now, the problem begins. We have applied a uniform/homogeneous magnetic field (Bo). There are a lot of protons but we don’t know who’s who and residing where. That is why we also apply orthogonal magnetic field ‘gradients’ along the three (x, y, z) axes. [In a classroom, spray gradually ‘more’ yellow color in the front row and to the left than the back and to the right. In a similar way, spray blue color; hope your students don’t object. Now, every one of your students has a unique color: yellow, blue or green and with different hues]

Now that we get a decaying signal, which of high frequency; we mix it with a low frequency signal, in much the similar way as in heterodyning, to produce an ‘interferogram’. This interference map is digitized, which is called the Free Induction Decay (FID). Thus, we do find too many frequencies in ‘the low frequency map’ which occur in ‘almost’ the same time. So, what can we do?

Waka Waka! In this football World Cup 2010 at South Africa, audience seems to have a deafening organ, what they call ‘vuvuzela’. How are we going to analyze so many vuvuzelas when they are blowing at the ‘same time’? Just plot them in ‘frequency domain’ instead of ‘time domain’. Here’s Discrete Fourier Transform (DFT) which will do happily for you. [Simply put, it samples the different frequencies and plots them; not all vuvuzelas have the same frequency]

Now, that fuzzy picture of multiple frequencies has a 'spatial information' (owing to its orthogonal gradient magnetic field), contrast information (due to its ‘relaxation’ parameter), and foremost that it can be analyzed visually by humans, have enabled MRI to be a indispensable tool for the medical professional, as much as NMR has to the physicist or the discerning chemist. In MRI (NMR) it is not that important where or how energy is absorbed, but how quickly the excited protons revert back to its previous position is much more important, and hence the relevance of T1 and T2 relaxation times.

By the way, contrast depends on the t1 and t2 relaxation, the surrounding chemical environment affecting relaxation, and of course the water content of the tissues [gray matter contains 10-15% more water than white matter.]

Finally, the article wouldn't be resourceful enough if I do not post some MRI scans of the brain, this time, that of an epileptic patient (below).

(A sagittal section is obtained as the 'slice' takes a 'left to right' view (and vice versa); a coronal section means a 'front to back' view (or vice versa), and an axial slice means a virtual transverse section through the head.) Here's the picture of an actual MRI Machine below:

Naturally, the small tunnel may induce claustrophobia; the whirring acoustic noise from switched gradient coils may be troublesome to the patient; any implanted pacemaker may be subjected to interference from the electrical field resulting in dislodgement or malfunction (as in other ferromagnetic objects such as wrist watch, key rings etc.). Moreover, sudden movement by the patient may induce voltage in semicircular canal producing vertigo, a sensation of giddiness. Advances in MRI technology is happening fast. Claustrophobia may now be ameliorated with a wide bore MRI. A newly developed MRI scanner with Total Imaging Matrix (TIM) technology patients don't feel as claustrophobic, the imaging time is quick, quality of picture is better and even the acoustic noise is less (watch the YouTube video here). Whatever be the shortcomings of MRI, the benefits far outweigh the risks and it is here to stay and evolve.

References:
Magnetic Resonance Imaging: David D. Stark, William. G. Bradley, Jr.
NMR spectroscopy
Magnetic Resonance Imaging
MRI basics
Principles of NMR
William P. Dillon. Neuroimaging in Neurologic Disorders. In: Harrison's Principles of Internal Medicine, 17th Ed., Volume 2, McGraw Hill; 2008. p. 2491-2497.
Ian L. Pykett, Ph.D., Jeffrey H. Newhouse, M.D., Ferdinando S. Buonanno, M.D., Thomas J. Brady, M.D., Mark R. Goldman, M.D., J. Philip Kistler, M.D., & Gerald M. Pohost, M.D. (1982). Principles of Nuclear MagneticResonance Imaging Radiology
P.S. We will discuss T1 and T2 relaxation, fMRI, tractography and NMR spectroscopy later.

