Abstract

The achievable image quality in fluorescence microscopy and nanoscopy is usually limited by photobleaching. Reducing the light dose imposed on the sample is thus a challenge for all these imaging techniques. Various approaches like CLEM, RESCue, MINFIELD, DyMIN and smart RESOLFT have been presented in the last years and have proven to significantly reduce the required light dose in diffraction-limited as well as super-resolution imaging, thus resulting in less photobleaching and phototoxicity. None of these methods has so far been able to transfer the light dose reduction into a faster recording at pixel dwell times of a few ten microseconds. By implementing a scan system with low latency and large field of view we could directly convert the light dose reduction of RESCue into a shorter acquisition time for STED nanoscopy. In this way, FastRESCue speeds up the acquisition locally up to 10-fold and allows overall for a 5 times faster acquisition at only 20% of the light dose in biological samples.

© 2020 Optical Society of America under the terms of the OSA Open Access Publishing Agreement

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References

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2019 (1)

J. Dreier, M. Castello, G. Coceano, R. Cáceres, J. Plastino, G. Vicidomini, and I. Testa, “Smart scanning for low-illumination and fast RESOLFT nanoscopy in vivo,” Nat. Commun. 10(1), 556 (2019).
[Crossref]

2017 (4)

F. Göttfert, T. Pleiner, J. Heine, V. Westphal, D. Görlich, S. J. Sahl, and S. W. Hell, “Strong signal increase in STED fluorescence microscopy by imaging regions of subdiffraction extent,” Proc. Natl. Acad. Sci. U. S. A. 114(9), 2125–2130 (2017).
[Crossref]

J. Heine, M. Reuss, B. Harke, E. D’Este, S. J. Sahl, and S. W. Hell, “Adaptive-illumination STED nanoscopy,” Proc. Natl. Acad. Sci. U. S. A. 114(37), 9797–9802 (2017).
[Crossref]

F. Balzarotti, Y. Eilers, K. C. Gwosch, A. H. Gynnå, V. Westphal, F. D. Stefani, J. Elf, and S. W. Hell, “Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes,” Science 355(6325), 606–612 (2017).
[Crossref]

S. J. Sahl, S. W. Hell, and S. Jakobs, “Fluorescence nanoscopy in cell biology,” Nat. Rev. Mol. Cell Biol. 18(11), 685–701 (2017).
[Crossref]

2016 (1)

C. Boudreau, T.-L. E. Wee, Y.-R. S. Duh, M. P. Couto, K. H. Ardakani, and C. M. Brown, “Excitation Light Dose Engineering to Reduce Photo-bleaching and Photo-toxicity,” Sci. Rep. 6(1), 30892 (2016).
[Crossref]

2015 (1)

J. Jonkman and C. M. Brown, “Any Way You Slice It - A Comparison of Confocal Microscopy Techniques,” J. Biomol. Tech. 26(2), 54–65 (2015).
[Crossref]

2014 (1)

G. R. B. E. Römer and P. Bechtold, “Electro-optic and acousto-optic laser beam scanners,” Phys. Procedia 56, 29–39 (2014).
[Crossref]

2012 (1)

H. Schneckenburger, P. Weber, M. Wagner, S. Schickinger, V. Richter, T. Bruns, W. S. L. Strauss, and R. Wittig, “Light exposure and cell viability in fluorescence microscopy,” J. Microsc. 245(3), 311–318 (2012).
[Crossref]

2011 (1)

2010 (1)

C. A. Combs, “Fluorescence Microscopy: A Concise Guide to Current Imaging Methods,” Curr. Protoc. Neurosci. 50(1), 2.1.1–2.1.14 (2010).
[Crossref]

2009 (1)

G. Donnert, C. Eggeling, and S. W. Hell, “Triplet-relaxation microscopy with bunched pulsed excitation,” Photochem. Photobiol. Sci. 8(4), 481–485 (2009).
[Crossref]

2008 (1)

J. Vogelsang, R. Kasper, C. Steinhauer, B. Person, M. Heilemann, M. Sauer, and P. Tinnefeld, “A Reducing and Oxidizing System Minimizes Photobleaching and Blinking of Fluorescent Dyes,” Angew. Chem., Int. Ed. 47(29), 5465–5469 (2008).
[Crossref]

2007 (3)

S. W. Hell, “Far-Field Optical Nanoscopy,” Science 316(5828), 1153–1158 (2007).
[Crossref]

G. Donnert, C. Eggeling, and S. W. Hell, “Major signal increase in fluorescence microscopy through dark-state relaxation,” Nat. Methods 4(1), 81–86 (2007).
[Crossref]

R. A. Hoebe, C. H. Van Oven, T. W. J. Gadella Jr., P. B. Dhonukshe, C. J. F. Van Noorden, and E. M. M. Manders, “Controlled light-exposure microscopy reduces photobleaching and phototoxicity in fluorescence live-cell imaging,” Nat. Biotechnol. 25(2), 249–253 (2007).
[Crossref]

2006 (2)

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Lührmann, R. Jahn, C. Eggeling, and S. W. Hell, “Macromolecular-scale resolution in biological fluorescence microscopy,” Proc. Natl. Acad. Sci. U. S. A. 103(31), 11440–11445 (2006).
[Crossref]

R. T. Borlinghaus, “MRT letter: High Speed Scanning Has the Potential to Increase Fluorescence Yield and to Reduce Photobleaching,” Microsc. Res. Tech. 69(9), 689–692 (2006).
[Crossref]

2004 (1)

T. Bernas, M. Zarȩbski, R. R. Cook, and J. W. Dobrucki, “Minimizing photobleaching during confocal microscopy of fluorescent probes bound to chromatin: role of anoxia and photon flux,” J. Microsc. 215(3), 281–296 (2004).
[Crossref]

2003 (3)

D. J. Stephens and V. J. Allan, “Light Microscopy Techniques for Live Cell Imaging,” Science 300(5616), 82–86 (2003).
[Crossref]

C. L. Rieder and A. Khodjakov, “Mitosis Through the Microscope: Advances in Seeing Inside Live Dividing Cells,” Science 300(5616), 91–96 (2003).
[Crossref]

S. W. Hell, S. Jakobs, and L. Kastrup, “Imaging and writing at the nanoscale with focused visible light through saturable optical transitions,” Appl. Phys. A 77(7), 859–860 (2003).
[Crossref]

2001 (1)

T. 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(6), 066613 (2001).
[Crossref]

2000 (1)

T. A. Klar, S. Jakobs, M. Dyba, A. Egner, and S. W. Hell, “Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission,” Proc. Natl. Acad. Sci. U. S. A. 97(15), 8206–8210 (2000).
[Crossref]

1996 (1)

L. Song, C. A. G. O. Varma, J. W. Verhoeven, and H. J. Tanke, “Influence of the Triplet Excited State on the Photobleaching Kinetics of Fluorescein in Microscopy,” Biophys. J. 70(6), 2959–2968 (1996).
[Crossref]

1994 (1)

Allan, V. J.

