Abstract

We report replacement of one side of a static illumination, dual sided, thin-sheet laser imaging microscope (TSLIM) with an intensity modulated laser scanner in order to implement structured illumination (SI) and HiLo image demodulation techniques for background rejection. The new system is equipped with one static and one scanned light-sheet and is called a scanning thin-sheet laser imaging microscope (sTSLIM). It is an optimized version of a light-sheet fluorescent microscope that is designed to image large specimens (<15 mm in diameter). In this paper we describe the hardware and software modifications to TSLIM that allow for static and uniform light-sheet illumination with SI and HiLo image demodulation. The static light-sheet has a thickness of 3.2 µm; whereas, the scanned side has a light-sheet thickness of 4.2 µm. The scanned side images specimens with subcellular resolution (<1 µm lateral and <4 µm axial resolution) with a size up to 15 mm. SI and HiLo produce superior contrast compared to both the uniform static and scanned light-sheets. HiLo contrast was greater than SI and is faster and more robust than SI because as it produces images in two-thirds of the time and exhibits fewer intensity streaking artifacts.

© 2011 OSA

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  1. P. A. Santi, S. B. Johnson, M. Hillenbrand, P. Z. GrandPre, T. J. Glass, and J. R. Leger, “Thin-sheet laser imaging microscopy for optical sectioning of thick tissues,” Biotechniques46(4), 287–294 (2009).
    [PubMed]
  2. M. A. 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]
  3. 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]
  4. P. J. Keller and E. H. 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]
  5. 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]
  6. P. Schacht, S. B. Johnson, and P. A. Santi, “Implementation of a continuous scanning procedure and a line scan camera for thin-sheet laser imaging microscopy,” Biomed. Opt. Express1(2), 598–609 (2010).
    [CrossRef] [PubMed]
  7. J. A. N. Buytaert and J. J. J. Dirckx, “Design and quantitative resolution measurements of an optical virtual sectioning three-dimensional imaging technique for biomedical specimens, featuring two-micrometer slicing resolution,” J. Biomed. Opt.12(1), 014039 (2007).
    [CrossRef] [PubMed]
  8. L. H. Schaefer, D. Schuster, and J. Schaffer, “Structured illumination microscopy: artefact analysis and reduction utilizing a parameter optimization approach,” J. Microsc.216(2), 165–174 (2004).
    [CrossRef] [PubMed]

2010 (3)

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]

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. Schacht, S. B. Johnson, and P. A. Santi, “Implementation of a continuous scanning procedure and a line scan camera for thin-sheet laser imaging microscopy,” Biomed. Opt. Express1(2), 598–609 (2010).
[CrossRef] [PubMed]

2009 (1)

P. A. Santi, S. B. Johnson, M. Hillenbrand, P. Z. GrandPre, T. J. Glass, and J. R. Leger, “Thin-sheet laser imaging microscopy for optical sectioning of thick tissues,” Biotechniques46(4), 287–294 (2009).
[PubMed]

2008 (1)

P. J. Keller and E. H. 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]

2007 (1)

J. A. N. Buytaert and J. J. J. Dirckx, “Design and quantitative resolution measurements of an optical virtual sectioning three-dimensional imaging technique for biomedical specimens, featuring two-micrometer slicing resolution,” J. Biomed. Opt.12(1), 014039 (2007).
[CrossRef] [PubMed]

2004 (1)

L. H. Schaefer, D. Schuster, and J. Schaffer, “Structured illumination microscopy: artefact analysis and reduction utilizing a parameter optimization approach,” J. Microsc.216(2), 165–174 (2004).
[CrossRef] [PubMed]

1997 (1)

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]

Buytaert, J. A. N.

J. A. N. Buytaert and J. J. J. Dirckx, “Design and quantitative resolution measurements of an optical virtual sectioning three-dimensional imaging technique for biomedical specimens, featuring two-micrometer slicing resolution,” J. Biomed. Opt.12(1), 014039 (2007).
[CrossRef] [PubMed]

Dirckx, J. J. J.

J. A. N. Buytaert and J. J. J. Dirckx, “Design and quantitative resolution measurements of an optical virtual sectioning three-dimensional imaging technique for biomedical specimens, featuring two-micrometer slicing resolution,” J. Biomed. Opt.12(1), 014039 (2007).
[CrossRef] [PubMed]

Glass, T. J.

P. A. Santi, S. B. Johnson, M. Hillenbrand, P. Z. GrandPre, T. J. Glass, and J. R. Leger, “Thin-sheet laser imaging microscopy for optical sectioning of thick tissues,” Biotechniques46(4), 287–294 (2009).
[PubMed]

GrandPre, P. Z.

P. A. Santi, S. B. Johnson, M. Hillenbrand, P. Z. GrandPre, T. J. Glass, and J. R. Leger, “Thin-sheet laser imaging microscopy for optical sectioning of thick tissues,” Biotechniques46(4), 287–294 (2009).
[PubMed]

Hillenbrand, M.

P. A. Santi, S. B. Johnson, M. Hillenbrand, P. Z. GrandPre, T. J. Glass, and J. R. Leger, “Thin-sheet laser imaging microscopy for optical sectioning of thick tissues,” Biotechniques46(4), 287–294 (2009).
[PubMed]

Johnson, S. B.

