1. Introduction

Confocal and two-photon microscopes are currently the workhorses of most biomedical imaging facilities. However, owing to their slow imaging speed, high light dosage, poor penetration depth, and anisotropic resolution these microscopes are not suitable for imaging large tissues [1]. Light sheet fluorescence microscopy (LSFM) is a rapidly evolving microscopy technique that can acquire fast 3D images with high spatiotemporal resolutions across large field-of-views (FOVs) yet being gentle to the sample which greatly reduces photobleaching and phototoxicity [1,2]. Owing to its myriad of advantages LSFM has been used to image across various biomedical fields involving in vivo imaging of cells, spheroids, tissues, organs, embryos (worms, flies, zebrafish, etc.), and plants [1–11].

LSFM, in its simplest form, employs a Gaussian beam that is focused in only one direction through a cylindrical lens and excites fluorophores only in the in-focus plane of a detection objective. This is accomplished using two orthogonal objectives where an excitation objective generates a thin sheet of light at the focal plane of a detection objective. This plane is imaged directly onto a scientific Complementary Metal-Oxide-Semiconductor (sCMOS) camera, resulting in a massive parallelization of data collection compared to the point scanning microscopes [1]. It should be noted here that in LSFM the lateral-resolution is determined by the numerical aperture (NA) of the detection objective while the waist of the excitation beam governs the resolution in the third dimension (also called z-resolution or axial-resolution). Because of the beam's Gaussian nature, there exists a trade-off between the thickness of the beam-waist and the area over which the beam can be approximated as a sheet of light. Therefore, an everlasting impetus in the field of LSFM research circles around alleviating this problem: How to create a thin sheet of light over a large FOV?

Several methods have been proposed to extend the FOV without sacrificing the z-resolution. Propagation invariant beams, such as Bessel beams [12–15], Lattice light sheet [16], Airy beams [17], and Field synthesis light sheet [18], theoretically retain a narrow beam waist over an arbitrarily long distance. However, for one-photon illumination, these techniques do not provide a significant improvement over a Gaussian beam in confocal parameter and beam waist [19]. Another approach, classified as multi-view imaging, generates final images by computationally fusing the complementary views [11,20–25]. This is often carried out by rotating the sample in a conventional LSFM [21,26], swapping perpendicular illumination and detection arm sequentially (e.g., Dual-view iSPIM) [20], using two illuminations and detection objectives arranged along two perpendicular axes (e.g., MuVi-SPIM, SiMView) [22–24], and four orthogonal objectives where each objective has both illumination and detection arm (e.g., IsoView) [25]. The Multiview imaging technique improves the spatial resolution and makes resolution more isotropic across the FOV. Nevertheless, because one has to image the sample from multiple directions, this method reduces imaging speed and requires massive post-processing. Another approach called digitally-scanned-light sheet microcopy (DSLM) creates a time-averaged light sheet by rapidly moving the beam vertically and horizontally through the sample [27]. When the Bessel beam is scanned laterally, this method creates out-of-focus blur and reduces image resolution and contrast owing to the beam sidelobes [28]. Tiling light sheet selective plane illumination microscopy (TLS-SPIM) employs a similar scheme and tiles light sheets to multiple positions in multiple frames so that a large FOV can be imaged by repeating this process and stitiching all images together [29–31]. However, this technique suffers from low imaging speed, high computational post-processing and poor axial resolution compared to conventional LSFMs.

Lately, axially swept light sheet microscopy (ASLM) based microscopes have shown great promise when it came to volumetric imaging of large tissues at diffraction-limited isotropic resolution. In this technique, the waist of the light sheet is translated in the axial direction and synchronized with the rolling shutter readout of the sCMOS camera [32]. This ensures only the light sheet’s waist is captured while rejecting the out-of-focus blur thereby providing robust optical sectioning and high-resolution imaging over large FOVs [32]. Ever since its conception [32], several microscopes designed and built upon this core technique have come into existence [33–35]. For example, the Cleared-tissue ASLM (ctASLM) extended this idea to develop an instrument that was capable of imaging at sub-300 nm level isotropic resolution while maintaining compatibility with all tissue-clearing methods [33]. Another initiative is known as the mesoscale selective plane-illumination microscope (mesoSPIM), which integrated ASLM by employing an electrotuneable lens (ETL) showed that volumetric imaging of centimeter-sized cleared samples with 5-6 µm isotropic resolution was possible within minutes [34]. However, as explained further in the ‘Method’ section, ASLM suffers from two fundamental constraints: low detection signal and the limited frame-rate for full-FOV-imaging, both of which adversely affect the imaging speed. These further complicate matters such as fluorophore selection for the biological specimens, achievable temporal resolution, and ease of alignment while building the instrument.

