Abstract

Laser-scanning confocal fluorescence microscopy is an indispensable tool for biomedical research by virtue of its high spatial resolution. Its temporal resolution is equally important, but is still inadequate for many applications. Here we present a confocal fluorescence microscope that, for the first time to our knowledge, surpasses the highest possible frame rate constrained only by the fluorescence lifetime of fluorophores (typically a few to several nanoseconds). This microscope is enabled by integrating a broadband, spatially distributed, dual-frequency comb or spatial dual-comb and quadrature amplitude modulation for optimizing spectral efficiency into frequency-division multiplexing with single-pixel photodetection for signal integration. Specifically, we demonstrate confocal fluorescence microscopy at a record high frame rate of 16,000 frames/s. To show its broad biomedical utility, we use the microscope to demonstrate 3D volumetric confocal fluorescence microscopy of cellular dynamics at 104 volumes/s and confocal fluorescence imaging flow cytometry of hematological and microalgal cells at up to 2 m/s.

© 2018 Optical Society of America under the terms of the OSA Open Access Publishing Agreement

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References

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2017 (1)

2016 (3)

Y. Wakisaka, Y. Suzuki, O. Iwata, A. Nakashima, T. Ito, M. Hirose, R. Domon, M. Sugawara, N. Tsumura, H. Watarai, T. Shimobaba, K. Suzuki, K. Goda, and Y. Ozeki, “Probing the metabolic heterogeneity of live Euglena gracilis with stimulated Raman scattering microscopy,” Nat. Microbiol. 1, 16124 (2016).
[Crossref]

T. Blasi, H. Hennig, H. D. Summers, F. J. Theis, J. Cerveira, J. O. Patterson, D. Davies, A. Filby, A. E. Carpenter, and P. Rees, “Label-free cell cycle analysis for high-throughput imaging flow cytometry,” Nat. Commun. 7, 10256 (2016).
[Crossref]

I. Coddington, N. Newbury, and W. Swann, “Dual-comb spectroscopy,” Optica 3, 414–426 (2016).
[Crossref]

2015 (2)

J. P. Nguyen, F. B. Shipley, A. N. Linder, G. S. Plummer, M. Liu, S. U. Setru, J. W. Shaevitz, and A. M. Leifer, “Whole-brain calcium imaging with cellular resolution in freely behaving Caenorhabditis elegans,” Proc. Natl. Acad. Sci. USA 113, E1074–E1081 (2015).
[Crossref]

C.-S. Liao, P. Wang, P. Wang, J. Li, H. J. Lee, G. Eakins, and J.-X. Cheng, “Spectrometer-free vibrational imaging by retrieving stimulated Raman signal from highly scattered photons,” Sci. Adv. 1, e1500738 (2015).
[Crossref]

2014 (2)

P. W. Winter and H. Shroff, “Faster fluorescence microscopy: advances in high speed biological imaging,” Curr. Opin. Chem. Biol. 20, 46–53 (2014).
[Crossref]

P. K. Chattopadhyay, T. M. Gierahn, M. Roederer, and J. C. Love, “Single-cell technologies for monitoring immune systems,” Nat. Immunol. 15, 128–135 (2014).
[Crossref]

2013 (2)

S. Choi, P. Kim, R. Boutilier, M. Y. Kim, Y. J. Lee, and H. Lee, “Development of a high speed laser scanning confocal microscope with an acquisition rate up to 200 frames per second,” Opt. Express 21, 23611–23618 (2013).
[Crossref]

E. D. Diebold, B. W. Buckley, D. R. Gossett, and B. Jalali, “Digitally synthesized beat frequency multiplexing for sub-millisecond fluorescence microscopy,” Nat. Photonics 7, 806–810 (2013).
[Crossref]

2012 (3)

