Abstract

Three-dimensional super-resolution imaging in thick, semi-transparent biological specimens is hindered by light scattering, which increases background and degrades both contrast and optical sectioning. We describe a simple method that mitigates these issues, improving image quality in our recently developed two-photon instant structured illumination microscope without requiring any hardware modifications to the instrument. By exciting the specimen with three laterally-structured, phase-shifted illumination patterns and post-processing the resulting images, we digitally remove both scattered and out-of-focus emissions that would otherwise contaminate our raw data. We demonstrate the improved performance of our approach in biological samples, including pollen grains, primary mouse aortic endothelial cells cultured in a three–dimensional collagen matrix and live tumor-like cell spheroids.

© 2015 Optical Society of America

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References

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  1. R. S. Fischer, Y. Wu, P. Kanchanawong, H. Shroff, and C. M. Waterman, “Microscopy in 3D: a biologist’s toolbox,” Trends Cell Biol. 21(12), 682–691 (2011).
    [Crossref] [PubMed]
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    [Crossref] [PubMed]
  3. F. Helmchen and W. Denk, “Deep tissue two-photon microscopy,” Nat. Methods 2(12), 932–940 (2005).
    [Crossref] [PubMed]
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    [Crossref] [PubMed]
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    [Crossref] [PubMed]
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    [Crossref] [PubMed]
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    [Crossref] [PubMed]

2014 (1)

2013 (3)

2011 (1)

R. S. Fischer, Y. Wu, P. Kanchanawong, H. Shroff, and C. M. Waterman, “Microscopy in 3D: a biologist’s toolbox,” Trends Cell Biol. 21(12), 682–691 (2011).
[Crossref] [PubMed]

2010 (2)

C. B. Müller and J. Enderlein, “Image scanning microscopy,” Phys. Rev. Lett. 104(19), 198101 (2010).
[Crossref] [PubMed]

P. J. Keller, A. D. Schmidt, A. Santella, K. Khairy, Z. Bao, J. Wittbrodt, and E. H. K. Stelzer, “Fast, high-contrast imaging of animal development with scanned light sheet-based structured-illumination microscopy,” Nat. Methods 7(8), 637–642 (2010).
[Crossref] [PubMed]

2009 (1)

R. S. Fischer, M. L. Gardel, X. Ma, R. S. Adelstein, and C. M. Waterman, “Local cortical tension by myosin II guides 3D endothelial cell branching,” Curr. Biol. 19(3), 260–265 (2009).
[Crossref] [PubMed]

2007 (1)

2005 (1)

F. Helmchen and W. Denk, “Deep tissue two-photon microscopy,” Nat. Methods 2(12), 932–940 (2005).
[Crossref] [PubMed]

1999 (1)

R. Gauderon, P. B. Lukins, and C. J. R. Sheppard, “Effect of a confocal pinhole in two-photon microscopy,” Microsc. Res. Tech. 47(3), 210–214 (1999).
[Crossref] [PubMed]

1997 (1)

Adelstein, R. S.

R. S. Fischer, M. L. Gardel, X. Ma, R. S. Adelstein, and C. M. Waterman, “Local cortical tension by myosin II guides 3D endothelial cell branching,” Curr. Biol. 19(3), 260–265 (2009).
[Crossref] [PubMed]

Azar, L. N.

Bao, Z.

P. J. Keller, A. D. Schmidt, A. Santella, K. Khairy, Z. Bao, J. Wittbrodt, and E. H. K. Stelzer, “Fast, high-contrast imaging of animal development with scanned light sheet-based structured-illumination microscopy,” Nat. Methods 7(8), 637–642 (2010).
[Crossref] [PubMed]

Brandt, R. A. J.

Breedijk, R. M. P.

Breuninger, T.

Chandris, P.

A. G. York, P. Chandris, D. D. Nogare, J. Head, P. Wawrzusin, R. S. Fischer, A. Chitnis, and H. Shroff, “Instant super-resolution imaging in live cells and embryos via analog image processing,” Nat. Methods 10(11), 1122–1126 (2013).
[Crossref] [PubMed]

Chitnis, A.

P. W. Winter, A. G. York, D. D. Nogare, M. Ingaramo, R. Christensen, A. Chitnis, G. H. Patterson, and H. Shroff, “Two-photon instant structured illumination microscopy improves the depth penetration of super-resolution imaging in thick scattering samples,” Optica 1(3), 181–191 (2014).
[Crossref] [PubMed]

A. G. York, P. Chandris, D. D. Nogare, J. Head, P. Wawrzusin, R. S. Fischer, A. Chitnis, and H. Shroff, “Instant super-resolution imaging in live cells and embryos via analog image processing,” Nat. Methods 10(11), 1122–1126 (2013).
[Crossref] [PubMed]

Choi, H.

Christensen, R.

de Jong, B. E.

De Luca, G. M. R.

Denk, W.

F. Helmchen and W. Denk, “Deep tissue two-photon microscopy,” Nat. Methods 2(12), 932–940 (2005).
[Crossref] [PubMed]

Enderlein, J.

C. B. Müller and J. Enderlein, “Image scanning microscopy,” Phys. Rev. Lett. 104(19), 198101 (2010).
[Crossref] [PubMed]

Fantini, S.

Fischer, R. S.

A. G. York, P. Chandris, D. D. Nogare, J. Head, P. Wawrzusin, R. S. Fischer, A. Chitnis, and H. Shroff, “Instant super-resolution imaging in live cells and embryos via analog image processing,” Nat. Methods 10(11), 1122–1126 (2013).
[Crossref] [PubMed]

R. S. Fischer, Y. Wu, P. Kanchanawong, H. Shroff, and C. M. Waterman, “Microscopy in 3D: a biologist’s toolbox,” Trends Cell Biol. 21(12), 682–691 (2011).
[Crossref] [PubMed]

R. S. Fischer, M. L. Gardel, X. Ma, R. S. Adelstein, and C. M. Waterman, “Local cortical tension by myosin II guides 3D endothelial cell branching,” Curr. Biol. 19(3), 260–265 (2009).
[Crossref] [PubMed]

Gardel, M. L.

R. S. Fischer, M. L. Gardel, X. Ma, R. S. Adelstein, and C. M. Waterman, “Local cortical tension by myosin II guides 3D endothelial cell branching,” Curr. Biol. 19(3), 260–265 (2009).
[Crossref] [PubMed]

Gauderon, R.

R. Gauderon, P. B. Lukins, and C. J. R. Sheppard, “Effect of a confocal pinhole in two-photon microscopy,” Microsc. Res. Tech. 47(3), 210–214 (1999).
[Crossref] [PubMed]

Greger, K.

Hallacoglu, B.

Head, J.

A. G. York, P. Chandris, D. D. Nogare, J. Head, P. Wawrzusin, R. S. Fischer, A. Chitnis, and H. Shroff, “Instant super-resolution imaging in live cells and embryos via analog image processing,” Nat. Methods 10(11), 1122–1126 (2013).
[Crossref] [PubMed]

Helmchen, F.

F. Helmchen and W. Denk, “Deep tissue two-photon microscopy,” Nat. Methods 2(12), 932–940 (2005).
[Crossref] [PubMed]

Hoebe, R. A.

Ingaramo, M.

Juskaitis, R.

Kanchanawong, P.

R. S. Fischer, Y. Wu, P. Kanchanawong, H. Shroff, and C. M. Waterman, “Microscopy in 3D: a biologist’s toolbox,” Trends Cell Biol. 21(12), 682–691 (2011).
[Crossref] [PubMed]

Keller, P. J.

P. J. Keller, A. D. Schmidt, A. Santella, K. Khairy, Z. Bao, J. Wittbrodt, and E. H. K. Stelzer, “Fast, high-contrast imaging of animal development with scanned light sheet-based structured-illumination microscopy,” Nat. Methods 7(8), 637–642 (2010).
[Crossref] [PubMed]

Khairy, K.

P. J. Keller, A. D. Schmidt, A. Santella, K. Khairy, Z. Bao, J. Wittbrodt, and E. H. K. Stelzer, “Fast, high-contrast imaging of animal development with scanned light sheet-based structured-illumination microscopy,” Nat. Methods 7(8), 637–642 (2010).
[Crossref] [PubMed]

Lukins, P. B.

R. Gauderon, P. B. Lukins, and C. J. R. Sheppard, “Effect of a confocal pinhole in two-photon microscopy,” Microsc. Res. Tech. 47(3), 210–214 (1999).
[Crossref] [PubMed]

Ma, X.

