Rapid three-dimensional isotropic imaging of living cells using Bessel beam plane illumination

Transcription

1 Nature Methods Rapid three-dimensional isotropic imaging of living cells using beam plane illumination Thomas A Planchon, Liang Gao, Daniel E Milkie, Michael W Davidson, James A Galbraith, Catherine G Galbraith & Eric Betzig Note: Supplementary Videos 1 12 are available on the Nature Methods website. Supplementary figures and tables: Supplementary Figure 1 Supplementary Figure 2 Supplementary Figure 3 Supplementary Figure 4 Supplementary Figure 5 Supplementary Figure 6 Supplementary Figure 7 Supplementary Figure 8 Supplementary Figure 9 Supplementary Figure 10 Supplementary Figure 11 Supplementary Figure 12 Supplementary Figure 13 Supplementary Figure 14 beam cross-sectional characteristics for different imum numerical apertures of illumination. Theoretical and experimental curves of the longitudinal (y) extent of eleven beams of differing imum and minimum numerical apertures of illumination and one Gaussian beam of low numerical aperture Simplified schematic of the beam plane illumination microscope Virtual and actual views of the specimen chamber Optical sectioning capabilities of widefield microscopy and DSLM compared to the various modes of beam plane illumination microscopy Pre-deconvolution imum intensity projections in the xz plane for data shown in Fig. 2ae. Theoretical and experimental xz point spread functions for widefield microscopy, DSLM, and the various modes of beam plane illumination microscopy Axial theoretical and experimental point spread functions for widefield, confocal, and DSLM microscopy, as well as the various modes of beam plane illumination microscopy Theoretical and experimental intensity across a single beam, a swept sheet, and the overall axial PSF of the linear sheet mode Tradeoff between the width of the excitation profile of a swept sheet and the longitudinal extent of the beam Theoretical and experimental xz modulation transfer functions for widefield microscopy, DSLM, and the various modes of beam plane illumination microscopy Theoretical and experimental xz excitation point spread functions and corresponding modulation transfer functions for the single harmonic structured illumination mode as a function of the period of the beam exposure pattern Theoretical and experimental xz overall point spread functions and corresponding modulation transfer functions for the structured illumination mode with single harmonic excitation as a function of the period of the beam exposure pattern Planar ordered clusters of 304 nm diameter fluorescent beads resolved by single harmonic beam structured plane illumination

2 Supplementary Figure 15 Supplementary Figure 16 Supplementary Figure 17 Supplementary Figure 18 Supplementary Figure 19 Supplementary Figure 20 Supplementary Figure 21 Supplementary Figure 22 Supplementary Figure 23 Supplementary Figure 24 Supplementary Figure 25 Supplementary Figure 26 Supplementary Table 1 Three dimensional disordered groups of 352 nm diameter fluorescent beads resolved by single harmonic beam structured plane illumination Theoretical and experimental xz excitation patterns and corresponding modulation transfer functions for the multi- harmonic structured illumination mode as a function of the period of the beam exposure pattern Theoretical and experimental xz overall point spread functions and modulation transfer functions for the structured illumination mode with multi- harmonic excitation as a function of the period of the one-dimensional array of beams defining the excitation Image quality of antibody labeled microtubules in a fixed LLC-PK1 cell as a function of the fundamental period of excitation and number of phase-shifted images used in the structured illumination mode Pre- and post-deconvolution imum intensity projections in the xz plane of mitochondria in a fixed U2OS cell for the TPE-SI mode Comparison of post-deconvolution orthoslices in the xz plane of antibody-labeled microtubules in HeLa cells as obtained by confocal microscopy, DSLM, and various modes of beam plane illumination microscopy Schematic of the subsystem used to tile multiple images across the sensor of an scmos camera in the high speed configuration Three color multi-harmonic SI mode rendering of nuclear histones, the nuclear membrane, and the actin cytoskeleton in a fixed LLC-PK1 cell Two color TPE sheet mode rendering of filamentous actin and connexin-43 in a fixed HeLa cell Schematic of the instrument control architecture Timing diagrams showing waveforms for the swept sheet mode with tiling and the SI mode. Invariance of the three-dimensional PSF across an extended volume for different modes of beam plane illumination microscopy as demonstrated in xy and xz imum intensity projections of isolated fluorescent beads Full width at half ima (FWHM) of the axial point spread functions for various modes of beam plane illumination microscopy as compared to widefield, DSLM, and confocal microscopy Supplementary Table 2 Acquisition parameters for all images in Figures 2 6 Supplementary Table 3 Additional parameters for Supplementary Videos 2-12 Supplementary Table 4 Parts list for beam plane illumination microscopy

