Microscopy

Units of Measurement

1 m = 100 cm = 1000 mm

1 mm = 1000 µm (micro-)

1 µm = 1000 nm(nano-)

1 nm = 1000 pm(pico-)

Observing Microorganisms

Microscopy: The Instruments

A simple microscope has only one lens

Light Microscopy

Use of any kind of microscope that uses visible light to observe specimens

Types of light microscopy

Compound light microscopy

Darkfield microscopy

Phase-contrast microscopy

Differential interference contrast microscopy

Fluorescence microscopy

Confocal microscopy

The Compound Light Microscope

In a compound microscope, the  image from the objective lens is magnified again by the ocular lens

Total magnification =
objective lens ´ ocular lens

Compound Light Microscopy

Resolution is the ability of the lenses to distinguish two points

A microscope with a resolving power of 0.4 µm can distinguish between two points ≥ 0.4 µm

Shorter wavelengths of light provide greater resolution

Compound Light Microscopy

The refractive index is a measure of the light-bending ability of a medium

The light may bend in air so much that it misses the small high-magnification lens

Immersion oil is used to keep light from bending

Refraction in the Compound Microscope

Brightfield Illumination

Dark objects are visible against a bright background

Light reflected off the specimen does not enter the objective lens

Darkfield Illumination

Light objects are visible against a dark background

Light reflected off the specimen enters the objective lens

Phase-Contrast Microscopy

Emphasizes diffraction of the light that passes through a specimen

Differential Interference Contrast Microscopy

Accentuates diffraction of the light that passes through a specimen; uses two beams of light

Fluorescence Microscopy

Uses UV light

Fluorescent substances absorb UV light and emit visible light

Cells may be stained with fluorescent dyes (fluorochromes)

Confocal Microscopy

Cells stained with fluorochrome dyes

Short wavelength (blue) light used to excite the dyes

The light illuminates each plane in a specimen to produce a three-dimensional image

Up to 100 µm deep

Confocal Microscopy

Two-Photon Microscopy

Cells stained with fluorochrome dyes

Two photons of long- wavelength (red) light used to excite the dyes

Used to study cells attached to a surface

Up to 1 mm deep

Scanning Acoustic Microscopy (SAM)

Measures sound waves that are reflected back from an object

Used to study cells attached to a  surface

Resolution 1 µm

Electron Microscopy

Uses electrons instead of light

The shorter wavelength of electrons gives greater resolution

Transmission Electron Microscopy (TEM)

Ultrathin sections of specimens

Light passes through specimen, then an electromagnetic lens,
to a screen or film

Specimens may be stained with heavy metal salts

Transmission Electron Microscopy (TEM)

10,000–300,000´; resolution 2.5 nm

There are two common types of Electron Microscopes:

The Transmission electron microscope (TEM) produces images by detecting electrons that are transmitted through the sample.

The Scanning electron microscope (SEM) produces images by detecting secondary electrons which are emitted from the surface due to excitation by the primary electron beam.

Scanning Electron Microscopy (SEM)

An electron gun produces a beam of electrons that scans the surface of a whole specimen

Secondary electrons emitted from the specimen produce the image

 When using SEM, bulk biological samples are first coated with precious metal (gold or platinum) that readily reflects electrons. An image is formed by the electrons that bounce off the surface of the specimen and are then collected onto the imaging screen. The observer sees the three-dimensional picture of the surface of the sample without any internal information.

Scanned-Probe Microscopy

Scanning tunneling microscopy (STM) uses a metal probe to scan a specimen

Resolution 1/100 of an atom

Atomic force microscopy (AFM) uses a metal- and-diamond probe inserted into the specimen.

Produces three-dimensional images.

Staining: Coloring the microbe with a dye that emphasizes certain structures

Smear: A thin film of a solution of microbes on a slide

A smear is usually fixed to attach the microbes to the slide and to kill the microbes

Preparing Smears for Staining

Live or unstained cells have little contrast with the surrounding medium. Researchers do make discoveries about cell behavior by observing live specimens.

Preparing Smears for Staining

Stains consist of a positive and negative ion

In a basic dye, the chromophore is a cation

In an acidic dye, the chromophore is an anion

Staining the background instead of the cell is called negative staining

Simple Stains

Simple stain: Use of a single basic dye

A mordant (intensify the stain) may be used to hold the stain or coat the specimen to enlarge it

Differential Stains

Used to distinguish between bacteria

Gram stain

Acid-fast stain

Classifies bacteria into gram-positive
or gram-negative

Gram-positive bacteria tend to be killed by penicillin and detergents

Gram-negative bacteria are more resistant to antibiotics

Acid-Fast Stain

Stained waxy cell wall is not decolorized by  acid-alcohol

Mycobacterium

Nocardia

Special Stains

Used to distinguish parts of cells

Capsule stain

Endospore stain

Flagella stain

Negative Staining for Capsules

Cells stained

Negative stain

Endospore Staining

Primary stain: Malachite green, usually with heat

Decolorize cells: Water

Counterstain: Safranin

Flagella Staining

Mordant on flagella

Carbolfuchsin simple stain