Effects of tissue autofluorescence on FRET efficiency estimates

D. J. Pleshinger; Naga Annamdevula; Silas J. Leavesley; Thomas C. Rich

doi:10.1117/12.2583284

5 March 2021 Effects of tissue autofluorescence on FRET efficiency estimates

D. J. Pleshinger, Naga Annamdevula, Silas J. Leavesley, Thomas C. Rich

Proceedings Volume 11649, Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XXVIII; 1164919 (2021) https://doi.org/10.1117/12.2583284
Event: SPIE BiOS, 2021, Online Only

Conference Poster

Abstract

F¨orster resonance energy transfer (FRET) is a tool used for studying various biological process as well as for measuring molecular distances. This process can occur when the emission spectrum of the donor fluorophore overlaps with the excitation spectrum of the acceptor, and the fluorophores are in close enough distance for the energy to pass from donor to acceptor non-radiatively. The efficiency of this energy transfer is dependent on the distance and orientation of the fluorophores, in addition to their overlapping spectra. Here we present a study to assess the impact of tissue autofluorescence on estimates of FRET efficiency and fluorophore abundance within experimental cellular images in tissue. To accomplish this we performed a theoretical sensitivity analysis on FRET rat kidney control images with varying ranges of donor and acceptor fluorophores to observe their pixel by pixel responses. In the experimental data, the donor was the Turquoise fluorescent protein and the acceptor fluorophore was the Venus fluorescent protein. Detection of the acceptor was more difficult due to its excitation spectrum closely resembling the autofluorescence spectrum from the base image while the emission spectrum of the Turquoise donor was more unique and easier to detect. In addition, variable FRET efficiencies were added to data at different fluorophore levels to compare the visible abundance of FRET to the autofluorescence in the resultant images. This analysis can benefit future work by furthering the understanding of the donor and acceptor concentrations needed for strong FRET measurements.

Conference Presentation

Citation Download Citation

D. J. Pleshinger, Naga Annamdevula, Silas J. Leavesley, and Thomas C. Rich "Effects of tissue autofluorescence on FRET efficiency estimates", Proc. SPIE 11649, Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XXVIII, 1164919 (5 March 2021); https://doi.org/10.1117/12.2583284

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