Non-Telecentric 2P microscopy for 3D random access mesoscale imaging at single cell resolution
In neuroscience, diffraction limited two-photon (2P) microscopy is a cornerstone technique that permits minimally invasive optical monitoring of neuronal activity. However, most conventional 2P microscopes impose significant constraints on the size of the imaging field-of-view and the specific shape of the effective excitation volume, thus limiting the scope of biological questions that can be addressed and the information obtainable. Here, employing a non-telecentric (nTC) optical design, we present an ultra-low-cost, easily implemented and flexible solution to address these limitations, offering a several-fold expanded three-dimensional field of view that also maintains single-cell resolution. We show that this implementation also allows for straight-forward tailoring of the point-spread-function, increases effective excitation power, and achievable image brightness. Moreover, rapid laser-focus control via an electrically tunable lens allows near-simultaneous imaging of remote regions separated in three dimensions and permits the bending of imaging planes to follow natural curvatures in biological structures. Crucially, our core design is readily implemented (and reversed) within a matter of hours, and compatible with a wide range of existing 2P customizations, making it highly suitable as a base platform for further development. We demonstrate the application of our system for imaging neuronal activity in a variety of examples in zebrafish, mice and fruit flies.
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Due to technical limitations, full-text HTML conversion of this manuscript could not be completed. However, the latest manuscript can be downloaded and accessed as a PDF.
This is a list of supplementary files associated with this preprint. Click to download.
Supplementary Video S1, related to Figure 1 | Z-stack through three larval zebrafish
Supplementary Video S2, related to Figure 2 | Scan-profiles
Supplementary Video S3, related to Figures 3,6 and Fig. S4 | Scan-profiles during ETL shifts
Supplementary Video S4, related to Figure 4 | Mesoscale imaging of 2 zebrafish brains at the same time
Supplementary Video S5, related to Figure 6 | Half-pipe imaging of larval zebrafish brain
Supplementary Video S6 related to Figure S4 | Half-pipe multiplane imaging of larval zebrafish brain
Supplementary Video S7, related to Figure 7 | Mesoscale imaging of mouse brain slice
Supplementary Video S8, related to Figure 8 | Mesoscale imaging of mouse cortex in vivo
Supplementary Video S9, related to Figure 9 | Multiplane optogenetics in Drosophila larva
Posted 15 Dec, 2020
Non-Telecentric 2P microscopy for 3D random access mesoscale imaging at single cell resolution
Posted 15 Dec, 2020
In neuroscience, diffraction limited two-photon (2P) microscopy is a cornerstone technique that permits minimally invasive optical monitoring of neuronal activity. However, most conventional 2P microscopes impose significant constraints on the size of the imaging field-of-view and the specific shape of the effective excitation volume, thus limiting the scope of biological questions that can be addressed and the information obtainable. Here, employing a non-telecentric (nTC) optical design, we present an ultra-low-cost, easily implemented and flexible solution to address these limitations, offering a several-fold expanded three-dimensional field of view that also maintains single-cell resolution. We show that this implementation also allows for straight-forward tailoring of the point-spread-function, increases effective excitation power, and achievable image brightness. Moreover, rapid laser-focus control via an electrically tunable lens allows near-simultaneous imaging of remote regions separated in three dimensions and permits the bending of imaging planes to follow natural curvatures in biological structures. Crucially, our core design is readily implemented (and reversed) within a matter of hours, and compatible with a wide range of existing 2P customizations, making it highly suitable as a base platform for further development. We demonstrate the application of our system for imaging neuronal activity in a variety of examples in zebrafish, mice and fruit flies.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
Figure 9
Due to technical limitations, full-text HTML conversion of this manuscript could not be completed. However, the latest manuscript can be downloaded and accessed as a PDF.