Brightfield and Phase Contrast Microscopy

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Brightfield and Phase Contrast Microscopy
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Real and virtual image formation by biconvex
lenses

• Lens focal point
• For an object further away than the lens focal
  point, an inverted, real image will be formed on
  the opposite side of the lens
• For an object closer than the focal point, a
  virtual image will be formed on the same side of
  the lens
• http//micro.magnet.fsu.edu/primer/java/lens/bi-co
  nvex.html

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Compound Microscope

• The compound microscope uses at least two lens
  systems
• The objective forms an intermediate real image of
  the object at the objective tube length
• The ocular forms a virtual image of that
  intermediate image to the retina of the eye
• If we are dealing with a photodetector, we must
  use a projection lens to form a real image from
  the intermediate image

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Ray Tracings in the microscope
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Current microscope objective tend to be infinity
corrected

• Infinite tube length
• Require an additional lens in objective to
  converge beam
• Advantages
• Objectives are simpler
• Optical path is parallel through the microscope
  body

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Infinity correction
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Other lenses

• Collecter
• Condenser
• Allow us to use point light sources instead of
  parallel illumination
• Also (later) increase the resolution of the
  microscope
• Ironically, van Leeuwenhoek, who used simple
  non-compound, single-lens microscopes, was using
  the lens of his eye as a projection lens!

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Lens Resolution

• Geometric optics predicts lenses of infinite
  resolution
• However, because of the phenomenon of
  diffraction, every point in the object is
  converted into an Airy disc
• Diameter of Airy disc
• D 1.22 X ? / n sin a, or
• D 1.22 X ? / NA

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Airy disc
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We cannot resolve objects whose Airy discs
overlap by 20
As a consequence, Abbes rule is that d?/NA
http//micro.magnet.fsu.edu/primer/java/microscopy
/airydiscs/index.html
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Objective markings
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Reading an objective
http//micro.magnet.fsu.edu/primer/anatomy/specifi
cations.html
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For a typical 1.3 NA lens at 525 nm, the limit of
resolution is 400 nm

• How to improve?
• Larger NA (lenses, immersion fluid)
• Shorter ?
• Add a condensor
• D ? / (NAobj. NAcond.)
• So, for a 1.3 NA lens and condensor, D drops to
  200 nm

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Condensor
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Abberations

• Spherical aberration
• Most severe
• Immersion fluid
• Field curvature
• Chromatic aberration
• Astigmatism, coma
• http//micro.magnet.fsu.edu/primer/lightandcolor/o
  pticalaberrations.html

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Brightfield microscopy

• Generally only useful for stained biological
  specimens
• Unstained cells are virtually invisible

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HE stain From Wikipedia, the free
encyclopedia HE stained lung tissue sample from
an end-stage emphysema patient. RBCs are red,
nuclei are blue-purple, and other cellular and
extracellular material is pink. HE stain, or
haematoxylin and eosin stain, is a popular
staining method in histology. It is the most
widely used stain in medical diagnosis for
example when a pathologist looks at a biopsy of a
suspected cancer, the histological section is
likely to be stained with HE and termed HE
section, HE section, or HE section. The staining
method involves application of the basic dye
haematoxylin, which colors basophilic structures
with blue-purple hue, and alcohol-based acidic
eosin Y, which colors eosinophilic structures
bright pink. The basophilic structures are
usually the ones containing nucleic acids, such
as the ribosomes and the chromatin-rich cell
nucleus, and the cytoplasmatic regions rich in
RNA. The eosinophilic structures are generally
composed of intracellular or extracellular
protein. The Lewy bodies and Mallory bodies are
examples of eosinophilic structures. Most of the
cytoplasm is eosinophilic. Red blood cells are
stained intensely red. The structures do not have
to be acidic or basic to be called basophilic and
eosinophilic. The terminology is based on the
affinity to the dyes. Other colors, e.g. yellow
and brown, can be present in the sample they are
caused by intrinsic pigments, e.g. melanin. Some
structures do not stain well. Basal laminae need
to be stained by PAS stain or some silver stains,
if they have to be well visible. Reticular fibers
also require silver stain. Hydrophobic structures
also tend to remain clear these are usually rich
in fats, eg. adipocytes, myelin around neuron
axons, and Golgi apparatus membranes.
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Oblique illumination
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Oblique
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Darkfield
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Radiolarian in Darkfield
http//micro.magnet.fsu.edu/primer/techniques/dark
field.html
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Phase contrast
http//microscopy.fsu.edu/primer/techniques/phaseg
allery/chocells.html