Showing posts with label biophysics. Show all posts
Showing posts with label biophysics. Show all posts

February 07, 2014

Revisiting the Sternocleidomastoids: Accessory Muscles of Respiration


We know that sternocleidomastoids (strap muscles of the neck), scalene muscles and alae nasi are considered accessory muscles of breathing, although some controversy exists. If you saw an asthmatic individual in his desperate attempts at breathing or an agitated person or a person exercising vigorously, you could watch these muscles in action. However, that wasn't exactly what I had in mind while I was doing this experiment. I was really thrilled to chance upon it. Kind of a serendipitous discovery in its own right. You too can figure this out easily. The placement of the surface leads were as shown on the right. Red dots were for the reference and recording electrodes whereas the blue dot represents the ground (as usual). 

The neck muscles of each side were tested one at a time. For example, This wav file (open it Audacity or BYB neuron recorder) was obtained from EMG recording from the right sternocleidomastoid; the leads were placed on the right side and the ground lead on the manubrium. The head was at mid-line (neutral position) to start with. Next, it was turned to the right, then midline again, then to the left (without any external resistance applied), then against resistance applied by the left hand. The muscle of the right side moves the head to the left. Finally, the head was again restored in its neutral position. An improvised notation could be N to R to N to L to L+ to N (Legend: N=neutral, R = right, L=left, L+ = left against resistance). 

Similarly, the left side was tested in a likewise manner (N to L to N to R to R+ to N). And this is the waveform obtained. The surprise awaited me, I was in for an ambush!

While the leads were still on the strap muscles of the left side of my neck, I observed the EMG waveform in real-time. I noticed that as I was taking a deep breath, the EMG activity increased significantly. I didn't have to turn to my head to the right anymore! I then maintained this position by holding my breath. The activity continued. Here's the recorded .wav file. The associated camera recording will speak for it (see below: Youtube). Yes, it proved that it was indeed an accessory muscle called in to address forceful inhalation. I then did a forceful exhalation, but no increase in EMG activity was observed, buttressing my observation. 



As you may have noticed that I have used the terms inhalation and exhalation, in lieu of, inspiration and expiration. Well, inspiration, it definitely is! Expiration? No way!
Further analysis still awaits.
Spikerbox recordings may also possibly illustrate simultaneous EMG activities in protagonist and antagonist muscles. More of these later.

To be continued............
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Making a Human Interface Device Using SpikerBox

Q said to Bond: It's activated by nerve impulses from the wrist muscles. This dialog from the 1979 James Bond movie, Moonraker, seems to fit into place when we talk about interfacing biosignals (surface EMG, in this case) with an actuator. [Watch a clip from the movie below]




In order to achieve this human-machine interface using our good not-so-old spikerbox, we need to estimate the output signal amplitude coming from the extensors of the wrist (around 5 mV), amplify the signal by some 300 times by cascading two LM 386 IC (or any opamp taking care that the device doesn't go into saturation) to about 1500 mV. We can then connect the output from pin 5 of the IC via the 250 MFD to a *1K resistor (* = value to be experimented with) to ground. Next we connect a wire from negative terminal of the electrolytic capacitor to pin 13 of CD 4066, a quad bilateral switch IC. The Boolean output (on/off logic) signal may then be driven into terminals of a 'laser pointer', whose push button terminals will have to be replaced by the output from pins 1 and 2 of CD4016/4066. A sketch of it is shown below. Pardon my bad drawing 'skill'! Interfacing should not be a problem as the power supply is 5V and CMOS ICs are happy with that.






Output level of LM386 is at half the supply voltage level (i.e. at 2.5 V). At +5mV signal level, voltage at pin 5 becomes 4.0 V [2.5 + (5*300*10^-3)], making the control pin of 4066 go logic high. A low resistance (250 ohm) path is established between pin 1 and 2 of this IC, hopefully switching the laser on. We better not play with darts at this moment!

Another idea is to hook up an optical mouse in such a way that its internal potentiometer is swapped with a twin light-dependent resistor (LDR) as shown below. The picture at the left/top panel shows the component side of the mouse and the soldered side at the right. The three leads in the rectangle 'marked' in the right/bottom picture panel on the soldered side may be replaced by the new pot using a twin LDR, as shown in the middle panel (its middle two leads connected to form the central/common lead of the new three terminal pot as shown in red arrows). 











