ArticleInternational journal of molecular sciences2013
Development and experimental testing of an optical micro-spectroscopic technique incorporating true line-scan excitation.
Article in International journal of molecular sciences, 2013. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 42 papers.
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42 citing papers in PubMed.
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- Dielectric Spectroscopy Based Detection of Specific and Nonspecific Cellular Mechanisms.Sensors (Basel, Switzerland) · 2021Article
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- Quantifying the strength of heterointeractions among receptor tyrosine kinases from different subfamilies: Implications for cell signaling.The Journal of biological chemistry · 2020Article
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- A general method to quantify ligand-driven oligomerization from fluorescence-based images.Nature methods · 2019Article
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8 authors.
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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.
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