Abstract

In light sheet fluorescence microscopy optical sectioning is achieved by illuminating the sample orthogonally to the detection pathway with a thin, focused sheet of light. However, light scattering within the sample often deteriorates the optical sectioning effect. Here, we demonstrate that contrast and degree of confocality can greatly be increased by combining scanned light sheet fluorescence excitation and confocal slit detection. A high frame rate was achieved by using the “rolling shutter” of a scientific CMOS camera as a slit detector. Synchronizing the “rolling shutter” with the scanned illumination beam results in confocal line detection. Acquiring image data with selective plane illumination minimizes photo-damage while simultaneously enhancing contrast, optical sectioning and signal-to-noise ratio. Thus the imaging principle presented here merges the benefits of scanned light sheet microscopy and line-scanning confocal imaging.

© 2012 OSA

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References

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  1. A. H. Voie, D. H. Burns, and F. A. Spelman, “Orthogonal-plane fluorescence optical sectioning: three-dimensional imaging of macroscopic biological specimens,” J. Microsc.170(3), 229–236 (1993).
    [CrossRef] [PubMed]
  2. J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. K. Stelzer, “Optical sectioning deep inside live embryos by selective plane illumination microscopy,” Science305(5686), 1007–1009 (2004).
    [CrossRef] [PubMed]
  3. J. Huisken and D. Y. R. Stainier, “Even fluorescence excitation by multidirectional selective plane illumination microscopy (mSPIM),” Opt. Lett.32(17), 2608–2610 (2007).
    [CrossRef] [PubMed]
  4. H.-U. Dodt, U. Leischner, A. Schierloh, N. Jährling, C. P. Mauch, K. Deininger, J. M. Deussing, M. Eder, W. Zieglgänsberger, and K. Becker, “Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain,” Nat. Methods4(4), 331–336 (2007).
    [CrossRef] [PubMed]
  5. P. J. Keller and E. H. K. Stelzer, “Quantitative in vivo imaging of entire embryos with digital scanned laser light sheet fluorescence microscopy,” Curr. Opin. Neurobiol.18(6), 624–632 (2008).
    [CrossRef] [PubMed]
  6. P. J. Keller, A. D. Schmidt, J. Wittbrodt, and E. H. K. Stelzer, “Reconstruction of zebrafish early embryonic development by scanned light sheet microscopy,” Science322(5904), 1065–1069 (2008).
    [CrossRef] [PubMed]
  7. J. Mertz and J. Kim, “Scanning light-sheet microscopy in the whole mouse brain with HiLo background rejection,” J. Biomed. Opt.15(1), 016027 (2010).
    [CrossRef] [PubMed]
  8. F. O. Fahrbach and A. Rohrbach, “Propagation stability of self-reconstructing Bessel beams enables contrast-enhanced imaging in thick media,” Nat Commun3(632), 632 (2012).
    [CrossRef] [PubMed]
  9. H. K. A. Spiecker, “Method and arrangement for microscopy,” PCT Patent 2011/120629 (2011).
  10. J. G. Ritter, R. Veith, A. Veenendaal, J. P. Siebrasse, and U. Kubitscheck, “Light sheet microscopy for single molecule tracking in living tissue,” PLoS ONE5(7), e11639 (2010).
    [CrossRef] [PubMed]
  11. T. Wilson and C. Sheppard, Theory and Practice of Scanning Optical Microscopy (Academic Press, London, 1984), Chap. 2.
  12. J. T. Bushberg, J. A. Seibert, E. M. Leidholdt, Jr., and J. M. Boone, The Essential Physics of Medical Imaging (Lippincott Williams & Wilkins, Philadelphia, 2002).
  13. A. A. Michelson, Studies in Optics (University of Chicago Press 1927).
  14. P. J. Keller, A. D. Schmidt, A. Santella, K. Khairy, Z. Bao, J. Wittbrodt, and E. H. Stelzer, “Fast, high-contrast imaging of animal development with scanned light sheet-based structured-illumination microscopy,” Nat. Methods7(8), 637–642 (2010).
    [CrossRef] [PubMed]

2012 (1)

