MICROSCOPY

Tutorial 1

Types of Microscopy

• Light

• Fluorescence

• Confocal

• Electron– Transmission Electron Microscopy (TEM)– Scanning Electron Microscopy (SEM)

LIGHT MICROSCOPY

• Advantages– Live cells can be viewed, color can be seen

• Disadvantages– Limit of resolution is 0.2 microns

• Organelles viewable– Nucleus, sometimes mitochondria and the

Golgi apparatus

Light Microscopy cont.

• Example: Onion epidermal cells. You can see the nucleus.

Fluorescence Microscopy

• Fluorescent dyes (ie fluorochromes) (used for staining cells) are detected with the aid of a fluorescence microscope

• Dyed objects show up in bright color on a dark background

• Advantage – Can see live cells– Can highlight particular structures or molecules

Fluorescence Microscopy cont.

• Example: DAPI stained to show DNA during cell division.

• Top: (Interphase) Only the nucleus is visible

• Bottom: (Mitosis) chromosomes lined up at centre of cell.

Transmission Electron Microscopy

• Advantages– Good resolution (200 nm to 0.2 nm; size from

organelles to macromolecules• Disadvantages

– Samples subject to electron bombardment and vacuum

– Lots of sample preparation is required (fixation, resin embedding, sectioning into slices 50-100 nm thick, heavy metal stain)

– Hard to construct 3-D structure from 2-D slices– TEM cannot be used with living material

TEM cont.

Example– Liver hepatocytes

of a rat showing rough endoplasmic reticulum (RER) and mitochondria

Scanning Electron Microscopy

• Advantages– Show surface structures – 3-D images– Great depth of field

• Disadvantages– Electrons require a vacuum so most samples

have to be fixed and dried– Only topography (surface structure) can be

seen

SEM cont

• A male wolf spider

• These teeth contain openings that release poison. They not only grasp the prey but immobilize it and inject it with poison.

Image Collection

• The following slides contain images from the image database

https://www.biomedia.cellbiology.ubc.ca/cellbiol/

Can you tell what technique was used for each of the following

images?

https://www.biomedia.cellbiology.ubc.ca/cellbiol/user/scripts/qry_image.php?media_id=148&file_name=MinakoK2b.jpg&size=2&popup=0