Abstract

We present a multi-contrast microscope based on color-coded illumination and computation. A programmable three-color light-emitting diode (LED) array illuminates a specimen, in which each color corresponds to a different illumination angle. A single color image sensor records light transmitted through the specimen, and images at each color channel are then separated and utilized to obtain bright-field, dark-field, and differential phase contrast (DPC) images simultaneously. Quantitative phase imaging is also achieved based on DPC images acquired with two different LED illumination patterns. The multi-contrast and quantitative phase imaging capabilities of our method are demonstrated by presenting images of various transparent biological samples.

© 2015 Optical Society of America

Full Article  |  PDF Article
OSA Recommended Articles
Single-exposure quantitative phase imaging in color-coded LED microscopy

Wonchan Lee, Daeseong Jung, Suho Ryu, and Chulmin Joo
Opt. Express 25(7) 8398-8411 (2017)

Efficient quantitative phase microscopy using programmable annular LED illumination

Jiaji Li, Qian Chen, Jialin Zhang, Yan Zhang, Linpeng Lu, and Chao Zuo
Biomed. Opt. Express 8(10) 4687-4705 (2017)

Quantitative differential phase contrast imaging in an LED array microscope

Lei Tian and Laura Waller
Opt. Express 23(9) 11394-11403 (2015)

References

  • View by:
  • |
  • |
  • |

  1. J. Mertz, Introduction to Optical Microscopy (Roberts, 2010).
  2. K. Summers and M. W. Kirschner, “Characteristics of the polar assembly and disassembly of microtubules observed in vitro by darkfield light microscopy,” J. Cell Biol. 83(1), 205–217 (1979).
    [Crossref] [PubMed]
  3. S. Kudo, Y. Magariyama, and S. Aizawa, “Abrupt changes in flagellar rotation observed by laser dark-field microscopy,” Nature 346(6285), 677–680 (1990).
    [Crossref] [PubMed]
  4. F. Zernike, “How I discovered phase contrast,” Science 121(3141), 345–349 (1955).
    [Crossref] [PubMed]
  5. C. Burch and J. Stock, “Phase-contrast microscopy,” J. Sci. Instrum. 19(5), 71–75 (1942).
    [Crossref]
  6. G. Nomarski, “Differential microinterferometer with polarized light,” Phys. Radium 16, 9–13 (1955).
  7. E. D. Salmon and P. Tran, “High-resolution video-enhanced differential interference contrast light microscopy,” Methods Cell Biol. 72, 289–318 (2003).
    [Crossref] [PubMed]
  8. G. Zheng, C. Kolner, and C. Yang, “Microscopy refocusing and dark-field imaging by using a simple LED array,” Opt. Lett. 36(20), 3987–3989 (2011).
    [Crossref] [PubMed]
  9. L. Tian, J. Wang, and L. Waller, “3D differential phase-contrast microscopy with computational illumination using an LED array,” Opt. Lett. 39(5), 1326–1329 (2014).
    [Crossref] [PubMed]
  10. L. Tian and L. Waller, “Quantitative differential phase contrast imaging in an LED array microscope,” Opt. Express 23(9), 11394–11403 (2015).
    [Crossref] [PubMed]
  11. Z. Liu, L. Tian, S. Liu, and L. Waller, “Real-time brightfield, darkfield, and phase contrast imaging in a light-emitting diode array microscope,” J. Biomed. Opt. 19(10), 106002 (2014).
    [Crossref] [PubMed]
  12. W. B. Amos, S. Reichelt, D. M. Cattermole, and J. Laufer, “Re-evaluation of differential phase contrast (DPC) in a scanning laser microscope using a split detector as an alternative to differential interference contrast (DIC) optics,” J. Microsc. 210(2), 166–175 (2003).
    [Crossref] [PubMed]
  13. D. Hamilton and C. Sheppard, “Differential phase contrast in scanning optical microscopy,” J. Microsc. 133(1), 27–39 (1984).
    [Crossref]
  14. Y. Kawata, R. Juškaitis, T. Tanaka, T. Wilson, and S. Kawata, “Differential phase-contrast microscope with a split detector for the readout system of a multilayered optical memory,” Appl. Opt. 35(14), 2466–2470 (1996).
    [Crossref] [PubMed]
  15. T. N. Ford, K. K. Chu, and J. Mertz, “Phase-gradient microscopy in thick tissue with oblique back-illumination,” Nat. Methods 9(12), 1195–1197 (2012).
    [Crossref] [PubMed]
  16. B. Kachar, “Asymmetric illumination contrast: a method of image formation for video light microscopy,” Science 227(4688), 766–768 (1985).
    [Crossref] [PubMed]
  17. M. R. Arnison, K. G. Larkin, C. J. Sheppard, N. I. Smith, and C. J. Cogswell, “Linear phase imaging using differential interference contrast microscopy,” J. Microsc. 214(1), 7–12 (2004).
    [Crossref] [PubMed]
  18. A. B. Parthasarathy, K. K. Chu, T. N. Ford, and J. Mertz, “Quantitative phase imaging using a partitioned detection aperture,” Opt. Lett. 37(19), 4062–4064 (2012).
    [Crossref] [PubMed]
  19. R. Barankov and J. Mertz, “Single-exposure surface profilometry using partitioned aperture wavefront imaging,” Opt. Lett. 38(19), 3961–3964 (2013).
    [Crossref] [PubMed]
  20. D. Hamilton, C. J. Sheppard, and T. Wilson, “Improved imaging of phase gradients in scanning optical microscopy,” J. Microsc. 135(3), 275–286 (1984).
    [Crossref]
  21. S. B. Mehta and C. J. Sheppard, “Quantitative phase-gradient imaging at high resolution with asymmetric illumination-based differential phase contrast,” Opt. Lett. 34(13), 1924–1926 (2009).
    [Crossref] [PubMed]
  22. K. Guo, Z. Bian, S. Dong, P. Nanda, Y. M. Wang, and G. Zheng, “Microscopy illumination engineering using a low-cost liquid crystal display,” Biomed. Opt. Express 6(2), 574–579 (2015).
    [Crossref] [PubMed]
  23. L. Tian and L. Waller, “3D intensity and phase imaging from light field measurements in an LED array microscope,” Optica 2(2), 104–111 (2015).
    [Crossref]
  24. G. Zheng, R. Horstmeyer, and C. Yang, “Wide-field, high-resolution Fourier ptychographic microscopy,” Nat. Photonics 7(9), 739–745 (2013).
    [Crossref] [PubMed]
  25. L. Tian, X. Li, K. Ramchandran, and L. Waller, “Multiplexed coded illumination for Fourier Ptychography with an LED array microscope,” Biomed. Opt. Express 5(7), 2376–2389 (2014).
    [Crossref] [PubMed]

