Proposal of a new method to measure FRET quantitatively in living or fixed biomedical specimens on a laser microscope

P. Johannes Helm; Ole Petter Ottersen M.D.

doi:10.1117/12.874073

22 February 2011 Proposal of a new method to measure FRET quantitatively in living or fixed biomedical specimens on a laser microscope

P. Johannes Helm, Ole Petter Ottersen M.D.

Author Affiliations +

Proceedings Volume 7903, Multiphoton Microscopy in the Biomedical Sciences XI; 790331 (2011) https://doi.org/10.1117/12.874073
Event: SPIE BiOS, 2011, San Francisco, California, United States

Abstract

"Förster Resonance Energy Transfer", abbreviated "FRET", is a fluorescence phenomenon, which can be used to study and map co-localizations and dynamics of co-localizations at nanometer precision on a light microscope. FRET has been described as a "spectroscopic ruler". The efficiency of the radiationless energy transfer from an excited chromophore, the "donor", to another chromophore, the "acceptor", the excitation energy of which approximately matches the energy to be released by the donor, is dependent on the sixth power of the mutual distance between the two molecules in space. We propose a new, non-destructive technique for measuring FRET quantitatively and at high spatial and temporal resolution on a laser scanning microscope: Two laser beams of wavelengths suitable for the mutually exclusive excitation of the donor and the acceptor, the "donor beam" and the "acceptor beam", respectively, are intensity modulated by means of two electro optical modulators (EOM). The modulation patterns are rectangular at duty cycle 1/2. The modulation frequencies differ slightly. The acceptor beam is saturating the acceptor so that it cannot accept energy from the donor. The saturation is modulated in the same way as the acceptor beam. Since the donor beam also is modulated, though at a frequency slightly different from that of the acceptor beam, the intensity of the released donor fluorescence is modulated with the beat frequency of the frequencies of the two laser beam modulations and can be detected and interpreted in quantitative terms by means of a lock in amplifier.

Citation Download Citation

P. Johannes Helm and Ole Petter Ottersen M.D. "Proposal of a new method to measure FRET quantitatively in living or fixed biomedical specimens on a laser microscope", Proc. SPIE 7903, Multiphoton Microscopy in the Biomedical Sciences XI, 790331 (22 February 2011); https://doi.org/10.1117/12.874073

ACCESS THE FULL ARTICLE

INSTITUTIONAL
Select your institution to access the SPIE Digital Library.

SELECT YOUR INSTITUTION

PERSONAL
Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.

PERSONAL SIGN IN

No SPIE Account? Create one

PURCHASE THIS CONTENT

SUBSCRIBE TO DIGITAL LIBRARY

50 downloads per 1-year subscription

Members: $195

Non-members: $335 ADD TO CART

25 downloads per 1 - year subscription

Members: $145

Non-members: $250 ADD TO CART

PURCHASE SINGLE ARTICLE

Includes PDF, HTML & Video, when available