Fast multicolor 3D imaging using aberration-corrected multifocus microscopy

Journal name:
Nature Methods
Volume:
10,
Pages:
60–63
Year published:
DOI:
doi:10.1038/nmeth.2277
Received
Accepted
Published online

Conventional acquisition of three-dimensional (3D) microscopy data requires sequential z scanning and is often too slow to capture biological events. We report an aberration-corrected multifocus microscopy method capable of producing an instant focal stack of nine 2D images. Appended to an epifluorescence microscope, the multifocus system enables high-resolution 3D imaging in multiple colors with single-molecule sensitivity, at speeds limited by the camera readout time of a single image.

At a glance

Figures

  1. Aberration-corrected multifocus microscopy (MFM).
    Figure 1: Aberration-corrected multifocus microscopy (MFM).

    (a) Multifocus optical elements are appended to a wide-field fluorescence microscope after the primary image plane (at the camera port). Two relay lenses (f1 and f2, first and second relay lens, with focal lengths f1 = 150 and f2 = 200 mm) create a conjugate pupil plane (Fourier plane) and the final image plane. The multifocus grating (MFG) is placed in the Fourier plane and followed by the chromatic correction grating (CCG) and prism. A dichroic mirror (purple) splits the color channels onto separate cameras. (b) The MFG splits and shifts the focus of the sample emission light to form an instant focal series, in which each focal plane corresponds to a diffractive order of the MFG. Ray colors denote individual focal planes (diffractive orders). The CCG and prism correct the chromatic dispersion, illustrated by rays of wavelengths λmax and λmin, introduced by the MFG. (c) The instant focal stack recorded on the camera is computationally assembled into a 3D volume. (d) Schematics of the MFG, a phase-only diffractive grating with etch depth π; the grating function (basic grating pattern) of the MFG, optimized to distribute light evenly into the central 3 × 3 diffractive orders that form the nine focal planes; the CCG, with panels containing blazed diffractive gratings that reverse the dispersion of the MFG (central panel is blank); and the prism, which directs the images to their positions on the camera. Note the geometrical distortion of the MFG pattern, which introduces a phase shift that is dependent on diffractive order (mx, my) and gives rise to the focus shift in each plane of the multifocus image. (e) Raw, multifocus image of 200-nm fluorescent beads. As illustrated in c, the central tile is the (nondiffracted) traditional microscope image of the nominal focal plane. The surrounding eight tiles are the duplicate focus-shifted images, formed by diffraction in the MFG. The focus step between successive planes is Δz = 380 nm. (f) Axial (xz) point-spread function (PSF), radially averaged and displayed in log scale. (g) Gaussian curves fitted to the bead signal of each plane at different z positions of the stage. Best focus position is estimated as the maximum of the fitted curve (Supplementary Video 1). (h) Plot of the best focus position of each plane. The linear curve verifies the constant focus step Δz between planes. Scale bars, 1 μm.

  2. Multifocus imaging of yeast centromeres.
    Figure 2: Multifocus imaging of yeast centromeres.

    (a,b) Multifocus transmission (a) and fluorescence (b) images of S. cerevisiae expressing Cse4-GFP. (c) Separation of the centromeres during anaphase over time (maximum-intensity projections). (d) Movement in 3D of the two centromere clusters (black and gray). Bottom right, separation between the centromere clusters over time. Rapid movement (phase I) is followed by slow movement (phase II). Inset, average speed during phases I and II (n = 5 cells). Scale bars, 5 μm.

  3. Single-molecule tracking of RNA polymerase II.
    Figure 3: Single-molecule tracking of RNA polymerase II.

    (a) Maximum-intensity projection (MIP) of the 3D volume of the first frame of Supplementary Video 5 on the indicated planes. Purple spots correspond to single Halo-tagged RNA polymerase II molecules in U2OS cells. The nuclear membrane (green) is visualized using lamin B1–GFP. Scale bar, 5 μm. (b) Movement of the single molecule marked by the dashed frame in a, visualized in MIP in the xz plane (scale as in a). (c) Histogram of diffusion coefficient (n = 109 molecules from seven cells). (d) Examples of individual trajectories of RNA polymerase II, showing (left to right) a bound molecule, a diffusing molecule and a molecule with mixed dynamics. Dimensions are in micrometers. Corresponding 3D temporal sequences are available in Supplementary Video 6.

