Abstract

Localization based super-resolution microscopy techniques require precise drift correction methods because the achieved spatial resolution is close to both the mechanical and optical performance limits of modern light microscopes. Multi-color imaging methods require corrections in addition to those dealing with drift due to the static, but spatially-dependent, chromatic offset between images. We present computer simulations to quantify this effect, which is primarily caused by the high-NA objectives used in super-resolution microscopy. Although the chromatic offset in well corrected systems is only a fraction of an optical wavelength in magnitude (<50 nm) and thus negligible in traditional diffraction limited imaging, we show that object colocalization by multi-color super-resolution methods is impossible without appropriate image correction. The simulated data are in excellent agreement with experiments using fluorescent beads excited and localized at multiple wavelengths. Finally we present a rigorous and practical calibration protocol to correct for chromatic optical offset, and demonstrate its efficacy for the imaging of transferrin receptor protein colocalization in HeLa cells using two-color direct stochastic optical reconstruction microscopy (dSTORM).

©2013 Optical Society of America

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    [Crossref] [PubMed]

2012 (5)

E. J. Rees, M. Erdelyi, D. Pinotsi, A. Knight, D. Metcalf, and C. F. Kaminski, “Blind assessment of localization microscopy image resolution,” Opt. Nanoscopy 1(1), 12 (2012).
[Crossref]

M. Bates, G. T. Dempsey, K. H. Chen, and X. Zhuang, “Multicolor super-resolution fluorescence imaging via multi-parameter fluorophore detection,” ChemPhysChem 13(1), 99–107 (2012).
[Crossref] [PubMed]

A. Löschberger, S. van de Linde, M. C. Dabauvalle, B. Rieger, M. Heilemann, G. Krohne, and M. Sauer, “Super-resolution imaging visualizes the eightfold symmetry of gp210 proteins around the nuclear pore complex and resolves the central channel with nanometer resolution,” J. Cell Sci. 125(3), 570–575 (2012).
[Crossref] [PubMed]

M. Ahn, E. De Genst, G. S. Kaminski Schierle, M. Erdelyi, C. F. Kaminski, C. M. Dobson, and J. R. Kumita, “Analysis of the native structure, stability and aggregation of biotinylated human lysozyme,” PLoS ONE 7(11), e50192 (2012).
[Crossref] [PubMed]

S. H. Lee, M. Baday, M. Tjioe, P. D. Simonson, R. Zhang, E. Cai, and P. R. Selvin, “Using fixed fiduciary markers for stage drift correction,” Opt. Express 20(11), 12177–12183 (2012).
[Crossref] [PubMed]

2011 (4)

M. J. Mlodzianoski, J. M. Schreiner, S. P. Callahan, K. Smolková, A. Dlasková, J. Santorová, P. Ježek, and J. Bewersdorf, “Sample drift correction in 3D fluorescence photoactivation localization microscopy,” Opt. Express 19(16), 15009–15019 (2011).
[Crossref] [PubMed]

G. S. Kaminski Schierle, S. van de Linde, M. Erdelyi, E. K. Esbjörner, T. Klein, E. Rees, C. W. Bertoncini, C. M. Dobson, M. Sauer, and C. F. Kaminski, “In situ measurements of the formation and morphology of intracellular β-Amyloid fibrils by super-resolution fluorescence imaging,” J. Am. Chem. Soc. 133(33), 12902–12905 (2011).
[Crossref] [PubMed]

G. T. Dempsey, J. C. Vaughan, K. H. Chen, M. Bates, and X. Zhuang, “Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging,” Nat. Methods 8(12), 1027–1036 (2011).
[Crossref] [PubMed]

D. Baddeley, D. Crossman, S. Rossberger, J. E. Cheyne, J. M. Montgomery, I. D. Jayasinghe, C. Cremer, M. B. Cannell, and C. Soeller, “4D super-resolution microscopy with conventional fluorophores and single wavelength excitation in optically thick cells and tissues,” PLoS ONE 6(5), e20645 (2011).
[Crossref] [PubMed]

2010 (3)

A. Pertsinidis, Y. Zhang, and S. Chu, “Subnanometre single-molecule localization, registration and distance measurements,” Nature 466(7306), 647–651 (2010).
[Crossref] [PubMed]

I. Testa, C. A. Wurm, R. Medda, E. Rothermel, C. von Middendorf, J. Fölling, S. Jakobs, A. Schönle, S. W. Hell, and C. Eggeling, “Multicolor fluorescence nanoscopy in fixed and living cells by exciting conventional fluorophores with a single wavelength,” Biophys. J. 99(8), 2686–2694 (2010).
[Crossref] [PubMed]

S. Stallinga and B. Rieger, “Accuracy of the Gaussian point spread function model in 2D localization microscopy,” Opt. Express 18(24), 24461–24476 (2010).
[Crossref] [PubMed]

2009 (2)

M. Heilemann, S. van de Linde, A. Mukherjee, and M. Sauer, “Super-resolution imaging with small organic fluorophores,” Angew. Chem. Int. Ed. Engl. 48(37), 6903–6908 (2009).
[Crossref] [PubMed]

S. van de Linde, U. Endesfelder, A. Mukherjee, M. Schüttpelz, G. Wiebusch, S. Wolter, M. Heilemann, and M. Sauer, “Multicolor photoswitching microscopy for subdiffraction-resolution fluorescence imaging,” Photochem. Photobiol. Sci. 8(4), 465–469 (2009).
[Crossref] [PubMed]

2008 (1)

J. Fölling, M. Bossi, H. Bock, R. Medda, C. A. Wurm, B. Hein, S. Jakobs, C. Eggeling, and S. W. Hell, “Fluorescence nanoscopy by ground-state depletion and single-molecule return,” Nat. Methods 5(11), 943–945 (2008).
[Crossref] [PubMed]

2007 (2)

H. Bock, C. Geisler, C. A. Wurm, C. von Middendorff, S. Jacobs, A. Schonle, A. Egner, S. W. Hell, and C. Eggeling, “Two-color far-field fluorescence nanoscopy based on photoswitchable emitters,” Appl. Phys. B 88(2), 161–165 (2007).
[Crossref]

S. W. Hell, “Far-field optical nanoscopy,” Science 316(5828), 1153–1158 (2007).
[Crossref] [PubMed]

2006 (3)

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science 313(5793), 1642–1645 (2006).
[Crossref] [PubMed]

S. T. Hess, T. P. K. Girirajan, and M. D. Mason, “Ultra-high resolution imaging by fluorescence photoactivation localization microscopy,” Biophys. J. 91(11), 4258–4272 (2006).
[Crossref] [PubMed]

M. J. Rust, M. Bates, and X. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3(10), 793–796 (2006).
[Crossref] [PubMed]

2002 (1)

R. E. Thompson, D. R. Larson, and W. W. Webb, “Precise nanometer localization analysis for individual fluorescent probes,” Biophys. J. 82(5), 2775–2783 (2002).
[Crossref] [PubMed]

Ahn, M.

M. Ahn, E. De Genst, G. S. Kaminski Schierle, M. Erdelyi, C. F. Kaminski, C. M. Dobson, and J. R. Kumita, “Analysis of the native structure, stability and aggregation of biotinylated human lysozyme,” PLoS ONE 7(11), e50192 (2012).
[Crossref] [PubMed]

Baday, M.

Baddeley, D.

D. Baddeley, D. Crossman, S. Rossberger, J. E. Cheyne, J. M. Montgomery, I. D. Jayasinghe, C. Cremer, M. B. Cannell, and C. Soeller, “4D super-resolution microscopy with conventional fluorophores and single wavelength excitation in optically thick cells and tissues,” PLoS ONE 6(5), e20645 (2011).
[Crossref] [PubMed]

Bates, M.

M. Bates, G. T. Dempsey, K. H. Chen, and X. Zhuang, “Multicolor super-resolution fluorescence imaging via multi-parameter fluorophore detection,” ChemPhysChem 13(1), 99–107 (2012).
[Crossref] [PubMed]

G. T. Dempsey, J. C. Vaughan, K. H. Chen, M. Bates, and X. Zhuang, “Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging,” Nat. Methods 8(12), 1027–1036 (2011).
[Crossref] [PubMed]

M. J. Rust, M. Bates, and X. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3(10), 793–796 (2006).
[Crossref] [PubMed]

Bertoncini, C. W.

G. S. Kaminski Schierle, S. van de Linde, M. Erdelyi, E. K. Esbjörner, T. Klein, E. Rees, C. W. Bertoncini, C. M. Dobson, M. Sauer, and C. F. Kaminski, “In situ measurements of the formation and morphology of intracellular β-Amyloid fibrils by super-resolution fluorescence imaging,” J. Am. Chem. Soc. 133(33), 12902–12905 (2011).
[Crossref] [PubMed]

Betzig, E.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science 313(5793), 1642–1645 (2006).
[Crossref] [PubMed]

Bewersdorf, J.

Bock, H.

J. Fölling, M. Bossi, H. Bock, R. Medda, C. A. Wurm, B. Hein, S. Jakobs, C. Eggeling, and S. W. Hell, “Fluorescence nanoscopy by ground-state depletion and single-molecule return,” Nat. Methods 5(11), 943–945 (2008).
[Crossref] [PubMed]

H. Bock, C. Geisler, C. A. Wurm, C. von Middendorff, S. Jacobs, A. Schonle, A. Egner, S. W. Hell, and C. Eggeling, “Two-color far-field fluorescence nanoscopy based on photoswitchable emitters,” Appl. Phys. B 88(2), 161–165 (2007).
[Crossref]

Bonifacino, J. S.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science 313(5793), 1642–1645 (2006).
[Crossref] [PubMed]

Bossi, M.

J. Fölling, M. Bossi, H. Bock, R. Medda, C. A. Wurm, B. Hein, S. Jakobs, C. Eggeling, and S. W. Hell, “Fluorescence nanoscopy by ground-state depletion and single-molecule return,” Nat. Methods 5(11), 943–945 (2008).
[Crossref] [PubMed]

Cai, E.

Callahan, S. P.

Cannell, M. B.

D. Baddeley, D. Crossman, S. Rossberger, J. E. Cheyne, J. M. Montgomery, I. D. Jayasinghe, C. Cremer, M. B. Cannell, and C. Soeller, “4D super-resolution microscopy with conventional fluorophores and single wavelength excitation in optically thick cells and tissues,” PLoS ONE 6(5), e20645 (2011).
[Crossref] [PubMed]

Chen, K. H.

M. Bates, G. T. Dempsey, K. H. Chen, and X. Zhuang, “Multicolor super-resolution fluorescence imaging via multi-parameter fluorophore detection,” ChemPhysChem 13(1), 99–107 (2012).
[Crossref] [PubMed]

G. T. Dempsey, J. C. Vaughan, K. H. Chen, M. Bates, and X. Zhuang, “Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging,” Nat. Methods 8(12), 1027–1036 (2011).
[Crossref] [PubMed]

Cheyne, J. E.

D. Baddeley, D. Crossman, S. Rossberger, J. E. Cheyne, J. M. Montgomery, I. D. Jayasinghe, C. Cremer, M. B. Cannell, and C. Soeller, “4D super-resolution microscopy with conventional fluorophores and single wavelength excitation in optically thick cells and tissues,” PLoS ONE 6(5), e20645 (2011).
[Crossref] [PubMed]

Chu, S.

A. Pertsinidis, Y. Zhang, and S. Chu, “Subnanometre single-molecule localization, registration and distance measurements,” Nature 466(7306), 647–651 (2010).
[Crossref] [PubMed]

Cremer, C.

D. Baddeley, D. Crossman, S. Rossberger, J. E. Cheyne, J. M. Montgomery, I. D. Jayasinghe, C. Cremer, M. B. Cannell, and C. Soeller, “4D super-resolution microscopy with conventional fluorophores and single wavelength excitation in optically thick cells and tissues,” PLoS ONE 6(5), e20645 (2011).
[Crossref] [PubMed]

Crossman, D.

D. Baddeley, D. Crossman, S. Rossberger, J. E. Cheyne, J. M. Montgomery, I. D. Jayasinghe, C. Cremer, M. B. Cannell, and C. Soeller, “4D super-resolution microscopy with conventional fluorophores and single wavelength excitation in optically thick cells and tissues,” PLoS ONE 6(5), e20645 (2011).
[Crossref] [PubMed]

Dabauvalle, M. C.

A. Löschberger, S. van de Linde, M. C. Dabauvalle, B. Rieger, M. Heilemann, G. Krohne, and M. Sauer, “Super-resolution imaging visualizes the eightfold symmetry of gp210 proteins around the nuclear pore complex and resolves the central channel with nanometer resolution,” J. Cell Sci. 125(3), 570–575 (2012).
[Crossref] [PubMed]

Davidson, M. W.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science 313(5793), 1642–1645 (2006).
[Crossref] [PubMed]

De Genst, E.

M. Ahn, E. De Genst, G. S. Kaminski Schierle, M. Erdelyi, C. F. Kaminski, C. M. Dobson, and J. R. Kumita, “Analysis of the native structure, stability and aggregation of biotinylated human lysozyme,” PLoS ONE 7(11), e50192 (2012).
[Crossref] [PubMed]

Dempsey, G. T.

M. Bates, G. T. Dempsey, K. H. Chen, and X. Zhuang, “Multicolor super-resolution fluorescence imaging via multi-parameter fluorophore detection,” ChemPhysChem 13(1), 99–107 (2012).
[Crossref] [PubMed]

G. T. Dempsey, J. C. Vaughan, K. H. Chen, M. Bates, and X. Zhuang, “Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging,” Nat. Methods 8(12), 1027–1036 (2011).
[Crossref] [PubMed]

Dlasková, A.

Dobson, C. M.

M. Ahn, E. De Genst, G. S. Kaminski Schierle, M. Erdelyi, C. F. Kaminski, C. M. Dobson, and J. R. Kumita, “Analysis of the native structure, stability and aggregation of biotinylated human lysozyme,” PLoS ONE 7(11), e50192 (2012).
[Crossref] [PubMed]

G. S. Kaminski Schierle, S. van de Linde, M. Erdelyi, E. K. Esbjörner, T. Klein, E. Rees, C. W. Bertoncini, C. M. Dobson, M. Sauer, and C. F. Kaminski, “In situ measurements of the formation and morphology of intracellular β-Amyloid fibrils by super-resolution fluorescence imaging,” J. Am. Chem. Soc. 133(33), 12902–12905 (2011).
[Crossref] [PubMed]

Eggeling, C.

I. Testa, C. A. Wurm, R. Medda, E. Rothermel, C. von Middendorf, J. Fölling, S. Jakobs, A. Schönle, S. W. Hell, and C. Eggeling, “Multicolor fluorescence nanoscopy in fixed and living cells by exciting conventional fluorophores with a single wavelength,” Biophys. J. 99(8), 2686–2694 (2010).
[Crossref] [PubMed]

J. Fölling, M. Bossi, H. Bock, R. Medda, C. A. Wurm, B. Hein, S. Jakobs, C. Eggeling, and S. W. Hell, “Fluorescence nanoscopy by ground-state depletion and single-molecule return,” Nat. Methods 5(11), 943–945 (2008).
[Crossref] [PubMed]

H. Bock, C. Geisler, C. A. Wurm, C. von Middendorff, S. Jacobs, A. Schonle, A. Egner, S. W. Hell, and C. Eggeling, “Two-color far-field fluorescence nanoscopy based on photoswitchable emitters,” Appl. Phys. B 88(2), 161–165 (2007).
[Crossref]

Egner, A.

H. Bock, C. Geisler, C. A. Wurm, C. von Middendorff, S. Jacobs, A. Schonle, A. Egner, S. W. Hell, and C. Eggeling, “Two-color far-field fluorescence nanoscopy based on photoswitchable emitters,” Appl. Phys. B 88(2), 161–165 (2007).
[Crossref]

Endesfelder, U.

S. van de Linde, U. Endesfelder, A. Mukherjee, M. Schüttpelz, G. Wiebusch, S. Wolter, M. Heilemann, and M. Sauer, “Multicolor photoswitching microscopy for subdiffraction-resolution fluorescence imaging,” Photochem. Photobiol. Sci. 8(4), 465–469 (2009).
[Crossref] [PubMed]

Erdelyi, M.

E. J. Rees, M. Erdelyi, D. Pinotsi, A. Knight, D. Metcalf, and C. F. Kaminski, “Blind assessment of localization microscopy image resolution,” Opt. Nanoscopy 1(1), 12 (2012).
[Crossref]

M. Ahn, E. De Genst, G. S. Kaminski Schierle, M. Erdelyi, C. F. Kaminski, C. M. Dobson, and J. R. Kumita, “Analysis of the native structure, stability and aggregation of biotinylated human lysozyme,” PLoS ONE 7(11), e50192 (2012).
[Crossref] [PubMed]

G. S. Kaminski Schierle, S. van de Linde, M. Erdelyi, E. K. Esbjörner, T. Klein, E. Rees, C. W. Bertoncini, C. M. Dobson, M. Sauer, and C. F. Kaminski, “In situ measurements of the formation and morphology of intracellular β-Amyloid fibrils by super-resolution fluorescence imaging,” J. Am. Chem. Soc. 133(33), 12902–12905 (2011).
[Crossref] [PubMed]

Esbjörner, E. K.

G. S. Kaminski Schierle, S. van de Linde, M. Erdelyi, E. K. Esbjörner, T. Klein, E. Rees, C. W. Bertoncini, C. M. Dobson, M. Sauer, and C. F. Kaminski, “In situ measurements of the formation and morphology of intracellular β-Amyloid fibrils by super-resolution fluorescence imaging,” J. Am. Chem. Soc. 133(33), 12902–12905 (2011).
[Crossref] [PubMed]

Fölling, J.

I. Testa, C. A. Wurm, R. Medda, E. Rothermel, C. von Middendorf, J. Fölling, S. Jakobs, A. Schönle, S. W. Hell, and C. Eggeling, “Multicolor fluorescence nanoscopy in fixed and living cells by exciting conventional fluorophores with a single wavelength,” Biophys. J. 99(8), 2686–2694 (2010).
[Crossref] [PubMed]

J. Fölling, M. Bossi, H. Bock, R. Medda, C. A. Wurm, B. Hein, S. Jakobs, C. Eggeling, and S. W. Hell, “Fluorescence nanoscopy by ground-state depletion and single-molecule return,” Nat. Methods 5(11), 943–945 (2008).
[Crossref] [PubMed]

Geisler, C.

H. Bock, C. Geisler, C. A. Wurm, C. von Middendorff, S. Jacobs, A. Schonle, A. Egner, S. W. Hell, and C. Eggeling, “Two-color far-field fluorescence nanoscopy based on photoswitchable emitters,” Appl. Phys. B 88(2), 161–165 (2007).
[Crossref]

Girirajan, T. P. K.

S. T. Hess, T. P. K. Girirajan, and M. D. Mason, “Ultra-high resolution imaging by fluorescence photoactivation localization microscopy,” Biophys. J. 91(11), 4258–4272 (2006).
[Crossref] [PubMed]

Heilemann, M.

A. Löschberger, S. van de Linde, M. C. Dabauvalle, B. Rieger, M. Heilemann, G. Krohne, and M. Sauer, “Super-resolution imaging visualizes the eightfold symmetry of gp210 proteins around the nuclear pore complex and resolves the central channel with nanometer resolution,” J. Cell Sci. 125(3), 570–575 (2012).
[Crossref] [PubMed]

M. Heilemann, S. van de Linde, A. Mukherjee, and M. Sauer, “Super-resolution imaging with small organic fluorophores,” Angew. Chem. Int. Ed. Engl. 48(37), 6903–6908 (2009).
[Crossref] [PubMed]

S. van de Linde, U. Endesfelder, A. Mukherjee, M. Schüttpelz, G. Wiebusch, S. Wolter, M. Heilemann, and M. Sauer, “Multicolor photoswitching microscopy for subdiffraction-resolution fluorescence imaging,” Photochem. Photobiol. Sci. 8(4), 465–469 (2009).
[Crossref] [PubMed]

Hein, B.

J. Fölling, M. Bossi, H. Bock, R. Medda, C. A. Wurm, B. Hein, S. Jakobs, C. Eggeling, and S. W. Hell, “Fluorescence nanoscopy by ground-state depletion and single-molecule return,” Nat. Methods 5(11), 943–945 (2008).
[Crossref] [PubMed]

Hell, S. W.

I. Testa, C. A. Wurm, R. Medda, E. Rothermel, C. von Middendorf, J. Fölling, S. Jakobs, A. Schönle, S. W. Hell, and C. Eggeling, “Multicolor fluorescence nanoscopy in fixed and living cells by exciting conventional fluorophores with a single wavelength,” Biophys. J. 99(8), 2686–2694 (2010).
[Crossref] [PubMed]

J. Fölling, M. Bossi, H. Bock, R. Medda, C. A. Wurm, B. Hein, S. Jakobs, C. Eggeling, and S. W. Hell, “Fluorescence nanoscopy by ground-state depletion and single-molecule return,” Nat. Methods 5(11), 943–945 (2008).
[Crossref] [PubMed]

S. W. Hell, “Far-field optical nanoscopy,” Science 316(5828), 1153–1158 (2007).
[Crossref] [PubMed]

H. Bock, C. Geisler, C. A. Wurm, C. von Middendorff, S. Jacobs, A. Schonle, A. Egner, S. W. Hell, and C. Eggeling, “Two-color far-field fluorescence nanoscopy based on photoswitchable emitters,” Appl. Phys. B 88(2), 161–165 (2007).
[Crossref]

Hess, H. F.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science 313(5793), 1642–1645 (2006).
[Crossref] [PubMed]

Hess, S. T.

S. T. Hess, T. P. K. Girirajan, and M. D. Mason, “Ultra-high resolution imaging by fluorescence photoactivation localization microscopy,” Biophys. J. 91(11), 4258–4272 (2006).
[Crossref] [PubMed]

Jacobs, S.

H. Bock, C. Geisler, C. A. Wurm, C. von Middendorff, S. Jacobs, A. Schonle, A. Egner, S. W. Hell, and C. Eggeling, “Two-color far-field fluorescence nanoscopy based on photoswitchable emitters,” Appl. Phys. B 88(2), 161–165 (2007).
[Crossref]

Jakobs, S.

I. Testa, C. A. Wurm, R. Medda, E. Rothermel, C. von Middendorf, J. Fölling, S. Jakobs, A. Schönle, S. W. Hell, and C. Eggeling, “Multicolor fluorescence nanoscopy in fixed and living cells by exciting conventional fluorophores with a single wavelength,” Biophys. J. 99(8), 2686–2694 (2010).
[Crossref] [PubMed]

J. Fölling, M. Bossi, H. Bock, R. Medda, C. A. Wurm, B. Hein, S. Jakobs, C. Eggeling, and S. W. Hell, “Fluorescence nanoscopy by ground-state depletion and single-molecule return,” Nat. Methods 5(11), 943–945 (2008).
[Crossref] [PubMed]

Jayasinghe, I. D.

D. Baddeley, D. Crossman, S. Rossberger, J. E. Cheyne, J. M. Montgomery, I. D. Jayasinghe, C. Cremer, M. B. Cannell, and C. Soeller, “4D super-resolution microscopy with conventional fluorophores and single wavelength excitation in optically thick cells and tissues,” PLoS ONE 6(5), e20645 (2011).
[Crossref] [PubMed]

Ježek, P.

Kaminski, C. F.

E. J. Rees, M. Erdelyi, D. Pinotsi, A. Knight, D. Metcalf, and C. F. Kaminski, “Blind assessment of localization microscopy image resolution,” Opt. Nanoscopy 1(1), 12 (2012).
[Crossref]

M. Ahn, E. De Genst, G. S. Kaminski Schierle, M. Erdelyi, C. F. Kaminski, C. M. Dobson, and J. R. Kumita, “Analysis of the native structure, stability and aggregation of biotinylated human lysozyme,” PLoS ONE 7(11), e50192 (2012).
[Crossref] [PubMed]

G. S. Kaminski Schierle, S. van de Linde, M. Erdelyi, E. K. Esbjörner, T. Klein, E. Rees, C. W. Bertoncini, C. M. Dobson, M. Sauer, and C. F. Kaminski, “In situ measurements of the formation and morphology of intracellular β-Amyloid fibrils by super-resolution fluorescence imaging,” J. Am. Chem. Soc. 133(33), 12902–12905 (2011).
[Crossref] [PubMed]

Kaminski Schierle, G. S.

M. Ahn, E. De Genst, G. S. Kaminski Schierle, M. Erdelyi, C. F. Kaminski, C. M. Dobson, and J. R. Kumita, “Analysis of the native structure, stability and aggregation of biotinylated human lysozyme,” PLoS ONE 7(11), e50192 (2012).
[Crossref] [PubMed]

G. S. Kaminski Schierle, S. van de Linde, M. Erdelyi, E. K. Esbjörner, T. Klein, E. Rees, C. W. Bertoncini, C. M. Dobson, M. Sauer, and C. F. Kaminski, “In situ measurements of the formation and morphology of intracellular β-Amyloid fibrils by super-resolution fluorescence imaging,” J. Am. Chem. Soc. 133(33), 12902–12905 (2011).
[Crossref] [PubMed]

Klein, T.

G. S. Kaminski Schierle, S. van de Linde, M. Erdelyi, E. K. Esbjörner, T. Klein, E. Rees, C. W. Bertoncini, C. M. Dobson, M. Sauer, and C. F. Kaminski, “In situ measurements of the formation and morphology of intracellular β-Amyloid fibrils by super-resolution fluorescence imaging,” J. Am. Chem. Soc. 133(33), 12902–12905 (2011).
[Crossref] [PubMed]

Knight, A.

E. J. Rees, M. Erdelyi, D. Pinotsi, A. Knight, D. Metcalf, and C. F. Kaminski, “Blind assessment of localization microscopy image resolution,” Opt. Nanoscopy 1(1), 12 (2012).
[Crossref]

Krohne, G.

A. Löschberger, S. van de Linde, M. C. Dabauvalle, B. Rieger, M. Heilemann, G. Krohne, and M. Sauer, “Super-resolution imaging visualizes the eightfold symmetry of gp210 proteins around the nuclear pore complex and resolves the central channel with nanometer resolution,” J. Cell Sci. 125(3), 570–575 (2012).
[Crossref] [PubMed]

Kumita, J. R.

M. Ahn, E. De Genst, G. S. Kaminski Schierle, M. Erdelyi, C. F. Kaminski, C. M. Dobson, and J. R. Kumita, “Analysis of the native structure, stability and aggregation of biotinylated human lysozyme,” PLoS ONE 7(11), e50192 (2012).
[Crossref] [PubMed]

Larson, D. R.

R. E. Thompson, D. R. Larson, and W. W. Webb, “Precise nanometer localization analysis for individual fluorescent probes,” Biophys. J. 82(5), 2775–2783 (2002).
[Crossref] [PubMed]

Lee, S. H.

Lindwasser, O. W.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science 313(5793), 1642–1645 (2006).
[Crossref] [PubMed]

Lippincott-Schwartz, J.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science 313(5793), 1642–1645 (2006).
[Crossref] [PubMed]

Löschberger, A.

A. Löschberger, S. van de Linde, M. C. Dabauvalle, B. Rieger, M. Heilemann, G. Krohne, and M. Sauer, “Super-resolution imaging visualizes the eightfold symmetry of gp210 proteins around the nuclear pore complex and resolves the central channel with nanometer resolution,” J. Cell Sci. 125(3), 570–575 (2012).
[Crossref] [PubMed]

Mason, M. D.

S. T. Hess, T. P. K. Girirajan, and M. D. Mason, “Ultra-high resolution imaging by fluorescence photoactivation localization microscopy,” Biophys. J. 91(11), 4258–4272 (2006).
[Crossref] [PubMed]

Medda, R.

I. Testa, C. A. Wurm, R. Medda, E. Rothermel, C. von Middendorf, J. Fölling, S. Jakobs, A. Schönle, S. W. Hell, and C. Eggeling, “Multicolor fluorescence nanoscopy in fixed and living cells by exciting conventional fluorophores with a single wavelength,” Biophys. J. 99(8), 2686–2694 (2010).
[Crossref] [PubMed]

J. Fölling, M. Bossi, H. Bock, R. Medda, C. A. Wurm, B. Hein, S. Jakobs, C. Eggeling, and S. W. Hell, “Fluorescence nanoscopy by ground-state depletion and single-molecule return,” Nat. Methods 5(11), 943–945 (2008).
[Crossref] [PubMed]

Metcalf, D.

E. J. Rees, M. Erdelyi, D. Pinotsi, A. Knight, D. Metcalf, and C. F. Kaminski, “Blind assessment of localization microscopy image resolution,” Opt. Nanoscopy 1(1), 12 (2012).
[Crossref]

Mlodzianoski, M. J.

Montgomery, J. M.

D. Baddeley, D. Crossman, S. Rossberger, J. E. Cheyne, J. M. Montgomery, I. D. Jayasinghe, C. Cremer, M. B. Cannell, and C. Soeller, “4D super-resolution microscopy with conventional fluorophores and single wavelength excitation in optically thick cells and tissues,” PLoS ONE 6(5), e20645 (2011).
[Crossref] [PubMed]

Mukherjee, A.

S. van de Linde, U. Endesfelder, A. Mukherjee, M. Schüttpelz, G. Wiebusch, S. Wolter, M. Heilemann, and M. Sauer, “Multicolor photoswitching microscopy for subdiffraction-resolution fluorescence imaging,” Photochem. Photobiol. Sci. 8(4), 465–469 (2009).
[Crossref] [PubMed]

M. Heilemann, S. van de Linde, A. Mukherjee, and M. Sauer, “Super-resolution imaging with small organic fluorophores,” Angew. Chem. Int. Ed. Engl. 48(37), 6903–6908 (2009).
[Crossref] [PubMed]

Olenych, S.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science 313(5793), 1642–1645 (2006).
[Crossref] [PubMed]

Patterson, G. H.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science 313(5793), 1642–1645 (2006).
[Crossref] [PubMed]

Pertsinidis, A.

A. Pertsinidis, Y. Zhang, and S. Chu, “Subnanometre single-molecule localization, registration and distance measurements,” Nature 466(7306), 647–651 (2010).
[Crossref] [PubMed]

Pinotsi, D.

E. J. Rees, M. Erdelyi, D. Pinotsi, A. Knight, D. Metcalf, and C. F. Kaminski, “Blind assessment of localization microscopy image resolution,” Opt. Nanoscopy 1(1), 12 (2012).
[Crossref]

Rees, E.

G. S. Kaminski Schierle, S. van de Linde, M. Erdelyi, E. K. Esbjörner, T. Klein, E. Rees, C. W. Bertoncini, C. M. Dobson, M. Sauer, and C. F. Kaminski, “In situ measurements of the formation and morphology of intracellular β-Amyloid fibrils by super-resolution fluorescence imaging,” J. Am. Chem. Soc. 133(33), 12902–12905 (2011).
[Crossref] [PubMed]

Rees, E. J.

E. J. Rees, M. Erdelyi, D. Pinotsi, A. Knight, D. Metcalf, and C. F. Kaminski, “Blind assessment of localization microscopy image resolution,” Opt. Nanoscopy 1(1), 12 (2012).
[Crossref]

Rieger, B.

A. Löschberger, S. van de Linde, M. C. Dabauvalle, B. Rieger, M. Heilemann, G. Krohne, and M. Sauer, “Super-resolution imaging visualizes the eightfold symmetry of gp210 proteins around the nuclear pore complex and resolves the central channel with nanometer resolution,” J. Cell Sci. 125(3), 570–575 (2012).
[Crossref] [PubMed]

S. Stallinga and B. Rieger, “Accuracy of the Gaussian point spread function model in 2D localization microscopy,” Opt. Express 18(24), 24461–24476 (2010).
[Crossref] [PubMed]

Rossberger, S.

D. Baddeley, D. Crossman, S. Rossberger, J. E. Cheyne, J. M. Montgomery, I. D. Jayasinghe, C. Cremer, M. B. Cannell, and C. Soeller, “4D super-resolution microscopy with conventional fluorophores and single wavelength excitation in optically thick cells and tissues,” PLoS ONE 6(5), e20645 (2011).
[Crossref] [PubMed]

Rothermel, E.

I. Testa, C. A. Wurm, R. Medda, E. Rothermel, C. von Middendorf, J. Fölling, S. Jakobs, A. Schönle, S. W. Hell, and C. Eggeling, “Multicolor fluorescence nanoscopy in fixed and living cells by exciting conventional fluorophores with a single wavelength,” Biophys. J. 99(8), 2686–2694 (2010).
[Crossref] [PubMed]

Rust, M. J.

M. J. Rust, M. Bates, and X. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3(10), 793–796 (2006).
[Crossref] [PubMed]

Santorová, J.

Sauer, M.

A. Löschberger, S. van de Linde, M. C. Dabauvalle, B. Rieger, M. Heilemann, G. Krohne, and M. Sauer, “Super-resolution imaging visualizes the eightfold symmetry of gp210 proteins around the nuclear pore complex and resolves the central channel with nanometer resolution,” J. Cell Sci. 125(3), 570–575 (2012).
[Crossref] [PubMed]

G. S. Kaminski Schierle, S. van de Linde, M. Erdelyi, E. K. Esbjörner, T. Klein, E. Rees, C. W. Bertoncini, C. M. Dobson, M. Sauer, and C. F. Kaminski, “In situ measurements of the formation and morphology of intracellular β-Amyloid fibrils by super-resolution fluorescence imaging,” J. Am. Chem. Soc. 133(33), 12902–12905 (2011).
[Crossref] [PubMed]

S. van de Linde, U. Endesfelder, A. Mukherjee, M. Schüttpelz, G. Wiebusch, S. Wolter, M. Heilemann, and M. Sauer, “Multicolor photoswitching microscopy for subdiffraction-resolution fluorescence imaging,” Photochem. Photobiol. Sci. 8(4), 465–469 (2009).
[Crossref] [PubMed]

M. Heilemann, S. van de Linde, A. Mukherjee, and M. Sauer, “Super-resolution imaging with small organic fluorophores,” Angew. Chem. Int. Ed. Engl. 48(37), 6903–6908 (2009).
[Crossref] [PubMed]

Schonle, A.

H. Bock, C. Geisler, C. A. Wurm, C. von Middendorff, S. Jacobs, A. Schonle, A. Egner, S. W. Hell, and C. Eggeling, “Two-color far-field fluorescence nanoscopy based on photoswitchable emitters,” Appl. Phys. B 88(2), 161–165 (2007).
[Crossref]

Schönle, A.

I. Testa, C. A. Wurm, R. Medda, E. Rothermel, C. von Middendorf, J. Fölling, S. Jakobs, A. Schönle, S. W. Hell, and C. Eggeling, “Multicolor fluorescence nanoscopy in fixed and living cells by exciting conventional fluorophores with a single wavelength,” Biophys. J. 99(8), 2686–2694 (2010).
[Crossref] [PubMed]

Schreiner, J. M.

Schüttpelz, M.

S. van de Linde, U. Endesfelder, A. Mukherjee, M. Schüttpelz, G. Wiebusch, S. Wolter, M. Heilemann, and M. Sauer, “Multicolor photoswitching microscopy for subdiffraction-resolution fluorescence imaging,” Photochem. Photobiol. Sci. 8(4), 465–469 (2009).
[Crossref] [PubMed]

Selvin, P. R.

Simonson, P. D.

Smolková, K.

Soeller, C.

