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Biomedical Imaging. Eugen Kvasnak, PhD. Department of Medical Biophysics and Informatics 3rd Medical Faculty of Charles University. Content. Microscopy Ultrasound & Sonography SPECT & Gamma Camera CT NMR & fMRI PET. Microscopy.

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Biomedical Imaging

Eugen Kvasnak, PhD.

Department of Medical Biophysics and Informatics

3rd Medical Faculty of Charles University


  • Microscopy

  • Ultrasound & Sonography

  • SPECT & Gamma Camera

  • CT

  • NMR & fMRI

  • PET


  • main branches:optical, electron and scanning probe microscopy. (+ less used X-ray microscopy)

  • Optical and electron microscopy involves the diffraction, reflection, or refraction of radiation incident upon the subject of study, and the subsequent collection of this scattered radiation in order to build up an image.

  • Scanning probe microscopy involves the interaction of a scanning probe with the surface or object of interest.

Optical microscopy- definition

  • Optical or light microscopy involves passing visible light transmitted through or reflected from the sample through a single or multiple lenses to allow a magnified view of the sample.

  • The resulting image can be detected directly by the eye, imaged on a photographic plate or captured digitally.

  • The single lens with its attachments, or the system of lenses and imaging equipment, along with the appropriate lighting equipment, sample stage and support, makes up the basic light microscope.

Optical microscopy - magnification

Optical microscopy l imitations
Optical microscopy- limitations

OM can only image dark or strongly refracting objects effectively.

Out of focus light from points outside the focal plane reduces image clarity. Compound optical microscopes are limited in their ability to resolve fine details by the properties of light and the refractive materials used to manufacture lenses. A lens magnifies by bending light. Optical microscopes are restricted in their ability to resolve features by a phenomenon called diffraction which, based on the numerical aperture AN of the optical system and the wavelengths of light used (λ), sets a definite limit (d) to the optical resolution. Assuming that optical aberrations are negligible, the resolution (d) is given by:

In case of λ = 550 nm(green light), with air as medium, the highest practical AN is 0.95, with oil, up to 1.5.

Due to diffraction, even the best optical microscope is limited to a resolution of around 0.2 micrometres.

Optical microscopy- types

  • Optical microscopy techniques

  • Bright field optical microscopy

  • Oblique illumination

  • Dark field optical microscopy

  • Phase contrast optical microscopy

  • Differential interference contrast microscopy

  • Fluorescence microscopy

  • Confocal laser scanning microscopy

  • Deconvolution microscopy

  • Near-field Scanning OM

Electron microscopy definition and types
Electron Microscopy-definition and types

  • developed in the 1930s that use electron beams instead of light.

  • because of the much lower wavelength of the electron beam than of light, resolution is far higher.


  • Transmission electron microscopy (TEM) is principally quite similar to the compound light microscope, by sending an electron beam through a very thin slice of the specimen. The resolution limit (in 2005) is around 0.05 nanometer.

  • Scanning electron microscopy (SEM) visualizes details on the surfaces of cells and particles and gives a very nice 3D view. The magnification is in the lower range than that of the transmission electron microscope.

Transmission e lectron m icroscopy tem
Transmission Electron Microscopy (TEM)

  • beam of electrons is transmitted through a specimen, then an image is formed, magnified and directed to appear either on a fluorescent screen or layer of photographic film or to be detected by a sensor (e.g. charge-coupled device, CCDcamera.

  • involves a high voltage electron beam emitted by a cathode, usually a tungsten filament and focused by electrostatic and electromagnetic lenses.

  • electron beam that has been transmitted through a specimen that is in part transparent to electrons carries information about the inner structure of the specimen in the electron beam that reaches the imaging system of the microscope.

  • spatial variation in this information (the "image") is then magnified by a series of electromagnetic lenses until it is recorded by hitting a fluorescent screen, photographic plate, or CCD camera. The image detected by the CCD may be displayed in real time on a monitor or computer.

Transmission Electron Microscopy (TEM)

Neuron growing on astroglia

Black Ant

House Fly

House Fly

Human stem cells

Human red blood cells

Neurons CNS

Scanning Electron Microscopy (SEM)

  • type of electron microscope capable of producing high-resolution images of a sample surface.

  • due to the manner in which the image is created, SEM images have a characteristic 3D appearance and are useful for judging the surface structure of the sample.


  • depends on the size of the electron spot, which in turn depends on the magnetic electron-optical system which produces the scanning beam.

  • is not high enough to image individual atoms, as is possible in the TEM … so that, it is 1-20 nm

X ray microscopy
X-ray microscopy

  • less common,

  • developed since the late 1940s,

  • resolution of X-ray microscopy lies between that of light microscopy and the electron microscopy.

