Fluorescence Spectroscopy, Biochemical Applications

Fluorescence Spectroscopy, Biochemical Applications

Fluorescence Spectroscopy, Biochemical Applications Jason B Shear, University of Texas at Austin, TX, USA ã 2017 Elsevier Ltd. All rights reserved. l...

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Fluorescence Spectroscopy, Biochemical Applications Jason B Shear, University of Texas at Austin, TX, USA ã 2017 Elsevier Ltd. All rights reserved.

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molar extinction coefficient

Introduction A remarkable range of fluorescence spectroscopy techniques has been developed in the last fifty years to characterize fundamental properties of biological systems. The rapid progress over this period has led to strategies for monitoring events that transpire on the femtosecond time scale, for discerning features smaller than the Rayleigh resolution limit of light and for detecting individual molecules. More routinely, fluorescence spectroscopy has provided invaluable insights into the structure, function and concentrations of macromolecules, small molecules, lipids and inorganic ions. Moreover, fluorescence analysis of living systems has begun to reveal directly how these biochemicals function and interact in situ. The initial section of this article presents an overview of the measurement formats used in fluorescence studies of biochemical systems. In the sections that follow, the application of diverse spectroscopic techniques to the study of biomolecular systems is examined, considering both the properties and uses of intrinsic biological fluorophores and also of fluorescent probes that have been designed to report on the presence or condition of biochemicals. In the Conclusion, several fluorescence approaches that may offer new capabilities in future biochemical studies are discussed. A fully comprehensive treatment of the diverse applications of fluorescence in biological studies is not possible in this article. For example, fluorescence studies of biological porphyrins–a vast field of spectroscopy – is mentioned in the most cursory fashion. Readers are encouraged to refer to other articles as listed in Further reading section, for additional information, as well as to the sources in the Further reading list at the end of this article.

Overview of Measurement Formats In Situ Measurements In general, when information is sought regarding the spatial distribution of chemicals within cells or tissue, some variant of fluorescence microscopy is used – often in combination with transmission or Nomarski imaging. From an optical standpoint, fluorescence microscopy measurements either use a wide-field geometry, in which an entire field-of-view is illuminated simultaneously, or rely on raster scanning of a small resolution element to sequentially generate a fluorescence image. In the first approach, an arc lamp is commonly used This article is reproduced from the previous edition, Copyright 1999, Elsevier Ltd.

Encyclopedia of Spectroscopy and Spectrometry, Third Edition

wavelength quantum yield

as the excitation source and large-format images can be acquired at video rates using an array detector. In scanning fluorescence microscopy, a laser typically provides the excitation light, which is focused critically to diffraction spot sizes as small as 0.2 mm using a high numerical aperture (NA) microscope objective. The sequential nature of image formation in scanning microscopy makes this procedure relatively slow. Although large format two-dimensional (2D) images typically require half a second or more to acquire, small 2D images or line scans can be produced much more rapidly. A fundamental advantage of the two scanning techniques most commonly used–confocal and multiphoton microscopies – is an ability to acquire 3D images of various biological specimens by scanning the laser focal spot in a given plane, then shifting the fine focus of the microscope to repeat the process in a new plane. In confocal microscopy, out-of-plane fluorescence is prevented from reaching a single-element detector by placing a small aperture in a plane conjugate to the focal spot; multiphoton microscopy achieves a similar degree of axial resolution through an inherent 3D localization of the excitation focal volume.

