Future Perspective of Single-Molecule FRET Biosensors and Intravital FRET Microscopy

Abstract

Förster (or fluorescence) resonance energy transfer (FRET) is a nonradiative energy transfer process between two fluorophores located in close proximity to each other. To date, a variety of biosensors based on the principle of FRET have been developed to monitor the activity of kinases, proteases, GTPases or lipid concentration in living cells. In addition, generation of biosensors that can monitor physical stresses such as mechanical power, heat, or electric/magnetic fields is also expected based on recent discoveries on the effects of these stressors on cell behavior. These biosensors can now be stably expressed in cells and mice by transposon technologies. In addition, two-photon excitation microscopy can be used to detect the activities or concentrations of bioactive molecules in vivo. In the future, more sophisticated techniques for image acquisition and quantitative analysis will be needed to obtain more precise FRET signals in spatiotemporal dimensions. Improvement of tissue/organ position fixation methods for mouse imaging is the first step toward effective image acquisition. Progress in the development of fluorescent proteins that can be excited with longer wavelength should be applied to FRET biosensors to obtain deeper structures. The development of computational programs that can separately quantify signals from single cells embedded in complicated three-dimensional environments is also expected. Along with the progress in these methodologies, two-photon excitation intravital FRET microscopy will be a powerful and valuable tool for the comprehensive understanding of biomedical phenomena.

Figure 1

Schematic of single-molecule FRET biosensors. Schematics of constructions and operating principles are shown for Cameleon (A), the general backbone of single-molecule FRET biosensors (B), and EKAREV-NLS (C). Note that the order of donor/acceptor FPs and sensor/ligand domains from the N-terminus (N′) to the C-terminus (C′) is arbitrary, and optimization is required for each biosensor. LS, localization signal.

Figure 2

Application of a FRET biosensor to 3D live imaging. (A) Representative kymographs of ERK activity in melanoma cells and MAFs. Cells stably expressing EKAREV-NLS were embedded in 3D gels, treated with a BRAF inhibitor at the indicated time point (black arrowhead), and imaged for 13 h in total. Shown are pseudo-colored ratiometric FRET images depicted with an intensity-modulated display (IMD) mode of 8-ratio 32-intensity. High ERK activities are depicted in red (warm) hues and low activities in blue (cold) hues. (B) WM266.4 human melanoma cells stably expressing EKAREV-NLS were subcutaneously injected into nude mice and treated with DMSO (control) or PLX4720 (BRAF inhibitor) for 13 and 11 days, respectively. Shown are ratiometric FRET images merged with signals of second harmonic generation (white). Please note that cells treated with BRAF inhibitors still exhibit high levels of ERK activities within reorganized nest-like structures consisting of thick collagen fibers. Scale, 200 μm. (This image was modified from Hirata et al. (42))

Figure 3

Aspiration fixation system for mouse intravital imaging. (A) Overview of the aspiration fixation system, which consists of a stage (a), a chamber dish (b), a cover glass (c), an arm for x-y adjustment (d), a height adjustment device (e), and an aspiration tube (f), which is connected to a pump. (B) Intravital intestinal imaging of an anesthetized mouse. The red arrow indicates the mouse intestine appressed beneath the coverglass by negative pressure. (C) Schematic of the aspiration fixation system.

Figure 4

Cancer dormancy in brain tissue. (A) A schematic illustration of mouse intravital FRET microscopy in the future. (B and C) Cancer cells were injected into the left ventricle of a nude mouse and the brain was excised after 32 days. The brain was made transparent by the tissue-clearing CUBIC method (75), stained with first/second antibodies, and imaged under a two-photon excitation microscope. The white rectangle in (B) is magnified in (C). Red, cancer cells; green, blood vessels; cyan, activated astrocytes. Scale, (B) 1 mm; (C) 100 μm.