  • X-rays are a form of electromagnetic radiation with a wavelength in the range of 10 to 0.01 nanometers, corresponding to frequencies in the range 30 PHz to 30 EHz.

Ultrasound sonography basics
Ultrasound (Sonography) - basics

It is used to visualize muscles, tendons, and many internal organs, their size, structure and any pathological lesions with real time tomographic images. They are also used to visualize a fetus during routine and emergency prenatal care.

The technology is relatively inexpensive and portable, especially when compared with modalities such as magnetic resonance imaging(MRI) and computed tomography (CT).

It poses no known risksto the patient, it is generally described as a "safe test" because it does not use ionizing radiation, which imposes hazards (e.g. cancer production and chromosome breakage).

However, it has two potential physiological effects: it enhances inflammatory response; and it can heat soft tissue.

Ultrasound – how does it work?

  • the same principles involved in the sonar used by bats, ships and fishermen.

  • when a soundwave (frequency 2.0 to 10.0 megahertz) strikes an object, it bounces backward or echoes.

  • by measuring these echo waves it is possible to determine how far away the object is and its size, shape, consistency (solid, filled with fluid, or both) and uniformity.

  • a transducer both sends the sound waves and records the echoing waves. When the transducer is pressed against the skin, it directs a stream of inaudible, high-frequency sound waves into the body. As the sound waves bounce off of internal organs, fluids and tissues, the sensitive microphone in the transducer recordstiny changes in the sound's pitch and direction. These signature waves are instantly measured and displayed by a computer, which in turn creates a real-time picture on the monitor.

U ltrasound b iomedical applications
Ultrasound - biomedical applications

  • heart and blood vessels, incl. the abdominal aorta and its major branches

  • liver

  • gallbladder

  • spleen

  • pancreas

  • kidneys

  • bladder

  • uterus, ovaries, and unborn child (fetus) in pregnant patients

  • eyes

  • thyroid and parathyroid glands

  • scrotum (testicles)

Ultrasound – limitations

Ultrasound waves are reflected by air or gas; therefore ultrasound is not an ideal imaging technique for the bowel.

Ultrasound waves do not pass through air; therefore an evaluation of the stomach, small intestine and large intestine may be limited. Intestinal gas may also prevent visualization of deeper structures such as the pancreas and aorta.

Patients who are obese are more difficult to image because tissue attenuates (weakens) the sound waves as they pass deeper into the body.

Ultrasound has difficulty penetrating bone and therefore can only see the outer surface of bony structures and not what lies within.


  • Single Photon Emission Computed Tomography.

  • gamma ray emissions are the source of information

    (contrary to X-ray transmissions used in conventional CT)

  • allows to visualize functional information about a patient's specific organ or body system (similarly to X-ray Computed Tomography (CT) or Magnetic Resonance Imaging (MRI)

SPECT- how does it work?

  • Internal radiation is administered by means of a pharmaceutical which is labeled with a radioactive isotope / tracer /radiopharmaceutical, is either injected, ingested, or inhaled.

  • The radioactive isotope decays, resulting in the emission of gamma rays. These gamma rays give us a picture of what's happening inside the patient's body.

SPECT /Gamma camera - how does it work?

  • The Gamma camera collects gamma rays that are emitted from within the patient, enabling us to reconstruct a picture of where the gamma rays originated. From this, we can determine how a particular organ or system is functioning.

  • The gamma camera can be used in planar imaging to acquire 2-dimensional images, or in SPECT imaging to acquire 3-dimensionalimages.

Gamma Camera

Once a radiopharmaceutical has been administered, it is necessary to detect the gamma ray emissions in order to attain the functional information. 

The instrument used in Nuclear Medicine for the detection of gamma rays is known as the Gamma camera.The components making up the gamma camera are the collimator, detector crystal, photomultiplier tube array, position logic circuits, and the data analysiscomputer. 

Gamma Camera - how does it work?

Gamma Camera - Collimator

- the first object that an emitted gamma photon encounters after exiting the body. The collimator is a pattern of holes through gamma ray absorbing material, usually lead or tungsten, that allows the projection of the gamma ray image onto the detector crystal.  The collimator achieves this by only allowing those gamma rays traveling along certain directions to reach the detector.

Gamma Camera - Scintillation Detector

  • In order to detect the gamma photon we use scintillation detectors.  A Thallium-activated Sodium Iodide [NaI(Tl)] detector crystal is generally used in Gamma cameras.  This is due to this crystal's optimal detection efficiency for the gamma ray energies of radionuclide emission common to Nuclear Medicine. 