R. S. Fischer, M. L. Gardel, X. Ma, R. S. Adelstein, and C. M. Waterman, “Local cortical tension by myosin II guides 3D endothelial cell branching,” Curr. Biol. 19(3), 260–265 (2009).
[Crossref] [PubMed]

Manders, E. M. M.

Müller, C. B.

C. B. Müller and J. Enderlein, “Image scanning microscopy,” Phys. Rev. Lett. 104(19), 198101 (2010).
[Crossref] [PubMed]

Neil, M. A. A.

Nogare, D. D.

P. W. Winter, A. G. York, D. D. Nogare, M. Ingaramo, R. Christensen, A. Chitnis, G. H. Patterson, and H. Shroff, “Two-photon instant structured illumination microscopy improves the depth penetration of super-resolution imaging in thick scattering samples,” Optica 1(3), 181–191 (2014).
[Crossref] [PubMed]

A. G. York, P. Chandris, D. D. Nogare, J. Head, P. Wawrzusin, R. S. Fischer, A. Chitnis, and H. Shroff, “Instant super-resolution imaging in live cells and embryos via analog image processing,” Nat. Methods 10(11), 1122–1126 (2013).
[Crossref] [PubMed]

Patterson, G. H.

Santella, A.

P. J. Keller, A. D. Schmidt, A. Santella, K. Khairy, Z. Bao, J. Wittbrodt, and E. H. K. Stelzer, “Fast, high-contrast imaging of animal development with scanned light sheet-based structured-illumination microscopy,” Nat. Methods 7(8), 637–642 (2010).
[Crossref] [PubMed]

Schmidt, A. D.

P. J. Keller, A. D. Schmidt, A. Santella, K. Khairy, Z. Bao, J. Wittbrodt, and E. H. K. Stelzer, “Fast, high-contrast imaging of animal development with scanned light sheet-based structured-illumination microscopy,” Nat. Methods 7(8), 637–642 (2010).
[Crossref] [PubMed]

Sheppard, C. J. R.

Shroff, H.

P. W. Winter, A. G. York, D. D. Nogare, M. Ingaramo, R. Christensen, A. Chitnis, G. H. Patterson, and H. Shroff, “Two-photon instant structured illumination microscopy improves the depth penetration of super-resolution imaging in thick scattering samples,” Optica 1(3), 181–191 (2014).
[Crossref] [PubMed]

A. G. York, P. Chandris, D. D. Nogare, J. Head, P. Wawrzusin, R. S. Fischer, A. Chitnis, and H. Shroff, “Instant super-resolution imaging in live cells and embryos via analog image processing,” Nat. Methods 10(11), 1122–1126 (2013).
[Crossref] [PubMed]

R. S. Fischer, Y. Wu, P. Kanchanawong, H. Shroff, and C. M. Waterman, “Microscopy in 3D: a biologist’s toolbox,” Trends Cell Biol. 21(12), 682–691 (2011).
[Crossref] [PubMed]

So, P. T. C.

Stallinga, S.

Stelzer, E. H. K.

P. J. Keller, A. D. Schmidt, A. Santella, K. Khairy, Z. Bao, J. Wittbrodt, and E. H. K. Stelzer, “Fast, high-contrast imaging of animal development with scanned light sheet-based structured-illumination microscopy,” Nat. Methods 7(8), 637–642 (2010).
[Crossref] [PubMed]

T. Breuninger, K. Greger, and E. H. K. Stelzer, “Lateral modulation boosts image quality in single plane illumination fluorescence microscopy,” Opt. Lett. 32(13), 1938–1940 (2007).
[Crossref] [PubMed]

Timmermans, W.

Waterman, C. M.

R. S. Fischer, Y. Wu, P. Kanchanawong, H. Shroff, and C. M. Waterman, “Microscopy in 3D: a biologist’s toolbox,” Trends Cell Biol. 21(12), 682–691 (2011).
[Crossref] [PubMed]

R. S. Fischer, M. L. Gardel, X. Ma, R. S. Adelstein, and C. M. Waterman, “Local cortical tension by myosin II guides 3D endothelial cell branching,” Curr. Biol. 19(3), 260–265 (2009).
[Crossref] [PubMed]

Wawrzusin, P.

A. G. York, P. Chandris, D. D. Nogare, J. Head, P. Wawrzusin, R. S. Fischer, A. Chitnis, and H. Shroff, “Instant super-resolution imaging in live cells and embryos via analog image processing,” Nat. Methods 10(11), 1122–1126 (2013).
[Crossref] [PubMed]

Wilson, T.