Measurements with chemical separations Though microscopy can be used to quantify simultaneously up to several chemical species by acquiring detailed spectroscopic information, when analysis of many, potentially unidentified, cellular components is desired, a separation procedure typically is used in combination with fluorescence detection. Commonly used techniques include high-pressure liquid chromatography (HPLC), capillary electrophoresis (CE) and related capillary chromatography techniques and gel electrophoresis. In HPLC and CE, trace analysis frequently is performed by labelling non-fluorescent components either pre- or post-separation using fluorogenic reagents. Capillary techniques frequently offer several advantages over HPLC, including faster and more efficient separations, lower sample volume requirements and alleviation of the need for highpressure equipment. In DNA gel electrophoresis, fluorescent intercalating dyes can be used to stain oligonucleotide bands in gels after electrophoresis. For DNA sequencing in agarose gels, the Sanger method can be used to generate fluorescent fragments by labelling primers with fluorophores whose emission spectra encode a specific terminating dideoxyribo-nucleotide. Analysis of proteins using SDS-polyacrylamide slab gel electrophoresis or various capillary techniques can be accomplished by fluorescently labelling analytes before separation.

http://dx.doi.org/10.1016/B978-0-12-803224-4.00106-0

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Cuvette, flow and ‘chip’ measurements A large number of studies on isolated biochemical systems can be performed in cuvettes or deep-well slides. In particular, characterizations of macro-molecular structure are frequently conducted using thermostatted cells with capabilities for stopped-flow exchange of various solution modifiers (e.g. ligands, denaturants) on a millisecond timescale. In many cases, instruments used for such studies also can perform nanosecond or microsecond time-resolved measurements of emission intensity or polarization in addition to standard steady-state detection after polarization. In many instances, it is desirable to rapidly count or isolate a subpopulation of cells exhibiting a particular chemical or characteristic. In flow cytometry, this property is linked to a corresponding difference in cellular fluorescence (e.g. by selectively labelling a relevant gene product), which provides a means for measuring relatively large numbers of cells in a flowing stream using automated instrumentation. Fluorescence-activated cell sorting (FACS) uses a flow cytometer in combination with a shunting system to isolate cells exhibiting characteristic fluorescence. The presence of various oligonucleotides, peptides and small molecules in solution can be analysed using solid state ‘chip’ based devices, in which receptor molecules (often antibodies or complementary oligonucleotides) immobilized on the chip surface bind the analyte(s) of interest. As a result of analyte binding, a characteristic fluorescence signal is generated from the receptor, analyte or some other interacting species. In ELISA (enzyme-linked immunosorbent assay), the binding of analyte is ultimately linked to the enzymatic production of a fluorescent solution-phase molecule.

Analyses Based on Intrinsic Biological Fluorescence Ideally, all biological chemicals would carry highly specific spectroscopic signatures that define not only their identities but also every aspect of their physical states and environments, and they would yield optical signals intense enough to reveal the smallest changes relevant to a particular experiment. Biological analyses, of course, are not so straightforward and generally provide very limited information in the absence of exogenous ‘probe’ chemicals. Nevertheless, there are several classes of intrinsically fluorescent biological molecules, and a host of elegant spectroscopic techniques are used to investigate the properties of these species. The aromatic amino acids – tryptophan (Trp), tyrosine (Tyr) and phenylalanine (Phe) – have strong deep-UV absorption bands (lex < 230 nm) corresponding to S0 ! S2 transitions, but commonly are excited to the S1 state in fluorescence studies (lex260–280 nm) to minimize photoreaction and enhance fluorescence quantum yields (Ff). At these longer wavelengths, Trp has the largest molar extinction coefficient (emax  5600) and quantum yield (Ff  0.2) of the three amino acids; for Phe, the values of e and Ff are so poor that this species is rarely useful in fluorescence studies. When subjected to UV irradiation, proteins with both Trp and Tyr (Figure 1) typically exhibit emission spectra whose shape is characteristic

