Microscopes

Micrographia

• Robert HookeRobert Hooke

• 16651665

• First report of cell First report of cell structurestructure

• corkcork

First illustrated First illustrated book on book on

microscopymicroscopy

Binocular Microscope

Early MicroscopeEarly compound microscopes were

beautifully crafted but had

severe chromatic distortion

Outline

• Units of Measurement

• Microscopes: features & uses– Light– Electron

Units of Measurement

Light Microscopes

• Brightlight

• Darkfield

• Phase Contrast

• Differential Interference Contrast

• Fluorescence

• Confocal

Parts of the Microscope

Changes in light as it passes through a cell

Bright Light Microscopy

• FEATURES• structures > 0.2 um• specimen dark against

bright background• cheap and easy

• USES• Stained samples• Simple morphology• Count cells

AKA Bright field microscopy

Stained bacteria

Shape is easier to see after staining

Granules & Capsules

Darkfield Microscopy

• FEATURES• Special light source

for indirect reflected light– condenser disk

• object bright against black background

• improved resolution

• USES• living organisms• fragile organism• small microbes

– Treponema pallidum

Flagella can be seen by Dark field microscopy

Dark field Microscopy

Click on image to start video

• Light reflected

• Objects bright against dark background

• Slightly improved resolution

Phase Contrast Microscopy

• FEATURES• Special condenser • Special objective lens• Uses diffracted light• No stains

• USES• Living Structures• Motility• Internal cell Structures

Phase Light

Effect of edges on phase light

Bacteria by Phase Contrast

Phase Contrast Microscopy

• Live cells

• internal organelles visible

• Dynamic processes

Click on image to show Video

Differential Interference Contrast Microscopy

• FEATURES• Two Light Beams• Sample appears

coloured

• USES• 3D images

First developed by Nomarski in 1952

World’s Largest Bacterium

• Visible to naked eye

• In gut of Surgeon fish

• Eats alga

Procloron under DIG Microscopy

Procloron dividing

Four Types of Light Microscopy

Brightfield Phase contrast

Nomarski Darkfield

Fluorescence Microscopy

• FEATURES• UV light source• Special lenses• objects fluorscent

against black background

• USES• Immunofluorescent• labelled antibodies• rapid identification

Light path in a flourescent Scope

Fluorecent cells

Fluorescent Molecules

Double fluorescent labelling

Confocal Microscopy

• FEATURES• Laser light• multiple images• computer generated

• USES• 2D and 3D of fine

structural detail

Confocal Microscopy

Fluorescence Microscopy

Conventional Confocal

3D Reconstruction ViaConfocal Microscopy

Electron Microscopy

• Transmission Electron Microscopy

• Scanning Electron Microscopy

Lens in Microscopes

Plasma Membrane: Glycocalyx

Glycocalyx of plasma membrane of a T lymphocyte stained with ruthenium red

Scanning EM

Bacteria on Skin

Flagella & Pili

Scanning EM

Scanning EM

TEM

SEM

DICComparison of EM methods