Abstract

Nowadays, there is a hot debate among industry and academic researchers that whether the newly developed scientific-grade Complementary Metal Oxide Semiconductor (sCMOS) cameras could become the image sensors of choice in localization-based super-resolution microscopy. To help researchers find answers to this question, here we reported an experimental methodology for quantitatively comparing the performance of low-light cameras in single molecule detection (characterized via image SNR) and localization (via localization accuracy). We found that a newly launched sCMOS camera can present superior imaging performance than a popular Electron Multiplying Charge Coupled Device (EMCCD) camera in a signal range (15-12000 photon/pixel) more than enough for typical localization-based super-resolution microscopy.

© 2012 OSA

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2012

2011

Z. L. Huang, H. Y. Zhu, F. Long, H. Q. Ma, L. S. Qin, Y. F. Liu, J. P. Ding, Z. H. Zhang, Q. M. Luo, and S. Q. Zeng, “Localization-based super-resolution microscopy with an sCMOS camera,” Opt. Express19(20), 19156–19168 (2011).
[CrossRef] [PubMed]

J. W. Lichtman and W. Denk, “The big and the small: Challenges of imaging the brain’s circuits,” Science334(6056), 618–623 (2011).
[CrossRef] [PubMed]

J. R. Joubert and D. K. Sharma, “EMCCD vs. sCMOS for microscopic imaging,” Photon. Spectra45, 46–50 (2011).

S. A. Jones, S. H. Shim, J. He, and X. Zhuang, “Fast, three-dimensional super-resolution imaging of live cells,” Nat. Methods8(6), 499–505 (2011).
[CrossRef] [PubMed]

G. T. Dempsey, J. C. Vaughan, K. H. Chen, M. Bates, and X. W. Zhuang, “Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging,” Nat. Methods8(12), 1027–1036 (2011).
[CrossRef] [PubMed]

2010

K. I. Mortensen, L. S. Churchman, J. A. Spudich, and H. Flyvbjerg, “Optimized localization analysis for single-molecule tracking and super-resolution microscopy,” Nat. Methods7(5), 377–381 (2010).
[CrossRef] [PubMed]

M. C. DeSantis, S. H. DeCenzo, J. L. Li, and Y. M. Wang, “Precision analysis for standard deviation measurements of immobile single fluorescent molecule images,” Opt. Express18(7), 6563–6576 (2010).
[CrossRef] [PubMed]

T. W. Quan, P. C. Li, F. Long, S. Q. Zeng, Q. M. Luo, P. N. Hedde, G. U. Nienhaus, and Z. L. Huang, “Ultra-fast, high-precision image analysis for localization-based super resolution microscopy,” Opt. Express18(11), 11867–11876 (2010).
[CrossRef] [PubMed]

B. Fowler, C. A. Liu, S. Mims, J. Balicki, W. Li, H. Do, J. Appelbaum, and P. Vu, “A 5.5Mpixel 100 frames/sec wide dynamic range low noise CMOS image sensor for scientific applications,” Proc. SPIE7536, 753607, 753607-12 (2010).
[CrossRef]

M. A. Thompson, J. S. Biteen, S. J. Lord, N. R. Conley, and W. E. Moerner, “Molecules and methods for super-resolution imaging,” Methods Enzymol.475, 27–59 (2010).
[CrossRef] [PubMed]

T. W. Quan, S. Q. Zeng, and Z. L. Huang, “Localization capability and limitation of electron-multiplying charge-coupled, scientific complementary metal-oxide semiconductor, and charge-coupled devices for superresolution imaging,” J. Biomed. Opt.15(6), 066005 (2010).
[CrossRef] [PubMed]

B. Huang, H. Babcock, and X. W. Zhuang, “Breaking the diffraction barrier: Super-resolution imaging of cells,” Cell143(7), 1047–1058 (2010).
[CrossRef] [PubMed]

2009

T. J. Gould, V. V. Verkhusha, and S. T. Hess, “Imaging biological structures with fluorescence photoactivation localization microscopy,” Nat. Protoc.4(3), 291–308 (2009).
[CrossRef] [PubMed]

G. Holst, “Scientific CMOS image sensors,” Laser Photon.5, 18–21 (2009).

2008

M. Fernández-Suárez and A. Y. Ting, “Fluorescent probes for super-resolution imaging in living cells,” Nat. Rev. Mol. Cell Biol.9(12), 929–943 (2008).
[CrossRef] [PubMed]

2007

B. Moomaw, “Camera technologies for low light imaging: overview and relative advantages,” Methods Cell Biol.81, 251–283 (2007).
[CrossRef] [PubMed]

2006

M. Bigas, E. Cabruja, J. Forest, and J. Salvi, “Review of CMOS image sensors,” Microelectron. J.37(5), 433–451 (2006).
[CrossRef]

M. F. Snoeij, A. J. P. Theuwissen, K. A. A. Makinwa, and J. H. Huijsing, “A CMOS imager with column-level ADC using dynamic column fixed-pattern noise reduction,” IEEE J. Solid-st. Circulation41, 3007–3015 (2006).

