Light Microscope Flashcards
Types of Light Microscopes
- Bright Field Microscope
- Dark Field Microscope
- Phase Contrast Optics
- Polarized Microscope
- Differential Interference Contrast (DIC) or Nomarski
- Fluorescence Microscope
- Confocal Microscopes
- The stereo microscope
Bright field microscopy
– light from an incandescent source is aimed toward a lens beneath or above the stage called the condenser,
– through or over the specimen,
– through an objective lens, and to
– the eye through a second magnifying lens, the ocular or eyepiece.
what does a good quality bright field microscope have?
– a built-in illuminator,
– adjustable condenser with aperture diaphragm (contrast) control, The condenser is used to focus light on the specimen through an opening in the stage.
– mechanical stage, and binocular eyepiece tube.
Image formation of bright field microscopy
– The magnification of the image is simply the objective lens magnification multiplied by eyepiece magnification.
– The condenser is used to focus light on the specimen through an opening in the stage.
– After passing through (or reflected on) the specimen, the light is displayed to the eye with an apparent field that is much larger than the area illuminated - times the ocular magnification.
– Adjustments to the condenser can affect resolution and contrast.
– The bright field condenser usually contains an aperture diaphragm, a device that controls the diameter of the light beam coming up through the condenser
– When the diaphragm is stopped down (nearly closed) the light comes straight up through the center of the condenser lens and contrast is high.
– When the diaphragm is wide open the image is brighter and contrast is low.
– Adjust the condenser by starting with the aperture diaphragm stopped down (high contrast).
Dark Field illumination
– An opaque disc is placed ahead of the condenser lens, so that only light that is scattered by objects can reach the eye.
– The contrast is completely reversed from that obtained by bright field illumination. Objects are often seen in “false colors,” that is, the reflected light is of a color different than the color of the object.
– Features that are light in bright filed will be dark in the dark field and those that are dark in bright-filed will be bright in dark field. This highlights angles of surfaces (pits, cracks, and etched grain boundaries) allows more positive identification of their nature that images from the bright-field illumination.
– Better resolution can be obtained using dark field as opposed to bright field viewing.
Oblique illumination
– The surface relief of a metallographic specimen can often be enhanced by using oblique illumination.
– This involves offsetting the condenser lens system, usually by positioning the condenser aperture slightly off the optical axis, which has its limitation.
– This gives the image a 3-dimensional appearance and can highlight otherwise invisible features.
– Oblique illumination suffers from the same limitations as bright field microscopy (low contrast and low apparent resolution due to out of focus objects), but may highlight otherwise invisible structures.
– The apparent three-dimensional effect afforded by oblique illumination techniques does not represent the actual specimen geometry or topography, and should not be employed to conduct measurements of specimen dimensions.
Rheinberg illumination
– Rheinberg illumination was developed by Rheinberg around 1895. He used colored filters to make distinctions between parts of the optical system.
– The Rheinberg filters work very well on fibers and plant material etc. It may produce a clearer view of the texture of a subject.
– The concentric filters of the zoom system give a better idea of the three-dimensional properties of a subject, a bit like a satellite picture showing different heights in different colors.
– Rheinberg illumination is based on dark-field illumination. With colored filters instead of the dark ‘patch stop’ wonderful effects can be created.
– Because there is less contrast it is easier for the eyes. A green outer filter may help to see more details since the human eye is especially good in seeing greens!
– It is important that the central filter is much darker than the outer filter!
– The use of several colors will create a distinction between different areas in the subject. You can use any color you like. In this case the primary light colors of red, green, and blue are used. The combination of these colors with a proper subject will produce all colors.
– The different refractive indexes and angles in a subject will light up in a different colors. This way the colors will give information about the three dimensional aspects of the subject.
– It is possible to make oblique darkfield or oblique Rheinberg!
Chromatic Aberration
Chromatic aberration or “color fringing” is caused when the lens not focusing different wavelengths of light onto the exact same focal plane (the focal length for different wavelengths is different) and/or by the lens magnifying different wavelengths differently. The amount of chromatic aberration depends on the dispersion of the glass.
Chromatic aberration can be minimized by using an achromatic lens, in which materials with differing dispersion are assembled together to form a compound lens. The most common type is an achromatic doublet with elements made of crown glass (has low refractive Index) and flint glass (high refractive index) . This reduces the amount of chromatic aberration over a certain range of wavelengths, though it does not produce perfect correction. By combining more than two lenses of different composition, the degree of correction can be further increased.
Polarized Light Microscope
– Since many metals and metallic and non-metallic phases are optically anisotropic, polarized light is particularly useful in metallography.
– Polarized light is obtained by placing a polarizer in front of the condenser lens of the microscope and placing an analyzer behind the eyepiece, as shown.
– The polarizer produces plane-polarized light which strikes the surface and is reflected through the analyzer to the eyepieces. If an anisotropic metal is examined with the analyzer set 90o to the polarizer the grain structure will be visible.
– However, viewing an isotropic metal (cubic metals) under such conditions will produce a dark, “extinguished” condition.
– Polarized light reveals grain structure and twinning in anisotropic metals and alloys and for identifying phases and inclusions. (look at diagram)
Differential Interference Contrast (Nomarski, DIC)
– Differential interference contrast (DIC), produces considerable depth of resolution and a three dimensional effect.
– D.I.C. and related optics give a specimen a three dimensional appearance that is not unlike the appearance of a specimen in a scanning electron microscope. These methods enhance depth of focus so that thicker specimens can be observed at higher magnifications.
– Light from an incandescent source is passed through a polarizer, so that all of the light getting through must vibrate in a single plane.
– The beam is then passed through a prism (placed between objective and vertical illuminator) that separates it into components that are separated by a very small distance. The two l9ight beams produced that exhibit coherent interference on the image plane.
– This leads to slightly displaced (laterally) images differing in phase by (l/2) which produce height contrast. When the contrast is optimized one can obtain a very distinct image that appears three dimensional.
– The effect is very much like what you see when a subject is shadowed by a strong light coming from one side. The colors are similar to those obtained by bright filed.
Principles and Applications of Two-Beam Interferometry
– A beam emitted by the light source is split into two beams of nearly equal intensity by a half mirror (beam splitter), one of these beams being directed onto a flat reference mirror and the other onto the specimen surface. The light produced by reflection of these two beams is then made to interfere.
– When observed from the viewing port, interference occurs between the image of the reference mirror and the image of the specimen surface. Since the light waves reflected by the specimen and the reference mirror originate from the splitting of a beam emitted by the same light source, these waves are mutually coherent, and consequently a two-beam interference pattern is obtained.
– If the two beam reinforce each other (where the optical-path difference between them is equal to or multiple of half the wavelength of the monochromatic light)
– If the two beam interfere with each other (where the optical-path difference does not satisfy the above conditions).
– Contour lines will form from these effects.
Light microscope Contrast, what is it? how is it generated
Contrast – relative differences of intensities of objects within the field.
Contrast is generated by:
1. Absorption - of light (stain)
2. Diffraction - scattering of light
3. Refraction - bending of light between two mediums
– Amplitude Objects: affect light intensity by scattering or absorption (satin absorb light at specific wavelengths)
Amplitude objects are easily discernable since the human eye is sensitive to brightness differences.
– Phase Objects: change the phase of illuminating light but not the intensity
Phase objects are not discernable and must be converted to intensity differences with a phase contracts or differential interference Contrast (DIC) Microscope.
Resolution
Resolution, d, is the minimum distance at which two points are resolvable (appears as two distinct pints) (look at equation)
