Abstract

Owing to the advantage of being non-invasive in observing living samples, far-field optical microscopy is widely used in the life sciences, but the existence of the diffraction barrier leads to the poor imaging of samples with spatial features smaller than approximately half the wavelength of the probes. This limit has been overcome by a number of pointwise scanning optical imaging techniques, such as stimulated emission depletion microscopy (STED) and saturated excitation microscopy (SAX). Here, we introduce the concept of saturated absorption competition (SAC) microscopy as a simple means of providing sub-diffraction spatial resolution in fluorescence imaging. Our approach can be physically implemented in a confocal microscope by dividing the input laser source into a time-modulated primary excitation beam and a doughnut-shaped saturation beam and subsequently employing a homodyne detection scheme to select the modulated fluorescence signal. Herein, we provide both a physico-chemical model of SAC and experimentally demonstrate a transverse spatial resolution of 1.5- to 2-fold that of confocal.

© 2017 Optical Society of America

Full Article  |  PDF Article
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References

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2015 (1)

B. Yang, J. B. Trebbia, R. Baby, P. Tamarat, and B. Lounis, Nat. Photonics 9, 658 (2015).
[Crossref]

2014 (1)

S. W. Chu, T. Y. Su, R. Oketani, Y. Huang, H. Wu, Y. Yonemaru, M. Yamanaka, H. Lee, G. Zhuo, M. Lee, S. Kawata, and K. Fujita, Phys. Rev. Lett. 112, 017402 (2014).
[Crossref]

2013 (2)

R. P. Nieuwenhuizen, K. A. Lidke, M. Bates, D. L. Puig, D. Grünwald, S. Stallinga, and B. Rieger, Nat. Methods 10, 557 (2013).
[Crossref]

P. Wang, M. N. Slipchenko, J. Mitchell, C. Yang, E. O. Potma, X. Xu, and J. X. Cheng, Nat. Photonics 7, 449 (2013).
[Crossref]

2012 (1)

D. Wildanger, B. R. Patton, H. Schill, L. Marseglia, J. P. Hadden, S. Knauer, A. Schönle, J. G. Rarity, J. L. O’Brien, S. W. Hell, and J. M. Smith, Adv. Mater. 24, OP309 (2012).
[Crossref]

2011 (1)

2010 (1)

2009 (2)

E. Rittweger, K. Y. Han, S. E. Irvine, C. Eggeling, and S. W. Hell, Nat. Photonics 3, 144 (2009).
[Crossref]

E. Rittweger, D. Wildanger, and S. Hell, Europhys. Lett. 86, 14001 (2009).
[Crossref]

2007 (4)

J. Keller, A. Schönle, and S. W. Hell, Opt. Express 15, 3361 (2007).
[Crossref]

K. Fujita, M. Kobayashi, S. Kawano, M. Yamanaka, and S. Kawata, Phys. Rev. Lett. 99, 228105 (2007).
[Crossref]

K. I. Willig, B. Harke, R. Medda, and S. W. Hell, Nat. Methods 4, 915 (2007).
[Crossref]

R. Heintzmann, Micron 38, 136 (2007).
[Crossref]

2005 (1)

C. Eggeling, A. Volkmer, and C. A. Seidel, Chem. Phys. Chem. 6, 791 (2005).
[Crossref]

2003 (1)

S. W. Hell, Nat. Biotechnol. 21, 1347 (2003).
[Crossref]

1995 (1)

S. W. Hell and M. Kroug, Appl. Phys. B 60, 495 (1995).
[Crossref]

1994 (1)

Baby, R.

B. Yang, J. B. Trebbia, R. Baby, P. Tamarat, and B. Lounis, Nat. Photonics 9, 658 (2015).
[Crossref]

Bates, M.

R. P. Nieuwenhuizen, K. A. Lidke, M. Bates, D. L. Puig, D. Grünwald, S. Stallinga, and B. Rieger, Nat. Methods 10, 557 (2013).
[Crossref]

Chang, H. C.

Cheng, J. X.

P. Wang, M. N. Slipchenko, J. Mitchell, C. Yang, E. O. Potma, X. Xu, and J. X. Cheng, Nat. Photonics 7, 449 (2013).
[Crossref]

Chu, S. W.

S. W. Chu, T. Y. Su, R. Oketani, Y. Huang, H. Wu, Y. Yonemaru, M. Yamanaka, H. Lee, G. Zhuo, M. Lee, S. Kawata, and K. Fujita, Phys. Rev. Lett. 112, 017402 (2014).
[Crossref]

Eggeling, C.