2015 (3)

2014 (3)

2013 (2)

G. Zheng, R. Horstmeyer, and C. Yang, “Wide-field, high-resolution Fourier ptychographic microscopy,” Nat. Photonics 7(9), 739–745 (2013).
[Crossref] [PubMed]

R. Barankov and J. Mertz, “Single-exposure surface profilometry using partitioned aperture wavefront imaging,” Opt. Lett. 38(19), 3961–3964 (2013).
[Crossref] [PubMed]

2012 (2)

A. B. Parthasarathy, K. K. Chu, T. N. Ford, and J. Mertz, “Quantitative phase imaging using a partitioned detection aperture,” Opt. Lett. 37(19), 4062–4064 (2012).
[Crossref] [PubMed]

T. N. Ford, K. K. Chu, and J. Mertz, “Phase-gradient microscopy in thick tissue with oblique back-illumination,” Nat. Methods 9(12), 1195–1197 (2012).
[Crossref] [PubMed]

2011 (1)

2009 (1)

2004 (1)

M. R. Arnison, K. G. Larkin, C. J. Sheppard, N. I. Smith, and C. J. Cogswell, “Linear phase imaging using differential interference contrast microscopy,” J. Microsc. 214(1), 7–12 (2004).
[Crossref] [PubMed]

2003 (2)

E. D. Salmon and P. Tran, “High-resolution video-enhanced differential interference contrast light microscopy,” Methods Cell Biol. 72, 289–318 (2003).
[Crossref] [PubMed]

W. B. Amos, S. Reichelt, D. M. Cattermole, and J. Laufer, “Re-evaluation of differential phase contrast (DPC) in a scanning laser microscope using a split detector as an alternative to differential interference contrast (DIC) optics,” J. Microsc. 210(2), 166–175 (2003).
[Crossref] [PubMed]

