A spherical aberration-free microscopy system for live brain imaging

Abstract

The high-resolution in vivo imaging of mouse brain for quantitative analysis of fine structures, such as dendritic spines, requires objectives with high numerical apertures (NAs) and long working distances (WDs). However, this imaging approach is often hampered by spherical aberration (SA) that results from the mismatch of refractive indices in the optical path and becomes more severe with increasing depth of target from the brain surface. Whereas a revolving objective correction collar has been designed to compensate SA, its adjustment requires manual operation and is inevitably accompanied by considerable focal shift, making it difficult to acquire the best image of a given fluorescent object. To solve the problems, we have created an objective-attached device and formulated a fast iterative algorithm for the realization of an automatic SA compensation system. The device coordinates the collar rotation and the Z-position of an objective, enabling correction collar adjustment while stably focusing on a target. The algorithm provides the best adjustment on the basis of the calculated contrast of acquired images. Together, they enable the system to compensate SA at a given depth. As proof of concept, we applied the SA compensation system to in vivo two-photon imaging with a 25 × water-immersion objective (NA, 1.05; WD, 2 mm). It effectively reduced SA regardless of location, allowing quantitative and reproducible analysis of fine structures of YFP-labeled neurons in the mouse cerebral cortical layers. Interestingly, although the cortical structure was optically heterogeneous along the z-axis, the refractive index of each layer could be assessed on the basis of the compensation degree. It was also possible to make fully corrected three-dimensional reconstructions of YFP-labeled neurons in live brain samples. Our SA compensation system, called Deep-C, is expected to bring out the best in all correction-collar-equipped objectives for imaging deep regions of heterogeneous tissues.

In vivo two-photon microscopic observation and ablation in deeper brain regions realized by modifications of excitation beam diameter and immersion liquid

“…The minimized focal volume indicated that the RI mismatch between the immersion liquid and the specimens might also be reduced. The result suggests that the average value of the RI in the living mouse cortex ranges from 1.35 to 1.36, which is consistent with a result from a recent report [23]. This consistency also suggests that the method developed here may evaluate the average value of the RI in various biological specimens.…”

“…In Fig 3 , the immersion liquid with an RI value of 1.35 or 1.36 minimized the focal volume in deep regions of the living mouse cortex mainly by compensation of the spherical aberration caused by the specimens ( S2 Fig ; S1 Table ) [ 21 , 23 , 31 ]. The minimized focal volume indicated that the RI mismatch between the immersion liquid and the specimens might also be reduced.…”

“…Moreover, optical aberrations of the focusing excitation laser light beam are usually induced by heterogeneous distributions of the refractive index (RI) in the specimen, preventing tighter focusing that expands the focal volume especially in deeper regions. To compensate for optical aberrations, sophisticated adaptive optical (AO) techniques have been proposed and applied for two-photon imaging of fixed or living mouse brains [ 19 – 23 ]. However, incompatibilities of such technologies with conventional two-photon microscopy have prevented their widespread use.…”

Pathological application of carbocyanine dye-based multicolour imaging of vasculature and associated structures

“…Another choice is to use an objective with a correction collar. In this case, the physical adjustment of the collar by hand or an automated system is required 45 .…”

Design framework for high-speed 3D scanning tools and development of an axial focusing micromirror-based array

“…In addition, while in principle only 3 modes of control are required for 3D scanning (Figure 1), additional degrees of freedom may be required for a given application to address target position-dependent aberrations stemming from the scanning tool itself, from another component of the optical system being employed, or from the sample/region of interest. For instance, additional radial phase control beyond defocusing may be needed to address target depth-dependent spherical aberrations, 8 and off-axis conditions introduced by beam-steering across large lateral fields may require addressing coma and field curvature aberrations. 9 Altogether, the simultaneously prevalent and application-specific nature of optical 3D scanning tools warrants an efficient and systematic method of designing such tools using the same versatile platform.…”