Chapter 2: Microscopy

MCB 3020C

unit: 1 centimeter (cm)

value: 10^-2 meter

unit: 1 millimeter (mm)

value: 10^-3 meter

unit: 1 micrometer (um)

value: 10^-6 meter

unit: 1 nanometer (nm)

value: 10^-9 meter

unit: 1 angstrom (A)

value: 10^-10 meter

viruses are measured in

nanometers (nm)

protozoa are measured in

about 200 micrometers (um)

what can be seen with an electron microscope?

• 1nm to 100 um

• molecules, viruses, small bacteria, bacteria, unicellular algae, cyanobacteria, colonial algae, protozoa

what can be seen with a light microscope?

• 100 nm to 1mm

• small bacteria, unicellular algae, cyanobacteria, bacteria, colonial algae, protozoa, fungi

what can be seen with the naked eye?

• 1mm to 10 m

• fungi and multicellular organism

refractive index

measure of how greatly a substance slows the velocity of light

focal point

focus light rays at a specific place

focal length

distance between center of lens and focal point

how does lens and focal length relate?

strength of lens related to focal length

• short focal length (less light needed) = more magnification)

media: vacuum

index of refraction: 1.00

media: all air

index of refraction: 1.0003

media: pure carbon dioxide gas

index of refraction: 1.0005

media: ice

index of refraction: 1.31

media: pure water

index of refraction: 1.33

media: oil

index of refraction: 1.48

media: glass

index of refraction: 1.52

refractive index (n)

ratio of velocity of light in a vacuum

vacuum (c )

velocity in a specified medium (v)

refractive index formula

n=c/v

• n: refractive index

• c: vacuum

• v: medium

the light microscope

varieties:

• bright field microscope

• dark field microscope

• phase contrast microscope

• fluorescence microscope

• confocal microscope

compound microscopes, image formed by the action of >2 lenses

the bright field microscope

• dark image against a brighter background

• several objective lenses

  • parfocal microscopes remain in focus when objectives are cahnges

• total magnification

total magnification

product of the magnifications of the ocular lenses and the objective lenses

microscope resolution

ability of a lens to separate or distinguish between small objects that are close together

major factor determining resolution

based on the wavelength of light used = shorter the wavelength the greater the resolution

objective lens determining resolution

numerical aperture of the objective lens (ability to gather light) = larger the numerical aperture, the greater the resolution and the shorter the working distance of the lens

how to increase resolution of the microscope: oil immersion

light rays that did not enter the objective due to reflection and refraction at the surfaces will now show = increases resolution and numerical aperture

working distance

distance between the front surface of lens and surface of cover glass or specimen in sharp focus

resolving power

smaller resolving power number = better resolution for the microscope

resolving power = wavelength (nm) / numerical aperture of objective + numerical aperture of condenser

dark field microscope

• image formed by light reflected or refracted by specimen

• bright image of an object against a dark background

• used to observe living, unstained samples

what is dark field microscope used to observe

• to observe internal structures in eukaryotic microorganisms (living organisms)

• to identify bacteria such as Treponema pallidum, causative agent of syphilis

• look for shadows in the cells of living organisms

phase-contrast microscope

• converts slight differences in refractive index and cell density into easily detected variations in light intensity

• light rays from hollow cone of light passing through an unstained cell are slowed down and out of phase and dark compared to the bright background

• excellent way to observe living cells

what is phase-contrast microscope used to observe

• studying microbial motility

• detecting bacterial structures such as:

  • endospores

  • inclusion bodies

  • refractive indices different from that of water

fluorescence microscope

• exposed specimen to ultraviolet, violet or blue light

• specimens stained with fluorochromes

• shows a bright image of the object resulting from the fluorescent light emitted by the specimen

what is fluorescence microscope used to observe

• in medical microbiology and microbial ecology studies

• fluorochrome-labeled probes (antibodies) or fluorochromes tag specific cell constituents for the identification of unknown pathogens

• localization of specific proteins in cells (green fluorescence protein)

confocal microscopy

• confocal scanning laser microscopy (CLSM) creates sharp, composite 3D image of specimens by using laser beams, aperture to eliminate stray light, and computer interface

• a computer combines optical z-sections, the digitized signals form a 3D image with excellent contrast and resolution

what is confocal microscope used to observe

valuable for examining living biofilms

electron microscopy

• transmission electron microscope (TEM)

• scanning electron microscope (SEM)

• electron cryotomography

transmission electron microscope

• resolution about 1000 times better than that of the light microscope (0.5nm vs 0.2um) = due to short wavelength of the electron beam used to create the image

• electrons scatter wehn they pass through thin sections of a specimen - transmitted electrons are used to produce an image of electron dense objects on a fluorescent screen

• preparation: cutting thin sections chemical fixation, drying, embedding in plastics, and staining with heavy atoms

  • also negative staining, shadowing, and freeze-etching

scanning electron microscope

• electrons reflected from the surface of a specimen to produce a 3D image of its surface features

• resolution of 7nm or less

• preparation: chemical fixation, drying, and coating with metals

electron cryotomography

• samples rapidly frozen to extremely low temperatures, preserving internal features

• sample is viewed from many angles to create 3D images

characteristics of light microscope

• about 1000-15000 maginification

• 0.2 um at best resolution

• visible light

• travel through air

• glass lens

• differential light absorption

• specimen on a glass slide

characteristics of transmission electron microscope

• over 10,000 magnification

• 0.5 nm at best resolution

• electron beam

• travels through high vacuum

• electromagnet lens

• scattering of electrons

• specimen on a metal grid (copper)