Developments in Imaging Technology and Staining Techniques

Unit C: Section 1.3

Science 10

Light Microscopes

Light microscopes magnify cells through the use of one or more curved lenses and a light source.

Two important aspects of microscopy are contrast and resolution.

Which dot grabs your attention?

The dark one due to the sharp contrast against the white background.  In this example, the contrast even overrides the size of the larger circle in its ability to focus the user’s visual awareness. 

Contrast Contrast (pg 253 – 254) Contrast is the ability to see differences in structure due

to differences in light absorption

- With Brightfield microscopy, most of the cells arecolorless because light passes directly through them.

Manipulating the light source by closing the iris diaphragm will alter the contrast.

Staining for Contrast Stains may be used to enhance the contrast.

Stains may attach to particular parts of the cell, improving the contrast between internal structures and producing better images. There are two types:

• Simple staining stains all structures. • Differential staining stains only certain structures.

One disadvantage of these techniques is that fixation and staining kill the cells, so it is not possible to view living tissue.

See Fig. 1.13 and 1.14 on pg. 254-255 for comparison*

Resolution (Pg 255 – 256)

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

• The eye alone can distinguish images as small as 0.1 mm

• The limit with a light microscope is about 0.2 um (2 x 10-7 m)

The efficiency of the light microscope is limited because as magnification increases, the image becomes blurred. • Approximate maximum magnification is 1500X

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Developments in Imaging Technology

Contrast Enhancing Techniques & Fluorescence Microscopy

(pg 256 – 257)

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Techniques were developed to improve images by altering the light path through the specimen.

These systems of illumination include darkfield, phase contrast, and differential interference contrast illumination (see page 256 for examples).

Confocal Technology • In the confocal laser scanning microscope (CLSM), a

laser concentrates light onto a specimen. The reflection is passed through a tiny opening called the confocal pinhole and reaches an electronic detector that converts the light into an image.

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Shown here are optical slices through a pollen grain as viewed by a CLSM. The last image was created by stacking the optical slices.

The advantage is that all of the out of focus light is eliminated

Fluorescence Microscopy – was also developed to improve images but also gave information about the molecules on a cell’s surface

GFP (Green Fluorescent Protein)- This protein was first discovered in a luminous jellyfish.- It would glow green under UV light.

- It has been used to study where molecules attach themselves in tissues.

• The GFP gene (which codes for the protein) has been inserted into other organisms’ genomes.• E.g. mice, rabbits

Other “dyes” are now used in order to get fluorescence.

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Normal mice

Mice with the GFP gene inserted

Different dyes will glow different colours

An example would be using different dyes to highlight the various stages of cell division.

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Stage of Mitosis

Dyes

Spectral Karyotyping

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Normal Spectral Karyotypes

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Definitely not Normal!!

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Electron Microscopy pg 258 – 260)

At the University of Toronto, James Hillier and Albert Prebus developed the first functional electron microscope (1930’s).

An electron microscope uses a beam of electrons instead of a light wave.

The image is formed by the absorption or scattering of the electron beam caused by electron-dense material.

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Transmission Electron Microscope (TEM)

It depends on a beam of electrons passed through a very thin section of fixed and stained tissue embedded in plastic. - The material has to be dead.

The electrons pass through the specimen, through a tube and then fall on a fluorescent screen or on photographic film (producing black-and-white photographs). - The air inside the tube must be particle

free and dry.

One of the drawbacks of the TEM is the difficulty of building up a three-dimensional picture of the cell from very thin sections and make any sense out of it.

Scanning Electron Microscope (SEM).

• This technology gives information about the surface features of a specimen.

• Specimens are fixed and covered with an electron dense material like gold, which reflects electrons.

• When the investigator scans the surface of the specimen, the electrons bouncing off the surface are picked up by a sensor and a three-dimensional image is formed.

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SEM TEM

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SEM TEM

Pollen

Moth Eye

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IMAGES

Can you identify these “things” scanned using an SEM?

You have all seen these things many times! All belong to animals!

Butterfly scales Fly Foot Aphid

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How about these?

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Mosquito head

Dust mite

Mite on a human hair

Water fleaDaphnia

Bird Feathers

Foraminifera

Head of a Tapeworm

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AFM STM

**Do Check & Reflect P. 260 # 1 - 6

Electron Microscopes A form of the SEM has been developed that allows the

use of live material. The recently developed Scanning Tunnelling Microscope

(STM) and Atomic Force Microscope (AFM) are able to reveal even smaller structures than the transmission or scanning electron microscopes can.

See figure C1.24 page 261

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Microscopy ComparisonsContrast Enhancing Improve images by altering the light path

through the specimen (includes darkfield, phase contrast and differential interference)

Confocal Laser beam concentrates on specimen; only light that is in focus reaches the detector

Fluorescence Fluorescent molecules emitted light of a different wavelength, causing particular areas to glow

TEM Uses a beam of electrons; tissue must be thin and stained. Gives great detail but no 3-D image

SEM Specimen is fixed and covered in electron dense material which reflects electrons which are picked up by a sensor. Gives great 3-D images