3 SUPPLEMENTARY FIGURE 1 minimum numerical apertures of illumination. beam cross-sectional characteristics for different imum and SUPPLEMETARY FIGURE 1. Theoretical and experimental beam intensity cross-sections (left), z axis linecuts (center), and modulation transfer functions (right) as functions of the imum and minimum numerical apertures of illumination shown at far left.

4 SUPPLEMENTARY FIGURE 2 Theoretical and experimental curves of the longitudinal (y) extent of eleven beams of differing imum and minimum numerical apertures of illumination and one Gaussian beam of low numerical aperture. SUPPLEMENTARY FIGURE 2. Theoretical (blue) and experimental (red) curves of the longitudinal (y) extent of eleven beams of differing imum and minimum numerical apertures of illumination and one Gaussian beam of low numerical aperture.

5 SUPPLEMENTARY FIGURE 3 Simplified schematic of the beam plane illumination microscope. SUPPLEMENTARY FIGURE 3. Simplified schematic of the beam plane illumination microscope. Light from laser (L) is reflected from x-axis galvanometer (XG) and transmitted in turn by relay lenses (RL) to z-axis galvanometer (ZG) and annular apodization mask (AM). XG, ZG, and AM are all at conjugate planes, so that the Gaussian beam falling on AM does not oscillate as XG and ZG are scanned. Similarly, AM is conjugate to the rear pupil plane of excitation objective (XO) so that the thin annular illumination transmitted through AM produces a beam within specimen (S) that translates along x and z without tilting. The light sheet created by scanning XG creates fluorescence at the focal plane of detection objective (DO), which is imaged at camera (C) by tube lens (TL). Different planes within S are imaged by translating DO with z-axis piezoelectric collar (ZP) in synchronization with the z axis motion of the beam provided by ZG. XO, DO and S reside in medium-filled specimen chamber (SC), and epiobjective (EO) provides a conventional view of specimen (S), for view finding purposes.

6 SUPPLEMENTARY FIGURE 4 Virtual and actual views of the specimen chamber. SUPPLEMENTARY FIGURE 4. Virtual and actual views of the specimen chamber. (a) Virtual view through translucent specimen chamber (SC) showing orthogonal excitation and detection objectives (EO, DO) and specimen holder (SH) at 45 to each. (b) Actual view through epi-port (EP) after removal of epi-objective, showing converging and then expanding light cone (LC) and region of beam excitation (BB) near the focus, as well water surface (WS) within the chamber.

7 SUPPLEMENTARY FIGURE 5 Optical sectioning capabilities widefield microscopy and DSLM compared to the various modes of beam plane illumination microscopy. SUPPLEMENTARY FIGURE 5. Optical sectioning capabilities of widefield microscopy and digital scanned light sheet microscopy (DSLM, NAexc 0.2 ) compared to that of various modes of beam plane illumination microscopy ( NA 0.53 ), at three different planes through mitochondria-labeled fixed exc LLC-PK1 cells. The illuminated region shifts in y at different z planes, since the sample is mounted at 45 to the y and z axes. Scale bar: 10 m.