E. Rittweger, K. Y. Han, S. E. Irvine, C. Eggeling, and S. W. Hell, Nat. Photonics 3, 144 (2009).
[Crossref]

C. Eggeling, A. Volkmer, and C. A. Seidel, Chem. Phys. Chem. 6, 791 (2005).
[Crossref]

Fujita, K.

S. W. Chu, T. Y. Su, R. Oketani, Y. Huang, H. Wu, Y. Yonemaru, M. Yamanaka, H. Lee, G. Zhuo, M. Lee, S. Kawata, and K. Fujita, Phys. Rev. Lett. 112, 017402 (2014).
[Crossref]

M. Yamanaka, Y. K. Tzeng, S. Kawano, N. I. Smith, S. Kawata, H. C. Chang, and K. Fujita, Biomed. Opt. Express 2, 1946 (2011).
[Crossref]

K. Fujita, M. Kobayashi, S. Kawano, M. Yamanaka, and S. Kawata, Phys. Rev. Lett. 99, 228105 (2007).
[Crossref]

Grünwald, D.

R. P. Nieuwenhuizen, K. A. Lidke, M. Bates, D. L. Puig, D. Grünwald, S. Stallinga, and B. Rieger, Nat. Methods 10, 557 (2013).
[Crossref]

Guo, H.

Hadden, J. P.

D. Wildanger, B. R. Patton, H. Schill, L. Marseglia, J. P. Hadden, S. Knauer, A. Schönle, J. G. Rarity, J. L. O’Brien, S. W. Hell, and J. M. Smith, Adv. Mater. 24, OP309 (2012).
[Crossref]

Han, K. Y.

E. Rittweger, K. Y. Han, S. E. Irvine, C. Eggeling, and S. W. Hell, Nat. Photonics 3, 144 (2009).
[Crossref]

Harke, B.

K. I. Willig, B. Harke, R. Medda, and S. W. Hell, Nat. Methods 4, 915 (2007).
[Crossref]

Heintzmann, R.

R. Heintzmann, Micron 38, 136 (2007).
[Crossref]

Hell, S.

E. Rittweger, D. Wildanger, and S. Hell, Europhys. Lett. 86, 14001 (2009).
[Crossref]

Hell, S. W.

D. Wildanger, B. R. Patton, H. Schill, L. Marseglia, J. P. Hadden, S. Knauer, A. Schönle, J. G. Rarity, J. L. O’Brien, S. W. Hell, and J. M. Smith, Adv. Mater. 24, OP309 (2012).
[Crossref]

E. Rittweger, K. Y. Han, S. E. Irvine, C. Eggeling, and S. W. Hell, Nat. Photonics 3, 144 (2009).
[Crossref]

K. I. Willig, B. Harke, R. Medda, and S. W. Hell, Nat. Methods 4, 915 (2007).
[Crossref]

J. Keller, A. Schönle, and S. W. Hell, Opt. Express 15, 3361 (2007).
[Crossref]

S. W. Hell, Nat. Biotechnol. 21, 1347 (2003).
[Crossref]

S. W. Hell and M. Kroug, Appl. Phys. B 60, 495 (1995).
[Crossref]

S. W. Hell and J. Wichmann, Opt. Lett. 19, 780 (1994).
[Crossref]

Huan, C. H.

Huang, Y.

S. W. Chu, T. Y. Su, R. Oketani, Y. Huang, H. Wu, Y. Yonemaru, M. Yamanaka, H. Lee, G. Zhuo, M. Lee, S. Kawata, and K. Fujita, Phys. Rev. Lett. 112, 017402 (2014).
[Crossref]

Irvine, S. E.

E. Rittweger, K. Y. Han, S. E. Irvine, C. Eggeling, and S. W. Hell, Nat. Photonics 3, 144 (2009).
[Crossref]

Kawano, S.

M. Yamanaka, Y. K. Tzeng, S. Kawano, N. I. Smith, S. Kawata, H. C. Chang, and K. Fujita, Biomed. Opt. Express 2, 1946 (2011).
[Crossref]

K. Fujita, M. Kobayashi, S. Kawano, M. Yamanaka, and S. Kawata, Phys. Rev. Lett. 99, 228105 (2007).
[Crossref]

Kawata, S.

