Transcription of Introduction to 1 Fluorescence - medicalexposedownloads.com
1 During the past 20 years there has been a remarkablegrowth in the use of Fluorescence in the biological spectroscopy and time-resolved fluorescenceare considered to be primarily research tools in biochem-istry and biophysics. This emphasis has changed, and theuse of Fluorescence has expanded. Fluorescence is now adominant methodology used extensively in biotechnology,flow cytometry, medical diagnostics, DNA sequencing,forensics, and genetic analysis, to name a few. Fluorescencedetection is highly sensitive, and there is no longer the needfor the expense and difficulties of handling radioactive trac-ers for most biochemical measurements. There has beendramatic growth in the use of Fluorescence for cellular andmolecular imaging.
2 Fluorescence imaging can reveal thelocalization and measurements of intracellular molecules,sometimes at the level of single-molecule technology is used by scientists frommany disciplines. This volume describes the principles offluorescence that underlie its uses in the biological andchemical sciences. Throughout the book we have includedexamples that illustrate how the principles are used in dif-ferent PHENOMENA OF FLUORESCENCEL uminescence is the emission of light from any substance,and occurs from electronically excited states. Lumines-cence is formally divided into two categories fluores-cence and phosphorescence depending on the nature ofthe excited state.
3 In excited singlet states, the electron in theexcited orbital is paired (by opposite spin) to the secondelectron in the ground-state orbital. Consequently, return tothe ground state is spin allowed and occurs rapidly by emis-sion of a photon. The emission rates of Fluorescence aretypically 108s 1, so that a typical Fluorescence lifetime isnear 10 ns (10 x 10 9s). As will be described in Chapter 4,the lifetime ( ) of a fluorophore is the average time betweenits excitation and return to the ground state. It is valuable toconsider a 1-ns lifetime within the context of the speed oflight. Light travels 30 cm, or about one foot, in onenanosecond. Many fluorophores display subnanosecondlifetimes.
4 Because of the short timescale of Fluorescence ,measurement of the time-resolved emission requiressophisticated optics and electronics. In spite of the addedcomplexity, time-resolved Fluorescence is widely usedbecause of the increased information available from thedata, as compared with stationary or steady-state measure-ments. Additionally, advances in technology have madetime-resolved measurements easier, even when using is emission of light from triplet excit-ed states, in which the electron in the excited orbital has thesame spin orientation as the ground-state electron. Transi-tions to the ground state are forbidden and the emissionrates are slow (103to 100s 1), so that phosphorescence life-times are typically milliseconds to seconds.
5 Even longerlifetimes are possible, as is seen from "glow-in-the-dark"toys. Following exposure to light, the phosphorescence sub-stances glow for several minutes while the excited phospho-rs slowly return to the ground state. Phosphorescence isusually not seen in fluid solutions at room temperature. Thisis because there exist many deactivation processes thatcompete with emission, such as non-radiative decay andquenching processes. It should be noted that the distinctionbetween Fluorescence and phosphorescence is not alwaysclear. Transition metal ligand complexes (MLCs), whichcontain a metal and one or more organic ligands, displaymixed singlet triplet states. These MLCs display interme-diate lifetimes of hundreds of nanoseconds to severalmicroseconds.
6 In this book we will concentrate mainly onthe more rapid phenomenon of typically occurs from aromatic mole-cules. Some typical fluorescent substances (fluorophores)are shown in Figure One widely encountered fluo-rophore is quinine, which is present in tonic water. If oneobserves a glass of tonic water that is exposed to sunlight, afaint blue glow is frequently visible at the surface. Thisglow is most apparent when the glass is observed at a right1 Introduction toFluorescence1angle relative to the direction of the sunlight, and when thedielectric constant is decreased by adding less polar sol-vents like alcohols. The quinine in tonic water is excited bythe ultraviolet light from the sun.
7 Upon return to the groundstate the quinine emits blue light with a wavelength near450 nm. The first observation of Fluorescence from a qui-nine solution in sunlight was reported by Sir John FrederickWilliam Herschel (Figure ) in following is anexcerpt from this early report:On a case of superficial colour presented by a homo-geneous liquid internally colourless. By Sir JohnFrederick William Herschel, Philosophical Translationof the Royal Society of London (1845) 135:143 January 28, 1845 Read February 13, 1845."The sulphate of quinine is well known to be ofextremely sparing solubility in water. It is howevereasily and copiously soluble in tartaric acid.
8 Equalweights of the sulphate and of crystallised tartaricacid, rubbed up together with addition of a very littlewater, dissolve entirely and immediately. It is thissolution, largely diluted, which exhibits the opticalphenomenon in question. Though perfectly transparentand colourless when held between the eye and thelight, or a white object, it yet exhibits in certainaspects, and under certain incidences of the light, anextremely vivid and beautiful celestial blue colour,which, from the circumstances of its occurrence,would seem to originate in those strata which the lightfirst penetrates in entering the liquid, and which, if notstrictly superficial, at least exert their peculiar powerof analysing the incident rays and dispersing thosewhich compose the tint in question, only through avery small depth within the see the colour in question to advantage.
9 Allthat is requisite is to dissolve the two ingredientsabove mentioned in equal proportions, in about ahundred times their joint weight of water, andhaving filtered the solution, pour it into a tall nar-row cylindrical glass vessel or test tube, which isto be set upright on a dark coloured substancebefore an open window exposed to strong day-light or sunshine, but with no cross lights, or anystrong reflected light from behind. If we lookdown perpendicularly into the vessel so that thevisual ray shall graze the internal surface of theglass through a great part of its depth, the wholeof that surface of the liquid on which the lightfirst strikes will appear of a lively blue.
10 If the liquid be poured out into another vessel,the descending stream gleams internally from all2 Introduction TO FLUORESCENCEF igure Structures of typical fluorescent Sir John Fredrich William Herschel, March 7, 1792 toMay 11, 1871. Reproduced courtesy of the Library and InformationCentre, Royal Society of undulating inequalities, with the same livelyyet delicate blue colour, .. thus clearly demon-strating that contact with a denser medium has noshare in producing this singular thinnest film of the liquid seems quite aseffective in producing this superficial colour as aconsiderable thickness. For instance, if in pour-ing it from one glass into another, .. the end ofthe funnel be made to touch the internal surfaceof the vessel well moistened, so as to spread thedescending stream over an extensive surface, theintensity of the colour is such that it is almostimpossible to avoid supposing that we have ahighly coloured liquid under our view.