2004

R. J. Ober, S. Ram, and E. S. Ward, “Localization accuracy in single-molecule microscopy,” Biophys. J.86(2), 1185–1200 (2004).
[CrossRef] [PubMed]

2003

M. S. Robbins and B. J. Hadwen, “The noise performance of electron multiplying charge-coupled devices,” IEEE Trans. Electron. Dev.50(5), 1227–1232 (2003).
[CrossRef]

2002

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]

1998

A. El Gamal, B. Fowlera, H. Min, and X. Q. Liu, “Modeling and estimation of FPN components in CMOS image,” Proc. SPIE3301, 168–177 (1998).
[CrossRef]

Appelbaum, J.

B. Fowler, C. A. Liu, S. Mims, J. Balicki, W. Li, H. Do, J. Appelbaum, and P. Vu, “A 5.5Mpixel 100 frames/sec wide dynamic range low noise CMOS image sensor for scientific applications,” Proc. SPIE7536, 753607, 753607-12 (2010).
[CrossRef]

Babcock, H.

B. Huang, H. Babcock, and X. W. Zhuang, “Breaking the diffraction barrier: Super-resolution imaging of cells,” Cell143(7), 1047–1058 (2010).
[CrossRef] [PubMed]

Balicki, J.

B. Fowler, C. A. Liu, S. Mims, J. Balicki, W. Li, H. Do, J. Appelbaum, and P. Vu, “A 5.5Mpixel 100 frames/sec wide dynamic range low noise CMOS image sensor for scientific applications,” Proc. SPIE7536, 753607, 753607-12 (2010).
[CrossRef]

Bates, M.

G. T. Dempsey, J. C. Vaughan, K. H. Chen, M. Bates, and X. W. Zhuang, “Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging,” Nat. Methods8(12), 1027–1036 (2011).
[CrossRef] [PubMed]

Bigas, M.

M. Bigas, E. Cabruja, J. Forest, and J. Salvi, “Review of CMOS image sensors,” Microelectron. J.37(5), 433–451 (2006).
[CrossRef]

Biteen, J. S.

M. A. Thompson, J. S. Biteen, S. J. Lord, N. R. Conley, and W. E. Moerner, “Molecules and methods for super-resolution imaging,” Methods Enzymol.475, 27–59 (2010).
[CrossRef] [PubMed]

Bruchez, M. P.

Cabruja, E.

M. Bigas, E. Cabruja, J. Forest, and J. Salvi, “Review of CMOS image sensors,” Microelectron. J.37(5), 433–451 (2006).
[CrossRef]

Chen, K. H.

G. T. Dempsey, J. C. Vaughan, K. H. Chen, M. Bates, and X. W. Zhuang, “Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging,” Nat. Methods8(12), 1027–1036 (2011).
[CrossRef] [PubMed]

Churchman, L. S.

K. I. Mortensen, L. S. Churchman, J. A. Spudich, and H. Flyvbjerg, “Optimized localization analysis for single-molecule tracking and super-resolution microscopy,” Nat. Methods7(5), 377–381 (2010).
[CrossRef] [PubMed]

Conley, N. R.

M. A. Thompson, J. S. Biteen, S. J. Lord, N. R. Conley, and W. E. Moerner, “Molecules and methods for super-resolution imaging,” Methods Enzymol.475, 27–59 (2010).
[CrossRef] [PubMed]

DeCenzo, S. H.

Dempsey, G. T.

G. T. Dempsey, J. C. Vaughan, K. H. Chen, M. Bates, and X. W. Zhuang, “Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging,” Nat. Methods8(12), 1027–1036 (2011).
[CrossRef] [PubMed]

Denk, W.

J. W. Lichtman and W. Denk, “The big and the small: Challenges of imaging the brain’s circuits,” Science334(6056), 618–623 (2011).
[CrossRef] [PubMed]

DeSantis, M. C.