1996 (1)

1990 (1)

S. Kudo, Y. Magariyama, and S. Aizawa, “Abrupt changes in flagellar rotation observed by laser dark-field microscopy,” Nature 346(6285), 677–680 (1990).
[Crossref] [PubMed]

1985 (1)

B. Kachar, “Asymmetric illumination contrast: a method of image formation for video light microscopy,” Science 227(4688), 766–768 (1985).
[Crossref] [PubMed]

1984 (2)

D. Hamilton and C. Sheppard, “Differential phase contrast in scanning optical microscopy,” J. Microsc. 133(1), 27–39 (1984).
[Crossref]

D. Hamilton, C. J. Sheppard, and T. Wilson, “Improved imaging of phase gradients in scanning optical microscopy,” J. Microsc. 135(3), 275–286 (1984).
[Crossref]

1979 (1)

K. Summers and M. W. Kirschner, “Characteristics of the polar assembly and disassembly of microtubules observed in vitro by darkfield light microscopy,” J. Cell Biol. 83(1), 205–217 (1979).
[Crossref] [PubMed]

1955 (2)

G. Nomarski, “Differential microinterferometer with polarized light,” Phys. Radium 16, 9–13 (1955).

F. Zernike, “How I discovered phase contrast,” Science 121(3141), 345–349 (1955).
[Crossref] [PubMed]

1942 (1)

C. Burch and J. Stock, “Phase-contrast microscopy,” J. Sci. Instrum. 19(5), 71–75 (1942).
[Crossref]

Aizawa, S.

S. Kudo, Y. Magariyama, and S. Aizawa, “Abrupt changes in flagellar rotation observed by laser dark-field microscopy,” Nature 346(6285), 677–680 (1990).
[Crossref] [PubMed]

Amos, W. B.

W. B. Amos, S. Reichelt, D. M. Cattermole, and J. Laufer, “Re-evaluation of differential phase contrast (DPC) in a scanning laser microscope using a split detector as an alternative to differential interference contrast (DIC) optics,” J. Microsc. 210(2), 166–175 (2003).
[Crossref] [PubMed]

Arnison, M. R.

M. R. Arnison, K. G. Larkin, C. J. Sheppard, N. I. Smith, and C. J. Cogswell, “Linear phase imaging using differential interference contrast microscopy,” J. Microsc. 214(1), 7–12 (2004).
[Crossref] [PubMed]

Barankov, R.

Bian, Z.

Burch, C.

C. Burch and J. Stock, “Phase-contrast microscopy,” J. Sci. Instrum. 19(5), 71–75 (1942).
[Crossref]

Cattermole, D. M.

W. B. Amos, S. Reichelt, D. M. Cattermole, and J. Laufer, “Re-evaluation of differential phase contrast (DPC) in a scanning laser microscope using a split detector as an alternative to differential interference contrast (DIC) optics,” J. Microsc. 210(2), 166–175 (2003).
[Crossref] [PubMed]

Chu, K. K.

A. B. Parthasarathy, K. K. Chu, T. N. Ford, and J. Mertz, “Quantitative phase imaging using a partitioned detection aperture,” Opt. Lett. 37(19), 4062–4064 (2012).
[Crossref] [PubMed]

T. N. Ford, K. K. Chu, and J. Mertz, “Phase-gradient microscopy in thick tissue with oblique back-illumination,” Nat. Methods 9(12), 1195–1197 (2012).
[Crossref] [PubMed]

Cogswell, C. J.

M. R. Arnison, K. G. Larkin, C. J. Sheppard, N. I. Smith, and C. J. Cogswell, “Linear phase imaging using differential interference contrast microscopy,” J. Microsc. 214(1), 7–12 (2004).
[Crossref] [PubMed]

Dong, S.

Ford, T. N.

A. B. Parthasarathy, K. K. Chu, T. N. Ford, and J. Mertz, “Quantitative phase imaging using a partitioned detection aperture,” Opt. Lett. 37(19), 4062–4064 (2012).
[Crossref] [PubMed]

T. N. Ford, K. K. Chu, and J. Mertz, “Phase-gradient microscopy in thick tissue with oblique back-illumination,” Nat. Methods 9(12), 1195–1197 (2012).
[Crossref] [PubMed]

Guo, K.

