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Cell Biology Lecture No. 2.docx

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Biology 2382B
Robert Cumming

Cell Biology Lecture No. 2: Imaging In Cell Biology th Monday January 14 , 2013 Anton Van Leeuwenhoek: • Anton van Leeuwenhoek was the first person to construct a microscope adequate enough to observe live cells, back when people didn’t even know cells existed. • His microscope was simplistic, but shares many features of modern microscopes. • He would set drop of cell culture on the pin of the microscope and examine live cells in sunlight by moving the stage closer or further away from view. • The reason for Leeuwenhoek’s success at protozoa and bacteria (“animalcules”) as well as erythrocytes and sperm cells was due to o His skill at grinding lenses (maximizing the lens’ magnification and resolution) o And his naturally acute eyesight (achieving a total magnification of 200x). Features Of A Modern Compound Microscope: • As opposed to Leeuwenhoek’s single lens microscope, a compound microscope is a light microscope using two or more lenses (increasing the magnification properties). • The condenser helps to focus the light from the light source on to a specimen, which is usually mounted on a glass coverslip or microscope slide. • As the light passes through that specimen, it is collected and focused via an objective lens (a magnifying lens). • Another magnifying lens is located in the eyepiece or the ocular. • The resultant image of the specimen is magnified 100x at the objective stage (maximum in light microscopy) and 10x at the ocular stage, giving a total 1000x magnification to the observer. • -*The ability to see a specimen is really dependent on the properties of the specimen itself and the refractive index observed. As light refraction is the ability to bend light (or slow it down), only structures with a high refractive index are observable. Resolution Of Microscopes: • Magnification of a specimen is meaningless without the ability to observe detail, which is dependent upon resolution. o Resolution is the ability to distinguish between two very closely positioned objects as separate entities. The maximum resolution of various lenses include: the naked eye (0.2mm), light microscopes (0.2 μm), electron microscopes (0.1 nm). o A conventional microscope can never resolve objects or cellular features that are less than ~0.2 microns apart. o Thus, *smaller resolution is more optimal for observing better detail. • -Resolution (D) can be calculated in the following formula: o D = 0.61 λ / N sin α o where D is the distance resolved between 2 points, λ is the wavelength of light used, N is the refractive index of medium between the specimen and the objective lens, α is the 1/2 angle of light entering the objective, and N sin α is the numerical aperture. o In order to minimize (optimize) resolution, we can increase the refractive index of the medium (using oil or water instead of air), decrease the wavelength, or more importantly, increase the angle of light entering the objective (making objective really close to the specimen). This limits the resolution to about 0.2 microns. Obtaining Contrast In Light Microscopy: • There is inherent variability in the refractive properties of certain cellular structures. • Cellular constituents (oluşturan parçalardan her biri) with high refractive properties can slow the passage of a light beam by a quarter wavelength at most. • To get the most contrast (observe dark areas of the cell), o we need the light to dim in that part of the cell and have very low peaks and troughs (bright light is indicative of very high peaks and troughs). • So when the two wavelengths recombine at the objective lens, there will be that shows contrast, but not completely as light microscopy is limited by ¼ wavelength maximum refraction. Phase Contrast Microscopy: • Phase contrast microscopy limits the amount of light that passes through the specimen as opposed to bright field microscopy. • It effectively focuses a cone of light through the specimen (slowing the wavelength down by a quarter) as well as past a phase plate that has inner coating that slows down the wavelength of light down by another quarter. • Cumulatively, the light is slowed down by a half (the wavelengths cancel each other out when they recombine) and dim light is produced. • Live, unstained cells are now given further detail by the dimmer features. Differential Interference Contrast (DIC)/Normarski Microscopy: • Differential Interference Contrast (DIC) microscopy takes advantage of the polarity of light (different angles) and not the phases of light wavelengths as in phase contrast. • By using different polarizers that allow specific planes of light through (horizontal or vertical), DIC microscopy recombines the different polar wavelengths of light to provide a different degree of definition that shows the three-dimensional shape (im portant in outlining large organelles like the nucleus and the vacuole). • This is different to phase microscopy, which produces a halo effect on observed cells. • In summary, phase contrast and DIC are two forms of light microscopy used to generate good resolution. Fluorescence Microscopy: • Fluorescence microscopy uses certain chemicals that will emit light when they absorb a certain wavelength of light like invisible UV light. • The use of these chemical dyes can be located on specific cellular structures and can be imaged according to the colour of light they emit. • A fluorophore is chemical that when it’s outer electrons are excited by a certain wavelength of light, they consecutively return to their original ground state, emitting a certain wavelength of light in doing so. • Fluorophores have an excitation wavelength (which excites the fluorophore) and an emission wavelength (which is what the excited fluorophore emits). • The difference between the optimal and excitation wavelengths is known as the Stokes shift. Obtaining Fluorescent Images: • Fluorescence microscopy can be used by the same microscopes used for light micro
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