D. J. Stephens and V. J. Allan, “Light Microscopy Techniques for Live Cell Imaging,” Science 300(5616), 82–86 (2003).
[Crossref]

Andrei, M. A.

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Lührmann, R. Jahn, C. Eggeling, and S. W. Hell, “Macromolecular-scale resolution in biological fluorescence microscopy,” Proc. Natl. Acad. Sci. U. S. A. 103(31), 11440–11445 (2006).
[Crossref]

Ardakani, K. H.

C. Boudreau, T.-L. E. Wee, Y.-R. S. Duh, M. P. Couto, K. H. Ardakani, and C. M. Brown, “Excitation Light Dose Engineering to Reduce Photo-bleaching and Photo-toxicity,” Sci. Rep. 6(1), 30892 (2016).
[Crossref]

Balzarotti, F.

F. Balzarotti, Y. Eilers, K. C. Gwosch, A. H. Gynnå, V. Westphal, F. D. Stefani, J. Elf, and S. W. Hell, “Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes,” Science 355(6325), 606–612 (2017).
[Crossref]

Bechtold, P.

G. R. B. E. Römer and P. Bechtold, “Electro-optic and acousto-optic laser beam scanners,” Phys. Procedia 56, 29–39 (2014).
[Crossref]

Bernas, T.

T. Bernas, M. Zarȩbski, R. R. Cook, and J. W. Dobrucki, “Minimizing photobleaching during confocal microscopy of fluorescent probes bound to chromatin: role of anoxia and photon flux,” J. Microsc. 215(3), 281–296 (2004).
[Crossref]

Borlinghaus, R. T.

R. T. Borlinghaus, “MRT letter: High Speed Scanning Has the Potential to Increase Fluorescence Yield and to Reduce Photobleaching,” Microsc. Res. Tech. 69(9), 689–692 (2006).
[Crossref]

Boudreau, C.

C. Boudreau, T.-L. E. Wee, Y.-R. S. Duh, M. P. Couto, K. H. Ardakani, and C. M. Brown, “Excitation Light Dose Engineering to Reduce Photo-bleaching and Photo-toxicity,” Sci. Rep. 6(1), 30892 (2016).
[Crossref]

Brown, C. M.

C. Boudreau, T.-L. E. Wee, Y.-R. S. Duh, M. P. Couto, K. H. Ardakani, and C. M. Brown, “Excitation Light Dose Engineering to Reduce Photo-bleaching and Photo-toxicity,” Sci. Rep. 6(1), 30892 (2016).
[Crossref]

J. Jonkman and C. M. Brown, “Any Way You Slice It - A Comparison of Confocal Microscopy Techniques,” J. Biomol. Tech. 26(2), 54–65 (2015).
[Crossref]

Bruns, T.

H. Schneckenburger, P. Weber, M. Wagner, S. Schickinger, V. Richter, T. Bruns, W. S. L. Strauss, and R. Wittig, “Light exposure and cell viability in fluorescence microscopy,” J. Microsc. 245(3), 311–318 (2012).
[Crossref]

Cáceres, R.

J. Dreier, M. Castello, G. Coceano, R. Cáceres, J. Plastino, G. Vicidomini, and I. Testa, “Smart scanning for low-illumination and fast RESOLFT nanoscopy in vivo,” Nat. Commun. 10(1), 556 (2019).
[Crossref]

Castello, M.

J. Dreier, M. Castello, G. Coceano, R. Cáceres, J. Plastino, G. Vicidomini, and I. Testa, “Smart scanning for low-illumination and fast RESOLFT nanoscopy in vivo,” Nat. Commun. 10(1), 556 (2019).
[Crossref]

Coceano, G.

J. Dreier, M. Castello, G. Coceano, R. Cáceres, J. Plastino, G. Vicidomini, and I. Testa, “Smart scanning for low-illumination and fast RESOLFT nanoscopy in vivo,” Nat. Commun. 10(1), 556 (2019).
[Crossref]

Combs, C. A.

C. A. Combs, “Fluorescence Microscopy: A Concise Guide to Current Imaging Methods,” Curr. Protoc. Neurosci. 50(1), 2.1.1–2.1.14 (2010).
[Crossref]

Cook, R. R.

T. Bernas, M. Zarȩbski, R. R. Cook, and J. W. Dobrucki, “Minimizing photobleaching during confocal microscopy of fluorescent probes bound to chromatin: role of anoxia and photon flux,” J. Microsc. 215(3), 281–296 (2004).
[Crossref]

Couto, M. P.

C. Boudreau, T.-L. E. Wee, Y.-R. S. Duh, M. P. Couto, K. H. Ardakani, and C. M. Brown, “Excitation Light Dose Engineering to Reduce Photo-bleaching and Photo-toxicity,” Sci. Rep. 6(1), 30892 (2016).
[Crossref]

D’Este, E.

J. Heine, M. Reuss, B. Harke, E. D’Este, S. J. Sahl, and S. W. Hell, “Adaptive-illumination STED nanoscopy,” Proc. Natl. Acad. Sci. U. S. A. 114(37), 9797–9802 (2017).
[Crossref]

Dhonukshe, P. B.

R. A. Hoebe, C. H. Van Oven, T. W. J. Gadella Jr., P. B. Dhonukshe, C. J. F. Van Noorden, and E. M. M. Manders, “Controlled light-exposure microscopy reduces photobleaching and phototoxicity in fluorescence live-cell imaging,” Nat. Biotechnol. 25(2), 249–253 (2007).
[Crossref]

Dobrucki, J. W.