8 SUPPLEMENTARY FIGURE 6 data shown in Fig. 2a-e. Pre-deconvolution imum intensity projections in the xz plane for the SUPPLEMENTARY FIGURE 6. Pre-deconvolution imum intensity projections in the xz plane for the data shown in Fig. 2a-e. Excitation parameters: min NA 0.60, NA 0.58 for the linear sheet, 3 phase single harmonic SI, and 9 phase multi-harmonic SI cases; min NA 0.53, NA 0.49 for the TPE and TPE-SI cases; NA 0.20 for the DSLM case. NAdetect 0.80 in all cases. Scale bar: 10 m.

9 SUPPLEMENTARY FIGURE 7 Theoretical and experimental xz point spread functions for widefield microscopy, DSLM, and the various modes of beam plane illumination microscopy. SUPPLEMENTARY FIGURE 7. Theoretical and experimental xz point spread functions for widefield microscopy, DSLM, and the various modes of beam plane illumination microscopy. Excitation parameters: min NA 0.60, NA 0.58, 488 nm for the linear sheet, 3 phase single harmonic SI, and 9 phase multi-harmonic SI cases; exc min NA 0.53, NA 0.49, 910 nm for the TPE and TPE-SI cases; NA 0.20, 488 nm for the DSLM case. SI TPE excitation period = 0.79 and 2.4 m for the 3 and 9 phase SI cases, respectively, and 1.0 for the TPE SI 5 phase case. NAdetect 0.80 in all cases. exc

10 SUPPLEMENTARY FIGURE 8 Axial theoretical and experimental point spread functions for widefield, confocal, and DSLM microscopy, as well as the various modes of beam plane illumination microscopy. SUPPLEMENTARY FIGURE 8. Axial theoretical and experimental point spread functions for widefield, confocal, and DSLM microscopy, as well as the various modes of beam plane illumination microscopy. Excitation and detection conditions for widefield, DSLM, and all modes is the same as in Supplementary Fig. 7; condition are NA 1.20, 488 nm for the confocal LSM 510 and LSM 5 LIVE. Measurements in (a), (b) are from 100 nm beads, 200 nm beads were used in (c), and curves in (d) represent averages across 40 microtubules each as described in Fig 3. Full widths at half ima measured from these graphs are summarized in Supplementary Table 1. exc

11 SUPPLEMENTARY FIGURE 9 Theoretical and experimental intensity across a single beam, a swept sheet, and the overall axial PSF of the linear sheet mode SUPPLEMENTARY FIGURE 9. Theoretical and experimental intensity across: (a) a single beam; (b) a swept sheet; and (c) the overall axial PSF of the linear sheet mode, showing the excitation tails in the swept sheet resulting from the side lobes, and the mitigation of these tails in the overall PSF due to the axial envelope of the detection PSF. exc 488 nm and NAdetect min NA 0.60, NA 0.58,

12 SUPPLEMENTARY FIGURE 10 sheet and the longitudinal extent of the beam. Tradeoff between the width of the excitation profile of a swept SUPPLEMENTARY FIGURE 10. Tradeoff between (a) the width of the excitation profile of a swept sheet and (b) the longitudinal extent of the beam. Beam illumination parameters: min NA 0.60 in all cases, NA 0.58 (blue), 0.56 (green), 0.52 (red), 0.45 (purple), 0.30 (olive), and 0.00 (cyan, Gaussian beam case).

13 SUPPLEMENTARY FIGURE 11 Theoretical and experimental xz modulation transfer functions for widefield microscopy, DSLM, and the various modes of beam plane illumination microscopy. SUPPLEMENTARY FIGURE 11. Theoretical and experimental xz modulation transfer functions for widefield microscopy, DSLM, and the various modes of beam plane illumination microscopy, calculated from the corresponding point spread functions in Supplementary Fig. 7. All MTFs are normalized to the imum frequency Abbe s Law, with exc or k NA k NA 2 exc / NA 0.8 for the excitation objective. exc 2 exc / TPE / 2 set by

