MICROSCOPY Tutorial 1 Types of Microscopy Light Fluorescence

  • Slides: 19
Download presentation
MICROSCOPY Tutorial 1

MICROSCOPY Tutorial 1

Types of Microscopy • • Light Fluorescence Confocal Electron – Transmission Electron Microscopy (TEM)

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

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.

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

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. •

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;

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)

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 –

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

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.

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?