1. IPC Friedrich-Schiller-Universität Jena 1 http://biology.about.com 2. Contrast modes in light microscopy: Bright field

2. IPC Friedrich-Schiller-Universität Jena 2 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

3. IPC Friedrich-Schiller-Universität Jena 3 Dark Field Transmission Objective Lense Tube Lense CCD Bright object on dark background Useful for life-cell imaging of vesicles (Richardson Microscope) Ring-like condensor aperture at NA condensor > NA objective 2. Contrast modes in light microscopy: Dark field

4. IPC Friedrich-Schiller-Universität Jena 4 Bright FieldDark Field http://biology.about.com 2. Contrast modes in light microscopy: Dark field

5. IPC Friedrich-Schiller-Universität Jena 5 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

6. IPC Friedrich-Schiller-Universität Jena 6 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

7. IPC Friedrich-Schiller-Universität Jena 7 Fourier-transformation & Optics Plane Waves are simple points in reciprocal space A lens performs a Fourier-transform between its Foci Fourier-transformation of Amplitude

8. IPC Friedrich-Schiller-Universität Jena 8 Fourier-transformation & Optics Fourier- plane Object Image f f f f Laser

9. IPC Friedrich-Schiller-Universität Jena 9 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

10. IPC Friedrich-Schiller-Universität Jena 10 The Light Wave - Phase Contrast imaginary real time Ampitude Scattered Phase Changed 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

11. IPC Friedrich-Schiller-Universität Jena 11 Make it 90 deg extra Phase! imaginary real Scattered Result Zernike Phase Contrast! + 90 deg 2. Contrast modes in light microscopy: Phase contrast

12. IPC Friedrich-Schiller-Universität Jena 12 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

13. IPC Friedrich-Schiller-Universität Jena 13 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

14. IPC Friedrich-Schiller-Universität Jena 14 2. Contrast modes in light microscopy: Polarisation contrast back focal plane High angle (high NA) depolarisation Maltese Cross

15. IPC Friedrich-Schiller-Universität Jena 15 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.

16. IPC Friedrich-Schiller-Universität Jena 16 Bright field microscopyPhase contrast microscopy DIC microscopyDark field microscopy 2. Contrast modes in light microscopy: DIC

17. IPC Friedrich-Schiller-Universität Jena 17 2. Contrast modes in light microscopy: DIC