Magnetic Resonance Imaging (MRI) Physics & Instrumentation



Name of Student: Mohamad Al-Hafiz bin Ibrahim

2.1 RF leakage
2.2 Aliasing
2.3 Patient motion
2.4 Gibbs & Truncation
2.5 Chemical Shift
2.6 Magnetic Susceptibility
2.7 Flow Motion
Figure 1: Zipper Artifact may appear as horizontal line across the image
Figure 2: The part of the body that outside the FOV is mismapped within the FOV.
Figure 3:The appearances of ghost lines at the anterior to the abdominal wall
Figure 4: Image shown the effect of head movement or motion during MR scanning
Figure 5: Bright and dark lines are visible in image
Figure 6: Arrow show dark line at the interface of fat and water .
Figure 7: MR image shown massive distortion of magnetic field .
Figure 8: (a) CSF pulsation-related artifact in the phase encoding direction in T2-weighted image while (b) show reduction of flow artefact
Magnetic Resonance Imaging (MRI) is one of the medical imaging and diagnosis technique which widely used due to its capability to produce high resolution of cross- sectional anatomical images and high tissues contrast. Eventhough MRI has various advantageous features, but still there are numerous sources of artifacts either patient-related, signal processing-dependent and hardware (machine) related (Erasmus, Hurter, Naudé, Kritzinger, & Acho, 2004).

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Definitely, artifacts can degrade the image quality and may mimicking pathology or obscure the abnormalities which can lead to misdiagnosis of MRI images. The MRI artifact can be defined as a structure or feature appearing in MRI image produced by artificial means which is not originate within the scanned object (Erasmus et al., 2004). Commonly, MRI artifacts can be caused by RF leakage, aliasing, patient motion, Gibbs, truncation, chemical shift, magnetic susceptibility and flow motion.
2.1 RF leakage
This artifact also known as Zipper artifact. It occurs when there are leakage of RF or electromagnetic energy generated from certain equipment into MRI system (Stadler, Schima, Ba-Ssalamah, Kettenbach, & Eisenhuber, 2007). This extrinsic RF came at a certain frequency then interferes with MRI signal produced by patient. The potential sources of the extrinsic RF are due to penetration of the RF into the shielded scanning room especially when the door is open during images acquisition (Ruan, 2013). After that, the RF will be picked up by the receiver chain of the image sub system (Zhuo & Gullapalli, 2006). This RF perhaps generated by radio, illumination or electronic device such as monitoring equipment in the surrounding (Stadler et al., 2007).
In order to overcome this artifact, the operator should identified and eliminate the possible source of the penetration. It can be done by ensure the door of the MR room remain closed during scanning, use only MR compatible MR monitor equipment, and remove the external RF source from the surrounding (Ruan, 2013).

Figure 1: Zipper Artifact may appear as horizontal line across the image (Allen, n.d.).
2.2 Aliasing
Aliasing or wrap around artifacts can be describe as an artifacts that caused by anatomy that lies outside of field of view (FOV) mismapped within the FOV (Westbrook, Roth, & Talbot, 2011). This is because of improper selection of parameter in MR systems especially FOV. The FOV in MRI means the anatomical area that should be covered or imaged during scanning (Morelli et al., 2011). When the selected FOV is smaller than the size of area that should be imaged means the data are under-sampled (Ruan, 2013). Therefore, there are high chances for signals from the outside FOV falsely detect then create an interference with signal within FOV and encode on the reconstructed images thus ‘wrap around’ to the opposite side of image which become aliasing artifacts (Erasmus et al., 2004). Westbrook et al., (2011) state that aliasing artefact can happen along frequency encoding axis (frequency wrap) and phase encoding axis (phase wrap).
Basically, this aliasing artifacts can be eliminated through increase the sampling rate or oversampling along the frequency direction (Westbrook et al., 2011). However, high pass and low pass filter should be used as well in order to filter out frequency outside the FOV which can increase noise in image (Hiroshi, Schlechtweg, & Kose, 2009). Besides that, selection of receiver coil which unable to excite or detect the signals from anatomical tissues that lying outside the FOV also important to minimise the artifacts (Ruan, 2013). Lastly, No Phase Wrap (NPW), Phase oversampling or Fold Over supression techniques is also preferred to avoid aliasing artefact by oversamples in phase direction, thus, the phase curve get to extends and cover wider signal producing structures (Westbrook et al., 2011).

Figure 2: The part of the body that outside the FOV is mismapped within the FOV and located at the opposite side of the image (Prashant, 2014).
2.3 Patient motion
Patient motion artifact is a very most common artefact in MRI. It is caused by movement of anatomical structure during imaging sequence (Zhuo & Gullapalli, 2006). There is a broad range of examples of structure movement such as heart or arterial pulsations, respiration process, peristalsis, tremor (Parkinson’s disease) and gross movement of patient (Stadler et al., 2007). Hence, if there is a scanned anatomical part moved during the scanning, the phase gradient cannot predict and encode the signal, thus, that structures will be misplaced in phased encoding direction. As a result, it will causes MR images shown the appearances of mismapping, blurring and ghosting artefact within it (Westbrook et al., 2011).
There are several ways to eliminate or avoid the patient motion artifacts. The remedies are nullifies signal by applying pre-saturation pulses over the area which have potential to produce artifacts (Stadler et al., 2007). This way is more effective to prevent ghosting during patient swallowing. Besides that, Westbrook et al (2011) proposed that attaching a set of bellows over patient’s chest in respiratory compensation which is also known as respiratory ordered phase encoding (ROPE) might help to minimize ghosting in longer sequences while in short sequences, cooperation from patient to hold their breath during scanning is preferred.
Next,cardiac gating also plays role in reducing this kind of artefact. For example, electrocardiogram (ECG) gating used to monitors cardiac motion that trigger the excitation pulse. Hence, each excitation pulse in each slice can be timed and acquired at the same phase of cardiac cycle (Westbrook et al., 2011). In the other hand, asking for patient cooperation for keeping still, clear explanation about procedures, and optimize the patient’s comfortability are important to make them immobilize during scanning (Hiroshi et al., 2009).

Figure 3:The appearances of ghost lines at the anterior to the abdominal wall indicate as motion artifact because of breathing (Zhuo & Gullapalli, 2006).

Figure 4: Image shown the effect of head movement or motion during MR scanning (Hornak, n.d.)
2.4 Gibbs & Truncation
Truncation artefact also can be called as Gibbs Ringing artefact (Czervionke, Czervionke, Daniels, & Hauhgton, 1988). Its happen as result of It is causes by abrupt undersampling of data that results in incorrect representation of high and low signals interfaces (Westbrook et al., 2011). That problems lead to visibility of fine lines in MR image and also respectively caused by incomplete digitization of the echo (Ruan, 2013). However, according to Erasmus et al.,(2004), alternating dark and bright lines may visible in image due to a sharp transition in signal intensity.
In order to correct this type of artefact, there are several ways that can be used. For example, increase the matrix size, 256 x 256 instead of 256 x 128 (Westbrook et al., 2011). Next, applying various filters to k-space data before Fourier transform also should be considered (Erasmus et al., 2004). Besides that, provide more phase encoding steps also preferred to make truncation or gibbs artifacts less intense and narrower (, n.d.).

Figure 5: Bright and dark lines are visible in image parallel and adjacent to the outer convexity of calvaria (Prashant, 2014).
2.5 Chemical Shift
This type of artifact commonly found in MRI image of abdominal and spine imaging. Since fat and water each consist of hydrogen protons but different combination of molecules, fat contain hydrogen binds with carbon,while in water, hydrogen combine with oxygen (Westbrook et al., 2011). Hence, that different chemical environment exist shown that there are different precession frequency between fat and water which fat has lower precessional frequency rather than water (Erasmus et al., 2004). Based on the Larmor equation, precessional frequency is proportional to the strength of magnetic field (Westbrook et al., 2011). Therefore, this chemical shift can become artifact due to that difference of the precessional frequency between fat and water at higher field of magnetic strengths during the frequency encoding or slice-select directions (Ruan, 2013). That frequency is basically used to encode their spatial positions, thus, any chemical shift can lead to spatial misregistration of the MR signal (Morelli et al., 2011). MR images will show the bright or dark outlines at fat-water interfaces as the artefact.
To avoid this artefact , a few remedies should be considered such as perform scanning at low magnetic field strength, increase the receive bandwidth in keeping with good signal-noise-ratio (SNR) (Westbrook et al., 2011) . It is also suggested to use minimum FOV as possible. Lastly, swapping phase and encoding direction also may useful to reduce this artefact (Hiroshi et al., 2009).

