A single-laser source all-optical holographic microscope for brain investigation – SLALLOM

Optical means for stimulating and monitoring neuronal activity have provided a lot of insight in neurophysiology lately toward our understanding on how brain works. Optogenetic actuators, calcium or voltage imaging probes and other molecular tools combined with advanced microscopies allowed ‘all-optical’ readout and manipulation of neural circuits. Yet, important challenges remain to be overcome to achieve full optical neuronal control, concerning reliable delivery and expression of sensors and actuators in the same neurons, elimination of cross-talk between the imaging and manipulation channels, and achieving recording and manipulation each with single-neuron and single-action-potential precision. SLALLOM is a concerted attempt between two academic (Wavefront-Engineering Microscopy group; WEM, Neurophotonics Lab. and Lasers group, Charles Fabry Laboratory; LCF) and two industrial partners (Amplitude Systemes; AS, ALPhANOV) aiming to remedy the last two challenges.
The central idea of SLALLOM is to develop a novel single-light-source ‘all-optical’ two-photon computer-generated holography (CGH) microscope using an innovative frequency-converted dual-output directly diode pumped Thulium (Tm)-doped fiber amplifier for three-dimensional (3D) multicell excitation and monitoring.
The proposed laser system aims at the disruption of current laser technology used for 2-photon imaging and activation in optogenetic studies. Indeed, mature laser technologies suffer from drawbacks (e.g. lack of energy, absence of repetition rate tunability, excitation wavelength not matching the 2-photon absorption spectra peak of most molecular tools) that prevent their use for massive parallelization of neuron manipulation. We therefore propose a cutting-edge dual-branch ultrafast fiber laser system operating in the 920-975 nm wavelength range. This laser system steps away from conventional laser technologies (e.g. Ti:sapphire laser, Ytterbium fiber laser) and builds upon frequency upconversion of Tm-doped ultrafast fiber amplifiers seeded by a frequency-shifted Erbium oscillator. The two branch parameters will be optimized for their respective goal: imaging with >5 W, 40 MHz and 100 fs and photoactivation with >5 W, 10 µJ and 100 fs.
A 3D-CGH microscope appropriately modified for addressing a large excitation field, will be assembled together with a 2-photon scanning system for 3D structure or functional imaging of neuronal activity, with genetic reporters. The developed laser will be used as a single-laser source for both imaging and stimulation, aiming to treat the cross-talk between these modalities by exploiting the superior temporal resolution provided by CGH in combination with highly-efficient fast-kinetic opsins. The microscope will be used to follow brain complexity in the visual cortex in vivo at high spatiotemporal resolution.
The project, led with the WEM group, forerunner in developing advanced optical methods for neuronal stimulation, gathers specific and complementary skills from four partners whose expertise is recognized at international level. The WEM group has proposed about ten years ago the application of spatiotemporal light patterning with CGH and temporal focusing as a means of precisely parallel targeting cells groups, enabling photostimulation at high spatio-temporal precision. LCF is widely acknowledged as a major actor of the research in diode-pumped ultrafast lasers. ALPhANOV is a French technological center specialized in the development of innovative high-power fiber laser, especially for ultra-short pulse amplification. Finally, AS is the world leading company providing integrated, industrial-grade ultrafast laser systems, and has a long-standing collaboration with LCF through a common laboratory.
SLALLOM consortium will demonstrate a reliable ground-breaking ultrafast laser source adapted to a 3D-CGH microscope to study the brain activity in vivo at high spatiotemporal resolution with scientific and industrial outcomes.