Introduction to Fluorescence Techniques, Lecture notes of Chemistry

Download Introduction to Fluorescence Techniques and more Lecture notes Chemistry in PDF only on Docsity! Introduction to Fluorescence Techniques 3 Figure 1 Jablonski diagram illustrating the processes involved in the creation of an excited electronic singlet state by optical absorption and subsequent emission of uorescence. The labeled stages 1, 2 and 3 are explained in the adjoining text. S0 E ne rg y S1′ S1 hνEM 2 3hνEX 1 Fluorescent probes enable researchers to detect particular com- ponents of complex biomolecular assemblies, such as live cells, with exquisite sensitivity and selectivity. e purpose of this introduction is to briey outline uorescence principles and techniques for newcomers to the eld. The Fluorescence Process Fluorescence is the result of a three-stage process that occurs in certain molecules (generally polyaromatic hydrocarbons or hetero- cycles) called uorophores or uorescent dyes. A uorescent probe is a uorophore designed to respond to a specic stimulus or to localize within a specic region of a biological specimen. e process respon- sible for the uorescence of uorescent probes and other uorophores is illustrated by the simple electronic-state diagram (Jablonski diagram) shown in Figure 1. Stage 1: Excitation A photon of energy hνEX is supplied by an external source such as an incandescent lamp or a laser and absorbed by the uorophore, creat- ing an excited electronic singlet state (S1́ ). is process distinguishes uorescence from chemiluminescence, in which the excited state is populated by a chemical reaction. Stage 2: Excited-State Lifetime e excited state exists for a nite time (typically 1–10 nano- seconds). During this time, the uorophore undergoes conformational changes and is also subject to a multitude of possible interactions with its molecular environment. ese processes have two important con- sequences. First, the energy of S1́ is partially dissipated, yielding a re- laxed singlet excited state (S1) from which uorescence emission origi- nates. Second, not all the molecules initially excited by absorption (Stage 1) return to the ground state (S0) by uorescence emission. Other process- es such as collisional quenching, uorescence resonance energy trans- fer (FRET) (Fluorescence Resonance Energy Transfer (FRET)—Note 1.2) and intersystem crossing may also depopulate S1. e uorescence Introduction to Fluorescence Techniques quantum yield, which is the ratio of the number of uorescence photons emitted (Stage 3) to the number of photons absorbed (Stage 1), is a mea- sure of the relative extent to which these processes occur. Stage 3: Fluorescence Emission A photon of energy hνEM is emitted, returning the uorophore to its ground state S0. Due to energy dissipation during the excited-state life- time, the energy of this photon is lower, and therefore of longer wave- length, than the excitation photon hνEX. e dierence in energy or wavelength represented by (hνEX – hνEM) is called the Stokes shi. e Stokes shi is fundamental to the sensitivity of uorescence techniques because it allows emission photons to be detected against a low back- ground, isolated from excitation photons. In contrast, absorption spec- trophotometry requires measurement of transmitted light relative to high incident light levels at the same wavelength. Fluorescence Spectra e entire uorescence process is cyclical. Unless the uorophore is irreversibly destroyed in the excited state (an important phenomenon known as photobleaching), the same uorophore can be repeatedly ex- cited and detected. e fact that a single uorophore can generate many thousands of detectable photons is fundamental to the high sensitiv- ity of uorescence detection techniques. For polyatomic molecules in solution, the discrete electronic transitions represented by hνEX and hνEM in Figure 1 are replaced by rather broad energy spectra called the uorescence excitation spectrum and uorescence emission spectrum, respectively (Table 1). e bandwidths of these spectra are parameters of particular importance for applications in which two or more dier- ent uorophores are simultaneously detected . e uorescence excita- tion spectrum of a single uorophore species in dilute solution