Winter, P. W.

Wittbrodt, J.

P. J. Keller, A. D. Schmidt, A. Santella, K. Khairy, Z. Bao, J. Wittbrodt, and E. H. K. Stelzer, “Fast, high-contrast imaging of animal development with scanned light sheet-based structured-illumination microscopy,” Nat. Methods 7(8), 637–642 (2010).
[Crossref] [PubMed]

Wu, Y.

R. S. Fischer, Y. Wu, P. Kanchanawong, H. Shroff, and C. M. Waterman, “Microscopy in 3D: a biologist’s toolbox,” Trends Cell Biol. 21(12), 682–691 (2011).
[Crossref] [PubMed]

Yew, E. Y. S.

York, A. G.

P. W. Winter, A. G. York, D. D. Nogare, M. Ingaramo, R. Christensen, A. Chitnis, G. H. Patterson, and H. Shroff, “Two-photon instant structured illumination microscopy improves the depth penetration of super-resolution imaging in thick scattering samples,” Optica 1(3), 181–191 (2014).
[Crossref] [PubMed]

A. G. York, P. Chandris, D. D. Nogare, J. Head, P. Wawrzusin, R. S. Fischer, A. Chitnis, and H. Shroff, “Instant super-resolution imaging in live cells and embryos via analog image processing,” Nat. Methods 10(11), 1122–1126 (2013).
[Crossref] [PubMed]

Zeelenberg, C. H. C.

Biomed. Opt. Express (2)

Curr. Biol. (1)

R. S. Fischer, M. L. Gardel, X. Ma, R. S. Adelstein, and C. M. Waterman, “Local cortical tension by myosin II guides 3D endothelial cell branching,” Curr. Biol. 19(3), 260–265 (2009).
[Crossref] [PubMed]

Microsc. Res. Tech. (1)

R. Gauderon, P. B. Lukins, and C. J. R. Sheppard, “Effect of a confocal pinhole in two-photon microscopy,” Microsc. Res. Tech. 47(3), 210–214 (1999).
[Crossref] [PubMed]

Nat. Methods (3)

F. Helmchen and W. Denk, “Deep tissue two-photon microscopy,” Nat. Methods 2(12), 932–940 (2005).
[Crossref] [PubMed]

A. G. York, P. Chandris, D. D. Nogare, J. Head, P. Wawrzusin, R. S. Fischer, A. Chitnis, and H. Shroff, “Instant super-resolution imaging in live cells and embryos via analog image processing,” Nat. Methods 10(11), 1122–1126 (2013).
[Crossref] [PubMed]

P. J. Keller, A. D. Schmidt, A. Santella, K. Khairy, Z. Bao, J. Wittbrodt, and E. H. K. Stelzer, “Fast, high-contrast imaging of animal development with scanned light sheet-based structured-illumination microscopy,” Nat. Methods 7(8), 637–642 (2010).
[Crossref] [PubMed]

Opt. Lett. (2)

Optica (1)

Phys. Rev. Lett. (1)

C. B. Müller and J. Enderlein, “Image scanning microscopy,” Phys. Rev. Lett. 104(19), 198101 (2010).
[Crossref] [PubMed]

Trends Cell Biol. (1)

R. S. Fischer, Y. Wu, P. Kanchanawong, H. Shroff, and C. M. Waterman, “Microscopy in 3D: a biologist’s toolbox,” Trends Cell Biol. 21(12), 682–691 (2011).
[Crossref] [PubMed]

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Figures (5)

Fig. 1
Fig. 1 Incoherent structured illumination improves contrast in 2P ISIM. (a) Raw images of a 20 μm diameter ragweed pollen grain when using patterned excitation in 2P ISIM. The relative phase shift (0°, 120°, or 240°) is indicated above each image. Raw images may be subsequently summed (bottom left) to recreate the normal image or processed according to Eq. (1) to yield an image with improved contrast (bottom right). Images were acquired 4 μm from the coverslip surface. (b) Comparative maximum intensity projections after summing, or (c) otherwise processing each slice in the imaging volume, and (d) line profiles derived from single slices in (a) confirm the superior contrast afforded by combining incoherent structured illumination with 2P ISIM. Pattern spacing: 1.6 μm. All scalebars: 4 μm. These images have not been deconvolved.
Fig. 2
Fig. 2 Using incoherent structured illumination to improve sectioning and contrast in thick samples. (a) Exemplary patterns at different spatial frequencies, as viewed in a thick fluorescent lake. Scalebar: 10 μm. ‘NA' corresponds to the case without a pattern, i.e. scanning conventional point-illumination through the sample. Patterns are shown at 0° phase offset. (b) and (c) Sectioning response in a thick fluorescent lake. In (b), images corresponding to different phases were summed, normalized according to their intensity at the thick lake interface (defined as the axial position at which the derivative of the change in intensity as a function of axial position is maximized) and compared. (c) The same data shown in (b) were processed according to Eq. (1), and normalized as before. Using higher frequency patterns results in better optical sectioning (less fluorescence) when imaging deeper into the lake (positive values of axial position in the graph). (d) XZ slice through a single 100 nm diameter fluorescent bead in a scattering matrix, as imaged conventionally (upper) and with 1.6 μm spaced pattern (lower). Note the reduction in background after applying patterned illumination. Scalebar: 500 nm. Bead data are deconvolved. (e) The FWHM of 100 nm subdiffractive fluorescent beads placed in the same scattering matrix as in (d), without (no pattern) and with incoherent structured illumination (1.6 μm spacing). Mean and standard deviations from 10 beads, after deconvolution, are shown. Beads appear the same size regardless of imaging condition. (f) Improvement in contrast, comparing different patterns at different depths in the scattering matrix used in (d) and (e).
Fig. 3
Fig. 3 Contrast improvement in densely tagged, thick biological specimens. (a) Partial maximum intensity projection (0-12μm) of a 30-μm thick volume, ~100 μm from the coverslip surface, highlighting phalloidin-labeled actin in a fixed primary culture mouse aortic endothelial cell in a 3D collagen matrix. Scalebar: 10 μm. (b) Digitally processed and (c) conventional 2P ISIM single slices (8 μm into volume), showing higher magnification views of yellow rectangle in (a). Note improved contrast in (b) compared to (c). Scale Bars: 5 μm. (d) Higher magnification view of yellow rectangle in (b), showing two actin bundles just resolved and (e) line profile (peaks are spaced 180 nm apart). Scalebar: 500 nm. Axial cuts through volume, corresponding to magenta dotted line in (a) are shown in digitally processed (f) and conventional (g) 2P ISIM. Blue arrows highlight void areas within cell that are visible in (f), due to improved sectioning, but masked in (g). Pattern spacing: 1.6 μm. Scalebar: 5 μm. These images have been deconvolved.
Fig. 4
Fig. 4 Incoherent structured illumination improves contrast and axial sectioning in thick, scattering biological specimens. (a) 3D rendering of a 35-μm thick volume showing MitoTracker Red-labeled mitochondria and GFP-tagged peroxisomes in a live tumor-like spheroid. The spheroid is a co-culture of transformed keratinocytes (HACAT) growing on human lung fibroblasts (MRL-TR). MitoTracker Red labels mitochondria in both cell types while peroxisomal targeted GFP is only expressed in the stromal layer fibroblasts. (b) Digitally processed and (c) conventional 2P ISIM single slices at indicated axial depth from the coverslip. Yellow arrows highlight specific structures displaying improved contrast in (b) compared to (c). (d) Digitally processed and (e) conventional XZ cuts through the spheroid volume at indicated Y position. Yellow arrows highlight specific structures displaying improved contrast in (d) compared to (e). Pattern spacing: 1.6 μm. All scalebars: 10 μm. These images have been deconvolved.
Fig. 5
Fig. 5 Stripe-correction algorithm. (a) Variable striping artifacts as seen in Alexa488-phalloidin stained primary mouse aortic endothelial cells are corrected on a slice-by-slice basis by (b) horizontally binning the image data, (c) expanding the binned image to match the dimensions of (a) and (d) dividing the original image (a) by (c) to obtain the corrected image. Scale bars: 4 μm. These images have not been deconvolved.

Equations (4)

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I( z )= ( ( I 0° I 120° ) 2 + ( I 120° I 240° ) 2 + ( I 240° I 0° ) 2 ) 2
σ= i ( c i × ( i I ¯ I ) 2 ) C1
I= i i ×  c i
C= i c i

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