of Trp residues (lmax350 nm) because of non-radiative energy transfer from Tyr to Trp. Despite the fact that Trp and Tyr are not highly fluorescent and undergo intersystem crossing and myriad photoreactions with large quantum yields, these species can be useful probes of protein structure. Emission from both Trp and Tyr is quenched to different extents by a variety of intramolecular polar moieties (including carboxylic acids, amino groups and imidazole groups), bound cofactors (e.g. NADþ), prosthetic groups (e.g. heme), and small molecules in solution, making determination of protein structural changes possible through measurement of excited-state lifetimes or steady-state fluorescence intensities. Unlike Tyr, Trp fluorescence often is found to be higher in proteins than in free solution. In molecules containing multiple fluorescent residues, the overall modulation of fluorescence associated with protein conformational changes can be small due to an averaging of advantageous and deleterious quantum yield changes. For this reason, timeresolved measurements of multiexponential excited state decay times sometimes can provide information unavailable from steady-state intensity measurements. The Stokes shift for Trp can also depend significantly on environment. When Trp is sequestered in the hydrophobic core of a protein, little or no reorientation of solvent takes place after excitation of the chromophore and emission is blue-shifted relative to a Trp residue on the exterior of a protein. Extreme examples include azurin, in which Trp appears to be completely isolated from H2O (lmax<310 nm), and denatured parvalbumin, in which the Trp emission spectrum is essentially identical to free Trp. For proteins that contain Tyr but not Trp, the shapes of the emission spectra closely match free Tyr (lmax305 nm), regardless of local chemical environment. A number of macromolecular diffusion and conformational properties can be studied using fluorescence anisotropy, fluorescence correlation spectroscopy (FCS) and fluorescence recovery after photobleaching (FRAP). These techniques most commonly are applied to proteins labelled with highly fluorescent probes, but can exploit intrinsic fluorescence in some instances. In fluorescence anisotropy studies, polarized light is used to selectively excite molecules whose transition dipole moments are aligned with the electric field vector. Steadystate measurements of fluorescence anisotropy can be accomplished using a continuous excitation source; provided that anisotropy decays monoexponentially after excitation, information can be obtained on properties such as rotational diffusion times in membranes and the existence of ligand–host

Figure 1 Fluorescent amino acids.

Fluorescence Spectroscopy, Biochemical Applications

interactions. Time-resolved measurements, in which anisotropy is measured as a function of time following pulsed excitation, can provide more detailed information on phenomena such as anisotropic rotational diffusion and can be used to study complex processes such as electron transfer in lightharvesting chlorophyll complexes. FCS is a steady-state technique for measuring the concentration and diffusion coefficient of a fluorescent molecule based on the magnitude of signal fluctuations as molecules diffuse through a finite probe volume. Diffusion coefficients can also be determined with FRAP, a technique in which fluorescent molecules within the probe volume are photo-bleached with a high-intensity pulse of light. After the bleaching event, the kinetics of fluorescence recovery (representing the diffusion of ‘fresh’ molecules into the probe volume) are monitored with low-intensity irradiation. Depending on the circumstances, FCS or FRAP can be useful for measuring the local viscosity of cellular or isolated biochemical environments, for monitoring ligand/host interactions and for determining changes in diffusion coefficients associated with macromolecular conformational changes. Identification and measurement of UV fluorescent proteins in complex biological samples requires mixtures to be fractionated into individual components, often with CE or HPLC. CE with on-column UV fluorescence detection has been shown to be useful in measuring attomole quantities of protein in individual red blood cells. Peptide mapping can be accomplished using HPLC with post-column UV fluorescence detection by purifying individual proteins, then subjecting various fractions to proteolytic digests before separating the resulting peptide segments with chromatography. Metabolic derivatives of Trp and Tyr also produce significant UV fluorescence. In some secretory cells, Trp is converted to the indolamine neurotransmitter, serotonin, which in turn can be processed into the pineal hormone, melatonin. Metabolic conversion of Tyr yields the catecholamines (dopamine, norepinephrine and epinephrine). Although the cellular concentration of transmitters is typically low, their concentrations within secretory granules can approach 1 molar. The fluorescence properties of these neurotransmitters are analogous to the parent amino acids, with indolamines exhibiting stronger emission and at longer excitation and emission wavelengths than the catecholamines. Intrinsic UV fluorescence has been used to perform in situ measurements of serotonin. Loss of serotonin from astrocytes can be monitored using wide-field UV fluorescence microscopy after various chemical treatments. In addition, threephoton scanning fluorescence microscopy has been shown to be useful for tracking secretion of serotonin from granules in cultured cells in response to cross-linking of cell surface IgE receptors. Fluorescence from indolamines and catecholamines separated with CE has provided a means to measure these species in the low- to mid-attomole range. In some instances, protein fluorescence does not derive directly from the aromatic amino acids. Two such proteins native to the jellyfish genus Aequorea –aequorin and the green fluorescent protein (GFP) – have been particularly useful tools in biochemical studies. Aequorin emits light (lmax470 nm) from the bound luminophore coelenterazine as the result of a Ca2þ-promoted oxidation reaction and hence has been useful as an intracellular Ca2þ probe both through microinjection