Videos

  1. Raw data of 200-nm beads mounted on a coverslip and displaced along the z axis in 100-nm steps using a piezoelectric stage.
    Video 1: Raw data of 200-nm beads mounted on a coverslip and displaced along the z axis in 100-nm steps using a piezoelectric stage.
  2. The reconstructed 3D rendered movie of the data stack of Supplementary Video 1, showing the beads realigned inside the imaging volume.
    Video 2: The reconstructed 3D rendered movie of the data stack of Supplementary Video 1, showing the beads realigned inside the imaging volume.
  3. Raw data of yeast cells expressing Cse4-GFP.
    Video 3: Raw data of yeast cells expressing Cse4-GFP.
    Lateral field of view 20×20 μm. Focus step between successive planes Δz = 380 nm. (Voxel size: x,y = 120 nm, z = 380 nm.) Data were acquired at a speed of one multifocus image (exposure time 100 ms) every 3 s. Scale bar, 1.5 μm.
  4. 3D reconstruction of Supplementary Video 3, visualizing the 3D dynamics of yeast centromeres during anaphase.
    Video 4: 3D reconstruction of Supplementary Video 3, visualizing the 3D dynamics of yeast centromeres during anaphase.
    Upper movie, xy view; lower movie, xz view. Scale bar, 1 μm.
  5. 3D view of single trajectories of RNA polymerase II (magenta) diffusing in the nucleus of U2OS cells transfected with lamin B1GFP as a nuclear membrane marker (green).
    Video 5: 3D view of single trajectories of RNA polymerase II (magenta) diffusing in the nucleus of U2OS cells transfected with lamin B1–GFP as a nuclear membrane marker (green).
    Upper movie, xy view; lower movie, xz view. Only trajectories longer than five frames are displayed as segments. Data were acquired at a speed of 35 volumes s–1. (Voxel size: x,y = 120 nm, z = 380 nm.)
  6. Examples of 3D trajectories, exhibiting slow diffusion, rapid diffusion and mixed behavior.
    Video 6: Examples of 3D trajectories, exhibiting slow diffusion, rapid diffusion and mixed behavior.
    Data were acquired at a speed of 35 volumes s–1.
  7. The unc-47 GABAergic motor neurons of this C. elegans embryo express a green fluorescent protein.
    Video 7: The unc-47 GABAergic motor neurons of this C. elegans embryo express a green fluorescent protein.
    This movie shows the raw multifocus data, a set of nine simultaneously formed focal planes (60×60 μm at 2-μm separation covering a depth of 18 μm) arranged in a 3×3 array on the camera. Data were recorded at 9 Hz for 5.5 min and are displayed at 10× this speed. Scale bar, 20 μm.
  8. Average-intensity projection along z of the 3D assembled data of the C. elegans embryo in Supplementary Video 7.
    Video 8: Average-intensity projection along z of the 3D assembled data of the C. elegans embryo in Supplementary Video 7.

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Author information

  1. These authors contributed equally to this work.

    • Jiji Chen &
    • Bassam Hajj

Affiliations

  1. Joint Graduate Group in Bioengineering, University of California, San Francisco (UCSF)/University of California, Berkeley, San Francisco, California, USA.

    • Sara Abrahamsson
  2. Howard Hughes Medical Institute, Janelia Farm Research Campus, Ashburn, Virginia, USA.

    • Sara Abrahamsson,
    • Gaku Mizuguchi,
    • Carl Wu &
    • Mats G L Gustafsson
  3. Laboratory for Neural Circuits and Behavior, The Rockefeller University, New York, New York, USA.

    • Sara Abrahamsson,
    • Alexander Y Katsov &
    • Cornelia I Bargmann
  4. Transcription Imaging Consortium, HHMI, Janelia Farm Research Campus, Ashburn, Virginia, USA.

    • Jiji Chen,
    • Bassam Hajj,
    • Jan Wisniewski,
    • Gaku Mizuguchi,
    • Claire Dugast Darzacq,
    • Xavier Darzacq,
    • Carl Wu &
    • Maxime Dahan
  5. Department of Imaging Science and Technology, Delft University of Technology, Delft, The Netherlands.

    • Sjoerd Stallinga &
    • Carl Wu
  6. Laboratory of Biochemistry and Molecular Biology, National Cancer Institute, Bethesda, Maryland, USA.

    • Jan Wisniewski &
    • Gaku Mizuguchi
  7. Functional Imaging of Transcription, Institut de Biologie de l'École Normale Supérieure, Centre National de la Recherche Scientifique (CNRS) Unité Mixte de Recherche (UMR) 8197, Paris, France.