14 SUPPLEMENTARY FIGURE 12 Theoretical and experimental xz excitation patterns and corresponding modulation transfer functions for the single harmonic structured illumination mode as a function of the period of the beam exposure pattern. SUPPLEMENTARY FIGURE 12. Theoretical and experimental xz excitation patterns and corresponding modulation transfer functions for the single harmonic structured illumination mode as a function of the period of the beam exposure pattern. beam illumination parameters: min NA 0.60, NA All MTFs are normalized to the imum frequency k 2 NAexc / set by Abbe s Law, with NA 0.8 for the excitation objective. exc

15 SUPPLEMENTARY FIGURE 13 Theoretical and experimental xz overall point spread functions and corresponding modulation transfer functions for the structured illumination mode with single harmonic excitation as a function of the period of the the beam exposure pattern. SUPPLEMENTARY FIGURE 13 Theoretical and experimental xz overall point spread functions and corresponding modulation transfer functions for the structured illumination mode with single harmonic excitation as a function of the period of the the beam exposure pattern. The axial resolution improves as the excitation period increases from the minimum of allowed by diffraction /2NA (0.407 m for 488 nm, NA 0.6 in this example) up to the imum of NA for / which the excitation pattern still contains only a single harmonic in the scan direction. Furthermore, as the period increases, the axial resolution increasingly exceeds the classical diffraction limit ( k k here, since all MTFs are normalized to k 2 NA / for the NA 0.8 z / z 0.75 excitation objective). z exc exc

16 SUPPLEMENTARY FIGURE 14 by single harmonic beam structured plane illumination. Planar ordered clusters of 304 nm diameter fluorescent beads resolved SUPPLEMENTARY FIGURE 14 Planar ordered clusters of 304 nm diameter fluorescent beads resolved by single harmonic beam structured plane illumination. Top row: xy (left) and xz (right) imum intensity projections and volume rendering (center) in the x sample plane at 45 to the y and z microscope coordinate axes, after filtering to remove noise beyond the Abbe limit in xy, and twice the Abbe limit in z. Middle row: xy (left), x (center), and xz (right) orthoslices through the 3D MTF calculated from raw 3D image stack. Green ovals represent the Abbe limit in xyz space, and blue ovals represent an extended limit based on the Abbe limit in xy (0.32 m for det 0.52 m, NAdet 0.8 ), and twice the Abbe limit in z (0.474 m for exc m, NA ). Bottom row: corresponding views after deconvolution using a theoretical PSF. Scale bar at bottom center: 2 m.

17 SUPPLEMENTARY FIGURE 15 Three dimensional disordered groups of 352 nm diameter fluorescent beads resolved by single harmonic beam structured plane illumination. SUPPLEMENTARY FIGURE 15 Three dimensional disordered groups of 352 nm diameter fluorescent beads resolved by single harmonic beam structured plane illumination. Top row: volume renderings as viewed nearly parallel to the sample plane for both the original and deconvolved data (scale bar: 2 m). Bottom row: Orthoslices through the 3D MTF calculated from the 3D image stack. Imaging conditions and figure descriptions are identical to Supplementary Fig. XX.

18 SUPPLEMENTARY FIGURE 16 Theoretical and experimental xz excitation patterns and corresponding modulation transfer functions for the multi-harmonic structured illumination mode as a function of the period of the beam exposure pattern. SUPPLEMENTARY FIGURE 16 Theoretical and experimental xz excitation patterns and corresponding modulation transfer functions for the multi-harmonic structured illumination mode as a function of the period of the beam exposure pattern. A new harmonic is introduced into the excitation pattern whenever the period increases by the Abbe limit of /2NA. beam illumination parameters: min NA 0.60, NA All MTFs are normalized to the imum frequency 2 exc / k NA set by Abbe s Law, with NA 0.8 for the excitation objective. exc