Here, we propose to upgrade the core ASLM technique to create a new imaging platform that not only improves the detection signal but also allows these microscopes to image faster. Furthermore, our suggested technique simplifies the process of determining ASLM parameters, making this new version far superior to the previous one. We demonstrate the performance of our microscope by imaging fluorescent beads and a chemically cleared mouse brain.

2. Method

LSFMs that employ Gaussian illumination suffer from a fundamental problem: the axial resolution decreases, thereby resulting in blurred images, as one moves away from the waist of the light sheet, as shown in Fig. 1 .a. ASLM overcomes this by creating a virtual light sheet with a diffraction-limited waist over the entire FOV of the camera. The working principle of ASLM can be seen in Fig. 1 .b. The waist of the light sheet is swept along the propagation direction of the light such that it is synchronized with the camera readout using the rolling shutter feature of sCMOS cameras [32,33]. Since the light sheet is imaged directly onto the 2D array of the sCMOS camera, high optical sectioning is achieved by restricting the active pixels of the 2D array to roughly twice the Rayleigh length of the beam [32]. By rejecting the out-of-focus blur this scheme allows one to maintain a high NA beam waist over a large FOV.

 

Fig. 1. Optical principle of a traditional LSFM and ASLM in single and dual-focus configurations. (a) The variable thickness of a Gaussian light sheet causes uneven illumination across the FOV in a traditional LSFM which limits the axial resolution only to the waist of the light sheet. (b) The ASLM works on the premise of scanning a high NA light sheet in its axial direction. The light sheet scan is synchronized with the rolling shutter readout of an sCMOS camera, which is tuned to the width of the beam waist. This decouples the FOV from beam-waist limited axial resolution. (c) In dual-focus ASLM mode, two foci are staggered and scanned simultaneously only over half of the sCMOS chip. (d) In a dual focus setup, the incoming beam is split by PBS2 into two paths with S and P polarizations. The combination of a concave and a convex lens, (L9 and L10) for S and (L11 and L12) for P, alters the wavefronts of two beams differently. When combined using PBS3 and fed into objective lens 2 (OBJ2), it generates two axially staggering spots. Flip mirrors (FM1 and FM2) were engaged to create a standard single focus ASLM for benchmarking.

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However, the ASLM concept has two fundamental drawbacks. First, in ASLM, as explained above, to capture one frame within a certain acquisition time, a strip of active pixels is rolled over the entire camera chip, as shown in Fig. 1 .b. Therefore, the effective exposure time at each pixel is only a fraction of the acquisition time which ultimately lowers the signal-to-noise ratio (SNR). As a result, the user at this point should either increase the excitation power to generate higher fluorescence signal which may result in an adverse phototoxic effect, or capture the images at a lower frame-rate which effectively increases the exposure time at each pixel but slows down imaging speed. Therefore, ASLM-based microscopes make a compromise of either improving imaging speed or SNR. Second, all ASLM-based microscopes use some sort of actuator that sweeps the light sheet along the axial direction. For ctASLM this is the linear-focus-actuator (LFA) located in the remote focusing arm while for mesoSPIM an ETL is used for this purpose. Because the light sheet is scanned over the entire camera chip, these actuators must oscillate with larger strokes to cover the entire FOV. For sub-100 ms acquisition times, non-linear phenomena start to creep in through the actuators which makes the scan of the light sheet non-uniform. In practice, this non-uniformity not only limits scanning to about 10 frames/sec over the full FOV but also makes finding ASLM parameters to synchronize rolling-shutter and the sweeping focus difficult.