is usu- ally identical to its absorption spectrum. e absorption spectrum can therefore be used as a surrogate excitation spectrum data set. Under the same conditions, the uorescence emission spectrum is independent of the excitation wavelength, due to the partial dissipation of excitation energy during the excited-state lifetime, as illustrated in Figure 1. e emission intensity is proportional to the amplitude of the uorescence excitation spectrum at the excitation wavelength (Figure 2). Figure 2 Excitation of a uorophore at three dierent wavelengths (EX 1, EX 2, EX 3) does not change the emission prole but does produce variations in uorescence emission inten- sity (EM 1, EM 2, EM 3) that correspond to the amplitude of the excitation spectrum. Excitation spectrum Emission spectrum EX 3 EX 1 EX 2 EM 3 EM 1 EM 2 Wavelength Fl uo re sc en ce e xc ita tio n Fl uo re sc en ce e m is si on Introduction to Fluorescence Techniques 4 www.invitrogen.com/probes IMPORTANT NOTICE: The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. The Molecular Probes® Handbook: A Guide to Fluorescent Probes and Labeling Technologies Fluorescence Detection Fluorescence Instrumentation Four essential elements of uorescence detection systems can be identied from the preceding discussion: 1) an excitation source, 2) a uorophore, 3) wavelength lters to isolate emission photons from excitation photons, 4) a detector that registers emission photons and produces a recordable output, usually as an electrical signal. Regardless of the application, compatibility of these four elements is essential for optimizing uorescence detection. Fluorescence instruments are primarily of four types, each provid- ing distinctly dierent information: • Spectrouorometers and microplate readers measure the average properties of bulk (µL to mL) samples. • Fluorescence microscopes resolve uorescence as a function of spa- tial coordinates in two or three dimensions for microscopic objects (less than ~0.1 mm diameter). • Fluorescence scanners, including microarray readers, resolve uo- rescence as a function of spatial coordinates in two dimensions for macroscopic objects such as electrophoresis gels, blots and chromatograms. • Flow cytometers measure uorescence per cell in a owing stream, allowing subpopulations within a large sample to be identied and quantitated. Other types of instrumentation that use uorescence detection include capillary electrophoresis apparatus, DNA sequencers 1 and microuidic devices.2,3 Each type of instrument produces dierent Table 1 Spectroscopic proterties of uorescent dyes. Property Denition Signicance Fluorescence excitation spectrum * An X,Y plot of excitation wavelength versus number of uorescence photons generated by a uorophore. Optimum instrument setup should deliver excitation light as close to the peak of the excitation spectrum of the uorophore as possible. Absorption spectrum An X,Y plot of wavelength versus absorbance of a chromophore or uorophore. To a rst approximation, the absorption spectrum of a uorophore is equivalent to the uorescence excitation spectrum.† To the extent that this approximation holds, the absorption spectrum can be used as a surrogate for the uorescence excitation spectrum. Fluorescence emission spectrum * An X,Y plot of emission wavelength versus number of uorescence photons generated by a uorophore. Fluorescence emission spectral discrimination is the most straightforward basis for multiplex detection ‡ and for resolving probe uorescence from background autouorescence. Extinction coecient (EC) Capacity for light absorption at a specic wavelength.§ Fluorescence output per uorophore (“brightness”) is proportional to the product of the extinction coecient (at the relevant excitation wavelength) and the uorescence quantum yield. Fluorescence quantum yield (QY) Number of uorescence photons emitted per excitation photon absorbed. See “Extinction coecient.” Quenching Loss of uorescence signal due to short-range interactions between the uorophore and the local molecular environment, including other uorophores (self-quenching). Loss of uorescence is reversible to the extent that the causative molecular interactions can be controlled.