Abstract

We present a multichannel fluorescence microscopy technique for high throughput imaging applications. A microlens array with over 140,000 elements is used to image centimeter-scale samples at up to 18.1 megapixels per second. Large field-of-view multichannel fluorescent imaging is demonstrated in both sequential and parallel geometries. The extended dynamic range of this approach is also discussed.

© 2014 Optical Society of America

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  1. R. Pepperkok and J. Ellenberg, “High-throughput fluorescence microscopy for systems biology,” Nat. Rev. Mol. Cell Biol. 7(9), 690–696 (2006).
    [CrossRef] [PubMed]
  2. P. Lang, K. Yeow, A. Nichols, and A. Scheer, “Cellular imaging in drug discovery,” Nat. Rev. Drug Discov. 5(4), 343–356 (2006).
    [CrossRef] [PubMed]
  3. M. Bickle, “The beautiful cell: high-content screening in drug discovery,” Anal. Bioanal. Chem. 398(1), 219–226 (2010).
    [CrossRef] [PubMed]
  4. Olympus ScanR specifications website, http://www.olympus-europa.com/microscopy/en/microscopy/components/component_details/component_detail_21320.jsp . Accessed 22 January 2014.
  5. Molecular Devices ImageXpress Micro XLS specifications website, http://www.moleculardevices.com/Products/Instruments/High-Content-Screening/ImageXpress-Micro.html . Accessed 28 January 2014.
  6. J. Wu, X. Cui, G. Zheng, Y. M. Wang, L. M. Lee, and C. Yang, “Wide field-of-view microscope based on holographic focus grid illumination,” Opt. Lett. 35(13), 2188–2190 (2010).
    [CrossRef] [PubMed]
  7. S. Pang, C. Han, M. Kato, P. W. Sternberg, and C. Yang, “Wide and scalable field-of-view Talbot-grid-based fluorescence microscopy,” Opt. Lett. 37(23), 5018–5020 (2012).
    [CrossRef] [PubMed]
  8. S. Pang, C. Han, J. Erath, A. Rodriguez, and C. Yang, “Wide field-of-view Talbot grid-based microscopy for multicolor fluorescence imaging,” Opt. Express 21(12), 14555–14565 (2013).
    [CrossRef] [PubMed]
  9. S. A. Arpali, C. Arpali, A. F. Coskun, H. H. Chiang, and A. Ozcan, “High-throughput screening of large volumes of whole blood using structured illumination and fluorescent on-chip imaging,” Lab Chip 12(23), 4968–4971 (2012).
    [CrossRef] [PubMed]
  10. B. Hulsken, D. Vossen, and S. Stallinga, “High NA diffractive array illuminators and application in a multi-spot scanning microscope,” J. Eur. Opt. Soc. Rapid Publ. 7, 12026 (2012).
    [CrossRef]
  11. A. Orth and K. B. Crozier, “Microscopy with microlens arrays: high throughput, high resolution and light-field imaging,” Opt. Express 20(12), 13522–13531 (2012).
    [CrossRef] [PubMed]
  12. A. Orth and K. B. Crozier, “Gigapixel fluorescence microscopy with a water immersion microlens array,” Opt. Express 21(2), 2361–2368 (2013).
    [CrossRef] [PubMed]
  13. J. B. Pawley, Handbook of Biological Confocal Microscopy, 3rd ed. (Springer, 2006).
  14. S. Preibisch, S. Saalfeld, and P. Tomancak, “Globally optimal stitching of tiled 3D microscopic image acquisitions,” Bioinformatics 25(11), 1463–1465 (2009).
    [CrossRef] [PubMed]
  15. F. T. O’Neill and J. T. Sheridan, “Photoresist reflow method of microlens production Part II: Analytic models,” Optik 113(9), 405–420 (2002).
    [CrossRef]
  16. J. W. Goodman, Introduction to Fourier optics (McGraw-Hill International Editions, 1996), Chap. 2.
  17. D. Cai, K. B. Cohen, T. Luo, J. W. Lichtman, and J. R. Sanes, “Improved tools for the Brainbow toolbox,” Nat. Methods 10(6), 540–547 (2013).
    [CrossRef]