Figure 6: Arrow show dark line at the interface of fat and water indicate as chemical shift artefact (Javan, Rear, & Machin, 2011).
2.6 Magnetic Susceptibility
Susceptibilty can be refer as characteristic of substance which be magnetized when exposed to magnetic field (Gary, n.d.). MRI physics explain magnetic susceptibility artifacts normally happens because of substance or material especially ferromagnetic materials and also at air-tissues interface which have different degree of magnetic susceptibility that can distort the external magnetic field when placed in large magnetic field. Besides that, the differences also lead to magnetic field inhomogeneity at the scanner region resulting in spins dephase faster and frequency shift between surrounding tissues (Zhuo & Gullapalli, 2006). Artifacts in the image will appear as bright and dark areas with spatial distortion of anatomical structures (Stadler et al., 2007).
Generally, these artifacts can be reduced by ensure all metal objects that attached to the patient has been removed before the scan. Next, spin echo sequences are more preferred to be used instead of gradient echo because it use 180° RF rephasing pulse which ideal at compensating for phase differentiation between fat and water (Westbrook et al., 2011). Since fast spin echo techniques also contribute in reduction of this type of artefact, hence, short TE is used along with spin echo (Stadler et al., 2007). Metal Artefact Reduction Sequence (MARS) technique can be used in order to minimize the size and intensity of this artifact which developed by magnetic field distortion by introducing an additional gradient following the slice gradient during frequency encoding gradient is used (Olsen, Munk, & Lee, 2000).

Figure 7: MR image shown massive distortion of magnetic field due to implanted dental retention system (Schubert, 2012).
2.7 Flow Motion
Flow artefact can be categorized as one kind of motion artefact which mainly caused by natural motion of liquids such as blood or cerebrospinal fluid (CSF) in the body. For example, hydrogen nuclei in blood flow within the scanned slice may trigger excitation from an incoming RF pulse, however, the signal perhaps cannot be readout due to possibility of that flowing blood have left the slice (Hiroshi et al., 2009). As a result, vessels image appear empty or low signal intensity (less bright). Generally, there are reasons of low signal intensity such as intravascular signal void by time of flight effects, first echo dephasing and fast flow (Hiroshi et al., 2009).
Nevertheless, this artifacts also can appear bright or high signal intensity. This is because of the slow blood flow (flow related enhancement), even echo rephrasing and diastolic pseudogating (Hiroshi et al., 2009).
The preferred solutions as remedies for flow motion artifacts are by reduction of phase shifts using flow compensation in order to produce gradient moment nulling, suppress the blood signal by apply saturation pulses parallel to slices and synchronization of imaging sequences with cardiac cycle using cardiac triggering (Zhuo & Gullapalli, 2006).

Figure 8: (a) CSF pulsation-related artifact in the phase encoding direction in T2-weighted image while (b) show reduction of flow artefact with gradient moment nulling (Morelli et al., 2011).
It is important for all operators, radiologist and engineers in MRI are able to recognize common MRI artifacts because there are a broad of range of cause that contributing to artefact. Eventhough, artifacts are unable to be totally eliminated but it can be minimized or avoided with specifics remedies in order to improve the MR image quality (Morelli et al., 2011). Therefore, basic knowledge of MRI artifacts should be learned and all MRI system operators should familiar with their MRI unit in department.
Allen, E. D. (n.d.). Zipper and Related Artifacts. Retrieved May 9, 2015, from
Czervionke, L. F., Czervionke, J. M., Daniels, D. L., & Hauhgton, V. M. (1988). Characteristic features of MR truncation artifacts. American Journal of Roentgenology, 151, 1219–1228.
Erasmus, L. J., Hurter, D., Naudé, M., Kritzinger, H. G., & Acho, S. (2004). REVIEW ARTICLE: A Short Overview of MRI Artefacts. SA Journal of Radiology, 8(August), 13–17.
Gary, P. L. (n.d.). What is MRI ? Magnetic Resonance Imaging ( MRI ).
Hiroshi, Y., Schlechtweg, P., & Kose, K. (2009). Magnetic Resonance Imaging. Imaging of Arthritis and Metabolic Bone Disease:Expert Consult – Online and Print, p34–48.
Hornak, J. P. (n.d.). The Basics of MRI: Image Artifacts. Retrieved May 9, 2015, from
Javan, R., Rear, J. R. O., & Machin, J. E. (2011). Fundamentals Behind the 10 Most Common Magnetic Resonance Imaging Artifacts with Correction Strategies and. European Society of Radiology, 1–78.
Morelli, J. N., Runge, V. M., Ai, F., Attenberger, U., Vu, L., Schmeets, S. H., … Kirsch, J. E. (2011). An image-based approach to understanding the physics of MR artifacts. Radiographics : A Review Publication of the Radiological Society of North America, Inc, 31, 849–866.
Olsen, R. V, Munk, P. L., & Lee, M. J. (2000). Metal Artifact Reduction Sequence: Early Clinical Applications. Radiographics : A Review Publication of the Radiological Society of North America, Inc, 20, 699–712.
Prashant, M. (2014). Aliasing artifacts. Retrieved May 11, 2015, from
Ruan, C. (2013). MRI Artifacts : Mechanism and Control. Personal Conclusion, 1–9.
Schubert, R. (2012). Magnetic susceptibility artifact. Retrieved May 9, 2015, from
Stadler, A., Schima, W., Ba-Ssalamah, A., Kettenbach, J., & Eisenhuber, E. (2007). Artifacts in body MR imaging: Their appearance and how to eliminate them. European Radiology, 17, 1242–1255.
Westbrook, C., Roth, C. K., & Talbot, J. (2011). MRI In Practice (4th Editio, pp. 225–260). United Kingdom: Blackwell Publishing Ltd. (n.d.). MRI Artifacts. Retrieved May 8, 2015, from Artifact
Zhuo, J., & Gullapalli, R. P. (2006). AAPM/RSNA physics tutorial for residents: MR artifacts, safety, and quality control. Radiographics : A Review Publication of the Radiological Society of North America, Inc, 26, 275–297.

Imaging Techniques in Medical Science

Electrodiagnosis is the field of study that utilizes the science of electrophysiology. Specifically, electrodiagnostics study the human neurophysiology through the utilization of electrical technology. Neurodiagnostics, evoked potentials and electromyography are aspects of electro diagnosis.
Electromyography was the first electrodiagnostic examination to be developed. The procedure involves the placement of needles to several muscles to record various stages of muscle activity, minimal contraction, maximal activity and even rest. A normal muscle is electrically silent when at rest. Spontaneous depolarization of individual muscle fibers results from damaged muscle tissue. The mentioned alterations can be detected through the needle examination portion of electrodiagnostic examination. [122]
No special preparation is generally necessary. Avoid using any lotions or creams on the day of the examination. Temperature could affect the result of the test hence if the temperature is cold; the patient should wait in a warm room for a while before the test is conducted.
One concern with electromyographic testing is that needles are utilized and it could be painful. However, the new computerized technology permits the usage of needles that can records so that small insertion of it feels lesser painful than the insertion of a normal size needle. Needles with small gauge can be used, because nothing is aspirated or injected. A troublesome trend is the performance of nonphysician health care personnel in electromyographic testing. Interpretation of electromyograms and performance of electromyography needs enough technical skill and capability to assimilate physician’s understanding. [121] In a study conducted by Rathinaraj and colleagues regarding the efficacy of spinal segmental stabilization exercise program and the efficiency to improve the muscular activity and pain reduction which is assessed through electromyography because limited studies are conducted using electromyography as an assessment parameter of muscular activity. Their study showed that exercise play a vital role in alleviating low back pain particularly in the mechanical back pain brought by spinal instability, which needs spinal segmental stabilization exercise program. Positive progress in muscular activity and pain reduction proves the exercise program.
History of low back pain is associated with higher baseline muscle activation and that electromyography responses are modulated from this activated state, rather than showing acute burst activity from inactive state, perhaps to prevent trunk displacements.
Nerve conduction studies are essential part of the complete electrodiagnostic examination. [123] In a nerve conduction studies, the contraction is caused by the electrical charge distributed to the nerves in the periphery. An electrode capable of recording is posited on a muscle innervated by the specific nerve, and information about impulse can be recorded including its latency. Latency is the time required for an impulse to travel from stimulus to the recording. Nerve conduction velocity and the distanced traveled can also be computed. The said measures are important gauge of damage to the nerve which specifically tests the integrity of the myelin sheath of the nerves. The nerve conduction studies and needle examination are key components of a complete electromyographic examination. The amplitude of the contraction of the muscle can be compared signal’s initial size thus providing information regarding the number of functional neurons that consists the nerve.

