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Unformatted text preview: Two-photon Excitation Fluorescence Bioassays P EKKA H ANNINEN , a J ORI S OUKKA , b AND J UHANI T. S OINI c a Department of Cell Biology and Anatomy, Laboratory of Biophysics, University of Turku, Turku, Finland b ArcDia, Inc., Turku, Finland c Turku University of Applied Sciences, Turku, Finland Application of two-photon excitation of fluorescence in microscopy is one of the major discoveries of the renaissance of light microscopy that started in the 1980s. The technique derives its advan- tages from the biologically smooth wavelength of the excitation light and the confinement of the excitation. Difficult, and seemingly nontransparent, samples may be imaged with the technique with good resolution. Although the bioresearch has been concentrating mostly on the positive properties of the technique for imaging, the same properties may be applied successfully to non- imaging bioassays. This article focuses on the development path of two-photon excitationbased assay system. Key words: two-photon excitation; bioassay; in vitro diagnostics Introduction Fluorescence imaging and imaging-related tech- niques are among the most important tools in mod- ern biosciences. The field has been evolving at an un- precedented rate over the last two decades, driven by advances in biosciences, chemistry, physics, and en- gineering. Among the tools that have evolved dur- ing this time are two- or (more broadly) multiphoton excitationbased methods and technologies. Since the postulation, 1 the first proof of two-photon excitation, 2 and the advent of two-photon scanning microscopy, 3 the technique has developed into one of the standard tools of biomedical imaging, especially of thick and dense specimens and recently even within living animals. 4 The two-photon excitation technique has some distinct advantages over its single-photon counterpart, confocal microscopy. The use of red to near-infrared excitation light reduces absorption and scattering within biological material; the excitation is spatially limited to the most intense focal area of illumi- nation, making the use of the confocal pinhole unnec- essary and allowing the detector to be placed virtually anywhere along the optical path of emission light. Fur- ther, the background signals from out-of-focus areas can easily be reduced to practically zero, and several Address for correspondence: Pekka Hanninen, Department of Cell Biology and Anatomy, Laboratory of Biophysics, University of Turku, 20014 Turku, Finland. firstname.lastname@example.org labels with distinct emission spectra may efficiently be excited with one two-photon excitation laser line. 5 The last advantage obviously becomes a disadvantage for fluorescence resonance energy transfer experiments....
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This note was uploaded on 07/11/2010 for the course SPECTOGRAP 545 taught by Professor Gdf during the Spring '10 term at AIB College of Business.
- Spring '10