F. O. Fahrbach and A. Rohrbach, “Propagation stability of self-reconstructing Bessel beams enables contrast-enhanced imaging in thick media,” Nat Commun3(632), 632 (2012).
[CrossRef] [PubMed]

2010 (3)

J. G. Ritter, R. Veith, A. Veenendaal, J. P. Siebrasse, and U. Kubitscheck, “Light sheet microscopy for single molecule tracking in living tissue,” PLoS ONE5(7), e11639 (2010).
[CrossRef] [PubMed]

P. J. Keller, A. D. Schmidt, A. Santella, K. Khairy, Z. Bao, J. Wittbrodt, and E. H. Stelzer, “Fast, high-contrast imaging of animal development with scanned light sheet-based structured-illumination microscopy,” Nat. Methods7(8), 637–642 (2010).
[CrossRef] [PubMed]

J. Mertz and J. Kim, “Scanning light-sheet microscopy in the whole mouse brain with HiLo background rejection,” J. Biomed. Opt.15(1), 016027 (2010).
[CrossRef] [PubMed]

2008 (2)

P. J. Keller and E. H. K. Stelzer, “Quantitative in vivo imaging of entire embryos with digital scanned laser light sheet fluorescence microscopy,” Curr. Opin. Neurobiol.18(6), 624–632 (2008).
[CrossRef] [PubMed]

P. J. Keller, A. D. Schmidt, J. Wittbrodt, and E. H. K. Stelzer, “Reconstruction of zebrafish early embryonic development by scanned light sheet microscopy,” Science322(5904), 1065–1069 (2008).
[CrossRef] [PubMed]

2007 (2)

J. Huisken and D. Y. R. Stainier, “Even fluorescence excitation by multidirectional selective plane illumination microscopy (mSPIM),” Opt. Lett.32(17), 2608–2610 (2007).
[CrossRef] [PubMed]

H.-U. Dodt, U. Leischner, A. Schierloh, N. Jährling, C. P. Mauch, K. Deininger, J. M. Deussing, M. Eder, W. Zieglgänsberger, and K. Becker, “Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain,” Nat. Methods4(4), 331–336 (2007).
[CrossRef] [PubMed]

2004 (1)

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. K. Stelzer, “Optical sectioning deep inside live embryos by selective plane illumination microscopy,” Science305(5686), 1007–1009 (2004).
[CrossRef] [PubMed]

1993 (1)

A. H. Voie, D. H. Burns, and F. A. Spelman, “Orthogonal-plane fluorescence optical sectioning: three-dimensional imaging of macroscopic biological specimens,” J. Microsc.170(3), 229–236 (1993).
[CrossRef] [PubMed]

Bao, Z.

P. J. Keller, A. D. Schmidt, A. Santella, K. Khairy, Z. Bao, J. Wittbrodt, and E. H. Stelzer, “Fast, high-contrast imaging of animal development with scanned light sheet-based structured-illumination microscopy,” Nat. Methods7(8), 637–642 (2010).
[CrossRef] [PubMed]

Becker, K.

H.-U. Dodt, U. Leischner, A. Schierloh, N. Jährling, C. P. Mauch, K. Deininger, J. M. Deussing, M. Eder, W. Zieglgänsberger, and K. Becker, “Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain,” Nat. Methods4(4), 331–336 (2007).
[CrossRef] [PubMed]

Burns, D. H.

A. H. Voie, D. H. Burns, and F. A. Spelman, “Orthogonal-plane fluorescence optical sectioning: three-dimensional imaging of macroscopic biological specimens,” J. Microsc.170(3), 229–236 (1993).
[CrossRef] [PubMed]

Deininger, K.

H.-U. Dodt, U. Leischner, A. Schierloh, N. Jährling, C. P. Mauch, K. Deininger, J. M. Deussing, M. Eder, W. Zieglgänsberger, and K. Becker, “Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain,” Nat. Methods4(4), 331–336 (2007).
[CrossRef] [PubMed]

Del Bene, F.

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. K. Stelzer, “Optical sectioning deep inside live embryos by selective plane illumination microscopy,” Science305(5686), 1007–1009 (2004).
[CrossRef] [PubMed]

Deussing, J. M.

