2 Contrast modes in light microscopy Bright field

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

2. Contrast modes in light microscopy: Dark 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 2 Kardioid condenser IPC Friedrich-Schiller-Universität Jena

2. Contrast modes in light microscopy: Dark field 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 NAcondensor > NAobjective 3 IPC Friedrich-Schiller-Universität Jena

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

2. Contrast modes in light microscopy: Dark field 2. 2 Dark field (light refraction = real part of refractive index) § Without specimen: light rays do not arrive at objective field of view dark 5 IPC Friedrich-Schiller-Universität Jena

2. Contrast modes in light microscopy: Dark field 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 6 IPC Friedrich-Schiller-Universität Jena

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

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

2. Contrast modes in light microscopy: Phase contrast 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 Phase difference § Such specimen are called phase objects § Phase objects are not visible in the bright field Refractive indices Phase contrast via refractive index differences 9 IPC Friedrich-Schiller-Universität Jena

2. Contrast modes in light microscopy: Phase contrast Ampitude ge an Ch t Ph as e d ere real att Sc d The Light Wave - Phase Contrast imaginary time A small phase change can be described by interference of unscattered light with 90 deg out of phase light 10 IPC Friedrich-Schiller-Universität Jena

2. Contrast modes in light microscopy: Phase contrast real Make it 90 deg extra Phase! + d e ter g e d 90 at c S ult s e R imaginary Zernike Phase Contrast! 11 IPC Friedrich-Schiller-Universität Jena

2. Contrast modes in light microscopy: Phase contrast 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 p/2 (l/4 -plate) so that primary beam interferes with diffracted light with maximum contrast phase ring typical "halo" internal epidermis of an onion 12 Bright field image Phase contrast ring aperture IPC Friedrich-Schiller-Universität Jena

2. Contrast modes in light microscopy: Polarisation contrast 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 Analizer § Biology visible (edge birifringence) Polarizer Glucose crystals 13 IPC Friedrich-Schiller-Universität Jena

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

2. Contrast modes in light microscopy: DIC 2. 5 Differential interference contrast 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. 15 IPC Friedrich-Schiller-Universität Jena

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

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