    • Pierre Soule,
    • Florian Mueller,
    • Claire Dugast Darzacq &
    • Xavier Darzacq
  8. Institut Pasteur, Imaging and Modeling Group, CNRS, Unité de Recherche Associée 2582, Paris, France.

    • Florian Mueller
  9. Université Paris Diderot, Paris, France.

    • Claire Dugast Darzacq
  10. Howard Hughes Medical Institute (HHMI), The Rockefeller University, New York, New York, USA.

    • Cornelia I Bargmann
  11. HHMI, UCSF, San Francisco, California, USA.

    • David A Agard
  12. Department of Biochemistry & Biophysics, UCSF, San Francisco, California, USA.

    • David A Agard
  13. Laboratoire Kastler Brossel, CNRS UMR 8552, Institut de Biologie et Département de Physique, École Normale Supérieure, Paris, France.

    • Maxime Dahan
  14. Present address: Institut Curie, CNRS UMR 168, Paris, France.

    • Maxime Dahan
  15. Deceased.

    • Mats G L Gustafsson

Contributions

The optical layout was conceived by M.G.L.G. Optical design and optimizations were made by M.G.L.G. and S.A. S.A. built the system and implemented the hardware control electronics. J.C., B.H. and M.D. performed single-molecule and yeast experiments. S.A., B.H., J.C. and M.D. developed image processing tools, and B.H. and J.C. analyzed the data. S.A. and A.Y.K. acquired the C. elegans data. J.W. and G.M. constructed the yeast strain; J.W. and C.W. participated in centromere imaging. P.S., C.D.D. and X.D. constructed and characterized the RPB1 cellular system. S.S. contributed to the theoretical performance evaluation of the microscope. F.M. and X.D. provided the 3D single-emitter detection algorithm. M.G.L.G., D.A.A. and C.I.B. supervised the project. S.A. and M.D. prepared the manuscript.

Competing financial interests

The authors declare no competing financial interests.

Corresponding authors

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Author details

Supplementary information

Video

  1. Video 1: Raw data of 200-nm beads mounted on a coverslip and displaced along the z axis in 100-nm steps using a piezoelectric stage. (3 MB, Download)
  2. Video 2: The reconstructed 3D rendered movie of the data stack of Supplementary Video 1, showing the beads realigned inside the imaging volume. (1.61 MB, Download)
  3. Video 3: Raw data of yeast cells expressing Cse4-GFP. (18.88 MB, Download)
    Lateral field of view 20×20 μm. Focus step between successive planes Δz = 380 nm. (Voxel size: x,y = 120 nm, z = 380 nm.) Data were acquired at a speed of one multifocus image (exposure time 100 ms) every 3 s. Scale bar, 1.5 μm.
  4. Video 4: 3D reconstruction of Supplementary Video 3, visualizing the 3D dynamics of yeast centromeres during anaphase. (19.71 MB, Download)
    Upper movie, xy view; lower movie, xz view. Scale bar, 1 μm.
  5. Video 5: 3D view of single trajectories of RNA polymerase II (magenta) diffusing in the nucleus of U2OS cells transfected with lamin B1–GFP as a nuclear membrane marker (green). (17.2 MB, Download)
    Upper movie, xy view; lower movie, xz view. Only trajectories longer than five frames are displayed as segments. Data were acquired at a speed of 35 volumes s–1. (Voxel size: x,y = 120 nm, z = 380 nm.)
  6. Video 6: Examples of 3D trajectories, exhibiting slow diffusion, rapid diffusion and mixed behavior. (7.22 MB, Download)
    Data were acquired at a speed of 35 volumes s–1.
  7. Video 7: The unc-47 GABAergic motor neurons of this C. elegans embryo express a green fluorescent protein. (2.79 MB, Download)
    This movie shows the raw multifocus data, a set of nine simultaneously formed focal planes (60×60 μm at 2-μm separation covering a depth of 18 μm) arranged in a 3×3 array on the camera. Data were recorded at 9 Hz for 5.5 min and are displayed at 10× this speed. Scale bar, 20 μm.
  8. Video 8: Average-intensity projection along z of the 3D assembled data of the C. elegans embryo in Supplementary Video 7. (1.79 MB, Download)

PDF files

  1. Supplementary Text and Figures (7 MB)

    Supplementary Figures 1–3 and Supplementary Notes 1–4

Additional data