IPC Friedrich-Schiller-Universität Jena 1 2. Contrast modes in light microscopy: Bright field

IPC Friedrich-Schiller-Universität Jena1

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2. Contrast modes in light microscopy: Bright field


2.2 Dark field (light scattering = real part of refractive index)

Light microscopy with instrumental contrast enhancement = optical contrasting:

E.g. dark field, phase contrast, polarization, differential interference contrast

Investigation of living objects possible (great for vesicles)

Dark field microscopy

For transparent unstained samples light will be scattered at phase boundaries i.e. between structures of different refractive indices

Dark filed utilizes this to visualize boundaries

Illumination with special high NA condenser units

Paraboloid condenser Kardioid condenser

2. Contrast modes in light microscopy: Dark field


Dark FieldTransmission

ObjectiveLense

TubeLense CCD

Bright object on dark background

Useful for life-cell imaging of vesicles (Richardson Microscope)

Ring-like condensor aperture at NAcondensor > NAobjective



Bright Field Dark Field

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2.2 Dark field (light refraction = real part of refractive index)

Without specimen: light rays do not arrive at objective field of view dark



2.2 Dark field (light refraction = real part of refractive index)

With specimen: light rays will be refracted at sample edges arrive at objective bright sample edges on dark background

Transparent specimen in dark filed



Fourier-transformation & Optics

•Plane Waves are simple points in reciprocal space

•A lens performs a Fourier-transformbetween its Foci

Fourier-transformation of Amplitude


Fourier-transformation & Optics

Fourier-plane

Object Image

f f f f

Laser


2.3 Phase contrast microscopy

Most cell compartments are no amplitude objects

Many organelles exhibit different refractive indices and therefore diffract light beams differently leading to a phase shift compared to a undisturbed reference beam

Such specimen are called phase objects

Phase objects are not visible in the bright field

Phase contrast via refractive index differences

Phase difference

Refractive indices

2. Contrast modes in light microscopy: Phase contrast


The Light Wave - Phase Contrast

imaginary

real

timeA

mpi

tude

Scattered

Phase

Cha

nged

time

A small phase change can be described by interference of unscattered light with 90 deg out of phase light



Make it 90 deg extra Phase!

imaginary

real

Scattered

Result

Zernike Phase Contrast!

+ 90 deg



2.3 Phase contrast microscopy Illuminating beam hits annular ring

Non diffracted beam (primary beam) hits phase ring within objective after specimen

Phase ring is conjugated complement to annular aperture:

Phase ring attenuates primary beam(to balance with scattered light)

Phase ring shifts beam by /2 (/4-plate) so that primary beam interferes with diffracted light with maximum contrast


ring aperture

phase ring

Bright field image Phase contrast

typical"halo"

internal epidermis of an onion


2.4 Polarization contrast Specimen is placed between two

crossed polarizer

Many specimen like e.g. birefringent materials (crystals) rotate polarization plane and can be observed by a polarization microscope

Biology visible (edge birifringence)

Glucose crystals

2. Contrast modes in light microscopy: Polarisation contrast

Polarizer

Analizer


2. Contrast modes in light microscopy: Polarisation contrast

back focal plane

High angle (high NA) depolarisation

Maltese Cross


2.5 Differential interference contrast microscopy (DIC)

2. Contrast modes in light microscopy: DIC

DIC works by separating a polarized light source into two beams which take slightly different paths through the sample. Where the length of each optical path (i.e. the product of refractive index and geometric path length) differs, the beams interfere when they are recombined.


Bright field microscopy Phase contrast microscopy

DIC microscopy Dark field microscopy




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IPC Friedrich-Schiller-Universität Jena 1 2. Contrast modes in light microscopy: Bright field