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Nerve conduction study is also referred as nerve conduction velocity test. During this procedure, two electrodes will be attached to the surface of the skin. One electrode will stimulate the nerve while the other one will record it. The speed of nerve conduction studies is associated to the degree of myelination and diameter of the nerve. A nerve functioning normally transmits a stronger and faster impulse than an altered nerve. It is like an electric wire with plastic or rubber insulation around it. Generally the range of normal conduction velocity is 50 to 60 meters per second. However, the normal conduction velocity may be different from one nerve to another and one individual to another. Nerve conduction velocity test is commonly conducted along with electromyography. A condition that may be examined with nerve conduction studies includes Carpal tunnel syndrome, Guillain-Barre Syndrome, Charcot-Mari-Tooth syndrome, herniated disc, neuropathy, polyneuropathy, sciatic nerve problems and peripheral nerve injuries. Nerve conduction study techniques specifically include motor nerve conduction studies and sensory nerve conduction studies. Sensory nerve conduction studies are normal when focal sensory loss is due to nerve root damage for the nerve roots are proximal to the nerve bodies in the dorsal ganglia. [33]
Evoked potentials or evoked responses, measures the electrophysiologic responses of the nervous system to different stimuli. Theoretically, almost any sensory modality can be tested, however in clinical practice only few are used in routine basis. [208] Evoked potentials demonstrate abnormal sensory function when the neurologic test results do not reveal abnormalities. It reveals clinically unsuspected pathology when demyelinating disease is suspected. It determines the anatomic distribution of a disease process and it objectively monitors the condition whether the patient is progressing or not. [125]
Visual evoked potential examines the function of the visual pathway beginning from the retina going to the occipital cortex. It specifically measures the capacity of the visual pathways to conduct from the optic nerve, to theoptic chiasm and optic radiations going to the occipital cortex. Brainstem auditory evoked potentials measure the function of the auditory nerve and auditory pathways in the brainstem. [124] Somatosensory evoked potential is a diagnostic test to assess the speed of impulse conduction across the spinal cord. The said methodology is consists of using electrical stimulus in the nerves of arms and legs measuring the impulse generated by different points in the body.
Electrodiagnostics is utilized to examine lumbosacral radiculopathy potentially underlying low back pain. The examinations serve as an extension of the physical examination and clinical history furthermore it complements the neuroimaging studies. Among the common low back pathologies amenable to electrodiagnostic studies include spinal stenosis and lumbosacral disc herniation. The electrodiagnostics can help in the decision making processes when considering surgical management. [126] Electrodiagnostic studies are essential part of the diagnostic evaluation when the physical examination or history suggests that neural structures may contribute as symptom generators. Lumbosacral radiculopathies, peripheral nerve injuries and plexopathies are of primary concern when examining patients having low back pain. The study assists in quantifying neurophysiologic injuries and alterations using the said techniques.
Bone Scan
A bone scan shows the images of metabolic activity of the skeleton. Conventionally, it is accomplished by imaging radionuclide whose physiology closely resembles a metabolic activity within the bone. Nuclear scintigraphy of the bone generally uses the radionuclides fluoride-18 (F-18) or technetium-99m (Tc-99m). Tc-99m is commonly attached to medronic acid (Tc-99m MDP) and F-18 incorporated into sodium fluoride (F-18 NaF). The molecules are injected intravenously while a nuclear camera that contains salt crystal captures the decay of photons from radioisotope. This is attained through the process of fluorescence or scintillation that occurs when the photon released by the radionuclide hits the salt crystals within the nuclear camera. The scintillations are converted to images for interpretation by nuclear medicine specialist. [127] A bone scan is used utilized to: [143]

Diagnose a bone tumor or neoplasms
Ascertain if a cancer already metastasizes to the bones. The common cancers that could spread to the bones include breast, lung, prostate, thyroid, and kidney.
Diagnose a fracture, especially if it cannot be seen on a plain x-ray
Rule-out osteomyelitis or bone infection
Determine or diagnose the etiology of bone pain, when no other cause has been recognized
Assess metabolic disorders, such as renal osteodystrophy, osteoporosis, osteomalacia, primary hyperparathyroidism, complex regional pain syndrome, and Paget’s disease

