Abstract

In this paper, we report a method for 3D visualization of a biological specimen utilizing a structured light wide-field microscopic imaging system. This method improves on existing structured light imaging modalities by reassigning fluorescence photons generated from off-focal plane excitation, improving in-focus signal strength. Utilizing a maximum likelihood approach, we identify the most likely fluorophore distribution in 3D that will produce the observed image stacks under structured and uniform illumination using an iterative maximization algorithm. Our results show the optical sectioning capability of tissue specimens while mostly preserving image stack photon count, which is usually not achievable with other existing structured light imaging methods.

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  1. D. A. Agard, Y. Hiraoka, P. Shaw, and J. W. Sedat, “Fluorescence microscopy in three dimensions,” Methods Cell Biol.30, 353–377 (1989).
    [CrossRef] [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. D. Karadaglić and T. Wilson, “Image formation in structured illumination wide-field fluorescence microscopy,” Micron39(7), 808–818 (2008).
    [CrossRef] [PubMed]
  4. M. G. Gustafsson, L. Shao, P. M. Carlton, C. J. R. Wang, I. N. Golubovskaya, W. Z. Cande, D. A. Agard, and J. W. Sedat, “Three-dimensional resolution doubling in wide-field fluorescence microscopy by structured illumination,” Biophys. J.94(12), 4957–4970 (2008).
    [CrossRef] [PubMed]
  5. D. Lim, K. K. Chu, and J. Mertz, “Wide-field fluorescence sectioning with hybrid speckle and uniform-illumination microscopy,” Opt. Lett.33(16), 1819–1821 (2008).
    [CrossRef] [PubMed]
  6. 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]
  7. J. W. Goodman, Introduction to Fourier Optics (McGraw-Hill, 1996).
  8. M. Gu, Principles of Three-Dimensional Imaging in Confocal Microscopes (World Scientific, 1996), Chap. 5.
  9. D. L. Snyder, Random Point Processes (Wiley, New York, 1975), Chap. 6.
  10. A.van den Bos, Parameter Estimation for Scientists and Engineers (Wiley-Interscience, 2007).
  11. B. Richards and E. Wolf, “Electromagnetic diffraction in optical systems. II. Structure of the image field in an aplanatic system,” Proc. R. Soc. Lond. A Math. Phys. Sci.253(1274), 358–379 (1959).
    [CrossRef]
  12. V. Krishnamurthi, Y.-H. Liu, S. Bhattacharyya, J. N. Turner, and T. J. Holmes, “Blind deconvolution of fluorescence micrographs by maximum-likelihood estimation,” Appl. Opt.34(29), 6633–6647 (1995).
    [CrossRef] [PubMed]

2010

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

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

M. G. Gustafsson, L. Shao, P. M. Carlton, C. J. R. Wang, I. N. Golubovskaya, W. Z. Cande, D. A. Agard, and J. W. Sedat, “Three-dimensional resolution doubling in wide-field fluorescence microscopy by structured illumination,” Biophys. J.94(12), 4957–4970 (2008).
[CrossRef] [PubMed]

D. Lim, K. K. Chu, and J. Mertz, “Wide-field fluorescence sectioning with hybrid speckle and uniform-illumination microscopy,” Opt. Lett.33(16), 1819–1821 (2008).
[CrossRef] [PubMed]

1997

1995

1989

D. A. Agard, Y. Hiraoka, P. Shaw, and J. W. Sedat, “Fluorescence microscopy in three dimensions,” Methods Cell Biol.30, 353–377 (1989).
[CrossRef] [PubMed]

1959

B. Richards and E. Wolf, “Electromagnetic diffraction in optical systems. II. Structure of the image field in an aplanatic system,” Proc. R. Soc. Lond. A Math. Phys. Sci.253(1274), 358–379 (1959).
[CrossRef]

Agard, D. A.

M. G. Gustafsson, L. Shao, P. M. Carlton, C. J. R. Wang, I. N. Golubovskaya, W. Z. Cande, D. A. Agard, and J. W. Sedat, “Three-dimensional resolution doubling in wide-field fluorescence microscopy by structured illumination,” Biophys. J.94(12), 4957–4970 (2008).
[CrossRef] [PubMed]

D. A. Agard, Y. Hiraoka, P. Shaw, and J. W. Sedat, “Fluorescence microscopy in three dimensions,” Methods Cell Biol.30, 353–377 (1989).
[CrossRef] [PubMed]

Bhattacharyya, S.

Cande, W. Z.

M. G. Gustafsson, L. Shao, P. M. Carlton, C. J. R. Wang, I. N. Golubovskaya, W. Z. Cande, D. A. Agard, and J. W. Sedat, “Three-dimensional resolution doubling in wide-field fluorescence microscopy by structured illumination,” Biophys. J.94(12), 4957–4970 (2008).
[CrossRef] [PubMed]

Carlton, P. M.

