VII Contrasting Techniques From Brightfield to Plas-DIC December 2008 Rudi Rottenfusser

C ONTRAST 50 – 0 / = 1 50 – 100 / = – 50 / = 0 50 Units0 Units100 Units 50 Units 50

Transmitted Light Brightfield Oblique Darkfield Modulation, Varel Contrast Phase Contrast Polarized Light DIC (Differential Interference Contrast) Fluorescence - not any more > Epi ! Incident Light Brightfield Oblique Darkfield Not applicable Not any more (DIC !) Polarized Light DIC (Differential Interference Contrast) Fluorescence (Epi) Illumination Techniques - Overview

Example

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

Brightfield Best Resolution when Condenser NA matches Objective NA! Minimum Contrast! Specimen Objective Condenser Resolution (minimum resolved distance between 2 details): d min d

Getting more contrast in the microscope: “Dropping” the condenser No more separation of controls for field size and aperture angle Higher contrast, but at the cost of NA Scattered light enters the objective Condenser not in proper position > spherical/chromatic aberrations Bad Idea!

Increases contrast Increases depth of field Reduces resolution Getting more contrast in the microscope: “Stopping down” the condenser (reducing the size of aperture diaphragm) Condenser Aperture matches Objective Condenser Aperture stopped down

Effect of Aperture Diaphragm NA Condenser = NA Objective

Paramecium bursaria Condenser diaphragm openCondenser Diaphragm almost closed

Paramecium bursaria Indian Ink StainingFeulgen StainingSilver Staining Different Staining Techniques

Contrasting Techniques Going more into details Brightfield Oblique Darkfield Phase Varel Hoffman Pol DIC Plas-DIC

Getting more contrast in the microscope: Oblique Illumination (moving the aperture diaphragm sideways) Increases contrast Increases depth of field 3-D effect Slightly reduces resolution

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

Highest contrast Detection of sub-resolution details possible No staining necessary Central Darkfield via “hollow cone” Oblique Darkfield via Illumination from the side Excellent technique to detect traces of contaminants Not useful for Measurements (sizes exaggerated)

Paramecium bursaria Polarized Light Darkfield

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 + New positive / negative Phase Contrast

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)

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

Phase Plate Objective Specimen Condenser Condenser Phase Ring Intermediate Image Phase Contrast Imaging Path { Diffraction Orders Non-diffracted wave  shifted by  /4) Diffracted wave  shifted by  /4)

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

Sales Training Oct More Information in Phase Contrast Positive and negative Phase Contrast in one Objective Objectives: LD Plan-Nefluoar 20x Ph1 Ph2- Korr LD Plan-Neofluar 40x Ph1 Ph2- Korr MDCK cells (dog) R. Nitschke and F. Kotsis, Life Imaging Center, Freiburg Positive Phase Contrast Negative Phase Contrast Observation

Paramecium bursaria Phase Contrast Condenser diaphragm open Brightfield

Rhipidodendron Phase Contrast Cochliopodium Phase Contrast

Lyngbya Bacteria Phase Contrast

Neurons Thin Phase Object in plastic vessel Varel Contrast

Varel Contrast ( Zeiss) For unstained (live) specimens Combination of oblique illumination and attenuation of non-diffracted light No “Halo”-effect as in Phase Contrast Complementary technique to Phase (easy switchover) Simulated 3-D image (similar to DIC) Less resolution than DIC Works with plastic dishes

Movable Ring Sector (Varel Ring) Required Components for Varel: 1.Objective with Varel- and Ph ring 2.Slider or Condenser with specific Varel 1 or Varel 2 ring sector Back Focal Plane of Varel / Phase Objective Brightfield / obliqueDarkfield “Varel”

Modulation Contrast (Hoffman) For unstained (live) specimens For unstained (live) specimens Simulated 3-D image (similar to DIC) Simulated 3-D image (similar to DIC) No Halo-effect (as in Phase Contrast) No Halo-effect (as in Phase Contrast) Usable with plastic dishes Usable with plastic dishes Less resolution as DIC Less resolution as DIC Note: Modulation Contrast Objectives are not recommended for fluorescence; due to potential damage of modulator and uneven illumination

3% transmittance Modulation Contrast Required Components for Modulation Contrast: Specially Modified Objective (With Built-in Modulator) Modified Condenser with off- axis slit (double slit with polarizer)

Polarized Light One starts out usually by crossing two polarizers (polarizer and “analyzer”) in a microscope. The specimen is located between them. Only birefringent particles (e.g. crystals) become visible, when they are rotated via rotating stage. Isotropic components will remain dark. Polarized Light looks sometimes just like Darkfield because edges become visible due to “edge birefringence”.

