Bessel beam plane illumination microscope
Four lasers of excitation wavelengths 405 nm, 488 nm, 561 nm and 635 nm are expanded to a common 1/e2 beam diameter of 2.0 mm and combined into a single co-linear beam using dichroic beamsplitters. An acousto-optic tunable filter (AOTF) is used to select one or more wavelengths, control intensities and provide on-off modulation for structured illumination. The combined beam is then sent to the x galvanometer.
In a separate path, the near-infrared beam from a Ti-sapphire laser passes through a Pockels cell, is expanded to a 6.0 mm 1/e2 diameter and is sent through a near-infrared achromat and axicon doublet to create an annular excitation pattern22 at the focal plane of the achromat. A pair of relay lenses then image the annular pattern at an outer diameter of 0.65 mm to 1.3 mm (depending on the desired NA of the eventual Bessel beam) onto the x galvanometer.
The visible or near-infrared light impingent on the x
galvanometer is imaged at 2× magnification by relay lenses onto the conjugate z
galvanometer, and then magnified an additional 3× by relay lenses and imaged onto a conjugate custom-fabricated annulus mask. The mask contains various transmissive annuli of differing outer and inner diameters etched into an opaque aluminum coating on a quartz substrate; each annulus defines a Bessel beam of unique central peak width and longitudinal extent (Supplementary Figs. 1 and 2
With appropriate relay lenses chosen to match the diameter of the annular near-infrared excitation pattern from the achromat and axicon doublet to the diameter of the desired annulus in the mask, >50% of the energy impingent on the mask is transmitted. Such an arrangement is needed in the two-photon mode to deliver enough average power (~50–250 mW) to the rear pupil of the excitation objective to permit imaging at frame rates of 5–30 ms over fields of view as large as 60 μm × 80 μm. However, for linear excitation, a simple Gaussian beam projected onto the annulus can transmit enough power (~5–100 μW) to image at 10–100 ms per frame. The spatially filtered annular beam emerging from the annulus mask is imaged via another pair of relay lenses onto the conjugate back focal plane (BFP) of water-dipping excitation objective. The light is then focused by the excitation objective to form the Bessel beam in the sample chamber.
Two different approaches are used to measure fluorescence generated by the beam in the sample. In the high-sensitivity version, light collected by the detection objective is imaged directly via a tube lens onto an EMCCD camera. In the high-speed version (Supplementary Fig. 21
), light collected by the detection objective is first imaged by a tube lens at an intermediate image plane, where an adjustable slit crops the image in the y
direction to sharply define the field of view. A pair of relay lenses transfers this image to an sCMOS camera. To exploit the full speed of the camera, a third, tiling galvanometer placed between the relay lenses at the plane conjugate to the BFP of the detection objective is rotated in discrete steps as successive planes in the specimen are scanned. As a result, several images are tiled across the width of the camera sensor and can be read out in parallel.
In general, an image at a single plane is created by using the x galvanometer to scan the Bessel beam across the focal plane of the detection objective while integrating the fluorescence signal collected by the detection objective at the camera. A 3D image stack is then acquired by repeating this process at multiple planes along the axis of the detection objective, using the z galvanometer to translate the beam while moving the detection objective by the same amount using a piezoelectric collar (100 μm range).
The specimen is placed on a 18-mm coverslip, clamped into a custom sample holder (Supplementary Fig. 4a
) and inserted from above into a cavity in a sample chamber filled with aqueous medium. Silicone rubber film (70–80 μm thick) stretched over the sides of the excitation, detection and epi-illumination objectives is clamped to the sides of the sample chamber to contain the media in the sample chamber while still permitting the objectives to move with minimal resistance. The sample and holder are mounted at a 45° angle in the y–z
plane defined by the axes of excitation and detection objectives, and translated with sample stages to place the desired part of the specimen in the imaging volume. An epi-illumination objective orthogonal to the sample holder provides a conventional, low-magnification view of the sample and serves as a viewfinder.
Information on specific part numbers and vendors for all key components is given in Supplementary Table 4
A control schematic for the microscope is shown in Supplementary Figure 24
. The camera, set to internal triggering, served as the master timing source. Timing pulses from the camera triggered a field-programmable gate array (FPGA) card which generated user-defined waveforms (Supplementary Fig. 25
) to control the hardware above. Analog outputs controlling the x
galvanometers, the piezoelectric collar, and the tiling galvanometer were conditioned by individual scaling amplifiers to match their 16-bit resolution to the control range of each device. During linear operation (for example, Bessel single or multiharmonic SI modes), additional analog output drove the AOTF to control the intensity of each laser wavelength individually. During two-photon operation, additional outputs drove a Pockels cell and a mechanical shutter to control the intensity of the excitation or block it completely. The FPGA and image capture cards reside in a control computer with dual Hexa-core processors and 96 gigabytes of RAM (random-access memory).
