Abstract

We present a novel approach to high-throughput Fluorescence Correlation Spectroscopy (FCS) which enables us to obtain one order of magnitude improvement in acquisition time. Our approach utilizes a liquid crystal on silicon spatial light modulator to generate dynamically adjustable focal spots, and uses an eight-pixel monolithic single-photon avalanche photodiode array. We demonstrate the capabilities of this system by showing FCS of Rhodamine 6G under various viscosities, and by showing that, with proper calibration of each detection channel, one order of magnitude improvement in acquisition speed is obtained. More generally, our approach will allow higher throughput single-molecule studies to be performed.

© 2010 OSA

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2010

F. Guerrieri, S. Tisa, A. Tosi, and F. Zappa, “Two-dimensional SPAD imaging camera for photon counting,” IEEE Photonics Journal 2(5), 759–774 (2010).
[CrossRef]

R. A. Colyer, G. Scalia, T. Kim, I. Rech, D. Resnati, S. Marangoni, M. Ghioni, S. Cova, S. Weiss, and X. Michalet, “High-throughput multispot single-molecule spectroscopy,” Proc. SPIE 7571, 75710G (2010).

T. Wohland, X. Shi, J. Sankaran, and E. H. K. Stelzer, “Single plane illumination fluorescence correlation spectroscopy (SPIM-FCS) probes inhomogeneous three-dimensional environments,” Opt. Express 18(10), 10627–10641 (2010).
[CrossRef] [PubMed]

2009

I. Rech, S. Marangoni, D. Resnati, M. Ghioni, and S. Cova, “Multipixel single-photon avalanche diode array for parallel photon counting applications,” J. Mod. Opt. 56(2), 326–333 (2009).
[CrossRef]

2008

X. Michalet, A. Cheng, J. Antelman, M. Suyama, K. Arisaka, and S. Weiss, “Hybrid photodetector for single-molecule spectroscopy and microscopy,” Proc. SPIE 6862, 68620F (2008).

C. Niclass, C. Favi, T. Kluter, M. Gersbach, and E. Charbon, “A 128 x 128 single-photon image sensor with column-level 10-bit time-to-digital converter array,” IEEE J. Solid-state Circuits 43(12), 2977–2989 (2008).
[CrossRef]

C. B. Müller, A. Loman, V. Pacheco, F. Koberling, D. Willbold, W. Richtering, and J. Enderlein, “Precise measurement of diffusion by multi-color dual-focus fluorescence correlation spectroscopy,” Europhys. Lett. 83(4), 46001 (2008).
[CrossRef]

2007

I. Rech, D. Resnati, S. Marangoni, M. Ghioni, and S. Cova, “Compact-eight channel photon counting module with monolithic array detector,” Proc. SPIE 6771, 677113 (2007).

M. J. Culbertson and D. L. Burden, “A distributed algorithm for multi-tau autocorrelation,” Rev. Sci. Instrum. 78(4), 044102 (2007).
[CrossRef] [PubMed]

E. Haustein and P. Schwille, “Fluorescence correlation spectroscopy: novel variations of an established technique,” Annu. Rev. Biophys. Biomol. Struct. 36(1), 151–169 (2007).
[CrossRef] [PubMed]

B. Kannan, L. Guo, T. Sudhaharan, S. Ahmed, I. Maruyama, and T. Wohland, “Spatially resolved total internal reflection fluorescence correlation microscopy using an electron multiplying charge-coupled device camera,” Anal. Chem. 79(12), 4463–4470 (2007).
[CrossRef] [PubMed]

2006

B. Kannan, J. Y. Har, P. Liu, I. Maruyama, J. L. Ding, and T. Wohland, “Electron multiplying charge-coupled device camera based fluorescence correlation spectroscopy,” Anal. Chem. 78(10), 3444–3451 (2006).
[CrossRef] [PubMed]

D. R. Sisan, R. Arevalo, C. Graves, R. McAllister, and J. S. Urbach, “Spatially resolved fluorescence correlation spectroscopy using a spinning disk confocal microscope,” Biophys. J. 91(11), 4241–4252 (2006).
[CrossRef] [PubMed]

X. Michalet, S. Weiss, and M. Jäger, “Single-molecule fluorescence studies of protein folding and conformational dynamics,” Chem. Rev. 106(5), 1785–1813 (2006).
[CrossRef] [PubMed]

C. Niclass, A. Rochas, P. A. Besse, R. Popovic, and E. Charbon, “A 4 mu s integration time imager based on CMOS single photon avalanche diode technology,” Sens. Actuators A Phys. 130-131, 273–281 (2006).
[CrossRef]

T. A. Laurence, S. Fore, and T. Huser, “Fast, flexible algorithm for calculating photon correlations,” Opt. Lett. 31(6), 829–831 (2006).
[CrossRef] [PubMed]

M. Burkhardt and P. Schwille, “Electron multiplying CCD based detection for spatially resolved fluorescence correlation spectroscopy,” Opt. Express 14(12), 5013–5020 (2006).
[CrossRef] [PubMed]

2005

X. W. Wang, H. Dai, and K. Xu, “Tunable reflective lens array based on liquid crystal on silicon,” Opt. Express 13(2), 352–357 (2005).
[CrossRef] [PubMed]

M. Polin, K. Ladavac, S. H. Lee, Y. Roichman, and D. G. Grier, “Optimized holographic optical traps,” Opt. Express 13(15), 5831–5845 (2005).
[CrossRef] [PubMed]

M. Polin, K. Ladavac, S. H. Lee, Y. Roichman, and D. G. Grier, “Optimized holographic optical traps,” Opt. Express 13(15), 5831–5845 (2005).
[CrossRef] [PubMed]

A. N. Kapanidis, E. Margeat, T. A. Laurence, S. Doose, S. O. Ho, J. Mukhopadhyay, E. Kortkhonjia, V. Mekler, R. H. Ebright, and S. Weiss, “Retention of transcription initiation factor sigma70 in transcription elongation: single-molecule analysis,” Mol. Cell 20(3), 347–356 (2005).
[CrossRef] [PubMed]

M. Gösch, H. Blom, S. Anderegg, K. Korn, P. Thyberg, M. Wells, T. Lasser, R. Rigler, A. Magnusson, and S. Hård, “Parallel dual-color fluorescence cross-correlation spectroscopy using diffractive optical elements,” J. Biomed. Opt. 10(5), 054008 (2005).
[CrossRef] [PubMed]

2004

M. Gösch, A. Serov, T. Anhut, T. Lasser, A. Rochas, P.-A. Besse, R. S. Popovic, H. Blom, and R. Rigler, “Parallel single molecule detection with a fully integrated single-photon 2x2 CMOS detector array,” J. Biomed. Opt. 9(5), 913–921 (2004).
[CrossRef] [PubMed]

H. Dai, K. Xu, Y. Liu, X. Wang, and J. Liu, “Characteristics of LCoS phase-only spatial light modulator and its applications,” Opt. Commun. 238(4-6), 269–276 (2004).
[CrossRef]

2003

S. Saffarian and E. L. Elson, “Statistical analysis of fluorescence correlation spectroscopy: the standard deviation and bias,” Biophys. J. 84(3), 2030–2042 (2003).
[CrossRef] [PubMed]

R. Verberk and M. Orrit, “Photon statistics in the fluorescence of single molecules and nanocrystals: correlation functions versus distributions of on- and off-times,” J. Chem. Phys. 119(4), 2214–2222 (2003).
[CrossRef]

2002

O. Krichevsky and G. Bonnet, “Fluorescence correlation spectroscopy: the technique and its applications,” Rep. Prog. Phys. 65(2), 251–297 (2002).
[CrossRef]

2001

E. R. Dufresne, G. C. Spalding, M. T. Dearing, S. A. Sheets, and D. G. Grier, “Computer-generated holographic optical tweezer arrays,” Rev. Sci. Instrum. 72(3), 1810–1816 (2001).
[CrossRef]

T. Wohland, R. Rigler, and H. Vogel, “The standard deviation in fluorescence correlation spectroscopy,” Biophys. J. 80(6), 2987–2999 (2001).
[CrossRef] [PubMed]

X. Michalet, T. D. Lacoste, and S. Weiss, “Ultrahigh-resolution colocalization of spectrally separable point-like fluorescent probes,” Methods 25(1), 87–102 (2001).
[CrossRef] [PubMed]

R. Heintzmann, Q. S. Hanley, D. Arndt-Jovin, and T. M. Jovin, “A dual path programmable array microscope (PAM): simultaneous acquisition of conjugate and non-conjugate images,” J. Microsc. 204(2), 119–135 (2001).
[CrossRef] [PubMed]

2000

S. Weiss, “Measuring conformational dynamics of biomolecules by single molecule fluorescence spectroscopy,” Nat. Struct. Biol. 7(9), 724–729 (2000).
[CrossRef] [PubMed]

D. N. Fittinghoff, P. W. Wiseman, and J. A. Squier, “Widefield multiphoton and temporally decorrelated multifocal multiphoton microscopy,” Opt. Express 7(8), 273–279 (2000).
[CrossRef] [PubMed]

1999

1998

A. H. Buist, M. Müller, J. A. Squier, and G. J. Brakenhoff, “Real time two-photon absorption microscopy using multipoint excitation,” J. Microsc. 192(2), 217–226 (1998).
[CrossRef]

J. Bewersdorf, R. Pick, and S. W. Hell, “Multifocal multiphoton microscopy,” Opt. Lett. 23(9), 655–657 (1998).
[CrossRef] [PubMed]

1997

P. Kask, R. Günther, and P. Axhausen, “Statistical accuracy in fluorescence fluctuation experiments,” Eur. Biophys. J. 25(3), 163–169 (1997).
[CrossRef]

1990

H. Qian, “On the statistics of fluorescence correlation spectroscopy,” Biophys. Chem. 38(1-2), 49–57 (1990).
[CrossRef] [PubMed]

Ahmed, S.

B. Kannan, L. Guo, T. Sudhaharan, S. Ahmed, I. Maruyama, and T. Wohland, “Spatially resolved total internal reflection fluorescence correlation microscopy using an electron multiplying charge-coupled device camera,” Anal. Chem. 79(12), 4463–4470 (2007).
[CrossRef] [PubMed]

Anderegg, S.

M. Gösch, H. Blom, S. Anderegg, K. Korn, P. Thyberg, M. Wells, T. Lasser, R. Rigler, A. Magnusson, and S. Hård, “Parallel dual-color fluorescence cross-correlation spectroscopy using diffractive optical elements,” J. Biomed. Opt. 10(5), 054008 (2005).
[CrossRef] [PubMed]

Anhut, T.

M. Gösch, A. Serov, T. Anhut, T. Lasser, A. Rochas, P.-A. Besse, R. S. Popovic, H. Blom, and R. Rigler, “Parallel single molecule detection with a fully integrated single-photon 2x2 CMOS detector array,” J. Biomed. Opt. 9(5), 913–921 (2004).
[CrossRef] [PubMed]

Antelman, J.

X. Michalet, A. Cheng, J. Antelman, M. Suyama, K. Arisaka, and S. Weiss, “Hybrid photodetector for single-molecule spectroscopy and microscopy,” Proc. SPIE 6862, 68620F (2008).

Arevalo, R.

D. R. Sisan, R. Arevalo, C. Graves, R. McAllister, and J. S. Urbach, “Spatially resolved fluorescence correlation spectroscopy using a spinning disk confocal microscope,” Biophys. J. 91(11), 4241–4252 (2006).
[CrossRef] [PubMed]

Arisaka, K.

X. Michalet, A. Cheng, J. Antelman, M. Suyama, K. Arisaka, and S. Weiss, “Hybrid photodetector for single-molecule spectroscopy and microscopy,” Proc. SPIE 6862, 68620F (2008).

Arndt-Jovin, D.

R. Heintzmann, Q. S. Hanley, D. Arndt-Jovin, and T. M. Jovin, “A dual path programmable array microscope (PAM): simultaneous acquisition of conjugate and non-conjugate images,” J. Microsc. 204(2), 119–135 (2001).
[CrossRef] [PubMed]

Axhausen, P.

P. Kask, R. Günther, and P. Axhausen, “Statistical accuracy in fluorescence fluctuation experiments,” Eur. Biophys. J. 25(3), 163–169 (1997).
[CrossRef]

Besse, P. A.

C. Niclass, A. Rochas, P. A. Besse, R. Popovic, and E. Charbon, “A 4 mu s integration time imager based on CMOS single photon avalanche diode technology,” Sens. Actuators A Phys. 130-131, 273–281 (2006).
[CrossRef]

Besse, P.-A.

M. Gösch, A. Serov, T. Anhut, T. Lasser, A. Rochas, P.-A. Besse, R. S. Popovic, H. Blom, and R. Rigler, “Parallel single molecule detection with a fully integrated single-photon 2x2 CMOS detector array,” J. Biomed. Opt. 9(5), 913–921 (2004).
[CrossRef] [PubMed]

Bewersdorf, J.

Blom, H.

M. Gösch, H. Blom, S. Anderegg, K. Korn, P. Thyberg, M. Wells, T. Lasser, R. Rigler, A. Magnusson, and S. Hård, “Parallel dual-color fluorescence cross-correlation spectroscopy using diffractive optical elements,” J. Biomed. Opt. 10(5), 054008 (2005).
[CrossRef] [PubMed]

M. Gösch, A. Serov, T. Anhut, T. Lasser, A. Rochas, P.-A. Besse, R. S. Popovic, H. Blom, and R. Rigler, “Parallel single molecule detection with a fully integrated single-photon 2x2 CMOS detector array,” J. Biomed. Opt. 9(5), 913–921 (2004).
[CrossRef] [PubMed]

Bonnet, G.

O. Krichevsky and G. Bonnet, “Fluorescence correlation spectroscopy: the technique and its applications,” Rep. Prog. Phys. 65(2), 251–297 (2002).
[CrossRef]

Brakenhoff, G. J.

A. H. Buist, M. Müller, J. A. Squier, and G. J. Brakenhoff, “Real time two-photon absorption microscopy using multipoint excitation,” J. Microsc. 192(2), 217–226 (1998).
[CrossRef]

Buist, A. H.

A. H. Buist, M. Müller, J. A. Squier, and G. J. Brakenhoff, “Real time two-photon absorption microscopy using multipoint excitation,” J. Microsc. 192(2), 217–226 (1998).
[CrossRef]

Burden, D. L.

M. J. Culbertson and D. L. Burden, “A distributed algorithm for multi-tau autocorrelation,” Rev. Sci. Instrum. 78(4), 044102 (2007).
[CrossRef] [PubMed]

Burkhardt, M.

Charbon, E.

C. Niclass, C. Favi, T. Kluter, M. Gersbach, and E. Charbon, “A 128 x 128 single-photon image sensor with column-level 10-bit time-to-digital converter array,” IEEE J. Solid-state Circuits 43(12), 2977–2989 (2008).
[CrossRef]

C. Niclass, A. Rochas, P. A. Besse, R. Popovic, and E. Charbon, “A 4 mu s integration time imager based on CMOS single photon avalanche diode technology,” Sens. Actuators A Phys. 130-131, 273–281 (2006).
[CrossRef]

Cheng, A.

X. Michalet, A. Cheng, J. Antelman, M. Suyama, K. Arisaka, and S. Weiss, “Hybrid photodetector for single-molecule spectroscopy and microscopy,” Proc. SPIE 6862, 68620F (2008).

Colyer, R. A.

R. A. Colyer, G. Scalia, T. Kim, I. Rech, D. Resnati, S. Marangoni, M. Ghioni, S. Cova, S. Weiss, and X. Michalet, “High-throughput multispot single-molecule spectroscopy,” Proc. SPIE 7571, 75710G (2010).

Cova, S.

R. A. Colyer, G. Scalia, T. Kim, I. Rech, D. Resnati, S. Marangoni, M. Ghioni, S. Cova, S. Weiss, and X. Michalet, “High-throughput multispot single-molecule spectroscopy,” Proc. SPIE 7571, 75710G (2010).

I. Rech, S. Marangoni, D. Resnati, M. Ghioni, and S. Cova, “Multipixel single-photon avalanche diode array for parallel photon counting applications,” J. Mod. Opt. 56(2), 326–333 (2009).
[CrossRef]

I. Rech, D. Resnati, S. Marangoni, M. Ghioni, and S. Cova, “Compact-eight channel photon counting module with monolithic array detector,” Proc. SPIE 6771, 677113 (2007).

Culbertson, M. J.

M. J. Culbertson and D. L. Burden, “A distributed algorithm for multi-tau autocorrelation,” Rev. Sci. Instrum. 78(4), 044102 (2007).
[CrossRef] [PubMed]

Dai, H.

X. W. Wang, H. Dai, and K. Xu, “Tunable reflective lens array based on liquid crystal on silicon,” Opt. Express 13(2), 352–357 (2005).
[CrossRef] [PubMed]

H. Dai, K. Xu, Y. Liu, X. Wang, and J. Liu, “Characteristics of LCoS phase-only spatial light modulator and its applications,” Opt. Commun. 238(4-6), 269–276 (2004).
[CrossRef]

Dearing, M. T.

E. R. Dufresne, G. C. Spalding, M. T. Dearing, S. A. Sheets, and D. G. Grier, “Computer-generated holographic optical tweezer arrays,” Rev. Sci. Instrum. 72(3), 1810–1816 (2001).
[CrossRef]

Ding, J. L.

B. Kannan, J. Y. Har, P. Liu, I. Maruyama, J. L. Ding, and T. Wohland, “Electron multiplying charge-coupled device camera based fluorescence correlation spectroscopy,” Anal. Chem. 78(10), 3444–3451 (2006).
[CrossRef] [PubMed]

Doose, S.

A. N. Kapanidis, E. Margeat, T. A. Laurence, S. Doose, S. O. Ho, J. Mukhopadhyay, E. Kortkhonjia, V. Mekler, R. H. Ebright, and S. Weiss, “Retention of transcription initiation factor sigma70 in transcription elongation: single-molecule analysis,” Mol. Cell 20(3), 347–356 (2005).
[CrossRef] [PubMed]

Dufresne, E. R.

E. R. Dufresne, G. C. Spalding, M. T. Dearing, S. A. Sheets, and D. G. Grier, “Computer-generated holographic optical tweezer arrays,” Rev. Sci. Instrum. 72(3), 1810–1816 (2001).
[CrossRef]

Ebright, R. H.

A. N. Kapanidis, E. Margeat, T. A. Laurence, S. Doose, S. O. Ho, J. Mukhopadhyay, E. Kortkhonjia, V. Mekler, R. H. Ebright, and S. Weiss, “Retention of transcription initiation factor sigma70 in transcription elongation: single-molecule analysis,” Mol. Cell 20(3), 347–356 (2005).
[CrossRef] [PubMed]

Elson, E. L.

S. Saffarian and E. L. Elson, “Statistical analysis of fluorescence correlation spectroscopy: the standard deviation and bias,” Biophys. J. 84(3), 2030–2042 (2003).
[CrossRef] [PubMed]

Enderlein, J.

C. B. Müller, A. Loman, V. Pacheco, F. Koberling, D. Willbold, W. Richtering, and J. Enderlein, “Precise measurement of diffusion by multi-color dual-focus fluorescence correlation spectroscopy,” Europhys. Lett. 83(4), 46001 (2008).
[CrossRef]

Favi, C.

C. Niclass, C. Favi, T. Kluter, M. Gersbach, and E. Charbon, “A 128 x 128 single-photon image sensor with column-level 10-bit time-to-digital converter array,” IEEE J. Solid-state Circuits 43(12), 2977–2989 (2008).
[CrossRef]

Fittinghoff, D. N.

Fore, S.

Gersbach, M.

C. Niclass, C. Favi, T. Kluter, M. Gersbach, and E. Charbon, “A 128 x 128 single-photon image sensor with column-level 10-bit time-to-digital converter array,” IEEE J. Solid-state Circuits 43(12), 2977–2989 (2008).
[CrossRef]

Ghioni, M.

R. A. Colyer, G. Scalia, T. Kim, I. Rech, D. Resnati, S. Marangoni, M. Ghioni, S. Cova, S. Weiss, and X. Michalet, “High-throughput multispot single-molecule spectroscopy,” Proc. SPIE 7571, 75710G (2010).

I. Rech, S. Marangoni, D. Resnati, M. Ghioni, and S. Cova, “Multipixel single-photon avalanche diode array for parallel photon counting applications,” J. Mod. Opt. 56(2), 326–333 (2009).
[CrossRef]

I. Rech, D. Resnati, S. Marangoni, M. Ghioni, and S. Cova, “Compact-eight channel photon counting module with monolithic array detector,” Proc. SPIE 6771, 677113 (2007).

Gösch, M.

M. Gösch, H. Blom, S. Anderegg, K. Korn, P. Thyberg, M. Wells, T. Lasser, R. Rigler, A. Magnusson, and S. Hård, “Parallel dual-color fluorescence cross-correlation spectroscopy using diffractive optical elements,” J. Biomed. Opt. 10(5), 054008 (2005).
[CrossRef] [PubMed]

M. Gösch, A. Serov, T. Anhut, T. Lasser, A. Rochas, P.-A. Besse, R. S. Popovic, H. Blom, and R. Rigler, “Parallel single molecule detection with a fully integrated single-photon 2x2 CMOS detector array,” J. Biomed. Opt. 9(5), 913–921 (2004).
[CrossRef] [PubMed]

Graves, C.

D. R. Sisan, R. Arevalo, C. Graves, R. McAllister, and J. S. Urbach, “Spatially resolved fluorescence correlation spectroscopy using a spinning disk confocal microscope,” Biophys. J. 91(11), 4241–4252 (2006).
[CrossRef] [PubMed]

Grier, D. G.

Guerrieri, F.

F. Guerrieri, S. Tisa, A. Tosi, and F. Zappa, “Two-dimensional SPAD imaging camera for photon counting,” IEEE Photonics Journal 2(5), 759–774 (2010).
[CrossRef]

Günther, R.

P. Kask, R. Günther, and P. Axhausen, “Statistical accuracy in fluorescence fluctuation experiments,” Eur. Biophys. J. 25(3), 163–169 (1997).
[CrossRef]

Guo, L.

B. Kannan, L. Guo, T. Sudhaharan, S. Ahmed, I. Maruyama, and T. Wohland, “Spatially resolved total internal reflection fluorescence correlation microscopy using an electron multiplying charge-coupled device camera,” Anal. Chem. 79(12), 4463–4470 (2007).
[CrossRef] [PubMed]

Haist, T.

Hanley, Q. S.

R. Heintzmann, Q. S. Hanley, D. Arndt-Jovin, and T. M. Jovin, “A dual path programmable array microscope (PAM): simultaneous acquisition of conjugate and non-conjugate images,” J. Microsc. 204(2), 119–135 (2001).
[CrossRef] [PubMed]

Har, J. Y.

B. Kannan, J. Y. Har, P. Liu, I. Maruyama, J. L. Ding, and T. Wohland, “Electron multiplying charge-coupled device camera based fluorescence correlation spectroscopy,” Anal. Chem. 78(10), 3444–3451 (2006).
[CrossRef] [PubMed]

Hård, S.

M. Gösch, H. Blom, S. Anderegg, K. Korn, P. Thyberg, M. Wells, T. Lasser, R. Rigler, A. Magnusson, and S. Hård, “Parallel dual-color fluorescence cross-correlation spectroscopy using diffractive optical elements,” J. Biomed. Opt. 10(5), 054008 (2005).
[CrossRef] [PubMed]

Haustein, E.

E. Haustein and P. Schwille, “Fluorescence correlation spectroscopy: novel variations of an established technique,” Annu. Rev. Biophys. Biomol. Struct. 36(1), 151–169 (2007).
[CrossRef] [PubMed]

Heintzmann, R.

R. Heintzmann, Q. S. Hanley, D. Arndt-Jovin, and T. M. Jovin, “A dual path programmable array microscope (PAM): simultaneous acquisition of conjugate and non-conjugate images,” J. Microsc. 204(2), 119–135 (2001).
[CrossRef] [PubMed]

Hell, S. W.

Ho, S. O.

A. N. Kapanidis, E. Margeat, T. A. Laurence, S. Doose, S. O. Ho, J. Mukhopadhyay, E. Kortkhonjia, V. Mekler, R. H. Ebright, and S. Weiss, “Retention of transcription initiation factor sigma70 in transcription elongation: single-molecule analysis,” Mol. Cell 20(3), 347–356 (2005).
[CrossRef] [PubMed]

Huser, T.

Jäger, M.

X. Michalet, S. Weiss, and M. Jäger, “Single-molecule fluorescence studies of protein folding and conformational dynamics,” Chem. Rev. 106(5), 1785–1813 (2006).
[CrossRef] [PubMed]

Jovin, T. M.

R. Heintzmann, Q. S. Hanley, D. Arndt-Jovin, and T. M. Jovin, “A dual path programmable array microscope (PAM): simultaneous acquisition of conjugate and non-conjugate images,” J. Microsc. 204(2), 119–135 (2001).
[CrossRef] [PubMed]

Kannan, B.

B. Kannan, L. Guo, T. Sudhaharan, S. Ahmed, I. Maruyama, and T. Wohland, “Spatially resolved total internal reflection fluorescence correlation microscopy using an electron multiplying charge-coupled device camera,” Anal. Chem. 79(12), 4463–4470 (2007).
[CrossRef] [PubMed]

B. Kannan, J. Y. Har, P. Liu, I. Maruyama, J. L. Ding, and T. Wohland, “Electron multiplying charge-coupled device camera based fluorescence correlation spectroscopy,” Anal. Chem. 78(10), 3444–3451 (2006).
[CrossRef] [PubMed]

Kapanidis, A. N.

A. N. Kapanidis, E. Margeat, T. A. Laurence, S. Doose, S. O. Ho, J. Mukhopadhyay, E. Kortkhonjia, V. Mekler, R. H. Ebright, and S. Weiss, “Retention of transcription initiation factor sigma70 in transcription elongation: single-molecule analysis,” Mol. Cell 20(3), 347–356 (2005).
[CrossRef] [PubMed]

Kask, P.

P. Kask, R. Günther, and P. Axhausen, “Statistical accuracy in fluorescence fluctuation experiments,” Eur. Biophys. J. 25(3), 163–169 (1997).
[CrossRef]

Kim, T.

R. A. Colyer, G. Scalia, T. Kim, I. Rech, D. Resnati, S. Marangoni, M. Ghioni, S. Cova, S. Weiss, and X. Michalet, “High-throughput multispot single-molecule spectroscopy,” Proc. SPIE 7571, 75710G (2010).

Kluter, T.

C. Niclass, C. Favi, T. Kluter, M. Gersbach, and E. Charbon, “A 128 x 128 single-photon image sensor with column-level 10-bit time-to-digital converter array,” IEEE J. Solid-state Circuits 43(12), 2977–2989 (2008).
[CrossRef]

Koberling, F.

C. B. Müller, A. Loman, V. Pacheco, F. Koberling, D. Willbold, W. Richtering, and J. Enderlein, “Precise measurement of diffusion by multi-color dual-focus fluorescence correlation spectroscopy,” Europhys. Lett. 83(4), 46001 (2008).
[CrossRef]

Korn, K.

M. Gösch, H. Blom, S. Anderegg, K. Korn, P. Thyberg, M. Wells, T. Lasser, R. Rigler, A. Magnusson, and S. Hård, “Parallel dual-color fluorescence cross-correlation spectroscopy using diffractive optical elements,” J. Biomed. Opt. 10(5), 054008 (2005).
[CrossRef] [PubMed]

Kortkhonjia, E.

A. N. Kapanidis, E. Margeat, T. A. Laurence, S. Doose, S. O. Ho, J. Mukhopadhyay, E. Kortkhonjia, V. Mekler, R. H. Ebright, and S. Weiss, “Retention of transcription initiation factor sigma70 in transcription elongation: single-molecule analysis,” Mol. Cell 20(3), 347–356 (2005).
[CrossRef] [PubMed]

Krichevsky, O.

O. Krichevsky and G. Bonnet, “Fluorescence correlation spectroscopy: the technique and its applications,” Rep. Prog. Phys. 65(2), 251–297 (2002).
[CrossRef]

Lacoste, T. D.

X. Michalet, T. D. Lacoste, and S. Weiss, “Ultrahigh-resolution colocalization of spectrally separable point-like fluorescent probes,” Methods 25(1), 87–102 (2001).
[CrossRef] [PubMed]

Ladavac, K.

Lasser, T.