Created: Jun 24, 2010; Last modified: Mar 10, 2014

Fourier Analysis: The Art and Science of Finding The Needle in a Haystack

Every time I listen to the heavy metal band Pantera my wife would invariably wonder aloud why I listen to all this ‘noise’. True, many music lovers would rather refer bands like Pantera as quintessential noise than music; there are persons like me who can dissect the melody from the apparent chaos of runaway frequencies of guitars, drums and so on. I can even analyze and follow individual instruments over time. This is what Fourier is about, or stated otherwise, my ear & brain can be said to be doing a Fourier transform on the said musical piece.

Joseph Fourier, a French mathematician, realized that all periodic waves could be ‘synthesized’ by mixing sine waves of right frequency, amplitude and phase. For example, a square wave could be prepared by ‘adding’ the fundamental frequency (the lowest frequency; say 70Hz) with an infinite number of its odd harmonics (e.g. 210Hz, 350Hz, 490Hz and so on. Harmonics are multiples of the fundamental frequency.) This is Fourier synthesis. Similarly, you could break down a periodic signal in which the amplitude varies over time into one of a frequency versus time graph. This is Fourier analysis, and it can be seen that here we are actually ‘decomposing’ the ‘signal’ into its frequency spectrum, over time. The process of decomposing a function into its constituent frequencies is known as Fourier transform. You can have a ‘hands on experience’ at what a square wave ‘looks’ or ‘sounds’ like and how a periodic wave is decomposed into its constituent parts here. Do experiment on the sine, cosine, triangle wave and square wave functions as well and turn on the sound of your PC while you are at this site!

While Fourier originally devised this to solve the problem of heat propagation, the impact of Fourier analysis can now be felt in almost every field of science, instrumentation, entertainment and telecommunications, and even arts. Whenever you use your audio graphic equalizer to suit a piece of musical performance to your taste, you are doing a Fourier. Here you are boosting some particular audio frequencies while suppressing others, obtained by a Fourier analysis of the audio signal. You are assigning relative weights to the frequencies by sliding those sliders. Likewise, when you compress a picture (graphic) file using software such as JPEG, an inbuilt program does a Fourier transform,--> eliminates the weaker components from the analysis and--> then saves the information in a compact way.

In Nuclear Magnetic Resonance Imaging (NMRI), the emitted radio frequency is Fourier transformed to give frequency versus time, throwing valuable information about nuclear spins. Fourier analysis may also be employed to remove mains AC hum frequencies, in mobile telephony and many other situations.

One day, we may expect, that Fourier analysis may be used to pick up the ‘right frequency’ in the brain EEG waves and may put the study of ‘mirror neuron’ and ‘thought controlled devices’ into a whole new domain.

Last modified: May15, 2013
Reference: Fourier analysis (Wikipedia),
Explained: The Discrete Fourier Transform

Phase Alignment of Neocortical Gamma Oscillations by Hippocampal Theta Waves

An empty brain is the devil’s workshop, goes the proverb. Actually, the brain is never empty. Even in our deepest slumber, the brain continues to weave waves of electrical rhythms that can be seen with the aid of electroencephalogram or EEG. When we place electrodes on the scalp or on the cortex (inside the skull), and amplify the faint signals via bioinstrumentation amplifier, we can lay our hands on these fluctuating rhythms. (More on the electronics of EEG may be found at the OpenEEG project site).

We have as many as 100 billion neurons in the brain. In the superficial layers of the cortex, the neurons have numerous dendrites branching out from the soma or cell body (shown in grey oval in this picture).These neurons have been compared to a forest of trees where the branches are the dendrites and the trunk the axon. These dendrites make extensive connections among each other. They also get connections from the axon collaterals of neighboring axons (i.e. the 'trunks' of other trees connect to these 'twigs' by offshoot from the trunks). Since there are a lot of axons converging on the dendrites of each neuron, and given the fact that these axons can be excitatory (red) or inhibitory (green) depending on the neurotransmitter, the sum of input may be either negative or positive (with respect to the cell body). Thus an alternating current (cortical dipole) will flow between the shifting dendrites and the soma. This along with thalamocortical oscillations produces the EEG waves.

The brain doesn’t churn out the rhythm just like that. Had the neurons fired randomly the oscillations would have cancelled out.EEG waves occur due to synchronous discharge of neurons producing the alpha, beta, theta, gamma and other telltale waves. Like all other electrical waves, they too have a frequency and amplitude. Alpha waves, for example, have a frequency of 8-12 Hz (cycles per second) and an amplitude ranging from 50-100 microvolt when recorded from the scalp, and it is found when a person is resting comfortably with eyes closed and the mind wandering. On the other hand, gamma rhythm has a frequency of 30-80 Hz, and it is found when a person is deeply engrossed on some work.

