title: Development and experimental testing of an optical micro-spectroscopic technique incorporating true line-scan excitation.
abstract: Multiphoton micro-spectroscopy, employing diffraction optics and electron-multiplying CCD (EMCCD) cameras, is a suitable method for determining protein complex stoichiometry, quaternary structure, and spatial distribution in living cells using Förster resonance energy transfer (FRET) imaging. The method provides highly resolved spectra of molecules or molecular complexes at each image pixel, and it does so on a timescale shorter than that of molecular diffusion, which scrambles the spectral information. Acquisition of an entire spectrally resolved image, however, is slower than that of broad-bandwidth microscopes because it takes longer times to collect the same number of photons at each emission wavelength as in a broad bandwidth. Here, we demonstrate an optical micro-spectroscopic scheme that employs a laser beam shaped into a line to excite in parallel multiple sample voxels. The method presents dramatically increased sensitivity and/or acquisition speed and, at the same time, has excellent spatial and spectral resolution, similar to point-scan configurations. When applied to FRET imaging using an oligomeric FRET construct expressed in living cells and consisting of a FRET acceptor linked to three donors, the technique based on line-shaped excitation provides higher accuracy compared to the point-scan approach, and it reduces artifacts caused by photobleaching and other undesired photophysical effects.
authors.affiliation: Physics Department, University of Wisconsin-Milwaukee, Milwaukee, WI 53211, USA. biener@uwm.edu.
authors.forename: Gabriel
authors.initials: G
authors.lastname: Biener
authors.affiliation: Physics Department, University of Wisconsin-Milwaukee, Milwaukee, WI 53211, USA. mstoneman@auroraspectral.com.
authors.forename: Michael R
authors.initials: MR
authors.lastname: Stoneman
authors.affiliation: Physics Department, University of Wisconsin-Milwaukee, Milwaukee, WI 53211, USA. acbas@uwm.edu.
authors.forename: Gheorghe
authors.initials: G
authors.lastname: Acbas
authors.affiliation: Physics Department, University of Wisconsin-Milwaukee, Milwaukee, WI 53211, USA. jdholz@uwm.edu.
authors.forename: Jessica D
authors.initials: JD
authors.lastname: Holz
authors.affiliation: Physics Department, University of Wisconsin-Milwaukee, Milwaukee, WI 53211, USA. orlova@uwm.edu.
authors.forename: Marianna
authors.initials: M
authors.lastname: Orlova
authors.affiliation: Physics Department, University of Wisconsin-Milwaukee, Milwaukee, WI 53211, USA. komarova@uwm.edu.
authors.forename: Liudmila
authors.initials: L
authors.lastname: Komarova
authors.affiliation: Physics Department, University of Wisconsin-Milwaukee, Milwaukee, WI 53211, USA. skuchin@uwm.edu.
authors.forename: Sergei
authors.initials: S
authors.lastname: Kuchin
authors.affiliation: Physics Department, University of Wisconsin-Milwaukee, Milwaukee, WI 53211, USA. vraicu@uwm.edu.
authors.forename: Valerică
authors.initials: V
authors.lastname: Raicu
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D004151:Dipeptides / Q000737:chemistry; D004867:Equipment Design; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence / Q000295:instrumentation* / Q000379:methods; D038761:Photobleaching; D017785:Photons; D012441:Saccharomyces cerevisiae / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D004151:Dipeptides; D008164:Luminescent Proteins; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins