Two-photon (2-P) all-optical approaches combine in vivo 2-P calcium imaging and 2-P optogenetic modulations. Here, firstly, we combined in vivo juxtacellular recordings and GCaMP6f-based 2-P calcium imaging in mouse visual cortex to tune our detection algorithm towards a 100% specific identification of action potential-related calcium transients. Secondly, we minimized photostimulation artifacts by using extended-wavelength-spectrum laser sources for optogenetic stimulation. We achieved artifact-free all-optical experiments performing optogenetic stimulation from 1100 nm to 1300 nm. Thirdly, we determined the spectral range for maximizing efficacy until 1300 nm. The rate of evoked transients in GCaMP6f/C1V1-co-expressing cortical neurons peaked already at 1100 nm. By refining spike detection and defining 1100 nm as the optimal wavelength for artifact-free and effective GCaMP6f/C1V1-based all-optical physiology, we increased the translational value of these approaches, e.g., for the development of network-based therapies.

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A wavelength tunable femtosecond optical parametric oscillator pumped by the second harmonic of a Yb: KGW solid state oscillator was investigated. The intracavity group delay dispersion was positive, and soliton condition was satisfied by introducing negative nonlinearity from cascaded quadratic nonlinearity (CQN). Two different approaches were investigated – CQN induced by the same amplifying nonlinear crystal or CQN induced by an additional second harmonic generating nonlinear crystal inside the same resonator. The second crystal was shown to correct the resonator misalignment induced by the rotation of the amplifying crystal as the wavelength was tuned in the range of 770-970 nm. It simultaneously compensated positive resonator GDD offsets of +/- 1000 fs2 with +/- 5% SHG power losses, simulating a method for compensation of GDD ripples in a broadband mirror.

Three synchronously pumped optical parametric oscillators (SPOPO's) based on different nonlinear optical materials are used to show the viability of Yb:KGW lasers for generation of pump radiation. Periodically poled lithium niobate (PPLN) is a convenient nonlinear optical material to begin the investigation, and typical of PPLN interesting additional phenomena are observed. Optical parametric amplification proves to be a useful technique for establishing SPOPO operation in lithium triborate (LBO) and beta barium borate (BBO) with a lower gain than PPLN. Numerical modelling helps in the analysis of SPOPO performance and indicates possible directions for future development such as non-collinear propagation to mitigate group velocity differences. The compact construction and efficient operation of femtosecond Yb:KGW lasers provide a favourable source of pump radiation for SPOPO's in these preliminary investigations.

contributing first authors One Sentence Summary: We maximize translational relevance of 2-P all-optical physiology by increasing specificity, minimizing artifacts and optimizing stimulation efficacy. Abstract:Two-photon (2-P) all-optical approaches combine in vivo 2-P calcium imaging and 2-P optogenetic modulations and have the potential to build a framework for network-based therapies, e.g. for rebalancing maladaptive activity patterns in preclinical models of neurological disorders. Here, our goal was to tailor these approaches for this purpose: Firstly, we combined in vivo juxtacellular recordings and GCaMP6f-based 2-P calcium imaging in layer II/III of mouse visual cortex to tune our detection algorithm towards a 100 % specific identification of APrelated calcium transients. False-positive-free detection was achieved at a sensitivity of approximately 73 %. To further increase specificity, secondly, we minimized photostimulation artifacts as a potential source for false-positives by using extended-wavelength-spectrum laser sources for optogenetic stimulation of the excitatory opsin C1V1. We achieved artifact-free all-optical experiments performing photostimulations at 1100 nm or higher and simultaneous calcium imaging at 920 nm in mouse visual cortex in vivo. Thirdly, we determined the spectral range for maximizing efficacy of optogenetic control by performing 2-P photostimulations of individual neurons with wavelengths up to 1300 nm. The rate of evoked transients in GCaMP6f/C1V1-coexpressing cortical neurons peaked already at 1100 nm. By refining spike detection and defining 1100 nm as the optimal wavelength for artifact-free and effective stimulations of C1V1 in GCaMP-based all-optical interrogations, we increased the translational value of these approaches, e.g. for the use in preclinical applications of network-based therapies.