Abstract

Laser sheet based microscopy has become widely accepted as an effective active illumination method for real time three-dimensional (3D) imaging of biological tissue samples. The light sheet geometry, where the camera is oriented perpendicular to the sheet itself, provides an effective method of eliminating some of the scattered light and minimizing the sample exposure to radiation. However, residual background noise still remains, limiting the contrast and visibility of potentially interesting features in the samples. In this article, we investigate additional structuring of the illumination for improved background rejection, and propose a new technique, “3D HiLo” where we combine two HiLo images processed from orthogonal directions to improve the condition of the 3D reconstruction. We present a comparative study of conventional structured illumination based demodulation methods, namely 3Phase and HiLo with a newly implemented 3D HiLo approach and demonstrate that the latter yields superior signal-to-background ratio in both lateral and axial dimensions, while simultaneously suppressing image processing artifacts.

© 2012 OSA

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References

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  1. C. J. Sheppard and T. Wilson, “The theory of the direct-view confocal microscope,” J. Microsc.124(2), 107–117 (1981).
    [CrossRef] [PubMed]
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    [CrossRef]
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    [CrossRef] [PubMed]
  8. 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. Methods7(8), 637–642 (2010).
    [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]

2012 (2)

2011 (4)

J. Mertz, “Optical sectioning microscopy with planar or structured illumination,” Nat. Methods8(10), 811–819 (2011).
[CrossRef] [PubMed]

T. Wilson, “Optical sectioning in fluorescence microscopy,” J. Microsc.242(2), 111–116 (2011).
[CrossRef] [PubMed]

G. Best, R. Amberger, D. Baddeley, T. Ach, S. Dithmar, R. Heintzmann, and C. Cremer, “Structured illumination microscopy of autofluorescent aggregations in human tissue,” Micron42(4), 330–335 (2011).
[CrossRef] [PubMed]

L. Gao, N. Bedard, N. Hagen, R. T. Kester, and T. S. Tkaczyk, “Depth-resolved image mapping spectrometer (IMS) with structured illumination,” Opt. Express19(18), 17439–17452 (2011).
[CrossRef] [PubMed]

2010 (2)

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. 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, 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]

D. Karadaglić and T. Wilson, “Image formation in structured illumination wide-field fluorescence microscopy,” Micron39(7), 808–818 (2008).
[CrossRef] [PubMed]

1997 (1)

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]

1981 (2)

C. J. Sheppard and T. Wilson, “The theory of the direct-view confocal microscope,” J. Microsc.124(2), 107–117 (1981).
[CrossRef] [PubMed]

R. R. Schram, “Light-scanning photomicrography,” Microscope29, 13–17 (1981).

1964 (1)

Ach, T.

G. Best, R. Amberger, D. Baddeley, T. Ach, S. Dithmar, R. Heintzmann, and C. Cremer, “Structured illumination microscopy of autofluorescent aggregations in human tissue,” Micron42(4), 330–335 (2011).
[CrossRef] [PubMed]

Amberger, R.

G. Best, R. Amberger, D. Baddeley, T. Ach, S. Dithmar, R. Heintzmann, and C. Cremer, “Structured illumination microscopy of autofluorescent aggregations in human tissue,” Micron42(4), 330–335 (2011).
[CrossRef] [PubMed]

Artigas, D.

Baddeley, D.

G. Best, R. Amberger, D. Baddeley, T. Ach, S. Dithmar, R. Heintzmann, and C. Cremer, “Structured illumination microscopy of autofluorescent aggregations in human tissue,” Micron42(4), 330–335 (2011).
[CrossRef] [PubMed]

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. Methods7(8), 637–642 (2010).
[CrossRef] [PubMed]

Bastiaens, P. I.

M. A. A. Neil, A. Squire, R. Juskaitis, R. Juskaitis, P. I. Bastiaens, and T. Wilson. “Wide-field optically sectioning fluorescence microscopy with laser illumination,” J. Microsc.-Oxford197, 1–4 (2000).

Bedard, N.

Best, G.

G. Best, R. Amberger, D. Baddeley, T. Ach, S. Dithmar, R. Heintzmann, and C. Cremer, “Structured illumination microscopy of autofluorescent aggregations in human tissue,” Micron42(4), 330–335 (2011).
[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]

Cremer, C.

G. Best, R. Amberger, D. Baddeley, T. Ach, S. Dithmar, R. Heintzmann, and C. Cremer, “Structured illumination microscopy of autofluorescent aggregations in human tissue,” Micron42(4), 330–335 (2011).
[CrossRef] [PubMed]

Dithmar, S.

