Technology Report pubs.acs.org/jchemeduc
FluAnisot: A Simulated Experiment in Fluorescence Anisotropy Measurement Stephen W. Bigger*,† and Andrew S. Bigger‡ †
College of Engineering and Science, Victoria University, P.O. Box 14428, Melbourne 8001, Australia Software Developer, Lonely Planet, P.O. Box 1, Footscray 3011, Australia
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S Supporting Information *
ABSTRACT: The FluAnisot educational software package is a fully contained tutorial on the technique of fluorescence anisotropy measurement as well as a simulator on which two experiments can be performed. The procedure for each of the experiments is also contained within the package along with example analyses of results that were obtained using the software.
KEYWORDS: Upper-Division Undergraduate, Second-Year Undergraduate, Physical Chemistry, Computer-Based Learning, Inquiry-Based/Discovery Learning, Biophysical Chemistry, Fluorescence Spectroscopy teady-state fluorescence anisotropy experiments1 can be considered to be an extension of the familiar fluorimetry technique2,3 and provide information about rotational dynamics of molecules in solution. Fluorescence anisotropy measurements can thus be applied to studying the rotational motion of proteins and synthetic polymers as well as probing the structures of bilayers and lipids. As long ago as the early 1920s, Perrin4,5 performed the original exploratory work in this field. Since then the technique has been used among other things in clinical studies including fetal and neonatal lung maturity6−8 as well as pregnancies complicated by hypertension.9 Furthermore, the more sophisticated time-resolved anisotropy measurements (TRAMS) have been used in the early detection of Alzheimer β-amyloid peptide aggregation.10 More recently, steady-state fluorescence anisotropy measurements have been widely adopted in the field of high-throughput screening in drug discovery,11 for studying biomolecular interactions12 as well as performing various clinical immunoassays.13 Fluorescence anisotropy has also been applied in modern imaging techniques.14 The FluAnisot desktop software will be most suitable for schools that do not have a fluorimeter or fluorescence polarimeter and perhaps instructors who are looking for another option to introduce their students to advanced fluorescence techniques and their applications. Furthermore, it is anticipated that the software will be suitable for upper-level undergraduate students in courses such as physical chemistry, biophysics, or polymer chemistry. Depending on the extent to which the user wishes to explore the options available in
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© 2013 American Chemical Society and Division of Chemical Education, Inc.
FluAnisot, it is estimated that the exercise can take between about 30 min (brief exploration) to about 90 min to complete (more complete exploration with error analysis, etc.). The software incorporates four sections that are accessed by tabs appearing on the user interface: Theory, Experiment, Flu Anisotropy, and Lab Book. It is supplied with a Supporting Information file that includes all theoretical and experimental material that can be printed from the software as well as answers to the questions appearing at the end of the experimental section. The Theory section presents a scrollable window containing discussion of the elementary theory and relevant equations1,15 pertaining to the study of fluorescence anisotropy. It also provides some examples of modern-day applications of the technique along with some relevant references for further reading. Furthermore, this section includes an animated demonstration that illustrates the calculation of the theoretical quantity known as the ″limiting anisotropy″ using a Monte Carlo approach that has been described elsewhere.1 Among the Theory text are links to ten information plates that provide further details and Web references on selected aspects. The software enables the user to obtain a printed version of the theory notes as well as any of the information plates. The Experiment section presents a scrollable window containing background information relating to two experiments1 that can be performed using the simulator contained within the Flu Anisotropy section of the software. Step-by-step Published: January 23, 2013 386
dx.doi.org/10.1021/ed300316w | J. Chem. Educ. 2013, 90, 386−387
Journal of Chemical Education
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instructions are provided in the window along with access to an information plate containing further details about the specific experiments and to another plate containing data that are required for the analysis of the experimental results. The software enables the user to obtain a printed version of the experimental notes, information, or data plates, if required. Nonetheless, the user can easily switch between the instructions and the experimental simulation without interruption to the progress of the experiment. The Flu Anisotropy section contains a graphic representing the functional elements of a standard laboratory fluorimeter that is equipped with polarizing filters along its excitation and emission optical axes. The user is able to separately set the excitation or emission polarizing filters to either a horizontal or a vertical orientation by clicking on the filter. The user can set the temperature of the test solution using a slider control and can enter the viscosity of the test solution via an edit field. One of the experiments requires the viscosity of water at a given temperature to be entered into the simulator. These data are preprogrammed in the simulator for convenience. As the experimental data are collected in the simulator they are automatically transferred to the Lab Book section of the program where they can be edited if necessary or copied to the clipboard for export as tab delimited data to a suitable spreadsheet program for processing. The Lab Book also contains model data obtained from each of the experiments and these data are displayed along with details of their corresponding analyses. Data in the Lab Book as well as the processed model data screen can be printed if required. The FluAnisot software is provided as a standalone application. Versions exist that run on standard Macintosh OSX or Windows environments. The software should be run in conjunction with a standard spreadsheet program for the plotting and labeling of the processed output data. This approach to handling the output data provides maximum flexibility with regard to its presentation and collation.