Bone scans are useful in a wide range of conditions. A common reason to have a bone scan is for examination of pain, in which bone scan can assist in identifying whether the source of the pain if from bone pathology or form soft tissue trauma. There are no specific preparations needed for radionuclide bone scan when using the tracer that map calcium metabolism, F-18 NaF or Tc-99m MDP. Patient should continue take their medications normally and eat normally. It is helpful to stay hydrated since the radiotracers are eliminated through the urine. Bone scans were known to emit much more radiation than CT and radiography. It must be kept in mind when considering whether or not to perform scans on a child. [108]
Before the bone scan, the patient should make it known if she is or might be pregnant and if she is breast feeding. The patient can use formula for 1 to 2 days after the scanning to wait until the radioactive tracer is gone from the body. The patient should report to the doctor if he or she have had an X-ray test utilizing barium as a contrast material, such as a barium enema or have taken a medication that contains bismuth within the past 4 days because barium and bismuth can interfere with test results of the scanning. The patient should limit his or her fluids for up to 4 hours before commencing the the test for the patient will be instructed to drink extra fluids after the injection of the tracer. The patient will empty his or her bladder right before the scan. Most probably the patient will have to wait for at least 1 to 3 hours after the injection of the tracer before your bone scan is done. [144]
The images produced by the bone scan should depict that the radioactive material has been distributed evenly all over the body. There must be no areas of increased or decreased distribution. “Hot spots” are portions with an increased distribution of the radioactive material. On the other hand “cold spots” are areas that show lesser of the amount of radioactive material. [143]
Many false-positive results can be expected among older adults. Discitis, osteomyelitis, metastatic disease, rheumatoid arthritis, degenerative spondylosis and ankylosing spondylodis may result in abnormal findings in the spine that are not directly related to acute trauma. False-negative results may occur in the first hours after acute trauma. If possible, 72 hours should be allowed to pass prior to nuclear bone scanning of the lumbar spine is attempted. [127]
Thermography is a noninvasive procedure that images infrared radiation (heat) released by the body surface. It is based on the principle that alterations in different of body functions alter the cutaneous vascular supply. Pain is a complicated phenomenon that cannot be simplified to a direct correlation with cutaneous heat production. Thermography. Thermography does not take a picture of pain itself; it does reveal pathophysiologic conditions related with soft tissue, circulatory neurovascular and musculoskeletal disorders.There are two type of thermography. It includes liquid crystal or contact and electroninc or noncontact thermography. [129]
The contact thermography utilizes cholesterol crystals that changes in color with the variations of surface temperature. The crystals are placed inside inflatable transparent boxes with one thermosensitive, flexible side that is applied to the body of the individual. Each of the boxes has a limited temperature range and its utilization for examination requires proper selection of the box with accompanying proper temperature range. An image is taken of the box to record the patterns of surface temperature. The box is chosen by trial and error method. The advantage of contact thermography includes the absence of radiation, much lower cost than electronic thermography and much easier to use. Electronic thermography uses an infrared radiation sensor that converts heat reading to electrical signals that are displayed on a black-and-white or colored monitor. A picture can be taken from the video screen. It can be also stored on a computer. This system has the advantage of viewing large areas of the body during a single examination. Examinations must be conducted in an air-conditioned, draft-free room. The ambient temperature must be between 68 degrees to 72 degrees Fahrenheit. The patient should also be instructed to refrain from cigarette smoking for the day of the test. Furthermore, the patient should refrain from taking pain medications, physical therapy and exposure to sunlight for at least 24 hours. The patient must be in equilibrium with room temperature for 30 to 60 minutes before the beginning of the procedure. The examination will be postponed if the patient is febrile. [129]
The examination of lumbosacral spine and lower legs with contact thermography consists of individual images of buttocks, posterior and lateral thighs, lower legs, dorsa of the feet, and toes. The examination requires 1 to 2 hours to complete. Abnomalities in the physiologic temperature distribution pattern also indicates alterations. Acute pain is said to be associated with increased heat whereas chronic pain is related to decreased temperature. Increased temperature is found over areas involved in an inflammatory process. Studies have stated a close correlation between abnormal thermograms and surgically proven discs. Investigators have also found that patients with disc herniation have a thermography and myelography accuracy rate of 95% and 84% correspondingly. [214] Thermographic findings correlated with magnetic resonance, myelography and computed tomography abnormalities in 94%, 80% and 84%. Twenty two magnetic resonance scan of patients with prolapsed of the disc associated with nerve root lesion, 95% of them had leg abnormalities on thermography. [129]
There is a good relationship between changes in symmetry of heat patterns and changes in pain intensity for most of the disorders that causes chronic pain. Thermography has been reported to useful in differentiating pain-free from pained subjects reporting back pain, knee pain, and leg pain. Thermography consistently indicates painful areas among patients with spinal cord injury.
Ultrasound Imaging
Ultrasound is a type of imaging which uses high-frequency sound waves to look at visceral organs and structures of the body. Ultrasound imaging of the musculoskeletal system is painless and safe. It is also called as ultrasound scanning or sonography. It involves the use of a probe or small transducer and ultrasound gel placed directly on the surface of the skin. The transducer transmits high-frequency sound waves through the gel into the body. Then, the transducer utilizes the sounds that bounce back and use them to create images in the computer. There is no risk for radiation because ultrasound imaging does not utilize ionizing radiation like what is used in radiography. Since sonographic images are captured in real-time, they can show the structure and the body’s internal organ movements, including the blood flow through the blood vessels. Ultrasound imaging is noninvasive medical test that aids physicians diagnose and treat medical disorders. Musculoskeletal ultrasound provides pictures of muscle, ligament, tendons, joints and soft tissue throughout the body. [142]
Ultrasound images are commonly used to help diagnose certain musculoskeletal conditions such as: tendon tears; muscle tears, masses or fluid collections; tears or sprain of ligaments; fluid effusion or inflammation; early alterations caused by rheumatoid arthritis; nerve entrapment; ganglionic cysts; benign and malignant soft tissue tumors; hernias; foreign bodies; and dislocations. [142]
Patients should be instructed to wear loose-fitting, comfortable clothing for the examination. The patient may be required to remove some of the clothing and accessories in the area to be examined. Ultrasound examinations are sensitive to motion. An active or crying child can lengthen the examination process. No other preparation is required. Musculoskeletal ultrasound evaluation is usually completed within 15 to 30 minutes but can take longer. Ultrasound may have difficulty penetrating the bone, hence only the outer surface can only be viewed. There are also limitations on the depth the sound waves can penetrate, thus deeper internal structures of larger patients may not be seen easily. [142]

Ultrasound Imaging Systems

An ultrasound scans also known as ultrasonography. Ultrasound will form the image by scanning using the high frequency sound waves. This device suitable to evaluate some part inside of the body. In physics, ultrasound is a sound with a frequency humans cannot hear. In diagnostic sonography, the ultrasound is usually between 2 and 18 MHz. (Anon 2012)

Figure 1 : The principal functional components of an ultrasound imaging system.(Perry Sprawls n.d.)

The ultrasound transducer converts an electrical signal into the ultrasound beam. The signal transmitted into the patient’s body, and then alters the returning echo into an electrical signal for processing and display. It use single-element circular disk to both transmit and receive ultrasound. (Hedrick et al. 2005)
Crystal of piezoelectric material with electrodes is the main part of the transducer. The electrodes are formed by plating a thin film of gold or silver on the crystal surface. The matching layer is located adjacent to the electrodes. The function is to improve the transfer of energy to and from the patient. All this part of the transducer is placed in an electrically insulating casing. This casing will give structural support. An acoustic insulator is made of rubber or cork it works to prevents the transmission of ultrasound energy into the casing. (Hedrick et al. 2005)

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When we change the transducer it will change the frequency too. A higher frequency transducer that produces a shorter wavelength has a thinner crystal. Normally the material that always almost used in transducer is lead zirconate titanate (PZT). PZT represents a piezoelectric ceramics with various extracts. It will change the properties to equal a particular application. In medical, PZT-5 is used because it has the properties of high electromechanical coupling coefficient, high dielectric constant, and ability to be formed in a particular size and shape. (Hedrick et al. 2005) BACKING MATERIAL
The functions of backing material will deliver the maximum amount of energy in the form of heat to the patient. This is will give a continuous output of ultrasound waves from the transducer. The other function is to absorb all the energy except for the one cycle of sound. Meaning’s that one that produced from the front face of the transducer. Backing materials should have acoustic impedance so that maximum energy transfer will occur. Besides that, backing material should have a high absorption coefficient. This is to prevent ultrasonic energy from reentering the crystal. In the backing material, they will use an epoxy resin and tungsten powder combination to damp the ultrasonic pulse. Next, the rear surface of the backing materials is slanted to prevent reflection of sound energy into the crystal. (Hedrick et al. 2005) MATCHING LAYER
The matching layer is placed in the transducer on the exit side of the crystal. This material with acoustic impedance is placed between the crystal and the patient. The function of the acoustic impedance to produced ultrasonic energy to be largely reflected at this interface. This creates a long pulse and reduces the beam intensity that enters the patient, which is we did not want it in the ultrasound. The reason why we need the matching layer is to shorten the pulse and the improve energy transfer across the crystal-tissue interface. However, the matching layer must have low-loss properties since high attenuation would stabilize the desired effect of high transmission. (Hedrick et al. 2005)
The pulse generator produces the electrical pulses. The size of the electrical pulses can be used to change the intensity and energy of the ultrasound beam. (Perry Sprawls n.d.)
After the echo is received by the transducer, amplification is used to intensification the size of the electrical pulses. Gain setting will controls the amount of amplification. The time gain compensation function is to alter the increase in relationship to the distance of echo sites inside the body. (Hedrick et al. 2005)
Controlling the scanning of the ultrasound beam is done by the scan generator. The way is by control the procedure when electrical pulses are functional to the piezoelectric elements in the transducer. (Perry Sprawls n.d.)
The digital imageis to produce the chosenforms for display. This includes giving it specific contrast characteristics and reformatting the image. (Perry Sprawls n.d.)
The digital ultrasound images are observed on the monitor and transmitted to work station. The other part of the ultrasound system is the digital storage device. The function is to store images for later viewing.(Perry Sprawls n.d.)