** Photobleaching Destruction of the excited uorophore due to photosensitized generation of reactive oxygen species (ROS), particularly singlet oxygen (1O2). Loss of uorescence signal is irreversible if the bleached uorophore population is not replenished (e.g., via diusion). Extent of photobleaching is dependent on the duration and intensity of exposure to excitation light. * Our online Fluorescence SpectraViewer (www.invitrogen.com/handbook/spectraviewer) provides an interactive utility for plotting and comparing uorescence excitation and emission spectra for over 250 uorophores (Using the Fluorescence SpectraViewer—Note 23.1). † Generally true for single uorophore species in homogeneous solutions but not in more complex heterogeneous samples. ‡ Multiplex detection refers to the process of simultaneously labeling a specimen with two or more uorescent probes to allow correlation of multiple structural or functional features. As well as specic association with their targets, the probes must have distinctive spectroscopic properties that can be discriminated by the detection instrument. § EC (units: cm–1 M–1) is dened by the Beer-Lambert law A = EC•c•l, where A = absorbance, c = molar concentration, l = optical pathlength.  EC values at the absorption maximum wavelength are listed in the Section Data Tables throughout The Molecular Probes® Handbook. ** In the case of self-quenching, this can be accomplished by disruption of uorophore compartmentalization, denaturation or fragmentation of biopolymer conjugates, or functionalization of the uorophore to produce increased electrostatic repulsion and water solubility. measurement artifacts and makes dierent demands on the uores- cent probe. For example, although photobleaching is oen a signicant problem in uorescence microscopy, it is not a major impediment in ow cytometry because the dwell time of individual cells in the excita- tion beam is short. Fluorescence Signals Fluorescence intensity is quantitatively dependent on the same pa- rameters as absorbance—dened by the Beer–Lambert law as the prod- uct of the molar extinction coecient, optical path length and solute concentration—as well as on the uorescence quantum yield of the dye and the excitation source intensity and uorescence collection ecien- cy of the instrument (Table 1). In dilute solutions or suspensions, uo- rescence intensity is linearly proportional to these parameters. When sample absorbance exceeds about 0.05 in a 1 cm pathlength, the rela- tionship becomes nonlinear and measurements may be distorted by artifacts such as self-absorption and the inner-lter eect.4,5 Because uorescence quantitation is dependent on the instrument, uorescent reference standards are essential for calibrating measure- ments made at dierent times or using dierent instrument congura- tions.6–8 To meet these requirements, we oer high-precision uores- cent microsphere reference standards for uorescence microscopy and ow cytometry and a set of ready-made uorescent standard solutions for spectrouorometry (Section 23.1, Section 23.2). A spectrouorometer is extremely exible, providing continuous ranges of excitation and emission wavelengths. Laser-scanning micro- scopes and ow cytometers, however, require probes that are excitable at a single xed wavelength. In contemporary instruments, the excita- tion source is usually the 488 nm spectral line of the argon-ion laser. As The Molecular Probes™ Handbook: A uide to Fluorescent Probes and Labeling Technologies IMPORTANT NOTICE : The products described in this manual are covered by o e or more Limited Use L bel License(s). Please refer to the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. thermofisher.com/probes Introduction to Fluorescence Techniques 7IMPORTANT NOTICE: The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. www.invitrogen.com/probes The Molecular Probes® Handbook: A Guide to Fluorescent Probes and Labeling Technologies Fluorophore–Fluorophore Interactions Fluorescence quenching can be dened as a bimolecular process that reduces the uores- cence quantum yield without changing the uorescence emission spectrum (Table 1); it can result from transient excited-state interactions (collisional quenching) or from formation of nonuorescent ground-state species. Self-quenching is the quenching of one uorophore by an- other; 34 it therefore tends to occur when high loading concentrations or labeling densities are used (Figure 7, Figure 8). DQ™ substrates (Section 10.4) are heavily labeled and therefore highly quenched biopolymers that exhibit dramatic uorescence enhancement upon enzymatic cleav- age 35 (Figure 9). Fluorescence resonance energy transfer (FRET) (Fluorescence Resonance Energy Transfer (FRET)—Note 1.2) is a strongly distance-dependent excited-state interaction in which emission of one uorophore is coupled to the excitation of another. Some excited uorophores interact to form excimers, which are excited-state dimers that exhibit altered emission spectra. Excimer formation by the polyaromatic hydrocarbon pyrene is described in Section 13.2 (Figure 10). Because they all depend on the interaction of adjacent uorophores, self-quenching, FRET and excimer formation can be exploited for monitoring a wide array of molecular assembly or fragmentation processes such as membrane fusion (Assays of Volume Change, Membrane Fusion and Membrane Permeability—Note 14.3), nucleic acid hybridization, ligand–receptor binding and polypeptide hydrolysis. Other Environmental Factors Many other environmental factors exert inuences on uorescence properties. e three most common are: • Solvent polarity (solvent in this context includes interior regions of cells, proteins, mem- branes and other biomolecular structures) • Proximity and concentrations of quenching species • pH of the aqueous medium Fluorescence spectra may be strongly dependent on solvent. is characteristic is most oen observed with uorophores that have large excited-state dipole moments, resulting in uores- cence spectral shis to longer wavelengths in polar solvents. Representative uorophores include the aminonaphthalenes such as prodan, badan (Figure 11) and dansyl, which are eective probes of environmental polarity in, for example, a protein’s interior.36 Binding of a probe to its target can dramatically aect its uorescence quantum yield (Monitoring Protein-Folding Processes with Environment-Sensitive Dyes—Note 9.1). Probes Figure 7 Comparison of relative uorescence as a func- tion of the number of uorophores attached per protein for goat anti–mouse IgG antibody conjugates prepared using Oregon Green® 514 carboxylic acid succinimidyl ester (O6139, j), Oregon Green® 488 carboxylic acid suc- cinimidyl ester (O6147, d), uorescein-5-EX succinimidyl ester (F6130, s) and uorescein isothiocyanate (FITC; F143, F1906, F1907; h). Conjugate uorescence is determined by measuring the uorescence quantum yield of the conjugat- ed dye relative to that of the free dye and multiplying by the number of uorophores per protein. 0 2 4 6 8 10 12 14 16 Fluorescein-EX FITC Oregon Green® 488 Fluorophores/protein (mol:mol) C on ju ga te  uo re sc en ce Oregon Green® 514 Figure 8 Comparison of the relative uorescence of goat anti–mouse IgG antibody conjugates of Rhodamine Red™-X succinimidyl ester (R6160, d) and Lissamine rhodamine B sulfonyl chloride (L20, L1908; s). Conjugate uorescence is determined by measuring the uorescence quantum yield of the conjugated dye relative to that of the free dye and multiplying by the number of uorophores per pro- tein. Higher numbers of uorophores attached per protein are attainable with Rhodamine Red™-X dye due to the lesser tendency of this dye to induce protein precipitation. Fluorophores/protein (mol:mol) Rhodamine Red™-X Lissamine™ rhodamine B C on ju ga te  uo re sc en ce 0 1 2 3 4 5 Figure 10 Excimer formation by pyrene in ethanol. Spectra are normalized to the 371.5 nm peak of the monomer. All spectra are essentially identical below 400 nm after normal- ization. Spectra are as follows: 1) 2 mM pyrene, purged with argon to remove oxygen; 2) 2 mM pyrene, air-equilibrated; 3) 0.5 mM pyrene (argon-purged); and 4) 2 µM pyrene (argon- purged). The monomer-to-excimer ratio (371.5/470 nm) is de- pendent on both pyrene concentration and the excited-state lifetime, which is variable because of quenching by oxygen. Fl uo re sc en ce e m is si on Wavelength (nm) 350 450400 500 550 600 1 2 3 4 Figure 11 Fluorescence emission spectra of the 2-mercapto- ethanol adduct of badan (B6057) in: 1) toluene, 2) chloroform, 3) acetonitrile, 4) ethanol, 5) methanol