Abstract

HiLo microscopy is a recently developed technique that provides both optical sectioning and fast imaging with a simple implementation and at a very low cost. The methodology combines widefield and speckled illumination images to obtain one optically sectioned image. Hence, the characteristics of such speckle illumination ultimately determine the quality of HiLo images and the overall performance of the method. In this work, we study how speckle contrast influence local variations of fluorescence intensity and brightness profiles of thick samples. We present this article as a guide to adjust the parameters of the system for optimizing the capabilities of this novel technology.

© 2011 OSA

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  1. 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]
  2. C. Ventalon, R. Heintzmann, and J. Mertz, “Dynamic speckle illumination microscopy with wavelet prefiltering,” Opt. Lett. 32(11), 1417–1419 (2007).
    [CrossRef] [PubMed]
  3. D. Lim, T. N. Ford, K. K. Chu, and J. Mertz, “Optically sectioned in vivo imaging with speckle illumination HiLo microscopy,” J. Biomed. Opt. 16(1), 016014 (2011).
    [CrossRef] [PubMed]
  4. 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]
  5. S. Santos, K. K. Chu, D. Lim, N. Bozinovic, T. N. Ford, C. Hourtoule, A. C. Bartoo, S. K. Singh, and J. Mertz, “Optically sectioned fluorescence endomicroscopy with hybrid-illumination imaging through a flexible fiber bundle,” J. Biomed. Opt. 14(3), 030502 (2009).
    [CrossRef] [PubMed]
  6. J. W. Goodman, Introduction to Fourier Optics (2nd Ed., McGraw-Hill, 1996), Chap. 6.
  7. C. Ventalon and J. Mertz, “Quasi-confocal fluorescence sectioning with dynamic speckle illumination,” Opt. Lett. 30(24), 3350–3352 (2005).
    [CrossRef] [PubMed]
  8. D. D. Duncan, S. J. Kirkpatrick, and R. K. Wang, “Statistics of local speckle contrast,” J. Opt. Soc. Am. A 25(1), 9–15 (2008).
    [CrossRef] [PubMed]
  9. J. Goodman, Speckle phenomena in optics (Roberts & Company, 2007).
  10. J. M. Bélisle, J. P. Correia, P. W. Wiseman, T. E. Kennedy, and S. Costantino, “Patterning protein concentration using laser-assisted adsorption by photobleaching, LAPAP,” Lab Chip 8(12), 2164–2167 (2008).
    [CrossRef] [PubMed]
  11. J. M. Belisle, D. Kunik, and S. Costantino, “Rapid multicomponent optical protein patterning,” Lab Chip 9(24), 3580–3585 (2009).
    [CrossRef] [PubMed]
  12. K. Singh, C. Dion, S. Costantino, M. Wajszilber, M. R. Lesk, and T. Ozaki, “Development of a novel instrument to measure the pulsatile movement of ocular tissues,” Exp. Eye Res. 91(1), 63–68 (2010).
    [CrossRef] [PubMed]
  13. K. Singh, C. Dion, M. R. Lesk, T. Ozaki, and S. Costantino, “Spectral-domain phase microscopy with improved sensitivity using two-dimensional detector arrays,” Rev. Sci. Instrum. 82(2), 023706 (2011).
    [CrossRef] [PubMed]

2011

D. Lim, T. N. Ford, K. K. Chu, and J. Mertz, “Optically sectioned in vivo imaging with speckle illumination HiLo microscopy,” J. Biomed. Opt. 16(1), 016014 (2011).
[CrossRef] [PubMed]

K. Singh, C. Dion, M. R. Lesk, T. Ozaki, and S. Costantino, “Spectral-domain phase microscopy with improved sensitivity using two-dimensional detector arrays,” Rev. Sci. Instrum. 82(2), 023706 (2011).
[CrossRef] [PubMed]

2010

K. Singh, C. Dion, S. Costantino, M. Wajszilber, M. R. Lesk, and T. Ozaki, “Development of a novel instrument to measure the pulsatile movement of ocular tissues,” Exp. Eye Res. 91(1), 63–68 (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]

2009

S. Santos, K. K. Chu, D. Lim, N. Bozinovic, T. N. Ford, C. Hourtoule, A. C. Bartoo, S. K. Singh, and J. Mertz, “Optically sectioned fluorescence endomicroscopy with hybrid-illumination imaging through a flexible fiber bundle,” J. Biomed. Opt. 14(3), 030502 (2009).
[CrossRef] [PubMed]

J. M. Belisle, D. Kunik, and S. Costantino, “Rapid multicomponent optical protein patterning,” Lab Chip 9(24), 3580–3585 (2009).
[CrossRef] [PubMed]

2008

2007

2005

Bartoo, A. C.

S. Santos, K. K. Chu, D. Lim, N. Bozinovic, T. N. Ford, C. Hourtoule, A. C. Bartoo, S. K. Singh, and J. Mertz, “Optically sectioned fluorescence endomicroscopy with hybrid-illumination imaging through a flexible fiber bundle,” J. Biomed. Opt. 14(3), 030502 (2009).
[CrossRef] [PubMed]

Belisle, J. M.

J. M. Belisle, D. Kunik, and S. Costantino, “Rapid multicomponent optical protein patterning,” Lab Chip 9(24), 3580–3585 (2009).
[CrossRef] [PubMed]

Bélisle, J. M.

J. M. Bélisle, J. P. Correia, P. W. Wiseman, T. E. Kennedy, and S. Costantino, “Patterning protein concentration using laser-assisted adsorption by photobleaching, LAPAP,” Lab Chip 8(12), 2164–2167 (2008).
[CrossRef] [PubMed]

Bozinovic, N.

S. Santos, K. K. Chu, D. Lim, N. Bozinovic, T. N. Ford, C. Hourtoule, A. C. Bartoo, S. K. Singh, and J. Mertz, “Optically sectioned fluorescence endomicroscopy with hybrid-illumination imaging through a flexible fiber bundle,” J. Biomed. Opt. 14(3), 030502 (2009).
[CrossRef] [PubMed]

Chu, K. K.

D. Lim, T. N. Ford, K. K. Chu, and J. Mertz, “Optically sectioned in vivo imaging with speckle illumination HiLo microscopy,” J. Biomed. Opt. 16(1), 016014 (2011).
[CrossRef] [PubMed]

S. Santos, K. K. Chu, D. Lim, N. Bozinovic, T. N. Ford, C. Hourtoule, A. C. Bartoo, S. K. Singh, and J. Mertz, “Optically sectioned fluorescence endomicroscopy with hybrid-illumination imaging through a flexible fiber bundle,” J. Biomed. Opt. 14(3), 030502 (2009).
[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]

Correia, J. P.

J. M. Bélisle, J. P. Correia, P. W. Wiseman, T. E. Kennedy, and S. Costantino, “Patterning protein concentration using laser-assisted adsorption by photobleaching, LAPAP,” Lab Chip 8(12), 2164–2167 (2008).
[CrossRef] [PubMed]

Costantino, S.

K. Singh, C. Dion, M. R. Lesk, T. Ozaki, and S. Costantino, “Spectral-domain phase microscopy with improved sensitivity using two-dimensional detector arrays,” Rev. Sci. Instrum. 82(2), 023706 (2011).
[CrossRef] [PubMed]

K. Singh, C. Dion, S. Costantino, M. Wajszilber, M. R. Lesk, and T. Ozaki, “Development of a novel instrument to measure the pulsatile movement of ocular tissues,” Exp. Eye Res. 91(1), 63–68 (2010).
[CrossRef] [PubMed]

J. M. Belisle, D. Kunik, and S. Costantino, “Rapid multicomponent optical protein patterning,” Lab Chip 9(24), 3580–3585 (2009).
[CrossRef] [PubMed]

J. M. Bélisle, J. P. Correia, P. W. Wiseman, T. E. Kennedy, and S. Costantino, “Patterning protein concentration using laser-assisted adsorption by photobleaching, LAPAP,” Lab Chip 8(12), 2164–2167 (2008).
[CrossRef] [PubMed]

Dion, C.

K. Singh, C. Dion, M. R. Lesk, T. Ozaki, and S. Costantino, “Spectral-domain phase microscopy with improved sensitivity using two-dimensional detector arrays,” Rev. Sci. Instrum. 82(2), 023706 (2011).
[CrossRef] [PubMed]

K. Singh, C. Dion, S. Costantino, M. Wajszilber, M. R. Lesk, and T. Ozaki, “Development of a novel instrument to measure the pulsatile movement of ocular tissues,” Exp. Eye Res. 91(1), 63–68 (2010).
[CrossRef] [PubMed]

Duncan, D. D.

Ford, T. N.

D. Lim, T. N. Ford, K. K. Chu, and J. Mertz, “Optically sectioned in vivo imaging with speckle illumination HiLo microscopy,” J. Biomed. Opt. 16(1), 016014 (2011).
[CrossRef] [PubMed]

S. Santos, K. K. Chu, D. Lim, N. Bozinovic, T. N. Ford, C. Hourtoule, A. C. Bartoo, S. K. Singh, and J. Mertz, “Optically sectioned fluorescence endomicroscopy with hybrid-illumination imaging through a flexible fiber bundle,” J. Biomed. Opt. 14(3), 030502 (2009).
[CrossRef] [PubMed]

Heintzmann, R.

Hourtoule, C.

S. Santos, K. K. Chu, D. Lim, N. Bozinovic, T. N. Ford, C. Hourtoule, A. C. Bartoo, S. K. Singh, and J. Mertz, “Optically sectioned fluorescence endomicroscopy with hybrid-illumination imaging through a flexible fiber bundle,” J. Biomed. Opt. 14(3), 030502 (2009).
[CrossRef] [PubMed]

Kennedy, T. E.

J. M. Bélisle, J. P. Correia, P. W. Wiseman, T. E. Kennedy, and S. Costantino, “Patterning protein concentration using laser-assisted adsorption by photobleaching, LAPAP,” Lab Chip 8(12), 2164–2167 (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]

Kirkpatrick, S. J.

Kunik, D.

J. M. Belisle, D. Kunik, and S. Costantino, “Rapid multicomponent optical protein patterning,” Lab Chip 9(24), 3580–3585 (2009).
[CrossRef] [PubMed]

Lesk, M. R.

K. Singh, C. Dion, M. R. Lesk, T. Ozaki, and S. Costantino, “Spectral-domain phase microscopy with improved sensitivity using two-dimensional detector arrays,” Rev. Sci. Instrum. 82(2), 023706 (2011).
[CrossRef] [PubMed]

K. Singh, C. Dion, S. Costantino, M. Wajszilber, M. R. Lesk, and T. Ozaki, “Development of a novel instrument to measure the pulsatile movement of ocular tissues,” Exp. Eye Res. 91(1), 63–68 (2010).
[CrossRef] [PubMed]

Lim, D.

D. Lim, T. N. Ford, K. K. Chu, and J. Mertz, “Optically sectioned in vivo imaging with speckle illumination HiLo microscopy,” J. Biomed. Opt. 16(1), 016014 (2011).
[CrossRef] [PubMed]

S. Santos, K. K. Chu, D. Lim, N. Bozinovic, T. N. Ford, C. Hourtoule, A. C. Bartoo, S. K. Singh, and J. Mertz, “Optically sectioned fluorescence endomicroscopy with hybrid-illumination imaging through a flexible fiber bundle,” J. Biomed. Opt. 14(3), 030502 (2009).
[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]

Mertz, J.

D. Lim, T. N. Ford, K. K. Chu, and J. Mertz, “Optically sectioned in vivo imaging with speckle illumination HiLo microscopy,” J. Biomed. Opt. 16(1), 016014 (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]

S. Santos, K. K. Chu, D. Lim, N. Bozinovic, T. N. Ford, C. Hourtoule, A. C. Bartoo, S. K. Singh, and J. Mertz, “Optically sectioned fluorescence endomicroscopy with hybrid-illumination imaging through a flexible fiber bundle,” J. Biomed. Opt. 14(3), 030502 (2009).
[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]

C. Ventalon, R. Heintzmann, and J. Mertz, “Dynamic speckle illumination microscopy with wavelet prefiltering,” Opt. Lett. 32(11), 1417–1419 (2007).
[CrossRef] [PubMed]

C. Ventalon and J. Mertz, “Quasi-confocal fluorescence sectioning with dynamic speckle illumination,” Opt. Lett. 30(24), 3350–3352 (2005).
[CrossRef] [PubMed]

Ozaki, T.

