FLIM

The FEMTOSmart for FLIM

The FEMTOSmart Galvo upgraded with the FLIM extension is suitable for performing in vivo and in vitro 2P-FLIM studies in a wide range of model organisms. In this complex system, up to two hybrid detectors (combination of GaAsP cathode and an avalanche detector) with two photon counting channels provide a high detection efficiency and a fast timing response, with less noise. As afterpulsing is essentially absent in these detectors, the dynamic range of fluorescence decay recordings is considerably increased. The special scanning modes and patterns of the FEMTOSmart Galvo provide high spatiotemporal resolution during measurements. The setup is perfectly suitable to monitor glutamate release and intracellular calcium concentration, to perform FRET measurements, or to quantify the ratio between reduced and oxidized NADH and FAD molecules, in the research of neurodegenerative diseases or to reveal metabolic changes during tumorigenesis in cancer research.

Tornado imaging for the direct monitoring of single-quantum, single-synapse glutamate release in situ

A FEMTOSmart Galvo with a FLIM extension is suitable to follow single action potential-evoked presynaptic Ca²⁺ dynamics, with nanomolar sensitivity, in individual small, presynaptic boutons in acute brain slices. Jensen et al., from the laboratory of Prof. Dmitri Rusakov, have advanced a two-photon excitation FLIM technique. They used the flexible Tornado scanning mode of our galvanometric scanner-based microscope, which provides pixel sampling in a spiral, recording the signal’s spatiotemporal dynamics more favourably, and improves the signal-to-noise ratio. With this method, they performed time-resolved imaging of Ca²⁺ sensitive fluorescence lifetime of Oregon Green BAPTA-1. In parallel, intensity Tornado imaging made it possible to monitor single-quantum, single-synapse glutamate release in situ directly, using the locally expressed extracellular optical glutamate sensor iGluSnFr. This study paves the way for simultaneous registration of presynaptic Ca²⁺ dynamics and transmitter release in an intact brain at the level of individual synapses.

(Related article: Monitoring single-synapse glutamate release and presynaptic calcium concentration in organised brain tissue, Thomas P. Jensen, Kaiyu Zheng, Olga Tyurikova, James P. Reynolds, Dmitri A. Rusakov, Cell Calcium, 2017)

FLIM imaging reveals that distinct sites in individual boutons release glutamate in a probabilistic fashion

In a subsequent publication from the same laboratory, Rama et al. describe how they used the FEMTOSmart Galvo with the FLIM extension to monitor spike-evoked glutamate release and presynaptic calcium entry in the mossy fiber boutons of granule cells, with the use of the optical glutamate sensor SF-iGluSnFR and the intracellular Ca²⁺ indicator Cal-590, and the above mentioned Tornado scanning mode. These presynaptic boutons feature multiple release sites and are densely packed with synaptic vesicles to sustain a highly facilitating “detonator” transmission. In this study, multiplexed imaging showed that distinct sites in individual boutons release glutamate in a probabilistic fashion, while also showing use-dependent short-term facilitation. The results help to reveal whether glutamate release sites act independently or rather synchronously, and to understand the fundamentals of synaptic transmission and hippocampal network function in a wider context.

(Related article: Glutamate Imaging Reveals Multiple Sites of Stochastic Release in the CA3 Giant Mossy Fiber Boutons, Sylvain Rama, Thomas P. Jensen, Dmitri A. Rusakov, Frontiers in Cellular Neuroscience, 2019)