2013 (3)

2012 (4)

S. Pang, C. Han, M. Kato, P. W. Sternberg, and C. Yang, “Wide and scalable field-of-view Talbot-grid-based fluorescence microscopy,” Opt. Lett. 37(23), 5018–5020 (2012).
[CrossRef] [PubMed]

S. A. Arpali, C. Arpali, A. F. Coskun, H. H. Chiang, and A. Ozcan, “High-throughput screening of large volumes of whole blood using structured illumination and fluorescent on-chip imaging,” Lab Chip 12(23), 4968–4971 (2012).
[CrossRef] [PubMed]

B. Hulsken, D. Vossen, and S. Stallinga, “High NA diffractive array illuminators and application in a multi-spot scanning microscope,” J. Eur. Opt. Soc. Rapid Publ. 7, 12026 (2012).
[CrossRef]

A. Orth and K. B. Crozier, “Microscopy with microlens arrays: high throughput, high resolution and light-field imaging,” Opt. Express 20(12), 13522–13531 (2012).
[CrossRef] [PubMed]

2010 (2)

2009 (1)

S. Preibisch, S. Saalfeld, and P. Tomancak, “Globally optimal stitching of tiled 3D microscopic image acquisitions,” Bioinformatics 25(11), 1463–1465 (2009).
[CrossRef] [PubMed]

2006 (2)

R. Pepperkok and J. Ellenberg, “High-throughput fluorescence microscopy for systems biology,” Nat. Rev. Mol. Cell Biol. 7(9), 690–696 (2006).
[CrossRef] [PubMed]

P. Lang, K. Yeow, A. Nichols, and A. Scheer, “Cellular imaging in drug discovery,” Nat. Rev. Drug Discov. 5(4), 343–356 (2006).
[CrossRef] [PubMed]

2002 (1)

F. T. O’Neill and J. T. Sheridan, “Photoresist reflow method of microlens production Part II: Analytic models,” Optik 113(9), 405–420 (2002).
[CrossRef]

Arpali, C.

S. A. Arpali, C. Arpali, A. F. Coskun, H. H. Chiang, and A. Ozcan, “High-throughput screening of large volumes of whole blood using structured illumination and fluorescent on-chip imaging,” Lab Chip 12(23), 4968–4971 (2012).
[CrossRef] [PubMed]

Arpali, S. A.

S. A. Arpali, C. Arpali, A. F. Coskun, H. H. Chiang, and A. Ozcan, “High-throughput screening of large volumes of whole blood using structured illumination and fluorescent on-chip imaging,” Lab Chip 12(23), 4968–4971 (2012).
[CrossRef] [PubMed]

Bickle, M.

M. Bickle, “The beautiful cell: high-content screening in drug discovery,” Anal. Bioanal. Chem. 398(1), 219–226 (2010).
[CrossRef] [PubMed]

Cai, D.

D. Cai, K. B. Cohen, T. Luo, J. W. Lichtman, and J. R. Sanes, “Improved tools for the Brainbow toolbox,” Nat. Methods 10(6), 540–547 (2013).
[CrossRef]

Chiang, H. H.

S. A. Arpali, C. Arpali, A. F. Coskun, H. H. Chiang, and A. Ozcan, “High-throughput screening of large volumes of whole blood using structured illumination and fluorescent on-chip imaging,” Lab Chip 12(23), 4968–4971 (2012).
[CrossRef] [PubMed]

Cohen, K. B.

D. Cai, K. B. Cohen, T. Luo, J. W. Lichtman, and J. R. Sanes, “Improved tools for the Brainbow toolbox,” Nat. Methods 10(6), 540–547 (2013).
[CrossRef]

Coskun, A. F.

S. A. Arpali, C. Arpali, A. F. Coskun, H. H. Chiang, and A. Ozcan, “High-throughput screening of large volumes of whole blood using structured illumination and fluorescent on-chip imaging,” Lab Chip 12(23), 4968–4971 (2012).
[CrossRef] [PubMed]

Crozier, K. B.