H.-U. Dodt, U. Leischner, A. Schierloh, N. Jährling, C. P. Mauch, K. Deininger, J. M. Deussing, M. Eder, W. Zieglgänsberger, and K. Becker, “Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain,” Nat. Methods4(4), 331–336 (2007).
[CrossRef] [PubMed]

Dodt, H.-U.

H.-U. Dodt, U. Leischner, A. Schierloh, N. Jährling, C. P. Mauch, K. Deininger, J. M. Deussing, M. Eder, W. Zieglgänsberger, and K. Becker, “Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain,” Nat. Methods4(4), 331–336 (2007).
[CrossRef] [PubMed]

Eder, M.

H.-U. Dodt, U. Leischner, A. Schierloh, N. Jährling, C. P. Mauch, K. Deininger, J. M. Deussing, M. Eder, W. Zieglgänsberger, and K. Becker, “Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain,” Nat. Methods4(4), 331–336 (2007).
[CrossRef] [PubMed]

Fahrbach, F. O.

F. O. Fahrbach and A. Rohrbach, “Propagation stability of self-reconstructing Bessel beams enables contrast-enhanced imaging in thick media,” Nat Commun3(632), 632 (2012).
[CrossRef] [PubMed]

Huisken, J.

J. Huisken and D. Y. R. Stainier, “Even fluorescence excitation by multidirectional selective plane illumination microscopy (mSPIM),” Opt. Lett.32(17), 2608–2610 (2007).
[CrossRef] [PubMed]

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. K. Stelzer, “Optical sectioning deep inside live embryos by selective plane illumination microscopy,” Science305(5686), 1007–1009 (2004).
[CrossRef] [PubMed]

Jährling, N.

H.-U. Dodt, U. Leischner, A. Schierloh, N. Jährling, C. P. Mauch, K. Deininger, J. M. Deussing, M. Eder, W. Zieglgänsberger, and K. Becker, “Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain,” Nat. Methods4(4), 331–336 (2007).
[CrossRef] [PubMed]

Keller, P. J.

P. J. Keller, A. D. Schmidt, A. Santella, K. Khairy, Z. Bao, J. Wittbrodt, and E. H. Stelzer, “Fast, high-contrast imaging of animal development with scanned light sheet-based structured-illumination microscopy,” Nat. Methods7(8), 637–642 (2010).
[CrossRef] [PubMed]

P. J. Keller and E. H. K. Stelzer, “Quantitative in vivo imaging of entire embryos with digital scanned laser light sheet fluorescence microscopy,” Curr. Opin. Neurobiol.18(6), 624–632 (2008).
[CrossRef] [PubMed]

P. J. Keller, A. D. Schmidt, J. Wittbrodt, and E. H. K. Stelzer, “Reconstruction of zebrafish early embryonic development by scanned light sheet microscopy,” Science322(5904), 1065–1069 (2008).
[CrossRef] [PubMed]

Khairy, K.

P. J. Keller, A. D. Schmidt, A. Santella, K. Khairy, Z. Bao, J. Wittbrodt, and E. H. Stelzer, “Fast, high-contrast imaging of animal development with scanned light sheet-based structured-illumination microscopy,” Nat. Methods7(8), 637–642 (2010).
[CrossRef] [PubMed]

Kim, J.

J. Mertz and J. Kim, “Scanning light-sheet microscopy in the whole mouse brain with HiLo background rejection,” J. Biomed. Opt.15(1), 016027 (2010).
[CrossRef] [PubMed]

Kubitscheck, U.

J. G. Ritter, R. Veith, A. Veenendaal, J. P. Siebrasse, and U. Kubitscheck, “Light sheet microscopy for single molecule tracking in living tissue,” PLoS ONE5(7), e11639 (2010).
[CrossRef] [PubMed]

Leischner, U.

H.-U. Dodt, U. Leischner, A. Schierloh, N. Jährling, C. P. Mauch, K. Deininger, J. M. Deussing, M. Eder, W. Zieglgänsberger, and K. Becker, “Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain,” Nat. Methods4(4), 331–336 (2007).
[CrossRef] [PubMed]

Mauch, C. P.

