Fluorescence lifetime imaging nanoscopy for measuring Förster resonance energy transfer in cellular nanodomains

Abstract

Microscopy methods used to measure Förster resonance energy transfer (FRET) between fluorescently labeled proteins can provide information on protein interactions in cells. However, these methods are diffraction-limited, thus do not enable the resolution of the nanodomains in which such interactions occur in cells. To overcome this limitation, we assess FRET with an imaging system combining fluorescence lifetime imaging microscopy with stimulated emission depletion, termed fluorescence lifetime imaging nanoscopy (FLIN). The resulting FRET-FLIN approach utilizes immunolabeling of proteins in fixed cultured neurons. We demonstrate the capacity to discriminate nanoclusters of synaptic proteins exhibiting variable degrees of interactions with labeled binding partners inside dendritic spines of hippocampal neurons. This method enables the investigation of FRET within nanodomains of cells, approaching the scale of molecular signaling. © The Authors.Published by SPIE under a Creative Commons Attribution 4.0 Unported License. Distribution or reproduction of this work in whole or in part requires full attribution of the original publication, including its DOI. Results FLIN Microscope SystemWe built a STED microscope with additional time-correlated single-photon counting (TCSPC) capacity to perform FRET-FLIN measurements in cultured hippocampal neurons (Sec. 4). 10,13,14 This system could perform STED imaging of ATTO 594 and confocal imaging of ATTO 647N. In STED nanoscopy, the resolution is improved using a donut-shaped depletion beam that typically lasts a few hundreds of picoseconds to ensure optimal depletion of the fluorescence

Imaging the Neuroimmune Dynamics Across Space and Time

“…Similar to other commonly used fluorescence microscopy approaches, STED involves the detection of fluorescence by point-scanning. A doughnut-like shape beam is used to deplete surrounding fluorophores in order to only read the fluorescence of the fluorophore of interest ( Hell and Wichmann, 1994 ; Tardif et al, 2019 ) STED microscopy’s nanoscale resolution (65 nm in x-y and 150 nm in z) resulted in multiple applications, including the structural imaging of microglial and synaptic dynamics in fixed and live thick brain slices ( Hein et al, 2008 ; Chéreau et al, 2015 ). Recent progresses further allowed to generate the first STED able to image efficiently in vivo , by adding components of a two-photon system.…”

A Guide to Fluorescence Lifetime Microscopy and Förster's Resonance Energy Transfer in Neuroscience

“…For example, in a recent report, a wide‐field TCSPC‐based fluorescence lifetime imaging was combined with a light‐sheet illumination configuration for rapid 3D lifetime imaging (Hirvonen et al., 2020). In another recent study, a STED super‐resolution microscope was augmented with TCSPC instrumentation to perform high‐resolution FLIM‐FRET measurements in cultured hippocampal neurons (Tardif et al., 2019). Finally, in our lab, FLIM‐FRET microscopy has been combined with time‐resolved anisotropy and fluorescence correlation spectroscopy (FCS) to enhance the ability to detect protein conformational changes (Nguyen et al., 2015; Sarkar et al., 2017; Thaler et al., 2009).…”

Graduate programs in biophotonics: unique transdisciplinary training in applied photonics for the life sciences

“…3 Projects involving three trainees of the biophotonics programs in the team of Prof. P. De Koninck (Biochemistry) developed innovative methods using Fluorescence Resonance Energy Transfer (FRET) and Fluorescence Lifetime Imaging Microscopy (FLIM) and super-resolution microscopy for the study of synaptic protein interactions. 4 The team of Prof. A. Saghatelyan (Neuroscience) in collaboration with the team of Prof. D. Côté (Physics) employed in vitro and in vivo two-photon microscopy for the study of spine relocation of adult-born interneurons in the olfactory bulb. 5 The collaboration between the teams of Prof. D. Côté (Physics) and Prof. Y.…”