Microscopy: looking into the mirror

Microscopy: looking into the mirror Yang and Xi Light: Science & Applications (2018) 7:4 Official journal of the CIOMP 2047-7538 DOI 10.1038/s41377-018-0010-4 www.nature.com/lsa NEWS AND VIEWS Open Access 1 1 Xusan Yang andPengXi Mirrors can create a virtual excitation source for requirements and reduced the number of objectives 2,3 optical microscopy, which can greatly enhance the from 4 to 2 . With a modified deconvolution algorithm, spatiotemporal resolution of different fluorescence a spatial resolution as high as 300 nm in the x, y, and z microscopy techniques, thus advancing toward long- dimensions was achieved. Mirror-enhanced dual-view term live cell imaging. LSFM enables high speed, high-resolution imaging of In recent decades, many new discoveries have been biological specimens. obtained using novel optical microscopic techniques, such Mirrors not only can reduce the number of objectives in as confocal, multiphoton, super resolution, and light sheet light sheet microscopy but also can fold optical path to microscopies, which have attracted intensive interest from reduce the size. Previously, the resolution of light sheet biologists working in various fields. However, advances in microscopy was limited by the size of the objectives live cell fluorescence microscopy are facing multiple chal- because they should be placed perpendicularly and as lenges, such as low resolution, poor signal-to-background close as possible to each other. This restriction can be ratio (SBR), insufficient imaging speed, and photo- solved by changing the “real” objective to an “imaginary” toxicity. Interestingly, these grand challenges share a objective by using mirror reflection. In reflected light common solution: mirrors. When placing a reflective sheet microscopy (RLSM) developed by Sunney Xie’s mirror after an objective, the beam can be reflected, and group at Harvard University , a mirror is used to bend the a “virtual” excitation source can be generated without light path such that the illumination and detection additional cost. This conceptually simple approach objectives are not orthogonal, as in other light sheet provides an easy solution to the abovementioned chal- microscopes, but are opposite to each other. In this case, lenges, such as greater signal, better contrast, improved the physical size of the objective is no longer a barrier for optical section ability at relative low cost, and facilitating the application of high-numerical aperture objectives. live cell imaging with improved spatial resolution at a RLSM possesses the advantages of superior SBR, fast high speed. image acquisition speed, low phototoxicity, and optical Recently, an article published by Hari Shroff’s group sectioning capability. A thin light sheet of 0.5 μm can be from National Institute of Biomedical Imaging and created, with fivefold better SBR, thereby enabling reso- Bioengineering reported an approach, in which mirror lution of DNA-binding dynamics with a temporal reso- reflective imaging improved the resolution, speed, and lution of 100 Hz. In addition, combining the blinking collection efficiency in dual-view light sheet fluorescence photophysics of rhodamine-based dyes, RLS illumination microscopy (dual-view LSFM), as shown in Fig. 1a . Using microscopy can be upgraded to reflective light sheet mirror-based reflective coverslips, images of four illumination super-resolution microscopy . complementary views were obtained in 250 ms simulta- Mirrors can benefit microscopy not only in geometrical neously, and the imaging efficiency and speed were optics but also in wave optics. For example, a mirror can boosted by a factor of 2. Notably, mirror-enhanced cause interference and standing waves, similar to those in dual-view LSFM improved the spatiotemporal resolution a laser cavity. Peng Xi’s group at Peking University and collection efficiency with no additional hardware generated an axial narrowed focal spot in laser scanning 6,7 microscopy by adding a mirror beneath the specimen . In this technique, termed MEANS, a confocal microscope Correspondence: Xusan Yang (xusanyang@pku.edu.cn)or can be converted