Rapid three-dimensional isotropic imaging of living cells ... · Rapid three-dimensional isotropic...

Nature Methods

Rapid three-dimensional isotropic imaging of living cells using Bessel 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 Bessel beam cross-sectional characteristics for different maximum numerical apertures of illumination.

Supplementary Figure 2 Theoretical and experimental curves of the longitudinal (y) extent of eleven Bessel beams of differing maximum and minimum numerical apertures of illumination and one Gaussian beam of low numerical aperture

Supplementary Figure 3 Simplified schematic of the Bessel beam plane illumination microscope

Supplementary Figure 4 Virtual and actual views of the specimen chamber

Supplementary Figure 5 Optical sectioning capabilities of widefield microscopy and DSLM compared to the various modes of Bessel beam plane illumination microscopy

Supplementary Figure 6 Pre-deconvolution maximum intensity projections in the xz plane for data shown in Fig. 2a-e.

Supplementary Figure 7 Theoretical and experimental xz point spread functions for widefield microscopy, DSLM, and the various modes of Bessel 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 Bessel beam plane illumination microscopy

Supplementary Figure 9 Theoretical and experimental intensity across a single Bessel beam, a swept Bessel sheet, and the overall axial PSF of the linear Bessel sheet mode

Supplementary Figure 10 Tradeoff between the width of the excitation profile of a swept Bessel sheet and the longitudinal extent of the Bessel beam

Supplementary Figure 11 Theoretical and experimental xz modulation transfer functions for widefield microscopy, DSLM, and the various modes of Bessel beam plane illumination microscopy

Supplementary Figure 12 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 Bessel 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 Bessel beam exposure pattern

Supplementary Figure 14 Planar ordered clusters of 304 nm diameter fluorescent beads resolved by single harmonic Bessel beam structured plane illumination

Nature Methods: doi.10.1038/nmeth.1586

Supplementary Figure 15 Three dimensional disordered groups of 352 nm diameter fluorescent beads resolved by single harmonic Bessel beam structured plane illumination

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 Bessel beam exposure pattern

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 Bessel beams defining the excitation

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 19 Pre- and post-deconvolution maximum intensity projections in the xz plane of mitochondria in a fixed U2OS cell for the Bessel TPE-SI mode

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 Bessel beam plane illumination microscopy

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 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 23 Two color TPE sheet mode rendering of filamentous actin and connexin-43 in a fixed HeLa cell

Supplementary Figure 24 Schematic of the instrument control architecture

Supplementary Figure 25 Timing diagrams showing waveforms for the swept sheet mode with tiling and the SI mode.

Supplementary Figure 26 Invariance of the three-dimensional PSF across an extended volume for different modes of Bessel beam plane illumination microscopy as demonstrated in xy and xz maximum intensity projections of isolated fluorescent beads

Supplementary Table 1 Full width at half maxima (FWHM) of the axial point spread functions for various modes of Bessel 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 Bessel beam plane illumination microscopy


SUPPLEMENTARY FIGURE 1 Bessel beam cross-sectional characteristics for different maximum and

minimum numerical apertures of illumination.

SUPPLEMETARY FIGURE 1. Theoretical and experimental Bessel beam intensity cross-sections (left), z

axis linecuts (center), and modulation transfer functions (right) as functions of the maximum and

minimum numerical apertures of illumination shown at far left.


SUPPLEMENTARY FIGURE 2 Theoretical and experimental curves of the longitudinal (y) extent of

eleven Bessel beams of differing maximum 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 Bessel beams of differing maximum and minimum numerical apertures of illumination

and one Gaussian beam of low numerical aperture.


SUPPLEMENTARY FIGURE 3 Simplified schematic of the Bessel beam plane illumination microscope.

SUPPLEMENTARY FIGURE 3. Simplified schematic of the Bessel 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 Bessel 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 Bessel beam provided by ZG. XO, DO and S reside in medium-filled specimen chamber (SC), and epi-

objective (EO) provides a conventional view of specimen (S), for view finding purposes.


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 Bessel beam

excitation (BB) near the focus, as well water surface (WS) within the chamber.