The input maybe obtained preferably in stereo from the sternocleidomastoids of both sides. And the output amplified and fed into two LEDs separated by a window which communicate in a 'line of sight' with the 'twin LDR' setup. This would couple the two circuits (the SpikerBox setup and the laptop via the 'doctored' mouse) optically. The scrolling function (up/down) may then be undertaken by moving the neck sideways. The experimental setup may be as per this link. Details to be updated at a later date. You too may contribute.

Disclaimer: This circuit is a mere prototype, liable to have errors. I will check it at a later date, got to hone my electronic skill a little and get out of any further procrastination.

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Last modified: Apr20, 2014
Reference: hyper-links, unless specifically mentioned

Surface EMG from Thumb: Strongly 'Opposed' ?

Lead placement for the experiment, as shown on the right.
As I have already mentioned, the ground electrode sits atop
the manubrium sterni, on the upper chest (breastbone).

The camera recording is shown below. You can see the Piezoelectric crystal's spark and also hear its sound on snapping. However, I haven't yet had time to analyze if the spark did contribute some 'useful' static on the trace (open & watch this .wav file in BYB software).






Method: Pressing on a piezo crytal (from a cigarette lighter) by 'opposing' the thumb against the base of the little finger (as shown by the figure). Electrode placement shown in red & green markings [the ground electrode sits atop manubrium sterni]. Muscle (mainly) contracting is "opponens pollicis", a small, triangular muscle in the hand, which functions to oppose the thumb. [Strictly speaking, opposition of the thumb refers to the tip of the thumb touching the tips of other fingers. But that way, generating enough force without creating an unnecessary torque that topples the piezo is quite a challenge! After all, I had no assistant, and had to hold the camera too with the other hand]

Concept: A fixed and constant amount of force is necessary each time before the spring yields and the piezo fires. This end point is supposed to be caught in the trace 1) as a sharp spike after the surface EMG pattern or, 2) can be 'used' as a 'static'.

EMG amplitude and rate of spikes will need to be analyzed in short, discrete time intervals by simple counting.

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February 06, 2014

Interpreting Rate Coding Data: Counting Spikes in a Tracing & Consequent Plotting of Firing Rate Versus Time

We acquire data by using the following device configuration (the green laptop cable will go to the laptop mic input, running on battery power) as shown below. Please see this link to learn about the anatomy of a coaxial cable, so that you can make a ground connection off a coaxial cable.

You can also take the ground off the reference point used for the Faraday cage clip, if you'd like. Incoming analog signals from the spikerbox will go into the internal ADC (Analog to Digital Converter) of the Conexant HD audio port of the laptop, whence the signal will be digitally processed. After a signal has been stored either in Audacity or in the BYB neuron recorder, we can open the'.aup' or the '.wav' file respectively later.

Here, I am attaching a typical wav file captured by striking the quadriceps tendon with a percussion hammer (knee jerk). This is a deep (tendon) reflex. I'll post more on this later. You need to open it using either of the two softwares listed. I am posting a screenshot on the right.
You can see three large spikes in the tracing. These spikes are actually not single/solitary, as they might innocuously suggest, but they are a conglomeration of many individual ones.

The below tutorial explains how to interpret these raw data.



By the way, there's no simple way to count spikes! First, we need to define spikes. We can fix an arbitrary threshold, beyond which we will label them as one. Here are some very rudimentary thoughts on counting:
  • Coupling/buffering the output from the laptop audio-out port via ICs like ULN 2004 Darlington transistor arrays to drive filament lamps [these filament lamps are a modest way to an integrator as the heat takes time to dissipate off]. The more the spike rates, greater will be the brightness. Then pick-up this optical signal by an LDR (Light Dependent Resistor) and then noting the resistance change [precalibration is necessary]. We can use capacitors too (using their exponential charging/discharging equations) or a linear optocoupler like MOC5010.
  • Software: writing some code snippets to Matlab/Python programs 
  • Using a Foster-Seeley phase discriminator: However, a new algorithm should be developed.
  • Converting the electrical signals to mechanical tracings on a rotating Kymograph [speed adjusted] may make it easier to read and count
  • Allowing only signals above a certain 'predefined' threshold voltage to pass through. We may use diodes for this cut-off estimation. Next, employ a CD 4520, a dual 4 BIT binary up counter that advances from LOW to High transition on clock input '0' (CK0) when CK1 is high, after the signal has been "gate"d . We can use an appropriate crystal to this counting purpose. Many optically based circuits are available on the net.
  • We can think of using a frequency to voltage converter IC like LM 2907
  • But presently, i'll be counting them manually. My time limitations and fading electronic experiences precludes any pursuits of above kind.
After we have successfully counted the 'rates' of 'action potential's, we can plot their number/spikes vs time plot in a linear or logarithmic scale. 


