1. Fluorescence Spectroscopy 1

2. Molecular Fluorescence Spectroscopy Fluorescence is a photoluminescence process in which atoms or molecules are excited by absorption of electromagnetic radiation. The excited species then relax to the ground state, giving up their excess energy as photons. One of the most attractive features of molecular fluorescence is its inherent sensitivity, which is often one to three orders of magnitude better than absorption spectroscopy. 2

3. Another advantage is the large linear concentration range of fluorescence methods, which is significantly greater than those encountered in absorption spectroscopy. Fluorescence methods are, however, much less widely applicable than absorption methods because of the relatively limited number of chemical systems that show appreciable fluorescence. 3

4. Principles of Molecular Fluorescence Molecular fluorescence is measured by exciting the sample at the absorption wavelength, also called excitation wavelength, and measuring the emission at a longer wavelength called the emission or fluorescence wavelength. Usually, fluorescence emission is measured at right angles to the incident beam so as to avoid measuring the incident radiation. The short-lived emission that occurs is called fluorescence, whereas luminescence that is much longer lasting is called phosphorescence. 4

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6. Excitation Spectra and Fluorescence Spectra Because the energy differences between vibrational states is about the same for both ground and excited states, the absorption, or excitation spectrum, and the fluorescence spectrum for a compound often appear as approximate mirror images of one another with overlap occurring near the origin transition (0 vibrational level of E 1 to 0 vibrational level of E 0 ). There are many exceptions to this mirror-image rule, particularly when the excited and ground states have different molecular geometries or when different fluorescence bands originate from different parts of the molecule. 6

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10. Principles Interaction of photons with molecules results in promotion of valence electrons from ground state orbitals to high energy levels. The molecules are said to be in excited state. Molecules in excited state do not remain there long but spontaneously relax to more stable ground state. 10

11. The relaxation process is brought about by collisional energy transfer to solvent or other molecules in the solution. Some excited molecules however return to the ground state by emitting the excess energy as light. This process is called fluorescence. 11

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14. The emitted light has two important characteristics : 1. It is usually of longer wavelength (lower energy) than the excited light. This is because part of the energy associated with S state is lost as heat energy. 2. The emitted light is composed of many wavelengths which results in fluorescence spectrum. 14

15. Quantam yield Q The fluorescence intensity is described in terms of quantum yield. The quantum yield Q is the ratio of the number of photons emitted to the number of photons absorbed. 15

16. Intrinsic Fluors Some biomolecules are intrinsic fluors ie., they are fluorescent themselves. The amino acids with aromatic groups eg phenylalamine, tyrosine, tryptophan are fluorescent. Hence proteins containing these amino acids have intrinsic fluorescence. The purine and pyrimidine bases and some coenzymes eg NAD and FAD are also intrinsic fluors. Intrinsic fluorescence is used to study protein conformation changes and to probe the location of active site and coenzymes in enzymes. 16

17. Extrinsic Fluors These are fluorescent molecules that are added in biochemical system under study. Extrinsic fluorescence has been used to study the binding of fatty acids to serum albumin, to characterize the binding sites for cofactors and substrates in enzyme molecules and to study the intercalation of small molecules into the DNA double helix. 17

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19. ANS, dansyl chloride, fluorescein are used for protein studies. Ethidium, proflavine and acridines are used for nucleic acid characterization. Ethidium bromide has enhanced fluorescence when bound to double stranded DNA but not single stranded DNA. 19

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21. Instrumentation The basic instrument is a spectrofluorometer. It contains a light source, two monochromators, a sample holder and a detector. There are two monochromators, one for selection of the excitation wavelength, another for analysis of the emitted light. The detector is at 90 degrees to the excitation beam. Upon excitation of the sample molecules, the fluorescence is emitted in all directions and is detected by photocell at right angles to the excitation light beam. 21

22. The lamp source used is a xenon arc lamp that emits radiation in the UV, visible and near-infrared regions. The light is directed by an optical system to the excitation monochromator, which allows either preselection of wavelength or scanning of certain wavelength range. 22

23. The exciting light then passes into the sample chamber which contains fluorescence cuvette A special fluorescent cuvette with four translucent quartz or glass sides is used. When the excited light impinges on the sample cell, molecules in the solution are excited and some will emit light. 23

24. Light emitted at right angles to the incoming beam is analyzed by the emission monochromator. The wavelength analysis of emitted light is carried out by measuring the intensity of fluorescence at preselected wavelength. The analyzer monochromator directs emitted light of the preselected wavelength to the detector. 24

25. A photomultiplier tube serves as the detector to measure the intensity of the light. The output current from the photomultiplier is fed to some measuring device that indicates the extent of fluorescence. 25