K. Singh, C. Dion, M. R. Lesk, T. Ozaki, and S. Costantino, “Spectral-domain phase microscopy with improved sensitivity using two-dimensional detector arrays,” Rev. Sci. Instrum. 82(2), 023706 (2011).
[CrossRef] [PubMed]

K. Singh, C. Dion, S. Costantino, M. Wajszilber, M. R. Lesk, and T. Ozaki, “Development of a novel instrument to measure the pulsatile movement of ocular tissues,” Exp. Eye Res. 91(1), 63–68 (2010).
[CrossRef] [PubMed]

Santos, S.

S. Santos, K. K. Chu, D. Lim, N. Bozinovic, T. N. Ford, C. Hourtoule, A. C. Bartoo, S. K. Singh, and J. Mertz, “Optically sectioned fluorescence endomicroscopy with hybrid-illumination imaging through a flexible fiber bundle,” J. Biomed. Opt. 14(3), 030502 (2009).
[CrossRef] [PubMed]

Singh, K.

K. Singh, C. Dion, M. R. Lesk, T. Ozaki, and S. Costantino, “Spectral-domain phase microscopy with improved sensitivity using two-dimensional detector arrays,” Rev. Sci. Instrum. 82(2), 023706 (2011).
[CrossRef] [PubMed]

K. Singh, C. Dion, S. Costantino, M. Wajszilber, M. R. Lesk, and T. Ozaki, “Development of a novel instrument to measure the pulsatile movement of ocular tissues,” Exp. Eye Res. 91(1), 63–68 (2010).
[CrossRef] [PubMed]

Singh, S. K.

S. Santos, K. K. Chu, D. Lim, N. Bozinovic, T. N. Ford, C. Hourtoule, A. C. Bartoo, S. K. Singh, and J. Mertz, “Optically sectioned fluorescence endomicroscopy with hybrid-illumination imaging through a flexible fiber bundle,” J. Biomed. Opt. 14(3), 030502 (2009).
[CrossRef] [PubMed]

Ventalon, C.

Wajszilber, M.

K. Singh, C. Dion, S. Costantino, M. Wajszilber, M. R. Lesk, and T. Ozaki, “Development of a novel instrument to measure the pulsatile movement of ocular tissues,” Exp. Eye Res. 91(1), 63–68 (2010).
[CrossRef] [PubMed]

Wang, R. K.

Wiseman, P. W.

J. M. Bélisle, J. P. Correia, P. W. Wiseman, T. E. Kennedy, and S. Costantino, “Patterning protein concentration using laser-assisted adsorption by photobleaching, LAPAP,” Lab Chip 8(12), 2164–2167 (2008).
[CrossRef] [PubMed]

Exp. Eye Res.

K. Singh, C. Dion, S. Costantino, M. Wajszilber, M. R. Lesk, and T. Ozaki, “Development of a novel instrument to measure the pulsatile movement of ocular tissues,” Exp. Eye Res. 91(1), 63–68 (2010).
[CrossRef] [PubMed]

J. Biomed. Opt.

D. Lim, T. N. Ford, K. K. Chu, and J. Mertz, “Optically sectioned in vivo imaging with speckle illumination HiLo microscopy,” J. Biomed. Opt. 16(1), 016014 (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]

S. Santos, K. K. Chu, D. Lim, N. Bozinovic, T. N. Ford, C. Hourtoule, A. C. Bartoo, S. K. Singh, and J. Mertz, “Optically sectioned fluorescence endomicroscopy with hybrid-illumination imaging through a flexible fiber bundle,” J. Biomed. Opt. 14(3), 030502 (2009).
[CrossRef] [PubMed]

J. Opt. Soc. Am. A

Lab Chip

J. M. Bélisle, J. P. Correia, P. W. Wiseman, T. E. Kennedy, and S. Costantino, “Patterning protein concentration using laser-assisted adsorption by photobleaching, LAPAP,” Lab Chip 8(12), 2164–2167 (2008).
[CrossRef] [PubMed]

J. M. Belisle, D. Kunik, and S. Costantino, “Rapid multicomponent optical protein patterning,” Lab Chip 9(24), 3580–3585 (2009).
[CrossRef] [PubMed]

Opt. Lett.