19 SUPPLEMENTARY FIGURE 17 Theoretical and experimental xz overall point spread functions and modulation transfer functions for the structured illumination mode with multi-harmonic excitation as a function of the period of the one dimensional array of beams defining the excitation. SUPPLEMENTARY FIGURE 17 Theoretical and experimental xz overall point spread functions and corresponding modulation transfer functions for the structured illumination mode with multi-harmonic excitation as a function of the period of the one dimensional array of beams defining the excitation. As shown, comparable axial resolution is obtained for all periods despite their differing number of harmonics H, provided that N H 2 images of equal phase shift are used to reconstruct each optically sectioned SI image. beam illumination parameters: NA 0.60, NA All MTFs are normalized to the imum frequency k 2 NAexc / set by Abbe s min Law, with NA 0.8 for the excitation objective. exc

20 SUPPLEMENTARY FIGURE 18 Image quality of antibody labeled microtubules in a fixed LLC-PK1 cell as a function of the fundamental period of excitation and number of phase-shifted images used in the structured illumination mode. SUPPLEMENTARY FIGURE 18. Image quality of antibody labeled microtubules in a fixed LLC-PK1 cell as a function of the fundamental period of excitation and number of phase-shifted images used in the structured illumination mode. All are imum intensity projections normalized to the same scale. The SI signal and signal-to-noise ratio increases with an increasing number N of phase shifted images, even though the total integration time across all N images is the same in each case. min NA 0.52, NA 0.50, NA detect Parameters:

21 SUPPLEMENTARY FIGURE 19 Pre- and post-deconvolution imum intensity projections in the xz plane of mitochondria in a fixed U2OS cell for the TPE-SI mode. SUPPLEMENTARY FIGURE 19. (a) Pre- and (b) post-deconvolution imum intensity projections in the xz plane of mitochondria in a fixed U2OS cell for the TPE-SI mode. NA 0.49, 910 nm. Scale bar: 10 m. min TPE NA 0.53,

22 SUPPLEMENTARY FIGURE 20 Comparison of post-deconvolution orthoslices in the xz plane of antibody-labeled microtubules in HeLa cells as obtained by confocal microscopy, DSLM, and various modes of beam plane illumination microscopy. SUPPLEMENTARY FIGURE 20. Comparison of post-deconvolution orthoslices in the xz plane of antibody-labeled microtubules in HeLa cells as obtained by confocal microscopy, DSLM, and various modes of beam plane illumination microscopy: (a) point scanning confocal microscopy (Zeiss LSM 510, 40x/1.20 W objective, 1 Airy unit filtering); (b) line scanning confocal microscopy (Zeiss LSM 5 Live, same objective and filtering); (c) DSLM ( NAexc 0.20 ); (d) beam single harmonic SI mode (3 phases, 0.9 m period, phases, 2.7 m period min NA 0.52, NA 0.50 ); (e) beam multi-harmonic SI mode (9 min mode ( NA 0.53, NA 0.49 ). min NA 0.52, NA 0.50 ); (f) beam two-photon swept sheet

23 SUPPLEMENTARY FIGURE 21 Schematic of the subsystem used to tile multiple images across the sensor of an scmos camera in the high speed configuration. SUPPLEMENTARY FIGURE 21. Schematic of the subsystem used to tile multiple images across the sensor of an scmos camera in the high speed configuration. Fluorescence collected by detection objective (DO) is focused by tube lens (TL) for form an image at the plane of adjustable slit (AS). The image cropped by AS is re-imaged by relay lenses (RL) onto scientific CMOS camera (C). A tiling galvanometer (TG) is placed at the plane between the relay lenses RL that is conjugate to the back focal plane of DO. Multiple images can be exposed across the sensor of C by changing the angle of the mirror of TG, and then read out in parallel to exploit the full speed of C.

