Abstract
Two-photon (2P) microscopy is widely used in neuroscience, but the optical properties of brain tissue are poorly understood. We have investigated the effect of brain tissue on the 2P point spread function (PSF2P) by imaging fluorescent beads through living cortical slices. By combining this with measurements of the mean free path of the excitation light, adaptive optics and vector-based modeling that includes phase modulation and scattering, we show that tissue-induced wavefront distortions are the main determinant of enlargement and distortion of the PSF2P at intermediate imaging depths. Furthermore, they generate surrounding lobes that contain more than half of the 2P excitation. These effects reduce the resolution of fine structures and contrast and they, together with scattering, limit 2P excitation. Our results disentangle the contributions of scattering and wavefront distortion in shaping the cortical PSF2P, thereby providing a basis for improved 2P microscopy.
Highlights
IntroductionReceived 26 Aug 2011; revised 7 Oct 2011; accepted 9 Oct 2011; published 26 Oct 2011 7 November 2011 / Vol 19, No 23 / OPTICS EXPRESS 22756
Received 26 Aug 2011; revised 7 Oct 2011; accepted 9 Oct 2011; published 26 Oct 2011 7 November 2011 / Vol 19, No 23 / OPTICS EXPRESS 22756(PSF2P), which sets the image resolution in conventional 2P microscopy
Our results show that in cortex the PSF2P decomposes into a central Gaussian region and a speckle pattern consisting of multiple surrounding lobes that can carry more than half the 2P excitation at a tissue depth of 150 μm
Summary
Received 26 Aug 2011; revised 7 Oct 2011; accepted 9 Oct 2011; published 26 Oct 2011 7 November 2011 / Vol 19, No 23 / OPTICS EXPRESS 22756. Scattering and wavefront distortion both occur in brain tissue, light propagation in 2P microscopy is often modeled as purely scattering, and is described in terms of modulation of the power of ballistic photons [12]. Received 26 Aug 2011; revised 7 Oct 2011; accepted 9 Oct 2011; published 26 Oct 2011 7 November 2011 / Vol 19, No 23 / OPTICS EXPRESS 22757 symmetric around the optical axis These effects set a fundamental limit to the depth of 2P microscopy [12]. We have investigated the respective effects of statistically homogeneous scattering and wavefront distortion at intermediate depth in acute slices of barrel cortex, under conditions similar to those used to study neuronal activity To do this we examined how the fluorescence emitted by objects and the shape of the PSF2P changed when imaging through living tissue. Tissue-induced wavefront distortion reduces the image quality, 2P excitation and signal to noise ratio (SNR) of fluorescence signals
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