All control software was written in LabView 2010, 64-bit version. A timing diagram is shown in Supplementary Figure 25a
for the exemplary case of 3D data acquisition using a continuously swept beam (for example, TPE sheet mode) and the high-speed configuration with an sCMOS camera and tiling galvanometer (Supplementary Fig. 21
). When tiling T
images, a sawtooth waveform of T
periods was generated for the x
galvanometer over one interval of camera exposure, thereby initiating T
sweeps of the Bessel beam across the field of view. At the same time, a square waveform of T
periods was applied to the AOTF or Pockels cell to ensure that the excitation was delivered to the sample only during the T
forward sweeps of the beam. At the beginning of each flyback, the voltages to the z
galvanometer and piezoelectric collar were incremented to ensure that the Bessel beam and focal plane of the detection objective settled to the next z
plane in the specimen before the next sweep commenced, and the voltage to the tiling galvanometer was incremented to ensure that the light from the next image plane fell on the next tiled section of the sCMOS sensor. After all T
images were exposed, all control voltages returned to their initial state whereas all T
images were read out in parallel.
In the single- and multiharmonic SI modes, the waveform for the x
galvanometer was not a continuous ramp but rather a series of discrete steps (Supplementary Fig. 25b
). At the start of each step, the AOTF or Pockels cell voltage was set low during the ~100 μs interval required for the x
galvanometer to settle to a new position but otherwise was kept high to expose the sample at a periodic set of discrete points in the x
dimension. For SI with N
phases, these waveforms were repeated for N
images at each z
plane except that a slight offset was added to the waveform for the x
galvanometer in each case to shift the exposure pattern by the desired phase.
Precise conjugation of the x
galvanometers and the annular mask to the BFP of the excitation objective was essential to achieve a uniform, high-quality Bessel beam across the entire image volume. Mutual conjugation of these elements was obtained by diverting the beam transmitted through the annulus mask to an inspection camera conjugated to the annulus mask and adjusting the axial positions of the various relay lenses until no motion of the annular beam was seen as the x
galvanometers were scanned. The annulus mask was conjugated to the BFP to the excitation objective by filling the sample chamber with H2
O, replacing the epi-illumination objective with a transparent view port and adjusting the axial positions of the relay lenses between the annulus mask and excitation objective until the annular excitation pattern expanding past the focus of the excitation objective (Supplementary Fig. 4b
) was in focus on a screen ~40 cm beyond the view port.
Uniform intensity across the annular excitation pattern is necessary to produce a symmetric Bessel function with maximal energy in the central peak. To achieve this, the x
galvanometers and the annulus mask were moved transversely with stages while measuring the intensity distribution at the inspection camera. For the near-infrared beam, the axicon was translated laterally to obtain a uniform annular illumination pattern before the annulus mask. Concentricity of the final annular pattern to the rear pupil of excitation objective is also critical, as transverse misalignment at the BFP corresponds to a tilt of the plane swept by the Bessel beam relative to the focal plane of the detection objective. To achieve parallelism of these planes, the sample chamber was filled with a fluorescent dye solution, and the image of the Bessel beam recorded by the camera was monitored while the z
position (Supplementary Fig. 3
) of the annular excitation in the BFP was adjusted with mirrors until the beam was in focus across its length. The x
position of the excitation was then adjusted until the beam was aligned along the y
axis (perpendicular to the scan direction).
To insure that the scan plane and detection focal plane were coincident at all times, the piezoelectric collar was used to move the detection objective to several z positions throughout the image volume, and the control voltage to the z galvanometer required to achieve a focused Bessel beam at each position was recorded. From these measurements, an interpolated control waveform was calculated for the z galvanometer such that, whenever a 3D image stack was acquired, the Bessel scan plane always remained in focus as observed by the detection objective. For cases in which drift later occured during cellular imaging, any point-like object in the sample could be used to apply a corrective offset voltage to the piezoelectric collar until symmetry was regained in the observed axial PSF.
When all of these alignment steps were completed, the PSF of the microscope was invariant throughout the entire image volume, regardless of the specific mode of operation used (Supplementary Fig. 26
). This was essential for the validity of subsequent deconvolution and to minimize artifacts in the final 3D images.