Here we propose to alleviate these problems by simultaneously scanning two staggered light sheets in the axial direction and simultaneously synchronizing two rolling shutters in the two halves. By doing so each light sheet covers only one-half of the FOV and together cover the entire FOV in one camera acquisition cycle. This concept is shown schematically in Fig. 1 .c. There are two main advantages of this method as opposed to scanning a single focus across the entire FOV. First, sweeping dual-focus over half FOV requires shorter travel for the LFA. This shorter travel eases the oscillatory motion of the LFA. This not only results in a more uniform scan of the foci across the FOV at shorter acquisition times but also makes finding the ASLM parameters a lot easier. Second, our method allows ASLMs to either improve the signal or the framerate for a particular peak illumination intensity. Signal improvement: since each light sheet covers only half of the FOV, the effective exposure time at the active pixels doubles, thereby improving the acquired signal strength by two-fold. Framerate improvement: for the same detection photon budget, scanning dual-focus will allow users to scan at twice the frame rate using same laser power in each waist (as that of single-focus case). This can be particularly useful, in terms of the total acquisition time, for samples where the strength of the fluorescence signal is not an issue.

Figure 1 (d) (and Fig. S1) shows the schematic diagram to implement our proposed idea. There are three main concepts that are essential to achieve this goal: (1) generation of two simultaneous light sheets, (2) fast, aberration-free, scanning of these light sheets across half of the FOV such that the entire camera chip is covered yet maintaining a fixed distance between the two foci, and (3) synchronizing the position of sub-array readout on the sCMOS camera with axial scanning of light sheet for the two halves independently. To generate dual-focus setup, the polarizing beam splitter (PBS2) is used to split the illuminating beam into two paths, which are then fed into a lens assembly each containing a concave (L9 and L11) and a convex lens (L10 and L12). Owing to a different distance between the two lenses in each arm, each of these pairs modify the wavefront of the beams differently in each path (Fig. 1 (d), Fig. S1 ). This generates two foci, one with ‘S’ and another with ‘P’, which are staggered in space in the axial direction. A mirror attached to the LFA located in the remote focusing arm, may move back and forth, to sweep the focus axially and therefore results in the ASLM with dual-focus configuration (Fig. 1 (d), Fig. S1).

Figure 2 elucidates the working principle behind this concept by experimentally simulating the idea using a traditional light focus (called 2D focus here onwards) employing a standard achromatic lens (not cylindrical lens) in fluorescein solution. As can be seen, when the 2D focus (Fig. 2 (a)) is scanned in the propagation direction with a synchronized rolling shutter it results in a sharp line (Fig. 2 (b)). This is because the active pixels on the camera reject the fluorescence arising from outside the focal volume. A 1D focus (waist of the light sheet) when scanned similarly would become a thin virtual sheet (the concept behind ASLM). Figure 2 (c)-(d) shows the working principle behind our dual-focus concept. When two 2D foci are scanned for only half of the FOV they cover the entire camera chip, and appear as two sharp lines. It should be noted that the purpose of 2D focus is to simply allow the visualization of the focal volume and the resulting ASLM-type-scan in fluorescein and is in no way different than a light sheet where the confinement of light happens in 1D. To ensure that the spurious contribution of unfocused light from the ‘tail’ of one light sheet does not bleed into the waist of the other, we plotted a line profile through the two foci depicting the intensity profile across the entire FOV (inset of Fig. 2 (c)). Owing to a separation of ∼335 µm between the two foci, the intensity of out-of-focus light has reduced to near zero. Because these two foci maintain their separation throughout the travel, we can ensure that out-of-focus fluorescence from one light sheet never affects the other thereby preserving the most important feature of ASLM: optical sectioning.

 

Fig. 2. Visualization of the proposed concept. (a) Sharp single 2D focus spot at the center of the FOV. (b) Scanning of the single focus. The sharp line is the indication of matched rolling shutter speed with the 2D focus scanning rate for the entire FOV. (c) Sharp dual 2D focus spots on the centers of the individual half FOV. In the inset, the intensity profile shows that the two focus spots are separated enough that there is no out-of-focus contribution of one light sheet into the other. (d) Corresponding image as in (b) with dual-foci. The straight line indicates that the two foci are perfectly aligned, maintain constant separation and synchronized with the rolling shutter for the two FOVs. Scale bar: 50 µm.

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It should be noted that the confocal detection of dual beams has been previously investigated in DSLM-based techniques, albeit in the lateral direction. Two counter propagating rolling shutter modes have been used to image two parallel light sheets generated using identical illumination objectives facing each other [36], and an acousto-optic deflector (AOD) [37]. However, this counter-propagating movement not only results in inhomogeneous lighting and striping artifacts throughout the whole FOV but also degrades image quality due to crosstalk between the two light sheets. In addition, because these techniques were designed to improve the light confinement in the lateral direction for DSLM, the axial resolution of the light sheet has always been an issue, as in sub-micron isotropic resolution has never been achieved. Our method on the other hand is developed particularly for ASLM and maintains its most salient feature: isotropic sub-micron resolution over a large FOV. Not only do we create two fixed-distance, high-NA, Gaussian light sheets capable of axial scanning, but our rolling shutters also aren’t counter-propagating, as a result, our method doesn’t have a crosstalk problem like others.