Figure 3 : The production of the ultrasound pulse. (Perry Sprawls n.d.)
Frequency is the number of wave cycles passing a given point in a given increase of time. The unit is cycles/ second or hertz. Frequency is the inverse of the period. (Hedrick et al. 2005)

Figure 4 : The ultrasound pulse frequency. (Perry Sprawls n.d.)
Velocity is the rate and direction at which sound propagates through a medium. The average velocity of sound in soft tissue is 1540 m/s. (Hedrick et al. 2005)

Figure 5 : The ultrasound of velocity. (Perry Sprawls n.d.)
Wavelength is a physical characteristic of a wave that is the distance for one complete wave cycle. (Hedrick et al. 2005)

Figure 6 : The wavelength of the ultrasound. (Perry Sprawls n.d.)
Amplitude used to refer to the particle displacement, particle velocity or acoustic pressure of a sound wave. Amplitude also show the strength of the detected echo or the voltage induced in a crystal by a pressure wave. (Hedrick et al. 2005)
Intensity is a physical parameter that describes the amount of energy flowing through a unit cross-sectional area of a beam each second. This is the rate at which the wave transmits the energy over a small area. The unit of intensity is the watt per square centimeter or joule per second per square centimeter. (Hedrick et al. 2005)
Power is a measure of the total energy transmitted summed over the entire cross-sectional area of the beam per unit time. The unit of power is the watt. (Hedrick et al. 2005)
As the transducer emits pulses, it causes large instabilities of intensity in the region through which the pulse move. Each pulse consists of multiple cycles that produce intensity variations within the pulse itself-the maximum intensity, designated temporal peak (TP). Pulse average (PA) will controls the intensity averaged over the duration of a single pulse. Temporal average (TA) will controls the intensity averaged over the longer interval of the pulse repetition period. The TA intensity is related to the PA intensity by the duty factor (DF):
TA = DF×PA or by the pulse duration (PD) and pulse repetition frequency (PRF):TA =PD × PRF × PA.(Hedrick et al. 2005)
The maximum intensity of all measured values within the sound field is designated as the spatial peak (SP). The designation of spatial peak is not well-defined. In some applications it refers to the maximum intensity in a plane perpendicular to the beam axis at a particular distance from the transducer. The maximum intensity throughout the ultrasonic field which usually occurs along the beam axis. The focusing of the transducer is the most important determinant of spatial peak.(Hedrick et al. 2005)
Spatial averaging over the cross-sectional area of the beam for each temporal intensity is also specified. A cutoff point of 0.25 times the SP intensity has been established to the limit area over which the intensity is averaged. These three combinations are possible to happen are I(SATP)-spatial average, temporal peak intensity, I(SAPA)-spatial average, pulse average intensity and I(SATA)-spatial average, temporal average intensity.(Hedrick et al. 2005)

Figure 7: The interaction within a body of ultrasound (Perry Sprawls n.d.).
Absorption is the procedure whereby energy is placed in a medium by converting ultrasonic energy into other energy forms, primarily heat. It is an exponentially decreasing function and is the major factor in the total attenuation of the beam. (Hedrick et al. 2005)
Attenuation is the decrease in intensity as a sound beam travels through the medium. Attenuation depends on all the interactions of ultrasound with tissues which include scattering, divergence, and absorption. (Hedrick et al. 2005) Scattering is the rerouting of sound energy resulting from the sound beam striking an interface whose physical dimension is less than one wavelength. It is also called non specular reflection. (Hedrick et al. 2005)
Reflection is an interaction that results when the sound being redirected into the medium after striking an acoustic interface. The angle of incidence equals the angle of reflection. The intensity of the reflected wave is depends on the composition of the interface. (Hedrick et al. 2005)
Refraction is a process whereby sound enters one medium from another that will result in a bending or deviation of a sound beam from the predictable straight-line path. Refraction obeys Snell’s law, which is based on the ratio of the velocity of the sound in the respective media. Refraction will make artifacts in the image by the misregistration of structures (Hedrick et al. 2005)
A low-Q transducer has a short pulse length and a broad bandwidth while a high-Q transducer has a long pulse length and narrow bandwidth. The objectives beam width is to transmit a beam that would be directional with a narrow beam width. An echo is created anyway of the lateral position of the object in the ultrasonic field. The lateral dimension of the object in the image is defined as the same size as the beam width. Multiple small objects equidistant from the transducer are not resolved when encompassed by the beam. Focusing reduces the beam width at specific depth to enhance the spatial mapping of received echoes.(Ding et al. 2014) Sampling is restricted laterally by the width of the beam. Objects located outside the beam do not contribute signals. (Small 1971)
The focusing transducer made-up with an indented active element exhibits much broader bandwidth and higher sensitivity. To fabricate focusing transducers, we can add a lens and shaping the piezoelectric element. Among the focusing transducer designing methods, the shaping element used in transducers was reported to be much effective for fabricating high sensitivity device. Hard pressing and pressure defection techniques are the usual ways to shape transducer elements. For the flexible composite and polymer materials, the focusing transducer can be easily fabricated using those techniques.(Chen et al. 2013)

Figure 8: The width and pulse diameter characteristics of both unfocused and focused transducer. (Perry Sprawls n.d.)
Transmit focusing happen when the depth of the focal zone is altered by varying the delay times between crystal excitations. (Wright 1997)The scanning of the region of interest is conducted with a depth of focus selected by the operator. After review of the real-time image, a new focal zone may be certain to rescan the same area with dissimilar focusing in the scan plane. The beam is focused to a new depth simply by changing the delay times. The transducers that have the capabilities of this focusing are phased linear arrays. (Kossoff & Eng 2000)Electronic phasing of the elements allows variable focusing along the scan line which in turn controls beam width in the plane direction. High resolution images with multiple focal zones throughout the images are also possible using this adjustment delay lines. Multi zone transmit focusing reduces the frame rate, because the data must be composed for all the lines of sight across the array with a set focal zone depth before the lines of sight are repetitive with a different focal zone depth.
Dynamic focusing is in the receive mode. It does will reduce the effective sampling volume.(Kossoff & Eng 2000) Dynamic focusing will operate at all depths. The wave front from the object appears to be in phase for all the crystals resulting in a focused beam from the depth of interest. Beam formation is the delay and sum of strategy. The master synchronizer sends timing messages to the receiver-delay lines to indicate the elapsed time from transmission to reception. The elapsed time determines the delay times for each crystal. The depth for receive focus is always known, and thus receive-delay times are constantly changed to yield continually focused beam at all depths. During acquisition of image data the receive times delays are varied dynamically to sweep the focal zone to each point along the scan line. (Hedrick et al. 2005)
In ultrasound, high frequencies provide better quality images, but cannot penetrate through skin and organ deeply. Low frequencies can penetrate deeper, but the image quality is poor. Ultrasound is useful to view part inside of the body. They may also be useful in helping the surgeon when carrying out some types of biopsies. Ultrasound is a one of the safe procedure in imaging department.

Magnetic Resonance Imaging: Overview and Applications

How does Magnetic Resonance imaging work and how can it influence the future?
An Introduction to Magnetic Resonance Imaging[1][2][3]:
Magnetic resonance imaging (MRI) is used as an accurate form of disease detection which is usually used to confirm a patients condition, as well as a method of looking at trauma to the brain, examples of which could be bleeding and swelling. Alongside these uses MRI can be used to look at the soft tissues, as well as information on the structure of joints.

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Prior to the introduction, the only method for diagnosis for many of these problems were invasive methods such as surgery, and x-rays or CT-scans which were less accurate and ionizing, which could have a lasting effect upon the body. The use of MRI scans is only a recent phenomenon, with the first image on a person being produced as late as 1977, by Paul Lauterbur and Sir Peter Mansfield who received a shared Nobel prize for their work on this matter. Both scientists were looking at how nuclear magnetic resonance imaging could be used to look at solids and liquids, and both formed the theory behind it all, but it was Sir Peter Mansfield who developed the method used in MRI by firstly solving how to define a slice of a matter, and also how to produce images from multiple-pulse experiments. Although the work on producing images for biological specimens only came about as a result of the fact that it would be too hard to produce images of a solid.
How does Magnetic Resonance Imaging work[4][5]?
Magnetic resonance imaging involves a series of steps which are all explained below, in order to produce the final image that is used for diagnostics.
Nuclear Spin
The body’s mass is approximately 10% hydrogen, of which 70% is contained in water, and due to the fact that protons produce a large signal to a MRI scanner a more accurate image is produced, as they are in such large abundance in the body in the water.