24 SUPPLEMENTARY FIGURE 22 Three-color multi-harmonic SI mode rendering of nuclear histones, the nuclear membrane, and the actin cytoskeleton in a fixed LLC-PK1 cell. SUPPLEMENTARY FIGURE 22. Three-color multi-harmonic SI mode rendering of nuclear histones (blue), the nuclear membrane (red), and the actin cytoskeleton (green) in a fixed LLC-PK1 cell. Histones are labeled with mneptune / H2B ( exc 561 nm, 9 phases, 2.7 m period); the nuclear envelope is labeled with memerald / lamin B1 ( exc 488 nm, 9 phases, 2.4 m period); and the actin cytoskeleton is labeled with Alexa 568 / phalloidin ( exc 561 nm, 9 phases, 2.7 m period). Scale bar: 10 m.

25 SUPPLEMENTARY FIGURE 23 Two-color TPE sheet mode rendering of filamentous actin and connexin- 43 in a fixed HeLa cell. SUPPLEMENTARY FIGURE 23. Two-color TPE sheet mode rendering of filamentous actin (orange) and connexin-43 (green) in a fixed HeLa cell. Gap junctions are labeled with mcerulean3 / connexin-43 ( 858 nm), and filamentous actin is labeled with memerald / Lifeact (orange, 910 nm). TPE TPE

26 SUPPLEMENTARY FIGURE 24 Schematic of the instrument control architecture. SUPPLEMENTARY FIGURE 24. Schematic of the instrument control architecture.

27 SUPPLEMENTARY FIGURE 25 Timing diagrams showing waveforms for the swept sheet mode with tiling and the SI mode. SUPPLEMENTARY FIGURE 25. Timing diagrams showing waveforms for (a) the swept sheet mode with tiling and (b) the SI mode.

28 SUPPLEMENTARY FIGURE 26 Invariance of the three-dimensional PSF across an extended volume for different modes of beam plane illumination microscopy as demonstrated in xy and xz imum intensity projections of isolated fluorescent beads. SUPPLEMENTARY FIGURE 26. Invariance of the three-dimensional PSF across an extended volume for different modes of beam plane illumination microscopy as demonstrated in xy and xz imum intensity projections of isolated fluorescent beads.

29 SUPPLEMENTARY TABLE 1 Full width at half ima (FWHM) of the axial point spread functions for various modes of beam plane illumination microscopy as compared to widefield, DSLM, and confocal microscopy. Imaging Modality Theoretical FWHM FWHM from 100 nm bead Averaged FWHM from 40 microtubules widefield (0.8 NA) 1.76 m 1.85 m -- point scan confocal (LSM 510, 1.2 NA) 0.44 m 0.68 m (1.41 ± 0.13) m line scan confocal (LSM 5 LIVE, 1.2 NA) 0.44 m 0.76 m (1.88 ± 0.23) m DSLM 1.02 m 1.20 m (1.42 ± 0.16) m swept sheet 0.47 m 1.02 m (2.12 ± 0.21) m TPE sheet 0.45 m 0.49 m (0.57 ± 0.09) m TPE SI, 5 phases 0.47 m 0.53 m -- single harmonic SI (3 phases) 0.26 m 0.27 m (0.37 ± 0.05) m multi-harmonic SI (9 phases) 0.27 m 0.29 m (0.47 ± 0.10) m

30 SUPPLEMENTARY TABLE 2 Acquisition parameters for all images in Figs Image Property Fig. 2a Fig. 2b Fig. 2c Fig. 2d modality Widefield sheet SI 3 phases SI 9 phases annulus NA / NA min / / /0.58 cell line U2OS (fixed) U2OS (fixed) U2OS (fixed) U2OS (fixed) fluorescent label memerald/mito memerald/mito memerald/mito memerald/mito excitation wavelength 488 nm 488 nm 488 nm 488 nm back focal plane power 300 W 30 W 80 W 70 W emission filter FF01-525/50-25 FF01-525/50-25 FF01-525/50-25 FF01-525/50-25 image volume ( m 3 ) voxel volume (nm 3 ) CCD frame rate 50 ms 50 ms 150 ms 100 ms single plane frame rate 50 ms 50 ms 450 ms 900 ms volume acquisition time 17.5 sec 17.5 sec 157 sec 315 sec Image Property Fig. 2e Fig. 2f Fig. 2g modality TPE sheet SI 9 phases TPE sheet annulus NA / NA min 0.53/ / /0.56 cell line U2OS (fixed) U2OS (live) LLC-PK1 (live) fluorescent label memerald/mito memerald/map4 memerald/mito excitation wavelength 910 nm 488 nm 920 nm back focal plane power 100 mw 10 W 60 mw emission filter FF01-525/50-25 FF01-523/ FF01-525/50-25 image volume ( m 3 ) voxel volume (nm 3 ) CCD frame rate 80 ms 25 ms 30 ms single plane frame rate 80 ms 225 ms 30 ms volume acquisition time 40 sec 72 sec 10.2 sec

