1. Agenda How to make the specimen visible – Definition of Contrast
Techniques:
Brightfield
Phase
Darkfield
Pol
DIC (Differential Interference Contrast)
Fluorescence
Optical Sectioning – an expansion of Fluorescence
Agenda

2. 0 Units
50 Units
100 Units
C ONTRAST
50 Units 50 50
50 – 100 / = -0.33
50 – 0 / = 1
50 – 50 / = 0

3. Common Illumination Techniques
Brightfield
Darkfield
Phase Contrast
Polarized Light
DIC (Differential Interference Contrast)
Fluorescence (and related techniques)
In both Transmitted and Reflected Light Modes; Fluorescence now only in Reflected Light (Epi); Phase Contrast only in Transmitted Light (Reflected Light Phase totally replaced by DIC); There are more techniques, but these are not as important as the ones mentioned.

5. Brightfield For naturally absorbing or stained samples
True Color Representation
Proper Technique for Measurements
Spectral
Dimensional

8. Bacillaria

9. Paramecium bursaria Condenser diaphragm open
Brightfield: Algae – location of macronucleus and mouth are evident
Closing the condenser diaphragm creates higher contrast. The contractile vacuole, cilia and more cell details can be seen.
Condenser diaphragm open
Condenser Diaphragm almost closed

10. Paramecium bursaria Different Staining Techniques Indian Ink Staining
Staining techniques help to bring out details in brightfield illumination;
Cell suspended in Indian Ink and preparatio allowed to dry out. The ink settles around cell and into the depressions from which the cilia emerge.
Cell stained with Feulgen, a stain associated with DNA. The macronucleus and microneclei are clearly visible, but also small nuclei can be seen.
Silver staining is used to show the bases of the cilia. Shows the arrangement of the kineties (rows of cilia) on the cell.
Indian Ink Staining
Feulgen Staining
Silver Staining

12. Phase Contrast (Frits Zernike 1934)
- “Halo” effect > Reduced resolution
+ No staining necessary
+ Good Depth of Field
+ Easy alignment
+ Orientation independent
+ Repeatable setup
+ Works with plastic dishes

13. 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)

14. 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
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

15. 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 
Affected rays from specimen, expressed by the higher diffraction orders, do not pass through phase ring of objective >¼ wave retarded 
Tube Lens
Objective Phase Ring a) attenuates the non-diffracted 0th Order b) shifts it ¼ wave forward 
Objective
Specimen
Condenser
Illumination from Condenser Phase Ring (“0” Order) > meets phase ring  of objective

16. Paramecium bursaria Phase Contrast
In Phase Contrast, phase retardations which are not visible to the human eye, are converted into amplitude differences, making it possible to study detals as the cilia here.
Phase Contrast

17. Rhipidodendron
Phase Contrast

18. Cochliopodium
Phase Contrast

19. Lyngbya Bacteria
Phase Contrast

21. 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)
“Detection” Term more appropriate than “resolution”; Example: Moon at Night

22. Required conditions for Darkfield: Illumination Aperture must be larger than objective aperture I.e. direct light must bypass observer
Low NA Objective
High NA Objective
Iris Diaphragm

23. Paramecium bursaria Darkfield Polarized Light
Darkfield Illumination: Angle of Illumination is sufficiently oblique, that no illuminating light can enter the objective. Only scattered or diffracted light is visible. Very high degree of detection!
Polarized light: Specimen is placed between 2 crossed polarizers. Only light produced by birefringent particles or on edges of particles (“edge birefringence”) is visible. Looks sometimes almost identical to darkfield
Darkfield
Polarized Light

24. 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

25. Birefringent Material
Color of
sample and background modified by wave plate
Background
Brightfield
Polarized Light
Pol + Red I

26. 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

27. 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

29. 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 > orient fine details perpendicular to DIC prism

30. DIC
Observing local differences in phase retardation

31. 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
2 Wollaston Prism before condenser
1 Polarizer

32. Wollaston Prism
Birefringence (Different refractive index for different polarization orientations)
Polarized beam, under 45˚ to prism, gets split into “ordinary” and “extraordinary” beam

33. 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)

34. Paramecium bursaria DIC Interference
DIC (Differential Interference Contrast), sometimes also called “Nomarski Contrast”; Density gradients in samples become visible in an (artificial) 3-D way.
Interference Techniques require special equipment which produces 2 separate beams, of which 1 passes through the medium and the other through the specimen. Elements with different optical properties are converted into different colors.
DIC
Interference

35. 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

36. Epi - Fluorescence Observation port Excitation Filter Emission Filter
Light Source
Dichromatic Mirror
Light Sources:
Mercury (Hg), Xenon, Xenon/Hg Combinations; Laser; LED’s, Tungsten Halogen
Example: Specimen containing green fluorescing Fluorochrome

37. Epi - Fluorescence Filter Sets
Example
Curve for a typical GFP filter set

38. Epi - Fluorescence
(Specimen containing green fluorescing Fluorochrome)
Observation port
Excitation Filter
Emission Filter FL
Light Source
Dichromatic Mirror
Specimen containing green fluorescing Fluorochrome

39. Paramecium bursaria
Fluorescence

40. How to improve Fluorescence Imaging in a major way:
Optical Sectioning

41. Optical sectioning – increased contrast and sharpness

42. 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)

43. Overview of Optical sectioning Methods - cont‘d -
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

44. Limited Depth of Field With Standard Microcopy
Amber fossil (Chironomide)
Thickness app. 300 µm
Conventional incident light

45. Optical Sectioning + Extended Focus Software
Amber fossil (Chironomide)
Thickness app. 300 µm
Conventional incident light
3D reconstruction

46. Break Period – move to lab
Setting up / adjusting the microscopes for Brightfield