S. W. Chu, T. Y. Su, R. Oketani, Y. Huang, H. Wu, Y. Yonemaru, M. Yamanaka, H. Lee, G. Zhuo, M. Lee, S. Kawata, and K. Fujita, Phys. Rev. Lett. 112, 017402 (2014).
[Crossref]

M. Yamanaka, Y. K. Tzeng, S. Kawano, N. I. Smith, S. Kawata, H. C. Chang, and K. Fujita, Biomed. Opt. Express 2, 1946 (2011).
[Crossref]

K. Fujita, M. Kobayashi, S. Kawano, M. Yamanaka, and S. Kawata, Phys. Rev. Lett. 99, 228105 (2007).
[Crossref]

Keller, J.

Knauer, S.

D. Wildanger, B. R. Patton, H. Schill, L. Marseglia, J. P. Hadden, S. Knauer, A. Schönle, J. G. Rarity, J. L. O’Brien, S. W. Hell, and J. M. Smith, Adv. Mater. 24, OP309 (2012).
[Crossref]

Kobayashi, M.

K. Fujita, M. Kobayashi, S. Kawano, M. Yamanaka, and S. Kawata, Phys. Rev. Lett. 99, 228105 (2007).
[Crossref]

Kroug, M.

S. W. Hell and M. Kroug, Appl. Phys. B 60, 495 (1995).
[Crossref]

Lee, H.

S. W. Chu, T. Y. Su, R. Oketani, Y. Huang, H. Wu, Y. Yonemaru, M. Yamanaka, H. Lee, G. Zhuo, M. Lee, S. Kawata, and K. Fujita, Phys. Rev. Lett. 112, 017402 (2014).
[Crossref]

Lee, M.

S. W. Chu, T. Y. Su, R. Oketani, Y. Huang, H. Wu, Y. Yonemaru, M. Yamanaka, H. Lee, G. Zhuo, M. Lee, S. Kawata, and K. Fujita, Phys. Rev. Lett. 112, 017402 (2014).
[Crossref]

Lidke, K. A.

R. P. Nieuwenhuizen, K. A. Lidke, M. Bates, D. L. Puig, D. Grünwald, S. Stallinga, and B. Rieger, Nat. Methods 10, 557 (2013).
[Crossref]

Lounis, B.

B. Yang, J. B. Trebbia, R. Baby, P. Tamarat, and B. Lounis, Nat. Photonics 9, 658 (2015).
[Crossref]

Marseglia, L.

D. Wildanger, B. R. Patton, H. Schill, L. Marseglia, J. P. Hadden, S. Knauer, A. Schönle, J. G. Rarity, J. L. O’Brien, S. W. Hell, and J. M. Smith, Adv. Mater. 24, OP309 (2012).
[Crossref]

Medda, R.

K. I. Willig, B. Harke, R. Medda, and S. W. Hell, Nat. Methods 4, 915 (2007).
[Crossref]

Mitchell, J.

P. Wang, M. N. Slipchenko, J. Mitchell, C. Yang, E. O. Potma, X. Xu, and J. X. Cheng, Nat. Photonics 7, 449 (2013).
[Crossref]

Nieuwenhuizen, R. P.

R. P. Nieuwenhuizen, K. A. Lidke, M. Bates, D. L. Puig, D. Grünwald, S. Stallinga, and B. Rieger, Nat. Methods 10, 557 (2013).
[Crossref]

O’Brien, J. L.

D. Wildanger, B. R. Patton, H. Schill, L. Marseglia, J. P. Hadden, S. Knauer, A. Schönle, J. G. Rarity, J. L. O’Brien, S. W. Hell, and J. M. Smith, Adv. Mater. 24, OP309 (2012).
[Crossref]

Oketani, R.

S. W. Chu, T. Y. Su, R. Oketani, Y. Huang, H. Wu, Y. Yonemaru, M. Yamanaka, H. Lee, G. Zhuo, M. Lee, S. Kawata, and K. Fujita, Phys. Rev. Lett. 112, 017402 (2014).
[Crossref]

Patton, B. R.

D. Wildanger, B. R. Patton, H. Schill, L. Marseglia, J. P. Hadden, S. Knauer, A. Schönle, J. G. Rarity, J. L. O’Brien, S. W. Hell, and J. M. Smith, Adv. Mater. 24, OP309 (2012).
[Crossref]

Potma, E. O.

P. Wang, M. N. Slipchenko, J. Mitchell, C. Yang, E. O. Potma, X. Xu, and J. X. Cheng, Nat. Photonics 7, 449 (2013).
[Crossref]

Puig, D. L.

R. P. Nieuwenhuizen, K. A. Lidke, M. Bates, D. L. Puig, D. Grünwald, S. Stallinga, and B. Rieger, Nat. Methods 10, 557 (2013).
[Crossref]

Rarity, J. G.