It was known for a long time that the hippocampus exerted a role in learning by fostering long term potentiation (LTP) by aligning the neocortex, where memories are stored. The mechanisms behind this are now emerging. Sirota et al and Siapas et al have analyzed rat brains and found out that there were many localized gamma oscillators within the brain that gave rise to neocortical gamma bursts. These oscillators had varying frequencies but they phase aligned themselves with the arrival of hippocampal theta waves. A large fraction of pyramidal cells and interneurons too were phase aligned to the hippocampal theta rhythm.This is similar to a bar magnet aligning iron dust or other ferromagnetic materials by virtue of its magnetic field. Apart from the cerebral cortex, the cerebellar cortex and the hippocampus too can generate brain waves. Such a mechanism may explain the orchestration of many parts of the cortex (and hence the memory engrams they contain); and data synchronization and downloading to the hippocampus for memory retrieval. It also shows how hippocampus does the ‘indexing’ of cortical contents. These experiments throw light on neuronal plasticity and information flow, and may be someday they could help clinicians in fighting memory loss as it occurs in neurodegenerative diseases like Alzheimer’s disease.

Last modified: never
References:

Prefrontal Phase Locking to Hippocampal Theta Oscillations
Athanassios G. Siapas, Evgueniy V. Lubenov and Matthew A. Wilson. doi:10.1016/j.neuron.2005.02.028
A SIROTA, S MONTGOMERY, S FUJISAWA, Y ISOMURA, M ZUGARO, G BUZSAKI (2008). Entrainment of Neocortical Neurons and Gamma Oscillations by the Hippocampal Theta Rhythm Neuron, 60 (4), 683-697 DOI: 10.1016/j.neuron.2008.09.014

PET Scan: Particle Physics And Electronics in Medical Imaging

Numerous imaging modalities are there to view anatomical structures in our body. They include X rays, Ultrasound imaging, MRI and many other procedures where we can see normal or diseased tissues in our body. They tell us ‘the location’ (position) in the body the image corresponds to. If we wanted to see what were happening in these locations, we would then need to perform functional imaging techniques like PET scan or fMRI.

In Positron Emission Tomography or PET, a radioactive isotope that decays by positron emission is introduced into the body. Positron emitting radioisotopes are prepared by bombarding stable atomic nuclei by protons. Protons are speeded up in a particle accelerator called cyclotron which then impinge upon the stable nuclei, and knocks out one neutron from its nucleus. The proton now occupies the position where the ousted neutron once stayed. But this atomic configuration is unstable, so the proton now decays. It decays by emitting positron, a particle resembling an electron in all aspects except that the charge is positive and not negative. In other words, a positron is an antimatter: an anti-electron.

Many different radioisotopes are there, such as Fluorine18, Oxygen15, and Carbon11. 18F is the most commonly used isotope. It replaces hydroxyl (OH) group in molecules of interest. We can use 18F Fluorodeoxyglucose (18FDG) an analog of glucose for probing the activity of brain. Our brain uses glucose for its metabolism, so when it encounters 18FDG, it stores them. The FDG in the brain begins emitting positrons. These particles travel only a short distance before they meet nearby electrons and annihilate. Two gamma ray photons, each having 511 keV of energy are produced--- Photons because they are electromagnetic waves, and gamma ray because the frequencies correspond to the gamma ray spectrum of electromagnetic waves.

So by detecting these photons, we can find out where they came from, since we know that these photons are emitted back to back, 180 degrees apart. (They aren’t exactly collinear as their initial velocity is not zero, and some computational error always creeps in). For the detection part, we need a detection array which will convert these photons into electrons. This is done by scintillators. Bismuth germanate, Luterium oxyorthosilicate (LSO) are some of them. Photons which are incident on them produce electrons by photoelectric effect. These electrons are then guided through a vacuum tube, which has many positive electrodes (dynodes) held at successively higher voltages. These dynodes of this photomultiplier tube accelerate these electrons, which in turn knock-off more electrons from the dynode plates. Thus we get more electrons than what we started with. The signal has now been amplified and we now have a measurable current.

