Fluorescence Spectroscopy
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- Abstract
- Table of Contents
- Materials
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- Literature Cited
Abstract
Fluorescence is an extremely powerful tool in modern biology, physics, and chemistry laboratories. This unit focuses on methods that can be applied to biological studies. Starting with the preparation of fluorescently conjugated antibodies, the first part explains the physical principles of fluorescence and the features in an absorption spectrum, and how these are used to measure the degree of labeling on conjugated antibodies. The protocols describe how to obtain and interpret a fluorescence emission spectrum and utilize this information as a fluorescence indicator for protein detection. Curr. Protoc. Essential Lab. Tech. 2:2.4.1?2.4.29. © 2009 by John Wiley & Sons, Inc.
Keywords: fluorescence; immunolabeling; absorption spectrum; emission spectrum; fluorometer; spectrometer; steady state anisotropy
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PDF or HTML at Wiley Online LibraryTable of Contents
- Introduction
- Strategic Planning
- Safety Considerations
- Protocols
- Basic Protocol 1: Labeling Proteins with Fluorescently Tagged NHS‐Esters
- Support Protocol 1: Measuring the Extinction Coefficient of Protein
- Support Protocol 2: Preparing the Reactive Dye Stock Solution
- Support Protocol 3: Purification of Samples Greater than 100 µg by Gravity Filtration
- Support Protocol 4: Purification of Samples Less than 100 µg by Spin‐Column Filtration
- Alternate Protocol 1: Detecting Protein Using ANS
- Support Protocol 5: Preparing ANS Solution
- Reagents and Solutions
- Understanding Results
- Troubleshooting
- Literature Cited
- Figures
- Tables
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Basic Protocol 1: Labeling Proteins with Fluorescently Tagged NHS‐Esters
Materials
Support Protocol 1: Measuring the Extinction Coefficient of Protein
Materials
Support Protocol 2: Preparing the Reactive Dye Stock Solution
Materials
Support Protocol 3: Purification of Samples Greater than 100 µg by Gravity Filtration
Materials
Support Protocol 4: Purification of Samples Less than 100 µg by Spin‐Column Filtration
Material
Alternate Protocol 1: Detecting Protein Using ANS
Materials
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Figure 2.4.1 Jablonski diagram illustrating the electronic transitions behind absorption, fluorescence, and phosphorescence. The black horizontal lines are energy states. The main states (thick black lines) can split into a few small, vibrational states. The incoming light can be absorbed by the molecule if the electron can move up to a higher state to accommodate the energy. This absorbed energy can be dissipated fully in thermal form or partially in thermal form (Stokes shift) but mostly re‐emitted as fluorescence or phosphorescence photons. In the case of phosphorescence, there is an additional transition to the triplet state. View Image -
Figure 2.4.2 Molecular structure and spectra of Cy3 (in green) and Cy5 (in red). The solid lines are the excitation spectra. The dashed lines are the emission spectra. The numbers in their names refer to the number of carbons in the middle chain. View Image -
Figure 2.4.3 A schematic showing the concept of a spectrophotometer. A single‐color light is selected from continuous spectrum light source by a monochromator. The monochromator usually includes a grating which separates different color light at different angle. A specific color (angle) light passes through a slit and exits the monochromator. The detector measures the intensity of the single color light. View Image -
Figure 2.4.4 A schematic of a basic spectrofluorometer. It has two monochromators. One analyzes the wavelength of the excitation, the other analyzes that of the emission. The detected signal can be plotted as the function of the two or just one of the two. The emission is collected at an angle 90° away from the incident excitation to prevent the excitation light from overpowering the fluorescence. View Image -
Figure 2.4.5 Screen shot of software for the emission scan mode of a spectrofluorometer. The ability to control the wavelength of the excitation beam and to scan through the emission spectrum (relevant controls circled), is the absolute basic requirement to acquire an emission spectrum. View Image -