T. Bernas, M. Zarȩbski, R. R. Cook, and J. W. Dobrucki, “Minimizing photobleaching during confocal microscopy of fluorescent probes bound to chromatin: role of anoxia and photon flux,” J. Microsc. 215(3), 281–296 (2004).
[Crossref]

Donnert, G.

G. Donnert, C. Eggeling, and S. W. Hell, “Triplet-relaxation microscopy with bunched pulsed excitation,” Photochem. Photobiol. Sci. 8(4), 481–485 (2009).
[Crossref]

G. Donnert, C. Eggeling, and S. W. Hell, “Major signal increase in fluorescence microscopy through dark-state relaxation,” Nat. Methods 4(1), 81–86 (2007).
[Crossref]

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Lührmann, R. Jahn, C. Eggeling, and S. W. Hell, “Macromolecular-scale resolution in biological fluorescence microscopy,” Proc. Natl. Acad. Sci. U. S. A. 103(31), 11440–11445 (2006).
[Crossref]

Dreier, J.

J. Dreier, M. Castello, G. Coceano, R. Cáceres, J. Plastino, G. Vicidomini, and I. Testa, “Smart scanning for low-illumination and fast RESOLFT nanoscopy in vivo,” Nat. Commun. 10(1), 556 (2019).
[Crossref]

Duh, Y.-R. S.

C. Boudreau, T.-L. E. Wee, Y.-R. S. Duh, M. P. Couto, K. H. Ardakani, and C. M. Brown, “Excitation Light Dose Engineering to Reduce Photo-bleaching and Photo-toxicity,” Sci. Rep. 6(1), 30892 (2016).
[Crossref]

Dyba, M.

T. A. Klar, S. Jakobs, M. Dyba, A. Egner, and S. W. Hell, “Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission,” Proc. Natl. Acad. Sci. U. S. A. 97(15), 8206–8210 (2000).
[Crossref]

E. Römer, G. R. B.

G. R. B. E. Römer and P. Bechtold, “Electro-optic and acousto-optic laser beam scanners,” Phys. Procedia 56, 29–39 (2014).
[Crossref]

Eggeling, C.

G. Donnert, C. Eggeling, and S. W. Hell, “Triplet-relaxation microscopy with bunched pulsed excitation,” Photochem. Photobiol. Sci. 8(4), 481–485 (2009).
[Crossref]

G. Donnert, C. Eggeling, and S. W. Hell, “Major signal increase in fluorescence microscopy through dark-state relaxation,” Nat. Methods 4(1), 81–86 (2007).
[Crossref]

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Lührmann, R. Jahn, C. Eggeling, and S. W. Hell, “Macromolecular-scale resolution in biological fluorescence microscopy,” Proc. Natl. Acad. Sci. U. S. A. 103(31), 11440–11445 (2006).
[Crossref]

Egner, A.

T. A. Klar, S. Jakobs, M. Dyba, A. Egner, and S. W. Hell, “Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission,” Proc. Natl. Acad. Sci. U. S. A. 97(15), 8206–8210 (2000).
[Crossref]

Eilers, Y.

F. Balzarotti, Y. Eilers, K. C. Gwosch, A. H. Gynnå, V. Westphal, F. D. Stefani, J. Elf, and S. W. Hell, “Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes,” Science 355(6325), 606–612 (2017).
[Crossref]

Elf, J.

F. Balzarotti, Y. Eilers, K. C. Gwosch, A. H. Gynnå, V. Westphal, F. D. Stefani, J. Elf, and S. W. Hell, “Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes,” Science 355(6325), 606–612 (2017).
[Crossref]

Engel, E.

T. 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(6), 066613 (2001).
[Crossref]

Engelhardt, J.

Engler, A.

Gadella Jr., T. W. J.

R. A. Hoebe, C. H. Van Oven, T. W. J. Gadella Jr., P. B. Dhonukshe, C. J. F. Van Noorden, and E. M. M. Manders, “Controlled light-exposure microscopy reduces photobleaching and phototoxicity in fluorescence live-cell imaging,” Nat. Biotechnol. 25(2), 249–253 (2007).
[Crossref]

Görlich, D.

F. Göttfert, T. Pleiner, J. Heine, V. Westphal, D. Görlich, S. J. Sahl, and S. W. Hell, “Strong signal increase in STED fluorescence microscopy by imaging regions of subdiffraction extent,” Proc. Natl. Acad. Sci. U. S. A. 114(9), 2125–2130 (2017).
[Crossref]

Göttfert, F.

F. Göttfert, T. Pleiner, J. Heine, V. Westphal, D. Görlich, S. J. Sahl, and S. W. Hell, “Strong signal increase in STED fluorescence microscopy by imaging regions of subdiffraction extent,” Proc. Natl. Acad. Sci. U. S. A. 114(9), 2125–2130 (2017).
[Crossref]

F. Göttfert, “STED microscopy with scanning fields below the diffraction limit,” PhD thesis (Georg-August-Universität Göttingen, Germany, 2015).

Gwosch, K. C.

F. Balzarotti, Y. Eilers, K. C. Gwosch, A. H. Gynnå, V. Westphal, F. D. Stefani, J. Elf, and S. W. Hell, “Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes,” Science 355(6325), 606–612 (2017).
[Crossref]

Gynnå, A. H.

F. Balzarotti, Y. Eilers, K. C. Gwosch, A. H. Gynnå, V. Westphal, F. D. Stefani, J. Elf, and S. W. Hell, “Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes,” Science 355(6325), 606–612 (2017).
[Crossref]

Harke, B.

J. Heine, M. Reuss, B. Harke, E. D’Este, S. J. Sahl, and S. W. Hell, “Adaptive-illumination STED nanoscopy,” Proc. Natl. Acad. Sci. U. S. A. 114(37), 9797–9802 (2017).
[Crossref]

T. Staudt, A. Engler, E. Rittweger, B. Harke, J. Engelhardt, and S. W. Hell, “Far-field optical nanoscopy with reduced number of state transition cycles,” Opt. Express 19(6), 5644–5657 (2011).
[Crossref]

Heilemann, M.