SUPPLEMENTARY FIGURE 5 Optical sectioning capabilities widefield microscopy and DSLM compared

to the various modes of Bessel beam plane illumination microscopy.

SUPPLEMENTARY FIGURE 5. Optical sectioning capabilities of widefield microscopy and digital scanned

light sheet microscopy (DSLM, 0.2excNA ) compared to that of various modes of Bessel beam plane

illumination microscopy ( max 0.53excNA ), at three different planes through mitochondria-labeled fixed

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.


SUPPLEMENTARY FIGURE 6 Pre-deconvolution maximum intensity projections in the xz plane for the

data shown in Fig. 2a-e.

SUPPLEMENTARY FIGURE 6. Pre-deconvolution maximum intensity projections in the xz plane for the

data shown in Fig. 2a-e. Excitation parameters: max 0.60BesselNA , min 0.58BesselNA for the Bessel linear

sheet, 3 phase single harmonic SI, and 9 phase multi-harmonic SI cases; max 0.53BesselNA ,

min 0.49BesselNA for the Bessel TPE and TPE-SI cases; 0.20NA for the DSLM case.

detect 0.80NA

in all cases. Scale bar: 10 m.


SUPPLEMENTARY FIGURE 7 Theoretical and experimental xz point spread functions for widefield

microscopy, DSLM, and the various modes of Bessel beam plane illumination microscopy.

SUPPLEMENTARY FIGURE 7. Theoretical and experimental xz point spread functions for widefield

microscopy, DSLM, and the various modes of Bessel beam plane illumination microscopy. Excitation

parameters: max 0.60BesselNA , min 0.58BesselNA , 488exc nm for the Bessel linear sheet, 3 phase

single harmonic SI, and 9 phase multi-harmonic SI cases; max 0.53BesselNA , min 0.49BesselNA ,

910TPE nm for the Bessel TPE and TPE-SI cases; 0.20NA , 488exc nm for the DSLM case. SI

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. detect 0.80NA in all cases.


SUPPLEMENTARY FIGURE 8 Axial theoretical and experimental point spread functions for widefield,

confocal, and DSLM microscopy, as well as the various modes of Bessel 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 Bessel beam plane illumination

microscopy. Excitation and detection conditions for widefield, DSLM, and all Bessel modes is the same

as in Supplementary Fig. 7; condition are 1.20NA , 488exc 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 maxima

measured from these graphs are summarized in Supplementary Table 1.


SUPPLEMENTARY FIGURE 9 Theoretical and experimental intensity across a single Bessel beam, a

swept Bessel sheet, and the overall axial PSF of the linear Bessel sheet mode

SUPPLEMENTARY FIGURE 9. Theoretical and experimental intensity across: (a) a single Bessel beam;

(b) a swept Bessel sheet; and (c) the overall axial PSF of the linear Bessel sheet mode, showing the

excitation tails in the swept sheet resulting from the Bessel side lobes, and the mitigation of these tails

in the overall PSF due to the axial envelope of the detection PSF. max 0.60BesselNA , min 0.58BesselNA ,

488exc nm and detect 0.80NA .


SUPPLEMENTARY FIGURE 10 Tradeoff between the width of the excitation profile of a swept Bessel

sheet and the longitudinal extent of the Bessel beam.

SUPPLEMENTARY FIGURE 10. Tradeoff between (a) the width of the excitation profile of a swept

Bessel sheet and (b) the longitudinal extent of the Bessel beam. Beam illumination parameters:

max 0.60BesselNA in all cases, min 0.58BesselNA (blue), 0.56 (green), 0.52 (red), 0.45 (purple), 0.30

(olive), and 0.00 (cyan, Gaussian beam case).


SUPPLEMENTARY FIGURE 11 Theoretical and experimental xz modulation transfer functions for

widefield microscopy, DSLM, and the various modes of Bessel beam plane illumination microscopy.