Figure 2.4.6 Simplified schematics of various fluorometers. (A ) The T‐form spectrofluorometer with polarizers. (B ) L‐form spectrofluorometer. (C ) A filter fluorometer. View Image -
Figure 2.4.7 The Raichu FRET construct. A GTPase (Rac) and its effector p21 activated kinase (PAK) are linked together with cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) on each end, respectively. When Rac and PAK are disassociated, an excitation light favoring CFP will produce mostly CFP emission. When Rac is phosphorylated, Rac binds to PAK and collapses the construct. CFP is brought close to YFP, and the excitation energy is released partially as emission from YFP. Thus the protein dynamics can be observed according to the color of the fluorescent emission. View Image -
Figure 2.4.8 An illustration of how the absorption of a fluorescently labeled protein can be selective for the polarization of the excitation light. The double‐headed arrows indicate the orientation of the polarization of the light and the dipole of the fluorophore. The polarization of the emission is shifted from that of the excitation by the degree of rotation. View Image -
Figure 2.4.9 (A ) If a fluorophore is kept in vertical orientation, it receives only vertically polarized excitation light. The emission is also vertically polarized and will pass through the same orientation polarizer to arrive at the detector. (B ) The emission polarizer is rotated by 90°. Although everything else is identical to (A), no signal is detected. (C ) The emission filter is in horizontal orientation, and the fluorophores are free to rotate. Fluorescence can pass through the emission polarizer and be detected, if the fluorophore rotates to the right orientation at the time of emission. View Image -
Figure 2.4.10 Spectra of filters for imaging fluorescence. An excitation band‐pass filter(475/50), its transmission spectrum outlined in blue, is used. Two possible choices for the emission filter are shown in the green dashed (520/40) and the lime green solid line (545/75). In the background, the purple and yellow patterns illustrate the excitation and emission spectra of Cy2, respectively. Although 520/40 covers the emission peak, it is not an ideal choice because of the possibility of leaking excitation light. View Image -
Figure 2.4.11 (A ) From left to right, shows a disposable semimicro cuvette, a standard disposable cuvette, and a ultramicro cuvette. The semimicro and ultramicro cuvettes have only two polished windows on opposite sides, indicated by the arrows. When placing these cuvettes in the spectrometer, make sure the orientation is correct such that the light travels through a 1‐cm path length in the cuvette. (B ) Bird's‐eye view of T‐ and L‐form spectrometers. The letter C denotes where the cuvette is placed. View Image -
Figure 2.4.12 Absorption spectra of an ultramicro cuvette containing PBS (blue), sample eluted in PBS (red), and the corrected spectrum generated by subtracting one from the other (green). The blue line has a discontinuity between 300 and 350 nm, which is where the spectrometer switches light source. This results in the small peak at the same wavelength in the uncorrected spectrum. The absorbance was off by almost 1 OD before correcting for the background. This is because the small optical window in the ultramicro cuvette was not able to pass through the whole beam. View Image -
Figure 2.4.13 (A ) A gravity flow‐filtration column illuminated by a UV handheld lamp. Two fluorescent bands are visible. The first band, indicated by the arrow, is the fluorescently conjugated protein and should be collected. This particular picture shows filtration of 0.15 mg of BSA conjugated with 2 to 3 Alexa 488 per molecule. If the amount of fluorophore is much lower, and two fluorescent bands cannot be visualized clearly, a spin column as shown in panel (B ), can be used. View Image -
Figure 2.4.14 The spectrum of 10 µM ANS with different concentrations of BSA. The fluorescence of ANS, once bound to BSA, has an obvious peak at about 475 nm. At a very low concentration (0.8 nM), the fluorescence is not distinguishable from the blank sample, which has some background fluorescence. At 8 nM, although the intensity of ANS fluorescence is similar to that of the background, it can be detected because of the difference in wavelength. View Image -