D. Wildanger, B. R. Patton, H. Schill, L. Marseglia, J. P. Hadden, S. Knauer, A. Schönle, J. G. Rarity, J. L. O’Brien, S. W. Hell, and J. M. Smith, Adv. Mater. 24, OP309 (2012).
[Crossref]

Rieger, B.

R. P. Nieuwenhuizen, K. A. Lidke, M. Bates, D. L. Puig, D. Grünwald, S. Stallinga, and B. Rieger, Nat. Methods 10, 557 (2013).
[Crossref]

Rittweger, E.

E. Rittweger, D. Wildanger, and S. Hell, Europhys. Lett. 86, 14001 (2009).
[Crossref]

E. Rittweger, K. Y. Han, S. E. Irvine, C. Eggeling, and S. W. Hell, Nat. Photonics 3, 144 (2009).
[Crossref]

Schill, H.

D. Wildanger, B. R. Patton, H. Schill, L. Marseglia, J. P. Hadden, S. Knauer, A. Schönle, J. G. Rarity, J. L. O’Brien, S. W. Hell, and J. M. Smith, Adv. Mater. 24, OP309 (2012).
[Crossref]

Schönle, A.

D. Wildanger, B. R. Patton, H. Schill, L. Marseglia, J. P. Hadden, S. Knauer, A. Schönle, J. G. Rarity, J. L. O’Brien, S. W. Hell, and J. M. Smith, Adv. Mater. 24, OP309 (2012).
[Crossref]

J. Keller, A. Schönle, and S. W. Hell, Opt. Express 15, 3361 (2007).
[Crossref]

Seidel, C. A.

C. Eggeling, A. Volkmer, and C. A. Seidel, Chem. Phys. Chem. 6, 791 (2005).
[Crossref]

Slipchenko, M. N.

P. Wang, M. N. Slipchenko, J. Mitchell, C. Yang, E. O. Potma, X. Xu, and J. X. Cheng, Nat. Photonics 7, 449 (2013).
[Crossref]

Smith, J. M.

D. Wildanger, B. R. Patton, H. Schill, L. Marseglia, J. P. Hadden, S. Knauer, A. Schönle, J. G. Rarity, J. L. O’Brien, S. W. Hell, and J. M. Smith, Adv. Mater. 24, OP309 (2012).
[Crossref]

Smith, N. I.

Stallinga, S.

R. P. Nieuwenhuizen, K. A. Lidke, M. Bates, D. L. Puig, D. Grünwald, S. Stallinga, and B. Rieger, Nat. Methods 10, 557 (2013).
[Crossref]

Su, T. Y.

S. W. Chu, T. Y. Su, R. Oketani, Y. Huang, H. Wu, Y. Yonemaru, M. Yamanaka, H. Lee, G. Zhuo, M. Lee, S. Kawata, and K. Fujita, Phys. Rev. Lett. 112, 017402 (2014).
[Crossref]

Sum, T. C.

Tamarat, P.

B. Yang, J. B. Trebbia, R. Baby, P. Tamarat, and B. Lounis, Nat. Photonics 9, 658 (2015).
[Crossref]

Trebbia, J. B.

B. Yang, J. B. Trebbia, R. Baby, P. Tamarat, and B. Lounis, Nat. Photonics 9, 658 (2015).
[Crossref]

Tzeng, Y. K.

Volkmer, A.

C. Eggeling, A. Volkmer, and C. A. Seidel, Chem. Phys. Chem. 6, 791 (2005).
[Crossref]

Wang, P.

P. Wang, M. N. Slipchenko, J. Mitchell, C. Yang, E. O. Potma, X. Xu, and J. X. Cheng, Nat. Photonics 7, 449 (2013).
[Crossref]

Wichmann, J.

Wildanger, D.

D. Wildanger, B. R. Patton, H. Schill, L. Marseglia, J. P. Hadden, S. Knauer, A. Schönle, J. G. Rarity, J. L. O’Brien, S. W. Hell, and J. M. Smith, Adv. Mater. 24, OP309 (2012).
[Crossref]

E. Rittweger, D. Wildanger, and S. Hell, Europhys. Lett. 86, 14001 (2009).
[Crossref]

Willig, K. I.

K. I. Willig, B. Harke, R. Medda, and S. W. Hell, Nat. Methods 4, 915 (2007).
[Crossref]

Wu, H.

