Physiological fluorescence lifetime imaging microscopy improves Förster resonance energy transfer detection in living cells

Abstract

Abstract. Accurate, unambiguous detection of molecular interactions in living cells via measurements of Förster ͑or fluorescence͒ resonance energy transfer ͑FRET͒ events is experimentally challenging. We develop and apply a physiological fluorescence lifetime imaging microscopy ͑physiological FLIM͒ system to significantly improve FRET detection in living cells. Multiple positive and negative cellular controls are implemented to validate the experimental method developed. FLIM measurement techniques were found to remove fluorescence intensity-based artifacts, resulting in a seven-fold improvement in fluorescence measurement precision. The addition of cellular environmental controls, including both temperature and CO 2 stabilization, for physiological FLIM eliminates nonspecific FRET in the live-cell system studied. Overall, only physiological FLIM results in statistically significant results that clearly indicated the presence of specific molecular interactions in the live-cell system. This approach can be applied generally to improve the accuracy and precision of FRET measurements in living cells. Förster ͑or fluorescence͒ resonance energy transfer ͑FRET͒ can be used as an in vivo nanoscale ruler to detect the distance between fluorophores or interacting proteins with attached fluorophores.1 In intensity-based FRET detection, sophisticated calibration and correction procedures are usually required to cope with intensity variations, spectral bleedthrough, 2 back-bleedthrough, 3 and photobleaching. 4 In addition, nonspecific FRET, arising from random collisions of donor and acceptor and/or other unexpected associations of donor and acceptor, affects FRET accuracy, and although it can be detected, it may not be easily removed. 5Fluorescence lifetime imaging microscopy ͑FLIM͒ retrieves fluorophore lifetime data from fluorescence emission, and can be used to detect FRET with some advantages over intensity-based FRET.6 Interest in FLIM-FRET is growing, especially as commercial FLIM modules become available for confocal and multiphoton microscopy. However, accurate, unambiguous detection of molecular interactions in living cells remains experimentally challenging due to the presence of nonspecific FRET.In this study, we develop and apply a physiological FLIM system with experimental controls for CO 2 and temperature stabilization to create a physiological environment for livecell FLIM studies. Our purpose was to investigate how physiological FLIM affects FRET detection in living cells. To accomplish this, we studied a live-cell system involving Ras Homology Protein C ͑RhoC͒ inactive-form interactions. RhoC has been identified as a specific oncogene marker of aggressive breast cancers.7 When active, it is anchored on the cell membrane, interacting with effectors. When inactive, it is bound to its inhibitor RhoGDI␥ in the cytoplasm.8 Interactions between RhoGDI␥ and RhoC caused FRET to occur from the donor ͑cerulean, attached to RhoGDI␥͒ to the acceptor ͑citrine, attached to RhoC͒ in our experimental cellular group, as i...

Vinculin tension distributions of individual stress fibers within cell-matrix adhesions

“…Consistent with our previous studies, SF irradiation led to retraction of the severed ends of the targeted SF (top row), and inspection of the donor and FRET intensity (middle rows) revealed that the VinTS localizes to FAs as expected. Because the FRET intensity also depends on factors unrelated to donoracceptor separation (Chang and Mycek, 2012;Chang et al, 2007;Chang et al, 2009;Zhong et al, 2007), such as the amount of localized VinTS, we created FRET ratio maps in which we masked the FRET intensity to show only FAs and normalized this intensity by the donor intensity (bottom row). In these maps, an increasing FRET ratio corresponds to an increasing FRET signal (actual degree of energy transfer) or decreasing tension, which can in turn be tracked on an FA-by-FA basis.…”

Enhancing precision in time-domain fluorescence lifetime imaging

“…24 In addition, this system had a large temporal dynamic range ͑750 ps to ϱ͒, 50 ps lifetime discrimination, and spatial resolution of 1.4 m, which made it very suitable for studying a variety of endogenous and exogenous fluorophores in biological samples. 2,4,[25][26][27][28] Fluorescence lifetime maps were determined by first acquiring fluorescence intensity images at four delays and then calculating the lifetime values from the intensity images on a pixel-by-pixel basis ͑described in Sec. 3.2͒.…”

“…1 However, low fluorescence signals from biological samples can be a challenge, causing poor lifetime precision, and this will affect, to a great extent, the quantitative applications of FLIM, such as the detection of Förster resonance energy transfer for molecular interactions and the sensing of fluorophore microenvironments. [2][3][4][5] When endogenous fluorophores are imaged, low fluorescence signals may result from low intrinsic fluorophore concentrations and/or unfavorable optical properties of fluorophores ͑e.g., fluorescence excitation and emission wavelengths, quantum yield, photobleaching rate͒. When exogenous fluorophores are imaged, low signals can result from the low fluorophore concentrations that are required to minimize effects on sample physiology and/or from the low transfer efficiency of fluorophores/fluorophore precursors ͑genes or different forms of fluorophores͒ from extracellular media into live cells.…”

Extending Förster resonance energy transfer measurements beyond 100 Å using common organic fluorophores: enhanced transfer in the presence of multiple acceptors

“…The detection and resolution limit depends on many variables. 1,6,7,36,38 We are certain that for any given system, the use of multiple, rather than single, acceptors will enhance the transfer efficiency and allow for larger distances to be measured. There are also several commercially available reactive Near Infra Red probes that can be used to create a R 0 of about 80 to 85 Å.…”

“…For larger donor-acceptor separations, potential artifacts and errors that are typically present in experiments can easily overwhelm the expected effects, especially when employing cellular microscopy. 6,7 Recently available reactive forms of far-red probes with both high extinction coefficients and good quantum yields can extend the practical R 0 to about 80 Å and the observation window to around 110 to 120 Å. 8,9 Presently, one typically labels specific sites using a single donor on a given macromolecule and a single acceptor on another macromolecule in order to monitor intermolecular distances during interactions (e.g., physiological processes).…”