J. Vogelsang, R. Kasper, C. Steinhauer, B. Person, M. Heilemann, M. Sauer, and P. Tinnefeld, “A Reducing and Oxidizing System Minimizes Photobleaching and Blinking of Fluorescent Dyes,” Angew. Chem., Int. Ed. 47(29), 5465–5469 (2008).
[Crossref]

Heine, J.

J. Heine, M. Reuss, B. Harke, E. D’Este, S. J. Sahl, and S. W. Hell, “Adaptive-illumination STED nanoscopy,” Proc. Natl. Acad. Sci. U. S. A. 114(37), 9797–9802 (2017).
[Crossref]

F. Göttfert, T. Pleiner, J. Heine, V. Westphal, D. Görlich, S. J. Sahl, and S. W. Hell, “Strong signal increase in STED fluorescence microscopy by imaging regions of subdiffraction extent,” Proc. Natl. Acad. Sci. U. S. A. 114(9), 2125–2130 (2017).
[Crossref]

Hell, S. W.

F. Balzarotti, Y. Eilers, K. C. Gwosch, A. H. Gynnå, V. Westphal, F. D. Stefani, J. Elf, and S. W. Hell, “Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes,” Science 355(6325), 606–612 (2017).
[Crossref]

F. Göttfert, T. Pleiner, J. Heine, V. Westphal, D. Görlich, S. J. Sahl, and S. W. Hell, “Strong signal increase in STED fluorescence microscopy by imaging regions of subdiffraction extent,” Proc. Natl. Acad. Sci. U. S. A. 114(9), 2125–2130 (2017).
[Crossref]

J. Heine, M. Reuss, B. Harke, E. D’Este, S. J. Sahl, and S. W. Hell, “Adaptive-illumination STED nanoscopy,” Proc. Natl. Acad. Sci. U. S. A. 114(37), 9797–9802 (2017).
[Crossref]

S. J. Sahl, S. W. Hell, and S. Jakobs, “Fluorescence nanoscopy in cell biology,” Nat. Rev. Mol. Cell Biol. 18(11), 685–701 (2017).
[Crossref]

T. Staudt, A. Engler, E. Rittweger, B. Harke, J. Engelhardt, and S. W. Hell, “Far-field optical nanoscopy with reduced number of state transition cycles,” Opt. Express 19(6), 5644–5657 (2011).
[Crossref]

G. Donnert, C. Eggeling, and S. W. Hell, “Triplet-relaxation microscopy with bunched pulsed excitation,” Photochem. Photobiol. Sci. 8(4), 481–485 (2009).
[Crossref]

G. Donnert, C. Eggeling, and S. W. Hell, “Major signal increase in fluorescence microscopy through dark-state relaxation,” Nat. Methods 4(1), 81–86 (2007).
[Crossref]

S. W. Hell, “Far-Field Optical Nanoscopy,” Science 316(5828), 1153–1158 (2007).
[Crossref]

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Lührmann, R. Jahn, C. Eggeling, and S. W. Hell, “Macromolecular-scale resolution in biological fluorescence microscopy,” Proc. Natl. Acad. Sci. U. S. A. 103(31), 11440–11445 (2006).
[Crossref]

S. W. Hell, S. Jakobs, and L. Kastrup, “Imaging and writing at the nanoscale with focused visible light through saturable optical transitions,” Appl. Phys. A 77(7), 859–860 (2003).
[Crossref]

T. 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(6), 066613 (2001).
[Crossref]

T. A. Klar, S. Jakobs, M. Dyba, A. Egner, and S. W. Hell, “Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission,” Proc. Natl. Acad. Sci. U. S. A. 97(15), 8206–8210 (2000).
[Crossref]

S. W. Hell and J. Wichmann, “Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy,” Opt. Lett. 19(11), 780–782 (1994).
[Crossref]

Hoebe, R. A.

R. A. Hoebe, C. H. Van Oven, T. W. J. Gadella Jr., P. B. Dhonukshe, C. J. F. Van Noorden, and E. M. M. Manders, “Controlled light-exposure microscopy reduces photobleaching and phototoxicity in fluorescence live-cell imaging,” Nat. Biotechnol. 25(2), 249–253 (2007).
[Crossref]

Jahn, R.

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Lührmann, R. Jahn, C. Eggeling, and S. W. Hell, “Macromolecular-scale resolution in biological fluorescence microscopy,” Proc. Natl. Acad. Sci. U. S. A. 103(31), 11440–11445 (2006).
[Crossref]

Jakobs, S.

S. J. Sahl, S. W. Hell, and S. Jakobs, “Fluorescence nanoscopy in cell biology,” Nat. Rev. Mol. Cell Biol. 18(11), 685–701 (2017).
[Crossref]

S. W. Hell, S. Jakobs, and L. Kastrup, “Imaging and writing at the nanoscale with focused visible light through saturable optical transitions,” Appl. Phys. A 77(7), 859–860 (2003).
[Crossref]

T. A. Klar, S. Jakobs, M. Dyba, A. Egner, and S. W. Hell, “Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission,” Proc. Natl. Acad. Sci. U. S. A. 97(15), 8206–8210 (2000).
[Crossref]

Jonkman, J.

J. Jonkman and C. M. Brown, “Any Way You Slice It - A Comparison of Confocal Microscopy Techniques,” J. Biomol. Tech. 26(2), 54–65 (2015).
[Crossref]

Kasper, R.

J. Vogelsang, R. Kasper, C. Steinhauer, B. Person, M. Heilemann, M. Sauer, and P. Tinnefeld, “A Reducing and Oxidizing System Minimizes Photobleaching and Blinking of Fluorescent Dyes,” Angew. Chem., Int. Ed. 47(29), 5465–5469 (2008).
[Crossref]

Kastrup, L.

S. W. Hell, S. Jakobs, and L. Kastrup, “Imaging and writing at the nanoscale with focused visible light through saturable optical transitions,” Appl. Phys. A 77(7), 859–860 (2003).
[Crossref]

Keller, J.

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Lührmann, R. Jahn, C. Eggeling, and S. W. Hell, “Macromolecular-scale resolution in biological fluorescence microscopy,” Proc. Natl. Acad. Sci. U. S. A. 103(31), 11440–11445 (2006).
[Crossref]

Khodjakov, A.