Ding, J. P.

Do, H.

B. Fowler, C. A. Liu, S. Mims, J. Balicki, W. Li, H. Do, J. Appelbaum, and P. Vu, “A 5.5Mpixel 100 frames/sec wide dynamic range low noise CMOS image sensor for scientific applications,” Proc. SPIE7536, 753607, 753607-12 (2010).
[CrossRef]

El Gamal, A.

A. El Gamal, B. Fowlera, H. Min, and X. Q. Liu, “Modeling and estimation of FPN components in CMOS image,” Proc. SPIE3301, 168–177 (1998).
[CrossRef]

Fernández-Suárez, M.

M. Fernández-Suárez and A. Y. Ting, “Fluorescent probes for super-resolution imaging in living cells,” Nat. Rev. Mol. Cell Biol.9(12), 929–943 (2008).
[CrossRef] [PubMed]

Flyvbjerg, H.

K. I. Mortensen, L. S. Churchman, J. A. Spudich, and H. Flyvbjerg, “Optimized localization analysis for single-molecule tracking and super-resolution microscopy,” Nat. Methods7(5), 377–381 (2010).
[CrossRef] [PubMed]

Forest, J.

M. Bigas, E. Cabruja, J. Forest, and J. Salvi, “Review of CMOS image sensors,” Microelectron. J.37(5), 433–451 (2006).
[CrossRef]

Fowler, B.

B. Fowler, C. A. Liu, S. Mims, J. Balicki, W. Li, H. Do, J. Appelbaum, and P. Vu, “A 5.5Mpixel 100 frames/sec wide dynamic range low noise CMOS image sensor for scientific applications,” Proc. SPIE7536, 753607, 753607-12 (2010).
[CrossRef]

Fowlera, B.

A. El Gamal, B. Fowlera, H. Min, and X. Q. Liu, “Modeling and estimation of FPN components in CMOS image,” Proc. SPIE3301, 168–177 (1998).
[CrossRef]

Gould, T. J.

T. J. Gould, V. V. Verkhusha, and S. T. Hess, “Imaging biological structures with fluorescence photoactivation localization microscopy,” Nat. Protoc.4(3), 291–308 (2009).
[CrossRef] [PubMed]

Hadwen, B. J.

M. S. Robbins and B. J. Hadwen, “The noise performance of electron multiplying charge-coupled devices,” IEEE Trans. Electron. Dev.50(5), 1227–1232 (2003).
[CrossRef]

He, J.

S. A. Jones, S. H. Shim, J. He, and X. Zhuang, “Fast, three-dimensional super-resolution imaging of live cells,” Nat. Methods8(6), 499–505 (2011).
[CrossRef] [PubMed]

Hedde, P. N.

Hess, S. T.

T. J. Gould, V. V. Verkhusha, and S. T. Hess, “Imaging biological structures with fluorescence photoactivation localization microscopy,” Nat. Protoc.4(3), 291–308 (2009).
[CrossRef] [PubMed]

Holst, G.

G. Holst, “Scientific CMOS image sensors,” Laser Photon.5, 18–21 (2009).

Huang, B.

B. Huang, H. Babcock, and X. W. Zhuang, “Breaking the diffraction barrier: Super-resolution imaging of cells,” Cell143(7), 1047–1058 (2010).
[CrossRef] [PubMed]

Huang, Z. L.

Huijsing, J. H.

M. F. Snoeij, A. J. P. Theuwissen, K. A. A. Makinwa, and J. H. Huijsing, “A CMOS imager with column-level ADC using dynamic column fixed-pattern noise reduction,” IEEE J. Solid-st. Circulation41, 3007–3015 (2006).

Jones, S. A.

S. A. Jones, S. H. Shim, J. He, and X. Zhuang, “Fast, three-dimensional super-resolution imaging of live cells,” Nat. Methods8(6), 499–505 (2011).
[CrossRef] [PubMed]

Joubert, J. R.

J. R. Joubert and D. K. Sharma, “EMCCD vs. sCMOS for microscopic imaging,” Photon. Spectra45, 46–50 (2011).

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]

Li, J. L.

Li, P. C.

Li, W.

B. Fowler, C. A. Liu, S. Mims, J. Balicki, W. Li, H. Do, J. Appelbaum, and P. Vu, “A 5.5Mpixel 100 frames/sec wide dynamic range low noise CMOS image sensor for scientific applications,” Proc. SPIE7536, 753607, 753607-12 (2010).
[CrossRef]

Lichtman, J. W.