S. W. Chu, T. Y. Su, R. Oketani, Y. Huang, H. Wu, Y. Yonemaru, M. Yamanaka, H. Lee, G. Zhuo, M. Lee, S. Kawata, and K. Fujita, Phys. Rev. Lett. 112, 017402 (2014).
[Crossref]

Xing, G.

Xu, X.

P. Wang, M. N. Slipchenko, J. Mitchell, C. Yang, E. O. Potma, X. Xu, and J. X. Cheng, Nat. Photonics 7, 449 (2013).
[Crossref]

Yamanaka, M.

S. W. Chu, T. Y. Su, R. Oketani, Y. Huang, H. Wu, Y. Yonemaru, M. Yamanaka, H. Lee, G. Zhuo, M. Lee, S. Kawata, and K. Fujita, Phys. Rev. Lett. 112, 017402 (2014).
[Crossref]

M. Yamanaka, Y. K. Tzeng, S. Kawano, N. I. Smith, S. Kawata, H. C. Chang, and K. Fujita, Biomed. Opt. Express 2, 1946 (2011).
[Crossref]

K. Fujita, M. Kobayashi, S. Kawano, M. Yamanaka, and S. Kawata, Phys. Rev. Lett. 99, 228105 (2007).
[Crossref]

Yang, B.

B. Yang, J. B. Trebbia, R. Baby, P. Tamarat, and B. Lounis, Nat. Photonics 9, 658 (2015).
[Crossref]

Yang, C.

P. Wang, M. N. Slipchenko, J. Mitchell, C. Yang, E. O. Potma, X. Xu, and J. X. Cheng, Nat. Photonics 7, 449 (2013).
[Crossref]

Yonemaru, Y.

S. W. Chu, T. Y. Su, R. Oketani, Y. Huang, H. Wu, Y. Yonemaru, M. Yamanaka, H. Lee, G. Zhuo, M. Lee, S. Kawata, and K. Fujita, Phys. Rev. Lett. 112, 017402 (2014).
[Crossref]

Zhang, X.

Zhuo, G.

S. W. Chu, T. Y. Su, R. Oketani, Y. Huang, H. Wu, Y. Yonemaru, M. Yamanaka, H. Lee, G. Zhuo, M. Lee, S. Kawata, and K. Fujita, Phys. Rev. Lett. 112, 017402 (2014).
[Crossref]

Adv. Mater. (1)

D. Wildanger, B. R. Patton, H. Schill, L. Marseglia, J. P. Hadden, S. Knauer, A. Schönle, J. G. Rarity, J. L. O’Brien, S. W. Hell, and J. M. Smith, Adv. Mater. 24, OP309 (2012).
[Crossref]

Appl. Phys. B (1)

S. W. Hell and M. Kroug, Appl. Phys. B 60, 495 (1995).
[Crossref]

Biomed. Opt. Express (1)

Chem. Phys. Chem. (1)

C. Eggeling, A. Volkmer, and C. A. Seidel, Chem. Phys. Chem. 6, 791 (2005).
[Crossref]

Europhys. Lett. (1)

E. Rittweger, D. Wildanger, and S. Hell, Europhys. Lett. 86, 14001 (2009).
[Crossref]

Micron (1)

R. Heintzmann, Micron 38, 136 (2007).
[Crossref]

Nat. Biotechnol. (1)

S. W. Hell, Nat. Biotechnol. 21, 1347 (2003).
[Crossref]

Nat. Methods (2)

K. I. Willig, B. Harke, R. Medda, and S. W. Hell, Nat. Methods 4, 915 (2007).
[Crossref]

R. P. Nieuwenhuizen, K. A. Lidke, M. Bates, D. L. Puig, D. Grünwald, S. Stallinga, and B. Rieger, Nat. Methods 10, 557 (2013).
[Crossref]

Nat. Photonics (3)

P. Wang, M. N. Slipchenko, J. Mitchell, C. Yang, E. O. Potma, X. Xu, and J. X. Cheng, Nat. Photonics 7, 449 (2013).
[Crossref]

B. Yang, J. B. Trebbia, R. Baby, P. Tamarat, and B. Lounis, Nat. Photonics 9, 658 (2015).
[Crossref]

E. Rittweger, K. Y. Han, S. E. Irvine, C. Eggeling, and S. W. Hell, Nat. Photonics 3, 144 (2009).
[Crossref]

Opt. Express (2)

Opt. Lett. (1)

Phys. Rev. Lett. (2)

S. W. Chu, T. Y. Su, R. Oketani, Y. Huang, H. Wu, Y. Yonemaru, M. Yamanaka, H. Lee, G. Zhuo, M. Lee, S. Kawata, and K. Fujita, Phys. Rev. Lett. 112, 017402 (2014).
[Crossref]

K. Fujita, M. Kobayashi, S. Kawano, M. Yamanaka, and S. Kawata, Phys. Rev. Lett. 99, 228105 (2007).
[Crossref]

Supplementary Material (1)

NameDescription
» Supplement 1: PDF (3263 KB)      Supplement 1

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Figures (5)

Fig. 1.
Fig. 1.