C. L. Rieder and A. Khodjakov, “Mitosis Through the Microscope: Advances in Seeing Inside Live Dividing Cells,” Science 300(5616), 91–96 (2003).
[Crossref]

Klar, T.

T. 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(6), 066613 (2001).
[Crossref]

Klar, T. A.

T. A. Klar, S. Jakobs, M. Dyba, A. Egner, and S. W. Hell, “Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission,” Proc. Natl. Acad. Sci. U. S. A. 97(15), 8206–8210 (2000).
[Crossref]

Ledig, C.

C. Ledig and D. Rueckert, “Chapter 14 - Semantic Parsing of Brain MR Images,” in Medical Image Recognition, Segmentation and ParsingS. K. Zhou, ed. (Academic, 2016).

Lührmann, R.

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Lührmann, R. Jahn, C. Eggeling, and S. W. Hell, “Macromolecular-scale resolution in biological fluorescence microscopy,” Proc. Natl. Acad. Sci. U. S. A. 103(31), 11440–11445 (2006).
[Crossref]

Manders, E. M. M.

R. A. Hoebe, C. H. Van Oven, T. W. J. Gadella Jr., P. B. Dhonukshe, C. J. F. Van Noorden, and E. M. M. Manders, “Controlled light-exposure microscopy reduces photobleaching and phototoxicity in fluorescence live-cell imaging,” Nat. Biotechnol. 25(2), 249–253 (2007).
[Crossref]

Marshall, G. F.

G. F. Marshall and G. E. Stutz, Handbook of Optical and Laser Scanning (CRC, New York, 2004).

Medda, R.

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Lührmann, R. Jahn, C. Eggeling, and S. W. Hell, “Macromolecular-scale resolution in biological fluorescence microscopy,” Proc. Natl. Acad. Sci. U. S. A. 103(31), 11440–11445 (2006).
[Crossref]

O. Varma, C. A. G.

L. Song, C. A. G. O. Varma, J. W. Verhoeven, and H. J. Tanke, “Influence of the Triplet Excited State on the Photobleaching Kinetics of Fluorescein in Microscopy,” Biophys. J. 70(6), 2959–2968 (1996).
[Crossref]

Pawley, J. B.

J. B. Pawley, Handbook Of Biological Confocal Microscopy (Springer, 2006).

Periasamy, A.

Y. Sun and A. Periasamy, “Fluorescence Microscopy Imaging in Biomedical Sciences,” in Biomedical Optical Imaging Technologies, R. Liang, ed. (Springer, 2013).

Person, B.

J. Vogelsang, R. Kasper, C. Steinhauer, B. Person, M. Heilemann, M. Sauer, and P. Tinnefeld, “A Reducing and Oxidizing System Minimizes Photobleaching and Blinking of Fluorescent Dyes,” Angew. Chem., Int. Ed. 47(29), 5465–5469 (2008).
[Crossref]

Plastino, J.

J. Dreier, M. Castello, G. Coceano, R. Cáceres, J. Plastino, G. Vicidomini, and I. Testa, “Smart scanning for low-illumination and fast RESOLFT nanoscopy in vivo,” Nat. Commun. 10(1), 556 (2019).
[Crossref]

Pleiner, T.

F. Göttfert, T. Pleiner, J. Heine, V. Westphal, D. Görlich, S. J. Sahl, and S. W. Hell, “Strong signal increase in STED fluorescence microscopy by imaging regions of subdiffraction extent,” Proc. Natl. Acad. Sci. U. S. A. 114(9), 2125–2130 (2017).
[Crossref]

Reuss, M.

J. Heine, M. Reuss, B. Harke, E. D’Este, S. J. Sahl, and S. W. Hell, “Adaptive-illumination STED nanoscopy,” Proc. Natl. Acad. Sci. U. S. A. 114(37), 9797–9802 (2017).
[Crossref]

Richter, V.

H. Schneckenburger, P. Weber, M. Wagner, S. Schickinger, V. Richter, T. Bruns, W. S. L. Strauss, and R. Wittig, “Light exposure and cell viability in fluorescence microscopy,” J. Microsc. 245(3), 311–318 (2012).
[Crossref]

Rieder, C. L.

C. L. Rieder and A. Khodjakov, “Mitosis Through the Microscope: Advances in Seeing Inside Live Dividing Cells,” Science 300(5616), 91–96 (2003).
[Crossref]

Rittweger, E.

Rizzoli, S. O.

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Lührmann, R. Jahn, C. Eggeling, and S. W. Hell, “Macromolecular-scale resolution in biological fluorescence microscopy,” Proc. Natl. Acad. Sci. U. S. A. 103(31), 11440–11445 (2006).
[Crossref]

Rueckert, D.

C. Ledig and D. Rueckert, “Chapter 14 - Semantic Parsing of Brain MR Images,” in Medical Image Recognition, Segmentation and ParsingS. K. Zhou, ed. (Academic, 2016).

Sahl, S. J.

S. J. Sahl, S. W. Hell, and S. Jakobs, “Fluorescence nanoscopy in cell biology,” Nat. Rev. Mol. Cell Biol. 18(11), 685–701 (2017).
[Crossref]

J. Heine, M. Reuss, B. Harke, E. D’Este, S. J. Sahl, and S. W. Hell, “Adaptive-illumination STED nanoscopy,” Proc. Natl. Acad. Sci. U. S. A. 114(37), 9797–9802 (2017).
[Crossref]

F. Göttfert, T. Pleiner, J. Heine, V. Westphal, D. Görlich, S. J. Sahl, and S. W. Hell, “Strong signal increase in STED fluorescence microscopy by imaging regions of subdiffraction extent,” Proc. Natl. Acad. Sci. U. S. A. 114(9), 2125–2130 (2017).
[Crossref]

Sauer, M.

J. Vogelsang, R. Kasper, C. Steinhauer, B. Person, M. Heilemann, M. Sauer, and P. Tinnefeld, “A Reducing and Oxidizing System Minimizes Photobleaching and Blinking of Fluorescent Dyes,” Angew. Chem., Int. Ed. 47(29), 5465–5469 (2008).
[Crossref]

Schickinger, S.

H. Schneckenburger, P. Weber, M. Wagner, S. Schickinger, V. Richter, T. Bruns, W. S. L. Strauss, and R. Wittig, “Light exposure and cell viability in fluorescence microscopy,” J. Microsc. 245(3), 311–318 (2012).
[Crossref]

Schneckenburger, H.

