A By staining the specimen B By changing how the microscope interacts with the

A by staining the specimen b by changing how the

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A. By staining the specimen B. By changing how the microscope interacts with the light that passes through the specimen C. By setting Köhler illumination D. All of the above
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Problem with stains Specimens prepared with stains usually are killed. Stains may add artifacts to cell structures that don’t normally exist in the live cells.
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The Phase Contrast Microscope to the rescue… Pros: Contrast is produced in apparently transparent cells without killing cells. Cons: Phase contrast condensers and objective lenses add considerable cost to a microscope
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Phase Contrast Microscope Light passing through a transparent part of a specimen travels slower and is shifted compared to the uninfluenced light. This difference in phase can be increased by a phase-plate. This makes the transparent object shine out in contrast to its surroundings.
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Human glial cells grown in culture Brightfield Phase Contrast Phase Contrast Microscopy
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Fluorescence Microscopy
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Fluorescence Excitation Emission For fluorescence to occur , a molecule must be capable of absorbing light of a relatively short exciting wavelength and emitting some of this energy as longer wavelength fluorescent light. We need molecules that can fluoresce! Absorbance
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Another way of staining a cell… A fluorophore is a molecule which has the property of fluorescence. A fluorophore absorbs electromagnetic radiation of some specific wave length and then emits radiation of some slightly longer, lower energy wave length. A fluorophore can mark the location of the structure of interest.
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Fluorescent Labels Fluorophores (or fluorochromes) are used as stains in fluorescent microscopy. These are molecules that “fluoresce”, i.e. they absorb a relatively short wavelength of light, become excited, and decay to the ground state by emitting a slightly longer wavelength of light.
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One way it can lose this energy is to emit a photon of slightly lower energy (i.e., longer wave length)light. DAPI (4',6-diamidino-2-phenylindole) Emitted light 420 nm Absorbed UV 365 nm
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