H.-U. Dodt, U. Leischner, A. Schierloh, N. Jährling, C. P. Mauch, K. Deininger, J. M. Deussing, M. Eder, W. Zieglgänsberger, and K. Becker, “Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain,” Nat. Methods4(4), 331–336 (2007).
[CrossRef] [PubMed]

Mertz, J.

J. Mertz and J. Kim, “Scanning light-sheet microscopy in the whole mouse brain with HiLo background rejection,” J. Biomed. Opt.15(1), 016027 (2010).
[CrossRef] [PubMed]

Ritter, J. G.

J. G. Ritter, R. Veith, A. Veenendaal, J. P. Siebrasse, and U. Kubitscheck, “Light sheet microscopy for single molecule tracking in living tissue,” PLoS ONE5(7), e11639 (2010).
[CrossRef] [PubMed]

Rohrbach, A.

F. O. Fahrbach and A. Rohrbach, “Propagation stability of self-reconstructing Bessel beams enables contrast-enhanced imaging in thick media,” Nat Commun3(632), 632 (2012).
[CrossRef] [PubMed]

Santella, A.

P. J. Keller, A. D. Schmidt, A. Santella, K. Khairy, Z. Bao, J. Wittbrodt, and E. H. Stelzer, “Fast, high-contrast imaging of animal development with scanned light sheet-based structured-illumination microscopy,” Nat. Methods7(8), 637–642 (2010).
[CrossRef] [PubMed]

Schierloh, A.

H.-U. Dodt, U. Leischner, A. Schierloh, N. Jährling, C. P. Mauch, K. Deininger, J. M. Deussing, M. Eder, W. Zieglgänsberger, and K. Becker, “Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain,” Nat. Methods4(4), 331–336 (2007).
[CrossRef] [PubMed]

Schmidt, A. D.

P. J. Keller, A. D. Schmidt, A. Santella, K. Khairy, Z. Bao, J. Wittbrodt, and E. H. Stelzer, “Fast, high-contrast imaging of animal development with scanned light sheet-based structured-illumination microscopy,” Nat. Methods7(8), 637–642 (2010).
[CrossRef] [PubMed]

P. J. Keller, A. D. Schmidt, J. Wittbrodt, and E. H. K. Stelzer, “Reconstruction of zebrafish early embryonic development by scanned light sheet microscopy,” Science322(5904), 1065–1069 (2008).
[CrossRef] [PubMed]

Siebrasse, J. P.

J. G. Ritter, R. Veith, A. Veenendaal, J. P. Siebrasse, and U. Kubitscheck, “Light sheet microscopy for single molecule tracking in living tissue,” PLoS ONE5(7), e11639 (2010).
[CrossRef] [PubMed]

Spelman, F. A.

A. H. Voie, D. H. Burns, and F. A. Spelman, “Orthogonal-plane fluorescence optical sectioning: three-dimensional imaging of macroscopic biological specimens,” J. Microsc.170(3), 229–236 (1993).
[CrossRef] [PubMed]

Stainier, D. Y. R.

Stelzer, E. H.

P. J. Keller, A. D. Schmidt, A. Santella, K. Khairy, Z. Bao, J. Wittbrodt, and E. H. Stelzer, “Fast, high-contrast imaging of animal development with scanned light sheet-based structured-illumination microscopy,” Nat. Methods7(8), 637–642 (2010).
[CrossRef] [PubMed]

Stelzer, E. H. K.

P. J. Keller, A. D. Schmidt, J. Wittbrodt, and E. H. K. Stelzer, “Reconstruction of zebrafish early embryonic development by scanned light sheet microscopy,” Science322(5904), 1065–1069 (2008).
[CrossRef] [PubMed]

P. J. Keller and E. H. K. Stelzer, “Quantitative in vivo imaging of entire embryos with digital scanned laser light sheet fluorescence microscopy,” Curr. Opin. Neurobiol.18(6), 624–632 (2008).
[CrossRef] [PubMed]

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. K. Stelzer, “Optical sectioning deep inside live embryos by selective plane illumination microscopy,” Science305(5686), 1007–1009 (2004).
[CrossRef] [PubMed]

Swoger, J.