Polarized Light Birefringent Material Polarizer Analyzer

Polarized Light Birefringent Material Polarizer Analyzer Polarizer Analyzer

Polarized Light Birefringent Material Polarizer Analyzer Polarizer Analyzer

Polarized Light Birefringent Material Polarizer Analyzer

Polarized Light Birefringent Material Polarizer Analyzer

When Polarizers are crossed, only items that rotate the plane of polarization reach the detector. Polarized Light Polarizer 1 Polarizer 2 (Analyzer) Specimen Wave plate adds color

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

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

The numerical difference between the maximum and minimum refractive indices of anisotropic substances. n γ - n α. Birefringence may be qualitatively expressed as low ( ), moderate (0.010 – 0.050) high (>0.050) extreme (>0.2) Birefringence may be determined by use of compensators, or estimated through use of a Michel-Lévy Interference Color Chart. Birefringence

An excellent introduction to this chart is provided at McCrone’s website LOW < Moderate – High > 0.050

3 rd Order Red 1 st Order Red 1 st Order Red 2 nd Order Red 2 nd Order Red 3 rd Order Red

Retardation (nm) I/I o alphagamma 1st Order Red Plate 550 nm Retardation Sensitive Tint Field of View

Orthoscopy / Conoscopy Analyzing minerals is based on such morphological and optical features as form, cracks, color, pleochroisms, and their characteristic interference colors. Orthoscopy and conoscopy are the most important techniques in classical transmitted light polarization microscopy. With their different ways of examining, they provide different options, e.g. in mineral diagnosis in geological microscopy. In orthoscopy, each pixel corresponds to a dot in the specimen. In conoscopy, each pixel corresponds to a direction in the specimen. This technique requires the use of the highest objective and condenser aperture possible. Conoscopy is used when additional information about the specimen is necessary for analysis. It provides interference images that can be seen through the eyepiece and enables differentiation according to 1 or 2 axes and with compensator λ (λ-lamda, Red I), according to 1-axis positive/negative or 2-axis positive/ negative. A Bertrand lens in the light path makes visible the interference or axial image in the back focal plane of the specimen.

Some Types of Birefringence Intrinsic or crystalline (Quartz, Calcite, Myosin Filaments, Chromosomes, Keratin, Cellulose Fibers) Form or Textural (Plasma membranes, Actin filaments, microtubules) Edge (resulting from diffraction at edges of objects embedded in a medium of different refractive Index) Strain (resulting from mechanical stress e.g. glass, plastic sheets) Circular –also known as- Optical Rotation (sugars, amino acids, proteins)

The wave exhibits electric (E) and magnetic (B) fields whose amplitudes oscillate as a sine function over dimensions of space or time. The amplitudes of the electric and magnetic components at a particular instant or location are described as vectors that vibrate in two planes perpendicular to each other and perpendicular to the direction of propagation. At any given time or distance the E and B vectors are equal in phase. For convenience it is common to show only the electric field vector (E vector) of a wave in graphs and diagrams. Light as an electromagnetic wave

E  y x z EyEy ExEx EE Polarized Light

Polarized Light and Birefringence

Interface with birefringent Material n  = higher refractive index > slower wave n  = lower refractive index > faster wave

Linear polarizer ¼ wave plate Unpolarized light linearly polarized Circularly polarized How to create circularly polarized light

x z EyEy ExEx EE  Circularly Polarized Light E

y x z EyEy ExEx EE  Sénarmont Compensator * ¼ wave plate, located beforeanalyzer, is oriented with itsbirefringence parallel to the polarizer or analyzer. Therefore,there will be no effect on thepolarized beam.Birefringence produced byspecimen (occurring at 45˚), willbe converted by ¼ wave plate intocircular polarized light which canpass through the analyzer.By rotating the analyzer, it ispossible to introduce “bias”birefringence because it will not beparallel to ¼ wave plate any more. * 1 st described by de Sénarmont in 1840

9 Image 8 Tube lens 7 Analyzer 7a Wave Plate) 6 Wollaston Prism Slider 5 Objective 4 Specimen 3 Condenser 2 Wollaston Prism 1 Polarizer DIC Principle (F.H.Smith, 1952)

