How to make the specimen visible

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• How to make the specimen visible
• CONTRAST!
• Definition of Contrast
• Techniques
• Brightfield
• Phase
• Darkfield
• Pol
• DIC (Differential Interference Contrast)
• Fluorescence
• Optical Sectioning an expansion of Fluorescence

Agenda
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C ONTRAST
50 Units
0 Units
100 Units
50
50
50 Units
50 100 / 50 100 -0.33
50 0 / 50 0 1
50 50 / 50 50 0
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Common Illumination Techniques

• Brightfield
• Darkfield
• Phase Contrast
• Polarized Light
• DIC (Differential Interference Contrast)
• Fluorescence (and related techniques)

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Brightfield

• For naturally absorbing or stained samples
• True Color Representation
• Proper Technique for Measurements
• Spectral
• Dimensional

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Bacillaria
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Paramecium bursaria
Condenser diaphragm open
Condenser Diaphragm almost closed
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Paramecium bursaria
Different Staining Techniques
Indian Ink Staining
Feulgen Staining
Silver Staining
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Phase Contrast (Frits Zernike 1934)

• - Halo effect gt Reduced resolution
• No staining necessary
• Good Depth of Field
• Easy alignment
• Orientation independent
• Repeatable setup
• Works with plastic dishes

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• Required Components for Phase Contrast
• Objective with built-in Phase Annulus
• Condenser or Slider with Centerable Phase Ring
  for illumination (Ph0, 1, 2 or 3)

Required Adjustment Superimpose Phase Ring of
condenser over (dark) phase plate of objective
(after Koehler Illumination)
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• Illumination bypasses Specimen gt no phase shift

• Illumination passes through thin part of Specimen
  gt small phase retardation

• Illumination passes through thick part of
  Specimen gt larger phase retardation

Phase Shifts Cells have higher n than water.
Light moves slower in higher n, consequently
resulting in a phase retardation Phase shift
depends on n and on thickness of specimen detail
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• Non-diffracted and diffracted light are focused
  via tube lens ?into intermediate image and
  interfere with each other ¼¼ ½ wave
  shift causes destructive interference i.e.
  Specimen detail appears dark ?

• Affected rays from specimen, expressed by the
  higher diffraction orders, do not pass through
  phase ring of objective gt¼ wave
  retarded ?

Tube Lens

• Objective Phase Ring
  a) attenuates the non-diffracted 0th Order
  b) shifts it ¼ wave forward ?

Objective
Specimen

• Illumination from Condenser Phase Ring ?(0
  Order) gt meets phase ring ? of objective

Condenser
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Paramecium bursaria
Phase Contrast
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Rhipidodendron
Phase Contrast
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Cochliopodium
Phase Contrast
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Lyngbya Bacteria
Phase Contrast
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Darkfield

• No staining necessary
• Detection of sub-resolution details possible
• Excellent, reversed contrast
• Central Darkfield via hollow cone
• Oblique Darkfield via Illumination from the side
  
• Not useful for Measurements (sizes exaggerated)

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Required conditions for DarkfieldIllumination
Aperture must be larger than objective aperture
I.e. direct light must bypass observer
Low NA Objective
High NA Objective
Iris Diaphragm
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Paramecium bursaria
Polarized Light
Darkfield
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Polarized Light

• Specimen is placed between 2 crossed polarizers.
• Only light produced by birefringent particles
  (e.g. crystals) or coming from the edges of
  particles (edge birefringence) is visible.
• Looks sometimes like Darkfield
• Orientation-specific (linear Pol)
• Linear / circular Polarized Light

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Birefringent Material
Color of sample and background modified by wave
plate
Background
Brightfield
Polarized Light
Pol Red I
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Polarized Light
When Polarizers are crossed, only items that
rotate the plane of polarization reach the
detector. Wave plate adds color
Polarizer 2 (Analyzer)
Specimen
Polarizer 1
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Required / Recommended Components