and 6) water. Each solu- tion contains the same concentration of the adduct. Excitation of all samples is at 380 nm. Fl uo re sc en ce e m is si on Wavelength (nm) 400 500 600 1 2 3 4 5 6 Ex = 380 nm 450 550 Figure 9 Principle of enzyme detection via the disruption of intramolecular self-quenching. Enzyme-catalyzed hy- drolysis of the heavily labeled and almost totally quenched substrates provided in the EnzChek® Assay Kits relieves the intramolecular self-quenching, yielding brightly uorescent reaction products. Intramolecularly quenched substrate Enzyme Fluorescent cleavage products The Molecular Probes™ Handbook: A Guide to Fluorescent Probes and Labeling Technologies IMPORTANT NOTICE : The products described in this manual are cove d by one or more Limited Use Label License(s). Please ref to the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. thermofisher.com/probes Introduction to Fluorescence Techniques 8 www.invitrogen.com/probes IMPORTANT NOTICE: The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. The Molecular Probes® Handbook: A Guide to Fluorescent Probes and Labeling Technologies that have a high uorescence quantum yield when bound to a particular target but are otherwise eectively nonuorescent yield extremely low reagent background signals. e ultrasensitive SYBR®, SYTO®, PicoGreen®, RiboGreen® and OliGreen® nucleic acid stains (Chapter 8) are prime examples of this strategy. Similarly, uoro- genic enzyme substrates, which are nonuorescent or have only short-wavelength emission until they are converted to uorescent products by enzymatic cleavage, allow sensitive detection of enzymatic activity (Chapter 10). Extrinsic quenchers, the most ubiquitous of which are paramagnetic species such as O2 and heavy atoms such as iodide, reduce uorescence quantum yields in a concentration-dependent manner. If quenching is caused by collisional interac- tions, as is usually the case, information on the proximity of the uorophore and quencher and their mutual diusion rate can be derived. is quenching eect has been used productively to measure chloride-ion ux in cells (Section 21.2). Many uorophores are also quenched by proteins. Examples are NBD, uorescein and BODIPY® dyes, in which the eect is apparently due to charge-transfer interac- tions with aromatic amino acid residues.37,38 Consequently, antibodies raised against these uorophores are eective and highly specic uorescence quench- ers 38 (Section 7.4). Fluorophores such as BCECF and carboxy SNARF®-1 that have strongly pH-de- pendent absorption and uorescence characteristics can be used as physiological pH indicators. Fluorescein and hydroxycoumarins (umbelliferones) are further exam- ples of this type of uorophore. Structurally, pH sensitivity is due to a recongura- tion of the uorophore’s π-electron system that occurs upon protonation. BODIPY® FL and Alexa Fluor® 488 uorophores, both of which lack proto lytically ionizable substituents, provide spectrally equivalent alternatives to uorescein for applica- tions requiring a pH-insensitive probe (Section 1.3, Section 1.4). REFERENCES 1. Annu Rev Genomics Hum Genet (2008) 9:387; 2. Science (2007) 315:81; 3. Anal Chem (2008) 80:7063; 4. Anal Chem (2001) 73:2070; 5. Analyst (1994) 119:417; 6. J Microsc (2007) 228:390; 7. J Res Natl Inst Stand Technol (2001) 106:381; 8. Methods Cell Biol (1994) 42B:605; 9. J Microsc (1994) 176:281; 10. Cytometry B Clin Cytom (2009) 76:295; 11. Nat Protoc (2009) 4:372; 12. Nat Rev Immunol (2004) 4:648; 13. J Immunol Methods (2009) 344:6; 14. J Neurosci Methods (2007) 162:119; 15. J Neurosci Methods (2009) 180:116; 16. BMC Cell Biol (2008) 9:13; 17. Nat Protoc (2007) 2:1152; 18. Nat Med (2006) 12:972; 19. Biophys J (1983) 43:383; 20. Methods Cell Biol (2007) 81:415; 21. J Microsc (2009) 233:192; 22. Nat Methods (2008) 5:197; 23. J Phys Chem A (2007) 111:429; 24. Chemphyschem (2008) 9:2019; 25. Nat Methods (2007) 4:81; 26. Org Lett (2004) 6:909; 27. Biophys J (1995) 68:2588; 28. J Cell Biol (1985) 100:1309; 29. J Org Chem (1973) 38:1057; 30. Mol Cell Probes (2008) 22:294; 31. J Histochem Cytochem (2007) 55:545; 32. Exp Cell Res (2007) 313:1943; 33. J Histochem Cytochem (1995) 43:77; 34. ACS Chem Biol (2009) 4:535; 35. Anal Biochem (1997) 251:144; 36. Nature (1986) 319:70; 37. Bioconjug Chem (2003) 14:1133; 38. Immunochemistry (1977) 14:533. Selected Books and Articles e preceding discussion has introduced some general principles to consider when selecting a uorescent probe. Application-specic details are addressed in subsequent chapters of e Molecular Probes® Handbook. For in-depth treatments of uorescence techniques and their biological applications, the reader is referred to the many excellent books and review articles listed below. Principles of Fluorescence Detection Albani, J.R., Absorption et Fluorescence: Principes et Applications, Lavoisier (2001). is book is the rst on absorption and uoresc- nece to be published in the French language. Albani, J.R., Principles and Applications of Fluorescence Spectroscopy, Wiley-Blackwell (2007). Brand, L. and Johnson, M.L., Eds., Fluorescence Spectroscopy (Methods in Enzymology, Volume 450), Academic Press (2008). Gell, C., Brockwell, D. and Smith, A., Handbook of Single Molecule Fluorescence Spectroscopy, Oxford University Press (2006). Goldys, E.M., Ed., Fluorescence Applications in Biotechnology and Life Sciences, Wiley-Blackwell (2009). Guilbault, G.G., Ed., Practical Fluorescence, Second Edition, Marcel Dekker (1990). Joo, C., Balci, H., Ishitsuka, Y., Buranachai, C. and Ha, T., “Advances in single-molecule uorescence methods for molecular biology,” Annu Rev Biochem (2008) 77:51–76. Lakowicz, J.R., Principles of Fluorescence Spectroscopy, ird Edition, Springer (2006). Mathies, R.A., Peck, K. and Stryer, L., "Optimization of high-sensitivity uorescence detection," Anal Chem (1990) 62:1786–1791. Royer, C.A., "Approaches to teaching uorescence spectroscopy," Biophys J (1995) 68:1191–1195. Selvin, P.R. and Ha, T., Eds., Single-Molecule Techniques: A Laboratory Manual, Cold Spring Harbor Laboratory Press (2007). Valeur, B., Molecular Fluorescence: Principles and Applications, John Wiley and Sons (2002). Fluorophores and Fluorescent Probes Berlman, I.B., Handbook of Fluorescence Spectra of Aromatic Molecules, Second Edition, Academic Press (1971). Burry, R.W., Immunocytochemistry: A Practical Guide for Biomedical Research, Springer (2009). Chale, M. and Kain, S.R., Green Fluorescent Protein: Properties, Applications and Protocols, Second Edition, John Wiley and Sons (2006). Drexhage, K.H., "Structure and properties of laser dyes" in Dye Lasers, ird Edition, F.P. Schäfer, Ed., Springer-Verlag, (1990) p. 155–200. Green, F.J., e Sigma-Aldrich Handbook of Stains, Dyes and Indicators, Aldrich Chemical Company (1990). Griths, J., Colour and Constitution of Organic Molecules, Academic Press (1976). The Molecular Probes™ Handbook: A Guide to Fluorescent Probes and Labeling Technologies IMPORTANT NOTICE : The products described in this manual are covered by o e or more Limited Use L bel License(s). Please refer to the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. thermofisher.com/probes Introduction to Fluorescence Techniques 9IMPORTANT NOTICE: The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. www.invitrogen.com/probes The Molecular Probes® Handbook: A Guide to Fluorescent Probes and Labeling Technologies Hermanson, G.T., Bioconjugate Techniques, Second Edition, Academic Press (2008). Hilderbrand, S.A., “Labels and probes for live cell imaging: Overview and selection guide”, Methods Mol Biol (2010) 581:17–45. Kobayashi, H., Ogawa, M., Alford, R., Choyke, P.L. and Urano, Y., “New strategies for uorescent probe design in medical diagnostic imag- ing,” Chem Rev (2010) 110:2620–2640. Kricka, L.J. and Fortina, P., “Analytical ancestry: "Firsts" in uorescent labeling of nucleosides, nucleotides, and nucleic acids,” Clin Chem (2009) 55:670–683. Liehr, T., Ed., Fluorescence In Situ Hybridization (FISH): Application Guide, Springer (2008). Mason, W.T., Ed., Fluorescent and Luminescent Probes for Biological Activity, Second Edition, Academic Press (1999). Oliver, C. and Jamur, M.C., Eds., Immunocytochemical Methods and Protocols, ird Edition (Methods in Molecular Biology, Volume 588), Humana Press (2010). Taraska, J.W. and Zagotta, W.N., “Fluorescence applications in molecu- lar neurobiology”, Neuron (2010) 66:170–189. Fluorescence Microscopy Frigault, M.M., Lacoste, J., Swi, J.L. and Brown, C.M., “Live-cell