M. Gösch, H. Blom, S. Anderegg, K. Korn, P. Thyberg, M. Wells, T. Lasser, R. Rigler, A. Magnusson, and S. Hård, “Parallel dual-color fluorescence cross-correlation spectroscopy using diffractive optical elements,” J. Biomed. Opt. 10(5), 054008 (2005).
[CrossRef] [PubMed]

M. Gösch, A. Serov, T. Anhut, T. Lasser, A. Rochas, P.-A. Besse, R. S. Popovic, H. Blom, and R. Rigler, “Parallel single molecule detection with a fully integrated single-photon 2x2 CMOS detector array,” J. Biomed. Opt. 9(5), 913–921 (2004).
[CrossRef] [PubMed]

Laurence, T. A.

T. A. Laurence, S. Fore, and T. Huser, “Fast, flexible algorithm for calculating photon correlations,” Opt. Lett. 31(6), 829–831 (2006).
[CrossRef] [PubMed]

A. N. Kapanidis, E. Margeat, T. A. Laurence, S. Doose, S. O. Ho, J. Mukhopadhyay, E. Kortkhonjia, V. Mekler, R. H. Ebright, and S. Weiss, “Retention of transcription initiation factor sigma70 in transcription elongation: single-molecule analysis,” Mol. Cell 20(3), 347–356 (2005).
[CrossRef] [PubMed]

Lee, S. H.

Liu, J.

H. Dai, K. Xu, Y. Liu, X. Wang, and J. Liu, “Characteristics of LCoS phase-only spatial light modulator and its applications,” Opt. Commun. 238(4-6), 269–276 (2004).
[CrossRef]

Liu, P.

B. Kannan, J. Y. Har, P. Liu, I. Maruyama, J. L. Ding, and T. Wohland, “Electron multiplying charge-coupled device camera based fluorescence correlation spectroscopy,” Anal. Chem. 78(10), 3444–3451 (2006).
[CrossRef] [PubMed]

Liu, Y.

H. Dai, K. Xu, Y. Liu, X. Wang, and J. Liu, “Characteristics of LCoS phase-only spatial light modulator and its applications,” Opt. Commun. 238(4-6), 269–276 (2004).
[CrossRef]

Loman, A.

C. B. Müller, A. Loman, V. Pacheco, F. Koberling, D. Willbold, W. Richtering, and J. Enderlein, “Precise measurement of diffusion by multi-color dual-focus fluorescence correlation spectroscopy,” Europhys. Lett. 83(4), 46001 (2008).
[CrossRef]

Magnusson, A.

M. Gösch, H. Blom, S. Anderegg, K. Korn, P. Thyberg, M. Wells, T. Lasser, R. Rigler, A. Magnusson, and S. Hård, “Parallel dual-color fluorescence cross-correlation spectroscopy using diffractive optical elements,” J. Biomed. Opt. 10(5), 054008 (2005).
[CrossRef] [PubMed]

Marangoni, S.

R. A. Colyer, G. Scalia, T. Kim, I. Rech, D. Resnati, S. Marangoni, M. Ghioni, S. Cova, S. Weiss, and X. Michalet, “High-throughput multispot single-molecule spectroscopy,” Proc. SPIE 7571, 75710G (2010).

I. Rech, S. Marangoni, D. Resnati, M. Ghioni, and S. Cova, “Multipixel single-photon avalanche diode array for parallel photon counting applications,” J. Mod. Opt. 56(2), 326–333 (2009).
[CrossRef]

I. Rech, D. Resnati, S. Marangoni, M. Ghioni, and S. Cova, “Compact-eight channel photon counting module with monolithic array detector,” Proc. SPIE 6771, 677113 (2007).

Margeat, E.

A. N. Kapanidis, E. Margeat, T. A. Laurence, S. Doose, S. O. Ho, J. Mukhopadhyay, E. Kortkhonjia, V. Mekler, R. H. Ebright, and S. Weiss, “Retention of transcription initiation factor sigma70 in transcription elongation: single-molecule analysis,” Mol. Cell 20(3), 347–356 (2005).
[CrossRef] [PubMed]

Maruyama, I.

B. Kannan, L. Guo, T. Sudhaharan, S. Ahmed, I. Maruyama, and T. Wohland, “Spatially resolved total internal reflection fluorescence correlation microscopy using an electron multiplying charge-coupled device camera,” Anal. Chem. 79(12), 4463–4470 (2007).
[CrossRef] [PubMed]

B. Kannan, J. Y. Har, P. Liu, I. Maruyama, J. L. Ding, and T. Wohland, “Electron multiplying charge-coupled device camera based fluorescence correlation spectroscopy,” Anal. Chem. 78(10), 3444–3451 (2006).
[CrossRef] [PubMed]

McAllister, R.

D. R. Sisan, R. Arevalo, C. Graves, R. McAllister, and J. S. Urbach, “Spatially resolved fluorescence correlation spectroscopy using a spinning disk confocal microscope,” Biophys. J. 91(11), 4241–4252 (2006).
[CrossRef] [PubMed]

Mekler, V.

A. N. Kapanidis, E. Margeat, T. A. Laurence, S. Doose, S. O. Ho, J. Mukhopadhyay, E. Kortkhonjia, V. Mekler, R. H. Ebright, and S. Weiss, “Retention of transcription initiation factor sigma70 in transcription elongation: single-molecule analysis,” Mol. Cell 20(3), 347–356 (2005).
[CrossRef] [PubMed]

Michalet, X.

R. A. Colyer, G. Scalia, T. Kim, I. Rech, D. Resnati, S. Marangoni, M. Ghioni, S. Cova, S. Weiss, and X. Michalet, “High-throughput multispot single-molecule spectroscopy,” Proc. SPIE 7571, 75710G (2010).

X. Michalet, A. Cheng, J. Antelman, M. Suyama, K. Arisaka, and S. Weiss, “Hybrid photodetector for single-molecule spectroscopy and microscopy,” Proc. SPIE 6862, 68620F (2008).

X. Michalet, S. Weiss, and M. Jäger, “Single-molecule fluorescence studies of protein folding and conformational dynamics,” Chem. Rev. 106(5), 1785–1813 (2006).
[CrossRef] [PubMed]

X. Michalet, T. D. Lacoste, and S. Weiss, “Ultrahigh-resolution colocalization of spectrally separable point-like fluorescent probes,” Methods 25(1), 87–102 (2001).
[CrossRef] [PubMed]

Mukhopadhyay, J.

A. N. Kapanidis, E. Margeat, T. A. Laurence, S. Doose, S. O. Ho, J. Mukhopadhyay, E. Kortkhonjia, V. Mekler, R. H. Ebright, and S. Weiss, “Retention of transcription initiation factor sigma70 in transcription elongation: single-molecule analysis,” Mol. Cell 20(3), 347–356 (2005).
[CrossRef] [PubMed]

Müller, C. B.

C. B. Müller, A. Loman, V. Pacheco, F. Koberling, D. Willbold, W. Richtering, and J. Enderlein, “Precise measurement of diffusion by multi-color dual-focus fluorescence correlation spectroscopy,” Europhys. Lett. 83(4), 46001 (2008).
[CrossRef]

Müller, M.

A. H. Buist, M. Müller, J. A. Squier, and G. J. Brakenhoff, “Real time two-photon absorption microscopy using multipoint excitation,” J. Microsc. 192(2), 217–226 (1998).
[CrossRef]

Niclass, C.

C. Niclass, C. Favi, T. Kluter, M. Gersbach, and E. Charbon, “A 128 x 128 single-photon image sensor with column-level 10-bit time-to-digital converter array,” IEEE J. Solid-state Circuits 43(12), 2977–2989 (2008).
[CrossRef]

C. Niclass, A. Rochas, P. A. Besse, R. Popovic, and E. Charbon, “A 4 mu s integration time imager based on CMOS single photon avalanche diode technology,” Sens. Actuators A Phys. 130-131, 273–281 (2006).
[CrossRef]

Orrit, M.

R. Verberk and M. Orrit, “Photon statistics in the fluorescence of single molecules and nanocrystals: correlation functions versus distributions of on- and off-times,” J. Chem. Phys. 119(4), 2214–2222 (2003).
[CrossRef]

Pacheco, V.

C. B. Müller, A. Loman, V. Pacheco, F. Koberling, D. Willbold, W. Richtering, and J. Enderlein, “Precise measurement of diffusion by multi-color dual-focus fluorescence correlation spectroscopy,” Europhys. Lett. 83(4), 46001 (2008).
[CrossRef]

Pick, R.

Polin, M.

Popovic, R.

C. Niclass, A. Rochas, P. A. Besse, R. Popovic, and E. Charbon, “A 4 mu s integration time imager based on CMOS single photon avalanche diode technology,” Sens. Actuators A Phys. 130-131, 273–281 (2006).
[CrossRef]

Popovic, R. S.

M. Gösch, A. Serov, T. Anhut, T. Lasser, A. Rochas, P.-A. Besse, R. S. Popovic, H. Blom, and R. Rigler, “Parallel single molecule detection with a fully integrated single-photon 2x2 CMOS detector array,” J. Biomed. Opt. 9(5), 913–921 (2004).
[CrossRef] [PubMed]

Qian, H.

H. Qian, “On the statistics of fluorescence correlation spectroscopy,” Biophys. Chem. 38(1-2), 49–57 (1990).
[CrossRef] [PubMed]

Rech, I.

R. A. Colyer, G. Scalia, T. Kim, I. Rech, D. Resnati, S. Marangoni, M. Ghioni, S. Cova, S. Weiss, and X. Michalet, “High-throughput multispot single-molecule spectroscopy,” Proc. SPIE 7571, 75710G (2010).

I. Rech, S. Marangoni, D. Resnati, M. Ghioni, and S. Cova, “Multipixel single-photon avalanche diode array for parallel photon counting applications,” J. Mod. Opt. 56(2), 326–333 (2009).
[CrossRef]

I. Rech, D. Resnati, S. Marangoni, M. Ghioni, and S. Cova, “Compact-eight channel photon counting module with monolithic array detector,” Proc. SPIE 6771, 677113 (2007).

Reicherter, M.

Resnati, D.

R. A. Colyer, G. Scalia, T. Kim, I. Rech, D. Resnati, S. Marangoni, M. Ghioni, S. Cova, S. Weiss, and X. Michalet, “High-throughput multispot single-molecule spectroscopy,” Proc. SPIE 7571, 75710G (2010).

I. Rech, S. Marangoni, D. Resnati, M. Ghioni, and S. Cova, “Multipixel single-photon avalanche diode array for parallel photon counting applications,” J. Mod. Opt. 56(2), 326–333 (2009).
[CrossRef]

I. Rech, D. Resnati, S. Marangoni, M. Ghioni, and S. Cova, “Compact-eight channel photon counting module with monolithic array detector,” Proc. SPIE 6771, 677113 (2007).

Richtering, W.

C. B. Müller, A. Loman, V. Pacheco, F. Koberling, D. Willbold, W. Richtering, and J. Enderlein, “Precise measurement of diffusion by multi-color dual-focus fluorescence correlation spectroscopy,” Europhys. Lett. 83(4), 46001 (2008).
[CrossRef]

Rigler, R.

M. Gösch, H. Blom, S. Anderegg, K. Korn, P. Thyberg, M. Wells, T. Lasser, R. Rigler, A. Magnusson, and S. Hård, “Parallel dual-color fluorescence cross-correlation spectroscopy using diffractive optical elements,” J. Biomed. Opt. 10(5), 054008 (2005).
[CrossRef] [PubMed]

M. Gösch, A. Serov, T. Anhut, T. Lasser, A. Rochas, P.-A. Besse, R. S. Popovic, H. Blom, and R. Rigler, “Parallel single molecule detection with a fully integrated single-photon 2x2 CMOS detector array,” J. Biomed. Opt. 9(5), 913–921 (2004).
[CrossRef] [PubMed]

T. Wohland, R. Rigler, and H. Vogel, “The standard deviation in fluorescence correlation spectroscopy,” Biophys. J. 80(6), 2987–2999 (2001).
[CrossRef] [PubMed]

Rochas, A.

C. Niclass, A. Rochas, P. A. Besse, R. Popovic, and E. Charbon, “A 4 mu s integration time imager based on CMOS single photon avalanche diode technology,” Sens. Actuators A Phys. 130-131, 273–281 (2006).
[CrossRef]

M. Gösch, A. Serov, T. Anhut, T. Lasser, A. Rochas, P.-A. Besse, R. S. Popovic, H. Blom, and R. Rigler, “Parallel single molecule detection with a fully integrated single-photon 2x2 CMOS detector array,” J. Biomed. Opt. 9(5), 913–921 (2004).
[CrossRef] [PubMed]

Roichman, Y.

Saffarian, S.

S. Saffarian and E. L. Elson, “Statistical analysis of fluorescence correlation spectroscopy: the standard deviation and bias,” Biophys. J. 84(3), 2030–2042 (2003).
[CrossRef] [PubMed]

Sankaran, J.

Scalia, G.

R. A. Colyer, G. Scalia, T. Kim, I. Rech, D. Resnati, S. Marangoni, M. Ghioni, S. Cova, S. Weiss, and X. Michalet, “High-throughput multispot single-molecule spectroscopy,” Proc. SPIE 7571, 75710G (2010).

Schwille, P.

E. Haustein and P. Schwille, “Fluorescence correlation spectroscopy: novel variations of an established technique,” Annu. Rev. Biophys. Biomol. Struct. 36(1), 151–169 (2007).
[CrossRef] [PubMed]

M. Burkhardt and P. Schwille, “Electron multiplying CCD based detection for spatially resolved fluorescence correlation spectroscopy,” Opt. Express 14(12), 5013–5020 (2006).
[CrossRef] [PubMed]

Serov, A.

M. Gösch, A. Serov, T. Anhut, T. Lasser, A. Rochas, P.-A. Besse, R. S. Popovic, H. Blom, and R. Rigler, “Parallel single molecule detection with a fully integrated single-photon 2x2 CMOS detector array,” J. Biomed. Opt. 9(5), 913–921 (2004).
[CrossRef] [PubMed]

Sheets, S. A.

E. R. Dufresne, G. C. Spalding, M. T. Dearing, S. A. Sheets, and D. G. Grier, “Computer-generated holographic optical tweezer arrays,” Rev. Sci. Instrum. 72(3), 1810–1816 (2001).
[CrossRef]

Shi, X.

Sisan, D. R.

D. R. Sisan, R. Arevalo, C. Graves, R. McAllister, and J. S. Urbach, “Spatially resolved fluorescence correlation spectroscopy using a spinning disk confocal microscope,” Biophys. J. 91(11), 4241–4252 (2006).
[CrossRef] [PubMed]

Spalding, G. C.

E. R. Dufresne, G. C. Spalding, M. T. Dearing, S. A. Sheets, and D. G. Grier, “Computer-generated holographic optical tweezer arrays,” Rev. Sci. Instrum. 72(3), 1810–1816 (2001).
[CrossRef]

Squier, J. A.