The hydrogen nuclei in the body spin about an axis, this is illustrated in figure 1.1. As a result of the spinning protons being charged, the spinning of a nuclei along an axis causes a small circulating electric current, which in turn causes a small magnetic field.
If a collection of these nuclei were to be placed in a stronger external magnetic field (BØ), most of the nuclei will align their spin in the same direction as the external field.
As you can see from figure 1.2 not all of the hydrogen nuclei are aligned with the direction of the magnetic field, and this is because both alignments are possible, but the one with the field is a lower energy state, whilst the protons against the field are at a higher energy state. The protons are continually oscillating back and forth between the two states, because there is a tendency for nuclei in a high energy state to return to a lower energy state by emitting some of its energy to surrounding nuclei. There is usually enough thermal energy in the material for the nuclei to be flipped back.

How do protons precess about an axis?[6]
Spinning protons when in the presence of an external magnetic field do not arrange themselves perfectly parallel or anti-parallel to the magnetic field, as the nuclei always have equal but opposite magnetic charges they cancel out when there is no magnetic field. The particles tend to precess about the magnetic field lines, and this is illustrated in figure 1.3.
The nuclei complete a full rotation around the magnetic field in a period that is directly proportional to the strength of the external magnetic field. As you can see from the figure 1.4 the period gets smaller for a larger magnetic field and thus the frequency of the precession increases. This frequency is fixed depending upon the strength of the magnetic field, and is called the Larmor frequency and the relationship between them is given by:
f = (ω)/ (2π)
Where Υ is a constant called the gyromagnetic frequency, which varies for each type of particle. In MRI the Larmor frequency is about 50 MHz, which is in the radio frequency part of the magnetic frequency spectrum and the magnetic field has a magnitude of 1 or 2 Teslas.
Why is the Larmor frequency is of that form?
TheLarmor frequencyinMRI is the rate of precession of the magnetic moment of the proton around the external magnetic field. The frequency of precession is related to the strength of the external magnetic field,BØ.
TheLarmor precession of nuclei of a substance placed in a magnetic field B0 is calculated from Larmor Equation, the Larmor precession is measured in Radians seconds-1:
ω = γBØ
Where Υ is a constant called the gyromagnetic frequency, which varies for each type of particle, but in the case of MRI is a constant as you are only affecting hydrogen nuclei. As the external magnetic field would be uniform and constant, to work out the Larmor frequency you need to divde the Larmor precession by 2πf.
The frequency is measured in Hertz which is s–1 and as ω is measured in Radians seconds-1 so to work out the frequency needed you divide this by 2πf which results in the Larmor frequency which is per second.
The Net Magnetization Vector
The precession of the nuclei only has a small effect upon the total magnetic field, which is only a small increase in magnetic field along the external field axis.
This is because there are slightly more nuclei in parallel to the external magnetic field, than nuclei which are anti parallel to the external magnetic field. Although all of the nuclei in the material will be precessing at the Larmor frequency, they all may not be in phase. So the tranverse waves created by the nuclei get cancelled out which means that there will only be a small increase in field strength in the direction of the external field as not all of the nuclei parallel to the external field will get cancelled out.
Why do we need superconducting magnets to make the protons resonate?
Superconducting magnets are used in magnetic resonance imaging of the human body because magnetic resonance imaging requires extremely uniform fields across the subject and extreme stability over time. By having the magnet coils in the superconducting state helps to achieve parts-per-million spacial uniformity over a space large enough to hold a person, and parts per million hour-1 stability with time. This is the reason for using superconducting magnets alongside the fact that they are able to produce a magnetic field of a magnituted of 1 or 2 Teslas.
How to make the protons resonate?
In order to produce a magnetic resonance image we need to make the protons precess about the external field lines in phase with each other ; which will produce a small net transverse field which rotates about the axis of the external field at the Larmor frequency. This is the magnetic field that can be detected and in turn produce a magnetic resonance image.
These three stages are all depend upon the nuclei precessing in phase with each other, this is done by making them absorb radio-frequency radiation of the same frequency of the Larmor frequency. The absorbtion of the energy causes low-energy state protons to flip into the high-energy state, which means that the protons are anti-parallel to the external field lines and also precessing in phase with the applied signal, which in turn mean that they are in phase with each other. The number of protons that flip depends upon the duration of the radio frequency pulse, which is applied.
How a magnetic resonance image is produced:
In order for a magnetic resonance image to be produced, you need to be able to locate the part of the body that has an ailment, in order to do this three smaller non uniform magnetic fields are added to the constant field. If you look at figure 1.5 you are able to see how these magnetic fields are applied, with one running the length of the patient’s body, this is the z-axis and this is used to define slices through the body. The body in this diagram is the sample, with the other magnetic fields being applied in the x-axis and y-axis, within the plane of a slice.
The magnetic field strength at any point is the sum of the four fields that sets a unique larmour frequency and phase at each point in the body.
To actually produce an image a pulse of electromagnetic radiation is sent through the body at a set radio frequency. The protons with a Larmor frequency will absorb energy from the pulse and flip into the higher energy state. As a result a transverse field, which is rotating at the Larmor frequency, is produced for the specific part of the body. As a result of Flemings Left hand rule, the rotating magnetic field that is actually an alternating field induces an electric current in the detector. The image is gradually formed by sending a sequence of radio-frequency pulses through the body at different frequencies to pick out the Larmor frequencies at different locations. The signals produced and the relaxation time involved are processed by computer to assemble the image.
How is a clear image is produced on the scan?
The relaxation time is the time taken for the protons to fall back down to their lower energy state after the radio-frequency pulse is turned off. The protons fall into their lower energy state by passing their energy on into neighboring atoms. The relaxation time is measured by the change in the intensity of the signal induced in the detector. The relaxation time is used to show the contrast in tissue within an image, with the relaxation time dependent upon the nature of atoms close to the stimulated protons. Thus a clear image of the tissue can be seen.
The safety of magnetic resonance imaging scanning:
MRI is regarded as one of the safest ways of confirming a diagnosis, although there are some exceptions where a patient has form of metal in their body, which interferes with the powerful superconducting magnets, which allows a magnetic resonance image to be produced. An example of something, which could cause a magnetic resonance image to not work, is tattoos that have metal fragments in the ink that is used to form the image. The metal is dangerous because metal objects can be forcefully drawn to the magnet; if these metal objects are embedded in your body, they can be drawn to the magnet and cause damage.The magnetic resonance image can also be dangerous for pregnant ladies.
Using magnetic resonance imaging to detect cancer in the body
A future use of MRI is to detect cancer; cancer cells need far more energy than most other types of tissues in the body, so a new technique has just been developed where a patient suspected of having cancer is injected with glucose. This results in a differing brightness on the image produced with the tumor appearing far brighter. Although this is currently only a concept that is still some way from being used on the general public, and this is in part due to the fact that the magnetic field strength used to produce these results is far higher than being used currently and we would need to see if the same results would be produced at a lower field strength.
In conclusion by taking the simple model already in place MRI has a bright future in diagnosis and detection of diseases, with MRI being the safest form of diagnosis for soft tissue as a result of its non-ionizing nature and clear results that it produces. The future for MRI will become brighter as the cost of using it falls over time, so that it will be more freely available and thus use for mass cancer detection could occur for example.
MRI has already had a major impact on practices undertaken by doctors in assessing a patients needs, and the use of MRI stands to continue these changes further as more uses are developed.
Introduction to MRI:

How does Magnetic Resonance imaging work?

The larmor Frequency
The safety of MRI scanning

Using MRI to detect cancer in the body:

Are there any modern alternatives to this technology?

