Image-based adaptive optics for in vivo imaging in the hippocampus

International audienceAdaptive optics is a promising technique for the improvement of microscopy in tissues. A large palette of indirect and direct wavefront sensing methods has been proposed for in vivo imaging in experimental animal models. Application of most of these methods to complex samples suffers from either intrinsic and/or practical difficulties. Here we show a theoretically optimized wavefront correction method for inhomogeneously labeled biological samples. We demonstrate its performance at a depth of 200 μm in brain tissue within a sparsely labeled region such as the pyramidal cell layer of the hippocampus, with cells expressing GCamP6. This method is designed to be sample-independent thanks to an automatic axial locking on objects of interest through the use of an image-based metric that we designed. Using this method, we show an increase of in vivo imaging quality in the hippocampus. In vivo imaging of neuronal calcium dynamics using two-photon microscopy is an increasingly used method of choice to study neuronal activity at microcircuit level. In the dorsal region CA1 of the hippocampus (the most optically accessible), this technique allows neuronal activity recording, in large fields of view containing hundreds of cells 1. It has led to pioneering discoveries of multineuron dynamics including, for example fear conditioning 2 , spatial navigation 3–5 , epilepsy 6 or quiet rest 7. However, the implementation of this technique remains challenging as it requires, prior to cranial window implantation, surgery to remove the overlaying cortex, which introduces a high variability of " optical access " to the tissue. The main issues are the presence of blood from the capillaries and sometimes from small hemorrhage as well as the quality of the interface between the glass window and the brain surface. The former causes optical absorption and can be reduced by performing the surgery following water restriction to increase the viscosity of the blood 1,5 , while the latter causes optical aberrations. Furthermore, the densely packed layer of CA1 pyramidal neurons is located 200 μ m below the glass window covering the brain; the incoming laser beam is also perturbed by light scattering and optical aberrations during the propagation within the tissue. This problem should be tackled in order to improve detection of calcium probes which is impaired by the lowered contrast of the aberrated images. Even a modest improvement in contrast should lead to the detection of neural activity that otherwise is masked by background fluorescence from brain tissue. Optical aberrations alter the quality of beam focusing, which in turn leads to reduced spatial resolution but also to lower signal and contrast. Thus, even when objects of interest are one order of magnitude larger than the diffraction limited laser focus (e.g. neurons' somata are 10–15 μ m in diameter), the reduction of optical aberrations is critical to increasing the contrast of the fluorescence images. This improvement can be achieved using adaptive optics, a promising tool increasingly used for microscopy 8. Adaptive optics is the process of quantifying optical aberrations through wavefront measurement and correcting them by the use of an adaptive correction element (deformable mirror DM or spatial light modulator SLM). Note that in point-scanning two-photon microscopy the correction is applied on the excitation beam alone and no correction is needed on the detection path. In such microscopes, the wavefront can either be directly measured or indirectly estimated. Direct wave-front measurement relies on introducing a wavefront sensor such as a Shack-Hartmann in the detection part of the microscope. A point source in the sample is then imaged on the sensor. Direct methods have been proposed for two-photon imaging in weakly scattering samples where auto-fluorescence signals can be used to generate a highly localized signal 9,10 , but more complex methods such as coherence gating 11 or near-IR guide stars 12 are required to avoid out-of-focus fluorescence in highly scattering samples. Indirect or sensorless wavefront estimation has the advantage of being easy-to-implement on existing systems as it relies on conventional imaging systems. Indeed, this technique, called image-based adaptive optics, relies on successive image measurements wit