Imaging Technology and Staining Techniques

CHAPTER 1.3

Three factors affect how well you can see an object in a microscope…1. magnification2. contrast3. resolution

In brightfield microscopy, light passes through cells which are seen as colourless.

Manipulating the light source can improve contrast

Stains and colouring agents also improve contrast (they kill the cells)

CONTRAST

Resolution, or resolving power, is the ability to distinguish between two structures that are very close together

RESOLUTION

The human eye can distinguish images of objects that are 0.1 mm or larger.

In the early 1950’s, techniques were developed to improve images by altering the light path through the specimen Darkfield

Phase contrast

Differential interference contrast illumination

CONTRAST ENHANCING TECHNIQUES

Gives information about molecules on the cell surfaceFluorescent molecules on specimen reflect UV light at

diff erent wavelengths, causing it to glow

FLUORESCENCE MICROSCOPY

A laser concentrates light onto a specimenThe reflection is passed through a tiny hole (confocal

pinhole)Reaches an electronic detector that converts light

into imageMany slices of images are combined to form a 3D

picture

CONFOCAL TECHNOLOGY

Uses beams of electrons instead of light waves (produces finer detail)

The image is formed by absorption or scattering of the electron beam

Electron-dense materials do not let the electrons pass through

Focus with electromagnetics

ELECTRON MICROSCOPY

Transmission electron microscope (TEM) depends on a beam of electrons passed through a very thin section of fixed and stained tissue embedded in plastic. The electrons fall on fi lm and photographs are produced

Can magnify specimen up to 1 500 000x

TEM

Developed in 1940’sGives information of the surface features of the

specimenShow structural arrangements like the double layer of

the cell membraneRecently allows the use of live material

SEM

Feature Light Microscope Electron Microscope

Source Lamp or laser Electron gun

Radiation UV or visible light Electron beam

Lenses Curved glass surfaces Electromagnets

Receiver Eye or digital image Fluorescent screen or digital image

Focus Up and down movement of lenses

Adjustment of magnetic field

COMPARISON OF LIGHT AND ELECTRON MICROSCOPES

Scanning Tunnelling Microscope and Atomic Force Microscope are able to reveal even smaller structures than the TEM or SEM

CHAPTER 1.4 – CELL RESEARCH

The mapping of DNA sequences in genes involves may techniques…Breaking cells down to release their DNA

Using Chemical techniques to make many copies of DNA

Finding the sequence of chemical subunits through computer analysis

GENE MAPPING

An open system is one that must interact with its environment to maintain its existence.

CELL COMMUNICATION

X-ray crystallography uses x-rays, special sensors that analyze patterns of x-ray scattering, and computer technology to allow scientists to learn the details of molecular structure to help them understand how the molecule works

3-D STRUCTURE