Rev. Sci. Instrum.

K. Singh, C. Dion, M. R. Lesk, T. Ozaki, and S. Costantino, “Spectral-domain phase microscopy with improved sensitivity using two-dimensional detector arrays,” Rev. Sci. Instrum. 82(2), 023706 (2011).
[CrossRef] [PubMed]

Other

J. W. Goodman, Introduction to Fourier Optics (2nd Ed., McGraw-Hill, 1996), Chap. 6.

J. Goodman, Speckle phenomena in optics (Roberts & Company, 2007).

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Figures (4)

Fig. 1
Fig. 1

(a) Scheme of HiLo microscope. A laser diode at 473nm (Laserglow, Toronto, CA) illuminates a ground glass diffuser. Two lenses L1 and L2 of 100mm and 300mm focal lengths are used to illuminate the back-focal-plane of an objective in an inverted fluorescence microscope. A CCD camera (Retiga 2000R) was used for imaging. (b) Output roughness as a function of |κc |/|κmax | (bottom axis). The parameter Λ/Δg, in the top axis, roughly represents the square root of the number of speckle grains that fits in each sampling window.

Fig. 2
Fig. 2

20×20μm2 planar object imaged in-focus with a 60X NA=1.35 objective. (a) HiLo Images obtained using sampling windows with Λ/Δg of 1.3, 4 and 8 as indicated. In the experiment Δg = 0.46μm. The image obtained with uniform illumination is also shown. (b) Intensity profiles of images in (a) traced over the vertical dashed line P. (c) Roughness of the experimental image for various values of Λ/Δg (upper scale). The corresponding values of |κc |/|κmax | are shown in the bottom scale. The dashed red line represents the roughness of iu .

Fig. 3
Fig. 3

(a) Numerical simulation of speckle produced with λ = 488 nm and NA=0.4 in 3 axial planes. (b) Contrast of speckle illuminated sample (c) at various axial widths. Each position of the sample was illuminated with objectives of 3 different NA. The discrete markers correspond to the experimental measurements while the lines represent numerical simulations. Estimated experimental uncertainties are smaller than the markers size. Note that experiment and numerical simulation results are plotted with different scales. (c) Scheme of wedge-shaped glass chamber filled with fluorescent solution. The sample was imaged in regions I and II. (d) Scheme depicting the position of the object plane in a region of sample in (c).

Fig. 4
Fig. 4

Images of a wedge-shaped chamber filled with dye solution in two regions: I is a thin region of main thickness ~40μm and II is a thicker region of main thickness ~160μm. Each panel is organized as follows: In the left column the images is , iu , Cs and iHiLo are shown while the three bottom plots in the right column show the corresponding average horizontal profiles (notice the transversal coordinate is displayed in the top scale and the bottom scale indicates thickness of the sample). The profiles of iHiLo including band-pass prefiltering with σw=κmax , κmax /2 and κmax /3 are displayed (with independent normalization factors) along with iHiLo obtained without prefiltering. The top graph of each right column shows the linear fit of iu vs. the thickness T demonstrating the linearity of the CCD in the measured range. iHiLo without band-pass filter was computed with Λ=11 pixels, and |κc|=1/2Λ. The objective is a 10X 0.4NA, and the CCD pixel size is 7.4 μm.

Equations (6)

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C S ( ρ ) = σ S Λ i S Λ ,
i s u ( ρ ) = C S ( ρ ) × i u ( ρ ) ,
κ c 1 2 Λ .
W ( κ ) = exp ( | κ | 2 2 σ w 2 ) exp ( | κ | 2 σ w 2 )
Λ Δ g 4.88 ~ κ max κ c .
R ( C S ) = σ ( C S ) C S × 100

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