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. K. Stelzer, “Optical sectioning deep inside live embryos by selective plane illumination microscopy,” Science305(5686), 1007–1009 (2004).
[CrossRef] [PubMed]

Veenendaal, A.

J. G. Ritter, R. Veith, A. Veenendaal, J. P. Siebrasse, and U. Kubitscheck, “Light sheet microscopy for single molecule tracking in living tissue,” PLoS ONE5(7), e11639 (2010).
[CrossRef] [PubMed]

Veith, R.

J. G. Ritter, R. Veith, A. Veenendaal, J. P. Siebrasse, and U. Kubitscheck, “Light sheet microscopy for single molecule tracking in living tissue,” PLoS ONE5(7), e11639 (2010).
[CrossRef] [PubMed]

Voie, A. H.

A. H. Voie, D. H. Burns, and F. A. Spelman, “Orthogonal-plane fluorescence optical sectioning: three-dimensional imaging of macroscopic biological specimens,” J. Microsc.170(3), 229–236 (1993).
[CrossRef] [PubMed]

Wittbrodt, J.

P. J. Keller, A. D. Schmidt, A. Santella, K. Khairy, Z. Bao, J. Wittbrodt, and E. H. Stelzer, “Fast, high-contrast imaging of animal development with scanned light sheet-based structured-illumination microscopy,” Nat. Methods7(8), 637–642 (2010).
[CrossRef] [PubMed]

P. J. Keller, A. D. Schmidt, J. Wittbrodt, and E. H. K. Stelzer, “Reconstruction of zebrafish early embryonic development by scanned light sheet microscopy,” Science322(5904), 1065–1069 (2008).
[CrossRef] [PubMed]

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. K. Stelzer, “Optical sectioning deep inside live embryos by selective plane illumination microscopy,” Science305(5686), 1007–1009 (2004).
[CrossRef] [PubMed]

Zieglgänsberger, W.

H.-U. Dodt, U. Leischner, A. Schierloh, N. Jährling, C. P. Mauch, K. Deininger, J. M. Deussing, M. Eder, W. Zieglgänsberger, and K. Becker, “Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain,” Nat. Methods4(4), 331–336 (2007).
[CrossRef] [PubMed]

Curr. Opin. Neurobiol. (1)

P. J. Keller and E. H. K. Stelzer, “Quantitative in vivo imaging of entire embryos with digital scanned laser light sheet fluorescence microscopy,” Curr. Opin. Neurobiol.18(6), 624–632 (2008).
[CrossRef] [PubMed]

J. Biomed. Opt. (1)

J. Mertz and J. Kim, “Scanning light-sheet microscopy in the whole mouse brain with HiLo background rejection,” J. Biomed. Opt.15(1), 016027 (2010).
[CrossRef] [PubMed]

J. Microsc. (1)

A. H. Voie, D. H. Burns, and F. A. Spelman, “Orthogonal-plane fluorescence optical sectioning: three-dimensional imaging of macroscopic biological specimens,” J. Microsc.170(3), 229–236 (1993).
[CrossRef] [PubMed]

Nat Commun (1)

F. O. Fahrbach and A. Rohrbach, “Propagation stability of self-reconstructing Bessel beams enables contrast-enhanced imaging in thick media,” Nat Commun3(632), 632 (2012).
[CrossRef] [PubMed]

Nat. Methods (2)

H.-U. Dodt, U. Leischner, A. Schierloh, N. Jährling, C. P. Mauch, K. Deininger, J. M. Deussing, M. Eder, W. Zieglgänsberger, and K. Becker, “Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain,” Nat. Methods4(4), 331–336 (2007).
[CrossRef] [PubMed]

P. J. Keller, A. D. Schmidt, A. Santella, K. Khairy, Z. Bao, J. Wittbrodt, and E. H. Stelzer, “Fast, high-contrast imaging of animal development with scanned light sheet-based structured-illumination microscopy,” Nat. Methods7(8), 637–642 (2010).
[CrossRef] [PubMed]

