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![Page 1: How to make the specimen visible – CONTRAST! Definition of Contrast Techniques: Brightfield Phase Darkfield Pol DIC (Differential Interference.](https://reader035.fdocuments.in/reader035/viewer/2022062314/56649eb65503460f94bc07de/html5/thumbnails/1.jpg)
How to make the specimen visible How to make the specimen visible ––
CONTRAST!CONTRAST!
Definition of ContrastDefinition of Contrast
Techniques: Techniques: BrightfieldBrightfield PhasePhase DarkfieldDarkfield PolPol DIC (Differential Interference Contrast)DIC (Differential Interference Contrast) FluorescenceFluorescence Optical Sectioning – an expansion of Optical Sectioning – an expansion of
FluorescenceFluorescence
Age
nda
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C ONTRAST
50 – 0
/ 50 +
0 =
1
50 – 1
00 / 5
0 +
100 =
-0.3
3
50 – 5
0 / 5
0 +
50 =
0
Background of BrightnessSpecimen of BrightnessBackground of Brightness-Specimen of Brightness
50 Units0 Units 100 Units
50 Units 50 50
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• Brightfield• Darkfield• Phase Contrast• Polarized Light• DIC (Differential Interference Contrast)• Fluorescence (and related techniques)
Common Illumination Techniques
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![Page 5: How to make the specimen visible – CONTRAST! Definition of Contrast Techniques: Brightfield Phase Darkfield Pol DIC (Differential Interference.](https://reader035.fdocuments.in/reader035/viewer/2022062314/56649eb65503460f94bc07de/html5/thumbnails/5.jpg)
Brightfield
• For naturally absorbing or stained samples
• True Color Representation
• Proper Technique for Measurements •Spectral•Dimensional
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![Page 7: How to make the specimen visible – CONTRAST! Definition of Contrast Techniques: Brightfield Phase Darkfield Pol DIC (Differential Interference.](https://reader035.fdocuments.in/reader035/viewer/2022062314/56649eb65503460f94bc07de/html5/thumbnails/7.jpg)
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Paramecium bursaria
Condenser diaphragm open Condenser Diaphragm almost closed
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Paramecium bursaria
Indian Ink Staining Feulgen Staining Silver Staining
Different Staining Techniques
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![Page 11: How to make the specimen visible – CONTRAST! Definition of Contrast Techniques: Brightfield Phase Darkfield Pol DIC (Differential Interference.](https://reader035.fdocuments.in/reader035/viewer/2022062314/56649eb65503460f94bc07de/html5/thumbnails/11.jpg)
Phase Contrast Phase Contrast (Frits Zernike 1934)(Frits Zernike 1934)
- “Halo” effect > Reduced resolution
+ No staining necessary
+ Good Depth of Field
+ Easy alignment
+ Orientation independent
+ Repeatable setup
+ Works with plastic dishes
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Required Adjustment:Superimpose Phase Ring of condenser over (dark) phase plate of objective (after Koehler Illumination)
Required Components for Phase Contrast:
1. Objective with built-in Phase Annulus
2. Condenser or Slider with Centerable Phase Ring for illumination (Ph0, 1, 2 or 3)
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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
•Illumination bypasses Specimen > no phase shift
•Illumination passes through thin part of Specimen > small phase retardation
•Illumination passes through thick part of Specimen > larger phase retardation
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1. Illumination from Condenser Phase Ring (“0” Order) > meets phase ring of objective
2. Objective Phase Ring a) attenuates the non-diffracted 0th Order b) shifts it ¼ wave forward
3. Affected rays from specimen, expressed by the higher diffraction orders, do not pass through phase ring of objective >¼ wave retarded
4. Non-diffracted and diffracted light are focused via tube lens into intermediate image and interfere with each other; ¼+¼= ½ wave shift causes destructive interference i.e. Specimen detail appears dark
Condenser
Objective
Specimen
Tube Lens
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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 Darkfield:
Illumination Aperture must be larger than objective aperture
I.e. direct light must bypass observer
Iris Diaphragm
Low NA Objectiv
e
High NA Objective
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Paramecium bursaria
Polarized LightDarkfield
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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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Brightfield
Background
Birefringent Material
Polarized Light Pol + Red I
Color of sample and
background modified by wave plate
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When Polarizers are crossed, only items that rotate the plane of polarization reach the detector.
Wave plate adds color
Polarized Light
Polarizer 1
Polarizer 2
(Analyzer)
Specimen
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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 DIC (Differential Interference Contrast (Differential Interference Contrast
after Nomarski)after Nomarski) HighHigh Contrast Contrast andand high resolution high resolution
Control of condenser aperture for optimum Control of condenser aperture for optimum contrastcontrast
Changes GRADIENTS into brightness differencesChanges GRADIENTS into brightness differences
3-D Image appearance3-D Image appearance
Color DIC by adding a wave plateColor DIC by adding a wave plate
Best contrast / resolution via different DIC slidersBest contrast / resolution via different DIC sliders
Orientation-specific > orient fine details Orientation-specific > orient fine details perpendicular to DIC prismperpendicular to DIC prism
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DICDICObserving local differences in phase retardation
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9 Image
8 Tube lens7 Analyzer (7a with Wave Plate)
6 Wollaston Prism behind objective5 Objective
4 Specimen
3 Condenser with receptacle for prisms2 Wollaston Prism before condenser1 Polarizer
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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
DICInterference
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Fluorescence
• Easy to set up > 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
Example: Specimen containing green fluorescing Fluorochrome
Dichromatic Mirror
Emission Filter
Excita
tion Filte
r
Observation port
FL
Light Source
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Epi - Fluorescence Filter Sets
Curve for a typical GFP filter set
Example
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Epi - Fluorescence
(Specimen containing green fluorescing Fluorochrome)
Dichromatic Mirror
Emission Filter
Excita
tion Filte
r
Observation port
FL
Light Source
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
1. 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)
2. 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)
3. 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
- cont‘d -
4. 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
5. 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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Amber fossil (Chironomide)
Thickness app. 300 µm
Conventional incident light
3D reconstruction
Optical Sectioning + Extended Focus
Software
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• Break Period – move to labBreak Period – move to lab
• Setting up / adjusting the Setting up / adjusting the microscopes for Brightfieldmicroscopes for Brightfield