into an effective 4Pi microscope without Peng Xi (xipeng@pku.edu.cn) additional cost. The interference greatly enhanced the Department of Biomedical Engineering, College of Engineering, Peking University, Beijing, China © The Author(s) 2018 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to theCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; Yang and Xi Light: Science & Applications (2018) 7:4 Page 2 of 3 Fig. 1 Mirror-enhanced optical microscopy. a Mirror-enhanced a Fluorescence (from reflected sheet) Fluorescence Reflected dual-view light sheet microscopy duplicates the conventional iSPIM to light sheet Illuminating tetrad objectives. b Reflected light sheet microscopy folds the upper light sheet objective’s excitation to form a thin light sheet. c Mirror-enhanced Objective lens super-resolution microscopy generates an interferometric focal spot. 1, 6 a and c inset are adapted from refs. Focal plane Focal plane Mirrored z Specimen local intensity, which makes MEANS-STED capable of covership z ′ x ′ Translation resolving the porous structure of the nuclear pore com- y x y ′ plex. Moreover, in such a configuration, axial information about the cellular organelle can be obtained through the fluorescence lifetime, which is modulated by the surface Mirrored 8 plasma resonance of the mirror . light sheet In the future, mirror-enhanced dual-view LSFM and RLSM can be combined with single-molecule localization microscopy, super-resolution optical fluctuation imaging, and Bayesian analysis localization microscopy to achieve three-dimensional nanoscopic dynamics imaging in living Illumination cells. The MEANS approach has potential applications in objective fluorescence correlation spectroscopy, fluorescence life- time imaging microscopy, fluorescence recovery after Reflected light sheet photobleaching, and pump-probe-based microscopy AFM cantilever technologies such as coherent anti-Stokes Raman scat- tering, stimulated Raman scattering, and transient Sample stage absorption microscopy . As a fundamental optical element, mirrors can greatly enhance the performance of optical microscopy. The Detection unique view and resolution can help biological scientists objective observe live cell structures at improved resolution in both the spatial and temporal dimensions. The improvements offered by using mirrors for microscopy can smoothly accelerate fluorescence imaging applications in the realm of live cell dynamics studies. Conflict of interest The authors declare that they have no conflict of interest. Objective Received: 20 December 2017 Revised: 8 February 2018 Accepted: 25 –1000 0.8 February 2018 –800 0.6 –600 0.4 –400 References 0.2 –200 113 nm 1. Wu, Y. C. et al. Reflective imaging improves spatiotemporal resolution Mirror and collection efficiency in light sheet microscopy. Nat. Commun. 8, 1452 0 0 –500 0 500 (2017). r/nm 2. Wu, Y. C. et al. Spatially isotropic four-dimensional imaging with dual- Virtual objective view plane illumination microscopy. Nat. Biotechnol. 31, 1032–1038 (2013). 3. Kumar, A. et al. Dual-view plane illumination microscopy for rapid and spatially isotropic imaging. Nat. Protoc. 9,2555–2573 (2014). 4. Gebhardt,J.C.M. et al. Single-molecule imaging of transcription factor binding to DNA in live mammalian cells. Nat. Methods 10,421–426 (2013). 5. Zhao, Z. W. et al. Spatial organization of RNA polymerase II inside a mammalian cell nucleus revealed by reflected light-sheet superresolution microscopy. Proc. Natl Acad. Sci. USA 111,681–686 (2014). z/nm Yang and Xi Light: Science & Applications (2018) 7:4 Page 3 of 3 6. Yang, X. S. et al. Mirror-enhanced super-resolution microscopy. Light Sci. Appl. 5, 8. Chizhik, A. I., Rother, J., Gregor, I., Janshoff, A. & Enderlein, J. Metal-induced e16134 (2016). energy transfer for live cell nanoscopy. Nat. Photonics 8,124–127 (2014). 7. Graydon, O. Microscopy: axial super-resolution. Nat. Photonics 10,431–431 9. Wang,P.etal. Far-field imaging of non-fluorescent species with subdiffraction (2016). resolution. Nat. Photonics 7, 449–453 (2013). http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Light: Science & Applications Springer Journals