J. W. Lichtman and W. Denk, “The big and the small: Challenges of imaging the brain’s circuits,” Science334(6056), 618–623 (2011).
[CrossRef] [PubMed]

Liu, C. A.

B. Fowler, C. A. Liu, S. Mims, J. Balicki, W. Li, H. Do, J. Appelbaum, and P. Vu, “A 5.5Mpixel 100 frames/sec wide dynamic range low noise CMOS image sensor for scientific applications,” Proc. SPIE7536, 753607, 753607-12 (2010).
[CrossRef]

Liu, X. Q.

A. El Gamal, B. Fowlera, H. Min, and X. Q. Liu, “Modeling and estimation of FPN components in CMOS image,” Proc. SPIE3301, 168–177 (1998).
[CrossRef]

Liu, Y. F.

Long, F.

Lord, S. J.

M. A. Thompson, J. S. Biteen, S. J. Lord, N. R. Conley, and W. E. Moerner, “Molecules and methods for super-resolution imaging,” Methods Enzymol.475, 27–59 (2010).
[CrossRef] [PubMed]

Luo, Q. M.

Ma, H. Q.

Maji, S.

Makinwa, K. A. A.

M. F. Snoeij, A. J. P. Theuwissen, K. A. A. Makinwa, and J. H. Huijsing, “A CMOS imager with column-level ADC using dynamic column fixed-pattern noise reduction,” IEEE J. Solid-st. Circulation41, 3007–3015 (2006).

Mims, S.

B. Fowler, C. A. Liu, S. Mims, J. Balicki, W. Li, H. Do, J. Appelbaum, and P. Vu, “A 5.5Mpixel 100 frames/sec wide dynamic range low noise CMOS image sensor for scientific applications,” Proc. SPIE7536, 753607, 753607-12 (2010).
[CrossRef]

Min, H.

A. El Gamal, B. Fowlera, H. Min, and X. Q. Liu, “Modeling and estimation of FPN components in CMOS image,” Proc. SPIE3301, 168–177 (1998).
[CrossRef]

Moerner, W. E.

M. A. Thompson, J. S. Biteen, S. J. Lord, N. R. Conley, and W. E. Moerner, “Molecules and methods for super-resolution imaging,” Methods Enzymol.475, 27–59 (2010).
[CrossRef] [PubMed]

Moomaw, B.

B. Moomaw, “Camera technologies for low light imaging: overview and relative advantages,” Methods Cell Biol.81, 251–283 (2007).
[CrossRef] [PubMed]

Mortensen, K. I.

K. I. Mortensen, L. S. Churchman, J. A. Spudich, and H. Flyvbjerg, “Optimized localization analysis for single-molecule tracking and super-resolution microscopy,” Nat. Methods7(5), 377–381 (2010).
[CrossRef] [PubMed]

Nienhaus, G. U.

Ober, R. J.

R. J. Ober, S. Ram, and E. S. Ward, “Localization accuracy in single-molecule microscopy,” Biophys. J.86(2), 1185–1200 (2004).
[CrossRef] [PubMed]

Qin, L. S.

Quan, T. W.

T. W. Quan, P. C. Li, F. Long, S. Q. Zeng, Q. M. Luo, P. N. Hedde, G. U. Nienhaus, and Z. L. Huang, “Ultra-fast, high-precision image analysis for localization-based super resolution microscopy,” Opt. Express18(11), 11867–11876 (2010).
[CrossRef] [PubMed]

T. W. Quan, S. Q. Zeng, and Z. L. Huang, “Localization capability and limitation of electron-multiplying charge-coupled, scientific complementary metal-oxide semiconductor, and charge-coupled devices for superresolution imaging,” J. Biomed. Opt.15(6), 066005 (2010).
[CrossRef] [PubMed]

Ram, S.

R. J. Ober, S. Ram, and E. S. Ward, “Localization accuracy in single-molecule microscopy,” Biophys. J.86(2), 1185–1200 (2004).
[CrossRef] [PubMed]

Robbins, M. S.

M. S. Robbins and B. J. Hadwen, “The noise performance of electron multiplying charge-coupled devices,” IEEE Trans. Electron. Dev.50(5), 1227–1232 (2003).
[CrossRef]

Salvi, J.