D. N. Fittinghoff, P. W. Wiseman, and J. A. Squier, “Widefield multiphoton and temporally decorrelated multifocal multiphoton microscopy,” Opt. Express 7(8), 273–279 (2000).
[CrossRef] [PubMed]

A. H. Buist, M. Müller, J. A. Squier, and G. J. Brakenhoff, “Real time two-photon absorption microscopy using multipoint excitation,” J. Microsc. 192(2), 217–226 (1998).
[CrossRef]

Stelzer, E. H. K.

Sudhaharan, T.

B. Kannan, L. Guo, T. Sudhaharan, S. Ahmed, I. Maruyama, and T. Wohland, “Spatially resolved total internal reflection fluorescence correlation microscopy using an electron multiplying charge-coupled device camera,” Anal. Chem. 79(12), 4463–4470 (2007).
[CrossRef] [PubMed]

Suyama, M.

X. Michalet, A. Cheng, J. Antelman, M. Suyama, K. Arisaka, and S. Weiss, “Hybrid photodetector for single-molecule spectroscopy and microscopy,” Proc. SPIE 6862, 68620F (2008).

Thyberg, P.

M. Gösch, H. Blom, S. Anderegg, K. Korn, P. Thyberg, M. Wells, T. Lasser, R. Rigler, A. Magnusson, and S. Hård, “Parallel dual-color fluorescence cross-correlation spectroscopy using diffractive optical elements,” J. Biomed. Opt. 10(5), 054008 (2005).
[CrossRef] [PubMed]

Tisa, S.

F. Guerrieri, S. Tisa, A. Tosi, and F. Zappa, “Two-dimensional SPAD imaging camera for photon counting,” IEEE Photonics Journal 2(5), 759–774 (2010).
[CrossRef]

Tiziani, H. J.

Tosi, A.

F. Guerrieri, S. Tisa, A. Tosi, and F. Zappa, “Two-dimensional SPAD imaging camera for photon counting,” IEEE Photonics Journal 2(5), 759–774 (2010).
[CrossRef]

Urbach, J. S.

D. R. Sisan, R. Arevalo, C. Graves, R. McAllister, and J. S. Urbach, “Spatially resolved fluorescence correlation spectroscopy using a spinning disk confocal microscope,” Biophys. J. 91(11), 4241–4252 (2006).
[CrossRef] [PubMed]

Verberk, R.

R. Verberk and M. Orrit, “Photon statistics in the fluorescence of single molecules and nanocrystals: correlation functions versus distributions of on- and off-times,” J. Chem. Phys. 119(4), 2214–2222 (2003).
[CrossRef]

Vogel, H.

T. Wohland, R. Rigler, and H. Vogel, “The standard deviation in fluorescence correlation spectroscopy,” Biophys. J. 80(6), 2987–2999 (2001).
[CrossRef] [PubMed]

Wagemann, E. U.

Wang, X.

H. Dai, K. Xu, Y. Liu, X. Wang, and J. Liu, “Characteristics of LCoS phase-only spatial light modulator and its applications,” Opt. Commun. 238(4-6), 269–276 (2004).
[CrossRef]

Wang, X. W.

Weiss, S.

R. A. Colyer, G. Scalia, T. Kim, I. Rech, D. Resnati, S. Marangoni, M. Ghioni, S. Cova, S. Weiss, and X. Michalet, “High-throughput multispot single-molecule spectroscopy,” Proc. SPIE 7571, 75710G (2010).

X. Michalet, A. Cheng, J. Antelman, M. Suyama, K. Arisaka, and S. Weiss, “Hybrid photodetector for single-molecule spectroscopy and microscopy,” Proc. SPIE 6862, 68620F (2008).

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X. Michalet, T. D. Lacoste, and S. Weiss, “Ultrahigh-resolution colocalization of spectrally separable point-like fluorescent probes,” Methods 25(1), 87–102 (2001).
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C. B. Müller, A. Loman, V. Pacheco, F. Koberling, D. Willbold, W. Richtering, and J. Enderlein, “Precise measurement of diffusion by multi-color dual-focus fluorescence correlation spectroscopy,” Europhys. Lett. 83(4), 46001 (2008).
[CrossRef]

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F. Guerrieri, S. Tisa, A. Tosi, and F. Zappa, “Two-dimensional SPAD imaging camera for photon counting,” IEEE Photonics Journal 2(5), 759–774 (2010).
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Anal. Chem.

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[CrossRef] [PubMed]

B. Kannan, J. Y. Har, P. Liu, I. Maruyama, J. L. Ding, and T. Wohland, “Electron multiplying charge-coupled device camera based fluorescence correlation spectroscopy,” Anal. Chem. 78(10), 3444–3451 (2006).
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T. Wohland, R. Rigler, and H. Vogel, “The standard deviation in fluorescence correlation spectroscopy,” Biophys. J. 80(6), 2987–2999 (2001).
[CrossRef] [PubMed]

Chem. Rev.

X. Michalet, S. Weiss, and M. Jäger, “Single-molecule fluorescence studies of protein folding and conformational dynamics,” Chem. Rev. 106(5), 1785–1813 (2006).
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Europhys. Lett.

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[CrossRef]

IEEE J. Solid-state Circuits

C. Niclass, C. Favi, T. Kluter, M. Gersbach, and E. Charbon, “A 128 x 128 single-photon image sensor with column-level 10-bit time-to-digital converter array,” IEEE J. Solid-state Circuits 43(12), 2977–2989 (2008).
[CrossRef]

IEEE Photonics Journal

F. Guerrieri, S. Tisa, A. Tosi, and F. Zappa, “Two-dimensional SPAD imaging camera for photon counting,” IEEE Photonics Journal 2(5), 759–774 (2010).
[CrossRef]

J. Biomed. Opt.

M. Gösch, H. Blom, S. Anderegg, K. Korn, P. Thyberg, M. Wells, T. Lasser, R. Rigler, A. Magnusson, and S. Hård, “Parallel dual-color fluorescence cross-correlation spectroscopy using diffractive optical elements,” J. Biomed. Opt. 10(5), 054008 (2005).
[CrossRef] [PubMed]

M. Gösch, A. Serov, T. Anhut, T. Lasser, A. Rochas, P.-A. Besse, R. S. Popovic, H. Blom, and R. Rigler, “Parallel single molecule detection with a fully integrated single-photon 2x2 CMOS detector array,” J. Biomed. Opt. 9(5), 913–921 (2004).
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[CrossRef]

Methods

X. Michalet, T. D. Lacoste, and S. Weiss, “Ultrahigh-resolution colocalization of spectrally separable point-like fluorescent probes,” Methods 25(1), 87–102 (2001).
[CrossRef] [PubMed]

Mol. Cell

A. N. Kapanidis, E. Margeat, T. A. Laurence, S. Doose, S. O. Ho, J. Mukhopadhyay, E. Kortkhonjia, V. Mekler, R. H. Ebright, and S. Weiss, “Retention of transcription initiation factor sigma70 in transcription elongation: single-molecule analysis,” Mol. Cell 20(3), 347–356 (2005).
[CrossRef] [PubMed]

Nat. Struct. Biol.

S. Weiss, “Measuring conformational dynamics of biomolecules by single molecule fluorescence spectroscopy,” Nat. Struct. Biol. 7(9), 724–729 (2000).
[CrossRef] [PubMed]

Opt. Commun.