With advancement in detection technology, silicon avalanche photodiodes (silicon APD) has now shown promise to replace the vacuum technology (photomultiplier tube). As the name suggests, APDs work in a similar way an avalanche gains its momentum as it descends from the mountain-avalanche effect. Detecting photons aren’t sufficient. We need to detect only co-linear (coincident) photons. Each collinear photon pair (i.e. 180 degrees apart) will constitute an event. All other photons (noise) must be rejected. About 10^7 to 10^8 or more ‘events’ must be registered in order to have a good signal to noise ratio. Image faithfulness varies proportionally with the square root of the number of events.

By acquiring a large number of events, the computer software is able to determine exactly where these radioactive tracers are located. This, in our case, means the locations where the neurons are accumulating (accumulation is a function of utilization of glucose) 18FDG. Thus we get a functional map. In order to know ‘what’ these structure were, we need to combine anatomical imaging like MRI or CT with PET. This combined PET-CT or PET-MRI let us know what structures are doing how much.

PET scan is very useful in neuroscience researches, clinical diagnoses like cancer detection, receptor analyses and even watching gene expression in molecular biology.

References: A good site with animation: PET animation

Physicsworld

Last Mod: 10 Mar, 2014

Electron Microscopy And Graphene

Look at the sieve on the left. The perforations have a fixed diameter. We can study these holes by throwing balls at it. If the balls pass through, then the diameter of the holes are obviously more than those of the balls, (at least in one axis, if the balls were a bit oblong). Likewise, when the balls bounce off, we can conclude that the diameter of the holes are less than those of the ball.

Electron Microscopy (EM) involves the use of electrons to visualize atoms and molecules. Optical microscopy does not yield much high resolution, since the wavelength of light is more, allowing them to cover ‘more than one hole’ in the above analogy. If we employ a 1 centimeter ruler to measure an object 12 cm long, we will do much better than if we employed a 6 cm ruler, isn't it? The resolution will be much better then, 12 pixels vs 2 !

Using electrons allow us to do just that! Electrons can be thought of as wave, whose wavelength is related to the Planck's constant divided by its momentum (de Broglie hypothesis). The more you accelerated an electron, its wavelength became smaller and smaller. But you can't play dice with light, Einstein might book you for breaking speed limit then. You can't go faster than light, they say.

Now look at the diagram, provided courtesy of Opensource Handbook of Nanoscience and Nanotechnology. [Real marvelous illustration, by Kristian Molhave.] While optical microscopy used lenses for magnification of the image; in electron microscopy, electromagnetic and electrostatic lenses are used. You have seen how your hair stood on end when you combed your hair: thats electrostatic force. I did another hair-raising experiment during my childhood. I brought a strong magnet near the picture tube of my color TV (not LCD type but a CRT one). I saw a rainbow kind of pattern near the magnet. Its electromagnetic force! (DON'T do it yourself, else you may ruin your TV and a procedure called de-gaussing may be necessary).

Next we need a platform on which the sample should be placed: strong enough to tolerate the fast and furious electrons, yet transparent enough to let the electrons pass through, in case of transmission electron microscopy (TEM). Graphene, a one atom, one dimensional layer of carbon
is now being given a serious consideration as the candidate substrate. Now, Jannik Meyer, Alex Zettl and colleagues at the University of California, Berkeley have been able to visualize single atoms, such as hydrogen and carbon atoms, using this graphene substrate. However, speeding electrons sometimes drilled holes in the layer, even imparted some of its energy to make some hydrocarbon molecules move. They even watched one such hole being repaired using carbon from the atmosphere. One interesting thing is that, the carbon atoms in the graphene layer don't show them up, the orderly lattice structure takes care of that.

In scanning electron microscopy, the electron beam is scanned, as shown. The deflected (not transmitted!) beam containing information of the object, is picked up, and amplified by an electronic op-amp circuit. Thus, while you see whats inside of a cell in TEM, SEM allows you to have a pick of what goes on on the surface. But, how do you generate electrons in the first place? Simple! a heated tungsten filament will happily do it for you. And to speed them up, attract them with a bait, a high voltage anode. Don't forget to regulate the voltage and be careful of stray magnetic fields.

