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

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

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.

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

Bacillaria

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

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

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

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)

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

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

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

Rhipidodendron Phase Contrast

Cochliopodium Phase Contrast

Lyngbya Bacteria Phase Contrast

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

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

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

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

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

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

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

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

DIC Observing local differences in phase retardation

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

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

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)

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

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

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

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

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

Paramecium bursaria Fluorescence

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

Optical sectioning – increased contrast and sharpness

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)

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

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

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

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