G. Best, R. Amberger, D. Baddeley, T. Ach, S. Dithmar, R. Heintzmann, and C. Cremer, “Structured illumination microscopy of autofluorescent aggregations in human tissue,” Micron42(4), 330–335 (2011).
[CrossRef] [PubMed]

Gao, L.

Gualda, E. J.

Hagen, N.

Heintzmann, R.

G. Best, R. Amberger, D. Baddeley, T. Ach, S. Dithmar, R. Heintzmann, and C. Cremer, “Structured illumination microscopy of autofluorescent aggregations in human tissue,” Micron42(4), 330–335 (2011).
[CrossRef] [PubMed]

John, K.

Johnson, S. B.

Juskaitis, R.

M. A. A. Neil, A. Squire, R. Juskaitis, R. Juskaitis, P. I. Bastiaens, and T. Wilson. “Wide-field optically sectioning fluorescence microscopy with laser illumination,” J. Microsc.-Oxford197, 1–4 (2000).

M. A. A. Neil, A. Squire, R. Juskaitis, R. Juskaitis, P. I. Bastiaens, and T. Wilson. “Wide-field optically sectioning fluorescence microscopy with laser illumination,” J. Microsc.-Oxford197, 1–4 (2000).

M. A. Neil, R. Juskaitis, and T. Wilson, “Method of obtaining optical sectioning by using structured light in a conventional microscope,” Opt. Lett.22(24), 1905–1907 (1997).
[CrossRef] [PubMed]

Karadaglic, D.

D. Karadaglić and T. Wilson, “Image formation in structured illumination wide-field fluorescence microscopy,” Micron39(7), 808–818 (2008).
[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. 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]

Kester, R. T.

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. 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]

Licea-Rodriguez, J.

Loza-Alvarez, P.

Mayer, J.

McLachlan, D.

Mertz, J.

J. Mertz, “Optical sectioning microscopy with planar or structured illumination,” Nat. Methods8(10), 811–819 (2011).
[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]

Neil, M. A.

Neil, M. A. A.

M. A. A. Neil, A. Squire, R. Juskaitis, R. Juskaitis, P. I. Bastiaens, and T. Wilson. “Wide-field optically sectioning fluorescence microscopy with laser illumination,” J. Microsc.-Oxford197, 1–4 (2000).

Olarte, O. E.

Palero, J. A.

Rangel-Rojo, R.

Rocha-Mendoza, I.

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. Methods7(8), 637–642 (2010).
[CrossRef] [PubMed]

Santi, P. A.

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. 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]

Schram, R. R.

R. R. Schram, “Light-scanning photomicrography,” Microscope29, 13–17 (1981).

Schröter, T. J.

Sharpe, J.

Sheppard, C. J.

C. J. Sheppard and T. Wilson, “The theory of the direct-view confocal microscope,” J. Microsc.124(2), 107–117 (1981).
[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]

Squire, A.

M. A. A. Neil, A. Squire, R. Juskaitis, R. Juskaitis, P. I. Bastiaens, and T. Wilson. “Wide-field optically sectioning fluorescence microscopy with laser illumination,” J. Microsc.-Oxford197, 1–4 (2000).

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. 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]

Swoger, J.

Tkaczyk, T. S.

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]

Wilson, T.

T. Wilson, “Optical sectioning in fluorescence microscopy,” J. Microsc.242(2), 111–116 (2011).
[CrossRef] [PubMed]

D. Karadaglić and T. Wilson, “Image formation in structured illumination wide-field fluorescence microscopy,” Micron39(7), 808–818 (2008).
[CrossRef] [PubMed]

M. A. A. Neil, A. Squire, R. Juskaitis, R. Juskaitis, P. I. Bastiaens, and T. Wilson. “Wide-field optically sectioning fluorescence microscopy with laser illumination,” J. Microsc.-Oxford197, 1–4 (2000).

M. A. Neil, R. Juskaitis, and T. Wilson, “Method of obtaining optical sectioning by using structured light in a conventional microscope,” Opt. Lett.22(24), 1905–1907 (1997).
[CrossRef] [PubMed]

C. J. Sheppard and T. Wilson, “The theory of the direct-view confocal microscope,” J. Microsc.124(2), 107–117 (1981).
[CrossRef] [PubMed]

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. 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]

Appl. Opt. (1)

Biomed. Opt. Express (2)

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. (3)

T. Wilson, “Optical sectioning in fluorescence microscopy,” J. Microsc.242(2), 111–116 (2011).
[CrossRef] [PubMed]

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]

C. J. Sheppard and T. Wilson, “The theory of the direct-view confocal microscope,” J. Microsc.124(2), 107–117 (1981).
[CrossRef] [PubMed]

J. Microsc.-Oxford (1)

M. A. A. Neil, A. Squire, R. Juskaitis, R. Juskaitis, P. I. Bastiaens, and T. Wilson. “Wide-field optically sectioning fluorescence microscopy with laser illumination,” J. Microsc.-Oxford197, 1–4 (2000).

