Four-dimensional multi-site photolysis of caged neurotransmitters

Abstract

Neurons receive thousands of synaptic inputs that are distributed in space and time. The systematic study of how neurons process these inputs requires a technique to stimulate multiple yet highly targeted points of interest along the neuron's dendritic tree. Three-dimensional multi-focal patterns produced via holographic projection combined with two-photon photolysis of caged compounds can provide for highly localized release of neurotransmitters within each diffraction-limited focus, and in this way emulate simultaneous synaptic inputs to the neuron. However, this technique so far cannot achieve time-dependent stimulation patterns due to fundamental limitations of the hologram-encoding device and other factors that affect the consistency of controlled synaptic stimulation. Here, we report an advanced technique that enables the design and application of arbitrary spatio-temporal photostimulation patterns that resemble physiological synaptic inputs. By combining holographic projection with a programmable high-speed light-switching array, we have overcome temporal limitations with holographic projection, allowing us to mimic distributed activation of synaptic inputs leading to action potential generation. Our experiments uniquely demonstrate multi-site two-photon glutamate uncaging in three dimensions with submillisecond temporal resolution. Implementing this approach opens up new prospects for studying neuronal synaptic integration in four dimensions.

Low-cost photo-responsive nanocarriers by one-step functionalization of flame-made titania agglomerates withl-Lysine

“…50,51 These results are in line with recent cytotoxicity studies on organic-coated TiO 2 agglomerates. No immediate toxic effect of the injected FITC-lysine-TiO 2 was observed.…”

Improved two-photon imaging of living neurons in brain tissue through temporal gating

“…Our work highlights the use of temporal gating for imaging fluorescence from living biological samples where cell viability and photodamage are issues of major concern. Ongoing studies are directed towards integrating the temporal gating technique with our existing 2P microscope [10,11] for multisite photochemical stimulation of neurons.…”

“…The custom-built 2P microscope used in the experiments is part of system described in our previous works [10,11]. Figure 1(a) describes the basic geometry of the system.…”

“…In neuroscience, 2P microscopes have been utilized for manipulation of neuronal activity, as well as for imaging neuronal activity using fluorescent indicators, both in vitro and in vivo [7][8][9]. With this technique, non-invasive photochemical stimulation of neurons can be achieved using methods such as photolysis of caged neurotransmitters, [10][11][12] Utilizing the 2P technique for imaging live biological samples also requires obtaining high fluorescence yield with reduced photobleaching of fluorophores. The biochemical processes in the cells together with the photochemical properties of the fluorophores result in photobleaching and reduced fluorescence signal during imaging.…”

Using light to probe neuronal function

“…Phase-only SLMs can also be used with the holographic projection technique where a computer-generated phase hologram produces reconfigurable patterned illumination in 3D. This technique has been demonstrated for 1PE [61] and 2PE [62][63][64] patterned uncaging of neurotransmitters in a plane. The holographic technique has also been used to simultaneously induce arbitrary synaptic inputs in 3D [63].…”