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in vivo monitoring of receptor engagement by near-infrared FRET fluorescence lifetime imaging using the phasor approach

16 Apr | By Xavier Michalet
in vivo monitoring of receptor engagement by near-infrared FRET fluorescence lifetime imaging using the phasor approach
Progressive accumulation of doubly labeled transferrin receptor dimers in the liver (left, red) is monitored in real time by following the evolution of the corresponding points in the phasor plot (right, black arrow). Inset shows the extracted FRET fraction in both the urinary bladder (green) and liver (red) as a function of time (in min). The vertical arrow indicates the time when acceptor-labeled Tf was injected in the mouse. The mouse image on the left shows the location of pixels whose phasors are circles in the phasor plot (red: liver, green: urinary bladder)
Image source: authors
By: Sez-Jade Chen, Nattawut Sinsuebphon, Alena Rudkouskaya, Margarida Barroso, Xavier Intes, Xavier Michalet

Researchers at the Rensselaer Polytechnic Institute, Albany Medical College and UCLA have reported a novel approach to monitor molecular interactions in vivo. The method relies on Fӧrster resonant energy transfer (FRET) between matching fluorescent dyes (the donor and the acceptor dye), which only occurs when the dyes are within a few nanometers from one another. They used this phenomenon to monitor the binding of a small ligand molecule (transferrin, Tf, involved in iron transport), to its receptor (TfR), which is present in the membrane of all cells as a dimer, but is preferentially found in actively replicating tissues such as the liver and tumors. Upon binding of two Tf molecules, the TfR dimers are internalized, making this system both a model of ligand-receptor interaction and an attractive method for drug delivery.

In their experiments, the authors first injected near-infrared (NIR) donor-labeled Tf into the blood stream of live mice, and observed the accumulation of fluorescence in most tissues, but preferentially in the liver and the urinary bladder (UB). A few minutes later, they injected NIR acceptor-labeled Tf and observed shortening of the lifetime of the donor fluorescence in the liver, while no change was observed elsewhere. This observation is consistent with the binding of both donor- and acceptor-labeled Tf molecules on TfR dimers in the liver. The absence of noticeable changes elsewhere, and in particular in the UB, means that the donor and acceptor molecules are not in close proximity from one another, and therefore are not bound to TfR in significant numbers.

Similar experiments had been previously reported by the Intes and Barroso labs, but the novelty of this report resides in the analysis used. Instead of using standard fluorescence decay fitting, which is time-consuming and requires high signal-to-noise ratio, the authors used a much faster and robust method, first introduced in fluorescence lifetime imaging microscopy (FLIM) by Gratton and collaborators, the phasor approach. In this method, the fluorescence decay at each pixel after excitation with a pulsed laser is reduced to two Fourier coefficients (g and s), and represented as a single point (g, s), the phasor. The corresponding phasors are then histogrammed, comprising the so-called phasor plot (figure, right panel). This simple transformation of decays into points has many useful properties, the most important being that, after proper calibration, fluorescence decays which are single exponential functions are located on a semi-circle (the so-called universal circle, UC, shown in black in the figure). Decays with short lifetimes are located close to the (1,0) point, while decays with long lifetimes are located close to the (0, 0) point, providing a very simple visual representation of potentially complex decays.

In their experiments, the authors took advantage of another property of the phasor analysis: the phasor of the sum of two decays (i.e. a mixture of two decays) with lifetime τ1 and τ2, is located along the segment connecting the two phasors of the pure τ1 and τ2 decays, each one located on the UC. This makes it possible to very easily infer the fraction of 1 and 2 present at each location, by simply measuring the position of the phasor along the segment.

In these in vivo experiments, “decay 1” was the pure donor decay, observed at the beginning of the experiment, when no acceptor was present, while “decay 2” corresponded to both donor and acceptor Tf bound to TfR, and was characterized by a much shorter donor lifetime, as expected from donor fluorescence quenching induced by FRET. Over time, the phasors of pixels in the liver visibly migrated from donor-only toward FRET-only, without any need for further processing. This visual nature of phasor analysis (and the fact that it can be computed almost instantaneously) makes it a very powerful method to follow the pharmacokinetics of drug delivery. Importantly, the method can also be quantitative, as illustrated by the kinetic curve shown as an inset in the figure, representing the fraction of FRET pairs detected in both urinary bladder (green: none) and liver (red: exponential increase over time).

In summary, a new rapid and powerful method to monitor molecular interactions by FLI-FRET in vivo using short lifetime near-infrared dyes was demonstrated, which should make this approach attractive for many researchers. 

The research was published in the March issue of the Journal of Biophotonics (doi: 10.1002/jbio.201800185)

Tags:
flim, fret, in vivo, phasor

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