  • A detector crystal may be circular or rectangular. It is typically 3/8" thick and has dimensions of 30-50 cm. A gamma ray photon interacts with the detector by means of the Photoelectric Effect or Compton Scattering with the iodide ions of the crystal.  This interaction causes the release of electrons which in turn interact with the crystal lattice to produce light, in a process known as scintillation.

Gamma Camera – Photoelectric effect

Gamma Camera – Compton Scattering

Gamma Camera - Scintillation

Gamma Camera - Photomultiplier

  • - instrument that detects and amplifies the electrons that are produced by the photocathode which, when stimulated by lightphotons ejects electrons.

  • For every 7 to 10 photons incident on the photocathode, only one electron is generated.  This electron from the cathode is focused on a dynode which absorbs this electron and re-emits many more electrons (6 to 10).

Gamma Camera - Planar Dynamic Imaging

  • Since the camera remains at a fixed position in a planar study, it is possible to observe the motion of a radiotracer through the body by acquiring a series of planar images of the patient over time. 

  • Each image is a result of summing data over a short time interval, typically 1-10 seconds. 

SPECT - Imaging

  • If one rotates the camera around the patient, the camera will acquire views of the tracer distribution at a variety of angles.

  • After all these angles have been observed, it is possible to reconstruct a threedimensional view of the radiotracer distribution within the body. 


A set of bone scan projections


SPECT - Applications

  • Heart Imaging

  • Brain Imaging

  • Kidney/Renal Imaging

  • Bone Scans


Ct basics
CT - basics

  • CT scans use a series of X-ray beams

  • It creates cross-sectional images,e.g. of the brain and shows the structure of the brain, but not its function.

  • Digital geometry processing is used to generate a three-dimensionalimage of the internals of an object from a large series of two-dimensional X-ray images taken around a single axis of rotation

Ct basics1
CT - basics

  • CT's primary benefit is the ability to separate anatomical structures at different depths within the body.

  • A form of tomography can be performed by moving the X-ray source and detector during an exposure.

  • Anatomy at the target level remains sharp, while structures at different levels are blurred.

  • By varying the extent and path of motion, a variety of effects can be obtained, with variable depth of field and different degrees of blurring of 'out of plane' structures.

Ct principle
CT - principle

  • Because contemporary CT scanners offer isotropic, or near isotropic, resolution, display of images does not need to be restricted to the conventional axial images.

  • Instead, it is possible for a software program to build a volume by 'stacking' the individual slices one on top of the other. The program may then display the volume in an alternative manner.

Ct d iagnostic use
CT - diagnostic use


  • diagnosis of cerebrovascular accidents and intracranial hemorrhage

  • CT generally does not excludeinfarct in the acute stage of a stroke. For detection of tumors, CT scanning with IV contrast is occasionally used but is less sensitive than magnetic resonance imaging (MRI).

Ct d iagnostic use1
CT - diagnostic use

  • Chest

  • CT is excellent for detecting both acute and chronic changes in the lung parenchyma.

  • A variety of different techniques are used depending on the suspected abnormality.

  • For evaluation of chronic interstitial processes (emphysema, fibrosis, and so forth), thin sections with high spatial frequency reconstructions are used - often scans are performed both in inspiration and expiration. This special technique is called High resolution CT (HRCT).

  • For detection of airspace disease (such as

  • pneumonia) or cancer, relatively thick

  • sections and general Purposeimage

  • reconstruction techniques may be adequate.

Ct d iagnostic use2
CT - diagnostic use


  • With the advent of subsecond rotation combined with multi-slice CT (up to 64-slice), high resolution and high speed can be obtained at the same time, allowing excellent imaging of the coronary arteries (cardiac CT angiography).

  • Images with an even higher temporal resolution can be formed usingretrospective ECG gating. In this technique, each portion of the heart is imaged more than once while an ECG trace is recorded. The ECG is then used to correlate the CT data with their corresponding phases of cardiac contraction. Once this correlation is complete, all data that were recorded while the heart was in motion (systole) can be ignored and images can be made from the remaining data that happened to be acquired while the heart was at rest (diastole). In this way, individual frames in a cardiac CT investigation have a better temporal resolution than the shortest tube rotation time.

Ct d iagnostic use3
CT - diagnostic use

Abdominal and pelvic

  • CT is a sensitive method for diagnosis of abdominal diseases. It is used frequently to determine stage of cancer and to follow progress. It is also a useful test to investigate acute abdominal pain.