M. G. Gustafsson, L. Shao, P. M. Carlton, C. J. R. Wang, I. N. Golubovskaya, W. Z. Cande, D. A. Agard, and J. W. Sedat, “Three-dimensional resolution doubling in wide-field fluorescence microscopy by structured illumination,” Biophys. J.94(12), 4957–4970 (2008).
[CrossRef] [PubMed]

Chu, K. K.

Golubovskaya, I. N.

M. G. Gustafsson, L. Shao, P. M. Carlton, C. J. R. Wang, I. N. Golubovskaya, W. Z. Cande, D. A. Agard, and J. W. Sedat, “Three-dimensional resolution doubling in wide-field fluorescence microscopy by structured illumination,” Biophys. J.94(12), 4957–4970 (2008).
[CrossRef] [PubMed]

Gustafsson, M. G.

M. G. Gustafsson, L. Shao, P. M. Carlton, C. J. R. Wang, I. N. Golubovskaya, W. Z. Cande, D. A. Agard, and J. W. Sedat, “Three-dimensional resolution doubling in wide-field fluorescence microscopy by structured illumination,” Biophys. J.94(12), 4957–4970 (2008).
[CrossRef] [PubMed]

Hiraoka, Y.

D. A. Agard, Y. Hiraoka, P. Shaw, and J. W. Sedat, “Fluorescence microscopy in three dimensions,” Methods Cell Biol.30, 353–377 (1989).
[CrossRef] [PubMed]

Holmes, T. J.

Juskaitis, R.

Karadaglic, D.

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

Krishnamurthi, V.

Lim, D.

Liu, Y.-H.

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]

D. Lim, K. K. Chu, and J. Mertz, “Wide-field fluorescence sectioning with hybrid speckle and uniform-illumination microscopy,” Opt. Lett.33(16), 1819–1821 (2008).
[CrossRef] [PubMed]

Neil, M. A. A.

Richards, B.

B. Richards and E. Wolf, “Electromagnetic diffraction in optical systems. II. Structure of the image field in an aplanatic system,” Proc. R. Soc. Lond. A Math. Phys. Sci.253(1274), 358–379 (1959).
[CrossRef]

Sedat, J. W.

M. G. Gustafsson, L. Shao, P. M. Carlton, C. J. R. Wang, I. N. Golubovskaya, W. Z. Cande, D. A. Agard, and J. W. Sedat, “Three-dimensional resolution doubling in wide-field fluorescence microscopy by structured illumination,” Biophys. J.94(12), 4957–4970 (2008).
[CrossRef] [PubMed]

D. A. Agard, Y. Hiraoka, P. Shaw, and J. W. Sedat, “Fluorescence microscopy in three dimensions,” Methods Cell Biol.30, 353–377 (1989).
[CrossRef] [PubMed]

Shao, L.

M. G. Gustafsson, L. Shao, P. M. Carlton, C. J. R. Wang, I. N. Golubovskaya, W. Z. Cande, D. A. Agard, and J. W. Sedat, “Three-dimensional resolution doubling in wide-field fluorescence microscopy by structured illumination,” Biophys. J.94(12), 4957–4970 (2008).
[CrossRef] [PubMed]

Shaw, P.

D. A. Agard, Y. Hiraoka, P. Shaw, and J. W. Sedat, “Fluorescence microscopy in three dimensions,” Methods Cell Biol.30, 353–377 (1989).
[CrossRef] [PubMed]

Turner, J. N.

Wang, C. J. R.

M. G. Gustafsson, L. Shao, P. M. Carlton, C. J. R. Wang, I. N. Golubovskaya, W. Z. Cande, D. A. Agard, and J. W. Sedat, “Three-dimensional resolution doubling in wide-field fluorescence microscopy by structured illumination,” Biophys. J.94(12), 4957–4970 (2008).
[CrossRef] [PubMed]

Wilson, T.

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, 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]

Wolf, E.

B. Richards and E. Wolf, “Electromagnetic diffraction in optical systems. II. Structure of the image field in an aplanatic system,” Proc. R. Soc. Lond. A Math. Phys. Sci.253(1274), 358–379 (1959).
[CrossRef]

Appl. Opt.

Biophys. J.

M. G. Gustafsson, L. Shao, P. M. Carlton, C. J. R. Wang, I. N. Golubovskaya, W. Z. Cande, D. A. Agard, and J. W. Sedat, “Three-dimensional resolution doubling in wide-field fluorescence microscopy by structured illumination,” Biophys. J.94(12), 4957–4970 (2008).
[CrossRef] [PubMed]

J. Biomed. Opt.

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]

Methods Cell Biol.

D. A. Agard, Y. Hiraoka, P. Shaw, and J. W. Sedat, “Fluorescence microscopy in three dimensions,” Methods Cell Biol.30, 353–377 (1989).
[CrossRef] [PubMed]

Micron

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

Opt. Lett.

Proc. R. Soc. Lond. A Math. Phys. Sci.