Hamilton, D.

D. Hamilton, C. J. Sheppard, and T. Wilson, “Improved imaging of phase gradients in scanning optical microscopy,” J. Microsc. 135(3), 275–286 (1984).
[Crossref]

D. Hamilton and C. Sheppard, “Differential phase contrast in scanning optical microscopy,” J. Microsc. 133(1), 27–39 (1984).
[Crossref]

Horstmeyer, R.

G. Zheng, R. Horstmeyer, and C. Yang, “Wide-field, high-resolution Fourier ptychographic microscopy,” Nat. Photonics 7(9), 739–745 (2013).
[Crossref] [PubMed]

Juškaitis, R.

Kachar, B.

B. Kachar, “Asymmetric illumination contrast: a method of image formation for video light microscopy,” Science 227(4688), 766–768 (1985).
[Crossref] [PubMed]

Kawata, S.

Kawata, Y.

Kirschner, M. W.

K. Summers and M. W. Kirschner, “Characteristics of the polar assembly and disassembly of microtubules observed in vitro by darkfield light microscopy,” J. Cell Biol. 83(1), 205–217 (1979).
[Crossref] [PubMed]

Kolner, C.

Kudo, S.

S. Kudo, Y. Magariyama, and S. Aizawa, “Abrupt changes in flagellar rotation observed by laser dark-field microscopy,” Nature 346(6285), 677–680 (1990).
[Crossref] [PubMed]

Larkin, K. G.

M. R. Arnison, K. G. Larkin, C. J. Sheppard, N. I. Smith, and C. J. Cogswell, “Linear phase imaging using differential interference contrast microscopy,” J. Microsc. 214(1), 7–12 (2004).
[Crossref] [PubMed]

Laufer, J.

W. B. Amos, S. Reichelt, D. M. Cattermole, and J. Laufer, “Re-evaluation of differential phase contrast (DPC) in a scanning laser microscope using a split detector as an alternative to differential interference contrast (DIC) optics,” J. Microsc. 210(2), 166–175 (2003).
[Crossref] [PubMed]

Li, X.

Liu, S.

Z. Liu, L. Tian, S. Liu, and L. Waller, “Real-time brightfield, darkfield, and phase contrast imaging in a light-emitting diode array microscope,” J. Biomed. Opt. 19(10), 106002 (2014).
[Crossref] [PubMed]

Liu, Z.

Z. Liu, L. Tian, S. Liu, and L. Waller, “Real-time brightfield, darkfield, and phase contrast imaging in a light-emitting diode array microscope,” J. Biomed. Opt. 19(10), 106002 (2014).
[Crossref] [PubMed]

Magariyama, Y.

S. Kudo, Y. Magariyama, and S. Aizawa, “Abrupt changes in flagellar rotation observed by laser dark-field microscopy,” Nature 346(6285), 677–680 (1990).
[Crossref] [PubMed]

Mehta, S. B.

Mertz, J.

Nanda, P.

Nomarski, G.

G. Nomarski, “Differential microinterferometer with polarized light,” Phys. Radium 16, 9–13 (1955).

Parthasarathy, A. B.

Ramchandran, K.

Reichelt, S.

W. B. Amos, S. Reichelt, D. M. Cattermole, and J. Laufer, “Re-evaluation of differential phase contrast (DPC) in a scanning laser microscope using a split detector as an alternative to differential interference contrast (DIC) optics,” J. Microsc. 210(2), 166–175 (2003).
[Crossref] [PubMed]

Salmon, E. D.

E. D. Salmon and P. Tran, “High-resolution video-enhanced differential interference contrast light microscopy,” Methods Cell Biol. 72, 289–318 (2003).
[Crossref] [PubMed]

Sheppard, C.

D. Hamilton and C. Sheppard, “Differential phase contrast in scanning optical microscopy,” J. Microsc. 133(1), 27–39 (1984).
[Crossref]

Sheppard, C. J.

