A Multifocal Multiphoton Volumetric Imaging Technique for High Speed Time-Resolved FRET Imaging in vivo

Abstract

The use of Fluorescence lifetime imaging microscopy (FLIM) for studying spatio-temporal protein-protein interactions in situ through the detection of FRET between protein-bound fluorophores has been well-established. For intermolecular FRET a key advantage of using donor FLIM is that fluorescence-lifetime measurements of donor emission are independent of acceptor concentration and is thus suited to studies that explore biological interactions in intact cells and tissues.Compared to other techniques, the use of time correlated single photon counting to image fluorescence lifetime has become the gold standard for measuring FRET interactions in cells. However, due to its slow acquisition time (minutes) when compared with other techniques, such as Frequency-domain, time-gated FLIM or anisotropy, this method is impractical for measuring dynamic protein interactions in cells.We have previously presented a massively parallel, fully addressable time-resolved multifocal multiphoton microscope capable of producing fluorescence lifetime images with 55ps time-resolution giving improvements in acquisition speed of a factor of 64 improving the acquisition time to the order of seconds. Parallelised TCSPC detection was achieved using a specialised 32x32 10-bit time-domain counter array (∼55ps) with integrated low dark-count Single Photon Avalanche Photodiodes.In this paper, we discuss development of a multifocal multiphoton system where 4 individual axial planes are acquired simultaneously, allowing acquisition of volumetric data without sequential scanning at different focal positions. Using a spatial light modulator a holographic beamlet array is projected within the sample volume with defined axial plane separation. We demonstrate live FRET imaging of proteins in the ErbB family of receptors in cancer cells and discuss possible applications for the technique. Whilst only 4 planes are demonstrated, this technique can be extended to a larger number of focal planes enabling full 3D acquisition and reconstruction with modest constraints.