Next Article in Journal
Previous Article in Journal
Previous Article in Special Issue
Int. J. Mol. Sci. 2014, 15(1), 261-276; doi:10.3390/ijms15010261
Article

Development and Experimental Testing of an Optical Micro-Spectroscopic Technique Incorporating True Line-Scan Excitation

1
, 2
, 1
, 1
, 3
, 1
, 3
 and 1,3,4,*
Received: 18 September 2013; in revised form: 15 December 2013 / Accepted: 23 December 2013 / Published: 27 December 2013
(This article belongs to the Special Issue Frontiers of Micro-Spectroscopy in Biological Applications)
View Full-Text   |   Download PDF [489 KB, uploaded 19 June 2014]   |   Browse Figures
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.
Keywords: optical micro-spectroscopy; fluorescence; two-photon excitation; multi-photon excitation; energy transfer optical micro-spectroscopy; fluorescence; two-photon excitation; multi-photon excitation; energy transfer
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Export to BibTeX |
EndNote


MDPI and ACS Style

Biener, G.; Stoneman, M.R.; Acbas, G.; Holz, J.D.; Orlova, M.; Komarova, L.; Kuchin, S.; Raicu, V. Development and Experimental Testing of an Optical Micro-Spectroscopic Technique Incorporating True Line-Scan Excitation. Int. J. Mol. Sci. 2014, 15, 261-276.

AMA Style

Biener G, Stoneman MR, Acbas G, Holz JD, Orlova M, Komarova L, Kuchin S, Raicu V. Development and Experimental Testing of an Optical Micro-Spectroscopic Technique Incorporating True Line-Scan Excitation. International Journal of Molecular Sciences. 2014; 15(1):261-276.

Chicago/Turabian Style

Biener, Gabriel; Stoneman, Michael R.; Acbas, Gheorghe; Holz, Jessica D.; Orlova, Marianna; Komarova, Liudmila; Kuchin, Sergei; Raicu, Valerică. 2014. "Development and Experimental Testing of an Optical Micro-Spectroscopic Technique Incorporating True Line-Scan Excitation." Int. J. Mol. Sci. 15, no. 1: 261-276.



Int. J. Mol. Sci. EISSN 1422-0067 Published by MDPI AG, Basel, Switzerland RSS E-Mail Table of Contents Alert