Cui, X.

Ellenberg, J.

R. Pepperkok and J. Ellenberg, “High-throughput fluorescence microscopy for systems biology,” Nat. Rev. Mol. Cell Biol. 7(9), 690–696 (2006).
[CrossRef] [PubMed]

Erath, J.

Han, C.

Hulsken, B.

B. Hulsken, D. Vossen, and S. Stallinga, “High NA diffractive array illuminators and application in a multi-spot scanning microscope,” J. Eur. Opt. Soc. Rapid Publ. 7, 12026 (2012).
[CrossRef]

Kato, M.

Lang, P.

P. Lang, K. Yeow, A. Nichols, and A. Scheer, “Cellular imaging in drug discovery,” Nat. Rev. Drug Discov. 5(4), 343–356 (2006).
[CrossRef] [PubMed]

Lee, L. M.

Lichtman, J. W.

D. Cai, K. B. Cohen, T. Luo, J. W. Lichtman, and J. R. Sanes, “Improved tools for the Brainbow toolbox,” Nat. Methods 10(6), 540–547 (2013).
[CrossRef]

Luo, T.

D. Cai, K. B. Cohen, T. Luo, J. W. Lichtman, and J. R. Sanes, “Improved tools for the Brainbow toolbox,” Nat. Methods 10(6), 540–547 (2013).
[CrossRef]

Nichols, A.

P. Lang, K. Yeow, A. Nichols, and A. Scheer, “Cellular imaging in drug discovery,” Nat. Rev. Drug Discov. 5(4), 343–356 (2006).
[CrossRef] [PubMed]

O’Neill, F. T.

F. T. O’Neill and J. T. Sheridan, “Photoresist reflow method of microlens production Part II: Analytic models,” Optik 113(9), 405–420 (2002).
[CrossRef]

Orth, A.

Ozcan, A.

S. A. Arpali, C. Arpali, A. F. Coskun, H. H. Chiang, and A. Ozcan, “High-throughput screening of large volumes of whole blood using structured illumination and fluorescent on-chip imaging,” Lab Chip 12(23), 4968–4971 (2012).
[CrossRef] [PubMed]

Pang, S.

Pepperkok, R.

R. Pepperkok and J. Ellenberg, “High-throughput fluorescence microscopy for systems biology,” Nat. Rev. Mol. Cell Biol. 7(9), 690–696 (2006).
[CrossRef] [PubMed]

Preibisch, S.

S. Preibisch, S. Saalfeld, and P. Tomancak, “Globally optimal stitching of tiled 3D microscopic image acquisitions,” Bioinformatics 25(11), 1463–1465 (2009).
[CrossRef] [PubMed]

Rodriguez, A.

Saalfeld, S.

S. Preibisch, S. Saalfeld, and P. Tomancak, “Globally optimal stitching of tiled 3D microscopic image acquisitions,” Bioinformatics 25(11), 1463–1465 (2009).
[CrossRef] [PubMed]

Sanes, J. R.

D. Cai, K. B. Cohen, T. Luo, J. W. Lichtman, and J. R. Sanes, “Improved tools for the Brainbow toolbox,” Nat. Methods 10(6), 540–547 (2013).
[CrossRef]

Scheer, A.

P. Lang, K. Yeow, A. Nichols, and A. Scheer, “Cellular imaging in drug discovery,” Nat. Rev. Drug Discov. 5(4), 343–356 (2006).
[CrossRef] [PubMed]

Sheridan, J. T.

F. T. O’Neill and J. T. Sheridan, “Photoresist reflow method of microlens production Part II: Analytic models,” Optik 113(9), 405–420 (2002).
[CrossRef]

Stallinga, S.

B. Hulsken, D. Vossen, and S. Stallinga, “High NA diffractive array illuminators and application in a multi-spot scanning microscope,” J. Eur. Opt. Soc. Rapid Publ. 7, 12026 (2012).
[CrossRef]

Sternberg, P. W.

Tomancak, P.

