![]() The fact that a single fluorophore can generate many thousands of detectable photons is fundamental to the high sensitivity of fluorescence detection techniques. Unless the fluorophore is irreversibly destroyed in the excited state (an important phenomenon known as photobleaching, see below), the same fluorophore can be repeatedly excited and detected. The entire fluorescence process is cyclical. In contrast, absorption spectrophotometry requires measurement of transmitted light relative to high incident light levels at the same wavelength. The Stokes shift is fundamental to the sensitivity of fluorescence techniques because it allows emission photons to be detected against a low background, isolated from excitation photons. The difference in energy or wavelength represented by (hν EX – hν EM) is called the Stokes shift. Due to energy dissipation during the excited-state lifetime, the energy of this photon is lower, and therefore of longer wavelength, than the excitation photon hν EX. The fluorescence quantum yield, which is the ratio of the number of fluorescence photons emitted (Stage 3) to the number of photons absorbed (Stage 1), is a measure of the relative extent to which these processes occur.Ī photon of energy hν EM is emitted, returning the fluorophore to its ground state S 0. Other processes such as collisional quenching, fluorescence resonance energy transfer (FRET) ( Fluorescence Resonance Energy Transfer (FRET)-Note 1.2) and intersystem crossing (see below) may also depopulate S 1. Second, not all the molecules initially excited by absorption (Stage 1) return to the ground state (S 0) by fluorescence emission. First, the energy of S 1' is partially dissipated, yielding a relaxed singlet excited state (S 1) from which fluorescence emission originates. These processes have two important consequences. During this time, the fluorophore undergoes conformational changes and is also subject to a multitude of possible interactions with its molecular environment. The excited state exists for a finite time (typically 1–10 nanoseconds). This process distinguishes fluorescence from chemiluminescence, in which the excited state is populated by a chemical reaction. The Molecular Probes Handbook, 11th Edition DownloadĪ photon of energy hν EX is supplied by an external source such as an incandescent lamp or a laser and absorbed by the fluorophore, creating an excited electronic singlet state (S 1').Antifades and Other Tools for Fluorescence Applications-Chapter 23.Probes for Membrane Potential-Chapter 22.Indicators for Na+, K+, Cl– and Miscellaneous Ions-Chapter 21.Indicators for Ca2+, Mg2+, Zn2+ and Other Metal Ions-Chapter 19.Probes for Reactive Oxygen Species, Including Nitric Oxide-Chapter 18.Probes for Signal Transduction-Chapter 17.Probes for Endocytosis, Receptors and Ion Channels-Chapter 16.Assays for Cell Viability, Proliferation and Function-Chapter 15.Fluorescent Tracers of Cell Morphology and Fluid Flow-Chapter 14.Probes for Lipids and Membranes-Chapter 13.Probes for Cytoskeletal Proteins-Chapter 11.Enzyme Substrates and Assays-Chapter 10.Protein Detection and Analysis-Chapter 9.Nucleic Acid Detection and Analysis-Chapter 8. ![]() Antibodies, Avidins and Lectins-Chapter 7.Ultrasensitive Detection Technology-Chapter 6.Crosslinking and Photoactivatable Reagents-Chapter 5.Biotin and Hapten Derivatives-Chapter 4.Click Chemistry and Other Functional Group Modifications-Chapter 3.Fluorophores and Their Amine-Reactive Derivatives-Chapter 1.Chapter 23-Antifades and Other Tools for Fluorescence Applications.Chapter 22-Probes for Membrane Potential.Chapter 21-Indicators for Na+, K+, Cl– and Miscellaneous Ions.Chapter 19-Indicators for Ca2+, Mg2+, Zn2+ and Other Metal Ions.Chapter 18-Probes for Reactive Oxygen Species, Including Nitric Oxide.Chapter 17-Probes for Signal Transduction.Chapter 16-Probes for Endocytosis, Receptors and Ion Channels.Chapter 15-Assays for Cell Viability, Proliferation and Function.Chapter 14-Fluorescent Tracers of Cell Morphology and Fluid Flow.Chapter 13-Probes for Lipids and Membranes.Chapter 11-Probes for Cytoskeletal Proteins.Chapter 10-Enzyme Substrates and Assays.Chapter 9-Protein Detection and Analysis.Chapter 8-Nucleic Acid Detection and Analysis.Chapter 7-Antibodies, Avidins and Lectins.Chapter 6-Ultrasensitive Detection Technology.Chapter 5-Crosslinking and Photoactivatable Reagents.Chapter 4-Biotin and Hapten Derivatives.Chapter 3-Click Chemistry and other Functional Group Modifications.Chapter 1-Fluorophores and Their Amine-Reactive Derivatives.
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |