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Article
Non-Telecentric 2P microscopy for 3D random access mesoscale imaging at single cell resolution
Filip Janiak
University of Sussex
Corresponding Author
ORCiD: https://orcid.org/0000-0002-9295-2740
Tom Baden
University of Sussex
Corresponding Author
ORCiD: https://orcid.org/0000-0003-2808-4210
License:
This work is licensed under a CC BY 4.0 License. Read Full License
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.
Keywords
two-photon (2P) microscopy, limitations, non-telecentric (nTC) optical design, three-dimensional field, single-cell resolution, imaging neuronal activity
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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.
- JaniaketalSupplementaryRevision.pdf
- VideoS13fish.mp4
Supplementary Video S1, related to Figure 1 | Z-stack through three larval zebrafish
- VideoS2PSFexpansions.mp4
Supplementary Video S2, related to Figure 2 | Scan-profiles
- VideoS3ETL.mp4
Supplementary Video S3, related to Figures 3,6 and Fig. S4 | Scan-profiles during ETL shifts
- VideoS42Fish.mp4
Supplementary Video S4, related to Figure 4 | Mesoscale imaging of 2 zebrafish brains at the same time
- VideoS5Planebending.mp4
Supplementary Video S5, related to Figure 6 | Half-pipe imaging of larval zebrafish brain
- VideoS6Planebending2.mp4
Supplementary Video S6 related to Figure S4 | Half-pipe multiplane imaging of larval zebrafish brain
- VideoS7Mousebrainslice.mp4
Supplementary Video S7, related to Figure 7 | Mesoscale imaging of mouse brain slice
- VideoS8Mousecortex.mp4
Supplementary Video S8, related to Figure 8 | Mesoscale imaging of mouse cortex in vivo
- VideoS9Drosophilaopticalcircuitmapping.mp4
Supplementary Video S9, related to Figure 9 | Multiplane optogenetics in Drosophila larva
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This preprint is under consideration at a Nature Research Journal. A preprint is a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Nature journals have partnered with Research Square to offer In Review, a journal-integrated preprint deposition service.
Article
Non-Telecentric 2P microscopy for 3D random access mesoscale imaging at single cell resolution
Filip Janiak
University of Sussex
Corresponding Author
ORCiD: https://orcid.org/0000-0002-9295-2740
Tom Baden
University of Sussex
Corresponding Author
ORCiD: https://orcid.org/0000-0003-2808-4210
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
Peer Review Timeline
CURRENT STATUS: Under Review
Version 1
Posted 15 Dec, 2020
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
License:
This work is licensed under a CC BY 4.0 License. Read Full License
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.