3. Results

As discussed previously, in ASLM, the axial position of the beam is deterministically modulated by an actuator through the remote focusing arm. However, this deterministic requirement can often be compromised by a few interdependent factors: (a) speed of imaging—the smallest acquisition time in which the waist of the light sheet must travel the entire FOV to capture one frame, (b) FOV requirement— which decides the maximum distance the focus needs to travel, and (c) inherent nonlinear response of the LFA to the applied voltage. In Fig. 3 we try to quantify and correct for this nonlinear response by making use of the 2D focus spots. Figure 3 (a) (left) shows this nonlinear relationship between the applied voltage and the resulting focus shift for single focus as it is translated across the FOV. This not only makes synchronizing the camera’s rolling shutter to the axially moving focus very difficult but also reduces the overall FOV coverage. To overcome this, we designed a correction routine in our control software which, when pre-calibrated, corrects for this nonlinear response by providing additional voltage to the LFA (Fig. 3 (a), right). One can see from Fig. 3 (b) that the voltage required to correct for the nonlinearity decreases significantly because the two focus spots now must move only half as much as the single focus case. It should be noted that these corrections are for the static case and act as a coarse adjustment only. When the LFA oscillates rapidly these numbers are adjusted further to generate a more continuous correction. Figure 3 (c) shows the corrected voltage for both single and dual-focus mode across the entire FOV. Volumetric scans were then taken by synchronizing these voltages along with digital modulation of lasers and camera trigger pulse train (Fig. 3 (d)).

 

Fig. 3. Position response of the 2D focus with respect to the applied LFA voltage for (a, left) single and (b, left) dual-focus configuration. (c) The corrected voltage plotted against the actual voltage for single (blue) and dual-focus (red). (a, right) Single-focus and (b, right) dual-focus position response of the 2D focus after applying the correction. One can see that for the dual focus case the correction required is about half of that required for the single focus case. (d) The corrected sawtooth signal for the LFA is synchronized precisely with a series of deterministic transistor-transistor logic (TTL) triggers signal for the camera, laser modulation to carry out ASLM.

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To evaluate how scanning dual-focus improved the imaging speed over traditional ASLM, we employed a 2D focus to carryout ASLM and visualized how well the coverage across the entire FOV was by drawing line profiles. For 100 ms acquisition time the single focus mode could cover the entire FOV with a sharp line, as measured by line-profiles (Fig. 4 (a) and Fig. S2 (a),(d)). When the camera exposure time was lowered to 50 ms, sharp focus could only be achieved for one-third of the FOV (Fig. 4 (b) and Fig. S2 (b),(e)), while the rest of the FOV appears blurry. Compared to this for the dual-focus setup with even 50 ms camera exposure time, each half of the FOV is scanned by sharp lines of the scanning beam, resulting in a sharp line over the entire FOV as shown in Fig. 4 (c) (Fig. S2 (c), (f)). With these optimized ASLM parameters we simply switched the achromatic lens with a cylindrical lens, to generate light sheet, and imaged a PEGASOS cleared, Thy1-GFP mouse brain (see Supplement 1) [38]. The maximum intensity projection (MIP) of the mouse brain in the XY and YZ directions are shown in Fig. 4 (d) using 50 ms acquisition time. Due to the uncompromised sub-micron resolution across the entire FOV, high-quality neuronal imaging of 670 × 670 × 200 µm³ volume could be performed in 30 sec which using a traditional ASLM would have taken over a minute. It should be noted that this two-fold improvement in imaging speed might not seem much for such a small volume but can reduce hours or even days of acquisition time when imaging larger samples.