S. Preibisch, S. Saalfeld, and P. Tomancak, “Globally optimal stitching of tiled 3D microscopic image acquisitions,” Bioinformatics 25(11), 1463–1465 (2009).
[CrossRef] [PubMed]

Vossen, D.

B. Hulsken, D. Vossen, and S. Stallinga, “High NA diffractive array illuminators and application in a multi-spot scanning microscope,” J. Eur. Opt. Soc. Rapid Publ. 7, 12026 (2012).
[CrossRef]

Wang, Y. M.

Wu, J.

Yang, C.

Yeow, K.

P. Lang, K. Yeow, A. Nichols, and A. Scheer, “Cellular imaging in drug discovery,” Nat. Rev. Drug Discov. 5(4), 343–356 (2006).
[CrossRef] [PubMed]

Zheng, G.

Anal. Bioanal. Chem. (1)

M. Bickle, “The beautiful cell: high-content screening in drug discovery,” Anal. Bioanal. Chem. 398(1), 219–226 (2010).
[CrossRef] [PubMed]

Bioinformatics (1)

S. Preibisch, S. Saalfeld, and P. Tomancak, “Globally optimal stitching of tiled 3D microscopic image acquisitions,” Bioinformatics 25(11), 1463–1465 (2009).
[CrossRef] [PubMed]

J. Eur. Opt. Soc. Rapid Publ. (1)

B. Hulsken, D. Vossen, and S. Stallinga, “High NA diffractive array illuminators and application in a multi-spot scanning microscope,” J. Eur. Opt. Soc. Rapid Publ. 7, 12026 (2012).
[CrossRef]

Lab Chip (1)

S. A. Arpali, C. Arpali, A. F. Coskun, H. H. Chiang, and A. Ozcan, “High-throughput screening of large volumes of whole blood using structured illumination and fluorescent on-chip imaging,” Lab Chip 12(23), 4968–4971 (2012).
[CrossRef] [PubMed]

Nat. Methods (1)

D. Cai, K. B. Cohen, T. Luo, J. W. Lichtman, and J. R. Sanes, “Improved tools for the Brainbow toolbox,” Nat. Methods 10(6), 540–547 (2013).
[CrossRef]

Nat. Rev. Drug Discov. (1)

P. Lang, K. Yeow, A. Nichols, and A. Scheer, “Cellular imaging in drug discovery,” Nat. Rev. Drug Discov. 5(4), 343–356 (2006).
[CrossRef] [PubMed]

Nat. Rev. Mol. Cell Biol. (1)

R. Pepperkok and J. Ellenberg, “High-throughput fluorescence microscopy for systems biology,” Nat. Rev. Mol. Cell Biol. 7(9), 690–696 (2006).
[CrossRef] [PubMed]

Opt. Express (3)

Opt. Lett. (2)

Optik (1)

F. T. O’Neill and J. T. Sheridan, “Photoresist reflow method of microlens production Part II: Analytic models,” Optik 113(9), 405–420 (2002).
[CrossRef]

Other (4)

J. W. Goodman, Introduction to Fourier optics (McGraw-Hill International Editions, 1996), Chap. 2.

J. B. Pawley, Handbook of Biological Confocal Microscopy, 3rd ed. (Springer, 2006).

Olympus ScanR specifications website, http://www.olympus-europa.com/microscopy/en/microscopy/components/component_details/component_detail_21320.jsp . Accessed 22 January 2014.

Molecular Devices ImageXpress Micro XLS specifications website, http://www.moleculardevices.com/Products/Instruments/High-Content-Screening/ImageXpress-Micro.html . Accessed 28 January 2014.