Gihan Fernando1

[4] Advanced Physics by Steve Adams and Jonathon Allday- Page 504, Magnetic Resonance Imaging

Calcium Imaging in Neurons: Watching neurons in action

Functional imaging of neurons enables the direct visualization of neural activity at the level of action potential, calcium fluxes, intracellular signals, neurotransmitter release, and synaptic inputs. In this review, we use functional imaging to refer genetically encoded indicators of neural activity that report changes in intracellular calcium, neurotransmitter release, or voltage as changes in fluorescence. A broader definition of functional imaging might include the use of neuroimaging technology to measure an aspect of brain function and detection of neural activity by other means (for example, positron emission tomography, electroencephalography, magnetic resonance imaging, single-photon emission computed tomography or chemical dyes). Here we restrict the discussion to the general mechanism of neuronal calcium signaling, overview of the chemical fluorescent calcium indicator, and protein-based calcium indicators mostly genetically encoded calcium indicators (GECIs), Calcium imaging devices including confocal and two-photon microscopy. Finally, briefly discussing functional imaging of neurons in drosophila using GECIs as an application example to introduce new development in the field and conclude by providing an outlook on the prospects of calcium imaging.

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Neuronal Calcium Signaling
Calcium (Ca2+) is an essential intracellular secondary messenger in neuron, which facilitates the neurotransmitter release in neurons, transmission of the depolarizing signal and contribute to synaptic activity (5). At rest, neuronal cells have a Ca2+ concentration of 100 nm. but are activated when this level rises to 1000 nm. (5). Two main contributing factors to cytosolic Ca2+ concentration is the equilibrium between Ca2+ influx and efflux and calcium exchange in the internal stores. Besides, the dynamics of free Ca2+ within the neuron are also determined by, calcium-binding proteins such as parvalbumin, calbindin – D28k, or calretinin, most of which serve as Ca2+ buffers (25). Multiple mechanisms cause the Ca2+ influx from extracellular space. Some of the significant contributors to neuronal calcium signaling are:
voltage-gated calcium channels (VGCC), which, based on their threshold of voltage-dependent activation, are categorized into high (HVA) and low-voltage gated channels (LVA) (9). Which group of VGCC is present in a specific neuron depends on the type of cell and the sub compartment of the cell. VGCC is effectively activated in the dendrites and spines of most central neurons through backpropagation of action potential (33) and synaptically mediated depolarization of dendritic spines (6). Since the recording of somatic Ca2+ signals are commonly used to track the action potential activity invitro (16) and invivo (30), it is essential to note that the primary determinant of these signals is VGCCs.
1) Ionotropic glutamate receptors, N-methyl-D-Aspartate receptors (NMDA) are ionotropic glutamate receptors and mediate a significant part of postsynaptic Ca2+ influx into the dendritic spines of different types of neuronal cells (35). The fraction of calcium ions that contribute to the total current via NMDA receptor channels is approximately 6% -12% (8)
2) Metabotropic Glutamate Receptors(mGluRs), this group mediates both an increase in intracellular calcium as well as a transient receptor potential type C (TRPC) channels dependent inward current (12)
3) Calcium release from internal stores, mostly the Endoplasmic reticulum (ER), regulated by triphosphate inositol receptors (IP3Rs) and ryanodine receptors (RyRS), may also include other intracellular organelles (4).
A major challenge in analyzing the different sources of neuronal Ca2+ signals is that they are not generally active at a time but overlap with strong interactions. For example, calcium influxes in the dendrites and spines of CA1 hippocampal neurons via NMDA receptors and VGCCs during strong synaptic activity are non-linear, and their combined signals serve as a coincidence detector between pre- and postsynaptic activity (35). Given these complexities, calcium imaging is often indispensable to dissect the specific mechanisms of signaling in neurons.
Calcium Indicators
Bioluminescent calcium indicator
Aequorin is a calcium-activated photoprotein isolated from the hydrozoan Aequorea Victoria (2). Bioluminescent calcium indicator aequorin is composed of the apoprotein apoaequorin and a non-covalently bound chromophore. It contains three calcium-binding sites, and upon binding of calcium ions, the protein undergoes a conformational change. The change in protein conformation results in coelenterazine oxidation to coelenteramide, resulting in photon emission (about 470 nm wavelength) due to the decline of coelenterazine from the excited state to the ground state (2). Aequorin is characterized by a high signalto-noise ratio and can monitor the concentration of cytosolic calcium between 10-7to 10-3M (2). Bioluminescent recordings of calcium signals using aequorin do not require external illumination, thereby preventing problems such as phototoxicity, photobleaching, autofluorescence, and unwanted activation of photobiological processes (27). However, each molecule performs only one cycle of emissions, and the process of recharging is relatively slow (7). Furthermore, calcium recoding based on aequorin suffers from low quantity yield to low protein stability (2).

Figure 1: Calcium Indicators (Adapted from Konnerth et al.)
Chemical Calcium Indicator
Fura-2, a representative example for the fluorescent chemical calcium indicator, is a combination of calcium chelator and fluorophore (32). Fura-2 is excitable by ultraviolet light (350/380 nm), and between 505 and 520 nm is its emission peak. Calcium ion binding causes changes in intramolecular conformation, which leads to fluorescence emitted change. Fura-2 has the advantage that it can be used with dual-wavelength excitation, with one-photon excitation. Thus fura-2 enables the quantitative determination of the calcium concentration in a neuron of interest to be independent of the concentration of intracellular dye (32). Another important advantage of chemical calcium indicator is that they exist in a membrane-permeable and membraneimpermeable form, allowing their use in combination with a variety of different loading techniques (32). A significant drawback is that the cellular localization of chemical Ca2+ indicators cannot be easily controlled or explicitly targeted to a specific organelle (14).
Genetically encoded calcium indicators (GECIs)
GECIs are available in two flavors, those involving Forster resonance energy transfer (FRET) and single fluorophore. Here we have chosen Yellow Cameleon (YC) as a representative to display the GECIs based on FRET (18). FRET is the phenomenon on which the Cameleon sensor family relies. It occurs between closely applied fluorophores (donor and acceptor) that overlap the spectrum of emissions and excitation (17). The extent of FRET depends on the degree of overlap between the two spectra, the fluorescence dipole orientation, and, crucially, the distance between the two spectra. The degree of overlap between two spectra is very responsive to the distance between fluorophores over the 1-10 nm scale (17). YC 3.60 is made up of two fluorescent proteins and belongs to the GECI Cameleon family (17). YC consists of the enhanced cyan fluorescent-cent protein (ECFP) as a donor and the circularly permuted Venus protein as an acceptor, a linker sequence composed of calcium-binding protein calmodulin, and calmodulin-binding peptide M13 binds these two proteins (17). In the absence of calcium ions, the blue ECFP fluorescence (480nm) dominates the emission. Upon calcium binding, intramolecular conformation changes lead to reduction, which refers to a form of non-radiative energy transfer between an excited fluorophore donor and a fluorophore acceptor. Thus, due to the occurrence of FRET, the Venus protein is excited and emits photons (530 nm). The blue fluorescence is declining in practice, while the yellow fluorescence is increasing. The calcium signal is represented as the Venus-ECFP fluorescence ratio. To stop potential associations with endogenous binding partners of calmodulin, two specific methods have been introduced. The calmodulin-M13-binding interfaces are mutated in D3cpV-type GECIs to minimize cellular target interactions (17) substantially. Calmodulin is replaced by troponin C variants in another type of FRET-based calcium indicators. Troponin C is the calcium-binding protein in the cells of the cardiac and skeletal muscles and, as such, has no endogenic binding neuron partners (15).
The archetypal GECI is GCaMP, is a genetically encoded calcium marker, GCaMP is produced from a fusion of green fluorescent protein (GFP) circularly permuted (i.e., provided new N- and Ctermini) and M13, a peptide sequence from myosin light chain kinase and fused to calcium-binding protein calmodulin; The structural rearrangement of the sensor removes the GFP barrel in the presence of calcium, which dramatically increases the fluorescence output (19 ) . The original version has been improvised and reduplicated (by multiple groups) through rational design and selective mutagenesis to increase the total fluorescence change in response to calcium, regulate calcium-binding affinity, and accelerate response time (kinetics)— resulting in the recent generation of jGCaMP7(10).
Dye Loading Approaches
The loading of calcium indicators into neurons would rely mainly on three factors, the form of calcium indicator, the biological preparation, and the accurate scientific topic being discussed. The three most commonly employed approaches for dye loading of individual neuron are:
a) Single-cell loading by hard microelectrode impalement, whole-cell patch-clamp mounting, and single-cell electroporation, which can be used to load chemical and genetically encoded calcium markers (23,32).
b) Acute network loading. Most neurons are marked by acetoxymethyl ester (AM), by dextranconjugated dye c and by bulk electroporation simultaneously (23,32).
c) Genetically encoded calcium indicators (GECI) are transmitted via viral transduction, in utero electroporation, and by generation of transgenic lines (23,32).
Common Calcium Imaging Devices
The main types of instruments used for calcium imaging are:
Widefield microscopy
Using a Photodiode array or a or a charged coupled device (CCD)-based camera. The light source is typically a mercury or xenon lamp in such cases, making a simple adjustment of the wavelengths of the excitation. Spin between two wavelengths of excitation, as seen in ratiometric measures of excitation, can be performed by using either a filter wheel or a monochromatic light source. Excitation and emission light usually are distinguished by a dichroic mirror located within the microscope. The photodiode arrays can be used to carry out calcium imaging. The traditional photodiode array comprises a series of photodiode (typically 124-1020 elements). Every photodiode is one pixel. Photodiode arrays are characterized by very high dynamic range and high speed but rather low spatial resolution. CCD-based cameras consist of many photodiodes densely packed on a chip with a serial readout of the signals. The newest generation of CCD-based cameras has an exquisitely high spatial and temporal resolution, but in some cameras, the noise level per pixel is high. The contrast and resolution of calcium imaging based on wide-field microscopy is limited by light dispersion, especially when attempting to image neurons more profound in the brain tissue (22,30). Such limitations make it more suitable for in vitro neuronal calcium imaging, like calcium imaging in neuronal cell cultures (30)
Laser scanning microscopy
Calcium imaging of neurons at deeper brain locations is usually done with confocal or two-photon microscopy. Using a laser beam in a raster pattern specimen is scanned. The fluorescent light emitted from each scan point (fluorescence value acquired for each pixel) is captured by a detector to create an image. Confocal microscopy, as a light source, uses a continuous wave laser. A scanner monitors the excitation position Confocal system across the sample.

