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

International Journal of Molecular Sciences

Gabriel Biener²  Michael R. Stoneman³  Gheorghe Acbas²  Jessica D. Holz²  Marianna Orlova¹  Liudmila Komarova²  Sergei Kuchin¹ 

[1] Department of Biological Sciences, University of Wisconsin–Milwaukee, Milwaukee, WI 53201, USA; E-Mails:;Physics Department, University of Wisconsin–Milwaukee, Milwaukee, WI 53211, USA; E-Mails:;Aurora Spectral Technologies, Milwaukee, WI 53211, USA; E-Mail:

关键词: optical micro-spectroscopy;    fluorescence;    two-photon excitation;    multi-photon excitation;    energy transfer;   

DOI  :  10.3390/ijms15010261

来源: mdpi

【 摘 要 】

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.

【 授权许可】

CC BY   
© 2014 by the authors; licensee MDPI, Basel, Switzerland