Microscope

City College of San Francisco

Microbiology 12

Microscopy

The intent of this page is to give you an overview of the major features and uses of different types of microscopes that are commonly employed today. For more details, I recommend that you visit each of the links. Some are a little technical but I have reviewed each of them and they are all accurate and informative.

Technical terms

A microscope glossary

It is critical that you understand the parts and functions of the compound microscope as you proceed through the semester. The glossary above will help you understand these key concepts. Review the following:

Contrast
Diffraction
Resolving power
Working distance
Field of view
Magnification (calculation of)
Refractive index
Reflection

Types of microscopes

Microscope type Major characteristics Main applications, advantages and limitations

What is a light microscope?

Brightfield

Uses visible light for illumination; specimen appears against a light background

Total magnification between 1,000-2,000 x

Optimal resolving power: 0.2 um

Most commonly used microscope for cell study (used by this class) and cell counts

Relatively easy to use

Specimens must typically be killed, fixed and stained; can lead to distortion.

Small bacteria and viruses are not resolved

Phase contrast

Magnification and resolution comparable to light microscope

Exploits differences in refractive indexes.

Direct light passes through a ring-shaped diaphragm and the condenser, focusing light on the specimen.

Diffracted light from the specimen is captured by a plate in the objective lens.

Both the direct and diffracted light rays are brought together at the eye

Living cells and internal components are contrasted against the background giving greater definition and detail of cell structure

Cells need not be fixed or stained

Differential interference contrast (DIC)

Comparable to phase-contrast in terms of using differences in refractive indexes but gives greater resolution

Uses two beams of light that are split by prisms adding contrasting colors to specimen

Light waves are out of phase when they recombine and give a 3D appearance to specimen

Living specimens can be examined with a 3-D appearance and do not require staining

Dark-field

Uses condenser with an opaque disc that blocks out light in the center of the beam

Only light reflected by specimen enters objective lens

Specimen is bright against a dark background

Study of microbes that are not visible by brightfield microscopy, or are distorted by staining

Major use in microbiology is the detection of living spirochetes such as Borrelia and Treponema spp.

Fluorescence

Uses ultraviolet light (short wavelength) that causes specimen to to fluoresce

Visualization of cells or structures that fluoresce, either naturally or by the addition of special dyes called fluorochromes

Often used to identify cells or structures that react with specific antibodies; a technique known as immunofluorescence

What is an electron microscope ?

Transmission electron

Uses electron beam (short wavelength) instead of visible light

Total magnification:

10,000-100,000X

Resolution ~3 nm

Magnets focus electron beams instead of condenser

Image formed on electron-sensitive screen

Detailed examination of cell ultrastructure and viruses

Internal features may be observed through freeze fracture of cells

Specimens must be killed, dried and fixed; often leads to distortion and artifacts (see mesosome)

Scanning electron

Similar properties to TEM

Electron bean scans surface of specimen

Total magnification :

1,000-10,000X

Resolution: 1-10 nm

Study of viruses and surfaces of cells

What is a confocal microscope ?

Uses laser to light one plane of a specimen at a time

Specimens stained with fluorochromes

Uses small, pinhole aperture to eliminate blurring of image and improved resolution

Usually used in conjunction with a computer to produce 3D images and sections of cells and components. Images can be viewed in different orientations.

Scanning tunneling microscope

Metal probe scans surface "landscape" of specimen (rather like reading brail).

Greater resolution than e.m.

Detailed views of computer chips and macromolecules such as DNA

No special preparation of specimen needed

Atomic force microscope

Metal and diamond probe moves along surface of specimen

Detailed 3d images of biological molecules

No special preparation of specimen needed.

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Microscope type	Major characteristics	Main applications, advantages and limitations
What is a light microscope?
Brightfield	Uses visible light for illumination; specimen appears against a light background Total magnification between 1,000-2,000 x Optimal resolving power: 0.2 um	Most commonly used microscope for cell study (used by this class) and cell counts Relatively easy to use Specimens must typically be killed, fixed and stained; can lead to distortion. Small bacteria and viruses are not resolved
Phase contrast	Magnification and resolution comparable to light microscope Exploits differences in refractive indexes. Direct light passes through a ring-shaped diaphragm and the condenser, focusing light on the specimen. Diffracted light from the specimen is captured by a plate in the objective lens. Both the direct and diffracted light rays are brought together at the eye	Living cells and internal components are contrasted against the background giving greater definition and detail of cell structure Cells need not be fixed or stained
Differential interference contrast (DIC)	Comparable to phase-contrast in terms of using differences in refractive indexes but gives greater resolution Uses two beams of light that are split by prisms adding contrasting colors to specimen Light waves are out of phase when they recombine and give a 3D appearance to specimen	Living specimens can be examined with a 3-D appearance and do not require staining
Dark-field	Uses condenser with an opaque disc that blocks out light in the center of the beam Only light reflected by specimen enters objective lens Specimen is bright against a dark background	Study of microbes that are not visible by brightfield microscopy, or are distorted by staining Major use in microbiology is the detection of living spirochetes such as Borrelia and Treponema spp.
Fluorescence	Uses ultraviolet light (short wavelength) that causes specimen to to fluoresce	Visualization of cells or structures that fluoresce, either naturally or by the addition of special dyes called fluorochromes Often used to identify cells or structures that react with specific antibodies; a technique known as immunofluorescence
What is an electron microscope ?
Transmission electron	Uses electron beam (short wavelength) instead of visible light Total magnification: 10,000-100,000X Resolution ~3 nm Magnets focus electron beams instead of condenser Image formed on electron-sensitive screen	Detailed examination of cell ultrastructure and viruses Internal features may be observed through freeze fracture of cells Specimens must be killed, dried and fixed; often leads to distortion and artifacts (see mesosome)
Scanning electron	Similar properties to TEM Electron bean scans surface of specimen Total magnification : 1,000-10,000X Resolution: 1-10 nm	Study of viruses and surfaces of cells
What is a confocal microscope ?	Uses laser to light one plane of a specimen at a time Specimens stained with fluorochromes Uses small, pinhole aperture to eliminate blurring of image and improved resolution	Usually used in conjunction with a computer to produce 3D images and sections of cells and components. Images can be viewed in different orientations.
Scanning tunneling microscope	Metal probe scans surface "landscape" of specimen (rather like reading brail). Greater resolution than e.m.	Detailed views of computer chips and macromolecules such as DNA No special preparation of specimen needed
Atomic force microscope	Metal and diamond probe moves along surface of specimen	Detailed 3d images of biological molecules No special preparation of specimen needed.