SUPPLEMENTARY FIGURE 11. Theoretical and experimental xz modulation transfer functions for

widefield microscopy, DSLM, and the various modes of Bessel beam plane illumination microscopy,

calculated from the corresponding point spread functions in Supplementary Fig. 7. All MTFs are

normalized to the maximum frequency max

max 2 /exc exck NA or max

max 2 / / 2exc TPEk NA set by

Abbe’s Law, with max 0.8excNA for the Bessel excitation objective.


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 Bessel 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 Bessel beam exposure pattern. Bessel beam illumination parameters: max 0.60BesselNA ,

min 0.58BesselNA . All MTFs are normalized to the maximum frequency max

max 2 /exck NA set by Abbe’s

Law, with max 0.8excNA for the Bessel excitation objective.


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 Bessel beam exposure pattern.


corresponding modulation transfer functions for the structured illumination mode with single harmonic

excitation as a function of the period of the the Bessel beam exposure pattern. The axial resolution

improves as the excitation period increases from the minimum of max/ 2 BesselNA allowed by diffraction

(0.407 m for 488 nm, max 0.6BesselNA in this example) up to the maximum of max/ BesselNA 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 (

max/ 0.75z zk k here, since all MTFs are normalized to max max2 /z exck NA for the max 0.8excNA Bessel

excitation objective).


SUPPLEMENTARY FIGURE 14 Planar ordered clusters of 304 nm diameter fluorescent beads resolved

by single harmonic Bessel beam structured plane illumination.

SUPPLEMENTARY FIGURE 14 Planar ordered clusters of 304 nm diameter fluorescent beads resolved

by single harmonic Bessel beam structured plane illumination. Top row: xy (left) and xz (right) maximum

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, det 0.8NA ),

and twice the Abbe limit in z (0.474 m for 0.488exc m, max 0.515BesselNA ). Bottom row:

corresponding views after deconvolution using a theoretical PSF. Scale bar at bottom center: 2 m.


SUPPLEMENTARY FIGURE 15 Three dimensional disordered groups of 352 nm diameter fluorescent

beads resolved by single harmonic Bessel beam structured plane illumination.

SUPPLEMENTARY FIGURE 15 Three dimensional disordered groups of 352 nm diameter fluorescent

beads resolved by single harmonic Bessel 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.



modulation transfer functions for the multi-harmonic structured illumination mode as a function of the

period of the Bessel beam exposure pattern.


modulation transfer functions for the multi-harmonic structured illumination mode as a function of the

period of the Bessel beam exposure pattern. A new harmonic is introduced into the excitation pattern

whenever the period increases by the Abbe limit of max/ 2NA . Bessel beam illumination parameters:

max 0.60BesselNA , min 0.58BesselNA . All MTFs are normalized to the maximum frequency

max

max 2 /exck NA set by Abbe’s Law, with max 0.8excNA for the Bessel excitation objective.



modulation transfer functions for the structured illumination mode with multi-harmonic excitation as a

function of the period of the one dimensional array of Bessel beams defining the excitation.


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 Bessel beams defining the

excitation. As shown, comparable axial resolution is obtained for all periods despite their differing

number of harmonics H, provided that 2N H images of equal phase shift are used to reconstruct

each optically sectioned SI image. Bessel beam illumination parameters: max 0.60BesselNA ,

min 0.58BesselNA . All MTFs are normalized to the maximum frequency max

max 2 /exck NA set by Abbe’s

Law, with max 0.8excNA for the Bessel excitation objective.


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 maximum 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. Parameters:

max 0.52BesselNA , min 0.50BesselNA , 0.80detectNA .


SUPPLEMENTARY FIGURE 19 Pre- and post-deconvolution maximum intensity projections in the xz

plane of mitochondria in a fixed U2OS cell for the Bessel TPE-SI mode.

SUPPLEMENTARY FIGURE 19. (a) Pre- and (b) post-deconvolution maximum intensity projections in the

xz plane of mitochondria in a fixed U2OS cell for the Bessel TPE-SI mode. max 0.53BesselNA ,

min 0.49BesselNA , 910TPE nm. Scale bar: 10 m.