H. Schneckenburger, P. Weber, M. Wagner, S. Schickinger, V. Richter, T. Bruns, W. S. L. Strauss, and R. Wittig, “Light exposure and cell viability in fluorescence microscopy,” J. Microsc. 245(3), 311–318 (2012).
[Crossref]

Song, L.

L. Song, C. A. G. O. Varma, J. W. Verhoeven, and H. J. Tanke, “Influence of the Triplet Excited State on the Photobleaching Kinetics of Fluorescein in Microscopy,” Biophys. J. 70(6), 2959–2968 (1996).
[Crossref]

Staudt, T.

Stefani, F. D.

F. Balzarotti, Y. Eilers, K. C. Gwosch, A. H. Gynnå, V. Westphal, F. D. Stefani, J. Elf, and S. W. Hell, “Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes,” Science 355(6325), 606–612 (2017).
[Crossref]

Steinhauer, C.

J. Vogelsang, R. Kasper, C. Steinhauer, B. Person, M. Heilemann, M. Sauer, and P. Tinnefeld, “A Reducing and Oxidizing System Minimizes Photobleaching and Blinking of Fluorescent Dyes,” Angew. Chem., Int. Ed. 47(29), 5465–5469 (2008).
[Crossref]

Stephens, D. J.

D. J. Stephens and V. J. Allan, “Light Microscopy Techniques for Live Cell Imaging,” Science 300(5616), 82–86 (2003).
[Crossref]

Strauss, W. S. L.

H. Schneckenburger, P. Weber, M. Wagner, S. Schickinger, V. Richter, T. Bruns, W. S. L. Strauss, and R. Wittig, “Light exposure and cell viability in fluorescence microscopy,” J. Microsc. 245(3), 311–318 (2012).
[Crossref]

Stutz, G. E.

G. F. Marshall and G. E. Stutz, Handbook of Optical and Laser Scanning (CRC, New York, 2004).

Sun, Y.

Y. Sun and A. Periasamy, “Fluorescence Microscopy Imaging in Biomedical Sciences,” in Biomedical Optical Imaging Technologies, R. Liang, ed. (Springer, 2013).

Tanke, H. J.

L. Song, C. A. G. O. Varma, J. W. Verhoeven, and H. J. Tanke, “Influence of the Triplet Excited State on the Photobleaching Kinetics of Fluorescein in Microscopy,” Biophys. J. 70(6), 2959–2968 (1996).
[Crossref]

Testa, I.

J. Dreier, M. Castello, G. Coceano, R. Cáceres, J. Plastino, G. Vicidomini, and I. Testa, “Smart scanning for low-illumination and fast RESOLFT nanoscopy in vivo,” Nat. Commun. 10(1), 556 (2019).
[Crossref]

Tinnefeld, P.

J. Vogelsang, R. Kasper, C. Steinhauer, B. Person, M. Heilemann, M. Sauer, and P. Tinnefeld, “A Reducing and Oxidizing System Minimizes Photobleaching and Blinking of Fluorescent Dyes,” Angew. Chem., Int. Ed. 47(29), 5465–5469 (2008).
[Crossref]

Van Noorden, C. J. F.

R. A. Hoebe, C. H. Van Oven, T. W. J. Gadella Jr., P. B. Dhonukshe, C. J. F. Van Noorden, and E. M. M. Manders, “Controlled light-exposure microscopy reduces photobleaching and phototoxicity in fluorescence live-cell imaging,” Nat. Biotechnol. 25(2), 249–253 (2007).
[Crossref]

Van Oven, C. H.

R. A. Hoebe, C. H. Van Oven, T. W. J. Gadella Jr., P. B. Dhonukshe, C. J. F. Van Noorden, and E. M. M. Manders, “Controlled light-exposure microscopy reduces photobleaching and phototoxicity in fluorescence live-cell imaging,” Nat. Biotechnol. 25(2), 249–253 (2007).
[Crossref]

Verhoeven, J. W.

L. Song, C. A. G. O. Varma, J. W. Verhoeven, and H. J. Tanke, “Influence of the Triplet Excited State on the Photobleaching Kinetics of Fluorescein in Microscopy,” Biophys. J. 70(6), 2959–2968 (1996).
[Crossref]

Vicidomini, G.

J. Dreier, M. Castello, G. Coceano, R. Cáceres, J. Plastino, G. Vicidomini, and I. Testa, “Smart scanning for low-illumination and fast RESOLFT nanoscopy in vivo,” Nat. Commun. 10(1), 556 (2019).
[Crossref]

Vogelsang, J.

J. Vogelsang, R. Kasper, C. Steinhauer, B. Person, M. Heilemann, M. Sauer, and P. Tinnefeld, “A Reducing and Oxidizing System Minimizes Photobleaching and Blinking of Fluorescent Dyes,” Angew. Chem., Int. Ed. 47(29), 5465–5469 (2008).
[Crossref]

Wagner, M.

H. Schneckenburger, P. Weber, M. Wagner, S. Schickinger, V. Richter, T. Bruns, W. S. L. Strauss, and R. Wittig, “Light exposure and cell viability in fluorescence microscopy,” J. Microsc. 245(3), 311–318 (2012).
[Crossref]

Weber, P.

H. Schneckenburger, P. Weber, M. Wagner, S. Schickinger, V. Richter, T. Bruns, W. S. L. Strauss, and R. Wittig, “Light exposure and cell viability in fluorescence microscopy,” J. Microsc. 245(3), 311–318 (2012).
[Crossref]

Wee, T.-L. E.

C. Boudreau, T.-L. E. Wee, Y.-R. S. Duh, M. P. Couto, K. H. Ardakani, and C. M. Brown, “Excitation Light Dose Engineering to Reduce Photo-bleaching and Photo-toxicity,” Sci. Rep. 6(1), 30892 (2016).
[Crossref]

Westphal, V.