mi- croscopy: Tips and tools,” J Cell Sci (2009) 122:753–767. Fujimoto, J.G. and Farkas, D., Eds., Biomedical Optical Imaging, Oxford University Press (2009). Goldman, R.D., Swedlow, J.R. and Spector, D.L., Eds., Live Cell Imaging: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (2009). Hell, S.W., “Microscopy and its focal switch,” Nat Methods (2009) 6:24–32. Herman, B., Fluorescence Microscopy, Second Edition, BIOS Scientic Publishers (1998). Hibbs, A.R., Confocal Microscopy for Biologists, Springer (2004). Huang, B., Bates, M. and Zhuang, X., “Super-resolution uorescence microscopy,” Annu Rev Biochem (2009) 78:993–1016. Inoué, S. and Spring, K.R., Video Microscopy, Second Edition, Plenum Publishing (1997). Lichtman, J.W. and Conchello, J.A., “Fluorescence microscopy,” Nat Methods (2005) 2:910–919. Masters, B.R., Confocal Microscopy and Multiphoton Excitation Microscopy: e Genesis of Live Cell Imaging, SPIE Press (2006). Matsumoto, B., Ed., Cell Biological Applications of Confocal Microscopy, Second Edition (Methods in Cell Biology, Volume 70), Academic Press (2003). Murphy, D.B., Fundamentals of Light Microscopy and Electronic Imaging, John Wiley and Sons (2001). Ntziachristos, V., "Fluorescence molecular imaging," Annu Rev Biomed Eng (2006) 8:1–32. Patterson, G., Davidson, M., Manley, S. and Lippincott-Schwartz, J., “Superresolution imaging using single-molecule localization,” Annu Rev Phys Chem (2010) 61:345–367. Pawley, J.B., Ed., Handbook of Biological Confocal Microscopy, ird Edition, Springer (2006). Periasamy, A., Ed., Methods in Cellular Imaging, Oxford University Press (2001). Rosenthal, E. and Zinn, K.R., Eds., Optical Imaging of Cancer: Clinical Applications, Springer (2009). Spector, D.L. and Goldman, R.D., Basic Methods in Microscopy, Cold Spring Harbor Laboratory Press (2005). Svoboda, K. and Yasuda, R., “Principles of two-photon excitation mi- croscopy and its applications to neuroscience,” Neuron (2006) 50:823–839. Tsien, R.Y., " Imagining imaging’s future," Nat Rev Mol Cell Biol (2003) 4:SS16–SS21. Yuste, R. and Konnerth, A., Eds., Imaging in Neuroscience and Development: A Laboratory Manual, Cold Spring Harbor Laboratory Press (2005). Flow Cytometry Darzynkiewicz, Z., Crissman, H.A. and Robinson, J.P., Eds., Cytometry, ird Edition Parts A and B (Methods in Cell Biology, Volumes 63 and 64), Academic Press (2001). Givan, A.L., Flow Cytometry: First Principles, Second Edition, John Wiley and Sons (2001). Herzenberg, L.A., Parks, D., Sahaf, B., Perez, O., Roederer, M. and Herzenberg, L.A., "e history and future of the uorescence ac- tivated cell sorter and ow cytometry: A view from Stanford," Clin Chem (2002) 48:1819–1827. Herzenberg, L.A., Tung, J., Moore, W.A., Herzenberg, L.A. and Parks, D.R., “Interpreting ow cytometry data: A guide for the perplexed,” Nat Immunol (2006) 7:681–685. Preer F. and Dombkowski, D. “Advances in complex multiparameter ow cytometry technology: Applications in stem cell research,” Cytometry B (2009) 76:295–314. Shapiro, H.M., "Optical measurement in cytometry: Light scattering, extinction, absorption and uorescence," Meth Cell Biol (2001) 63:107–129. Shapiro, H.M., Practical Flow Cytometry, Fourth Edition, Wiley-Liss (2003). Sklar, L.A., Ed., Flow Cytometry for Biotechnology, Oxford University Press (2005). Other Fluorescence Measurement Techniques Dorak, M.T., Ed., Real-Time PCR, Taylor and Francis (2006). Gore, M., Ed., Spectrophotometry and Spectrouorimetry: A Practical Approach, Second Edition, Oxford University Press (2000). Mardis, E.R. “Next-generation DNA sequencing methods,” Annu Rev Genomics Hum Genet (2008) 9:387–402. Patton, W.F., "A thousand points of light: e application of uorescence detection technologies to two-dimensional gel electrophoresis and proteomics," Electrophoresis (2000) 21:1123–1144. Shimomura, O., Bioluminescence: Chemical Principles and Methods, World Scientic Publishing (2006). Fluorophores and Fluorescent Probes, continued The Molecular Probes™ Handbook: A Guide to Fluorescent Probes and Labeling Technologies IMPORTANT NOTICE : The produc s described in this manual are overed by one or more Limited Use Label Lice se(s). Please refer to the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. thermofisher.com/probes