03Fluorescence - Lab 3 Fluorescence Microscopy Objective...

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Lab 3 Fluorescence Microscopy
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Objective for this lab… • Prepare slides to observe nucleic acids with direct fluorescence microscopy using DAPI on onion root tip cells & propidium iodide on cheek epithelial cells. • Compare the visualization of L8 cells under phase-contrast and those stained with DAPI and viewed under fluorescence microscopy.
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• Prepare and observe L8 cells double- stained. DAPI for their nuclear material and rhodamine phallodin for actin. • Prepare and observe L8 cells double- stained. DAPI for their nuclear material and indirect immunofluorescence to stain for tubulin. • Observe epithelial cells triple-stained for nuclear material, and the proteins tubulin and actin.
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Another way of staining a cell… • Attachment of a fluorophore to the molecules making up the structure of interest. • fluorophore is a molecule which has the property of fluorescence. • Absorbs electromagnetic radiation of some specific wave length and then emits radiation of some slightly longer, lower energy wave length.
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Recall! • Electrons of atoms are confined to orbitals of specific energy levels. • Usually, lower energy inner orbitals are occupied while the outer higher energy orbitals are unoccupied (ground state). • When photons of correct wavelength strike a molecule in ground state, it can transfer its energy to the electron and boost electron to a higher energy level (excited state).
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• Excited molecule usually doesn’t stay excited very long. It loses energy it has acquired. • One way it can lose this energy is to emit a photon of slightly lower energy (i.e., longer wave length)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.
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03Fluorescence - Lab 3 Fluorescence Microscopy Objective...

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