Microscopy: looking into the mirror

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Abstract

Yang and Xi Light: Science & Applications (2018) 7:4 Official journal of the CIOMP 2047-7538 DOI 10.1038/s41377-018-0010-4 www.nature.com/lsa NEWS AND VIEWS Open Access 1 1 Xusan Yang andPengXi Mirrors can create a virtual excitation source for requirements and reduced the number of objectives 2,3 optical microscopy, which can greatly enhance the from 4 to 2 . With a modified deconvolution algorithm, spatiotemporal resolution of different fluorescence a spatial resolution as high as 300 nm in the x, y, and z microscopy techniques, thus advancing toward long- dimensions was achieved. Mirror-enhanced dual-view term live cell imaging. LSFM enables high speed, high-resolution imaging of In recent decades, many new discoveries have been biological specimens. obtained using novel optical microscopic techniques, such Mirrors not only can reduce the number of objectives in as confocal, multiphoton, super resolution, and light sheet light sheet microscopy but also can fold optical path to microscopies, which have attracted intensive interest from reduce the size. Previously, the resolution of light sheet biologists working in various fields. However, advances in microscopy was limited by the size of the objectives live cell fluorescence microscopy are facing multiple chal- because they should be placed perpendicularly and as lenges, such as low resolution, poor signal-to-background close as possible to each other. This restriction can be ratio (SBR), insufficient imaging speed, and photo- solved by changing the “real” objective to an “imaginary” toxicity. Interestingly, these grand challenges share a objective by using mirror reflection. In reflected light common solution: mirrors. When placing a reflective sheet microscopy (RLSM) developed by Sunney Xie’s mirror after an objective, the beam can be reflected, and group at Harvard University , a mirror is used to bend the a “virtual” excitation source can be generated without light path such that the illumination and detection additional cost. This conceptually simple approach objectives are not orthogonal, as in other light sheet provides an easy solution to the abovementioned chal- microscopes, but are opposite to each other. In this case, lenges, such as greater signal, better contrast, improved the physical size of the objective is no longer a barrier for optical section ability at relative low cost, and facilitating the application of high-numerical aperture objectives. live cell imaging with improved spatial resolution at a RLSM possesses the advantages of superior SBR, fast high speed. image acquisition speed, low phototoxicity, and optical Recently, an article published by Hari Shroff’s group sectioning capability. A thin light sheet of 0.5 μm can be from National Institute of Biomedical Imaging and created, with fivefold better SBR, thereby enabling reso- Bioengineering reported an approach, in which mirror lution of DNA-binding dynamics with a temporal reso- reflective imaging improved the resolution, speed, and lution of 100 Hz. In addition, combining the blinking collection efficiency in dual-view light sheet fluorescence photophysics of rhodamine-based dyes, RLS illumination microscopy (dual-view LSFM), as shown in Fig. 1a . Using microscopy can be upgraded to reflective light sheet mirror-based reflective coverslips, images of four illumination super-resolution microscopy . complementary views were obtained in 250 ms simulta- Mirrors can benefit microscopy not only in geometrical neously, and the imaging efficiency and speed were optics but also in wave optics. For example, a mirror can boosted by a factor of 2. Notably, mirror-enhanced cause interference and standing waves, similar to those in dual-view LSFM improved the spatiotemporal resolution a laser cavity. Peng Xi’s group at Peking University and collection efficiency with no additional hardware generated an axial narrowed focal spot in laser scanning 6,7 microscopy by adding a mirror beneath the specimen . In this technique, termed MEANS, a confocal microscope Correspondence: Xusan Yang (xusanyang@pku.edu.cn)or can be converted