Abstract
.Conventional two-photon microscopy (TPM) is capable of imaging neural dynamics with subcellular resolution, but it is limited to a field-of-view (FOV) diameter . Although there has been recent progress in extending the FOV in TPM, a principled design approach for developing large FOV TPM (LF-TPM) with off-the-shelf components has yet to be established. Therefore, we present a design strategy that depends on analyzing the optical invariant of commercially available objectives, relay lenses, mirror scanners, and emission collection systems in isolation. Components are then selected to maximize the space-bandwidth product of the integrated microscope. In comparison with other LF-TPM systems, our strategy simplifies the sequence of design decisions and is applicable to extending the FOV in any microscope with an optical relay. The microscope we constructed with this design approach can image lateral and axial resolution over a 7-mm diameter FOV, which is a 100-fold increase in FOV compared with conventional TPM. As a demonstration of the potential that LF-TPM has on understanding the microarchitecture of the mouse brain across interhemispheric regions, we performed in vivo imaging of both the cerebral vasculature and microglia cell bodies over the mouse cortex.
Highlights
We modeled the performance of two microscopy systems both with relay magnification equal to 2 using Zemax: a conventional two-photon microscopy (TPM) system consisting of achromatic doublets L11 and L12 [Fig. 6(a)], and a high-throughput microscope consisting of L27 and L15 [Fig. 6(b)]
Both conventional TPM and mesoscopic optical imaging with planar illumination (MOIPI) have improved our understanding of the functional architecture of the mouse cortex
We have shown the potential that large FOV TPM (LF-TPM) has in studying the mouse brain over multiple spatial scales
Summary
Two-photon microscopy (TPM) has revolutionized in vivo studies on the microarchitecture of the mouse cerebral cortex, it has primarily been limited to measuring brain dynamics over a field of view (FOV) of around 500 × 500 μm[2] (i.e., an FOV diameter of 707 μm).[1,2] This limitation makes TPM impractical for the increasing number of studies on functional whole-brain imaging and mapping in mice that have been inspired by the effort to map the human connectome.[3,4] Researchers have, primarily utilized mesoscopic optical imaging with planar illumination (MOIPI) techniques to study brain function over large regions of the mouse cortex (up to 10 × 10 mm[2] FOV).[3,5,6] MOIPI techniques have relatively poor resolution (around 200- to 300-μm lateral resolution) and depth penetration in comparison with TPM.[7]. The current FOV in many commercially available and custom-built two-photon and confocal microscopes is limited due to the use of achromatic doublets as relay lenses, not the objective lens.[16,17]. Bumstead et al.: Designing a large field-of-view two-photon microscope using optical invariant analysis present a LF-TPM system constructed with off-the-shelf components and only a single scanning relay, which reduces the cost (
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