H. Choi, D. S. Tzeranis, J. W. Cha, P. Clémenceau, S. J. G. de Jong, L. K. van Geest, J. H. Moon, I. V. Yannas, and P. T. C. So, “3D-resolved fluorescence and phosphorescence lifetime imaging using temporal focusing wide-field two-photon excitation,” Opt. Express 20, 26219–26235 (2012).
[Crossref]

K. Goda, A. Ayazi, D. R. Gossett, J. Sadasivam, C. K. Lonappan, E. Sollier, A. M. Fard, S. C. Hur, J. Adam, C. Murray, C. Wang, N. Brackbill, D. Di Carlo, and B. Jalali, “High-throughput single-microparticle imaging flow analyzer,” Proc. Natl. Acad. Sci. USA 109, 11630–11635 (2012).
[Crossref]

S. S. Howard, A. Straub, N. G. Horton, D. Kobat, and C. Xu, “Frequency-multiplexed in vivo multiphoton phosphorescence lifetime microscopy,” Nat. Photonics 7, 33–37 (2012).
[Crossref]

2011 (1)

2010 (2)

B. Bernhardt, A. Ozawa, P. Jacquet, M. Jacquey, Y. Kobayashi, T. Udem, R. Holzwarth, G. Guelachvili, T. W. Hänsch, and N. Picqué, “Cavity-enhanced dual-comb spectroscopy,” Nat. Photonics 4, 55–57 (2010).
[Crossref]

M. Y. Berezin and S. Achilefu, “Fluorescence lifetime measurements and biological imaging,” Chem. Rev. 110, 2641–2684 (2010).
[Crossref]

2007 (3)

D. A. Basiji, W. E. Ortyn, L. Liang, V. Venkatachalam, and P. Morrissey, “Cellular image analysis and imaging by flow cytometry,” Clin. Chem. Lab. Med. 27, 653–670 (2007).
[Crossref]

D. C. Adler, Y. Chen, R. Huber, J. Schmitt, J. Connolly, and J. G. Fujimoto, “Three-dimensional endomicroscopy using optical coherence tomography,” Nat. Photonics 1, 709–716 (2007).
[Crossref]

C. Chang, D. Sud, and M. Mycek, “Fluorescence lifetime imaging microscopy,” Methods Cell Biol. 81, 495–524 (2007).
[Crossref]

2006 (3)

H. R. Petty, “Spatiotemporal chemical dynamics in living cells: from information trafficking to cell physiology,” Biosystems 83, 217–224 (2006).
[Crossref]

V. Bansal, S. Patel, and P. Saggau, “High-speed addressable confocal microscopy for functional imaging of cellular activity,” J. Biomed. Opt. 11, 34003 (2006).
[Crossref]

F. Wu, X. Zhang, J. Y. Cheung, K. Shi, Z. Liu, C. Luo, S. Yin, and P. Ruffin, “Frequency division multiplexed multichannel high-speed fluorescence confocal microscope,” Biophys. J. 91, 2290–2296 (2006).
[Crossref]

2005 (1)

A. L. Polglase, W. J. McLaren, S. A. Skinner, R. Kiesslich, M. F. Neurath, and P. M. Delaney, “A fluorescence confocal endomicroscope for in vivo microscopy of the upper- and the lower-GI tract,” Gastrointest. Endosc. 62, 686–695 (2005).
[Crossref]

2004 (1)

2002 (1)

2001 (1)

P. Herman, B. P. Maliwal, H.-J. Lin, and J. R. Lakowicz, “Frequency-domain fluorescence microscopy with the LED as a light source,” J. Microsc. 203, 176–181 (2001).
[Crossref]

1999 (1)

P. Bastiaens, “Fluorescence lifetime imaging microscopy: spatial resolution of biochemical processes in the cell,” Trends Cell Biol. 9, 48–52 (1999).
[Crossref]

1991 (1)

1988 (2)

C. J. R. Sheppard and X. Q. Mao, “Confocal microscopes with slit apertures,” J. Mod. Opt. 35, 1169–1185 (1988).
[Crossref]

