7/31/2017 1 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
7
Embed
7/31/2017 Compound Light Microscopy · PDF fileCompound Light Microscopy ... appear brighter • Light travels slower through thicker areas and structures ... • Mycobacterium...
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
7/31/2017
1
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
7/31/2017
2
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
7/31/2017
3
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