 

Fig. 4. XY view of 2D single and dual-focus beams in ASLM mode over 670 × 670 µm² FOV in fluorescein solution for different camera exposure times and orthogonal views of mouse brain. XY view of the FOV for single focus arrangement for (a) 100 ms and (b) 50 ms acquisition time. It can be seen that for the 100 ms acquisition time the rolling shutters maintain synchronous motion with the moving focus which is depicted by a sharp line. (b) For 50 ms exposure time, this synchronization becomes very difficult to maintain: depicted by sharp line only for one-third of the FOV and blurry regions towards the edges. (c) Using two foci the full FOV can be scanned even at 50 ms exposure time. This is because of the reduced load on the LFA which now has to travel only half as much distance as the single focus case. (d) Orthogonal views of the mouse brain in a dual-focus light sheet configuration 50 ms exposure time: twice as fast as a traditional ASLM. Scale bar: 50 µm.

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In order to characterize the resolution of the microscope, we imaged 500 nm green fluorescent beads embedded in a 2% agarose gel and measured the full-width-half-max (FWHM) of the point-spread-functions (PSF). Standard magnification calibration slides were used to access the actual magnification of these objectives since their focal lengths and the effective magnification change with the RI of the immersion medium. We found, for water, with a 200 mm focal length tube lens the effective magnification was 20.21× which ultimately decided the sCMOS pixel size to 0.32 µm. Figure 5 (a) and Fig. 5 (e) shows the XY view of MIP of stacks of beads over the entire FOV in single and dual-focus Gaussian light sheet mode, i.e., axial scanning turned off, respectively. The enclosed dashed yellow region depicts the waist of the light sheet since this is where the intensity of the emitted beads is the maximum. The inset, depicted by red boxes, shows an enlarged view of a randomly chosen region with several beads. In order to obtain quantitative evaluation of our microscope’s resolution, we measured the FWHM of the beads shown in these insets (Fig. 5 (c)-(d) and Fig. S3). The FWHMs for both cases (∼0.8 µm) suggest that we achieve aberration free diffraction-limited resolution. When the axial scanning is turned on, both single and dual-focus result in uniform coverage for the entire FOV (Fig. 5 (b) and Fig. 5 (f)). Similar FWHM measurements of the PSFs indicate that while we have diffraction limited performance in case of single-focus (Fig. 5 . c-d), the resolution of the microscope in dual-focus mode decreases slightly (Fig. 5 (g)-(j)). This is because of the aberrations induced by two extra lenses we used in the remote focusing arm in order to separate the two foci (L11/L12 and L9/L10 in Fig. 1 (d)). We would like to point out that these are raw, unprocessed images and routine deconvolution operations can certainly improve the final quality even further.

 

Fig. 5. Resolution and signal-strength assessment using 500 nm fluorescent beads embedded in agarose gel, imaged by single and dual-focus configurations in LSFM and ASLM mode. (a) The confined region within the dashed-yellow lines contain the light sheet waist in LSFM mode, and insets are the magnified view of the small red boxes. (b) The corresponding ASLM mode shows a uniform FOV coverage (inset: zoomed in view). (c, d) Plots depicting the lateral PSF (blue for LSFM and green for ASLM) (c) and axial PSF (d) of beads picked by arrows in insets. The peak of the ASLM (green) plot is normalized to LSFM intensities (blue). (e) and (f) are the corresponding images for LSFM and ASLM mode employing dual focus mode respectively. The insets show zoomed-in view of a few randomly chosen beads from both halves of the FOV. (g-j) The plots showing the lateral (g, i) and axial (h, j) PSF of beads shown with arrows in insets of (e) and (f). The FWHM of beads was calculated by Gaussian-curve fitting. Scale bar: (a, b, e, and f) 50 µm and (insets) 5 µm

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Next, we compared the detection signal performance of dual-focus with the traditional ASLM. For this, we use average intensity measurement from beads for both modes of illumination. So that the comparison can be made fairly the intensities of the PSFs for single and dual-focus ASLM are normalized to their respective Gaussian light sheet mode. This is to ensure that any discrepancies in the illumination laser power, between the two modes, did not affect the detection signal assessment. The total excitation laser power for single and dual-focus the laser power was set at 20mW and 40mW (with each arm receiving 20mW) respectively. As can be seen from Fig. 5 (c), the average intensity for single focus dropped from 1 a.u. for a Gaussian focus to 0.014 a.u. for ASLM. A similar plot involving two foci (Fig. 5 (g) and 5(i)) showed that the intensity changed from 1 a.u. for dual-focus Gaussian mode to 0.037 a.u for ASLM. This 2.5× improvement confirms our original hypothesis that the dual-focus arrangement enhances ASLM’s signal strength.