A. Draaijer and P. M. Houpt, “A standard video-rate confocal laser-scanning reflection and fluorescence microscope,” Scanning 10, 139–145 (1988).
[Crossref]

1975 (1)

R. Schantz, M.-L. Schantz, and H. Duranton, “Changes in amino acid and peptide composition of Euglena gracilis cells during chloroplast development,” Plant Sci. Lett. 5, 313–324 (1975).
[Crossref]

1971 (1)

S. Weinstein and P. Ebert, “Data transmission by frequency-division multiplexing using the discrete Fourier transform,” IEEE Trans. Commun. Technol. 19, 628–634 (1971).
[Crossref]

1952 (1)

M. Cramer and J. Myers, “Growth and photosynthetic characteristics of Euglena gracilis,” Arch. Mikrobiol. 17, 384–402 (1952).
[Crossref]

Achilefu, S.

M. Y. Berezin and S. Achilefu, “Fluorescence lifetime measurements and biological imaging,” Chem. Rev. 110, 2641–2684 (2010).
[Crossref]

Adam, J.

K. Goda, A. Ayazi, D. R. Gossett, J. Sadasivam, C. K. Lonappan, E. Sollier, A. M. Fard, S. C. Hur, J. Adam, C. Murray, C. Wang, N. Brackbill, D. Di Carlo, and B. Jalali, “High-throughput single-microparticle imaging flow analyzer,” Proc. Natl. Acad. Sci. USA 109, 11630–11635 (2012).
[Crossref]

Adler, D. C.

D. C. Adler, Y. Chen, R. Huber, J. Schmitt, J. Connolly, and J. G. Fujimoto, “Three-dimensional endomicroscopy using optical coherence tomography,” Nat. Photonics 1, 709–716 (2007).
[Crossref]

Arimoto, R.

Ayazi, A.

K. Goda, A. Ayazi, D. R. Gossett, J. Sadasivam, C. K. Lonappan, E. Sollier, A. M. Fard, S. C. Hur, J. Adam, C. Murray, C. Wang, N. Brackbill, D. Di Carlo, and B. Jalali, “High-throughput single-microparticle imaging flow analyzer,” Proc. Natl. Acad. Sci. USA 109, 11630–11635 (2012).
[Crossref]

Bansal, V.

V. Bansal, S. Patel, and P. Saggau, “High-speed addressable confocal microscopy for functional imaging of cellular activity,” J. Biomed. Opt. 11, 34003 (2006).
[Crossref]

Bartels, R. A.

Basiji, D. A.

D. A. Basiji, W. E. Ortyn, L. Liang, V. Venkatachalam, and P. Morrissey, “Cellular image analysis and imaging by flow cytometry,” Clin. Chem. Lab. Med. 27, 653–670 (2007).
[Crossref]

Bastiaens, P.

P. Bastiaens, “Fluorescence lifetime imaging microscopy: spatial resolution of biochemical processes in the cell,” Trends Cell Biol. 9, 48–52 (1999).
[Crossref]

Berezin, M. Y.

M. Y. Berezin and S. Achilefu, “Fluorescence lifetime measurements and biological imaging,” Chem. Rev. 110, 2641–2684 (2010).
[Crossref]

Bernhardt, B.

B. Bernhardt, A. Ozawa, P. Jacquet, M. Jacquey, Y. Kobayashi, T. Udem, R. Holzwarth, G. Guelachvili, T. W. Hänsch, and N. Picqué, “Cavity-enhanced dual-comb spectroscopy,” Nat. Photonics 4, 55–57 (2010).
[Crossref]

Blasi, T.

T. Blasi, H. Hennig, H. D. Summers, F. J. Theis, J. Cerveira, J. O. Patterson, D. Davies, A. Filby, A. E. Carpenter, and P. Rees, “Label-free cell cycle analysis for high-throughput imaging flow cytometry,” Nat. Commun. 7, 10256 (2016).
[Crossref]

Boutilier, R.