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and recombinant gene expression. The discovery and cloning of GFP has attracted much attention in part because of its utility as a reporter for various gene products when incorporated into fusion proteins. In GFP, the chromophoric unit has been identified as an imidazolone anion formed by cyclization and oxidation of tripeptide sequence (-Ser-Tyr-Gly-); fluorescence of this species is characterized by excitation maxima at 400 and 475 nm and peak emission at 510 nm. The fluorescence from GFP is intense enough to detect individual molecules of this species immobilized in aqueous gels. Both aequorin and GFP can be genetically targeted to accumulate in specific organelles to measure localized properties, such as [Ca2þ] and protein diffusion coefficients in mitochondria. A variety of protein cofactors have significant intrinsic fluorescence, including the reduced nicotinamide nucleotides (NADH and NADPH) and the oxidized flavins (Figure 2 ). The nicotinamides are excited in the near-UV and emit maximally at 470 nm; flavins exhibit both near-UV and visible excitation maxima, and have a peak emission at 515 nm. In both groups of cofactors, the adenine group partially quench fluorescence through collisions; in flavins, adenine also can quench fluorescence by forming an intramolecular complex with the fluorescent ring system. The significance of adenine quenching is underscored by the fact that flavin mononucleotide (FMN), a highly fluorescent cofactor that lacks adenine, has a fluorescence quantum yield 10-fold greater than that of flavin adenine dinucleotide (FAD). Flavins that are covalently or non-covalently bound to proteins display widely varying differences in fluorescence quantum yields, although

Figure 2 Three fluorescent cofactors.

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flavoproteins often are relatively non-fluorescent. In contrast, NADH bound to proteins sometimes has substantially greater fluorescence than free NADH because of a decreased ability of adenine to quench the nicotinamide group. In dehydrogenases, for example, crystallographic studies indicate that nicotinamide cofactors are bound in an extended fashion, with the adenine and nicotinamide groups bound to different pairs of b-sheets. Moreover, conformational changes in lactate dehydrogenase induced by effector molecules can be monitored as changes in NADH fluorescence. As a rule, the redox cofactors – like the fluorescent amino acids – have spectroscopic properties that are sensitive to environment and can be used to probe conformational changes in proteins. NADH and oxidized flavin (flavoprotein) fluorescence can be used as an indicator of the redox states of cells challenged with a variety of chemical effectors and has been used to correlate the oxidative status of cells in situ and in vivo to functions such as electrical activity in brain tissue. The fluorescence of NADH also has been used to characterize the in vitro activity of individual lactate dehydrogenase molecules, which reduce the non-fluorescent NADþ during oxidative production of pyruvate. This general strategy, in which single-enzyme molecules are characterized through the generation of a fluorescent product, can also be applied to reactions using engineered (non-biological) fluorogenic substrates. Most nucleic acids have poor fluorescence properties because of extremely short excited state lifetimes and are most often studied using non-biological intercalating or groove-binding dyes. One notable exception is the Y base, which has been used as an indicator of tRNA folding induced by divalent cation binding. In addition, some nucleotides exhibit moderate fluorescence under highly acidic conditions and can be measured in attomole to femtomole amounts using CE.