S. B. Mehta and C. J. Sheppard, “Quantitative phase-gradient imaging at high resolution with asymmetric illumination-based differential phase contrast,” Opt. Lett. 34(13), 1924–1926 (2009).
[Crossref] [PubMed]

M. R. Arnison, K. G. Larkin, C. J. Sheppard, N. I. Smith, and C. J. Cogswell, “Linear phase imaging using differential interference contrast microscopy,” J. Microsc. 214(1), 7–12 (2004).
[Crossref] [PubMed]

D. Hamilton, C. J. Sheppard, and T. Wilson, “Improved imaging of phase gradients in scanning optical microscopy,” J. Microsc. 135(3), 275–286 (1984).
[Crossref]

Smith, N. I.

M. R. Arnison, K. G. Larkin, C. J. Sheppard, N. I. Smith, and C. J. Cogswell, “Linear phase imaging using differential interference contrast microscopy,” J. Microsc. 214(1), 7–12 (2004).
[Crossref] [PubMed]

Stock, J.

C. Burch and J. Stock, “Phase-contrast microscopy,” J. Sci. Instrum. 19(5), 71–75 (1942).
[Crossref]

Summers, K.

K. Summers and M. W. Kirschner, “Characteristics of the polar assembly and disassembly of microtubules observed in vitro by darkfield light microscopy,” J. Cell Biol. 83(1), 205–217 (1979).
[Crossref] [PubMed]

Tanaka, T.

Tian, L.

Tran, P.

E. D. Salmon and P. Tran, “High-resolution video-enhanced differential interference contrast light microscopy,” Methods Cell Biol. 72, 289–318 (2003).
[Crossref] [PubMed]

Waller, L.

Wang, J.

Wang, Y. M.

Wilson, T.

Yang, C.

G. Zheng, R. Horstmeyer, and C. Yang, “Wide-field, high-resolution Fourier ptychographic microscopy,” Nat. Photonics 7(9), 739–745 (2013).
[Crossref] [PubMed]

G. Zheng, C. Kolner, and C. Yang, “Microscopy refocusing and dark-field imaging by using a simple LED array,” Opt. Lett. 36(20), 3987–3989 (2011).
[Crossref] [PubMed]

Zernike, F.

F. Zernike, “How I discovered phase contrast,” Science 121(3141), 345–349 (1955).
[Crossref] [PubMed]

Zheng, G.

Appl. Opt. (1)

Biomed. Opt. Express (2)

J. Biomed. Opt. (1)

Z. Liu, L. Tian, S. Liu, and L. Waller, “Real-time brightfield, darkfield, and phase contrast imaging in a light-emitting diode array microscope,” J. Biomed. Opt. 19(10), 106002 (2014).
[Crossref] [PubMed]

J. Cell Biol. (1)

K. Summers and M. W. Kirschner, “Characteristics of the polar assembly and disassembly of microtubules observed in vitro by darkfield light microscopy,” J. Cell Biol. 83(1), 205–217 (1979).
[Crossref] [PubMed]

J. Microsc. (4)

W. B. Amos, S. Reichelt, D. M. Cattermole, and J. Laufer, “Re-evaluation of differential phase contrast (DPC) in a scanning laser microscope using a split detector as an alternative to differential interference contrast (DIC) optics,” J. Microsc. 210(2), 166–175 (2003).
[Crossref] [PubMed]

D. Hamilton and C. Sheppard, “Differential phase contrast in scanning optical microscopy,” J. Microsc. 133(1), 27–39 (1984).
[Crossref]

M. R. Arnison, K. G. Larkin, C. J. Sheppard, N. I. Smith, and C. J. Cogswell, “Linear phase imaging using differential interference contrast microscopy,” J. Microsc. 214(1), 7–12 (2004).
[Crossref] [PubMed]

D. Hamilton, C. J. Sheppard, and T. Wilson, “Improved imaging of phase gradients in scanning optical microscopy,” J. Microsc. 135(3), 275–286 (1984).
[Crossref]

J. Sci. Instrum. (1)

C. Burch and J. Stock, “Phase-contrast microscopy,” J. Sci. Instrum. 19(5), 71–75 (1942).
[Crossref]

Methods Cell Biol. (1)

E. D. Salmon and P. Tran, “High-resolution video-enhanced differential interference contrast light microscopy,” Methods Cell Biol. 72, 289–318 (2003).
[Crossref] [PubMed]

Nat. Methods (1)