Bessel beam characterization
The longitudinal extent of the Bessel beam (Supplementary Fig. 2
) was measured directly from the image of the beam in dye solution, as above.
cross-sectional profile of the beam (Supplementary Fig. 1
) was measured by first positioning an isolated fluorescent bead to the center of the beam in the medium-filled sample chamber. Yellow-green 100-nm-diameter and 200-nm-diameter beads were used for linear and two-photon measurements, respectively. The piezoelectric collar was fixed at the position that provided an in-focus image of the bead on the camera. The x
galvanometers then scanned the beam across the bead, and an image was recorded of the bead at each beam position. From each image, the signal integrated in a window around the bead provided a measurement of the cross-sectional intensity of the beam at a specific x–z
position relative to its central peak. The set of all such measurements then yielded a 2D map of the x–z
cross-sectional intensity profile of the beam.
Point spread function measurements
Two types of PSF measurements were made: 2D excitation PSFs (Supplementary Figs. 1, 12 and 16
) and 3D overall PSFs (Supplementary Figs. 7, 13 and 17
). Each used 100-nm or 200-nm fluorescent beads, as above. For all excitation PSFs, the detection objective remained focused on the bead and only the Bessel beam excitation pattern was moved in x
dimensions. Each pixel in the resulting PSF then represented the integrated signal around the bead at the corresponding position of the excitation pattern. In contrast, for all overall PSFs, the detection objective was moved in concert with the z
motion of the excitation pattern, keeping the Bessel beam in focus at all times. The image of the bead obtained at each z
plane then gave the x–y
component of the overall PSF at that plane. In all cases, the experimental modulation transfer functions (Supplementary Figs. 11, 12, 13, 16 and 17
) were calculated from the absolute magnitude of the Fourier transform of the corresponding experimental PSF.
Theoretical point spread functions
All theoretical PSFs were calculated using a vector model of diffraction by an ideal lens of arbitrary NA23
. In particular, the excitation PSF of a single Bessel beam (Supplementary Fig. 1
) was calculated using integrals I1
in reference 23, except with the lower limit of integration changed from zero (that is, integration over the full solid angle defined by the NA) to the minimum illumination angle defined by the inner annulus diameter at the rear pupil. The two-photon single Bessel excitation PSF was calculated in the same manner, except using the longer excitation wavelength and squaring the result at the end. The theoretical wide-field PSF (Supplementary Fig. 7
) was calculated via integration over the full solid angle defined by the 0.8 NA of the detection objective.
The linear and two-photon theoretical sheet excitation PSFs were calculated by integrating the corresponding single Bessel beam PSFs along the x
scan axis. The corresponding overall PSFs (Supplementary Fig. 7
) were given by the products of these excitation PSFs with the wide-field detection PSF.
The linear (Supplementary Figs. 12 and 16
) and two-photon theoretical SI excitation PSFs were calculated by summing a periodic series of single Bessel beam PSFs, each offset by the stated period along the x
axis relative to its nearest neighbors. The sum extended 10 μm beyond the plotted field of view to account for the contribution of side lobes from beams outside the field. The linear and two-photon theoretical SI overall PSFs (Supplementary Fig. 7
) for N
phases were calculated by creating N
copies of the SI excitation PSF, each offset by 1/N
of the SI excitation period along the x
axis relative to one another, combining these copies according to equation (1)
, and then multiplying the result by the wide-field detection PSF.
Cells were imaged at 37 °C in DMEM with HEPES containing no phenol red. Temperature was maintained with a closed-loop system consisting of three Kapton heater tapes affixed to the exterior of the sample chamber, a resistance temperature detection probe inserted in the imaging medium and a proportional-integral-differential controller. After stabilization at 37 °C, PSFs measured from 100-nm beads were used to determine the offset voltage to the piezoelectric collar needed to align the focal plane of the detection objective to the scan plane of the Bessel beam. Cells could remain in the chamber more than 3 h and still undergo mitosis.
Cell imaging parameters
The parameters used to acquire the cellular images in , , , , and Supplementary Videos 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 are summarized in Supplementary Tables 2 and 3
Image deconvolution and display
Deconvolution of all images was performed in Amira version 5.3 (Visage Imaging) using an iterative maximum-likehood image restoration algorithm. Owing to the good agreement observed between the experimental and theoretical PSFs in all cases, the appropriate noise-free theoretical PSF, resampled to match the voxel size of the data, was used as the kernel for deconvolution. Typically, convergence to better than 2% was obtained in 12–15 iterations. Maximum intensity projections and volume renderings were performed using the ProjectionView and Voltex functions in Amira.