Analyses Using Non-biological Probes Although fluorimetric analysis of biological systems is experimentally simpler and less disruptive to inherent chemical properties when the use of synthetic fluorescent molecules can be avoided, in many instances the intrinsic properties of biological chemicals do not provide sufficient means for characterizing or quantifying desired properties. In the case of protein analysis, for example, many species lack Trp or Tyr residues altogether; in protein molecules that do contain these amino acids, the fluorescence characteristics may be inadequate to yield the needed information. Numerous probes have been developed for investigating biological molecules in vitro and in living cells, with greatly differing strategies for uncovering desired information. No common photophysical or photochemical characteristics can be ascribed to this entire assortment of non-biological fluorophores. Many probes have been engineered to have very large absorption cross sections and fluorescence quantum yields and to be resistant to photoreaction. The probes based on laser dyes such as fluorescein (emax>50 000; Ff > 0.9) or one of the rhodamines are good examples of this class of molecules and often are incorporated in a number of the calcium probes, fluorescent antibodies (see in subsequent paragraph) and reagents used to label proteins for conformational studies involving fluorescence anisotropy, FCS and FRAP. In addition, by

conjugating two spectroscopically distinct probes to different sites on a molecule, Fo¨rster energy transfer between the fluorophores can be used to measure intramolecular distances. Despite the advantages of these highly fluorescent molecules, probes having less optimal fluorescence properties are deliberately selected for some applications–often as a compromise to achieve a greater change in optical characteristics under different chemical environments or to avoid toxicity to living cells. For example, a dye whose fluorescence properties depend sensitively on the polarity and rigidity of its local environment may be more useful in many instances than a rhodamine for characterizing protein conformation. One such compound is NBD chloride, an amine-reactive reagent whose product has a markedly greater Ff value when occupying a hydrophobic site. Reactive fluorogenic reagents (e.g. NBD chloride, fluorescamine and CBQCA) are essentially non-fluorescent until they react with analyte molecules, and are commonly used to tag biological species that share a particular reactive group (e.g. a thiol or amine). This low-specificity labelling approach is extremely useful for identification and measurement of many low-concentration species in a mixture when some means exist for fractionating the multiple components (e.g. HPLC). Because of the low level of reagent fluorescence, a large excess of these species typically can be present in the reaction mixture without interfering with analysis. When characterizing samples containing many biomolecules, a probe that reacts with a particular chemical moiety sometimes does not provide adequate specificity, even when used with a highly efficient separation procedure. Analysis of the protein distribution in cellular specimens is perhaps the quintessential high-specificity requirement. Because of the value of such measurements in characterizing cellular composition, immunohistochemistry is one of the most ubiquitous biological applications of fluorescence. In routine determinations of gene products, cultured cellular samples or sectioned tissue are chemically fixed and subjected to sequential application of primary and secondary antibodies, followed by imaging with fluorescence microscopy. Primary antibodies can be raised against a tremendous diversity of cellular species, ranging from membrane proteins to small diffusable species such as neurotransmitters. Several species can be analysed in a single specimen by covalently attaching dyes with different excitation/emission spectra to different secondary antibodies. Most commonly, fixed and labelled samples are analysed using either wide-field or confocal laser scanning microscopy. When one seeks to analyse a solution sample for the presence of a known antigen, ELISA can be used. In one format, an immobilized antibody binds the antigen, removing it from solution. A second antibody which is conjugated to an enzyme capable of converting a non-fluorescent substrate into a fluorescent product is then applied that binds to the immobilized antigen. After a washing step, the enzymatic reaction is initiated. In this way, large signal amplifications provide extremely sensitive assays for analytes such as hormones and drug metabolites. Although immunological techniques can be useful for measuring analyte concentrations, uncertainties in crossreactivity and matrix effects on the antibody–antigen conjugation efficiency often limit this approach to semiquantitative determinations. Diffusable cytosolic species (e.g. second messengers) can be measured in living cells using highly specific fluorescent