T. N. Ford, K. K. Chu, and J. Mertz, “Phase-gradient microscopy in thick tissue with oblique back-illumination,” Nat. Methods 9(12), 1195–1197 (2012).
[Crossref] [PubMed]

Nat. Photonics (1)

G. Zheng, R. Horstmeyer, and C. Yang, “Wide-field, high-resolution Fourier ptychographic microscopy,” Nat. Photonics 7(9), 739–745 (2013).
[Crossref] [PubMed]

Nature (1)

S. Kudo, Y. Magariyama, and S. Aizawa, “Abrupt changes in flagellar rotation observed by laser dark-field microscopy,” Nature 346(6285), 677–680 (1990).
[Crossref] [PubMed]

Opt. Express (1)

Opt. Lett. (5)

Optica (1)

Phys. Radium (1)

G. Nomarski, “Differential microinterferometer with polarized light,” Phys. Radium 16, 9–13 (1955).

Science (2)

F. Zernike, “How I discovered phase contrast,” Science 121(3141), 345–349 (1955).
[Crossref] [PubMed]

B. Kachar, “Asymmetric illumination contrast: a method of image formation for video light microscopy,” Science 227(4688), 766–768 (1985).
[Crossref] [PubMed]

Other (1)

J. Mertz, Introduction to Optical Microscopy (Roberts, 2010).

Supplementary Material (1)

NameDescription
» Visualization 1: MOV (2132 KB)      Supplementary movie for Figure 5

Cited By

OSA participates in Crossref's Cited-By Linking service. Citing articles from OSA journals and other participating publishers are listed here.

Alert me when this article is cited.


Figures (8)

Fig. 1
Fig. 1 (a) cLEDscope schematic. A programmable color LED array is located at the Fourier plane of the specimen (S). The LED array illuminates the specimen, and a color image sensor records light transmitted through the specimen. Shown in the left of (b) are the patterns employed in cLEDscope imaging. The recorded color image for a given LED pattern is separated into images in red, blue, and green colors, which are then used to compute BF (IBF), DF (IDF), and DPC (IDPC) images. Representative multi-contrast images of a fish scale are shown on the right of (b). OBJ: Objective, NAOBJ: objective numerical aperture, TL: tube lens.
Fig. 2
Fig. 2 In cLEDscope, symmetric distribution of R and B patterns is produced at the pupil plane for a sample with no phase variation (a). If the sample exhibits spatially varying phase distribution, the distribution of R and B patterns will shift by an amount proportional to the phase gradient at the pupil plane.
Fig. 3
Fig. 3 Computed PGTFs for different illumination patterns. u ¯ and v ¯ denote spatial frequency normalized with N A OBj /λ in the x and y directions, respectively.
Fig. 4
Fig. 4 Single-shot multi-contrast images of onion cells (a-c) and scomber fish scales (d-f). Scale bar represents 100 μm.
Fig. 5
Fig. 5 Representative cLEDscope images of C. elegans acquired at 1, 2, and 3 seconds. Images were recorded at frame rate of 32 fps. Scale bar represents 100 μm.
Fig. 6
Fig. 6 (a) Polystyrene microspheres immersed in index matching liquid were imaged to evaluate phase measurement accuracy. (b) Quantitative phase image of the beads. A magnified view of the region indicated by the rectangle is shown in the inset. Scale bar denotes 50 μm. (c) Measured phase distribution along the dashed line in the inset of (b).
Fig. 7
Fig. 7 (a-b) DPC images along x and y directions. Images were obtained with two LED patterns in Fig. 1. (c) Quantitative phase image of the cells obtained with complex Fourier integration with two images in (a-b). (d) 3D representation of quantitative phase image of human epithelial cheek cells. The scalebar represents 50 μm.
Fig. 8
Fig. 8 Comparison of the phase measurements based on monochromatic and color-coded LED illumination. (a) and (b) shows the phase images obtained with monochromatic and color-coded LED illuminations, respectively. Shown in the top of each image is the LED illumination pattern utilized for image acquisition. Difference between the two measurements is presented in (c). The scalebar denotes 50 μm.

Equations (4)

Equations on this page are rendered with MathJax. Learn more.

I BF = I R + I B
I DF = I G
I DPC = I R I B I R + I B
C(u,v)= | P O (ξ,η) | 2 | P S (ξu,ηv) | 2 dξdη

Metrics