M. Bigas, E. Cabruja, J. Forest, and J. Salvi, “Review of CMOS image sensors,” Microelectron. J.37(5), 433–451 (2006).
[CrossRef]

Saurabh, S.

Sharma, D. K.

J. R. Joubert and D. K. Sharma, “EMCCD vs. sCMOS for microscopic imaging,” Photon. Spectra45, 46–50 (2011).

Shim, S. H.

S. A. Jones, S. H. Shim, J. He, and X. Zhuang, “Fast, three-dimensional super-resolution imaging of live cells,” Nat. Methods8(6), 499–505 (2011).
[CrossRef] [PubMed]

Snoeij, M. F.

M. F. Snoeij, A. J. P. Theuwissen, K. A. A. Makinwa, and J. H. Huijsing, “A CMOS imager with column-level ADC using dynamic column fixed-pattern noise reduction,” IEEE J. Solid-st. Circulation41, 3007–3015 (2006).

Spudich, J. A.

K. I. Mortensen, L. S. Churchman, J. A. Spudich, and H. Flyvbjerg, “Optimized localization analysis for single-molecule tracking and super-resolution microscopy,” Nat. Methods7(5), 377–381 (2010).
[CrossRef] [PubMed]

Theuwissen, A. J. P.

M. F. Snoeij, A. J. P. Theuwissen, K. A. A. Makinwa, and J. H. Huijsing, “A CMOS imager with column-level ADC using dynamic column fixed-pattern noise reduction,” IEEE J. Solid-st. Circulation41, 3007–3015 (2006).

Thompson, M. A.

M. A. Thompson, J. S. Biteen, S. J. Lord, N. R. Conley, and W. E. Moerner, “Molecules and methods for super-resolution imaging,” Methods Enzymol.475, 27–59 (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]

Ting, A. Y.

M. Fernández-Suárez and A. Y. Ting, “Fluorescent probes for super-resolution imaging in living cells,” Nat. Rev. Mol. Cell Biol.9(12), 929–943 (2008).
[CrossRef] [PubMed]

Vaughan, J. C.

G. T. Dempsey, J. C. Vaughan, K. H. Chen, M. Bates, and X. W. Zhuang, “Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging,” Nat. Methods8(12), 1027–1036 (2011).
[CrossRef] [PubMed]

Verkhusha, V. V.

T. J. Gould, V. V. Verkhusha, and S. T. Hess, “Imaging biological structures with fluorescence photoactivation localization microscopy,” Nat. Protoc.4(3), 291–308 (2009).
[CrossRef] [PubMed]

Vu, P.

B. Fowler, C. A. Liu, S. Mims, J. Balicki, W. Li, H. Do, J. Appelbaum, and P. Vu, “A 5.5Mpixel 100 frames/sec wide dynamic range low noise CMOS image sensor for scientific applications,” Proc. SPIE7536, 753607, 753607-12 (2010).
[CrossRef]

Wang, Y. M.

Ward, E. S.

R. J. Ober, S. Ram, and E. S. Ward, “Localization accuracy in single-molecule microscopy,” Biophys. J.86(2), 1185–1200 (2004).
[CrossRef] [PubMed]

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]

Zeng, S. Q.

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Figures (10)

Fig. 1
Fig. 1

Illustration for determining the localization accuracy from repeated single molecule imaging. (a) PSF of a point-like emitter and its center position (shown by the red cross) found by 2D Gaussian fitting. (b) Distribution of the center positions of the emitter from repeated imaging. (c) A representative histogram showing the center positions projected in x dimension. Note that the localization accuracy is averaged from the standard deviation of the histograms in x and y dimensions.

Fig. 2
Fig. 2

Simplified Image SNR for representative cameras. The read noise of the iXon 897 was set to be 0.5 e-, corresponding to a EM gain of 120 [24]. The inset in the right corner is an enlarged view of the region marked by the red rectangle. The signal values for the crossovers were shown by the numbers nearby.

Fig. 3
Fig. 3

Representative PTCs for the Flash 4.0 (a) and the iXon 897 with EM gain 120 (b). Note that: (i) Due to insufficient illumination intensity, image frames close to the Flash 4.0 camera’s saturation (signal > 7600 photon/pixel) were obtained by increasing exposure time from 30 ms to 200 ms. (ii) These are representative curves from three independent measurements which presented almost identical results. (iii) The PTC for the iXon 897 is similar to that reported in our pervious paper [13]. (iv) Theoretical Shot Noise was calculated by the square root of Signal*QE. The QE (quantum efficiency) in 580 nm was set to be 0.72 for the Flash 4.0, and 0.95 for the iXon 897, respectively. (v) The excess noise factor is 2 for the iXon 897. (vi) The red rectangles highlight typical signal ranges in localization microscopy.

