Universal light-sheet generation with field synthesis

Abstract

We introduce field synthesis, a theorem and method that can be used to synthesize any scanned or dithered light sheet, including those used in lattice light-sheet microscopy (LLSM), from an incoherent superposition of one-dimensional intensity distributions. Compared to LLSM, this user-friendly and modular approach offers a simplified optical design, higher light throughput and simultaneous multicolor illumination. Further, field synthesis achieves lower rates of photobleaching than light sheets generated by lateral beam scanning.

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Fig. 1: Light-sheet generation by field synthesis.
Fig. 2: Field synthesis reduces photobleaching and allows for simultaneous multicolor imaging.

Code availability

The MATLAB code used in this work is available as Supplementary Software; any updated versions can be found at https://github.com/AdvancedImagingUTSW/FieldSynthesis. The instrument control software can be requested for academic use from the corresponding authors and will be delivered under material transfer agreements with HHMI and UT Southwestern Medical Center.

Data availability

The datasets acquired for this study are available from the corresponding author upon request.

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Acknowledgements

We thank the Cancer Prevention Research Institute of Texas (RR160057 to R.F.), the National Institutes of Health (T32CA080621 to M.K., F32GM117793 to K.M.D., K25CA204526 to E.S.W.) and Human Frontier Science Program (LT000954/2015 to P.R.) for their generous support. We also thank K. Jaqaman (CPRIT R1216, UT Southwestern Endowed Scholars Program to K.J.) and R.D. Goldman (R01GM106023) for supporting M.K. We also thank M. Mettlen for preparing the SK-Mel2 cells.

Author information

R.F., K.M.D. and B.-J.C. designed the research. B.-J.C. and R.F. designed, built and operated the microscope. R.F., K.M.D., P.R. and B.-J.C. performed image analysis. E.S.W. provided the MV3 cancer cells. M.K. carried out mathematical derivations with P.R. and R.F., proved the theorems and prepared the MATLAB code. R.F., K.M.D., M.K. and B.-J.C. wrote the manuscript, and all authors provided feedback.

Correspondence to Reto Fiolka.

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Competing interests

R.F., B.-J.C. and M.K. have filed a patent for the field synthesis process and its applications to microscopy.

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Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Integrated supplementary information

Supplementary Figure 1 Basic components for conventional Lattice Light-Sheet and Field Synthesis Light-Sheet Generation.

Units to produce lattice-light-sheets conventionally and with Field Synthesis. Both units take a line shaped laser beam as an input and put out the final light-sheet into an image plane. PBS: Polarizing beam splitter, SLM: spatial light modulator; HWP: half wave plate.

Supplementary Figure 2 Sequential lattice experiment. This experiment enables testing the Field synthesis theorem using a conventional lattice light-sheet microscope.

In the sequential lattice illumination mode, which simulates the Field Synthesis process, three diffraction orders of a square lattice pattern were individually selected by a slit mask on the back aperture (Fourier space) of the objective. Each order was separately imaged in image space and the three datasets were added. The xz image shows the light-sheet on the focal plane. The yz image shows the light-sheet along the propagation direction (y). The xz and yz profiles of the light-sheets are the averages of six line-profiles from xz and yz images. The cross-sectional profiles show a comparison between dithered, normal square lattice light-sheets and the results obtained from the sequential lattice approach.

Supplementary Figure 3 Simulation of the Electric field and the Intensity of a square lattice light-sheet in Fourier and real space.

Top row from left to right: Electric field at the pupil created by the square lattice mask, Magnitude of the Fourier transform of the intensity of the square lattice, Magnitude of the Fourier transform of the intensity of the dithered square lattice. Bottom row from left to right: Electric field of the lattice at the focal plane, Intensity (square modulus) of the lattice, Intensity of the square lattice dithered over one period. The figure was generated by FieldSynthesisVersusLattice.m.

Supplementary Figure 4 Measurement of the intensity distribution of a square lattice and Field Synthesis light-sheet in the sample plane

A 50 nm fluorescent bead was scanned through the light-sheets and at every scan position, the measured fluorescence was summed up, resulting in the measurements shown in the top row. Bottom row shows intensity profiles along the propagation and axial direction of the light sheet. The experiment was repeated eight times for each mode with similar results.

Supplementary Figure 5 Simulation of conventional square Lattice profile and Field Synthesis square Lattice profile.

Top: Conventional lattice profile generated by dithering the lattice intensity. Middle: Field synthesis lattice profile generated by summing line scans over the pupil mask. Bottom: Overlay of the conventional and field synthesis lattice profiles. The figure was generated by FieldSynthesisVersusLattice.m.

