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Microscopy

Chapter 3

BIO 440

Compound Light Microscopy

• This is what we use in the laboratory

• A series of finely ground lenses form a

magnified image

• Specimen is illuminated with visible light

Fig. 3.1

Things to remember . . .

• Magnifying power – ratio of the object’s

apparent size when viewed through a lens

compared to the appearance of the object

when viewed with the naked eye from a

distance of 25 cm

• How to calculate the total magnification of a

specimen

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Things to remember . . .

• Resolution – ability of the lenses to distinguish

fine detail and structure (or, to distinguish

between objects that are close together)

– Our microscopes can distinguish between objects

that are as little as 0.2 μm apart

• Resolution is dependent on

– Wavelength of light (shorter the λ, greater the res)

– Numerical aperture (ability of lens to gather light)

Fig. 3.3

Things to remember . . .

• Contrast – refers to the differences in color

and light between the parts of an object or

between an object and its environment

• An unstained cell or organism has little

contrast with its surroundings and thus is

difficult to see

Brightfield illumination

• This is what we use in lab

• FOV is brightly illuminated

• Dark specimen on bright

background

Fig. 3.4a

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Darkfield Microscopy

• Specimen appears light on

a dark background

• Handy in situations where

we want to see live cells,

cells are hard to see in

light or do not stain well

• Treponema pallidum –

causative agent of syphilis

Fig. 3.4b

Phase-Contrast

• Not necessary to fix or stain specimens, so allows us to see living microbes

• Separates illuminating light from light refracted off of specimen

• Better cellular detail

• Light travels faster through thinner areas and structures appear brighter

• Light travels slower through thicker areas and structures appear darker

Fig. 3.4c

Differential Interference Contrast (DIC)

• Uses two beams of light which pass through

prisms

• Resolution better than standard phase-

contrast

• Image brightly colored and appears 3D

Fig. 3.5

Fluorescence Microscopy

• Takes advantage of fluorescence, the ability of substances to absorb short wavelengths of light (UV) and give off light at a longer spectrum

• Some organisms fluoresce naturally under UV light, those that do not can be stained with fluorochromes

– (i.e. fluorescein isothiocyanate - FITC)

• Bright microbes on dark background

• Fluorescent – antibody (FA) technique (aka immunofluorescence)

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Immunofluorescence

• Fluorescent antibodies can be used to detect

the presence of specific antigens

• Animal is injected with a specific antigen, and

then antibodies are harvested, then

Fig. 3.6

Confocal Microscopy

• Specimens are stained with fluorochromes

• Short wavelength (blue) laser light passes

through specimen

• Planes of the specimen are captured by the

microscope, and can then be used by a

computer to construct a 3D image

Electron Microscopy

• A beam of electrons, rather than beam of

light, is used to eliminate specimen

• Can be used to visualize objects smaller than

0.2 μm (viruses or internal cell structure)

• λ of electrons is much shorter than λ of light,

so MUCH higher resolution

• Uses electromagnetic versus glass lenses to

focus light on specimen

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Transmission electron microscopy

• Visualize viruses or internal intrastructure

• Ultrathin sections of specimen (100 nm)

• Image produced on fluorescent screen or

photographic plate

• 10,000X – 100,000X magnification

• High resolution (10 pm)

• Can use stains (mineral salts) to enhance

contrast

• Black and white images, 2D

Scanning electron microscopy

• Visualize surface features of intact cells and

viruses

• Image produced on viewing screen or

photographic plate

• 1,000X – 10,000X magnification

• High resolution (10 nm)

• Black and white images, 3D

Preparation of microscopic specimens

• Staining helps us localize and visualize small,

colorless microbes on microscope slides

• But before any staining occurs, we must first

“fix” the specimen

• Fixing the specimen

– Adhesion of the specimen to the slide

– Kills the specimen

– Preserves specimen with minimal distortion

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Heat fixation

• Make a smear

• Air dry/slide warmer

• Pass the slide (specimen side up) through the

flame of a Bunsen burner several times

• Once the heat fixation is completed, you can

go ahead and apply stain to the slide

Stains

• Stains are salts that are composed of a cation

and anion

• One of those ions is colored, and is referred to

as the chromophore

• Basic dyes are cationic, meaning that the

chromophore has a positive charge

– Crystal violet, methylene blue, safranin

• Acidic dyes are anionic, meaning that the

chromophore has a negative charge

– Eosin, nigrosin

Simple stains

• An aqueous or alcohol solution of a single

basic dye

• Good for visualizing cell shape and

arrangement

Differential stains

• Use more than one stain to differentiate

cellular components

• Used to visual structural differences between

different types of bacteria

– Gram stain

– Acid-fast stain

• Mycobacterium tuberculosis

• Mycobacterium leprae

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Gram stain

• Primary stain – Crystal violet

• Mordant – Gram’s iodine

• Decolorizer

• Counterstain - Safranin

Special stains

• Used to color parts of microorganisms

– Capsule stain

– Endospore stain

– Flagellar stain