Optical alignment device for two-photon microscopy
Two-photon excitation fluorescence microscopy has revolutionized our understanding of brain structure and function through the high resolution and large penetration depth it offers. Investigating neural structures in vivo requires gaining optical access to the brain, which is typically achieved by r...
| Main Authors: | , , , , , |
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| Format: | Journal article |
| Language: | English |
| Published: |
Optical Society of America
2018
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| _version_ | 1797101308915744768 |
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| author | Galiñanes, G Marchand, P Turcotte, R Pellat, S Ji, N Huber, D |
| author_facet | Galiñanes, G Marchand, P Turcotte, R Pellat, S Ji, N Huber, D |
| author_sort | Galiñanes, G |
| collection | OXFORD |
| description | Two-photon excitation fluorescence microscopy has revolutionized our understanding of brain structure and function through the high resolution and large penetration depth it offers. Investigating neural structures in vivo requires gaining optical access to the brain, which is typically achieved by replacing a part of the skull with one or several layers of cover glass windows. To compensate for the spherical aberrations caused by the presence of these layers of glass, collar-correction objectives are typically used. However, the efficiency of this correction has been shown to depend significantly on the tilt angle between the glass window surface and the optical axis of the imaging system. Here, we first expand these observations and characterize the effect of the tilt angle on the collected fluorescence signal with thicker windows (double cover slide) and compare these results with an objective devoid of collar-correction. Second, we present a simple optical alignment device designed to rapidly minimize the tilt angle in vivo and align the optical axis of the microscope perpendicularly to the glass window to an angle below 0.25°, thereby significantly improving the imaging quality. Finally, we describe a tilt-correction procedure for users in an in vivo setting, enabling the accurate alignment with a resolution of <0.2° in only few iterations. |
| first_indexed | 2024-03-07T05:50:01Z |
| format | Journal article |
| id | oxford-uuid:e8899d45-1d2a-4dfa-86be-3d097b69093a |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-07T05:50:01Z |
| publishDate | 2018 |
| publisher | Optical Society of America |
| record_format | dspace |
| spelling | oxford-uuid:e8899d45-1d2a-4dfa-86be-3d097b69093a2022-03-27T10:47:32ZOptical alignment device for two-photon microscopyJournal articlehttp://purl.org/coar/resource_type/c_dcae04bcuuid:e8899d45-1d2a-4dfa-86be-3d097b69093aEnglishSymplectic Elements at OxfordOptical Society of America2018Galiñanes, GMarchand, PTurcotte, RPellat, SJi, NHuber, DTwo-photon excitation fluorescence microscopy has revolutionized our understanding of brain structure and function through the high resolution and large penetration depth it offers. Investigating neural structures in vivo requires gaining optical access to the brain, which is typically achieved by replacing a part of the skull with one or several layers of cover glass windows. To compensate for the spherical aberrations caused by the presence of these layers of glass, collar-correction objectives are typically used. However, the efficiency of this correction has been shown to depend significantly on the tilt angle between the glass window surface and the optical axis of the imaging system. Here, we first expand these observations and characterize the effect of the tilt angle on the collected fluorescence signal with thicker windows (double cover slide) and compare these results with an objective devoid of collar-correction. Second, we present a simple optical alignment device designed to rapidly minimize the tilt angle in vivo and align the optical axis of the microscope perpendicularly to the glass window to an angle below 0.25°, thereby significantly improving the imaging quality. Finally, we describe a tilt-correction procedure for users in an in vivo setting, enabling the accurate alignment with a resolution of <0.2° in only few iterations. |
| spellingShingle | Galiñanes, G Marchand, P Turcotte, R Pellat, S Ji, N Huber, D Optical alignment device for two-photon microscopy |
| title | Optical alignment device for two-photon microscopy |
| title_full | Optical alignment device for two-photon microscopy |
| title_fullStr | Optical alignment device for two-photon microscopy |
| title_full_unstemmed | Optical alignment device for two-photon microscopy |
| title_short | Optical alignment device for two-photon microscopy |
| title_sort | optical alignment device for two photon microscopy |
| work_keys_str_mv | AT galinanesg opticalalignmentdevicefortwophotonmicroscopy AT marchandp opticalalignmentdevicefortwophotonmicroscopy AT turcotter opticalalignmentdevicefortwophotonmicroscopy AT pellats opticalalignmentdevicefortwophotonmicroscopy AT jin opticalalignmentdevicefortwophotonmicroscopy AT huberd opticalalignmentdevicefortwophotonmicroscopy |