Here's where I positioned the ground electrode, on the manubrium sterni, on the upper chest (breastbone). The overlying hairs had been shaved and cleaned with spirit to reduce the input impedance. In all the spikerbox experiments the placement of the ground remained the same unless otherwise stated. 

To be continued....

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Basic Concepts and some 'Assumptions' in SpikerBox Biopotential Experiments

Exploring signals of very low amplitude, as is usual in most bioelectrical signaling, is not an easy job. In many instances the power line hum often drowns these weak electromagnetic dialects. In fact, our body acts as an antenna in receiving these wayward disturbances. We can curb these interference by the following means:

  • Opting for battery operated power for the sensing device (the bio-instrumentation amplifier) as well as the data acquisition inputs, for example, a laptop that runs on battery. The brightness of the laptop display maybe set to maximum, as some laptops may emit significant stray noise when brightness is not saturated.
  • Switching off any fluorescent lights
  • By proper Earthing of the equipment
  • Using differential op-amp configuration for adequate noise suppression by eliminating common mode signals
  • Using a preamplifier; and coupler gels that reduce electrical impedance (resistance) between the electrode (lead) and the body surface
  • Choosing an operational amplifier having a high CMRR (common mode rejection ratio)
  • By using a unity gain amplifier (voltage follower) configuration that has a very high input impedance
  • Employing a noise cancelling mechanism like the 'right leg driver'
  • Using a Farady Cage for effective 'shielding' from electromagnetic interference (static)
  • Employing a hardware filter (band stop or Notch filter) or a software program to eliminate/reject a particular frequency band (e.g. 50 Hertz or 60 Hz cycle frequencies)
There's many more options to tackle the undesirable gatecrasher, the 'hum'. Yet it is so difficult to achieve. Easier said than done!

To probe a physiological or pathophysiological response, a stimulus requires that it be precise/ accurate in timing, amplitude, reproducible, relatively less damaging and that it leaves a 'desirable' stimulus artifact so that we can calculate the delay in response in the tracing. Of the various types of stimuli available we generally prefer electrical stimulation over others; such as, mechanical, thermal and chemical, for the above reasons. 

Yet, we sometimes need to take recourse to other forms; as I was kinda forced to use mechanical stimulation to elicit a 'knee jerk' or that I had to abandon an interesting project (Hoffmann's reflex) as the TENS (Transcutaneous Electrical Nerve Stimulation) unit would invariably inject more than enough electromagnetic static into the waveform to render it useless. Marking the exact instant of an applied mechanical stimulus is bothering. I tried to circumvent this by making an "induction circuit", the makeshift switch of which
would be placed on the patellar tendon and 'close' on tapping by a hammer, thus sending an intended static. Also tried to sandwich a piece of piezoelectric material between the percussion hammer and the quadriceps tendon. But none worked so far. I had to remain contented by recording with a camera simultaneously, so that it picked up the sound of patellar impact!

The experiments I have done so far were mostly on obtaining 'surface Electromyography (EMG)' and nerve conduction velocity studies (NCV or NCS). The signals were fed into the SpikerBox input through coaxial cables into its RCA jack inputs, where it was amplified by an integrated circuit (IC) INA 2126P, an Instrumentation Amplifier. There was also a provision for rendering the electrical impulses 'audible' following amplification of the signal by LM 386, which was set at its default gain of 20. [For a more complete detail, please visit "Backyard Brains Wiki"] Thus, we could hear the action potentials and also interface the output with a laptop or a smartphone by softwares such as Audacity, Backyard brains neuron recorder (my own personal favorite) etc for real-time viewing, as well as, for recording for later storage, retrieval and analysis. 