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 Bessel 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 Bessel 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 ( 0.20excNA ); (d) Bessel beam single harmonic SI mode (3

phases, 0.9 m period, max 0.52BesselNA , min 0.50BesselNA ); (e) Bessel beam multi-harmonic SI mode (9

phases, 2.7 m period max 0.52BesselNA , min 0.50BesselNA ); (f) Bessel beam two-photon swept sheet

mode ( max 0.53BesselNA , min 0.49BesselNA ).


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.


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 ( 561exc nm, 9 phases, 2.7 m period); the nuclear envelope is labeled

with mEmerald / lamin B1 ( 488exc nm, 9 phases, 2.4 m period); and the actin cytoskeleton is

labeled with Alexa 568 / phalloidin ( 561exc nm, 9 phases, 2.7 m period). Scale bar: 10 m.


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 (

858TPE nm), and filamentous actin is labeled with mEmerald / Lifeact (orange, 910TPE nm).


SUPPLEMENTARY FIGURE 24 Schematic of the instrument control architecture.

SUPPLEMENTARY FIGURE 24. Schematic of the instrument control architecture.


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.


SUPPLEMENTARY FIGURE 26 Invariance of the three-dimensional PSF across an extended volume for

different modes of Bessel beam plane illumination microscopy as demonstrated in xy and xz maximum

intensity projections of isolated fluorescent beads.

SUPPLEMENTARY FIGURE 26. Invariance of the three-dimensional PSF across an extended volume for

different modes of Bessel beam plane illumination microscopy as demonstrated in xy and xz maximum

intensity projections of isolated fluorescent beads.


SUPPLEMENTARY TABLE 1 Full width at half maxima (FWHM) of the axial point spread functions for

various modes of Bessel 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

Bessel swept sheet 0.47 m 1.02 m (2.12 ± 0.21) m

Bessel TPE sheet 0.45 m 0.49 m (0.57 ± 0.09) m

Bessel TPE SI, 5 phases 0.47 m 0.53 m --

single harmonic Bessel SI (3 phases) 0.26 m 0.27 m (0.37 ± 0.05) m

multi-harmonic Bessel SI (9 phases) 0.27 m 0.29 m (0.47 ± 0.10) m


SUPPLEMENTARY TABLE 2 Acquisition parameters for all images in Figs. 2-6.

Image Property Fig. 2a Fig. 2b Fig. 2c Fig. 2d

modality Widefield Bessel sheet Bessel SI 3 phases

Bessel SI 9 phases

annulus NAmax / NAmin - 0.60/0.58 0.60/0.58 0.60/0.58

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 (m3) 40 66 35 40 66 35 40 66 35 40 66 35

voxel volume (nm3) 133 133 100 133 133 100 133 133 100 133 133 100

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 Bessel TPE sheet Bessel SI 9 phases

Bessel TPE sheet

annulus NAmax / NAmin 0.53/0.49 0.62/0.59 0.6/0.56

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 FF01-523/610-25 FF01-525/50-25

image volume (m3) 53 80 50 40 40 40 40 40 40

voxel volume (nm3) 133 133 100 116 116 125 133 133 125

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


SUPPLEMENTARY TABLE 2 (cont’d) Acquisition parameters for all images in Figs. 2-6.

Image Property Fig. 3a Fig. 3b Fig. 3c Fig. 3d

modality Zeiss LSM 510 Zeiss LSM 5 LIVE DSLM Bessel SI 3 phases

annulus NAmax / NAmin 1.20/-- 1.20/-- 0.20/0.00 0.52/0.50

cell line HeLa (fixed) HeLa (fixed) HeLa (fixed) HeLa (fixed)

fluorescent label AF488 anti-

tubulin AF488 anti-tubulin AF488 anti-tubulin

AF488 anti-tubulin


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 (m3) 60 60 40 60 60 40 60 60 40 60 60 40

voxel volume (nm3) 133 133 100 130 130 100 133 133 100 130 130 100

CCD frame rate -- -- 50 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 Fig. 3e Fig. 3h