B. Richards and E. Wolf, “Electromagnetic diffraction in optical systems. II. Structure of the image field in an aplanatic system,” Proc. R. Soc. Lond. A Math. Phys. Sci.253(1274), 358–379 (1959).
[CrossRef]

Other

J. W. Goodman, Introduction to Fourier Optics (McGraw-Hill, 1996).

M. Gu, Principles of Three-Dimensional Imaging in Confocal Microscopes (World Scientific, 1996), Chap. 5.

D. L. Snyder, Random Point Processes (Wiley, New York, 1975), Chap. 6.

A.van den Bos, Parameter Estimation for Scientists and Engineers (Wiley-Interscience, 2007).

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Figures (5)

Fig. 1
Fig. 1

Schematic of structured light imaging based wide field fluorescence microscope system

Fig. 2
Fig. 2

Algorithm of photon reassignment process.

Fig. 3
Fig. 3

(a)–(c) Uniform and (d)–(f) structured images of fluorescence beads at object axial positions 24 μm, 48 μm, and 79 μm, respectively; (g)–(i) are the corresponding HiLo microscopy images, (j)–(l) are the reconstructed images using proposed model, and (m)–(o) are the deconvolution images.

Fig. 4
Fig. 4

(a) Line intensity plot showing the performance of the proposed method, (b) Analysis of the in-focus and background signals, (c) SNR and SBR plots.

Fig. 5
Fig. 5

(a) and (d) Wide field fluorescence uniform images of zebrafish intestine; (b) and (e) are the corresponding reconstructed images, for tissue axial positions 36 μm and 51 μm, respectively; (c) and (f) show the line profile of intensity values

Tables (1)

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Table 1 Evaluation of image parameters

Equations (10)

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E U (x,y,z)= e i k 1 z i λ 1 z exp[ i k 1 2z { (xξ) 2 + (yη) 2 } ]dξdη E (x,y,z) S = e i k 1 z i λ 1 z {1+cos( k g ξ)} exp[ i k 1 2z { (xξ) 2 + (yη) 2 } ]dξdη
f U (x',y', z ¯ )=( x',y',z' λ U (x,y,z, z ¯ ) | h 3D (x'x,y'y,z'z) | 2 ) | z=0
f S (x',y', z ¯ )=( x',y',z' λ S (x,y,z, z ¯ ) | h 3D (x'x,y'y,z'z) | 2 ) | z=0
l U (λ(x,y,z))= x,y, z ¯ [ N U (x',y', z ¯ )ln{ f U (x',y', z ¯ )} f U (x',y', z ¯ )]
l S (λ(x,y,z))= x,y, z ¯ [ N S (x',y', z ¯ )ln{ f S (x',y', z ¯ )} f S (x',y', z ¯ )]
l(λ(x,y,z))= x,y, z ¯ [ N U (x',y', z ¯ )ln{ f U (x',y', z ¯ )}+ N S (x',y', z ¯ )ln{ f S (x',y', z ¯ )} f U (x',y', z ¯ ) f S (x',y', z ¯ )]
λ ^ (k+1) (x,y,z)= λ ^ (k) (x,y,z) [ l{λ(x,y,z)}/λ(x,y,z) ] λ(x,y,z)= λ ^ (k) (x,y,z) [ 2 l{λ(x,y,z)}/ λ 2 (x,y,z) ] λ(x,y,z)= λ ^ (k) (x,y,z)
l(λ(x,y,z)) λ(x,y,z) = x,y, z ¯ [U(x,y, z ¯ )| h 3D (0,0, z ¯ ) | 2       ×{ N U (x',y', z ¯ ) ( x',y',z' λ U (x,y,z, z ¯ )| h 3D (x'x,y'y,z'z) | 2 ) | z=0 1}       +S(x,y, z ¯ )| h 3D (0,0, z ¯ ) | 2       ×{ N S (x',y', z ¯ ) ( x',y',z' λ S (x,y,z, z ¯ )| h 3D (x'x,y'y,z'z) | 2 ) | z=0 1}]
2 l ( λ ( x , y , z ) ) λ 2 ( x , y , z ) = x , y , z ¯ [ { U ( x , y , z ¯ ) | h 3 D ( 0 , 0 , z ¯ ) | 2 } 2        × { N U ( x ' , y ' , z ¯ ) { ( x ' , y ' , z ' λ U ( x , y , z , z ¯ ) | h 3 D ( x ' x , y ' y , z ' z ) | 2 ) | z = 0 } 2 }        + { S ( x , y , z ¯ ) | h 3 D ( 0 , 0 , z ¯ ) | 2 } 2        × { N S ( x ' , y ' , z ¯ ) { ( x ' , y ' , z ' λ S ( x , y , z , z ¯ ) | h 3 D ( x ' x , y ' y , z ' z ) | 2 ) | z = 0 } 2 } ]
SNR= I ¯ σ I ,

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