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27. Concentration and Fluorescence Intensity The radiant power of fluorescence F is proportional to the radiant power of the excitation beam absorbed: F = K(P 0 – P) where, P 0 is the radiant power of the beam incident on the sample and P is the radiant power after it traverses a pathlength b of the medium. Constant K depends on the Quantum efficiency. The efficiency of fluoresced to the number absorbed. 27

28. At low concentrations where fluorescence is most often employed F = K’P 0 c where, –c is the concentration of the fluorescent species and –K’ is a new proportionality constant. –F is directly proportional to analyte concentration. Thus, a plot of the fluorescent radiant power versus the concentration of the emitting species should be, and ordinarily is, linear at low concentrations. When c becomes great enough that the absorbance is larger than about 0.05, linearity is lost and F begins to reach a plateau with concentration. This effect is known as a primary absorption inner filter effect. In fact, at high concentrations, fluorescence radiant power can even begin to decrease with increasing concentration. 28

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31. Molecular Phosphorescence Spectroscopy Phosphorescence is a photoluminescence phenomenon that is quite similar to fluorescence. Understanding the difference between these two phenomena requires and understanding of electron spins and the difference between a singlet state and a triplet state. Ordinary molecules exist in the ground state with their electron spins paired. A molecular electronic state in which all electron spins are paired is said to be a singlet state. 31

32. When one of a pair of electrons in a molecule is excited to a higher-energy level, a singlet or a triplet state can be produced. In the excited singlet state the spin of the promoted electron is still opposite that of the remaining electron. In the triplet state, however, the spins of the two electrons become unpaired and are thus parallel. The excited triplet state is less energetic than the corresponding excited singlet state. 32

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34. Chemiluminescence Methods Chemiluminescence is produced when a chemical reaction yields an electronically excited molecule, which emits light as it returns to the ground state. Chemiluminescence reactions are encountered in a number of biological systems, where the process is often called bioluminescence. One attractive feature of chemiluminescence for analytical uses, is the very simple instrumentation. Since no external source of radiation is needed for excitation, the instrument may consist of only a reaction vessel and a photomultiplier tube. Generally, no wavelength selection device is needed because the only source of radiant is the chemical reaction. 34

35. Perrin- Jablonski diagram 35

36. S is singlet and T is triplet. The S 0 state is the ground state and the subscript numbers identify individual states. 36

37. n  →  *  →  *  n  →  *  →  *  →  * Energy level of MO 37

38. S0S0 Singlet & Triplet 38

39. Characteristics of Excited States Energy Lifetime Quantum Yield Polarization 39

40. Stokes shift The Stokes shift is the gap between the maximum of the first absorption band and the maximum of the fluorescence spectrum loss of vibrational energy in the excited state as heat by collision with solvent heat 40

41. Example Example: 7-amino-4-methylcoumarin (AMC) 41

42. Example 42

43. Example Example fluorophores fluorescein ethidium bromide bound to DNA. 43

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45. Lifetime 45

46. Lifetime Excited states decay exponentially with time – I = I 0 e -t/t I 0 is the initial intensity at time zero, I is the intensity at some later time t t is the lifetime of the excited state. k F = 1/ t, where k F is the rate constant for fluorescence. 46

47. Quantum Yield Quantum Yield =  F  F = number of fluorescence quanta emitted divided by number of quanta absorbed to a singlet excited state  F = ratio of photons emitted to photons absorbed Quantum yield is the ratio of photons emitted to photons absorbed by the system: 47

48. Quantum Yield 48

49. Quantum Yield & Structure rigidity 49

50. Polarization Molecule of interest is randomly oriented in a rigid matrix (organic solvent at low temperature or room temperature polymer). And plane polarized light is used as the excitation source. Degree of polarization is defined as P I || and I   are the intensities of the observed parallel and perpendicular components,  is the angle between thee mission and absorption transition moments. If  is 0° than P = +1/2, and if  is 90° than P = -1/3. 50

51. Steady-state measurements: , I Time-Resolved measurements:  Experimental Measurements 51

52. Instruments 52

53. Inner Filter Effect At low concentration the emission of light is uniform from the front to the back of sample cuvette. At high concentration more light is emitted from the front than theback. Since emitted light only from the middle of the cuvette is detected the concentration must be low to assure accurate  F measurements. 53

54. Inner Filter Effect 54

55. I f ( em) = I Abs ( ex).  f. f ( em). K I 0 ( ex ) em measured intensity of fluorescence at em absorbed intensity at ex fluorescence quantum yield fraction of intensity emitted at that particular wavelength fraction of total fluorescence that is detected If A  0 If we measure the sample and a standard under the same experimental conditions, keeping ex constant: Important : the index of refraction of the two solvents (sample and standard) must be the same Standards: Quinine sulfate in H 2 SO 4 1N:  f =0.55 Fluorescein in NaOH 0.1N:  f =0.93 Measurement of fluorescence quantum yields 55

