Current Optics and Photonics

  • 제8권4호
  • /
  • Pages.421-426
  • /
  • 2024
  • /
  • 2508-7266(pISSN)
  • /
  • 2508-7274(eISSN)
한국광학회 (Optical Society of Korea)
권호 표지
DOI QR코드

DOI QR Code

Development of an Alignment Method for Retarders in isoSTED Microscopy

  • Ilkyu Park (School of Mechanical Engineering, Soongsil University) ;
  • Dong-Ryoung Lee (School of Mechanical Engineering, Soongsil University)
  • 투고 : 2024.05.09
  • 심사 : 2024.06.05
  • 발행 : 2024.08.25
PDF 다운로드
⟨ 이전 논문 다음 논문 ⟩

초록

The use of stimulated emission depletion (STED) microscopy has significantly improved resolution beyond the limits imposed by diffraction; Furthermore, STED microscopy adopts a 4Pi-geometry to achieve an isotropic improvement in resolution. In isoSTED microscopy, a polarizing beam splitter and retarders are used in a 4Pi cavity to split beams of identical power, generating constructive and destructive interference for lateral and axial resolution improvements, respectively. The precise alignment of the retarders is crucial for optimizing the performance of isoSTED microscopy, because this orientation affects the quality of the depletion focus, necessitating zero intensity at the center. Incomplete destructive interference can lead to unwanted fluorescence inhibition, resulting in degraded resolution and contrast. However, measuring the intensity and polarization state in each optical path of the 4Pi cavity is complex and requires additional devices such as a power meter. Here, we propose a simple and accurate alignment method for the 4Pi cavity in isoSTED microscopy. Our approach demonstrates the equal allocation of power between upper and lower beam paths and achieves complete destructive interference using a polarizing beam displacer and a single CCD camera positioned outside the 4Pi cavity.

키워드

과제정보

This work was supported by the Soongsil University Research Fund (New Professor Support Research) of 2022.

참고문헌

  1. S. W. Hell and J. Wichmann, "Breaking the diffraction resolution limit by stimulated emission: Stimulated-emission-depletion fluorescence microscopy," Opt. Lett. 19, 780-782 (1994).  https://doi.org/10.1364/OL.19.000780
  2. T. A. Klar and S. W. Hell, "Subdiffraction resolution in far-field fluorescence microscopy," Opt. Lett. 24, 954-956 (1999).  https://doi.org/10.1364/OL.24.000954
  3. T. A. Klar, E. Engel, and S. W. Hell, "Breaking Abbe's diffraction resolution limit in fluorescence microscopy with stimulated emission depletion beams of various shapes," Phys. Rev. E 64, 066613 (2001). 
  4. S. W. Hell, "Microscopy and its focal switch," Nat. Methods. 6, 24-32 (2009).  https://doi.org/10.1038/nmeth.1291
  5. M. Dyba and S. W. Hell, "Focal spots of size λ/23 open up far-field fluorescence microscopy at 33 nm axial resolution," Phys. Rev. Lett. 88, 163901 (2002). 
  6. R. Schmidt, C. A. Wurm, S. Jakobs, J. Engelhardt, A. Egner, and S. W. Hell, "Spherical nanosized focal spot unravels the interior of cells," Nat. Methods 5, 539-544 (2008).  https://doi.org/10.1038/nmeth.1214
  7. X. Hao, E. S. Allgeyer, M. J. Booth, and J. Bewersdorf, "Point-spread function optimization in isoSTED nanoscopy," Opt. Lett. 40, 3627-3630 (2015).  https://doi.org/10.1364/OL.40.003627
  8. R. Siegmund, F. Werner, S. Jakobs, C. Geisler, and A. Egner, "isoSTED microscopy with water-immersion lenses and background reduction," Biophys. J. 120, P3303-3314 (2021).  https://doi.org/10.1016/j.bpj.2021.05.031
  9. S. Hell and E. H. K. Stelzer, "Properties of a 4Pi confocal fluorescence microscope," J. Opt. Soc. Am. A. 9, 2159-2166 (1992).  https://doi.org/10.1364/JOSAA.9.002159
  10. G. Shtengel, J. A. Galbraith, C. G. Galbraith, J. Lippincott-Schwartz, J. M. Gillette, S. Manley, R. Sougrat, C. M. Waterman, P. Kanchanawong, M. W. Davidson, R. D. Fetter, and H. F. Hess, "Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure," Proc. Natl. Acad. Sci. 106, 3125-3130 (2009).  https://doi.org/10.1073/pnas.0813131106
  11. X. Hao, E. S. Allgeyer, D. Lee, J. Antonello, K. Watters, J. A. Gerdes, L. K. Schroeder, F. Bottanelli, J. Zhao, P. Kidd, M. D. Lessard, J. E. Rothman, L. Cooley, T. Biederer, M. J. Booth, and J. Bewersdorf, "Three-dimensional adaptive optical nanoscopy for thick specimen imaging at sub-50-nm resolution," Nat. Methods 18, 688-693 (2021).  https://doi.org/10.1038/s41592-021-01149-9
  12. M. Schrader, S. W. Hell, and H. T. M. van der Voort, "Three-dimensional super-resolution with a 4Pi-confocal microscope using image restoration," J. Appl. Phys. 84, 4033-4042 (1998).  https://doi.org/10.1063/1.368616
  13. M. Varnham, D. Payne, A. Barlow, and R. Birch, "Analytic solution for the birefringence produced by thermal stress in polarization-maintaining optical fibers," J. Light. Technol. 1, 332-339 (1983). https://doi.org/10.1109/JLT.1983.1072123