Optical microscopy

advertisement
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
• 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 - scheme
Optical microscopy - magnification
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
• 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.
TYPES
• 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 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, CCD camera.
• 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 highresolution 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.
Resolution
• 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
• 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
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 risks to 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 sound wave (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 records tiny 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.
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 Positron Emission Computed
Tomography (SPECT)
SPECT
• 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 3dimensional images.
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 analysis computer.
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 light photons
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 three dimensional view of the radiotracer
distribution within the body.
SPECT - Applications
Heart
• Heart Imaging
• Brain Imaging
• Kidney/Renal Imaging
• Bone Scans
Brain
A set of bone scan
projections
• …
Kidney/Renal
Computed Tomography Scan (CT)
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 threedimensional image of the internals of an object from a large
series of two-dimensional X-ray images taken around a single
axis of rotation
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
• 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 - diagnostic use
Cranial
• diagnosis of cerebrovascular
accidents and intracranial
hemorrhage
• CT generally does not exclude
infarct 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 - 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 Purpose image
reconstruction techniques may be adequate.
CT - diagnostic use
Cardiac
• With the advent of subsecond rotation combined with multislice 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
using retrospective 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 - 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 step
CT – step by step
Magnetic Resonance Imaging (MRI)
MRI & fMRI - basics
• An MRI uses powerful magnets to excite hydrogen nuclei 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 - 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 nondestructive testing method to characterize the quality of
products such as produce and timber.
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 - 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 MRI detects 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
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.
Disadvantages:
•
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).
MRI & fMRI
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.
MRI & fMRI
• 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
trials.
MRI & fMRI - principle
Modern 3 Tesla clinical MRI scanner.
Medical MRI most frequently relies on the relaxation properties of
excited hydrogen nuclei 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 T2weighting. 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 (PET)
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)
Advantages:
• Provides an image of brain activity.
Disadvantages:
• Expensive to use.
• Radioactive material used.
For images thanks to:
• http://www.sprawls.org/ppmi2/
• http://www.sprawls.org/ppmi2/RADIOACT/
• http://www.sprawls.org/resources/CTIMG/module.htm#31
Diagnostic Medical Imaging
MRI
fMRI
SPECT
X-Ray
Ultrasound
CT
Thank you for your attention!
Download