Polarized light microscopy Polarized light microscopy uses plane-polarized light to analyze structures that are birefringent; unisotropic structures that have two different refractive indices, and capable of splitting a light wave into two unequally reflected or transmitted waves (e.g. cellulose microfibrils). Normal, un-polarized, light can be thought of as many waves, each oscillating at any one of an infinite number of orientations (planes) around the central axis. Plane-polarized light, produced by a polarizer, only oscillates in one plane because the polar only transmits light in that plane. The polarized light microscope must be equipped with both a polarizer, an optical filter that passes light of a specific polarization (oscillating plane) and blocks waves of other polarizations, positioned in the light path somewhere before the specimen, and an analyzer (a second polarizer), placed in the optical pathway after the objective. Image contrast arises from the interaction of plane-polarized light with a birefringent (or doubly-refracting) specimen to produce two individual 0
wave components that are each polarized in mutually perpendicular planes. The velocities of these components are different and vary with the propagation direction through the specimen. After exiting the specimen, the light components are recombined when they pass through the analyzer. Fluorescent Microscopy A fluorescence microscope is an optical microscope that uses fluorescence and phosphorescence instead of, or in addition to, reflection and absorption to study properties of organic or inorganic substances. In most cases the sample of interest is labeled with a fluorescent substance known as a fluorophore and then illuminated through the lens with the higher energy source. The illumination light is absorbed by the fluorophores (now attached to the sample) and causes them to emit a longer lower energy wavelength light. This fluorescent light can be separated from the surrounding radiation with filters designed for that 1
specific wavelength allowing the viewer to see only that which is fluorescing. Principle The basic task of the fluorescence microscope is to let excitation light radiate the specimen and then sort out the much weaker emitted light from the image. First, the microscope has a filter that only lets through radiation with the specific wavelength that matches your fluorescing material. The radiation interferes with the atoms in your specimen and electrons are excited to a higher energy level. When they relax to a lower level, they emit light. To become detectable (visible to the human eye) the fluorescence emitted from the sample is separated from the much brighter excitation light in a second filter. This works because the emitted light is of lower energy and has a longer wavelength than the light that is used for illumination. The majority of fluorescence microscopes, especially those used in the life sciences are of the epifluorescence design.light of the excitation wavelength is focused on the specimen through the objective lens. The fluorescence emitted by the specimen is focused to the detector by the same objective that is used for the excitation which for greatest sensitivity will have a very high numerical aperture. Since most of the excitation light is transmitted through the specimen, only reflected excitatory light reaches the objective together with the emitted light. An additional barrier filter between the objective and the detector can filter out the remaining excitation light from fluorescent light. 2
Applications: The refinement of epi-fluorescent microscopes and advent of more powerful focused light sources, such as lasers, has led to more technically advanced scopes such as the confocal laser scanning microscopes (CLSM) and total internal reflection fluorescence microscopes (TIRF). CLSM's are invaluable tools for producing high resolution 3-D images of subsurfaces in specimens such as microbes. Their advantage is that they are able to produce sharp images of thick samples at various depths by taking images point by point and reconstructing them with a computer These microscopes are often used for - Imaging structural components of small specimens, such as cells 3
Conducting viability studies on cell populations (are they alive or dead?) Imaging the genetic material within a cell (DNA and RNA) Viewing specific cells within a larger population with techniques such as FISH 4