Depth-enhanced molecular imaging with two-photon oblique plane microscopy
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arXiv
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| Autori principali: | , , , , , , , , |
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| Natura: | Preprint |
| Pubblicazione: |
2025
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| _version_ | 1866914179375431680 |
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| author | Keomanee-Dizon, Kevin Clenman, Yaakov Duran, Alejandra Ryabichko, Sergey Hansen, Pauline Borjigin, Tohn Thornton, Richard Toettcher, Jared E. McNamara, Harold M. |
| author_facet | Keomanee-Dizon, Kevin Clenman, Yaakov Duran, Alejandra Ryabichko, Sergey Hansen, Pauline Borjigin, Tohn Thornton, Richard Toettcher, Jared E. McNamara, Harold M. |
| contents | High-numerical-aperture (NA) oblique plane microscopy enables noninvasive fluorescence imaging of subcellular dynamics without requiring radical sample modification. However, performance degrades at depth in multicellular specimens as scattering and refractive-index heterogeneity raise out-of-focus background. We report a two-photon oblique plane microscope that improves resolution at depth by combining high-NA single-objective detection with multiphoton plane illumination. The microscope achieves $\sim\!300$ nm lateral and $\sim\!650$ nm axial resolution, with single-molecule sensitivity in vivo. Compared with two-photon point scanning, the lower illumination NA delivers an order of magnitude lower peak intensity, enabling $>\!5\times$ faster volumetric acquisition (up to $3.25 \times 10^6$ voxels s$^{-1}$) with reduced photodamage. In multicellular contexts, near-infrared nonlinear excitation enhances contrast throughout the illumination depth by $\sim\!2\times$ and restores volumetric resolving power by $>\!2\times$ relative to linear excitation. We demonstrate these capabilities through molecular imaging of epithelial tissue, stem-cell-derived gastruloids, and living fruit fly embryos, including multicolor transcription-factor dynamics, optogenetic subcellular control, and single-mRNA tracking, all using standard glass-based mounting. |
| format | Preprint |
| id |
arxiv_https___arxiv_org_abs_2511_09462 |
| institution | arXiv |
| publishDate | 2025 |
| record_format | arxiv |
| spellingShingle | Depth-enhanced molecular imaging with two-photon oblique plane microscopy Keomanee-Dizon, Kevin Clenman, Yaakov Duran, Alejandra Ryabichko, Sergey Hansen, Pauline Borjigin, Tohn Thornton, Richard Toettcher, Jared E. McNamara, Harold M. Optics Biological Physics Instrumentation and Detectors High-numerical-aperture (NA) oblique plane microscopy enables noninvasive fluorescence imaging of subcellular dynamics without requiring radical sample modification. However, performance degrades at depth in multicellular specimens as scattering and refractive-index heterogeneity raise out-of-focus background. We report a two-photon oblique plane microscope that improves resolution at depth by combining high-NA single-objective detection with multiphoton plane illumination. The microscope achieves $\sim\!300$ nm lateral and $\sim\!650$ nm axial resolution, with single-molecule sensitivity in vivo. Compared with two-photon point scanning, the lower illumination NA delivers an order of magnitude lower peak intensity, enabling $>\!5\times$ faster volumetric acquisition (up to $3.25 \times 10^6$ voxels s$^{-1}$) with reduced photodamage. In multicellular contexts, near-infrared nonlinear excitation enhances contrast throughout the illumination depth by $\sim\!2\times$ and restores volumetric resolving power by $>\!2\times$ relative to linear excitation. We demonstrate these capabilities through molecular imaging of epithelial tissue, stem-cell-derived gastruloids, and living fruit fly embryos, including multicolor transcription-factor dynamics, optogenetic subcellular control, and single-mRNA tracking, all using standard glass-based mounting. |
| title | Depth-enhanced molecular imaging with two-photon oblique plane microscopy |
| topic | Optics Biological Physics Instrumentation and Detectors |
| url | https://arxiv.org/abs/2511.09462 |