  • Renal/urinary stones, appendicitis, pancreatitis, diverticulitis, abdominal aortic aneurysm, and bowel obstruction are conditions that are readily diagnosed and assessed with CT.

  • CT is also the first line for detecting solid organ injury after trauma.

Ct step by step
CT – step by step

Ct step by step1
CT – step by step

Ct step by step2
CT – step by step

Ct step by step3
CT – step by step

Mri fmri basics
MRI& fMRI- basics

  • An MRI uses powerful magnets to excitehydrogennuclei in water molecules in human tissue, producing a detectable signal. Like a CT scan, an MRI traditionally creates a 2D image of a thin "slice" of the body.

  • The difference between a CT image and an MRI image is in the details. X-rays must be blocked by some form of dense tissue to create an image, therefore the image quality when looking at soft tissues will be poor.

  • An MRI can ONLY "see" hydrogen based objects, so bone, which is calcium based, will be a void in the image, and will not affect soft tissue views. This makes it excellent for peering into joints.

  • As an MRI does not use ionizing radiation, it is the preferred imaging method for children and pregnant women.

Mri fmri basics1
MRI& fMRI- basics

  • Magnetic resonance imaging (MRI), formerly referred to as magnetic resonance tomography (MRT) and, in scientific circles and as originally marketed by companies such as General Electric, nuclear magnetic resonance imaging (NMRI) or NMR zeugmatography imaging, is a non-invasive method using nuclear magnetic resonance to render images of the inside of an object.

  • It is primarily used in medical imaging to demonstrate pathological or other physiological alterations of living tissues.

  • MRI also has uses outside of the medical field, such as detecting rock permeability to hydrocarbons and as a non-destructive testing method to characterize the quality of products such as produce and timber.

Mri fmri basics2
MRI& fMRI- basics

  • MRI should not be confused with the NMR spectroscopy technique used in chemistry, although both are based on the same principles of nuclear magnetic resonance.

  • In fact MRI is a series of NMR experiments applied to the signal from nuclei (typified by the hydrogen nuclei in water) used to acquire spatial information in place of chemical information about molecules.

  • The same equipment, provided suitable probes and magnetic gradients are available, can be used for both imaging and spectroscopy.

Mri fmri basics3
MRI& fMRI- basics

  • The scanners used in medicine have a typical magnetic field strength of 0.2 to 3 Teslas. Construction costs approximately US$ 1 million per Tesla and maintenance an additional several hundred thousand dollars per year.

  • Medical Imaging MRI, or "NMR" as it was originally known, has only been in use since the 1980's. Effects from long term, or repeated exposure, to the intense magnetic field is not well documented.

  • Functional MRIdetects changes in blood flow to particular areas of the brain. It provides both an anatomical and a functional view of the brain.

  • MRI uses the detection of radio frequency signals produced by displaced radio waves in a magnetic field. It provides an anatomical view of the brain.

MRI& fMRI– dis/advantages


  • No X-rays or radioactive material is used.

  • Provides detailed view of the brain in different dimensions.

  • Safe, painless, non-invasive.

  • No special preparation (except the removal of all metal objects) is required from the patient. Patients can eat or drink anything before the procedure.


  • Expensive to use.

  • Cannot be used in patients with metallic devices (pacemakers).

  • Cannot be used with uncooperative patients because the patient must lie still.

  • Cannot be used with patients who are claustrophobic (unless new MRI systems with a more open design are used).


Functional MRI

  • A fMRI scan showing regions of activation in orange, including the primary visual cortex (V1, BA17).

  • Functional MRI (fMRI) measures signal changes in the brain that are due to changing neural activity. The brain is scanned at low resolution but at a rapid rate (typically once every 2-3 seconds). Increases in neural activity cause changes in the MR signal via T2* changes; this mechanism is referred to as the BOLD (blood-oxygen-level dependent) effect. Increased neural activity causes an increased demand for oxygen, and the vascular system actually overcompensates for this, increasing the amount of oxygenated hemoglobin (haemoglobin) relative to deoxygenated hemoglobin.


  • Because deoxygenated hemoglobin attenuates the MR signal, the vascular response leads to a signal increase that is related to the neural activity. The precise nature of the relationship between neural activity and the BOLD signal is a subject of current research. The BOLD effect also allows for the generation of high resolution 3D maps of the venous vasculature within neural tissue.

  • While BOLD signal is the most common method employed for neuroscience studies in human subjects, the flexible nature of MR imaging provides means to sensitize the signal to other aspects of the blood supply. Alternative techniques employ arterial spin labeling (ASL) or weight the MRI signal by cerebral blood flow (CBF) and cerebral blood volume

    (CBV). The CBV method requires injection of a class

    of MRI contrast agents that are now in human clinical


MRI& fMRI- principle

Modern 3 Tesla clinical MRI scanner.