Fig. 4
Fig. 4

Contribution of read noise (RN), shot noise (SN), excess noise (EN) and fixed pattern noise (FPN) to total noise under different incident signal intensities of the Flash 4.0 (a), Flash 2.8 (b) and iXon 897 (c). Note that the red rectangles highlight typical signal ranges in localization microscopy.

Fig. 5
Fig. 5

Imaging uniformity of the Flash 4.0 (a-d) and the iXon 897 (e-h) under different intensities of uniform illumination (shown by the values on the top of the images). Each image was overlaid from a set of 100 successive image frames. The mean values and standard deviations (S.D.) of individual images are shown in the right corner of the corresponding figures.

Fig. 6
Fig. 6

Dependence of different fixed pattern noises (a) and the ratio of pixel fixed pattern noise (pFPN) to column fixed pattern noise (cFPN) (b) on signal intensity in the Flash 4.0. In the cFPN curve in (a), the marked positions (1-4) correspond to the images in Fig. 5(a)-5(d). Note that the red rectangles highlight typical signal ranges in localization microscopy.

Fig. 7
Fig. 7

The variation of the peak signal intensity of the same fluorescent bead over time detected by the Flash 4.0 (a) and the iXon 897 (c). The data in (a) and (c) were further analyzed, and the results are presented in (b) and (d), respectively.

Fig. 8
Fig. 8

Dependence of Optimal Image SNR on incident signal intensity. Note that no photon background is assumed to accompany with the signal. The red rectangles highlight typical signal range in localization microscopy. The inset in the right corner is an enlarged view of the region marked by the red rectangle. The QE of the perfect camera was set to 1, while the QE of other cameras was set according to Table 1. The perfect camera was assumed to have no camera noise. The signal values for the crossovers were shown by the numbers nearby

Fig. 9
Fig. 9

The dependence of image SNR on different photon background intensity (a-d) and signal (e). Experimental Image SNR were determined from fluorescent beads with a total number of emitted photon of 1180 (a), 1550 (b), 1040 (c), and 2100 (d), respectively . But due to the pixel size difference of the cameras, the peak signal is 217 photon for the Flash 4.0 and 207 photon for the Flash 2.8 (a), 281 photon for the Flash 4.0 and 274 photon for the Flash 2.8 (b), 301 photon for the Flash 4.0 and 237 photon for the iXon 897(c), and 611 photon for the Flash 4.0 and 477 photon for the iXon 897 (d), respectively. The theoretical curves in (a-d) are Theoretical Image SNR which was calculated from Eq. (1). The curves in (e) were reproduced from Fig. 8. The isolated data points in (e) were obtained from (a-d). Each of the data points in (a-d) was averaged from 500 independent measurements. The standard deviations of the measurements are indicated by the error bars in (a-d).

Fig. 10
Fig. 10

Dependence of localization accuracy on photon background intensity. Localization accuracy was determined from fluorescent beads with a total number of emitted photon of 1180 (a), 1550 (b), 1040 (c), and 2100 (d), respectively. To aid data observation, smoothing spline fitting was used to generate the dotted lines. Each of the data points was averaged from 10 independent measurements. The error bars indicate the standard deviation. The pixel size at sample plane: (a-b) 108 nm for the Flash 4.0, 121 nm for the Flash 2.8; (c-d) 186 nm for the Flash 4.0 and 160 nm for the iXon 897.

Tables (2)

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Table 1 Some important parameters of the low-light cameras

Tables Icon

Table 2 Peak signal intensity of individual fluorescence emitters under different sampling densities

Equations (5)

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SN R theory = N sig ×QE ( N sig + N bkg )×QE× F n 2 + N camera 2
SN R optimal = N sig ×QE N sig ×QE× F n 2 + N camera 2
SN R simplified = N sig ×QE N sig ×QE× F n 2 +R N 2
SN R experiment = N sig ×QE N sig ×QE× F n 2 + σ bkg 2
SN R optimal N sig ×QE N sig ×QE× F n 2 = N sig × QE F n 2 N sig × QE F n 2 = N sig ×Q E eff N sig ×Q E eff

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