Phase contrast and DIC imaging

Transcription

1 Phase contrast and DIC imaging - Phase contrast - Differential-interferencecontrast (DIC) - Relief-phase contrast Optical microscopy course Kirsi Rilla 1 Key terms Contrast = the difference in color and light between parts of an image Amplitude = the magnitude of change in the oscillating light wave Interference = a phenomenon in which two waves superimpose to form a resultant wave of greater or lower amplitude 2 1

2 Interference and polarization Interference: Superimposition of two or more waves result in a new wave pattern. Waves in same phase Waves in different phase amplification attenuation Polarized light waves are light waves in which the vibrations occur in a single plane. 3 Phase contrast microscopy History - Fritz Fernicke, Dutch, physicist Nobel prize 1953 Principle - A contrast-enhancing optical technique - Utilized to produce high-contrast images of transparent specimens, such as living cells or microorganisms - Most living biological specimens are translucent. When viewed under transmitted light microscope, there is often little or no distinguishable contrast in the image. - When the light travels through a specimen, parts of the specimen are denser than other parts, creating a phase shift in the transmitted light (phase objects). - Our eyes are unable to detect these phase differences 4 2

3 Phase contrast microscopy - A phase disc of objective - A phase annulus ring in the light condenser - The phase disc have to be aligned with a phase annulus ring in the light condenser properly - Converts phase shifts in light passing through a transparent specimen to brightness changes in the image - Enhances the contrast 5 Phase contrast microscopy - Phase shift of light waves by utilizing phase rings - Enhances phase differences (¼ + ¼ = 1/2) better contrast - Even the smallest differences in the refractive index of the object become visible - The denser the object, the darker the image Phase object Phase shift Visible shift object visible 6 3

4 Phase contrast microscopy 1 = light source 2 = annular shaped light mask (condenser annulus) 3 = condenser 4 = specimen 5 = background light 6 = light bent by the specimen 7 = phase ring 8 = eyepiece with intermediate image 9 = eye Image: Alignment of condenser annulus (bright) and phase ring (dark) Copyright: Joerg Piper, Bad Bertrich, Germany, Phase contrast microscopy Advantages: + High contrast for unstained samples + Suitable for living cells and thin specimens + No need to be killed, fixed or stained + Ability to combine with other means of observation, such as fluorescence Disadvantages: - Not ideal for thick organisms or particles - The bright or dark halos around objects 8 4

5 Phase contrast microscopy OLYMPUS IX70 INVERTED MICROSCOPE IN THE INSTITUTE OF BIOMEDICINE EQUIPMENT: 1. OLYMPUS IX-70 inverted microscope 2. Olympus TH4-200 light source (brightfield light) 3. Olympus Cell MT-10 light source (fluorescent light) 4. ORCA-AG CCD (Charge-Coupled Device) cooled digital camera 5. Eppendorf InjectMan NI 2 micromanipulator 6. Eppendorf Femtojet microinjector 7. Nexus computer with Olympus Cell F imaging software 8. Evotec incubator, with temperature, CO2 and humidity control 9 Olympus IX70 inverted microscope 10 5

6 Objects suitable for phase contrast microcopy Phase contrast objects: Unstained specimens that do not absorb light: - Living cells (usually culture) - Microorganisms - Thin tissue slices - Subcellular particles - Cell nuclei and organelles 11 Microinjection - Injection of substances directly into a cell utilizing a glass micropipette and pressure - For DNA, antibodies, colors, drugs Disadvantages -Not for high cell numbers -Technically Challenging - Equipment necessary 12 6

7 Microinjection Advantages + Efficient (near to 100%) + Exact timing + Suitable for all cell types + Gentle method + Targeted manipulation (nucleus or cytoplasm) + Exact volumes + Economic + Several probes at the same time 13 DIC technique (Differential Interference Contrast) History: - Georges Nomarski, French physicist in the 1950s developed a Wollaston prism - Francis Smith developed the actual DIC microscope in 1955 Applications: - Live cell inspection and analysis like in phase contrast microscopy, Principle: - The light is divided into two bundles, whose polarization planes are perpendicular to each other. -After passing through the sample, the light bundles are combined again, and the sample gives rise to phase differences A three-dimensional illustration 14 7

8 DIC Eye 5. Analyzator 4. Wollaston prism Objective Specimen Condensor 2. Wollaston prism 1. Polarizator Light source 15 Principle of action: DIC 1. A polarizer to produce polarized light. 2. Wollaston prism splits the light into two bundles of light whose polarizations are perpendicular to each other. 3. Light bundles pass through the sample in a slightly different positions. They do not interfere with each other at this stage, because their polarization planes are reversed. 4. The second Wollaston prism to combine light bundles again, Making them interfere (gain or attenuation). 5. Polarizing analyzer inhibits the directly passing light, converting the original phase differences to darker and lighter shades. 16 8

9 DIC Advantages + High resolution + Good contrast + Suitable for thick specimen + No Halos Disadvantages - Not for birefringent objects - Not for plastic plates - Interpretation of images: pits and hills - Expensive and demands accuracy 17 Comparison between different techniques Mitotic spindle of metaphase Phase contrast microscopy Interference- Microscopy (DIC) 18 9

10 Relief-phase contrast-technique - A novel application of traditional phase contrast technique. - The transparent annulus is replaced by a sectoral light mask, which can be crescent-shaped or a small round aperture. - The specimen is only illuminated from one defined direction by oblique light beams. Advantages: - More contrast - Better focal depth - Three-dimensional appearance - No halo-artefacts 19 Relief-phase contrast 1 = light source 2 = modified condenser light mask 3 = condenser 4 = specimen 5 = background light 6 = light bent by the specimen 7 = phase ring 8 = eyepiece with intermediate image 9 = eye Image: Alignment of sectoral light mask (bright) and phase ring (dark) Copyright: Joerg Piper, Bad Bertrich, Germany,

11 Relief-phase contrast Phase contrast Relief-phase contrast 21 Traditional compared with relief-phase contrast Feature Objectives from different manufacturers Traditional Phase contrast no Relief- Phase contrast yes Light rays concentric exentric Condensor diaphragm open 3D effect no yes Open or smaller contrast good excellent sharpness good excellent depth accuracy narrow wider resolution high sometimes weaker halos strong weak 22 11

12 Relief-phase contrast Proliferating Breast cancer cells 23 12