Brackbill, N.

K. Goda, A. Ayazi, D. R. Gossett, J. Sadasivam, C. K. Lonappan, E. Sollier, A. M. Fard, S. C. Hur, J. Adam, C. Murray, C. Wang, N. Brackbill, D. Di Carlo, and B. Jalali, “High-throughput single-microparticle imaging flow analyzer,” Proc. Natl. Acad. Sci. USA 109, 11630–11635 (2012).
[Crossref]

Buckley, B. W.

E. D. Diebold, B. W. Buckley, D. R. Gossett, and B. Jalali, “Digitally synthesized beat frequency multiplexing for sub-millisecond fluorescence microscopy,” Nat. Photonics 7, 806–810 (2013).
[Crossref]

Carpenter, A. E.

T. Blasi, H. Hennig, H. D. Summers, F. J. Theis, J. Cerveira, J. O. Patterson, D. Davies, A. Filby, A. E. Carpenter, and P. Rees, “Label-free cell cycle analysis for high-throughput imaging flow cytometry,” Nat. Commun. 7, 10256 (2016).
[Crossref]

Cerveira, J.

T. Blasi, H. Hennig, H. D. Summers, F. J. Theis, J. Cerveira, J. O. Patterson, D. Davies, A. Filby, A. E. Carpenter, and P. Rees, “Label-free cell cycle analysis for high-throughput imaging flow cytometry,” Nat. Commun. 7, 10256 (2016).
[Crossref]

Cha, J. W.

Chan, G. C. F.

Chang, C.

C. Chang, D. Sud, and M. Mycek, “Fluorescence lifetime imaging microscopy,” Methods Cell Biol. 81, 495–524 (2007).
[Crossref]

Chattopadhyay, P. K.

P. K. Chattopadhyay, T. M. Gierahn, M. Roederer, and J. C. Love, “Single-cell technologies for monitoring immune systems,” Nat. Immunol. 15, 128–135 (2014).
[Crossref]

Chen, Y.

D. C. Adler, Y. Chen, R. Huber, J. Schmitt, J. Connolly, and J. G. Fujimoto, “Three-dimensional endomicroscopy using optical coherence tomography,” Nat. Photonics 1, 709–716 (2007).
[Crossref]

Cheng, J.-X.

C.-S. Liao, P. Wang, P. Wang, J. Li, H. J. Lee, G. Eakins, and J.-X. Cheng, “Spectrometer-free vibrational imaging by retrieving stimulated Raman signal from highly scattered photons,” Sci. Adv. 1, e1500738 (2015).
[Crossref]

Cheung, J. Y.

F. Wu, X. Zhang, J. Y. Cheung, K. Shi, Z. Liu, C. Luo, S. Yin, and P. Ruffin, “Frequency division multiplexed multichannel high-speed fluorescence confocal microscope,” Biophys. J. 91, 2290–2296 (2006).
[Crossref]

Choi, H.

Choi, S.

Clémenceau, P.

Coddington, I.

Connolly, J.

D. C. Adler, Y. Chen, R. Huber, J. Schmitt, J. Connolly, and J. G. Fujimoto, “Three-dimensional endomicroscopy using optical coherence tomography,” Nat. Photonics 1, 709–716 (2007).
[Crossref]

Cramer, M.

M. Cramer and J. Myers, “Growth and photosynthetic characteristics of Euglena gracilis,” Arch. Mikrobiol. 17, 384–402 (1952).
[Crossref]

Cristianini, N.

N. Cristianini and J. Shawe-Taylor, An Introduction to Support Vector Machines: And Other Kernel-Based Learning Methods (Cambridge University, 2000).

Davies, D.

T. Blasi, H. Hennig, H. D. Summers, F. J. Theis, J. Cerveira, J. O. Patterson, D. Davies, A. Filby, A. E. Carpenter, and P. Rees, “Label-free cell cycle analysis for high-throughput imaging flow cytometry,” Nat. Commun. 7, 10256 (2016).
[Crossref]

de Jong, S. J. G.