31 SUPPLEMENTARY TABLE 2 (cont d) Acquisition parameters for all images in Figs Image Property Fig. 3a Fig. 3b Fig. 3c Fig. 3d modality Zeiss LSM 510 Zeiss LSM 5 LIVE DSLM SI 3 phases annulus NA / NA min 1.20/ / / /0.50 cell line HeLa (fixed) HeLa (fixed) HeLa (fixed) HeLa (fixed) fluorescent label AF488 antitubulin AF488 anti-tubulin AF488 anti-tubulin AF488 antitubulin excitation wavelength 488 nm 488 nm 488 nm 488 nm back focal plane power 44 W 290 W 160 W 85 W emission filter FITC / GFP set FITC / GFP set FF01-525/50-25 FF01-525/50-25 image volume ( m 3 ) voxel volume (nm 3 ) CCD frame rate ms 200 ms single plane frame rate 586 ms 16.7 ms 50 ms 600 ms volume acquisition time 234 sec 6.7 sec 20 sec 240 s Image Property modality Fig. 3e TPE sheet Fig. 3h (top row) TPE sheet Fig. 3h (center row) Zeiss LSM 5 LIVE Fig. 3h (bottom row) Zeiss LSM 5 LIVE annulus NA / NA min 0.53/ / cell line HeLa (fixed) LLC-PK1 (live) LLC-PK1 (live) LLC-PK1 (live) fluorescent label AF488 antitubulin memerald/mito memerald/mito memerald/mito excitation wavelength 770 nm 920 nm 488 nm 488 nm back focal plane power 100 mw 60 mw 14 W 14 W emission filter FF01-525/50-25 FF01-525/50-25 FITC / GFP set FITC / GFP set image volume ( m 3 ) voxel volume (nm 3 ) CCD frame rate 50 ms 30 ms single plane frame rate 50 ms 30 ms 33 ms 33 ms volume acquisition time 20 sec 10.2 sec 9.8 sec 2.3 sec

32 SUPPLEMENTARY TABLE 2 (cont d) Acquisition parameters for all images in Figs Image Property Fig. 4a Fig. 4b Fig. 4c Fig. 5 modality SI 9 Phases TPE sheet TPE sheet TPE sheet annulus NA / NA min 0.62/ / / /0.57 cell line U2OS (live) HeLa (live) HeLa (live) LLC-PK1 (live) fluorescent label tdtomato/er memerald/lifeact memerald/c-src memerald/h2b excitation wavelength 561 nm 920 nm 920 nm 920 nm back focal plane power 5 W 200 mw 200 mw 220 mw emission filter FF01-525/50-25 FF01-525/50-25 FF01-525/50-25 FF01-525/50-25 image volume ( m 3 ) voxel volume (nm 3 ) CCD frame rate 17 ms 17 ms 37 ms 26 ms single plane frame rate 153 ms 17 ms 37 ms 5.2 ms volume acquisition time 50 sec 3.5 sec 10.2 sec 1.05 sec Image Property Fig. 6a Fig. 6b Sup Fig. 22 Sup Fig. 23 modality SI 9 phases TPE sheet SI 9 phases TPE sheet annulus NA / NA min 0.62/ / / /0.49 cell line U2OS (live) LLC-PK1 (live) LLC-PK1 (fixed) HeLa (fixed) fluorescent label memerald/map4 memerald/h2b memerald/h2b memerald/mann2 memerald/lamin B1 AF568 phalloidin mneptune/h2b mcerulean3/ Cx43 memerald/lifeact excitation wavelength 488/561nm 920 nm 488/561/561 nm 860/910 nm back focal plane power 10/5 W 50 mw 60/60/150 W 110/100 mw emission filter FF01-523/ FF01-525/50-25 FF01-525/50-25 FF01-593/40-25 LP02-633RS-25 FF01-470/ FF01-525/50-25 image volume ( m 3 ) voxel volume (nm 3 ) CCD frame rate 25 ms 33 ms 100 ms 80 ms single plane frame rate 225 ms 33 ms 900 ms 80 ms volume acquisition time 72 sec/color 10.7 sec 450 sec/color 40 sec/color