Fluorescence Spectroscopy, Biochemical Applications

probes. The calcium-sensitive dyes, a broad class of molecules whose excitation or emission properties are dependent on chelation of Ca2þ, are ubiquitous in cellular biology studies. For such compounds, it is important that affinity for Ca2þ is much greater than for other cationic species that may be present in the cytosol at much higher concentrations (e.g. Mg2þ). In general, cytosolic free Ca2þ concentrations vary over the approximate range 100 to 1000 nM, although much higher levels are sometimes reached transiently. Common examples of Ca2þ-sensitive dyes include fluo-3 (100-fold intensity increase when bound to Ca2þ), fura-2 (excitationlmax changes when bound to Ca2þ) and indo-1 (emissionlmax changes when bound to Ca2þ) (Figure 3 ). For these species, the Ca2þ dissociation constant has been designed to approximately match free cytosolic levels, thus maximizing the modulation in fluorescence for a given change in [Ca2þ]. Like TrP, indo-1 has an indole-based chromophore, but in this case the p-system is further delocalized to electronically interact with carboxylate groups responsible for Ca2þ chelation. In many instances, fura2 and indo-1 may offer advantages over fluo-3, as ratiometric measurements of excitation or emission intensities at two different wavelengths can help avoid errors associated with uncertainties in intracellular dye concentrations. Calcium probes have played an invaluable role in identifying a range of cytosolic phenomena, such as [Ca2þ] spiking after hormone binding to cell surface receptors and Ca2þ waves in oocytes

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following a fertilization event. Probes exist for other important cytosolic effectors, such as cyclic AMP (cAMP), which is monitored by the level of Fo¨rster energy transfer between fluorescein and rhodamine attached to a cAMP-binding enzyme. Although it is possible to microinject dyes for probing cytosolic milieus through patch or intracellular pipettes, it is more common to incubate many cultured cells in a medium containing low molecular mass dyes in an esterified form (e.g. acetoxymethyl or ‘AM’, ester). This parent species is hydrophobic and thus can diffuse freely through cell membranes. Once the compound is localized to the cytosol, non-specific esterases hydrolyse the hydrophobic group, generating a charged form of the probe that can no longer diffuse through the membrane. Large numbers of cells can also be loaded rapidly using electroporation. Structures in living cells often can be characterized using probes with specificity for classes of molecules rather than for particular target analytes. Hydrophobic or amphipathic dyes, for example, can be used to gauge cell membrane fluidity and diffusion, as well as exocytosis of secretory vesicles and subsequent membrane recycling. Examples of this class of dyes are 8-anilinonaphthalene sulfonate (ANS), which displays significant fluorescence only when bound to membranes (or protected hydrophobic regions of proteins), and FM-143, a compound used extensively to study regulated secretion from neurons. Some membrane dyes have been engineered (or serendipitously found) to alter their fluorescence properties based on the electric field across the phospholipid bilayer of cell or organelle membranes. Typically, such potentiometric dyes either respond with fast kinetics to electric field changes but display small modulations in fluorescence (e.g. the styrylpyridinium probes) or undergo large fluorescence changes but only very slowly. Because large, rapid changes in fluorescence generally are not obtained with existing probes, it is difficult to perform voltage measurements on individual neurons and other excitable cells. Other fluorogenic dyes, such as DAPI, ethidium bromide and the cyanine dyes, bind to DNA or RNA and undergo changes in fluorescence intensity of up to 1000-fold. Depending on the probe, these compounds can be used to image fixed cells or track mitotic events. Combinations of probes with different specificities for single-stranded, double-stranded and ribosomal RNA have been used to characterize the quantity and conformation of nucleic acids throughout the cell cycle.