DIC (Nomarski/Allen 1969) Differential Interference Contrast Changes GRADIENTS into brightness differences Changes GRADIENTS into brightness differences Eye-pleasing 3-D Image appearance Eye-pleasing 3-D Image appearance High Contrast and high resolution High Contrast and high resolution Control of condenser aperture for optimum contrast Control of condenser aperture for optimum contrast Great for “optical sectioning” due to small depth of field Great for “optical sectioning” due to small depth of field Color DIC by adding a wave plate Color DIC by adding a wave plate Best contrast / resolution via different DIC sliders Best contrast / resolution via different DIC sliders Orientation-specific > orient fine details perpendicular to DIC prism Orientation-specific > orient fine details perpendicular to DIC prism Requires strain-free elements, not for birefringent specimens Requires strain-free elements, not for birefringent specimens

y x z EyEy ExEx EE  Wollaston Prism Polarized beam, under 45˚ to prism,gets split into “ordinary” and“extraordinary” beam

DIC Observing local differences in phase retardation

IR-DIC IR increases depth of field – useful for thick tissues IR increases depth of field – useful for thick tissues Achieve Contrast in Electrophysiology applications Achieve Contrast in Electrophysiology applications Special Objective and Polarizer recommended Special Objective and Polarizer recommended Requires IR filter for transmitted light Requires IR filter for transmitted light For heat protection, special filter combination For heat protection, special filter combination

Special Filter Arrangement for IR-DIC RG 9 = IR Filter Calflex = Heat reflecting filter

62 PlasDIC PlasDIC Rainer Danz, 2004 (patented 2006) Most important before injection: sharp image of zona pellucida, tip of injection pipette and oolemma

Conventional DIC (Nomarski-Principle) : Note: Condenser, Specimen, Objectives are between polarizers, therefore they must not produce any birefringence for optimal results in DIC! Observing Back Focal Plane between crossed Polarizers (Only DIC Prism of condenser in place!) Observing Back Focal Plane between crossed Polarizers (Only DIC Slider of objective in place!) Homogeneous Exit Pupil at Back Focal Plane when both Wollaston prisms are in place Both prisms in place: Note: The fringes in the back focal plane are oriented at 45 o in the microscope. Polarizers are East-West, Analyzers South-North. It is impractical to “draw” a prism cross-section under 45 o to the drawing surface…

BFP Objective. Analyzer Objective Condenser Conventional DIC Slit Observing Back Focal Plane between crossed Polarizers: Françon- Yamamoto Polarizer Note: The fringes in the back focal plane are actually oriented at 45 o in the microscope. Polarizers are East-West, Analyzers South-North. This display takes into account that it is impractical to “draw” a prism cross-section under 45 o to the drawing surface…

BFP Objective. Analyzer Objective Condenser Conventional DIC Slit ZEISS Plas-DIC Polarizer Observing Back Focal Plane between crossed Polarizers: Note: The fringes in the back focal plane are actually oriented at 45 o in the microscope. Polarizers are East-West, Analyzers South-North. This display takes into account that it is impractical to “draw” a prism cross-section under 45 o to the drawing surface…

/4 Optimal Condition ! Contrast = f (Slit Width)  sinc  Slit Width as it is projected into Back Focal Plane (BFP) of Objective Sinc Function Distance between 0 and 1st order of birefringence Contrast

Required Components for Plas DIC 2) Slit diaphragm PlasDIC for condenser or slider 1) Nosepiece with receptacles for DIC sliders (AxioObserver or Axiovert 40 CFL) 3) Objective for available Plas-DIC Sliders 4) The right PlasDIC slider for each objective 5) Fixed Analyzer Slider or Analyzer in Cube Note: PlasDIC and Analyzer sliders should be removed during fluorescence imaging. They will reduce the intensity substantially if left in place!

68 PlasDIC - Advantages High optical resolution, close to regular DIC, at least equal to *Hoffmann Modulation contrast Excellent relief and three dimensional impression, large depth of field, great for work with manipulators and multiple probes Cost effective (no special objectives, no special condensor, no second prism) Plastic dishes, Ph objectives, birefringent specimens have no effect on image quality Very simple handling: no centering or change of diaphragm Easily upgradable: takes customers budget into account *Hoffmann Modulation contrast: Also very good contrast, but most users don´t know how to optimize the settings, which are much more complicated to establish. The first polarization- optical interference contrast designed for plastic vessels Patent No. DE

69 Comparison of Contrast Methods Phase VAREL Hoffmann PlasDIC DIC Contrast thick specimen _ _ Contrast thin specimen Resolution + _ _ Optical Sectioning capability _ _ _ Depth of focus in living cells _ _ Homogenity field of view ++ _ Reproducibility of setting _ _ _ + + Plastic Vessels + + (+) ++ _ Price 