into an effective 4Pi microscope without Peng Xi (xipeng@pku.edu.cn) additional cost. The interference greatly enhanced the Department of Biomedical Engineering, College of Engineering, Peking University, Beijing, China © The Author(s) 2018 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to theCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; Yang and Xi Light: Science & Applications (2018) 7:4 Page 2 of 3 Fig. 1 Mirror-enhanced optical microscopy. a Mirror-enhanced a Fluorescence (from reflected sheet) Fluorescence Reflected dual-view light sheet microscopy duplicates the conventional iSPIM to light sheet Illuminating tetrad objectives. b Reflected light sheet microscopy folds the upper light sheet objective’s excitation to form a thin light sheet. c Mirror-enhanced Objective lens super-resolution microscopy generates an interferometric focal spot. 1, 6 a and c inset are adapted from refs. Focal plane Focal plane Mirrored z Specimen local intensity, which makes MEANS-STED capable of covership z ′ x ′ Translation resolving the porous structure of the nuclear pore com- y x y ′ plex. Moreover, in such a configuration, axial information about the cellular organelle can be obtained through the fluorescence lifetime, which is modulated by the surface Mirrored 8 plasma resonance of the mirror . light sheet In the future, mirror-enhanced dual-view LSFM and RLSM can be combined with single-molecule localization microscopy, super-resolution optical fluctuation imaging, and Bayesian analysis localization microscopy to achieve three-dimensional nanoscopic dynamics imaging in living Illumination cells. The MEANS approach has potential applications in objective fluorescence correlation spectroscopy, fluorescence life- time imaging microscopy, fluorescence recovery after Reflected light sheet photobleaching, and pump-probe-based microscopy AFM cantilever technologies such as coherent anti-Stokes Raman scat- tering, stimulated Raman scattering, and transient Sample stage absorption microscopy . As a fundamental optical element, mirrors can greatly enhance the performance of optical microscopy. The Detection unique view and resolution can help biological scientists objective observe live cell structures at improved resolution in both the spatial and temporal dimensions. The improvements offered by using mirrors for microscopy can smoothly accelerate fluorescence imaging applications in the realm of live cell dynamics studies. Conflict of interest The authors declare that they have no conflict of interest. Objective Received: 20 December 2017 Revised: 8 February 2018 Accepted: 25 –1000 0.8 February 2018 –800 0.6 –600 0.4 –400 References 0.2 –200 113 nm 1. Wu, Y. C. et al. Reflective imaging improves spatiotemporal resolution Mirror and collection efficiency in light sheet microscopy. Nat. Commun. 8, 1452 0 0 –500 0 500 (2017). r/nm 2. Wu, Y. C. et al. Spatially isotropic four-dimensional imaging with dual- Virtual objective view plane illumination microscopy. Nat. Biotechnol. 31, 1032–1038 (2013). 3. Kumar, A. et al. Dual-view plane illumination microscopy for rapid and spatially isotropic imaging. Nat. Protoc. 9,2555–2573 (2014). 4. Gebhardt,J.C.M. et al. Single-molecule imaging of transcription factor binding to DNA in live mammalian cells. Nat. Methods 10,421–426 (2013). 5. Zhao, Z. W. et al. Spatial organization of RNA polymerase II inside a mammalian cell nucleus revealed by reflected light-sheet superresolution microscopy. Proc. Natl Acad. Sci. USA 111,681–686 (2014). z/nm Yang and Xi Light: Science & Applications (2018) 7:4 Page 3 of 3 6. Yang, X. S. et al. Mirror-enhanced super-resolution microscopy. Light Sci. Appl. 5, 8. Chizhik, A. I., Rother, J., Gregor, I., Janshoff, A. & Enderlein, J. Metal-induced e16134 (2016). energy transfer for live cell nanoscopy. Nat. Photonics 8,124–127 (2014). 7. Graydon, O. Microscopy: axial super-resolution. Nat. Photonics 10,431–431 9. Wang,P.etal. Far-field imaging of non-fluorescent species with subdiffraction (2016). resolution. Nat. Photonics 7, 449–453 (2013).

Journal

Light: Science & ApplicationsSpringer Journals

Published: May 18, 2018

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