Conclusion

Figure 3 Two common calcium probes.

In the 50 years since the intrinsic fluorescence of amino acids was first characterized, fluorescence spectroscopy has developed into one of the most versatile and powerful tools for investigating biochemical systems. In addition to the enormous range of applications that now exist, a variety of emerging analysis strategies provide evidence of a continued phase of rapid growth for fluorescence applications. Developments in instrumentation – laser sources, optics, and detectors – have made possible the characterization of individual enzyme molecules and highly fluorescent proteins such as GFP and b-phycoerythrin, a light-harvesting protein. Solid-state femtosecond sources recently have made it feasible to generate multiphoton-excited fluorescence of biological molecules in

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solution using relatively low integrated irradiation and have provided reproducibility necessary for experiments involving living specimens. Various technologies that rely on fluorescence for highsensitivity analysis have been made possible through advances in microfabrication and robotics. Examples include chip-based microarray sensors for detecting a multitude of ligands simultaneously, such as for gene expression profiling, and chips containing arrays of microscopic electrophoresis channels for performing high-throughput, rapid separations of DNA fragments. Other fluorescence techniques exploit the evanescent or near-field, properties of light to excite fluorescence in highly restricted regions of space, thereby improving measurement sensitivity or the spatial resolution of fluorescence imaging. Near-field scanning optical microscopy (NSOM) can image structures smaller than the diffraction limit of light using fluorescence generated by an evanescent field that escapes from the aperture at the end of a drawn, metallized fibre. The resolution obtained with this approach is determined by the dimensions of the aperture, which is usually limited by losses in throughput as the fibre tip diameter is reduced. Thus far, only a few biological applications of this powerful technique have been reported. Probably the most heavily used application of fluorescence today is in the proliferation of microchip arrays for gene expression profiling to evaluate changes caused by disease or drug therapy. However, new developments in the chemistry of fluorescent probes undoubtedly will open new applications for fluorescence in biochemistry. A growing number of companies and academic researchers are devising new biological probes and in some cases are using non-traditional strategies – such as combinatorial chemistry – to generate hosts with high specificity and large binding constants for desired ligands. Genetic manipulation of cultured cells offers particularly exciting opportunities for in situ biochemical measurements. Technology now exists for using an enzyme as a reporter for neurotransmitter-activated transcription in individual mammalian cells, with sensitivities capable of measuring fewer than 50 gene product molecules. In an initial demonstration of this strategy, large amplifications of gene product expression were obtained by localizing an engineered substrate to the cytosol whose fluorescence properties change when it is degraded by the reporter enzyme. Through the judicious use of molecular and cellular biology, chemistry and fluorescence spectroscopy, eventually it may be feasible to track the production and breakdown of individual molecules in living cells.

See also: Atomic Emission and Fluorescence Theory; Atomic Fluorescence, Methods and Instrumentation; Biological Applications of Hyperpolarized 13C NMR; Circular Dichroism and ORD, Biomacromolecular Applications; Circularly Polarized Luminescence and Fluorescence Detected Circular Dichroism; Electron Paramagnetic Resonance of Membrane Proteins; Fluorescence Microscopy, Applications; Fluorescence Polarization and Anisotropy; Fluorescence Spectroscopy, Organic Chemistry Applications; Fluorescence Theory; Fluorescence up-conversion Methods and Applications; Fluorescent Molecular Probes; IR, Biological Applications; Ligand–Protein Binding and Screening Using NMR Spectroscopy; Luminescence