Figure 2: Scanning Confocal and spinning disk
Upon passing a pinhole that blocks out-of-focus fluorescence, the emission light is detected and enters the photomultiplier tube. A dichroic mirror distinguishes the excitation and emission light. A significant limitation is that confocal aperture often blocks photons that are generated in the focal plane but are distributed on the way back through the optical path. Confocal microscopy, like wide-field microscopy, is therefore often limited to in vitro preparations, such as cultured neurons or brain slices. Eventually, some applications benefit from the use of disc-based confocal imagery, which requires the use of a spinning disc with many fine pinholes, each of which functions as an individual confocal aperture. A CCD -based camera can be used to detect images. Due to simultaneous sampling from several focal points, the spinning disc confocal approach can achieve higher image acquisition rates than laser scanning confocal microscopes (14.,34). Figure 2 provides a schematic image of the confocal microscope and the spinning discs confocal microscope.
A significant step forward in the field of neuroscience was the development of two-photon microscopy that enables high-resolution and high-sensitivity fluorescence microscopy in widespread in vivo brain tissue (20,29). Through two-photon microscopy, two low-energy near-IR photons come together to create a transition from the ground to the excited state in a fluorescent molecule. Figure 3, schematic image of the twophoton microscope The advantage is that the usual wavelengths of excitation are within the near-IR spectrum, with better tissue penetration than the visible light used in single-photon microscopy(20).It makes measurements of fluorescence concerning neuronal or vascular brain activity at > 100 micrometers deeper than standard objectives(20,31 ).

Figure 3: Two-Photon microscopy
Another significant advantage is that the fluorescence background frequency is shallow. For all these reasons, two-photon calcium imaging has become the preferred method of monitoring in deeper brain regions. Cortical circuits with well-preserved connections can be studied in vivo. Standard twophoton lasers can be tuned from 700 nm to 1000 nm or more and can be used to excite most commercially available fluorophores (20,29,31).
Functional imaging of neurons employing GECI in Drosophila
While cellular calcium transients are slower than the underlying electrical activity, all variants of GCaMP respond over relevant time scales to detect neural activity in fly neurons. GECIs have been optimized for chemical dyes that are sensitive to voltage or calcium (28). Comparisons between GCaMP responses and simultaneous electrophysiological stimulation or fly neuromuscular junction (NMJ) and olfactory lobe neurons demonstrate that fluorescence variations in GCaMP consistently represent a decrease and increases in neural activity in the neuronal populations in which it is expressed. Under certain conditions and in some neurons, GCaMP6 is even capable of monitoring single action potentials in single tests in live drosophila larvae (11), and jGCaMP7 sensors were shown to image a population of compass neurons in adult drosophila during behavior (10). Synaptically targeted calcium indicators have also been used to compare the activity levels of the different fly NMJ buttons (28). The generic fluorescence protein can be fused directly to the GCaMP, for example, with the Green-OrangeMatryoshka-GCaMP6 fusion “nested doll” (1), CaMPARI (11), and Photocon- vertible calcium indicators (28) enable user-specific classification of specific cells of interest, e.g., in areas where neuron populations are intertwined. GECIs are not promising “micro-scope electrophysiology,” but are new tools with their applications. Experiments requiring electrophysiological accuracy with GECIs should not be attempted realistically. GECIs enable a different type of experiment to visualize neural activity in many recognizable neurons simultaneously (3,10,11). GECIs are well suited to classify brain areas with corresponding activity, map regions that react to stimuli in more intact and behavioral animals, agnostically monitor for linked brain areas, and see how activity is progressing across sequential layers in the neural circuit. Several calcium sources may cause changes in cellular calcium levels. As GCaMP records calcium-binding, regardless of the origin of calcium, alternative sources should always be considered. Adding a voltage-gated calcium channel blocker or an actionpotential blocker could be used to verify that most of the fluorescence shift recorded by the GECI was due to action potential related influx of Ca2+ (28).
Imaging analysis
Typical usable image data is obtained as movies or Z-stacks in software supplied by microscope vendors. These files are often large and, by selecting precise regions of interest, files can be compressed, downsampled, or reduced. Analytics software packages are available on the ImageJ / FIJI platform (24) or custom scripts written by individual lab in MATLAB, R, or Python. Data is usually reported as a fluorescence shift, divided by the baseline (y-axis) versus the time (x-axis), with an image of the interest area where the signal is measured.
Fly line recommendations
There are numerous GECI fly lines available at the Bloomington Drosophila Stock Center ( infused with the newest generation of GCaMPs for imaging in presynaptic terminals. Many laboratories produce and optimize genetically encoded indicators for use in flies; one site that is recommended for use in fly’s potential users check is the Howard Hughes Medical Institute Janelia Genetically Encoded Neuronal Indicator and Effector (GENIE) Project site (
New generation of calcium indicators are being developed and published at a brisk pace, with each new generation offering brightness, photostability, sensitivity, and kinetics improvements. Specialized GECIs that could provide a better resolution of action potential acts and the timing of fast-spiking neurons. GECIs for the entire complement of neurotransmitters, neuromodulators, and secondary messengers are being created, potentially allowing complete investigation of molecular signaling in the neural circuit. What are the next significant challenges in calcium neuronal imaging? Another critical area that is likely to expand sharply in the coming years is calcium imaging in specified types of neurons awake, animals behaving. Such experiments are not limited to drosophila, c. elegans, but are likely to be applied to other types as well, such as ferrets, dogs, and primates. The use of calcium imaging in molecular medicine for a detailed analysis of signaling pathways in the explosively increasing number of disease models, particularly neurodegenerative diseases like Alzheimer’s and Parkinson’s. Lastly, calcium imaging in neurons can benefit significantly from the improved GECIs with higher signal sensitivity and better characteristics of transient response, and times were never better for quick, efficient circuit mapping in any model organism.
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