Supplementary Figure 6 Simulation and Proof of Concept for Field Synthesis.

Top from left to right: Original intensity pattern created by a complex mask, Sum of the square modulus of slices of the pupil mask as in the field synthesis method, Smearing of the intensity pattern as done in dithering. The bottom plot shows the overlay of several methods of calculating the vertical profile created by slicing or smearing. Projection is the profile created by averaging each row of the original intensity. Slice is the profile created by the field synthesis method. Smearing is the profile created by dithering the intensity. 1D FT is the profile created by taking a one-dimensional Fourier transform of the pupil mask in the z-direction and then summing the square modulus in the x-direction as in the above proof. Created by FieldSythesisTheorem.m.

Supplementary Figure 7 Stationary and dithered optical lattices.

Square (top) and hexagonal (bottom) lattice light-sheets as measured in transmission produced by our LLSM setup shown in Supplemenatry Figure 2. Undithered lattices are shown in the left column and the dithered lattices in the right column. The undithered lattices are shown here as a quality control for our experimental LLSM setup. Typical interference patterns of a 2D optical lattice are clearly visible (see also Chen et al1). Scale bar: 10 microns. The experiment was repeated eight times with similar results.

Supplementary Figure 8 Numerical simulation of the effect of line broadening in Field Synthesis.

The top row shows a simulation of Field Synthesis with a linewidth of one pixel and no blurring, which simulates scanning the pupil with a delta function. A conventional Bessel beam light-sheet was simulated by numerically scanning the Bessel beam over the full numerical domain and summing the intensities. Second and third row show simulations where a sinc function was used to blur the line in Field synthesis, such that the lateral extend of the light-sheet shrinks to half and one quarter of the field of view, respectively. The corresponding conventional Bessel beam light-sheet was simulated by numerically scanning the beam along a tophat scan profile. Fourth and fifth row show simulations where Gaussian blurring (sigma of 0.5 and 1 pixel, respectively) was applied to the linescan in Field Synthesis. Bessel beam light-sheets were simulated by scanning the Bessel beam along a Gaussian line profile (i.e. a scaling factor was applied at each scan position that followed a Gaussian function).

Supplementary Figure 9 Comparison of photo-bleaching of 50nm fluorescent nanonspheres.

Volumetric imaging by dithered square lattice (magenta curve) and Field Synthesis square lattice light sheet microscopy. Shaded bands represent the 95th percentile of each photobleaching curve, center line shows mean value (11 beads were measured for each condition). Laser power was adjusted such that both imaging modes yielded comparable counts when consecutively imaging the same group of beads.

Supplementary Figure 10 Measurement of axial resolution of square lattice and Field Synthesis square lattice light sheet microscopy on clathrin-coated pits.

Human SK-MEL-2 cells labeled with Clathrin light-chain RFP, as imaged with square lattice in Field Synthesis (a) and in LLSM (b). Individual axial cross sections and maximum intensity projections are shown. In each case, one cross-sectional profile (i.e. from one measurent) and the Gaussian fitting curve are shown as an example. For Field Synthesis, the axial FWHM is 0.768 + −0.0255 um (mean and standard deviation, computed from 10 measurements). For conventional square lattice, the axial FWHM is 0.749 + −0.0302 um (mean and standard deviation, computed from 10 measurements). The experiment was repeated five times for each imaging mode, yielding similar results.

Supplementary Figure 11 Schematic drawing of the experimental setup.

(a) Microscope in the fluorescence imaging mode. (b) Microscope in the transmission imaging mode. The setup consists of two illumination paths, one (along the blue arrows) for conventional LLSM1 and the other (along the red arrows) for Field Synthesis LSFM. The optical path is selected with flip mirror 1 and 2. For routine inspection of the illumination wavefront, flip mirror 3 (not shown in a) redirects the light to camera 4, which is conjugate to the back-pupil plane of the excitation objective. PBS: Polarizing Beam Splitter; ND: Neutral Density; PZT: piezo actuator.

Supplementary Figure 12 Alternative beam expander for smaller field of view.