Spectroscopy, Inorganic Condensed Matter Applications; Luminescence, Theory; Mass Spectrometry: Nucleic Acids and Nucleotides Studied Using MS; MRI Applications, Biological; NMR Spectroscopy of Nucleic Acids, Historical Overview; Nucleic Acids Studied by NMR Spectroscopy; Overview of Biochemical Applications of Mass Spectrometry; Peptides and Proteins Studied Using Mass Spectrometry; Plasmon-Controlled Fluorescence Methods and Applications; Protein Structure Analysis by CD, FTIR, and Raman Spectroscopies; Proteins Studied by NMR; Raman Optical Activity, Macromolecule and Biological Molecule Applications; Raman Spectroscopy, Biochemical Applications; Solid-State NMR of Membrane Proteins in Phospholipid Bilayers; Spectroscopy in Biotechnology Research and Development; Super-Resolution Fluorescence Microscopy, Localization Microscopy; SurfaceEnhanced Raman Scattering (SERS) Biochemical Applications; Thermoluminescence Theory and Analysis: Advances and Impact on Applications; UV-Visible Absorption Spectroscopy, Biomacromolecular Applications; UV–Visible Fluorescence Spectrometers; X-Ray Crystallography, Biomolecular Structure Determination Methods; X-Ray Fluorescence Spectrometers; X-Ray Fluorescence Spectroscopy, Applications.

Further Reading Cantor CR and Schimmel PR (1980) Biophysical Chemistry. Parts I–III. New York: Freeman. Chalfie M, Tu Y, Euskirchen G, Ward WW, and Prasher DC (1994) Green fluorescent protein as a marker for gene expression. Science 263: 802–805. Cobbold PH and Rink TJ (1987) Fluorescence and bioluminescence measurement of cytoplasmic free calcium. Biochemical Journal 248: 313–328. Craig DB, Arriaga E, Wong JCY, Lu H, and Dovichi NJ (1998) Life and death of a single enzyme molecule. Analytical Chemistry 70: 39A–43A. Creed D (1984) The photophysics and photochemistry of the near-UV absorbing amino acids– I. Tryptophan and its simple derivatives. Photochemistry and Photobiology 39: 537–562. Creed D (1984) The photophysics and photochemistry of the near-UV absorbing amino acids– II. Tyrosine and its simple derivatives. Photochemistry and Photobiology 39: 563–575. Everse J, Anderson B, and You K-S (1982) The Pyridine Nucleotide Coenzymes. New York: Academic Press. Freifelder D (1982) Physical Biochemistry, 2nd edn New York: W.H. Freeman. Giepmans BNG, Adams SR, Ellisman MH, and Tsien RY (2006) The fluorescent toolbox for assessing protein location and function. Science 312: 217–224. Hoagland RP (1996) Handbook of Fluorescent Probes and Research Chemicals, 6th edn Eugene, OR: Molecular Probes. Lakowicz JR (1983) Principles of Fluorescence Spectroscopy. New York: Plenum Press. Lakowicz JR (ed.) (1991) Topics in Fluorescence Spectroscopy, vol. 1 Techniques. New York: Plenum Press. Lakowicz JR (ed.) (1992) Topics in Fluorescence Spectroscopy, vol. 3 Biochemical Applications. New York: Plenum Press. Lillard SJ, Yeung ES, Lautamo RMA, and Mao DT (1995) Separation of hemoglobin variants in single human erythrocytes by capillary electrophoresis with laserinduced native fluorescence detection. Journal of Chromatography A 718: 397–404. Permyakov EA (1993) Luminescent Spectroscopy of Proteins. Boca Raton, FA: CRC Press. Rutter GA, Burnett P, Rizzuto R, et al. (1996) Subcellular imaging of intramitochondrial Ca2þ with recombinant targeted aequorin: significance for the regulation of pyruvate dehydrogenase activity. Proceedings of the National Academy of Science, USA 93: 5489–5494. Tsien RY (1994) Fluorescence imaging creates a window on the cell. Chemical Engineering News 72: 34–44. Zlokarnik G, Negulescu PA, Knapp TE, et al. (1998) Quantitation of transcription and clonal selection of single living cells with beta-lactamase as reporter. Science 279: 84–88.