BME 210 Lecture 3 Fluorescence Microscopy

BME 210 Lecture 3 Fluorescence Microscopy - 3. Fluorescence...

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3. Fluorescence Microscopy Fluorescence absorption of light of one wavelength followed by emission of light with a longer wavelength Fluorophores – molecules exhibiting fluorescence effect
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Fluorophore absorption and emission spectra Absorption Emission wavelength Stokes’ shift ex em Δ λ = λ em - λ ex - Stokes shift
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Mechanism of fluorescence Fluorescence is a quantum-mechanical effect From the quantum mechanics: 1. Energy of a molecule is discrete (quantized) 2. Energy of light is quantized: Quantum of energy – photon Photon energy E = hν = hc/λ 3. Energy is exchanged between light and molecules in discrete portions (photons)
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Mechanism of fluorescence S o S 1 1 3 2 Molecule Energy ground electronic state excited electronic state vibrational levels vibrational levels hv em < hv ex λ em > λ ex Fluorescence steps: 1. Excitation: absorption of a photon with energy h v ex 2. Relaxation: dissipation of part of the energy 3. Emission: return to one of the ground states with emission of a photon with energy h v em Energy states and transitions in a fluorophore molecule (Jablonski diagram) hv ex hv em
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Properties of fluorescence Fluorescence is highly specific: absorption and emission spectra strongly depend on molecule structure Different molecules can be distinguished by their spectra Fluorescence is highly sensitive to the environment: standard fluorophore molecules can be linked to bio-molecules reporting their presence and changes in their state
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Fluorescence microscope The standard transmission microscope + two optical filters: 1. Excitation filter 2. Emission filter This design is never used in biology because the emission filter doesn’t block the excitation light (green) completely; the small part, which goes through, is still brighter than the fluorescence (red) Excitation filter Emission filter Light source Objective lens Condenser lens Detector Object Simple design
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This note was uploaded on 04/28/2010 for the course BME eng. biolo taught by Professor Fast during the Spring '10 term at University of Alabama at Birmingham.

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BME 210 Lecture 3 Fluorescence Microscopy - 3. Fluorescence...

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