Principle model of SAC. (a) Five-level molecular electronic state model used to calculate the relationship between the excitation-emission intensities. (b) Illustration of the saturation of S1 state and the process of competition during the excitation. “a” denotes the high-intensity competition beam (solid lines), while “b” denotes the low-intensity primary excitation beam (dashed lines). (c) Decrease of the normalized fluorescence value (Is) in the S1 state of SAC (black curve) and STED (green curve) as a result of competition. To differentiate Is of the two methods, we take as ISC the IS of SAC and ISD as that of STED, which are represented by the green and black (inset) lines in (d), respectively.

Fig. 2.
Fig. 2.

Conceptual schematic of saturated absorption competition microscopy. PMT, photomultiplier; DM, dichroic mirror; OL, objective lens; M, reflective lens; WP, wave plate. Solid arrow and dashed arrow represent the sub-beams with phase modulation and the other with frequency modulation, respectively. The λ/2 WP is used here to tune the intensity ratio of the two beams. The sample in (a)–(d) is a 200-nm diameter fluorescent bead (FluoSpheres Carboxylate-Modified Microspheres, 0.2 μm, dark red fluorescent (660/680), 2% solids). Scale bar in (a) is 200 nm.

Fig. 3.
Fig. 3.

Comparison of experimental results obtained from conventional confocal microscopy and SAC microscopy. (a) The 2D intensity distribution of 100-nm diameter fluorescent nanoparticles (field of view is 7.68  μm×7.68  μm) obtained from (i) standard confocal microscopy using an input power of 5 μW, and (ii)–(vii) SAC microscopy as a function of input saturation-beam power (noted in the top right hand corner of each image). (b) The measured normalized fluorescence intensity as a function of the increase in doughnut beam power P. Inserted dashed lines reveal Ps, where the fluorescence drops down to half of its original amount. (c) Measured lateral FWHM of the detected fluorescence as a function of input saturation beam power; the trend appears to follow an inverse square law. Note that all the data are taken by counting the average value of Gaussian fitted beads’ sizes. (d,i) Magnified views of the fluorescence from a single bead in (a,i,iv,vi, and vii) (indicated by yellow boxes). (d,ii) Intensity profiles along the blue double arrow in (d,i). (e) Histogram depicting the distribution of bead sizes in confocal (a,i) and SAC (a,vii) with the highest doughnut beam power. The pixel size is 30 nm with a dwell time of 0.2 ms.

Fig. 4.
Fig. 4.

Imaging results of 40 nm fluorescent nanoparticles with confocal and SAC microscopy. Images of (a), confocal at 10 μW, (b), SAC for saturation power at 180 μW, (c), Richardson–Lucy (RL) deconvolution result of (a) with 10 iterations, (d), RL deconvolution result of (b) with 10 iterations. (e) Magnified views of areas indicated by green boxes in (a)–(d), scale bar, 300 nm. (f) Intensity profiles along the white lines in (e). (g) Magnified views of areas indicated by write dashed boxes in (a)–(d), scale bar, 300 nm. (h) Intensity profiles along the green lines in (g). Pixel per size is 30 nm and the per dwell time is 0.5 ms.

Fig. 5.
Fig. 5.

Imaging results of vero cells (Vero PFA, STAR-635P) with confocal and SAC microscopy. Vero cell was stained with primary antibodies against the nuclear pore complex protein Nup153 and secondary antibodies conjugated with STAR-635P. Images of (a) confocal at 1.5 μW, (b) SAC for saturation power of 40 μW, and (c) RL deconvolution result of (b) with 20 iterations. Pixel per size is 30 nm and the per dwell time is 0.5 ms. Inset scale bar, 500 nm.

Equations (3)

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S1eq=kT(kexc+kexc)(kexc+kexc)(kisc+kT)+k0kT.
S1eq=(kexc+kexc)/(3.245×(kexc+kexc)+k0).
S1eff=kexc/(3.245×(kexc+kexc)+k0),

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