lab #3 3-1

Bright-field microscopy

The most commonly used type of light microscopy in introductory microbiology and clinical laboratories.

Types of light microscopy

The four major types discussed in the manual are bright-field, dark-field, fluorescence, and phase-contrast microscopy.

Image production in bright-field microscopy

It uses light transmitted through the specimen to produce an image.

Specimen appearance in bright-field microscopy

A specimen appears shadowy against a bright background.

Use of stains in bright-field microscopy

Stains are commonly used because most biological specimens are transparent, and staining improves contrast between the specimen and background.

Disadvantage of staining microorganisms

The staining process usually kills the cells.

Condenser lens

It concentrates light and makes illumination of the specimen more uniform.

Objective lens

It uses refracted light from the specimen to produce a magnified real image.

Ocular lens

It magnifies the real image again to produce a virtual image seen by the eye.

Total magnification formula

Total magnification = objective magnification × ocular magnification.

Binocular microscope magnification

The magnifications of both ocular lenses aren't multiplied together because each eye views the image through one ocular lens.

Practical limit of magnification

Approximately 1300× for a light microscope.

Greater magnification utility

Greater magnification isn't always useful because image clarity becomes increasingly difficult to maintain.

Resolution

The clarity of an image and the ability to distinguish two points as separate objects.

Limit of resolution

The minimum distance two points must be apart for the microscope to distinguish them as separate.

Resolution and limit of resolution relationship

Resolution is better when the limit of resolution is smaller.

Best limit of resolution for light microscope

About 0.2 μm.

Wavelength's effect on resolution

Shorter wavelengths produce better resolution than longer wavelengths when other variables remain constant.

Wavelength as limiting factor for resolution

The wavelength of light limits how closely spaced two points can be while still being distinguished as separate.

Numerical aperture (NA)

A measure of a lens's ability to capture light from the specimen and use it to form an image.

Units of numerical aperture

Numerical aperture has no units.

Effect of increasing numerical aperture on resolution

It improves resolution by making the limit of resolution smaller.

Immersion oil

It increases the numerical aperture of the oil-immersion objective, improving resolution.

Objective lens for immersion oil

The oil-immersion objective is designed to be used with immersion oil.

Purpose of the iris diaphragm

To adjust the amount of light entering the specimen and help optimize illumination, contrast, and image quality.

Action with iris diaphragm as magnification increases

It should generally be opened more because a smaller portion of the light beam enters the lens.

Coarse-focus knob use

It is used for bringing the image into approximate focus.

Fine-focus knob use

It is used for bringing the image into sharpest focus.

Nosepiece ring usage

The nosepiece ring should be used to change objectives to avoid damaging them.

Parfocal definition

The objectives are designed so that once a specimen is focused with one objective, only a slight adjustment of fine focus should be needed after switching objectives.