BIOMEDICAL IMAGING

Week 9, Winter 12 ENGR 213 Aubrey Smith, M.S.

White Light

Microscope Anatomy

Theme

Name
base stand objective

Description
holds sample still holds viewer eye piece rotating part that gives magnification transmits the light to the eyes will have a magnification to it sample into view sample into perfect focus tungsten filament controls the intensity of the lamp lens system to align and focus light to be opened and closed for more or less light into the samples viewing specimen through the opposite side of the light source

Anatomy eye piece Coarse focus fine lamp rheostat condenser aperture Light transmittance

Depth of Field
Vertical distance from below the focal plane to above the focal plane where the image is in focus (greater the depth of field, the more things will be in focus) Be aware of:
Field of view Focal length Focus

Depth of the Field

Magnification
Degree of which

enlargement occurs
Numerical aperture (NA)

measure of light collecting ability

scale: small # = less able large # more able

This give NA = nsin = half

angle and n = refractive index

Objectives

Contrast
Different light intensity between images and adjacent

background Need at least 2% contrast to see with the naked eye

White Light

White light/ Bright Field

Bright field illumination, sample contrast comes from

absorbance of light in the sample.

White light/ Bright Field

Advantages Simplicity of setup with only basic equipment required. Limitations Very low contrast Low optical resolution The sample often has to be stained before viewing Enhancements Reducing or increasing the amount of the light source via the iris diaphragm. Use of a colored (usually blue) or polarizing filter on the light source to highlight features not visible under white light.

Common Stains
Hematoxlyin and Eosin Biotin

DIC Differential Interference Contrast

An excellent mechanism for rendering contrast in

transparent specimens Where optical path length gradients (in effect, the rate of change in the direction of wavefront shear) are primarily responsible for contrast
Beam-shearing interference system Produces a monochromatic shadow-cast image that effectively

displays the gradient of optical paths for both high and low spatial frequencies present in the specimen.

Steep gradients in path length generate excellent

contrast, and images display the pseudo threedimensional relief shading, which is characteristic of the DIC technique.

DIC

Phase Contrast
Takes advantage of minute refractive index differences Within cellular components and between unstained cells and their surrounding aqueous medium Producing contrast in transparent specimens. Phase contrast microscopy produces image intensity

(amplitude) values that vary as a function of specimen optical path length magnitude
Dense regions (those having large path lengths) appearing darker

than the background. Low thickness values, or a refractive index less than the surrounding medium, are rendered much lighter

Phase Contrast
Difference between the direct

and diffracted light is increased Undiffracted light, or direct light, in this cone speeds up as it passes through a phase-shifting element in the objective lens. Light passing through the specimen (and diffracted) is combined with direct light that passed through the phaseshifting element. The result of combining the two beams of light, is a darker or brighter spot in the image

Phase Contrast and DIC

The most fundamental
Characteristic
Image Brightness (Brightfield = 100 Percent) Epi-Fluorescence Light Loss (Brightfield = 0 Percent) Lateral Resolution Axial Resolution (Depth Discrimination) Illuminating Aperture Phase Shift Detection Limit Utility at High Phase Shifts Azimuthal Effects Halos and Shade-Off Stained Specimens Birefringent Specimens Birefringent Specimen Containers Brightfield Image Deterioration Cost

Phase Contrast
1.3 Percent

DIC
0.36 - 2.3 Percent 73 Percent

distinction is the optical basis upon which images are formed by the complementary techniques.
Specimens examined

28 Percent Condenser Annulus Restricted Poor 10 Percent of Objective NA < /100 Not Useful No Yes Not Useful Useful Yes Slight Moderate

Superior Superior Variable < /100 Useful Yes No Useful Not Useful No None High

by these contrastenhancing methods produce images that are often quite different in appearance and character when objectively compared.

