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