F. Göttfert, T. Pleiner, J. Heine, V. Westphal, D. Görlich, S. J. Sahl, and S. W. Hell, “Strong signal increase in STED fluorescence microscopy by imaging regions of subdiffraction extent,” Proc. Natl. Acad. Sci. U. S. A. 114(9), 2125–2130 (2017).
[Crossref]

F. Balzarotti, Y. Eilers, K. C. Gwosch, A. H. Gynnå, V. Westphal, F. D. Stefani, J. Elf, and S. W. Hell, “Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes,” Science 355(6325), 606–612 (2017).
[Crossref]

Wichmann, J.

Wittig, R.

H. Schneckenburger, P. Weber, M. Wagner, S. Schickinger, V. Richter, T. Bruns, W. S. L. Strauss, and R. Wittig, “Light exposure and cell viability in fluorescence microscopy,” J. Microsc. 245(3), 311–318 (2012).
[Crossref]

Zar?bski, M.

T. Bernas, M. Zarȩbski, R. R. Cook, and J. W. Dobrucki, “Minimizing photobleaching during confocal microscopy of fluorescent probes bound to chromatin: role of anoxia and photon flux,” J. Microsc. 215(3), 281–296 (2004).
[Crossref]

Angew. Chem., Int. Ed. (1)

J. Vogelsang, R. Kasper, C. Steinhauer, B. Person, M. Heilemann, M. Sauer, and P. Tinnefeld, “A Reducing and Oxidizing System Minimizes Photobleaching and Blinking of Fluorescent Dyes,” Angew. Chem., Int. Ed. 47(29), 5465–5469 (2008).
[Crossref]

Appl. Phys. A (1)

S. W. Hell, S. Jakobs, and L. Kastrup, “Imaging and writing at the nanoscale with focused visible light through saturable optical transitions,” Appl. Phys. A 77(7), 859–860 (2003).
[Crossref]

Biophys. J. (1)

L. Song, C. A. G. O. Varma, J. W. Verhoeven, and H. J. Tanke, “Influence of the Triplet Excited State on the Photobleaching Kinetics of Fluorescein in Microscopy,” Biophys. J. 70(6), 2959–2968 (1996).
[Crossref]

Curr. Protoc. Neurosci. (1)

C. A. Combs, “Fluorescence Microscopy: A Concise Guide to Current Imaging Methods,” Curr. Protoc. Neurosci. 50(1), 2.1.1–2.1.14 (2010).
[Crossref]

J. Biomol. Tech. (1)

J. Jonkman and C. M. Brown, “Any Way You Slice It - A Comparison of Confocal Microscopy Techniques,” J. Biomol. Tech. 26(2), 54–65 (2015).
[Crossref]

J. Microsc. (2)

H. Schneckenburger, P. Weber, M. Wagner, S. Schickinger, V. Richter, T. Bruns, W. S. L. Strauss, and R. Wittig, “Light exposure and cell viability in fluorescence microscopy,” J. Microsc. 245(3), 311–318 (2012).
[Crossref]

T. Bernas, M. Zarȩbski, R. R. Cook, and J. W. Dobrucki, “Minimizing photobleaching during confocal microscopy of fluorescent probes bound to chromatin: role of anoxia and photon flux,” J. Microsc. 215(3), 281–296 (2004).
[Crossref]

Microsc. Res. Tech. (1)

R. T. Borlinghaus, “MRT letter: High Speed Scanning Has the Potential to Increase Fluorescence Yield and to Reduce Photobleaching,” Microsc. Res. Tech. 69(9), 689–692 (2006).
[Crossref]

Nat. Biotechnol. (1)

R. A. Hoebe, C. H. Van Oven, T. W. J. Gadella Jr., P. B. Dhonukshe, C. J. F. Van Noorden, and E. M. M. Manders, “Controlled light-exposure microscopy reduces photobleaching and phototoxicity in fluorescence live-cell imaging,” Nat. Biotechnol. 25(2), 249–253 (2007).
[Crossref]

Nat. Commun. (1)

J. Dreier, M. Castello, G. Coceano, R. Cáceres, J. Plastino, G. Vicidomini, and I. Testa, “Smart scanning for low-illumination and fast RESOLFT nanoscopy in vivo,” Nat. Commun. 10(1), 556 (2019).
[Crossref]

Nat. Methods (1)

G. Donnert, C. Eggeling, and S. W. Hell, “Major signal increase in fluorescence microscopy through dark-state relaxation,” Nat. Methods 4(1), 81–86 (2007).
[Crossref]

Nat. Rev. Mol. Cell Biol. (1)

S. J. Sahl, S. W. Hell, and S. Jakobs, “Fluorescence nanoscopy in cell biology,” Nat. Rev. Mol. Cell Biol. 18(11), 685–701 (2017).
[Crossref]

Opt. Express (1)

Opt. Lett. (1)

Photochem. Photobiol. Sci. (1)

G. Donnert, C. Eggeling, and S. W. Hell, “Triplet-relaxation microscopy with bunched pulsed excitation,” Photochem. Photobiol. Sci. 8(4), 481–485 (2009).
[Crossref]

Phys. Procedia (1)

G. R. B. E. Römer and P. Bechtold, “Electro-optic and acousto-optic laser beam scanners,” Phys. Procedia 56, 29–39 (2014).
[Crossref]

Phys. Rev. E (1)

T. 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(6), 066613 (2001).
[Crossref]

Proc. Natl. Acad. Sci. U. S. A. (4)

T. A. Klar, S. Jakobs, M. Dyba, A. Egner, and S. W. Hell, “Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission,” Proc. Natl. Acad. Sci. U. S. A. 97(15), 8206–8210 (2000).
[Crossref]

F. Göttfert, T. Pleiner, J. Heine, V. Westphal, D. Görlich, S. J. Sahl, and S. W. Hell, “Strong signal increase in STED fluorescence microscopy by imaging regions of subdiffraction extent,” Proc. Natl. Acad. Sci. U. S. A. 114(9), 2125–2130 (2017).
[Crossref]

J. Heine, M. Reuss, B. Harke, E. D’Este, S. J. Sahl, and S. W. Hell, “Adaptive-illumination STED nanoscopy,” Proc. Natl. Acad. Sci. U. S. A. 114(37), 9797–9802 (2017).
[Crossref]

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Lührmann, R. Jahn, C. Eggeling, and S. W. Hell, “Macromolecular-scale resolution in biological fluorescence microscopy,” Proc. Natl. Acad. Sci. U. S. A. 103(31), 11440–11445 (2006).
[Crossref]