(top row) Fig. 3h

(center row) Fig. 3h

(bottom row)

modality Bessel TPE

sheet Bessel TPE

sheet Zeiss LSM 5 LIVE Zeiss LSM 5 LIVE

annulus NAmax / NAmin 0.53/0.49 0.6/0.56 -- --

cell line HeLa (fixed) LLC-PK1 (live) LLC-PK1 (live) LLC-PK1 (live)

fluorescent label AF488 anti-

tubulin mEmerald/mito mEmerald/mito mEmerald/mito


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 (m3) 60 60 40 40 40 40 41.4 41.4 38 41.4 41.4 17

voxel volume (nm3) 133 133 100 133 133 125 136 136 130 136 136 250

CCD frame rate 50 ms 30 ms -- --


volume acquisition time 20 sec 10.2 sec 9.8 sec 2.3 sec


SUPPLEMENTARY TABLE 2 (cont’d) Acquisition parameters for all images in Figs. 2-6.

Image Property Fig. 4a Fig. 4b Fig. 4c Fig. 5

modality Bessel SI 9 Phases

Bessel TPE sheet

Bessel TPE sheet Bessel TPE

sheet

annulus NAmax / NAmin 0.62/0.59 0.62/0.57 0.62/0.57 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


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 (m3) 40 40 40 30 30 30 48 46 42 33 33 30

voxel volume (nm3) 144 144 125 116 116 150 116 116 150 116 116 150


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 Bessel SI 9 phases

Bessel TPE sheet

Bessel SI 9 phases

Bessel TPE sheet

annulus NAmax / NAmin 0.62/0.59 0.6/0.56 0.60/0.58 0.53/0.49

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 FF01-523/610-25 FF01-525/50-25 FF01-525/50-25 FF01-593/40-25 LP02-633RS-25

FF01-470/100-25 FF01-525/50-25

image volume (m3) 40 40 40 40 40 40 53 67 50 53 80 50

voxel volume (nm3) 116 116 125 133 133 125 133 133 100 133 133 100



volume acquisition time

72 sec/color 10.7 sec 450 sec/color 40 sec/color


SUPPLEMENTARY TABLE 3 Additional parameters for Supplementary Videos 2-12.

Supplementary Video Corresponding

Figure Camera Parameters

No. of

Tiles

Supplementary Video 1 Supp. Fig. 16 Andor iXon 885 Single stack 1

Supplementary Video 2 Fig. 2f Hamamatsu ORCA-flash

2.8 Single stack 1

Supplementary Video 3 Fig. 3h Andor iXon 885 10.7 sec/stack, every 20 sec

300 stacks 1

Supplementary Video 4 Fig. 4a Andor iXon 885 50 sec/stack, every 60 sec

45 stacks 1

Supplementary Video 5 Fig. 4b Hamamatsu ORCA-flash

2.8

3.5 sec/stack, every 6 sec 100 stacks

1

Supplementary Video 6 Fig. 4c Hamamatsu ORCA-flash

2.8

10.2 sec/stack, every 12 sec 70 stacks

1

Supplementary Video 7 Fig. 5 Hamamatsu ORCA-flash

2.8

1.05 sec/stack 1-15 stack, every 21 sec 16-115 stack, continuous

116-170 stack, every 2 sec 171-201 stack, every 21 sec

201 stacks total

5

Supplementary Video 8 -- Hamamatsu ORCA-flash

2.8

7.3 ms/plane, continuous 7025 planes

2000 planes are shown in the movie 3

Supplementary Video 9 Fig. 6a Hamamatsu ORCA-flash

2.8 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 1

Supplementary Video 12 Fig. 6b Andor IXon 885 10.7 sec/stack, every 20 sec

122 stacks

1


SUPPLEMENTARY TABLE 4 Parts list for Bessel beam plane illumination microscopy

Parts in Supp. Figs. 3 and 21 Vendor Part number

laser L Coherent, Inc.

Cube-405-100C Sapphire 488 HP Sapphire 561 LP Cube-635-30E

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-400.650-TN

Pockels cell Conoptics 350-80-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 X5680 3.33 GHz processor

Kapton heater tapes Omega KHLV-0502/5-P

RTD probe Omega RTD-2-1PT100KN2515

PTD controller Omega CN4216-R1-R2


Rapid three-dimensional isotropic imaging of living cells ... · Rapid three-dimensional isotropic...

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