Light microscopy: topics

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Light microscopy topics

• Properties of light
• Compound microscope
• Resolution contrast
• Contrast modes in light microscopy
• Koehler Illumination
• Class exercises

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General References

• Salmon, E. D. and J. C. Canman. 1998. Proper
  Alignment and Adjustment of the Light Microscope.
  Current Protocols in Cell Biology 4.1.1-4.1.26,
  John Wiley and Sons, N.Y.
• Murphy, D. 2001. Fundamentals of Light Microscopy
  and Electronic Imaging. Wiley-Liss, N.Y.
• Keller, H.E. 1995. Objective lenses for confocal
  microscopy. In Handbook of biological confocal
  microsocpy, J.B.Pawley ed. , Plenum Press, N.Y.
• Microscopes Basics Beyond pdf available

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On line resource Molecular Expressions, a
Microscope Primer at http//www.microscopy.fs
u.edu/primer/index.html
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Some properties of light important for the
microscope
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Light as electromagnetic wave with mutually
perpendicular E, B components characterized by
wavelength,?, and frequency, ?, in cycles/s.
Wave velocity ? x ?. ?500nm--gt ?6x1014
cycles/s
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Defining wavefronts and rays
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Velocity of light in different media

• Index of refraction, n c/v
• Cspeed of light in vacuum3x108 m/s v velocity
  in media
• Light travels slower in more dense media

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Index of refraction for different media at 546 nm

• Air 1.0
• Water 1.3333
• Cytoplasm 1.38
• Glycerol 1.46
• Crown Glass 1.52
• Immersion Oil 1.515
• Protein 1.51-1.53
• Flint Glass 1.67

n increases with decreasing ?
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Note electronic cameras do not have same
spectral response as eyes
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The simplest microscope a magnifier
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The compound microscope
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The purpose of the microscope is to create
magnification so that structures can be
resolved by eye and to create contrast to make
objects visible.
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In the compound microscope, the objective forms a
real, inverted image at the eyepiece front
focal plane (the primary image plane)
The optical tube length (OTL), typically 160mm,
is the distance between the rear focal plane of
the objective and the intermediate image plane
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Total magnification in the compound microscope
Mt Mobj x Mep
Max Mt 1000xNA gt 1000NA, empty mag.
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Modern microscope component identification
Prisms Used to Re-Direct Light In Imaging
Path While Mirrors Are Used in Illumination
Path
E.D.Salmon
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In the compound microscope, the objective forms a
real, inverted image at the eyepiece front
focal plane (the primary image plane)
The optical tube length (OTL), typically 160mm,
is the distance between the rear focal plane of
the objective and the intermediate image plane
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A word about infinity corrected optics and its
advantages.object is set at front focal plane
of objective
Eliminates ghost images caused by con- verging
light, allows filters and polarizers to be
inserted in infinity space without corrections
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Key component the objective
--gt Aberrations
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Chromatic aberration and its correction
Achromat
Fluorite
Apochromat
Lens designer, using various glasses elements,
tries to bring all colors to common focus
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Spherical Aberration
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Objective Classes
Achromats corrected for chromatic aberration for
red, blue Fluorites chromatically corrected for
red, blue spherically corrected for 2
colors Apochromats chromatically corrected for
red, green blue spherically corrected for 2
colors Plan- further corrected to provide flat
field
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The 3 Classes of Objectives
Chromatic and Mono-Chromatic Corrections
E.D. Salmon
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Multilayer anti-reflection coatings

• Highly corrected objectives may have 15 elements.
  Each uncoated glass-air interface can reflect
  4-5, dropping objective thruput to as low as
  50.
• Multi layer AR coatings suppress reflections
  increasing transmission gt 99.9 as well as
  reducing ghosts and flare to preserve contrast.