P. Schacht, S. B. Johnson, and P. A. Santi, “Implementation of a continuous scanning procedure and a line scan camera for thin-sheet laser imaging microscopy,” Biomed. Opt. Express1(2), 598–609 (2010).
[CrossRef] [PubMed]

P. A. Santi, S. B. Johnson, M. Hillenbrand, P. Z. GrandPre, T. J. Glass, and J. R. Leger, “Thin-sheet laser imaging microscopy for optical sectioning of thick tissues,” Biotechniques46(4), 287–294 (2009).
[PubMed]

Juskaitis, R.

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. 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]

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]

Leger, J. R.

P. A. Santi, S. B. Johnson, M. Hillenbrand, P. Z. GrandPre, T. J. Glass, and J. R. Leger, “Thin-sheet laser imaging microscopy for optical sectioning of thick tissues,” Biotechniques46(4), 287–294 (2009).
[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]

Neil, M. A. A.

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]

Santi, P. A.

P. Schacht, S. B. Johnson, and P. A. Santi, “Implementation of a continuous scanning procedure and a line scan camera for thin-sheet laser imaging microscopy,” Biomed. Opt. Express1(2), 598–609 (2010).
[CrossRef] [PubMed]

P. A. Santi, S. B. Johnson, M. Hillenbrand, P. Z. GrandPre, T. J. Glass, and J. R. Leger, “Thin-sheet laser imaging microscopy for optical sectioning of thick tissues,” Biotechniques46(4), 287–294 (2009).
[PubMed]

Schacht, P.

Schaefer, L. H.

L. H. Schaefer, D. Schuster, and J. Schaffer, “Structured illumination microscopy: artefact analysis and reduction utilizing a parameter optimization approach,” J. Microsc.216(2), 165–174 (2004).
[CrossRef] [PubMed]

Schaffer, J.

L. H. Schaefer, D. Schuster, and J. Schaffer, “Structured illumination microscopy: artefact analysis and reduction utilizing a parameter optimization approach,” J. Microsc.216(2), 165–174 (2004).
[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]

Schuster, D.

L. H. Schaefer, D. Schuster, and J. Schaffer, “Structured illumination microscopy: artefact analysis and reduction utilizing a parameter optimization approach,” J. Microsc.216(2), 165–174 (2004).
[CrossRef] [PubMed]

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]

P. J. Keller and E. H. 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]

Wilson, T.

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]

Biomed. Opt. Express (1)

Biotechniques (1)

P. A. Santi, S. B. Johnson, M. Hillenbrand, P. Z. GrandPre, T. J. Glass, and J. R. Leger, “Thin-sheet laser imaging microscopy for optical sectioning of thick tissues,” Biotechniques46(4), 287–294 (2009).
[PubMed]

Curr. Opin. Neurobiol. (1)

P. J. Keller and E. H. 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. (2)

J. A. N. Buytaert and J. J. J. Dirckx, “Design and quantitative resolution measurements of an optical virtual sectioning three-dimensional imaging technique for biomedical specimens, featuring two-micrometer slicing resolution,” J. Biomed. Opt.12(1), 014039 (2007).
[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]

J. Microsc. (1)

L. H. Schaefer, D. Schuster, and J. Schaffer, “Structured illumination microscopy: artefact analysis and reduction utilizing a parameter optimization approach,” J. Microsc.216(2), 165–174 (2004).
[CrossRef] [PubMed]

Nat. Methods (1)

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)

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Figures (5)

Fig. 1
Fig. 1

A model of sTSLIM showing assembly of the device. The right side is the static illumination side and the left is the scanned illumination side. The following parts are included in this model: lasers, beam splitter (BS), acousto optic modulator (AOM), beam expander (BE), scanning galvanometer mirror system (galvano) (GM), F-Theta lens (F-Theta), tube lens (TL), microscope objective (MO), detection objective (DO) and cylindrical lens (CL).

Fig. 2
Fig. 2

Optical model of the illumination pathway. The galvano mirror is positioned at the focal point of the F-theta lens in order to produce a scan angle. The tube lens is mounted afocally to the F-Theta lens and the microscope objective to decrease the spot diameter of the F-Theta lens and illuminate the specimen.

Fig. 3
Fig. 3

Comparison images of the scala media from a mouse cochlea. The arrows indicate the illumination direction. The images are recorded and processed as follows: (a) static, uniform illumination, (b) scanned, uniform illumination, (c) structured illumination, and (d) HiLo background rejection. Scale bar = 100 µm.

Fig. 4
Fig. 4

Graph of image contrast measured as pixel intensity standard deviation and calculated from whole image energy-normalized histograms using Eq. (6) in Ref. [5].

Fig. 5
Fig. 5

Artifacts introduced by interruptions of the grid pattern. Arrowhead indicates a tissue heterogeneity that blurred the uniform (a) and grid pattern (b) images, which in turn led to a darkened region in the SI processed image (c) and, to a lesser extent, in the HiLo processed image (d). Scale bar = 50 µm.

Tables (1)

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Table 1 Comparison between structured illumination and HiLo background rejection

Equations (5)

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y'= f 1 '×Θ
Θ''=arctan( y' f 2 ' )
y'''= f 3 '×tan(Θ'')
y'''= f 3 '× f 1 '×Θ f 2 '
Θ= y'''× f 2 f 3 '× f 1 '

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