Medical MRI most frequently relies on the relaxation properties of excited hydrogennuclei in water and lipids. When the object to be imaged is placed in a powerful, uniform magnetic field, the spins of atomic nuclei with a resulting non-zero spin have to arrange in a particular manner with the applied magnetic field according to quantum mechanics. Nuclei of hydrogen atoms (protons) have a simple spin 1/2 and therefore align either parallel or antiparallel to the magnetic field.

MRI& fMRI- principle

The spin polarization determines the basic MRI signal strength. For protons, it refers to the population difference of the two energy states that are associated with the parallel and antiparallel alignment of the proton spins in the magnetic field and governed by Boltzmann statistics. In a 1.5 T magnetic field (at room temperature) this difference refers to only about one in a million nuclei since the thermal energy far exceeds the energy difference between the parallel and antiparallel states. Yet the vast quantity of nuclei in a small volume sum to produce a detectable change in field. Most basic explanations of MRI will say that the nuclei align parallel or anti-parallel with the static magnetic field; however, because of quantum mechanical reasons, the individual nuclei are actually set off at an angle from the direction of the static magnetic field. The bulk collection of nuclei can be partitioned into a set whose sum spin are aligned parallel and a set whose sum spin are anti-parallel.

MRI& fMRI- principle

The magnetic dipole moment of the nuclei then precesses around the axial field. While the proportion is nearly equal, slightly more are oriented at the low energy angle. The frequency with which the dipole moments precess is called the Larmor frequency. The tissue is then briefly exposed to pulses of electromagnetic energy (RF pulses) in a plane perpendicular to the magnetic field, causing some of the magnetically aligned hydrogen nuclei to assume a temporary non-aligned high-energy state. Or in other words, the steady-state equilibrium established in the static magnetic field becomes perturbed and the population difference of the two energy levels is altered. The frequency of the pulses is governed by the Larmor equation to match the required energy difference between the two spin states.

MRI& fMRI- applications

Clinical practice, MRI is used to distinguish pathologic tissue (such as a brain tumor) from normal tissue. One advantage of an MRI scan is that it is thought to be harmless to the patient. It uses strong magnetic fields and non-ionizing radiation in the radio frequency range. Compare this to CT scans and traditional X-rays which involve doses of ionizing radiation and may increase the risk of malignancy, especially in a fetus.

While CT provides good spatial resolution (the ability to distinguish two structures an arbitrarily small distance from each other as separate), MRI provides comparable resolution with far better contrast resolution (the ability to distinguish the differences between two arbitrarily similar but not identical tissues). The basis of this ability is the complex library of pulse sequences that the modern medical MRI scanner includes, each of which is optimized to provide image contrast based on the chemical sensitivity of MRI.

For example, with particular values of the echo time (TE) and the repetition time (TR), which are basic parameters of image acquisition, a sequence will take on the property of T2-weighting. On a T2-weighted scan, fat-, water- and fluid-containing tissues are bright (most modern T2 sequences are actually fast T2 sequences). Damaged tissue tends to develop edema, which makes a T2-weighted sequence sensitive for pathology, and generally able to distinguish pathologic tissue from normal tissue. With the addition of an additional radio frequency pulse and additional manipulation of the magnetic gradients, a T2-weighted sequence can be converted to a FLAIR sequence, in which free water is now dark, but edematous tissues remain bright. This sequence in particular is currently the most sensitive way to evaluate the brain for demyelinating diseases, such as multiple sclerosis.

The typical MRI examination consists of 5-20 sequences, each of which are chosen to provide a particular type of information about the subject tissues. This information is then synthesized by the interpreting physician.

Positron emission tomography pet1
Positron Emission Tomography(PET)

  • A scanner detects radioactive material that is injected or inhaled to produce an image of the brain.

  • Commonly used radioactively-labeled material includes oxygen, fluorine, carbon and nitrogen.

  • When this material gets into the bloodstream, it goes to areas of the brain that use it. So, oxygen and glucose accumulate in brain areas that are metabolically active.

  • When the radioactive material breaks down, it gives off a neutron and a positron.

  • When a positron hits an electron, both are destroyed and two gamma rays are released.

  • Gamma ray detectors record the brain area where the gamma rays are emitted. This method provides a functional view of the brain.

Positron Emission Tomography(PET)


  • Provides an image of brain activity.


  • Expensive to use.

  • Radioactive material used.

For images thanks to
For images thanks to:




Diagnostic medical imaging