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Objective Specifications
E.D. Salmon
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What is numerical aperature (NA)?
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Usually, higher magnification objectives have
greater NAs
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Sample specifications
Working distance separation between top of
coverslip and front element of objective when
specimen is in focus
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Resolution
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Airy Disk Formation by Finite Objective Aperture
The width of central maximum prop. to ?
and inversly prop. to objective aperature
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Lateral Resolution in Fluorescence Depends on
Resolving Overlapping Airy Disks
Rayleigh Criteria Overlap by r, then dip in
middle is 26 below Peak intensity
(2px/l)NAobj
E.D.Salmon
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Minimum resolvable distance, dmin
Fluorescence dmin 0.61l/NAobj self-luminous
object Trans-Illumination dmin l/(NAobj
NAcond) note that resolution depends on
condenser NA too for maximum resolution NAcond
should equal or exceed NAobj
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Why oil immersion lenses provide greater
resolutionthey have a larger NA (nsin?)
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Resolution is better at shorter wavelengths
higher objective NA and/or higher condenser NA
E.D. Salmon
High NA and/or shorter ? Low
NA and/or longer ?
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Rayleigh Criterion for the resolution of two
adjacent spots dlim 0.61 lo /
NAobj Examples (lo 550 nm) Mag f(mm) n
? NA dlim (mm) (NAcondNAobj) high dry
10x 16 1.00 15 0.25 1.10 40x 4 1.00 40 0.65 0.42
oil 100x 1.6 1.52 61 1.33 0.204 63x 2.5 1.52 67
.5 1.40 0.196
For dry objectives NA lt 0.95 for oil objectives
NA lt 1.52 with oil of n1.52
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Depth of field (vertical) resolution
D 0.61 ? cos ? / n(NA)
Low power, NA 0.25 D 8 ?m
Hi, dry, NA0.5 D 2 ?m
Oil immersion, NA 1.3 D0.4 ?m
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Higher NA means

• Brighter image NA2
• Greater lateral resolution
• Smaller depth of field

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Contrast
All the resolution in the world wont do you any
good, if there is no contrast to visualize the
specimen.
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Contrast
E.D.Salmon
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Phase contrast microscopy
diI-C16
HA
Thy-1
H-2
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Ridges in The Surface of Cheek Cells for
Resolution Tests
High Resolution DIC Microscopy
E.D.Salmon
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Keratocyte Differential Interference Contrast
(DIC) microscopy (from a goldfish scale, 3 times
real time)
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From Ted Salmon
Walker et al, Nature 347 780-782
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Dark field microscopy
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Interference reflection microscopy (IRM)
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Illumination for the microscope
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Purpose of Koehler Illumination

• To obtain even specimen illumination for
  photomicrography, video microscopy etc.
• To use field diaphragm alone to control
  illuminated area of specimen.
• To control the angle of the cone of
  illumination(contrast and resolution) by varying
  condenser diaphragm.

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A Lamp Collector Lens and Microscope Condenser
Lens are Used to Concentrate Light on the Specimen
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Optical Principle
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Summary of Köhler Illumination

• Focus specimen at low magnification
• Focus and center field diaphragm by adjusting
  condenser height and diaphragm position.
• Focus lamp filament on condenser iris diaphragm.
• Adjust condenser diaphragm appropriately.
• For visual observation, set condenser diaphragm
  to
• 70-90 of objective aperature.
• To enhance contrast, reduce condenser diaphragm
  to 40-50 of objective aperature.
• For video microscopy, set condenser aperature to
  objective aperature.

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Condenser is Translated Along Optical Axis to
Bring Field Diaphragm into Focus
Condenser X-Y Translation Screws Are Used
to Center Image of Field-Diaphragm
Condenser Focus Knob
Now, the field diaphragm controls the area
illuminated on the specimen
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Summary of Köhler Illumination

• Focus specimen at low magnification
• Focus and center field diaphragm by adjusting
  condenser height and diaphragm position.
• Focus lamp filament on condenser iris diaphragm.
• Adjust condenser diaphragm appropriately.
• For visual observation, set condenser diaphragm
  to
• 70-90 of objective aperature.
• To enhance contrast, reduce condenser diaphragm
  to 40-50 of objective aperature.
• For video microscopy, set condenser aperature to
  objective aperature.

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The Condenser Diaphragm Controls the Illumination
NA
An image of the Condenser Diaphragm is in-focus
in the Objective Back Focal Plan (Aperture). As
the condenser diaphragm is opened, the
illumination NA increases without changing the
area of specimen Illuminated (area controlled by
Field Diaphragm).
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Taking care of microscope optics

• Never dry clean a lens
• Use a solvent like Windex that will remove most
  everything. Use xylene under a hood as last
  resort.
• Use best quality lens tissue available.

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Diatom Resolution Test Specimens