Micron (2)

D. Karadaglić and T. Wilson, “Image formation in structured illumination wide-field fluorescence microscopy,” Micron39(7), 808–818 (2008).
[CrossRef] [PubMed]

G. Best, R. Amberger, D. Baddeley, T. Ach, S. Dithmar, R. Heintzmann, and C. Cremer, “Structured illumination microscopy of autofluorescent aggregations in human tissue,” Micron42(4), 330–335 (2011).
[CrossRef] [PubMed]

Microscope (1)

R. R. Schram, “Light-scanning photomicrography,” Microscope29, 13–17 (1981).

Nat. Methods (2)

J. Mertz, “Optical sectioning microscopy with planar or structured illumination,” Nat. Methods8(10), 811–819 (2011).
[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. Methods7(8), 637–642 (2010).
[CrossRef] [PubMed]

Opt. Express (1)

Opt. Lett. (1)

Science (1)

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 (1)

J. B. Pawley, Handbook of Confocal Microscopy (Plenum: New York, 1995).

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Figures (6)

Fig.1
Fig.1

Experimental schematics of digital laser sheet microscopy using structured illumination for simultaneous acquisition of HiLo and 3Phase methodologies.

Fig. 2
Fig. 2

Directional view of lateral XY-HiLo (lateral) and YZ-HiLo (axial).

Fig. 3
Fig. 3

Quantitative bead imaging to understand result of uniformly illuminated imaging, 3Phase imaging, conventional HiLo imaging and Combined axial and lateral Hilo imaging. (a-i) Simulated bead images-object, uniformly illuminated images, grid projected images, conventional HiLo and combined lateral and axial HiLo image (XY and XZ projected images respectively). (a-ii) Line profiles along Z, X and X directions of normal images, conventional HiLo images, axial HiLo images, and combined HiLo images respectively. (b-i) Experimental bead images for uniformly illuminated images, grid projected images, conventional HiLo and combined HiLo image (XY and XZ projected images respectively). (b-ii) Line profiles along Z, X and X directions of experimental normal images, conventional HiLo images, axial HiLo images, and combined HiLo images respectively.

Fig.4
Fig.4

Images of the same membrane of zebrafish embryo (EGFP) at 30 μm depth acquired with uniform illumination as well as different programs of structured illumination. The excitation is incident from the left direction in all experiments. (i) Normalized images of (from left to right): conventional (uniform illuminated) DSLM, 3Phase, conventional XY-HiLo and 3D HiLo (in this and ii, iii, we used the Variance Weighted method for 3D HiLo). (ii) Normalized intensity cross-sections of the images from line A (near the excitation) in Fig. 4(i). (iii) Normalized intensity cross-sections of the images from line B (far from the excitation) in Fig. 4(i). (iv) Comparison of the orthogonal HiLo images (XY, YZ) with conventional DSLM and the different methods described in Section 3 to combine the XY and YZ HiLo to obtain the 3D HiLo image.

Fig. 5
Fig. 5

(a): Imaging spine structure of larval zebrafish using both uniformly illuminated images and 3D HiLo method (Variance Weighted). Here nuclei are tagged with EGFP. (b) Zoom in to the region showed as dotted line in (a), from a deeper fluorescent plane (Z = 110 μm), for uniformly illuminated, 3Phase, conventional HiLo and combined HiLo methods, from left to right, respectively. Scale bar = 100 μm.

Fig. 6
Fig. 6

Zebrafish embryo images acquired and post-processed with different programs of structured illumination. Nuclei are stained with Histone H2B-mRFP (RED) and Cell membranes are stained with membrane-EGFP (pCAG-mGFP-GREEN). Inset: Membrane images of single fluorescent planes at 30μm depth image of the sample for corresponding images. Merged images are shown in right most columns. (Scale bar = 100 μm).

Equations (3)

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I 3D (x,y,z)=α(x) I XY (x,y;z)+{1α(x)} I YZ (y,z;x).
α(x)= x x max .
α(x)= σ XY (x) σ XY (x)+ σ YZ (x) .

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