For the image shown in , fixed HeLa cells transfected with plasmids encoding mEmerald–histone H2B were used. For consistency, a 60 μm × 60 μm × 40 μm imaging volume and a voxel of 0.13 μm × 0.13 μm × 0.10 μm was used for each method, and excitation power and detector gain was adjusted to typical levels in each case yielding 3D image stacks of similar SNR in typical acquisition times.
For each method, a selected volume encompassing a nucleus was repeatedly imaged, until the initial fluorescence intensity dropped by at least 80%. The integrated fluorescence in a 10 μm × 10 μm × 10 μm subvolume was then calculated for each image stack in the series. An initial photobleaching curve was obtained by plotting this integrated fluorescence, normalized to that in the first stack, across all stacks.
As the bleaching rate will increase as more signal is demanded from each image stack, comparison across methods requires that these results be normalized to account for small differences in SNR as well24. The signal for the initial stack was estimated for each method by: (i) Fourier transforming the raw stack in 3D; (ii) applying a mask to remove all frequency components beyond the Abbe limit; (iii) transforming back to obtain a filtered image stack; (iv) applying a mask to select only the top 20% brightest voxels; and (v) denoting the average value of these brightest voxels as the signal S. The noise for the initial stack for each method was estimated by: (vi) subtracting the raw and filtered 3D image stacks to obtain a noise stack; (vii) applying the same mask from item iv above to select only a subset of noise voxels; and (viii) denoting the RMS value of these voxels as the noise N. Finally, the x axes of the initial photobleaching curves were rescaled according to the relative initial SNR values obtained using each technique.
We cleaned 18-mm-diameter coverslips via immersion for 12 h in a continuously stirred 1:1:5 mixture by volume of 50% H2O2, 30% NH4OH and ultrapure H2O. Coverslips were then rinsed with ultrapure H2O, dipped in CH3OH and passed briefly through a gas flame. Coverslips destined for cell culture were coated with 10 μg ml−1 fibronectin in 1× PBS (pH 7.4), overnight at 4 °C.
Preparation of fluorescent bead samples
We pipetted 20 μl of a 10 mg ml−1 solution of poly(D-lysine) hydrobromide in H2O onto an uncoated, cleaned coverslip, and allowed it to dry for 30 min. After rinsing in H2O, 20 μl of a solution of fluorescent beads of the desired concentration and bead diameter was pipetted onto the coverslip and dried on a hot plate at 40 °C. Finally, unattached beads were removed by repeated washing in ultrapure H2O.
Cell culture and transfection
LLC-PK1, U2OS, HeLa or COS-7 (American Type Culture Collection) cells were grown to ~55–85% confluency in Dulbecco’s modified medium with high glucose and no phenol red supplemented with 15% FBS and 4 mM L-glutamine. Cells were transiently transfected with an Amaxa Nucleofector 96-well shuttle system per the manufacturer’s guidelines, using the following Nucleofector programs for each of the different cells lines: Kit SEM program FF-138 (LLC-PK1); Kit SG program EH-100 (U2OS); Kit SEM program CN-114 (HeLa); and Kit SEM program FF-104 (COS-7). Cells were then grown on fibronectin-coated coverslips for 24–30 h after transfection before fixation or live-cell imaging.
Cells were fixed for 15 min at 37 °C in a solution of 4% paraformaldehyde and 0.1% glutaraldehyde (pH 7.4). After fixation cells were rinsed 3× in PBS.
Fixed cells were permeabilized in 0.5% Triton X-100 in PBS for 10 min and then rinsed 3× with 0.1% Triton X-100 in PBS for 5 min each followed by Abdil blocking buffer (BRB80 (80 mM PIPES, 1 mM EGTA and 1 mM MgCl2; pH 6.9) with 0.1% Triton X-100 and 2% (w/v) BSA) for 10 min. Cells were then incubated in a 1:40 mixture of fluorescent phalloidin (1 μg ml−1) in Abdil for 20 min, rinsed 3× with 0.1% Triton X-100 in PBS for 5 min each and finally rinsed in PBS.
Cells were first extracted in BRB80 with 4 mM EGTA and 0.5% Triton X-100 for 30 s and then fixed. After three rinses with 0.1% Triton X-100 in PBS for 5 min each, a quenching solution of 1 mg ml−1 sodium borohydride in PBS was added for 10 min, followed by Abdil for 10 min. Primary antibodies to α-tubulin (clone DM1A) were then added for 30 min and, after washing with BRB80-T, incubated with fluorescent secondary antibody overnight at 4 °C.