• Polarizer (fixed or rotatable)
• Analyzer (fixed or rotatable)
• Strain-free Condenser and Objective
• Rotating, centerable Stage
• Wave plate and/or Compensator
• Crossline Eyepiece

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DIC (Differential Interference Contrast after
Nomarski)

• High Contrast and high resolution
• Control of condenser aperture for optimum
  contrast
• Changes GRADIENTS into brightness differences
• 3-D Image appearance
• Color DIC by adding a wave plate
• Best contrast / resolution via different DIC
  sliders
• Orientation-specific gt orient fine details
  perpendicular to DIC prism

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DIC
Observing local differences in phase retardation
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• 9 Image
• 8 Tube lens
• 7 Analyzer (7a with Wave Plate)
• 6 Wollaston Prism behind objective
• 5 Objective
• 4 Specimen
• 3 Condenser with receptacle for prisms

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Wollaston Prism
Birefringence (Different refractive index for
different polarization orientations) Polarized
beam, under 45 to prism, gets split into
ordinary and extraordinary beam
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Required Components for DIC

• Nosepiece with DIC receptacles
• Polarizer (or Sénarmont Polarizer)
• Low Strain Condenser and Objective
• DIC Prisms for Condenser ( I or II or III)
• Appropriate DIC Slider for each objective
• Analyzer (or Sénarmont Analyzer)
• Not needed for New Plas-DIC (up to 40x)

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Paramecium bursaria
DIC
Interference
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Fluorescence

• Easy to set up gt Objective Condenser
• Highly specific technique, wide selection of
  markers
• Detection and Identification of Proteins,
  Bacteria, Viruses
• Basics for
• Special Techniques eg. TIRF, FRET, FRAP etc.
• 3-D imaging
• Deconvolution
• Structured Illumination
• Confocal Techniques

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Epi - Fluorescence
Observation port
Excitation Filter
Emission Filter
FL Light Source
Dichromatic Mirror



Example Specimen containing green fluorescing
Fluorochrome
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Epi - Fluorescence Filter Sets
Example
Curve for a typical GFP filter set
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Epi - Fluorescence
(Specimen containing green fluorescing
Fluorochrome)
Observation port
Excitation Filter
Emission Filter
FL Light Source
Dichromatic Mirror



Specimen containing green fluorescing Fluorochrome
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Paramecium bursaria
Fluorescence
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How to improve Fluorescence Imaging in a major
way

• Optical Sectioning

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Optical sectioning increased contrast and
sharpness
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Overview of Optical
sectioning Methods

• Confocal and Multi-photon
  Laser Scanning Microscopy
• Pinhole prevents out-of-focus light getting to
  the sensor(s) (PMT - Photomultiplier) (30 70
  µm)
• Multi Photon does not require pinhole (90 500
  µm)
• Spinning disk systems
• A large number of pinholes (used for excitation
  and emission) is used to prevent out-of-focus
  light getting to the camera
• E.g. Perkin Elmer, Solamere ( up to 30 µm)
• Structured Illumination
• Moving grid represents the reference for in-focus
  information
• Zeiss Apotome (10-50 µm)

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Overview of Optical
sectioning Methods- contd -

• Total Internal Reflection Fluorescence (TIRF)
• High NA Objective projects beam at angle which
  exceeds critical angle.
• Area touching cover slip (evanescent field) is
  typically smaller than 200 nm
• Deconvolution
• Point-Spread function (PSF) information is used
  to calculate light back to its origin
• Post processing of an image stack

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Limited Depth of Field With Standard Microcopy

• Amber fossil (Chironomide)
• Thickness app. 300 µm
• Conventional incident light

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Optical Sectioning Extended Focus Software

• Amber fossil (Chironomide)
• Thickness app. 300 µm
• Conventional incident light
• 3D reconstruction

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• Break Period move to lab
• Setting up / adjusting the microscopes for
  Brightfield