Looks like there's plenty of room at the bottom.
Last modified: Jun 26, 2010
Reference: Silcox, J. (2008). Microscopy: Spot the atom Nature, 454 (7202), 283-284 DOI: 10.1038/454283a

How About Letting A Capsule Videograph Your Intestine?

In electronics we use integrated circuits (IC) to digitize analog signals, amplify them, and encode and transmit them where necessary. Digital logic ICs come in handy where generation of oscillator frequencies, for transmission of signals is needed.

These logic ICs come in broadly 2 types: CMOS-FET (Complementary Metal Oxide Semiconductor- Field Effect Transistor) and TTL (Transistor Transistor Logic). While TTL ICs offer higher speed, they also consume higher current and a fixed regulated voltage (typically 5V). CMOS ICs work happily anywhere between 3V and 18V. Voltage regulation is not necessary. Moreover, they consume much less current, making a battery last longer.

This is perhaps why scientists used CMOS devices in this capsule. They used a CMOS capsule (shown on the left) to film the innards of human beings. Patients were made to ingest (take orally) this capsule, which contained a tiny camera and was capable of transmitting the pictures, as it traveled through the gastrointestinal tract. It photographed noninvasively and unobtrusively. The wireless endoscope snapped and transmitted the films in several frames per second. A radio receiver was placed outside the body to pick the signals and the signals were processed. You get the picture effortlessly while the capsule toiled.

The capsule labored till the battery lasted or it was passed in the stool. The whole story sounds somewhat like voyager space probes. These little capsules are now routinely assisting surgeons in finding polyps, tumors and other pathologies like ulcerated mucosae. The picture on the left shows a real snap of small intestinal mucosal erosion (red area in the center). In a study in Imperial College, London, UK, by Hamdulay and others on Behcet syndrome, 10 out of 11 known cases showed intestinal ulcerations in this method.

May be in near future they will even perform surgeries through remote control.

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

Every Breath You Take, I'll Be Watching You

When The Beatles sang " What goes on in your heart? What goes on in your mind?" and The Police warned "Every breath you take", you thought they exaggerated. Now we know they didn't. Scientists have really advanced so much on picking up and interpreting faint magnetic signals, this could soon be a reality; reading and interpreting our minds, non-invasively.

As action potentials are generated in the heart or while we think, electromagnetic waves are generated. These waves are produced as a result of changing ionic fluxes in these organs. We have been capturing them through ECG, EEG and magneto-encephalograms (MEG) for quite some time. As science and technology advances, gadgets shrink, gadgets think, become more sensitive and specific.

When a electrically conducting piece of metal is kept in a magnetic field, the magnetic field will exert a force on the moving electrons that will push them to one side, creating a voltage differential in the conductor. This effect is known as Hall effect and has given the birth of many devices like Gaussmeter or magnetometer. Hall effect sensor ICs are used in many applications of electronics, requiring sensing of magnetic fields. (I found one Hall IC inside my floppy disk drive, at the periphery of the circular flywheel.) Sensing brain waves will not be easy using Hall ic though, as the device is not that sensitive.

Superconducting quantum interference devices (SQUID) are much more sensitive than the former. The intensity of brain's magnetic field just outside the skull varies from 0.1 to 1 picotesla, less than a hundred-millionth of Earth's magnetic field. SQUIDs consist of two liquid helium cooled superconductors sandwiching a piece of thin insulator, creating two Josephson junctions. The presence of any stray magnetic field gives rise to interference and is detected by SQUID probes. So sensitive is this technology that it is being used to probe biomagnetism, gravitational waves and other extremely weak phenomena. But it has its inherent weaknesses too. It is bulky, expensive, power hungry and needs to be cooled near absolute zero.

Now scientists are devising gadgets that can operate near room temperature, handy and relatively inexpensive. A cell containing an alkali gas (like rubidium) will practically transmit all of a circularly polarized laser beam, when the spin of rubidium atoms point in the same direction. An object when placed near it, will distort the spins and some laser beam will be absorbed. Such vapor cells has sensitivity in the femtotesla range. Got to be more careful next time you see your wife carrying a strange object in her hand and getting closer to you (or your head!).

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