Opt. Lett. (1)

PLoS ONE (1)

J. G. Ritter, R. Veith, A. Veenendaal, J. P. Siebrasse, and U. Kubitscheck, “Light sheet microscopy for single molecule tracking in living tissue,” PLoS ONE5(7), e11639 (2010).
[CrossRef] [PubMed]

Science (2)

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. K. Stelzer, “Optical sectioning deep inside live embryos by selective plane illumination microscopy,” Science305(5686), 1007–1009 (2004).
[CrossRef] [PubMed]

P. J. Keller, A. D. Schmidt, J. Wittbrodt, and E. H. K. Stelzer, “Reconstruction of zebrafish early embryonic development by scanned light sheet microscopy,” Science322(5904), 1065–1069 (2008).
[CrossRef] [PubMed]

Other (4)

H. K. A. Spiecker, “Method and arrangement for microscopy,” PCT Patent 2011/120629 (2011).

T. Wilson and C. Sheppard, Theory and Practice of Scanning Optical Microscopy (Academic Press, London, 1984), Chap. 2.

J. T. Bushberg, J. A. Seibert, E. M. Leidholdt, Jr., and J. M. Boone, The Essential Physics of Medical Imaging (Lippincott Williams & Wilkins, Philadelphia, 2002).

A. A. Michelson, Studies in Optics (University of Chicago Press 1927).

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Figures (7)

Fig. 1
Fig. 1

Illumination beam path. (A) Top view. The excitation beam exits a single mode fiber and is guided by mirrors onto a scanning mirror. The scanning position is imaged by a relay objective into the back focal plane of the illumination objective lens, which is located inside the objective housing. The relay lens assembly is composed of two lens systems with overlapping focal points. It also serves to expand the illumination beam. (B) Side view of the illumination optics.

Fig. 2
Fig. 2

Rolling shutter mode. An activated row was marked in red, and a read-out row was marked in yellow. The band of pixel rows in between is exposed to light simultaneously. This band moves from the top to the bottom of the sensor. Its width is defined by the single row exposure time, and can be adjusted from a minimum of one line up to the whole chip.

Fig. 3
Fig. 3

Image of the illumination beam entering from the left into a (A) homogeneously fluorescent solution of Alexa Fluor 647 in PBS buffer and (B) sample of fluorescent polysterene beads embedded in agarose containing Alexa Fluor 647. A significant amount of light was scattered leading to fluorescence excitation outside the directly illuminated probe region. Imaging conditions and image contrast settings were kept identical. Scale bar 100 μm.

Fig. 4
Fig. 4

Images of beads in an agarose gel demonstrating the contrast difference between global shutter and rolling shutter image acquisition. (A) Image taken in global shutter mode, (B) in rolling shutter mode with a slit width of 128 rows, (C) in rolling shutter mode with a slit width of 32 rows and (D) in rolling shutter mode with a slit width of 2 rows. The image acquisition time was 22 ms in each image. Scale bar, 50 μm.

Fig. 5
Fig. 5

3D reconstruction of a 120x120x50 μ m 3 volume of beads fixed in agarose. The sample volume was imaged in (A) global shutter mode and (B) with a rolling shutter of two lines. The reduction of background in the reconstructed rolling shutter data is evident. The rolling shutter data were averaged 40x to yield an SNR comparable to the global shutter images.

Fig. 6
Fig. 6

SNR and contrast. (A) SNR for different rolling shutter sizes in units of the illumination beam diameter, and for global shutter. (B) Contrast as a function of rolling shutter size, and for global shutter. The rolling shutter sizes corresponding to each point are 2, 4, 8, 16, 32, 64, 128 and 256 pixels, starting from the left.

Fig. 7
Fig. 7

Images of polytene chromosomes in C. tentans salivary gland cell nucleus. (A) Image taken in global shutter mode. The beam was swept once across the sample. (B) Nucleus imaged with a rolling shutter width corresponding to the illumination beam diameter. The bands are crisper and the background in dark areas of the sample is lower as compared to (A). Scale bar, 30 μm. (C) Normalized intensity along the blue and red line in (A) and (B), respectively.

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