Whole-cell multi-target single-molecule super-resolution imaging in 3D with microfluidics and a single-objective tilted light sheet
Abstract Multi-target single-molecule super-resolution fluorescence microscopy offers a powerful means of understanding the distributions and interplay between multiple subcellular structures at the nanoscale. However, single-molecule super-resolution imaging of whole mammalian cells is often hamper...
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| Format: | Article |
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Nature Portfolio
2024-11-01
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| Series: | Nature Communications |
| Online Access: | https://doi.org/10.1038/s41467-024-54609-z |
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| author | Nahima Saliba Gabriella Gagliano Anna-Karin Gustavsson |
| author_facet | Nahima Saliba Gabriella Gagliano Anna-Karin Gustavsson |
| author_sort | Nahima Saliba |
| collection | DOAJ |
| description | Abstract Multi-target single-molecule super-resolution fluorescence microscopy offers a powerful means of understanding the distributions and interplay between multiple subcellular structures at the nanoscale. However, single-molecule super-resolution imaging of whole mammalian cells is often hampered by high fluorescence background and slow acquisition speeds, especially when imaging multiple targets in 3D. In this work, we have mitigated these issues by developing a steerable, dithered, single-objective tilted light sheet for optical sectioning to reduce fluorescence background and a pipeline for 3D nanoprinting microfluidic systems for reflection of the light sheet into the sample. This easily adaptable microfluidic fabrication pipeline allows for the incorporation of reflective optics into microfluidic channels without disrupting efficient and automated solution exchange. We combine these innovations with point spread function engineering for nanoscale localization of individual molecules in 3D, deep learning for analysis of overlapping emitters, active 3D stabilization for drift correction and long-term imaging, and Exchange-PAINT for sequential multi-target imaging without chromatic offsets. We then demonstrate that this platform, termed soTILT3D, enables whole-cell multi-target 3D single-molecule super-resolution imaging with improved precision and imaging speed. |
| format | Article |
| id | doaj-art-aef8cfd0c2db405fb17a1cb93c7dfb45 |
| institution | Kabale University |
| issn | 2041-1723 |
| language | English |
| publishDate | 2024-11-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Nature Communications |
| spelling | doaj-art-aef8cfd0c2db405fb17a1cb93c7dfb452024-11-24T12:35:21ZengNature PortfolioNature Communications2041-17232024-11-0115111710.1038/s41467-024-54609-zWhole-cell multi-target single-molecule super-resolution imaging in 3D with microfluidics and a single-objective tilted light sheetNahima Saliba0Gabriella Gagliano1Anna-Karin Gustavsson2Department of Chemistry, Rice UniversityDepartment of Chemistry, Rice UniversityDepartment of Chemistry, Rice UniversityAbstract Multi-target single-molecule super-resolution fluorescence microscopy offers a powerful means of understanding the distributions and interplay between multiple subcellular structures at the nanoscale. However, single-molecule super-resolution imaging of whole mammalian cells is often hampered by high fluorescence background and slow acquisition speeds, especially when imaging multiple targets in 3D. In this work, we have mitigated these issues by developing a steerable, dithered, single-objective tilted light sheet for optical sectioning to reduce fluorescence background and a pipeline for 3D nanoprinting microfluidic systems for reflection of the light sheet into the sample. This easily adaptable microfluidic fabrication pipeline allows for the incorporation of reflective optics into microfluidic channels without disrupting efficient and automated solution exchange. We combine these innovations with point spread function engineering for nanoscale localization of individual molecules in 3D, deep learning for analysis of overlapping emitters, active 3D stabilization for drift correction and long-term imaging, and Exchange-PAINT for sequential multi-target imaging without chromatic offsets. We then demonstrate that this platform, termed soTILT3D, enables whole-cell multi-target 3D single-molecule super-resolution imaging with improved precision and imaging speed.https://doi.org/10.1038/s41467-024-54609-z |
| spellingShingle | Nahima Saliba Gabriella Gagliano Anna-Karin Gustavsson Whole-cell multi-target single-molecule super-resolution imaging in 3D with microfluidics and a single-objective tilted light sheet Nature Communications |
| title | Whole-cell multi-target single-molecule super-resolution imaging in 3D with microfluidics and a single-objective tilted light sheet |
| title_full | Whole-cell multi-target single-molecule super-resolution imaging in 3D with microfluidics and a single-objective tilted light sheet |
| title_fullStr | Whole-cell multi-target single-molecule super-resolution imaging in 3D with microfluidics and a single-objective tilted light sheet |
| title_full_unstemmed | Whole-cell multi-target single-molecule super-resolution imaging in 3D with microfluidics and a single-objective tilted light sheet |
| title_short | Whole-cell multi-target single-molecule super-resolution imaging in 3D with microfluidics and a single-objective tilted light sheet |
| title_sort | whole cell multi target single molecule super resolution imaging in 3d with microfluidics and a single objective tilted light sheet |
| url | https://doi.org/10.1038/s41467-024-54609-z |
| work_keys_str_mv | AT nahimasaliba wholecellmultitargetsinglemoleculesuperresolutionimagingin3dwithmicrofluidicsandasingleobjectivetiltedlightsheet AT gabriellagagliano wholecellmultitargetsinglemoleculesuperresolutionimagingin3dwithmicrofluidicsandasingleobjectivetiltedlightsheet AT annakaringustavsson wholecellmultitargetsinglemoleculesuperresolutionimagingin3dwithmicrofluidicsandasingleobjectivetiltedlightsheet |