MICROSCOPES

Microscopic Instruments differ in their lenses and the source of their illumination

Microscopes

• All microscopes operate on the same basic principles– Energy is projected toward an object (MO)– Energy bounces off of the object and

creates an impression on a sensing device– The device can be a TV screen, a piece of

film or the human eye– The image reveals the form, shape, size

and structural features of the object

I. LIGHT MICROSCOPE

Visible light illuminates the object

A. STEREO MICROSCOPE (SM)

• Light bulb for a light source

• Magnification of 35X

• Surface picture (3D)

Spore under a Stereo Microscope

B. COMPOUND MICROSCOPE (CM)

• Most commonly used – 2 lenses

• Ocular lens – 10X

• Objective lenses

• 10X, 40X, 100X

• Some can even go to

2000X

B. COMPOUND MICROSCOPE (CM)

• Light bulb for light source

• Light goes through object

• Small or thin slice object

• Size and shape of bacteria cell can be seen

II. ELECTRON MICROSCOPE

Thousand times better than a light

microscope.

II. ELECTRON MICROSCOPE

• German physicist Ernst Ruska

• Showed that electrons can flow in a sealed tube if a vacuum is maintained

• Magnets can be used to

pinpoint the flow of

electrons onto

an object

II. ELECTRON MICROSCOPE

• Depending upon the density of structures in the object, the electrons are either absorbed or deflected

• The electrons form an image that can be projected onto a screen and outlines the structures in the object

A. TRANSMISSION ELECTRON MICROSCOPE (TEM) 1931

• Used to see internal cell structures in detail

• Ultrathin sections of a specimen must be prepared because the electron beam can only penetrate matter a short distance

• Sections are floated in water and picked up on a wire grid

• Sections are inserted into the vacuum chamber of the microscope

• Electrons illuminate the specimen

A. TRANSMISSION ELECTRON MICROSCOPE (TEM) 1931

• Magnetic field focuses the beam of electrons (as condenser lens of light

• No ocular lens, electrons hit electron sensitive screen to create image

• 200,000X – Strongest

Microscope Transmission electron micrograph of E. coli

B. SCANNING ELECTRON MICROSCOPE (SEM) 1960’s

• Permits the surface of objects to be seen without having to make thin sections

• Specimens are placed in the vacuum chamber and coated with a thin layer of gold to increase conductivity

• The electron beam sweeps across the object and knocks loose showers of electrons

B. SCANNING ELECTRON MICROSCOPE (SEM) 1960’s

• A 3D image builds line by line like a TV receiver – can see surface detail

• Can magnify 50X – 100,000X

• See image on TV screen

SEM 3D Photo of E. Coli

B. SCANNING ELECTRON MICROSCOPE (SEM) 1960’s

E. Coli - TEME. Coli - SEM

III. VARIATIONS

Manipulation of Light

A. DARK-FIELD MICROSCOPE

• Highlights specimen against a black background – only object is illuminated

• Light scattered and hits object from different angles

• Like us seeing the moon at night because sunlight from behind the earth reflects off the moon

A. DARK-FIELD MICROSCOPE

• Good for living MOs to see size, shape and motility

• Helps in diagnosis of some diseases caused by spiral bacteria because they are so small

• Treponema palladum causes syhphilis and is identified from scrapings of the infected lesions

B. PHASE CONTRAST MICROSCOPE• Used in research labs for observing living MO

and their movement in medium where they are growing – no staining

• Same magnification as compound microscope but it detects small differences in density

• Compound microscope that increases the contrast between denser MOs and surrounding medium (MO denser than medium)

B. PHASE CONTRAST MICROSCOPE

Epithelial Cells - Phase Contrast Microscope Epithelial Cells -

Compound Microscope

C. FLUORESCENCE MICROSCOPE

• MO’s are coated with fluorescent dye and illuminated with UV light

• Coated MO’s appear to fluoresce

• Applications in

medical microbiology

tagged antibodies

D. SCANNING TUNNELING MICROSCOPE (STM) 1981

• Focus on surface of object, produce a map showing bumps and valleys of atoms

• Map surface as blind person with a cane (tap)

• No special prep to sample

Surface of Blue NickelSurface of CopperSurface of Blue Platinum