IPC Friedrich-Schiller-Universität Jena 1 2. Contrast modes in light microscopy: Bright field.
-
Upload
lola-footman -
Category
Documents
-
view
214 -
download
0
Transcript of IPC Friedrich-Schiller-Universität Jena 1 2. Contrast modes in light microscopy: Bright field.
IPC Friedrich-Schiller-Universität Jena1
http://biology.about.com
2. Contrast modes in light microscopy: Bright field
IPC Friedrich-Schiller-Universität Jena2
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
IPC Friedrich-Schiller-Universität Jena3
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
2. Contrast modes in light microscopy: Dark field
IPC Friedrich-Schiller-Universität Jena4
Bright Field Dark Field
http://biology.about.com
2. Contrast modes in light microscopy: Dark field
IPC Friedrich-Schiller-Universität Jena5
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. Contrast modes in light microscopy: Dark field
IPC Friedrich-Schiller-Universität Jena6
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
2. Contrast modes in light microscopy: Dark field
IPC Friedrich-Schiller-Universität Jena7
Fourier-transformation & Optics
•Plane Waves are simple points in reciprocal space
•A lens performs a Fourier-transformbetween its Foci
Fourier-transformation of Amplitude
IPC Friedrich-Schiller-Universität Jena8
Fourier-transformation & Optics
Fourier-plane
Object Image
f f f f
Laser
IPC Friedrich-Schiller-Universität Jena9
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
IPC Friedrich-Schiller-Universität Jena10
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
2. Contrast modes in light microscopy: Phase contrast
IPC Friedrich-Schiller-Universität Jena11
Make it 90 deg extra Phase!
imaginary
real
Scattered
Result
Zernike Phase Contrast!
+ 90 deg
2. Contrast modes in light microscopy: Phase contrast
IPC Friedrich-Schiller-Universität Jena12
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
2. Contrast modes in light microscopy: Phase contrast
ring aperture
phase ring
Bright field image Phase contrast
typical"halo"
internal epidermis of an onion
IPC Friedrich-Schiller-Universität Jena13
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
IPC Friedrich-Schiller-Universität Jena14
2. Contrast modes in light microscopy: Polarisation contrast
back focal plane
High angle (high NA) depolarisation
Maltese Cross
IPC Friedrich-Schiller-Universität Jena15
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.
IPC Friedrich-Schiller-Universität Jena16
Bright field microscopy Phase contrast microscopy
DIC microscopy Dark field microscopy
2. Contrast modes in light microscopy: DIC
IPC Friedrich-Schiller-Universität Jena17
2. Contrast modes in light microscopy: DIC