Phase Contrast and DIC

Fluorescence

Fluorescence Background
light interaction with molecules = the type of image you

are working with:

Absorption light goes into molecule light dissipates as heat

(white light) Fluorescence absorb the light energy and will emit the rest at a lower energy (higher )

Why use: 1. Improved contrast over transmitted light 2. Can detect low abundance structures 3. Can detect sub resolution structures molecules 4. You can have specificity of detection of more than one molecule at time

Fluorescence Background
Excited state lifetime Short half life of fluorophor Emission Absorbs to move to higher energy level Relaxes to release light Sensitivity Detect specific molecules Makes it qualitative Multicolor detection Can use multiple wavelengths of light See several structures at once Stability Will last for 6 months Low hazard Not radioactive Commercial availability Lower cost

Fluorescence Background

How to preform IHC

Fluorescence Terms
Components
strong white light source excitation filter beam splitter (to ensure only a

Imaging Issues
Bleed through: when looking at

specific wavelength of light goes through) emission filter filter cube long pass filter (transmits light longer than the given ) short pass filter (transmits light shorter than the given ) Band pass filter Different colors at once (based on the filters)

2 different fluorfluors, want to limit the overlap of the Brightness: too high get quenching (where all molecules are self absorbing E from adjacent molecule) Saturation: too many e- in the excited state and the intensity will not change based on the amount of light going through Photo bleaching: limited number of excitation/emission cycles Autofluorsecene: some native tissues do it!

How to preform Immunohistochemistry

Immunohistochemistry (IHC) uses the principle of

antibody/antigen binding to identify and localize proteins of interest within the cells of a tissue section. To enable this, the antibodies are directly or indirectly labelled with a chromophore or fluorophore. IHC is a key technique in research as well as investigative and diagnostic pathology, where there is a need to correlate the localization of antigens in specific cells with tissue morphology.

How to preform IHC

Direct
Uses one labeled antibody,

Indirect
Unlabelled primary antibody (first

which binds directly to the antigen being targeted Simple and quick

layer) which reacts with the tissue antigen, and a labelled secondary antibody (second layer) which reacts with the primary antibody More sensitive

Jablonskis - excitation
Light into an atom = excite e- to outer state (unstable) (absorbed a

photon( Eventually wants to be stable = release a photon and e- goes back to ground stated
Energy Given by Plancks Law

Stokes Shift
Is the difference between the 2 peaks, emission is usually

higher , lower energy

Excitation spectra the range of you can excite a photon Emission spectra the range of you can have light emitted from

Stokes Shift

Other Types of Imaging

Confocal
It enables the reconstruction of three-dimensional

structures from the obtained images. Can select out the excitation pin hole to get a very limited and length, by placing a pin hole over where the light line is for the line of the desired that you want to observe Advantages
controllable depth of field Elimination of image degrading out-of-focus information Collect serial optical sections from thick specimen

Use of spatial filtering to eliminate out-of-focus light or

flare in specimens that are thicker than the plane of focus

Confocal Specific Location in Sample

2 Photon
For each excitation, two photons of the infrared light are

absorbed
Due to the multiphoton absorption the background signal is strongly

suppressed. alternative to confocal microscopy due to its deeper tissue penetration, efficient light detection and reduced phototoxicity

2-photon imaging is particularly useful for live imaging of

thick samples.. Two photon imaging is also useful with UV excitable dyes. The infrared radiation used for excitation in 2-photon imaging penetrates into tissue more efficiently than shorter wavelengths.

2-Photon

2 Photon vs Confocal
Both achieve optical sectioning but they do so in different

ways.
Confocal, the size of the variable pinhole in front of the detector

(photomultiplier tube) determines, in part, the thickness of the optical section. two photon, optical sectioning results from the fact that the probability of a two photon event occurring (i.e. excitation) happens only at the focal plane where there is an extremely high photon density

As a result, in 2-photon imaging, excitation occurs only at

the plane of focus. Conversely, excitation (and bleaching) occurs throughout a significant portion of the sample depth with the confocal.

