Characterization and adaptive optical correction of aberrations during in vivo imaging in the mouse cortex

Abstract

The signal and resolution during in vivo imaging of the mouse brain is limited by sample-induced optical aberrations. We find that, although the optical aberrations can vary across the sample and increase in magnitude with depth, they remain stable for hours. As a result, two-photon adaptive optics can recover diffraction-limited performance to depths of 450 μm and improve imaging quality over fields of view of hundreds of microns. Adaptive optical correction yielded fivefold signal enhancement for small neuronal structures and a threefold increase in axial resolution. The corrections allowed us to detect smaller neuronal structures at greater contrast and also improve the signal-to-noise ratio during functional Ca 2 imaging in single neurons.T he ability to visualize biological systems in vivo has been a major attraction of optical microscopy, because studying biological systems as they evolve in their natural, physiological state provides relevant information that in vitro preparations often do not allow (1). However, for conventional optical microscopes to achieve their optimal, diffraction-limited resolution, the specimen needs to have identical optical properties to those of the immersion media for which the microscope objective is designed. For example, one of the most widely applied microscopy techniques for in vivo imaging, two-photon fluorescence microscopy, often uses water-dipping objectives. Because biological samples are comprised of structures (i.e., proteins, nuclear acids, and lipids) with refractive indices different from that of water, they induce optical aberrations to the incoming excitation wave and result in an enlarged focal spot within the sample and a concomitant deterioration of signal and resolution (2, 3). As a result, the resolution and contrast of optical microscopes is compromised in vivo, especially deep in tissue.Many questions related to how the brain processes information on both the neuronal circuit level and the cell biological level can be addressed by observing the morphology and activity of neurons inside a living and, preferably, awake and behaving mouse (1). In a typical experiment, an area of the skull is surgically removed and replaced with a cover glass to provide optical access to the underlying structure of interest (4). For imaging during behavior, the cover glass is often attached to an optically transparent plug embedded in the skull to improve mechanical stability and to prevent the skull from growing back and blocking the optical access (5, 6) (Fig. 1A). Before the excitation light of a two-photon microscope reaches the desired focal plane inside the brain, it has to traverse first the cranial window and then the brain tissue, both with optical properties different from water. Thus, they both impart optical aberrations on the excitation light, which leads to a distorted focus, even at the surface of the brain.These sample-induced aberrations can be corrected with adaptive optics (AO) to recover diffraction-limited resolution. In AO, a wavefront-shaping device m...

Optical Property Analyses of Plant Cells for Adaptive Optics Microscopy

“…[6,7] AO microscopy has successfully been used to cancel the disturbances caused by cellular structures in wide-field microscopy, [8] laser-scanning confocal microscopy, [9,10] multi-photon microscopy, [11][12][13] harmonic generation microscopy, [14,15] and superresolution stimulated emission depletion microscopy, [16,17] with or without the wavefront sensor. [18] So far, most AO microscopy has observed artificial samples and animal tissues; plant cells and tissues have not been examined by AO microscopy yet.…”

Adaptive optical versus spherical aberration corrections for in vivo brain imaging

“…Although the use of cranial windows has been paradigm shifting in neuroscience [2], the glass window introduces additional aberrations. In particular, light propagating through the glass window and the brain tissue accumulates spherical aberrations (SA) as these materials have a refractive index mismatch between themselves and with respect to the immersion media of standard objective lenses (e.g., air, water, oil) [3][4][5]. Several theoretical studies have modeled SA and their effects on image quality in confocal [6][7][8], widefield [8,9], confocal light sheet [10], and multiphoton [6] fluorescence microscopy.…”

Non-degenerate 2-photon excitation in scattering medium for fluorescence microscopy

“…Further studies will be required to determine the optimal combination of NIR and SWIR wavelengths and powers for achieving the maximum fluorescence signal while avoiding tissue damage. Additional improvements in the excitation efficiency can be achieved by implementation of Adaptive Optics (AO) to correct the phase distortions experienced by the NIR beam [45][46][47]. Specifically, the SWIR beam, which can be focused deep inside the tissue, can be used as a reference point ("guiding star") allowing us to adjust the phase of the NIR beam to achieve the maximum spatial overlap of their focal volumes.…”