56. The TCSPC measurement relies on the concept that the probability distribution for emission of a single photon after an excitation yields the actual intensity against time distribution of all the photons emitted as a result of the excitation. By sampling the single photon emission after a large number of excitation flashes, the experiment constructs this probability distribution. Time correlated single photon counting: #events........ t (nsec) different excitation flashes Start PMT Stop PMT sample exc. monochromator emission monochromator pulsed source tt Measurement of fluorescence lifetimes 56

57. Lifetime ns AbsorptionFluorescence Wavelength nm Absorptivity Wavelength nm Quantum Tryptophan 2.62805,6003480.20 Tyrosine 3.62741,4003030.14 Phenylalanine 6.42572002820.04 Intrinsic Fluorescence of Proteins and Peptides 57

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60. TryptophanTryptophan, the dominant intrinsic fluorophore, is generally present at about 1mol% in proteins. A protein may possess just one or a few Trp residues, which facilitates interpretation of the spectral data. TryptophanTryptophan is very sensitive to its local environment. It is possible to see changes in emission spectra in response to conformational changes, subunit association, substrate binding, denaturation, and anything that affects the local environment surronding the indole ring. Also, Trp appears to be uniquely sensitive to collisional quenching, either by externally added quenchers, or by nearby groups in the protein. TryptophanTryptophan fluorescence can be selectively excited at 295-305 nm. (to avoid excitation of Tyr)Tryptophan 60

61. IIIIII IVV Example Example: Tyrosine and its derivatives 61

62. I I II III IV II V V 62

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64. Emission spectra of Pseudomonas fluorescens azurin Pfl. For 275-nm excitation, a peak is observed due to the tyrosine residue(s) The position and structure of the fluorescence suggests that the indole residue is located in a completely nonpolar region of the protein. These results agree with X-ray studies, which show that the indole group is located in the hydrophobic core of the protein. In the presence of a denaturing agent, the TrpP emission loses its structure and shifts to 351nm, characteristic of a fully exposed Trp residue. Changes in emission spectra can be used to follow protein unfolding 64

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66. Resolution of the contributions of individual tryptophan residues in multi-tryptophan proteins. I(,t)=  i ( )exp(-t/  i ) i  1 =2ns,  2 = 5ns  1 =2ns  2 =5ns t (ns) Fluorescence intensity (A.U.) wavelength (nm)  em Example Time-resolved protein fluorescence 66

67. Isolated from the Pacific jellyfish Aequorea victoria and now plays central roles in biochemistry and cell biology due to its widespread use as an in vivo reporter of gene expression, cell lineage, protein ­ protein interactions and protein trafficking One of the most important attributes of GFP which makes it so useful in the life sciences is that the luminescent chromophore is formed in vivo, and can thus generate a labeled cellular macromolecule without the difficulties of labeling with exogenous agents. Green fluorescent protein (abbreviated GFP 67

68. The structure of GFP : eleven-strand beta- barrel wrapped around a central alpha-helix core. This central core contains the chromophore which is spontaneously formed from a chemical reaction involving residues Ser 65, Tyr 66, and Gly 67 (SYG) There is cyclization of the polypeptide backbone between Ser 65 and Gly 67 to form a 5-membered ring, followed by oxidation of Tyr 66. The high quantum yield of GFP fluorescence probably arises from the nearly complete protection of the fluorophore from quenching water or oxygen molecules by burial within the beta-barrel. Ribbon diagram of the Green Fluorescent Protein (GFP) drawn from the wild- type crystal structure. The buried chromophore, which is responsible for GFP's luminescence, is shown in full atomic detail. 68

69. Wild type GFP from jellyfish has two excitation peaks, a major one at 395 nm and a minor one at 475 nm with extinction coefficient of 30,000 and 7,000 M -1 cm -1, respectively. Its emission peak is at 509 nm in the lower green portion of the visible spectrum. For wild type GFP, exciting the protein at 395 nm leads to rapid quenching of the fluorescence with an increase in the 475 nm excitation band. This photoisomerization effect is prominent with irradiation of GFP by UV light. In a wide range of pH, increasing pH leads to a reduction in fluorescence by 395 nm excitation and an increased sensitivity to 475 nm excitation. 69

70. Melittin GIGAVLKVLT TGLPALISWI KRKRQQX 70

71. Carboxyfluorescence Example Carboxyfluorescence Biochemical Education 28 (2000) 171~173 71

72. Carboxyfluorescence Example Carboxyfluorescence Quenching Effect 72

73. Carboxyfluorescence Example Carboxyfluorescence pH Effect 73