Figure 6 (a)-(d) compares the MIP of XY and YZ view of a dense neuronal labeling within the cleared brain sample using single and dual-focus ASLM mode. The total excitation laser power for single and dual-focus the laser power was set at 20 mW and 40 mW (with each arm receiving 20 mW) respectively. When the lowest and highest intensities of both the images were adjusted to roughly similar levels, one can see that lower intensity regions in the brain section become more visible for dual-focus ASLM mode (depicted by blue arrows in Fig. 6 .c). In order to quantify the increase in signal-intensity between single and dual-focus modes, SNR calculations [39,40]. were performed on four randomly chosen regions of Fig. 6 (a)-(d) (as shown in Fig. 6 (e)). Details depicting the method of SNR calculations can be found in Supplementary section 6. As can be seen from Fig. 6 .f SNR improvements of up to 10 were observed between the two modes. As is evident from the intensity-ratios of Fig. 6 (g) a nearly two-fold improvement in average signal intensity results in 1.4-fold improvement in SNR between the two modes. We corroborated this finding by performing a similar analysis on a different section of the mouse brain, as shown in Fig. S4.

 

Fig. 6. Neuronal imaging of mouse brain acquired by single and dual-focus mode for 100 ms camera exposure time. (a-d) Orthogonal views of MIP. To visualize the intensity improvement over dual-focus imaging, the minimum and maximum intensity of both images are set equal. (e) Enlarged view of selected regions from (a-d). (f, g) Quantitative analysis of images shown in (e) in terms of SNR and signal intensities. As can be seen from the bar-charts of (f), dual-focus achieves higher SNR compared to single-focus. Bar charts of (g) depict that for 2× improvement in average signal intensity, for dual-focus, results in a 1.4× SNR improvement. Scale bar: (a-d) 50 µm (e) 10 µm.

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4. Discussion

In principle, while it is possible to scan an arbitrary number of light sheets for the gain of better SNR, in practice, there are two major considerations: First, the availability of an sCMOS camera that can roll the shutter independently in more than two regions. While some emerging sCMOSs have proposed to offer programmable scanning modes [41], these features are in their nascent stage and will need further investigation. Second is the spurious contribution of unfocused light from the ‘tail’ of one light sheet into the waist of another. This can adversely affect the optical sectioning capability of the ASLM. The actual number is dependent on the NA of the light sheet and the magnification of the detection arm. Our simulations suggest that a maximum of four 0.4 NA light sheets may fit within this FOV, without a significant bleed through.

Because we used a combination of concave and convex lenses to control the axial separation of the light sheets, the focal lengths and the effective magnification for the two states of polarization in the illumination arm changed slightly which not only made synchronizing the rolling shutter in two FOVs challenging, but also resulted in resolution-loss. Although we found this arrangement adequate and acceptable, in terms of the overall performance, several alternative schemes could make the light sheet separation control easier. For example, by using a step or a tilted mirror in the remote focusing arm [35] or binary SLM to generate tiled discontinuous light sheets [31] or, pLSFM scheme [42] axially staggered light sheets may be generated to carry out our scheme. We found that a more precise control over the light sheet positions could translate into faster imaging speed. As such at its current settings we could triple the speed of imaging (data not shown here) with 95% FOV coverage. Our future implementations will investigate these options to push the limits of ASLM even further.

In summary, we have demonstrated a new scheme whereby axially scanning two simultaneous light sheets improved the overall performance of ASLM both in terms of imaging speed and detected signal. We achieve this by splitting the sCMOS’s FOV into two and then running synchronized sub-array readout in the two halves independently. This enabled us to image a cleared mouse brain across a large FOV (670 × 670 µm²) with twice the frame rate (20 frames/sec) compared to the original ctASLM (10 frames/sec) at this FOV. Also, for a particular frame rate, compared to conventional ASLM, our concept illuminates the sample for twice the dwell time, thereby doubling the signal intensity as evaluated by SNR. Even though, this doubles the light dosage to the sample, since we maintain a constant peak illumination power in each focus, our method provides a gentler illumination compared to single focus where signal improvement requires one to increase the peak illumination power which may aggravate the non-linear photobleaching and photodamage effects. Additionally, because this new technique is developed upon the general ctASLM design, with several key innovations, it retains ctASLM’s most salient feature i.e. multicolor imaging across the entire refractive index range with diffraction-limited isotropic resolution. We therefore believe that our version of ASLM brings about the most advanced light sheet microscope that there is for large volume 3D imaging of tissues.