The EMG recordings was done when the muscles were at rest, and at exercise: isometric or isotonic. Since I do not have a 'force transducer', a 'dynamometer' or a Mosso's ergograph; I had to invent ways to measure force/'work done' by other ingenious (or ingenuous? only time will tell !) means. Here's some:
  • spring constant (Hooke's Law) of the device maybe obtained by calibrating with a known device and by measuring how far the cylinder moves into the outer barrel (there's a spring inside that snaps hard onto a piezo crystal when force is applied).
    Piezo based cigarette lighter producing high enough voltage causing a spark thus igniting the gas 
  • by coupling it to a linear potentiometer which have also been previously calibrated (see figure), and then displaying the resistance in a multimeter or through a linear dot/bar display using LM 3914 IC.
  • Work done = Force*Distance=m(mass)*g(acceleration due to Gravity)*displacement (see schematic diagram)
Although we would love a stereo output from a 2 channel SpikerBox, the one I have, I do not have a laptop that accepts stereo microphone input.  Stereo signals are more preferable to human interface devices than plain old mono input. A stereo signal 'may' still possibly be 'recorded' by a 
  • Desktop line-in (via Avance AC97 soundcard) input but that would entail 50 Hz hum to pass through as well. 
  • A stereo (tape/CD) recorder running on battery power is a better option. The data can later be played/digitized at a later time. 
  • A stereo FM transmitter or a 3 pin to USB adapter is yet another option. 

Meanwhile, here's some relevant issues regarding Two channel spikerbox:
  • The internal connection pattern of (4 ring TRRS vs 3 ring stereo) 'smartphone'-'laptop' cable foxed me  for some time to realize that the Left & Right channels were only capacitively coupled
  • That my low-cost Penta T-Pad tablet could NOT render waveforms in BYB app while my Samsung GT-S5360 gleefully obliged. However, the screen was way too small to allow any reasonable analysis & that data longer than >=2 seconds could not be saved in the mobile
  • There was significant 'clipping'/distortion of the waveform in my mobile phone [maybe I could have attached a resistor to the ground/common pin to attenuate the signal. But I opted for not modifying the device in any way.]
Hence, I used the other cable coming from Y splitter (audio splitter) for the measurement of output signal amplitude, since I was outsmarted by my smartphone!

My tryst with such experiments is not new. I have previously done amphibian nerve-muscle preparation experiments using Kymograph and Dubois-Reymond induction coils, in medical curriculum. This included studying the effects of external influences like temperature, load, repeated stimuli causing beneficial effect & fatigue etc. on the tissue. I also studied peristalsis & effects of external influences including drugs on Guinea-pig smooth muscle in Dale's tissue organ bath. Also being an electronics enthusiast, I tried to decode the language the heart speaks, in the honest pursuit of making biomedical instrumentation. It's tough! (fun intended)

Lastly, all that has been said above may be modified as we gain more insight and experience. Please feel free to use/modify this work, do your own research, give your feedback or point something wrong in this article. 

N.B.-Remember, electricity kills! Try to have a close friend, preferably a doc, by your side. Check that the 'Earth' outlet is really what it outta be!

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September 19, 2010

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’ blood brain barrier showing glial cell in blue and the vasculature in pinkimpermeability 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

September 11, 2010

fMRI, BOLD and the Beautiful

ResearchBlogging.orgWhen 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) Effect of scopolamine and lorazepam on memory using fMRIon 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

September 07, 2010

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?
a neuron showing an axon wrapped by myelin sheath
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)colorful world of tractography 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

July 21, 2010

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!). magnetization vector resolved into its components horizontal Mxy and a vertical MzRemember, 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. T1 and T2 weighted MRI scansNote: 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.

Last Modified: Aug 19, 2010

June 24, 2010

Understanding the Basic Principles of Nuclear Magnetic Resonance Imaging

ResearchBlogging.orgNuclear 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.

Animation of a Helium atom
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.

Precession of protons, animation

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 alignment of protons in a spinning nucleus in a static magnetic field(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 Magnetic
Resonance 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