Delaney, P. M.

A. L. Polglase, W. J. McLaren, S. A. Skinner, R. Kiesslich, M. F. Neurath, and P. M. Delaney, “A fluorescence confocal endomicroscope for in vivo microscopy of the upper- and the lower-GI tract,” Gastrointest. Endosc. 62, 686–695 (2005).
[Crossref]

Di Carlo, D.

K. Goda, A. Ayazi, D. R. Gossett, J. Sadasivam, C. K. Lonappan, E. Sollier, A. M. Fard, S. C. Hur, J. Adam, C. Murray, C. Wang, N. Brackbill, D. Di Carlo, and B. Jalali, “High-throughput single-microparticle imaging flow analyzer,” Proc. Natl. Acad. Sci. USA 109, 11630–11635 (2012).
[Crossref]

Diebold, E. D.

E. D. Diebold, B. W. Buckley, D. R. Gossett, and B. Jalali, “Digitally synthesized beat frequency multiplexing for sub-millisecond fluorescence microscopy,” Nat. Photonics 7, 806–810 (2013).
[Crossref]

Domon, R.

Y. Wakisaka, Y. Suzuki, O. Iwata, A. Nakashima, T. Ito, M. Hirose, R. Domon, M. Sugawara, N. Tsumura, H. Watarai, T. Shimobaba, K. Suzuki, K. Goda, and Y. Ozeki, “Probing the metabolic heterogeneity of live Euglena gracilis with stimulated Raman scattering microscopy,” Nat. Microbiol. 1, 16124 (2016).
[Crossref]

Draaijer, A.

A. Draaijer and P. M. Houpt, “A standard video-rate confocal laser-scanning reflection and fluorescence microscope,” Scanning 10, 139–145 (1988).
[Crossref]

Duranton, H.

R. Schantz, M.-L. Schantz, and H. Duranton, “Changes in amino acid and peptide composition of Euglena gracilis cells during chloroplast development,” Plant Sci. Lett. 5, 313–324 (1975).
[Crossref]

Eakins, G.

C.-S. Liao, P. Wang, P. Wang, J. Li, H. J. Lee, G. Eakins, and J.-X. Cheng, “Spectrometer-free vibrational imaging by retrieving stimulated Raman signal from highly scattered photons,” Sci. Adv. 1, e1500738 (2015).
[Crossref]

Ebert, P.

S. Weinstein and P. Ebert, “Data transmission by frequency-division multiplexing using the discrete Fourier transform,” IEEE Trans. Commun. Technol. 19, 628–634 (1971).
[Crossref]

Fard, A. M.

K. Goda, A. Ayazi, D. R. Gossett, J. Sadasivam, C. K. Lonappan, E. Sollier, A. M. Fard, S. C. Hur, J. Adam, C. Murray, C. Wang, N. Brackbill, D. Di Carlo, and B. Jalali, “High-throughput single-microparticle imaging flow analyzer,” Proc. Natl. Acad. Sci. USA 109, 11630–11635 (2012).
[Crossref]

Filby, A.

T. Blasi, H. Hennig, H. D. Summers, F. J. Theis, J. Cerveira, J. O. Patterson, D. Davies, A. Filby, A. E. Carpenter, and P. Rees, “Label-free cell cycle analysis for high-throughput imaging flow cytometry,” Nat. Commun. 7, 10256 (2016).
[Crossref]

Fujimoto, J. G.

D. C. Adler, Y. Chen, R. Huber, J. Schmitt, J. Connolly, and J. G. Fujimoto, “Three-dimensional endomicroscopy using optical coherence tomography,” Nat. Photonics 1, 709–716 (2007).
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Supplementary Material (3)

NameDescription
» Supplement 1       Supplement
» Visualization 1       Rotational motion of Euglena gracilis cells.
» Visualization 2       Flow of Euglena gracilis cells.

