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
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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
Nature Methods: doi.10.1038/nmeth.1586
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.
Nature Methods: doi.10.1038/nmeth.1586
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.
Nature Methods: doi.10.1038/nmeth.1586
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.
Nature Methods: doi.10.1038/nmeth.1586
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.