Intravital FRET: Probing Cellular and Tissue Function in Vivo

Abstract

The development of intravital Förster Resonance Energy Transfer (FRET) is required to probe cellular and tissue function in the natural context: the living organism. Only in this way can biomedicine truly comprehend pathogenesis and develop effective therapeutic strategies. Here we demonstrate and discuss the advantages and pitfalls of two strategies to quantify FRET in vivo—ratiometrically and time-resolved by fluorescence lifetime imaging—and show their concrete application in the context of neuroinflammation in adult mice.

A simple approach for measuring FRET in fluorescent biosensors using two-photon microscopy

“…The ratio images obtained from biosensors will be minimally affected by depth provided that the scattering and absorption does not vary significantly for the emission wavelengths of the probe. While we find no evidence of a significant effect of depth on FRET measurements obtained in vivo, minor effects were observed in other studies 32 . Therefore, biosensor measurements collected over a large range of depths should be evaluated for systematic effects of depth on the FRET ratio.…”

Intravital microscopy of biosensor activities and intrinsic metabolic states

“…Measurements collected at greater depths, however, will suffer from signal degradation, and wavelength-dependent differences in scattering can introduce depth-dependent effects on ratiometric measurements collected in biological tissues [6,7]. We find that useful signal in both the blue (480 nm) and yellow (530 nm) channel diminish before there is an obvious wavelength-dependent effect on the emission ratio, but others demonstrated minor effects of depth on ratiometric FRET measurements [7].…”

“…When imaging in highly scattering biological tissues, both the excitation light and the emission signal are attenuated in a wavelength- and depth-dependent fashion. This reduces the effective excitation power at the focal plane, while creating a higher detection threshold [6,7]. The loss of signal prevents the direct comparison of intensities acquired at different depths in a tissue.…”

New dimensions in intravital multi-photon imaging of immune reactions

“…Emission anisotropy measured at different polarized excitation provides information of rotational diffusion being sensitive to size, shape, orientation and motion of the molecules. A bright illustration of intravital application of FRET technology has been shown using Ca 2+ imaging of brain in transgenic mice CerTN L15, which contain a troponin-C FRET-based calcium biosensor [52][53][54] . Referring to cells of the immune system, FRET-based indicators of intracellular calcium have been used to measure lymphocyte activation …”