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Figures (4)

Fig. 1.
Fig. 1. FDM confocal fluorescence microscopy with the spatial dual-comb (SDC) beam assisted by quadrature amplitude modulation (QAM). See the main text for a complete description of the schematic and principles of the method. (a) Schematic of the microscope. (b) SDC beam generator. (c) Two frequency combs in the optical frequency domain and SDC beam in the radio frequency domain. (d) QAM-SDC beam generator. (e) Measured in-phase and quadrature components of the QAM-SDC beam. The insets show the equally spaced frequency comb lines.
Fig. 2.
Fig. 2. FDM confocal fluorescence microscopy at a high frame rate of 16,000 frames/s. BF, bright-field images; FL ch1, fluorescence images constructed from channel 1 (wavelength range: 509–580 nm); FL ch2, fluorescence images constructed from channel 2 (wavelength range: >580  nm). The dashed lines in the FL ch1 images represent cutoff frequencies where the modulation recovery falls down to 50% (obtained separately). The bottom plots show the phase delay (black solid curves and blue dots) and modulation recovery (dashed curves) of the used fluorescent probe in the FL ch1 images. The parameter τ represents the fluorescence lifetime of the fluorescent probe. The blue dots indicate the measured phase delay, while the black curves are the fits to the measured data. (a) Images of 6-μm fluorescent beads with the measured phase delay of the fluorescence signal. The fit to the measured data points indicates the fluorescence lifetime of 1.5 ns. Scale bar: 5 μm. (b) Images of MCF-7 cells stained by Calcein-AM with the measured phase delay of the fluorescence signal. The fit to the measured data points indicates the fluorescence lifetime of 2.9 ns. Scale bar: 10 μm. (c) Images of Euglena gracilis cells stained by SYTO16 with the measured phase delay of the fluorescence signal in ch1. The fit to the measured data points consists of 82% of 4.1 ns of the fluorescence lifetime and 18% of 0.33 ns of the fluorescence lifetime. Scale bar: 10 μm.
Fig. 3.
Fig. 3. 3D volumetric confocal fluorescence microscopy of freely moving motile Euglena gracilis cells at a high volume rate of 104 volumes/s. Box size: 70  μm(x)×80  μm(y)×90  μm(z). Green: nuclei stained by SYTO9. Red: chlorophyll (autofluorescence). The frame sequence shown here is a series of one out of every 10 frames to show the dynamical behavior (swimming with flagella) of the cells while the inset shows the consecutive frames with the full temporal resolution of 9.6 ms. A low-pass filter was employed for noise reduction. (a) Rotational motion of Euglena gracilis cells. (b) Flow of Euglena gracilis cells. The complete versions of the 3D movies are available in Visualization 1 and Visualization 2.
Fig. 4.
Fig. 4. Confocal fluorescence imaging flow cytometry. (a) Images libraries of neutrophils (top) and lymphocytes (bottom) isolated from murine bone marrow. A low-pass filter was employed for noise reduction. Green: nuclei stained by SYTO16. Gray: bright-field image in transmission mode. The arrows in the insets indicate the flow direction. The flow speed is 1 m/s. (b) Scatter plot of the cells (N=5600 for each). A SVM was applied to the image library to classify the cells. (c) Image libraries of Euglena gracilis cells. A low-pass filter was employed for noise reduction. Top: cells cultivated under a nitrogen-sufficient condition. Bottom: cells cultivated under a nitrogen-deficient condition. Green: intracellular lipids stained by BODIPY505/515. Red: intracellular chlorophyll (autofluorescence). Gray: bright-field image in transmission mode. The arrows in the insets indicate the flow direction. The flow speed is 2 m/s. (d) Scatter plot of the cells (N=1880 for each). A SVM was applied to the image library to classify the cells.

Equations (1)

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R(f)=eiarg[12πifτ]1+(2πfτ)2,

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