Sci. Rep. (1)

C. Boudreau, T.-L. E. Wee, Y.-R. S. Duh, M. P. Couto, K. H. Ardakani, and C. M. Brown, “Excitation Light Dose Engineering to Reduce Photo-bleaching and Photo-toxicity,” Sci. Rep. 6(1), 30892 (2016).
[Crossref]

Science (4)

S. W. Hell, “Far-Field Optical Nanoscopy,” Science 316(5828), 1153–1158 (2007).
[Crossref]

D. J. Stephens and V. J. Allan, “Light Microscopy Techniques for Live Cell Imaging,” Science 300(5616), 82–86 (2003).
[Crossref]

C. L. Rieder and A. Khodjakov, “Mitosis Through the Microscope: Advances in Seeing Inside Live Dividing Cells,” Science 300(5616), 91–96 (2003).
[Crossref]

F. Balzarotti, Y. Eilers, K. C. Gwosch, A. H. Gynnå, V. Westphal, F. D. Stefani, J. Elf, and S. W. Hell, “Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes,” Science 355(6325), 606–612 (2017).
[Crossref]

Other (5)

F. Göttfert, “STED microscopy with scanning fields below the diffraction limit,” PhD thesis (Georg-August-Universität Göttingen, Germany, 2015).

C. Ledig and D. Rueckert, “Chapter 14 - Semantic Parsing of Brain MR Images,” in Medical Image Recognition, Segmentation and ParsingS. K. Zhou, ed. (Academic, 2016).

G. F. Marshall and G. E. Stutz, Handbook of Optical and Laser Scanning (CRC, New York, 2004).

J. B. Pawley, Handbook Of Biological Confocal Microscopy (Springer, 2006).

Y. Sun and A. Periasamy, “Fluorescence Microscopy Imaging in Biomedical Sciences,” in Biomedical Optical Imaging Technologies, R. Liang, ed. (Springer, 2013).

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Figures (5)

Fig. 1.
Fig. 1. Implementation of FastRESCue-STED imaging. (a) The optical setup includes two EODs as fast scanning devices additionally to the galvanometer scanner. (Exc: excitation unit; APD: avalanche photo diode; STED: STED laser unit; $\lambda /2$, $\lambda /4$: half and quarter wave plate, respectively; TC1, TC2: telescopes; GT: Glan-Thompson-prism; GS: galvanometer scanner; OL: objective lens; S: sample). (b) Sketch of the combined scan system’s control. Two voltages for the scanner are output by the FPGA I/O device. They serve as inputs to the circuit board (red) and are subsequently linearly transformed. The monitoring output voltages of the scanner are used as additional inputs to derive the voltages for the EOD drivers.
Fig. 2.
Fig. 2. Performance of the scan system and impact on image quality. (a) Scan position displacement of the galvanometer scanner as a function of the pixel dwell time for a field of view of $3{\mu \textrm{m}}\times 3{\mu \textrm{m}}$ and $20\;\textrm{nm}\times 20$ nm pixel size, averaged over a line scan. The error bars denote ten times the standard error of the mean. For a pixel dwell time of $1{\mu \textrm{s}}$, the scan position displacement is more than $2{\mu \textrm{m}}$. (b-d) Imaging of fluorescent microspheres using only the galvanometer scanner with (b) STED and (c) RESCue-STED shows equivalent image quality, while the corresponding FastRESCue-STED image (d) exhibits clear distortions. (e) Only with the EODs as additional scanning devices for FastRESCue-STED, the scan position displacement is compensated and the image quality is restored. Note that for a better visualization, the resolution is only increased in the direction of the fast scan axis. The same color table is chosen for all images and the scale bar indicates a length of $500$ nm in (b-e).
Fig. 3.
Fig. 3. FastRESCue reduces light dose and frame time. (a-d) Images of fluorescent microspheres of $48$ nm diameter for (a) confocal, (b) STED and (c) FastRESCue-STED acquisition (scale bar $1{\mu \textrm{m}}$). The chosen RESCue parameters result in a light dose of $15.9\%$ and a frame time of $17.8\%$ compared to the standard STED acquisition. (d) shows the effective pixel dwell times in ${\mu \textrm{s}}$, illustrating the potential to speed up acquisition both in areas without structure as well as in areas of high signal. (e) The FastRESCue acquisition is repeated for varying RESCue parameters (see Table 2 in the appendix) at otherwise identical acquisition parameters, leading to a varying relative FastRESCue light dose. The resultant frame time compared to the standard STED acquisition is depicted as a function of this light dose, showing a direct translation of light dose saving into a faster acquisition. The red curve visualizes equal light dose and frame time saving.
Fig. 4.
Fig. 4. Advantage of FastRESCue on different spatial scales. Images of $\alpha$-tubulin in Vero cells (dye: Abberior STAR RED) recorded with (a) STED and (b) FastRESCue-STED (scale bar $1{\mu \textrm{m}}$). The global FastRESCue light dose and frame time is reduced to $20.3\%$ and $22.7\%$, respectively. The distribution of the effective pixel dwell time (in ${\mu \textrm{s}}$) is visualized in (c). Histograms of local relative acquisition times for different ROI sizes are displayed in (d), with the variance in dependence on the ROI size being depicted in the inset.
Fig. 5.
Fig. 5. FastRESCue-STED imaging of various cellular structures. Confocal overviews (a, c, e, g) and FastRESCue-STED images (b, d, f, h) of $\alpha$-tubulin in Vero cells (a,b), peroxisomes (PMP70) in Vero cells (c,d), vimentin in fibroblasts (e,f) and $\alpha$-tubulin in fibroblasts (g,h). For fluorescent labeling, the fluorophores Abberior STAR RED (a,b) and Abberior STAR 635P (c-h) were used. The length of the scale bar is $5{\mu \textrm{m}}$ (a,c,e,g) or $1{\mu \textrm{m}}$ (b,d,f,h). The percentage values in the upper right corner of the FastRESCue images denote the respective relative FastRESCue frame time.

Tables (2)

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Table 1. Acquisition parameters for the STED measurements displayed in Figs. 25.

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Table 2. RESCue parameters for the FastRESCue acquisitions analyzed in Fig. 3(e).