H. Dai, K. Xu, Y. Liu, X. Wang, and J. Liu, “Characteristics of LCoS phase-only spatial light modulator and its applications,” Opt. Commun. 238(4-6), 269–276 (2004).
[CrossRef]

Opt. Express

Opt. Lett.

Proc. SPIE

R. A. Colyer, G. Scalia, T. Kim, I. Rech, D. Resnati, S. Marangoni, M. Ghioni, S. Cova, S. Weiss, and X. Michalet, “High-throughput multispot single-molecule spectroscopy,” Proc. SPIE 7571, 75710G (2010).

X. Michalet, A. Cheng, J. Antelman, M. Suyama, K. Arisaka, and S. Weiss, “Hybrid photodetector for single-molecule spectroscopy and microscopy,” Proc. SPIE 6862, 68620F (2008).

I. Rech, D. Resnati, S. Marangoni, M. Ghioni, and S. Cova, “Compact-eight channel photon counting module with monolithic array detector,” Proc. SPIE 6771, 677113 (2007).

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Figures (13)

Fig. 3
Fig. 3

Schematic of the experimental setup using an LCOS-SLM. Blue lines represent the excitation light path. Green lines represent the emission light path towards the detectors (HPD for excitation profile measurements and SPAD array for FCS measurements). A first array of spots is generated in an intermediate image plane in front of the LCOS. A recollimating lens sends this pattern to the back of an objective lens, which focuses it into the sample. CS: Coverslip, Obj: objective lens, FM: flippable mirror. Top left inset: LCOS pattern degrees of freedom controllable by software. The pattern pitch can also be adjusted.

Fig. 1
Fig. 1

A schematic showing how the Huygens-Fresnel principle can be used to determine the desired phase delay at each point. Rays interfere constructively at a focal point when they all have the same total phase delay due to distance and phase delay applied by the LCOS.

Fig. 2
Fig. 2

Example LCOS pattern with a 20 pixel pitch used to generate 8x1 spots. Each pixel represents 20 μm on the LCOS screen. Gray levels correspond to the range available on the LCOS, and go from 0 (black) to 209 (pale gray) corresponding to a phase delay of 2π.

Fig. 4
Fig. 4

Autocorrelation functions of white light at 30kcps showing the afterpulsing behavior of each channel. The fits shown are power law functions. The exact amplitude depends on the count rate and is therefore channel-dependent.

Fig. 5
Fig. 5

Intensity maps of the 8 × 1 excitation spot pattern in the directions along (YZ) and perpendicular (XY) the optical axis. The profiles were recorded by stage scanning microscopy of an isolated 100 nm diameter bead using two different detectors, a wide active area single pixel (HPD), and a 8 × 1 SPAD array (SPAD). The same intensity scale was used in all images, where black = 0, red = 110, and white = 1100 counts. Scale bars are 5 µm.

Fig. 6
Fig. 6

Comparison between excitation (HPD) and excitation + detection (SPAD) bead scan profiles along the Y axis. The plot shows the clipping of the tails and background reduction for each peak in the SPAD measurement compared to the HPD, at the expense of an intensity loss in the outer channels.

Fig. 7
Fig. 7

Representative ACF and fit for R6G in 0% sucrose (channel 4). Fit residuals for all 8 channels are shown in the lower panel. The remaining small residuals are due to the slightly non-Gaussian aspects of the PSFs, as seen in Figs. 5 and 6.

Fig. 8
Fig. 8

R6G diffusion time as a function of viscosity for buffers with 0%, 10%, 20%, 30%, and 40% sucrose. A linear relationship (dashed line) is the expected result.

Fig. 9
Fig. 9

Channel dependences for the viscosity series of (a) d/η and (b) n/<n> (where for each viscosity value, <n> is averaged across the 8 channels).

Fig. 10
Fig. 10

The calibration process is demonstrated using the R6G 0% sample as a reference, where (a) shows the raw uncalibrated curves, and (b) shows the result of the calibration.

Fig. 11
Fig. 11

(a) Calibrated ACF curves obtained from 8 channels for 1 nM R6G in 0%, 10%, 20%, 30% and 40% sucrose. (b) Dependence of the diffusion coefficient on viscosity obtained by fitting the previous curves. The diffusion coefficients were determined by calibrating with the literature value of 414 µm2/s for R6G in water [34].

Fig. 12
Fig. 12

FCS curves obtained from many short acquisitions of (a) 1s and (b) 8s each, with the curves showing (a) merged acquisitions from all channels, and (b) acquisition from a single channel, with comparison to the full 248 second acquisition from a single channel.

Fig. 13
Fig. 13

One second time traces of R6G in 40% sucrose, shown for all 8 channels (1 ms binning). Single-molecule bursts with ~50-100 kHz emission rates are clearly visible.

Tables (1)

Tables Icon

Table 1 Excitation PSF characteristics. Beam-waist values wxy and wz were obtained by Gaussian fits of the PSF images acquired by XY and YZ scanning, respectively (wxy is the geometric mean of the waists in both X and Y directions). The excitation PSFs were imaged using a single-pixel HPD while the excitation + detection PSFs were imaged using the 8 × 1 SPAD array. All: mean and standard deviation of all spots, Diffr: diffraction limit for the setup characteristics [32].

Equations (21)

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Δ φ = 2πd Δ d λ f
φ = π ( 2 d 2 λ f )
G k ( τ ) = A k τ b k + γ k ( 1 B k I k ) 2 i = 1 S ( α k ( i ) ) 2 N k ( i ) ( 1 + 4 D ( i ) τ / w k , x y 2 ) 1 ( i = 1 S α k ( i ) N k ( i ) ) 2
γ k = Ω d r [ X k ( r ) D k ( r ) ] 2 Ω d r X k ( r ) D k ( r ) ,
G k ( τ ) = A k τ b k + i = 1 S n k ( i ) ( 1 + τ / d k ( i ) ) 1 ( i = 1 S n k ( i ) ) 2
d k ( i ) = w k , x y 2 4 D ( i ) n k ( i ) = γ k 1 ( 1 B k I k ) 2 N k ( i ) .
γ k 1 N k ( i ) = C ( i ) V k
D k ( i ) = w k , x y 2 4 d k ( i ) = d k ( 0 ) d k ( i ) D ( 0 ) C k ( i ) = ρ k ρ k ( 0 ) n k ( i ) n k ( 0 ) C ( 0 )
ρ k = ( 1 B k I k ) 2 ; ρ k ( 0 ) = ( 1 B k ( 0 ) I k ( 0 ) ) 2 .
β k = d k ( 0 ) d k ( 0 ) k g k = n k ( 0 ) n k ( 0 ) k ρ k ( 0 ) ρ k .
G ^ k ( τ ) = g k G k ( β k τ ) .
n ^ ( i ) = C ( i ) C ( 0 ) n k ( 0 ) k d ^ ( i ) = w k , x y 2 k 4 D ( i ) .
sin ( θ ) = 1.22 λ p
f max = p 2 1.22 λ
D = k B T 6 π η r
d k , η η = 3 π r 2 k B T w k , x y 2
n k ( i ) i = 1 M i = 1 M n k ( i )
w x y ~ 0.42 λ N A
d X w x y
M ~ r E w x y
d X = d E M ~ d E r E w x y

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