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

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

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

Common Illumination Techniques

Brightfield

• For naturally absorbing or stained samples

• True Color Representation

• Proper Technique for Measurements •Spectral•Dimensional

Paramecium bursaria

Condenser diaphragm open Condenser Diaphragm almost closed

Paramecium bursaria

Indian Ink Staining Feulgen Staining Silver Staining

Different Staining Techniques

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

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

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

Paramecium bursaria

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)

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

Paramecium bursaria

Polarized LightDarkfield

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

Brightfield

Background

Birefringent Material

Polarized Light Pol + Red I

Color of sample and

background modified by wave plate

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

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 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

DICDICObserving local differences in phase retardation

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

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

DICInterference

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

Example: Specimen containing green fluorescing Fluorochrome

Dichromatic Mirror

Emission Filter

Excita

tion Filte

r

Observation port

FL

Light Source

Epi - Fluorescence Filter Sets

Curve for a typical GFP filter set

Example

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

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

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)

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

Limited Depth of Field With Standard

Microcopy

Amber fossil (Chironomide)

Thickness app. 300 µm

Conventional incident light

Amber fossil (Chironomide)

Thickness app. 300 µm

Conventional incident light

3D reconstruction

Optical Sectioning + Extended Focus

Software

• Break Period – move to labBreak Period – move to lab

• Setting up / adjusting the Setting up / adjusting the microscopes for Brightfieldmicroscopes for Brightfield