Supplementary Material (1)

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Figures (7)

Fig. 1
Fig. 1

Schematic of the extended dynamic range microlens microscope. A microscope objective (OBJ) expands a laser beam (LB) that is focused into an array of focal spots by a microlens array (MLA) on a fluorescent sample (S). The sample sits on a raster scanning piezo stage (PS) and the MLA is imaged onto a camera (CAM) by a single lens reflex (SLR) lens. A quad-band dichroic mirror (DM) reflects the laser lines (473/532/658nm) while passing fluorescence wavelengths; an emission filter (EF) provides additional wavelength filtering. Inset i) Raw image of MLA as recorded by the camera. Scale bar is 1 mm. Inset ii) Zoom-in of green area outlined area in i). Representative N = 9 superpixels are outlined in red; N = 1 superpixels are outlined in blue. Inset iii) Representative focal spots created by the MLA with red (R), green (G) and blue (B) lasers at field angles of 0° and 2.5° Scale bar is 2μm.

Fig. 2
Fig. 2

a) Blue curve: Contact profilometry height trace along a 3cm line on a 96-well plate. Greyed out vertical bars are sections of plastic support on the well plate where there is no fluorescent sample. Insets: FOV of 5μm diameter fluorescent microspheres (Nile Red, green channel) at the focal distances indicated by the dotted horizontal lines. Positions are relative to the best focus plane. Negative distances are closer to the MLA and positive distances are farther away from the MLA. Scale bar is 20μm. b) Modulation transfer functions (MTFs) for the green laser channel at −6 (red), 0 (green) and 9μm (blue) from the best focus plane. Diffraction limited MTFs for 0.24 NA wide field and confocal microscopes with circular apertures are shown in purple and cyan, respectively. All MTFs are normalized to a maximum value of 1. Insets: Image of a 500nm bead (the PSF) placed at −6 (red), 0 (green) and 9μm (blue) from the best focus plane. Scale bar is 2μm.

Fig. 3
Fig. 3

a) Three channel image 16 well section of a 96-well plate, filled with fluorescent microspheres. Red, green and blue fluorescent microspheres are excited with red, green and blue laser lines, respectively. Red and blue microspheres have a nominal diameter of 7.3μm. Green microspheres have high spatial frequency features due to wrinkling and have a nominal diameter of 5μm. White boxes denote locations of (b)-(e). Scale bar is 8 mm. b) to e): Zoom-in of regions indicated in (a). Scale bars are 25μm.

Fig. 4
Fig. 4

a) Full field view of a dual channel image of a mouse kidney slice. Scale bar is 3mm. b) Zoomed-in view of boxed region in (a). Scale bar is 50μm. c) Further magnified view of boxed region in (b). Scale bar is 10μm. Media 1 is an animation starting with image (a), zooming into region (c) in the sample.

Fig. 5
Fig. 5

Parallel multichannel microscopy with a microlens array (MLA). The excitation geometry (not shown) is identical to that in Fig. 1. Fluorescence from the sample (S) - which sits on a piezo stage (PS) - enters a spectral splitting module (SSM) after the quad-band dichroic mirror (not shown, see Fig. 1). The SSM consists of: a long-pass dichoic mirror (LPDM [ edge  λ = 532 nm] ); a pair of broadband mirrors (M1 & M2); a pair of long-pass emission filters (LP1 [ λ > 575 nm] & LP2 [ λ > 500 nm] ). The SSM relays long- and short-pass spectral copies at opposing angles into the SLR lens, yielding spatially separated spectral images (inset) on the camera (CAM) sensor. The spectral copies are shown in false color in the inset (actual camera sensor is greyscale only).

Fig. 6
Fig. 6

a) Dual-channel mouse kidney slice image, acquired in the parallel configuration. Red color is the λ > 575 nm channel and green color is the 500 nm > λ > 532 nm channel. Scale bar is 2 mm. b) Magnified view of boxed region in (a). Inset: Corresponding area of the mouse kidney slice when channels are imaged sequentially (data set from Fig. 4). Scale bar is 50μm.

Fig. 7
Fig. 7

Signal-to-noise ratio (SNR) curves of microlens imaging system. White circles and red dots are SNR curves for superpixel sizes of N = 1 and N = 9 pixels, respectively. The dashed (solid) curve is a fit to the N = 1 (N = 9) data of the form SNR = a I / a I + n r 2 , where a and n r are fitting parameters and I is the pixel intensity (grey level). Dynamic range extension occurs for intensities (grey levels) lying between the vertical dashed lines.

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