Top: schematic overview of the small FOV beam expander (area shaded in light green). Middle: comparison of a Field Synthesis Bessel beam light-sheet generated with the beam expander shown in Supplemenatry Figure 11 and the small FOV beam expander. Left side shows a cross-sectional profile of the light-sheets, right shows the intensity distribution in the propagation direction. Bottom: comparison of a Field Synthesis square lattice light-sheet generated by the beam expander shown in Supplemenatry Figure 11 and the small FOV beam expander. Left side shows cross-sectional profiles of the light-sheets, right shows the intensity distribution in the propagation direction. The Bessel light sheet thickness/length is 0.37 ± 0.013 µm/13.79 ± 0.266 µm (mean and standard deviation, n = 30) and 0.39 ± 0.017/13.10 ± 0.103 µm (mean and standard deviation, n = 18) with Big and small expansion, respectively. The square lattice light sheet thickness/length is 0.90 ± 0.009 µm/15.73 ± 0.543 µm (mean and standard deviation, n = 24) and 0.92 ± 0.009/15.56 ± 0.393 µm (mean and standard deviation, n = 18) with Big and small expansion, respectively. The graphs show the mean values from these measurements. The experiment was repeated three times for each imaging mode, yielding similar results.

Supplementary Figure 13 Timing diagram for Field synthesis.

For Bessel beam light-sheets (or any light-sheet that has a continuous pupil function), the Galvo mirror in the Field Synthesis illumination train (see also Supplemenatry Fig 1 and 2) is driven with a sawtooth signal with fine step size. For lattice light-sheets, the galvo is driven by a sawtooth signal that consists of two steps.

Supplementary information

Supplementary Text and Figures

Supplementary Figs. 1–13, Supplementary Notes 1–4 and Supplementary Tables 1–3

Reporting Summary

Supplementary Video 1

Simulation of Bessel beam light-sheet generation. The top row of the simulation shows an annular mask that is placed in a Fourier plane, which produces a Bessel beam in sample space in this example. In the middle column on the top, the instantaneous intensity of a Bessel beam in real space is shown as it is scanned laterally. On the right, the time-averaged intensity is shown. On the bottom, the field synthesis process is shown. On the left, a line scan over the pupil mask is shown, and the resulting instantaneous interference pattern in real space is shown in the middle. On the right, the time-averaged intensity is shown.

Supplementary Video 2

Simulation of square lattice light-sheet generation. The top row of the simulation shows the intensity distribution in the pupil plane that produces a square lattice pattern. In the middle column on the top, the instantaneous intensity of the squared lattice pattern in real space is shown as it is scanned laterally. On the right, the time-averaged intensity is shown. On the bottom, the field synthesis process is shown. On the left, a discreet line scan over the pupil mask is shown, and the resulting instantaneous interference pattern in real space is shown in the middle. On the right, the time-averaged intensity is shown.

Supplementary Video 3

EB3 dynamics imaged with Bessel beam and field synthesis Bessel beam light-sheet microscopy. A U2OS cell was labeled with EB3-mNeonGreen. For imaging by scanned Bessel beam light-sheet microscopy (left), the camera exposure time was set to 20 ms and the volumetric acquisition rate was 7.5 s per volume. For imaging by field synthesis Bessel beam light-sheet microscopy (right), the camera exposure time was set to 20 ms and the volumetric acquisition rate was 6.5 s per volume. Scale bar, 10 μm. We repeated the experiment by imaging four different cells in each imaging mode, and obtained similar results.

Supplementary Video 4

Three-dimensional time-lapse imaging of an MV3 cell imaged with square lattice light-sheet microscopy. An MV3 cell labeled with GFP AktPH biosensor and CAAX-Tdtomato membrane. The exposure time was 10 ms per frame, and the total stack acquisition time was 4.43 s. The biosensor is shown in magenta, and membrane in green. Scale bar, 10 μm. We repeated the experiment by imaging seven different cells, and obtained similar results.

Supplementary Video 5

Three-dimensional time-lapse imaging of an MV3 with field synthesis operating in a square lattice light-sheet microscopy mode. The same MV3 cell as shown in Supplementary Video 3, imaged after traditional lattice light-sheet microscopy operating in the square lattice mode. The exposure time was 10 ms per frame and the total stack acquisition time was 2.13 s. Biosensor is shown in magenta, and membrane in green. Scale bar, 10 μm. We repeated the experiment by imaging seven different cells, and obtained similar results.

Supplementary Video 6

Bleb and filopodia dynamics imaged with square lattice and field synthesis square lattice light-sheet microscopy. The same MV3 cell as shown in Supplementary Videos 4 and 5, but with a zoomed-in view on the bottom right corner of the cell. The left side shows 14 time points acquired with square lattice, and the right side shows 28 time points acquired with field synthesis, spanning ~57 s. Scale bar, 5 μm. We repeated the experiment by imaging seven different cells, and obtained similar results.

Supplementary Software

MATLAB code.

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Chang, B., Kittisopikul, M., Dean, K.M. et al. Universal light-sheet generation with field synthesis. Nat Methods 16, 235–238 (2019). https://doi.org/10.1038/s41592-019-0327-9

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