D. Baddeley, D. Crossman, S. Rossberger, J. E. Cheyne, J. M. Montgomery, I. D. Jayasinghe, C. Cremer, M. B. Cannell, and C. Soeller, “4D super-resolution microscopy with conventional fluorophores and single wavelength excitation in optically thick cells and tissues,” PLoS ONE 6(5), e20645 (2011).
[Crossref] [PubMed]

Sougrat, R.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science 313(5793), 1642–1645 (2006).
[Crossref] [PubMed]

Stallinga, S.

Testa, I.

I. Testa, C. A. Wurm, R. Medda, E. Rothermel, C. von Middendorf, J. Fölling, S. Jakobs, A. Schönle, S. W. Hell, and C. Eggeling, “Multicolor fluorescence nanoscopy in fixed and living cells by exciting conventional fluorophores with a single wavelength,” Biophys. J. 99(8), 2686–2694 (2010).
[Crossref] [PubMed]

Thompson, R. E.

R. E. Thompson, D. R. Larson, and W. W. Webb, “Precise nanometer localization analysis for individual fluorescent probes,” Biophys. J. 82(5), 2775–2783 (2002).
[Crossref] [PubMed]

Tjioe, M.

van de Linde, S.

A. Löschberger, S. van de Linde, M. C. Dabauvalle, B. Rieger, M. Heilemann, G. Krohne, and M. Sauer, “Super-resolution imaging visualizes the eightfold symmetry of gp210 proteins around the nuclear pore complex and resolves the central channel with nanometer resolution,” J. Cell Sci. 125(3), 570–575 (2012).
[Crossref] [PubMed]

G. S. Kaminski Schierle, S. van de Linde, M. Erdelyi, E. K. Esbjörner, T. Klein, E. Rees, C. W. Bertoncini, C. M. Dobson, M. Sauer, and C. F. Kaminski, “In situ measurements of the formation and morphology of intracellular β-Amyloid fibrils by super-resolution fluorescence imaging,” J. Am. Chem. Soc. 133(33), 12902–12905 (2011).
[Crossref] [PubMed]

S. van de Linde, U. Endesfelder, A. Mukherjee, M. Schüttpelz, G. Wiebusch, S. Wolter, M. Heilemann, and M. Sauer, “Multicolor photoswitching microscopy for subdiffraction-resolution fluorescence imaging,” Photochem. Photobiol. Sci. 8(4), 465–469 (2009).
[Crossref] [PubMed]

M. Heilemann, S. van de Linde, A. Mukherjee, and M. Sauer, “Super-resolution imaging with small organic fluorophores,” Angew. Chem. Int. Ed. Engl. 48(37), 6903–6908 (2009).
[Crossref] [PubMed]

Vaughan, J. C.

G. T. Dempsey, J. C. Vaughan, K. H. Chen, M. Bates, and X. Zhuang, “Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging,” Nat. Methods 8(12), 1027–1036 (2011).
[Crossref] [PubMed]

von Middendorf, C.

I. Testa, C. A. Wurm, R. Medda, E. Rothermel, C. von Middendorf, J. Fölling, S. Jakobs, A. Schönle, S. W. Hell, and C. Eggeling, “Multicolor fluorescence nanoscopy in fixed and living cells by exciting conventional fluorophores with a single wavelength,” Biophys. J. 99(8), 2686–2694 (2010).
[Crossref] [PubMed]

von Middendorff, C.

H. Bock, C. Geisler, C. A. Wurm, C. von Middendorff, S. Jacobs, A. Schonle, A. Egner, S. W. Hell, and C. Eggeling, “Two-color far-field fluorescence nanoscopy based on photoswitchable emitters,” Appl. Phys. B 88(2), 161–165 (2007).
[Crossref]

Webb, W. W.

R. E. Thompson, D. R. Larson, and W. W. Webb, “Precise nanometer localization analysis for individual fluorescent probes,” Biophys. J. 82(5), 2775–2783 (2002).
[Crossref] [PubMed]

Wiebusch, G.

S. van de Linde, U. Endesfelder, A. Mukherjee, M. Schüttpelz, G. Wiebusch, S. Wolter, M. Heilemann, and M. Sauer, “Multicolor photoswitching microscopy for subdiffraction-resolution fluorescence imaging,” Photochem. Photobiol. Sci. 8(4), 465–469 (2009).
[Crossref] [PubMed]

Wolter, S.

S. van de Linde, U. Endesfelder, A. Mukherjee, M. Schüttpelz, G. Wiebusch, S. Wolter, M. Heilemann, and M. Sauer, “Multicolor photoswitching microscopy for subdiffraction-resolution fluorescence imaging,” Photochem. Photobiol. Sci. 8(4), 465–469 (2009).
[Crossref] [PubMed]

Wurm, C. A.

I. Testa, C. A. Wurm, R. Medda, E. Rothermel, C. von Middendorf, J. Fölling, S. Jakobs, A. Schönle, S. W. Hell, and C. Eggeling, “Multicolor fluorescence nanoscopy in fixed and living cells by exciting conventional fluorophores with a single wavelength,” Biophys. J. 99(8), 2686–2694 (2010).
[Crossref] [PubMed]

J. Fölling, M. Bossi, H. Bock, R. Medda, C. A. Wurm, B. Hein, S. Jakobs, C. Eggeling, and S. W. Hell, “Fluorescence nanoscopy by ground-state depletion and single-molecule return,” Nat. Methods 5(11), 943–945 (2008).
[Crossref] [PubMed]

H. Bock, C. Geisler, C. A. Wurm, C. von Middendorff, S. Jacobs, A. Schonle, A. Egner, S. W. Hell, and C. Eggeling, “Two-color far-field fluorescence nanoscopy based on photoswitchable emitters,” Appl. Phys. B 88(2), 161–165 (2007).
[Crossref]

Zhang, R.

Zhang, Y.

A. Pertsinidis, Y. Zhang, and S. Chu, “Subnanometre single-molecule localization, registration and distance measurements,” Nature 466(7306), 647–651 (2010).
[Crossref] [PubMed]

Zhuang, X.

M. Bates, G. T. Dempsey, K. H. Chen, and X. Zhuang, “Multicolor super-resolution fluorescence imaging via multi-parameter fluorophore detection,” ChemPhysChem 13(1), 99–107 (2012).
[Crossref] [PubMed]

G. T. Dempsey, J. C. Vaughan, K. H. Chen, M. Bates, and X. Zhuang, “Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging,” Nat. Methods 8(12), 1027–1036 (2011).
[Crossref] [PubMed]

M. J. Rust, M. Bates, and X. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3(10), 793–796 (2006).
[Crossref] [PubMed]

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

M. Heilemann, S. van de Linde, A. Mukherjee, and M. Sauer, “Super-resolution imaging with small organic fluorophores,” Angew. Chem. Int. Ed. Engl. 48(37), 6903–6908 (2009).
[Crossref] [PubMed]

Appl. Phys. B (1)

H. Bock, C. Geisler, C. A. Wurm, C. von Middendorff, S. Jacobs, A. Schonle, A. Egner, S. W. Hell, and C. Eggeling, “Two-color far-field fluorescence nanoscopy based on photoswitchable emitters,” Appl. Phys. B 88(2), 161–165 (2007).
[Crossref]

Biophys. J. (3)

R. E. Thompson, D. R. Larson, and W. W. Webb, “Precise nanometer localization analysis for individual fluorescent probes,” Biophys. J. 82(5), 2775–2783 (2002).
[Crossref] [PubMed]

S. T. Hess, T. P. K. Girirajan, and M. D. Mason, “Ultra-high resolution imaging by fluorescence photoactivation localization microscopy,” Biophys. J. 91(11), 4258–4272 (2006).
[Crossref] [PubMed]

I. Testa, C. A. Wurm, R. Medda, E. Rothermel, C. von Middendorf, J. Fölling, S. Jakobs, A. Schönle, S. W. Hell, and C. Eggeling, “Multicolor fluorescence nanoscopy in fixed and living cells by exciting conventional fluorophores with a single wavelength,” Biophys. J. 99(8), 2686–2694 (2010).
[Crossref] [PubMed]

ChemPhysChem (1)

M. Bates, G. T. Dempsey, K. H. Chen, and X. Zhuang, “Multicolor super-resolution fluorescence imaging via multi-parameter fluorophore detection,” ChemPhysChem 13(1), 99–107 (2012).
[Crossref] [PubMed]

J. Am. Chem. Soc. (1)

G. S. Kaminski Schierle, S. van de Linde, M. Erdelyi, E. K. Esbjörner, T. Klein, E. Rees, C. W. Bertoncini, C. M. Dobson, M. Sauer, and C. F. Kaminski, “In situ measurements of the formation and morphology of intracellular β-Amyloid fibrils by super-resolution fluorescence imaging,” J. Am. Chem. Soc. 133(33), 12902–12905 (2011).
[Crossref] [PubMed]

J. Cell Sci. (1)

A. Löschberger, S. van de Linde, M. C. Dabauvalle, B. Rieger, M. Heilemann, G. Krohne, and M. Sauer, “Super-resolution imaging visualizes the eightfold symmetry of gp210 proteins around the nuclear pore complex and resolves the central channel with nanometer resolution,” J. Cell Sci. 125(3), 570–575 (2012).
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Nat. Methods (3)

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Figures (6)

Fig. 1
Fig. 1 Calculated optical offset as a function of decentration of a point-like source at three different excitation wavelength pairs and under three different optical conditions: microscope objective only (A), tilted dichroic mirror (B) and wedged emission filter (C).

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Fig. 2
Fig. 2 Schematic view of the optical setup. Laser beams were expanded by lenses L1 (Thorlabs, AC127-025-A) and L2 (Thorlabs, AC254-150-A) and focused into the back focal plane of the microscope objective (O) by means of lens L3 (Thorlabs, AC508-250-A). Dotted and dashed lines depict the conjugate planes of the system. The image generated by the objective and tube lens (TL) was imaged into a CCD camera via a 1 × telescope formed by lenses L4 and L5 (Thorlabs, AC254-100-A). Multi-edge excitation (F1) and emission filters (F2) and a dichroic mirror (D) were applied to spectrally separate the excitation and emission light. The illumination condition was set via mirror M2 placed into the front focal plane of the focusing lens L3.

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Fig. 3
Fig. 3 Localization of a single fluorescent bead upon sequential excitation at 640(R), 561(G), 488(B) and 640 nm, respectively. a: Localization positions (dots) and errors (circles) for all the wavelengths calculated from the200-frame sub-stacks. Colocalized STORM images of R/G (b) and R/B (c) excitation pairs. The super-resolved pixel size is 16 nm by 16 nm.

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Fig. 4
Fig. 4 Experimental determination of spatial dependence of optical offset using excitation wavelengths 641 nm/561 nm (a, b, c) and 641 nm/491 nm (d, e, f). Raw (a, d) and averaged (b, e) data of four sequentially captured frames. Polynomial fitting (c, f) to the measured data provides a smooth distribution and allows determination of optical offset at an arbitrary point. Vectors representing optical offset were magnified for easier visualization (axes represent spatial position in pixels, and scale bars represent optical offset, also in camera pixels).

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Fig. 5
Fig. 5 Localization of a single multicolored bead from every sub-region using 488 nm (blue), 561 nm (green) and 640 nm (red) excitation before (a) and after (b) optical offset correction. Each tile corresponds to an area of 240 x 240 nm2. Adjacent tiles are separated by 10 μm in the image plane, to provide representative samples across the entire FOV of 41 μm × 41 μm. Scale bars: 50nm.

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Fig. 6
Fig. 6 dSTORM image of double labeled transferrin receptor proteins in HeLa cells using diffraction limited TIRF illumination (a, b), and super-resolved dSTORM imaging (c, d). The merged super-resolved image (e) shows mis-localization between the two images. The depicted four sub-regions before (e1-e4) and after (f1-f4) optical offset correction demonstrate the effectiveness of the correction method. Line-profiles through (e4) and (f4) sub-regions are also depicted.

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Tables (1)

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Table 1 Excitation and emission wavelengths used for experiments and simulations

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