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Technology Report
ASSOCIATED CONTENT
S Supporting Information *
FluAnisot software; fluorescence anisotropy theory; experimental setup; the Perrin equation; This material is available via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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REFERENCES
(1) Bigger, S. W.; Craig, R. A.; Ghiggino, K. P.; Scheirs, J. J. Chem. Educ. 1993, 70, A234−239. (2) Bigger, S. W.; Ghiggino, K. P.; Meilak, G. A.; Verity, B. J. Chem. Educ. 1992, 69, 675−677. (3) Bigger, S. W.; Watkins, P. J.; Verity, B. J. Chem. Educ. 2003, 80, 1191−1193. (4) Perrin, F. J. J. Phys. Radium 1926, 7 (12), 390. (5) Perrin, F. J. J. Phys. Radium 1936, 7 (1), 1. (6) Salmona, M.; Diomede, L.; Moro, G.; Minoli, I.; Bernini, S.; Agosti, S. J. Perinat. Med. 1993, 21, 349−354. (7) Henderson, C. E.; Gewolb, I. H.; Deutsch, J.; Cavalieri, R. L. J. Perinatol. 1993, 13, 428−432. (8) Liu, K. Z.; Shaw, R. A.; Dembinski, T. C.; Reid, G. J.; Ying, S. L.; Mantsch, H. H. Am. J. Obstet. Gynecol. 2000, 183, 181−187. (9) Barkai, G.; Reichman, B.; Kokia, E.; Segal, P.; Lusky, A.; Goldman, B.; Mashiach, S. J. Perinat. Med. 1994, 22, 107. (10) Allsop, D.; Swanson, L.; Moore, S.; Davies, Y.; York, A.; ElAgnaf, O. M.; Soutar, I. Biochem. Biophys. Res. Commun. 2001, 285, 58−63. (11) Owicki, J. C. J. Biomol. Screen. 2000, 5, 297−306. (12) Huang, X.; Aulabaugh, A. Methods Mol. Biol. 2009, 565, 127− 143. (13) Smith, D. S.; Eremin, S. A. Anal. Bioanal. Chem. 2008, 391, 1499−1507. (14) Roberti, M. J.; Jovin, T. M.; Jares-Erijman, E. PLoS One 2011, 6 (8), e23338. DOI: 10.1371/journal.pone.0023338 (15) Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 3rd ed.; Springer: New York, 2006; Vol. 1, pp 367−370.
SYSTEMS
It is anticipated the software will successfully run on a wide range of Macintosh and PC systems. The following systems are examples of ones that have been successfully trialed: 1. Macintosh • iMac 2.66 GHz Intel Core i5; Mac OS X 10.6.8 (Leopard); Fuji Xerox ApeosPort IV C5570 network printer • Macbook 2.16 GHz Intel Core 2 Duo; Mac OSX 10.7.2 (Lion); HP Deskjet F4400 Series and Apple PDF printer 2. PC Systems • Macbook 2.1.6 GHz Intel Core 2 Duo; Windows 7 Professional; HP Deskjet F4400 Series and Cute PDF Printer • Windows XP SP2 Virtual Machine running on Macbook 2.16 GHz Intel Core 2 Duo; HP Deskjet F4400 Series and Cute PDF Printer • Lenovo ESA4C406-86, Intel Core 2 DuoCPU, E8400 @ 3.00 GHz, 1.97 GHz, 3.25 GB of RAM, Microsoft Windows XP Professional Version 2002 Service Pack 3; Xerox Phaser 6360 Printer, Model P6360DT 387
dx.doi.org/10.1021/ed300316w | J. Chem. Educ. 2013, 90, 386−387
