Abstract
Recent progress in neuroscience to image and investigate brain function has been made possible by impressive developments in optogenetic and opto-molecular tools. Such research requires advances in optical techniques for the delivery of light through brain tissue with high spatial resolution. The tissue causes distortions to the wavefront of the incoming light which broadens the focus and consequently reduces the intensity and degrades the resolution. Such effects are detrimental in techniques requiring focal stimulation. Adaptive wavefront correction has been demonstrated to compensate for these distortions. However, iterative derivation of the corrective wavefront introduces time constraints that limit its applicability to probe living cells. Here, we demonstrate that we can pre-determine and generalize a small set of Zernike modes to correct for aberrations of the light propagating through specific brain regions. A priori identification of a corrective wavefront is a direct and fast technique that improves the quality of the focus without the need for iterative adaptive wavefront correction. We verify our technique by measuring the efficiency of two-photon photolysis of caged neurotransmitters along the dendrites of a whole-cell patched neuron. Our results show that encoding the selected Zernike modes on the excitation light can improve light propagation through brain slices of rats as observed by the neuron's evoked excitatory post-synaptic potential in response to localized focal uncaging at the spines of the neuron's dendrites.
Highlights
Laser microscopy is an important tool to understand the fundamental processes of neurons in brain tissue
The fixed brain slices were used for prior determination of the Zernike mode correction schematically described in Figure 1b, which illustrates an uncorrected beam propagating through the tissue and Figure 1c showing a wavefront corrected beam via a spatial light modulator (SLM)
Using fixed brain slice samples, we aimed to reduce the complexity for wavefront correction by identifying a few Zernike modes appropriate for optimizing the focus through different regions of the brain tissue
Summary
Laser microscopy is an important tool to understand the fundamental processes of neurons in brain tissue. While two-photon (2P) microscopy uses a less absorptive near-infrared (NIR) laser, refractive index inhomogeneity and scattering are still important factors that distort the point spread function (PSF) of the incident coherent light. Improving Focal Photostimulation of Cortical Neurons laser, such move can degrade living biological samples due to photo-toxicity and heating (Gautam et al, 2015), which can affect the physiological activity and are disruptive when probing fundamental cellular processes. The key to achieve minimally invasive imaging and probing of cellular processes is to build-up the capacity to rectify distortions of the incident light field, which optimizes the ability to achieve a near diffraction-limited focus at deep regions of the brain tissue
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