33 SUPPLEMENTARY TABLE 3 Additional parameters for Supplementary Videos Supplementary Video Corresponding Figure Camera Parameters Supplementary Video 1 Supp. Fig. 16 Andor ixon 885 Single stack 1 Supplementary Video 2 Fig. 2f Hamamatsu ORCA-flash 2.8 Supplementary Video 3 Fig. 3h Andor ixon 885 Supplementary Video 4 Fig. 4a Andor ixon 885 Supplementary Video 5 Supplementary Video 6 Fig. 4b Fig. 4c Supplementary Video 7 Fig. 5 Supplementary Video 8 -- Supplementary Video 9 Fig. 6a Hamamatsu ORCA-flash 2.8 Hamamatsu ORCA-flash 2.8 Hamamatsu ORCA-flash 2.8 Hamamatsu ORCA-flash 2.8 Hamamatsu ORCA-flash 2.8 No. of Tiles Single stack sec/stack, every 20 sec 300 stacks 50 sec/stack, every 60 sec 45 stacks 3.5 sec/stack, every 6 sec 100 stacks 10.2 sec/stack, every 12 sec 70 stacks 1.05 sec/stack 1-15 stack, every 21 sec stack, continuous stack, every 2 sec stack, every 21 sec 201 stacks total 7.3 ms/plane, continuous 7025 planes 2000 planes are shown in the movie Single stack 1 Supplementary Video 10 Supp. Fig. 22 Andor ixon 885 Single stack 1 Supplementary Video 11 Supp. Fig. 23 Andor IXon 885 Single stack Supplementary Video 12 Fig. 6b Andor IXon sec/stack, every 20 sec 122 stacks 1

34 SUPPLEMENTARY TABLE 4 Parts list for beam plane illumination microscopy Parts in Supp. Figs. 3 and 21 Vendor Part number laser L Coherent, Inc. Cube C Sapphire 488 HP Sapphire 561 LP Cube E Chameleon Ultra II galvanometers XG, ZG Cambridge Technology 6215HB annulus mask AM PhotoSciences custom design objectives XO, DO Nikon CFI APO 40x/0.80 W NIR objective EO Olympus UMPLFLN 10x/0.3 W objective piezo ZP Physik Instrumente P-726 PIFOC camera C Andor Hamamatsu ixon+ 885 Orca Flash 2.8 adjustable slit AS Thorlabs VA100 Other elements acousto-optic tunable filter AA Opto-Electronic AOTFnC TN Pockels cell Conoptics LA-02 axicon Thorlabs AX2505-B cover slip Warner Instruments CS-18R15 FPGA card National Instruments PCIe-7852R scaling amplifiers Stanford Research Systems SIM983 control computer Supermicro 7046GT-TRF chassis X8DTG-QF backplane Intel Xeon X GHz processor Kapton heater tapes Omega KHLV-0502/5-P RTD probe Omega RTD-2-1PT100KN2515 PTD controller Omega CN4216-R1-R2