Comprehensive quantitative evaluation of FLIM-FRET microscopy

Horst Wallrabe; Yuangsheng Sun; Zdenek Svindrych; Ammasi Periasamy

doi:10.1117/12.2180162

5 March 2015 Comprehensive quantitative evaluation of FLIM-FRET microscopy

Horst Wallrabe, Yuangsheng Sun, Zdenek Svindrych, Ammasi Periasamy

Proceedings Volume 9329, Multiphoton Microscopy in the Biomedical Sciences XV; 93293A (2015) https://doi.org/10.1117/12.2180162
Event: SPIE BiOS, 2015, San Francisco, California, United States

Abstract

Average lifetime between the usually bi-exponential double-label specimen and a mono-exponential single donor sample serves as a basis for the calculation of the average energy transfer efficiency (E). This semi-quantitative approach however does not fully explore cellular functions, such as endosomal pH differences, specific morphological features, examining sub-populations and the like. We applied a different, quantitative Region-of-Interest (ROI)-based method in 2 live-cell assays by TCSPC FLIM-FRET microscopy: a 5 amino-acid linked FRET standard and mouse pituitary cells expressing a dimerized C/EBPα-bZip transcription factor in the nucleus, both tagged with Cerulean (C) and Venus (V). ROIs with different selection thresholds were generated and compared. Average lifetimes are similar, but ratios between them and other subtle differences are revealed by comprehensive distribution information. Following published references, we also explored 3 different methods to calculate FLIM-FRET energy transfer efficiencies for the Cerulean- Venus constructs, producing differences and supporting the long-held notion that E is called 'apparent' efficiency. FRET's greatest contribution continues to be exploring changes taking place at the cellular level and quantifying differences in relative terms between control and variables.

Citation Download Citation

Horst Wallrabe, Yuangsheng Sun, Zdenek Svindrych, and Ammasi Periasamy "Comprehensive quantitative evaluation of FLIM-FRET microscopy", Proc. SPIE 9329, Multiphoton Microscopy in the Biomedical Sciences XV, 93293A (5 March 2015); https://doi.org/10.1117/12.2180162

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