Figure 2.4.15 A emission scan of unclean water excited at 350 nm. The range of the scan is intentionally broadened to show the elastically scattered excitation at 350 nm, the Raman‐scattered excitation near 400 nm, and a wide peak at around 446 nm from impurities in the water. Note that this plot has a logarithm scale. View Image -
Figure 2.4.16 A problematic spectrum showing possible discontinuity (indicated by the arrow) and lower‐than‐recommended minimal absorption at 280 nm. View Image
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Literature Cited
| Celli, A., Lee, C.Y., and Gratton, E. 2007. Fluorescence microscopy to study pressure between lipids in giant unilamellar vesicles. Methods Mol. Biol. 400:333‐339. | |
| Clegg, R.M. 1996. Fluorescence resonance energy transfer. In Fluorescence Imaging Spectroscopy and Microscopy (X.F. Wang and B. Herman, eds.) pp. 179‐252. John Wiley & Sons, Hoboken, N.J. | |
| Daniel, E. and Weber, G., 1966. Cooperative effects in binding by bovine serum albumin. I. The binding of 1‐anilino‐8‐naphthalene‐sulfonate. Fluorimetric titrations. Biochemistry 5:1893‐1900. | |
| Gaigalas, A.K., Li, L., Henderson, O., Vogt, R., Barr, J., Marti, G., Weaver, J., and Schwartz, A. 2001. The development of fluorescence intensity standards. J. Res. Natl. Inst. Stand. Technol. 106:381‐389 | |
| Itoh, R.E., Kurokawa, K., Ohba, Y., Yoshizaki, H., Mochizuki, N., and Matsuda, M. 2002. Activation of Rac and Cdc42 video imaged by fluorescent resonanace energy transfer‐based single‐molecule probes in the membrane of living cells. Mol. Cell. Biol. 22:6582‐6591. | |
| Karstens, T. and Kobs, K. 1980. Rhodamine B and rhodamine 101 as reference substances for fluorescence quantum yield measurements. J. Phys. Chem. 84:1871‐1872 | |
| Krasnowska, E.K., Gratton, E., and Parasassi, T. 1998. Prodan as a membrane surface fluorescence probe: Partitioning between water and phospholipid phases. Biophys. J. 74:1984‐1993. | |
| Loew, L.M. 1996. Potentiometric dyes: Imaging electrical activity of cell membranes. Pure Appl. Chem. 68:1405‐1409. | |
| Luby‐Phelps, K., Mujumdar, S., Mujumdar, R.B., Ernst, L.A., Galbraith, W., and Waggoner, A.S. 1993. A novel fluorescence ratiometric method confirms the low solvent viscosity of the cytoplasm. Biophys. J. 65:236‐242. | |
| MacGregor, R.B. and Weber, G. 1981. Fluorophores in polar media: Spectral effects of the Langevin distribution of electrostatic interactions. Ann. N.Y. Acad. Sci. 366:140‐154. | |
| Magde, D., Wong, R., and Seybold, P.G. 2002. Fluorescence quantum yields and their relation to lifetime of rhodamine 6G and fluorescein in nine solvents: Improved absolute standards for quantum yields. Photochem. Photobiol. 75:327‐334. | |
| Margulies, D., Melman, G., and Shanzer, A. 2005. Fluorescein as a model molecular calculator with reset capability. Nat. Mater. 4:768‐771. | |
| Mujumdar, R.B., Ernst, L.A., Mujumdar, S.R., Lewis, C.J., and Waggoner, A.S. 1993. Cyanine dye labeling reagents: Sulfoindocyanine succinimidyl esters. Bioconjug. Chem. 4:105‐111. | |
| Panchuk‐Voloshina, N., Haugland R.P., Bishop‐Stewart, J., Bhalgat, M.K., Millard, P.J., Mao, F., Leung, W., and Haugland, R.P. 1999. Alexa Dyes, a series of new fluorescent dyes that yield exceptionally bright, photostable conjugates. J. Histochem. Cytochem. 47:1179‐1188. | |
| Putney, J.W. 2006. Calcium Signaling, 2nd ed. CRC Press, Boca Raton, Fla. | |
| Stokes, G.G. 1852. On the change of refrangibility of light. Philos. Trans. R. Soc. 142:463‐562. | |
| Key Reference | |
| Lakowicz, J.R. 1999. Principles of Fluorescence Spectroscopy, 2nd ed. Kluwer Academic/Plenum Publishers, New York. | |
| The most complete textbook on fluorescence, covering the physics, history, and experiments. | |
| Internet Resources | |
| http://probes.invitrogen.com/handbook/ | |
| The Handbook from Molecular Probes, containing spectra and protocols for fluorescent probes. | |
| http://www.iss.com/resources/fluorophores.html | |
| A list of lifetime values for commonly used fluorophores. |
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