Scanning Electron Microscope (SEM)

Electron microscope that images a sample by scanning it

with a high-energy beam of electrons Electrons interact with the atoms that make up the sample producing signals that contain information about the sample's surface topography, composition, and other properties such as electrical conductivity. Unlike optical and transmission electron microscopes, image magnification in the SEM is not a function of the power of the objective lens Function is to focus the beam to a spot, and not to image the specimen

SEM

Medical Imaging

X-Rays
X-rays have smaller wavelengths and therefore higher

energy than ultraviolet waves. X-ray detectors collect actual photons of X-ray light X-ray technology uses electromagnetic radiation to make images.
The image is recorded on a film, called a radiograph. The parts of your body appear light or dark due to the different

rates that your tissues absorb the X-rays. Calcium in bones absorbs X-rays the most, so bones look white on the radiograph. Fat and other soft tissues absorb less, and look gray. Air absorbs least, so lungs look black.

X-rays

CT Scans
CT or CAT scans are special x-ray tests that produce cross-

sectional images of the body using x-rays and a computer. These images allow the radiologist, a medical doctor who specializes in images of the body, to look at the inside of the body just as you would look at the inside of a loaf of bread by slicing it. One of the best and fastest tools for studying the chest, abdomen and pelvis because it provides detailed, crosssectional views of all types of tissue.

CT Uses
Diagnosing many different cancers, including lung, liver,

kidney and pancreatic cancer, since the image allows a physician to confirm the presence of a tumor and measure its size, precise location and the extent of the tumor's involvement with other nearby tissue. Diagnosis and treatment of vascular diseases that can lead to stroke, kidney failure or even death. CT is commonly used to assess for pulmonary embolism (a blood clot in the lung vessels) as well as for abdominal aortic aneurysms (AAA). Diagnosingand treating spinal problems and injuries to the hands, feet and other skeletal structures because it can clearly show even very small bones as well as surrounding tissues such as muscle and blood vessels.

CT Scans

Magnetic Resonance Imaging (MRI)

Scan is a radiology technique that uses magnetism, radio

waves, and a computer to produce images of body structures. A tube surrounded by a giant circular magnet.
The magnet creates a strong magnetic field that aligns the protons

of hydrogen atoms, which are then exposed to a beam of radio waves. This spins the various protons of the body (generally water), and they produce a faint signal that is detected by the receiver portion of the MRI scanner

The image and resolution produced by MRI is quite

detailed and can detect tiny changes of structures within the body.

MRI

MRI Uses
Extremely accurate method of disease detection

throughout the body.

In the head, trauma to the brain can be seen as bleeding or

swelling. Other abnormalities often found include brain aneurysms, stroke, tumors of the brain, as well as tumors or inflammation of the spine.

Neurosurgeons use an MRI scan not only in defining brain

anatomy but in evaluating the integrity of the spinal cord after trauma. It provides valuable information on glands and organs within the abdomen, and accurate information about the structure of the joints, soft tissues, and bones of the body. Often, surgery can be deferred or more accurately directed after knowing the results of an MRI scan.

Differences between CT and MRI

X-ray vs Manget Increased Contrast with MRI MRI has clearer images CT is cheaper and easier to preform

Positron Emission Tomography (PET)

A type of nuclear medicine imaging Small amounts of radioactive material to diagnose and

determine the severity of or treat a variety of diseases,

including many types of cancers, heart disease, gastrointestinal,

endocrine, neurological disorders and other abnormalities within the body

use radioactive materials called radiopharmaceuticals or

radiotracers. measures important body functions, such as blood flow, oxygen use, and sugar (glucose) metabolism, to help doctors evaluate how well organs and tissues are functioning

PET Scan
1. As the radioisotope undergoes positron emission decay 2. 3. 4.

5.

it emits a positron The emitted positron travels in tissue for a short distance It loses kinetic energy, until it decelerates to a point where it can interact with an electron Then it annihilates both electron and positron, producing a pair of annihilation photons moving in approximately opposite directions. These are detected when they reach a scintillator in the scanning device, creating a burst of light

PET Scan
Because nuclear medicine procedures are able to

pinpoint molecular activity within the body, they offer the potential to identify disease in its earliest stages as well as a patients immediate response to therapeutic interventions. When combined with CT can get specific anatomical and disease locations

PET scans

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