Light-sheet microscopy with length-adaptive Bessel beams

Light-sheet microscopy with length-adaptive Bessel beams

TOBIAS MEINERT AND ALEXANDER ROHRBACH*

Laboratory for Bio-and Nano Photonics, Department of Microsystems Engineering - IMTEK, University of Freiburg, Freiburg 79110, Germany *[email protected]

Abstract: In light-sheet microscopy, a confined layer in the focal plane of the detection objective is illuminated from the side. The illumination light-sheet usually has a constant beam length independent of the shape of the biological object. Since the thickness and the length of the illumination light-sheet are coupled, a tradeoff between resolution, contrast and field of view has to be accepted. Here we show that scanned Bessel beams enable object adapted tailoring of the light-sheet defined by its beam length and position. The individual beam parameters are obtained from automatic object shape estimation by low-power laser light scattered at the object. Using Arabidopsis root tips, cell clusters and zebrafish tails, we demonstrate that Bessel beam light-sheet tailoring leads to a 50% increase in image contrast and a 50% reduction in photobleaching. Light-sheet tailoring requires only binary amplitude modulation, therefore allowing a real time illumination adaptation with little technical effort in the future.

© 2019 Optical Society of America under the terms of the OSA Open Access Publishing Agreement

1. Introduction

Light-sheet microscopy (LSM) illuminates only that part of the object that is in the focal plane and therefore offers three major advantages: first, high contrast images are captured because useful information from the focal plane is not superimposed by defocused background light [1–3]; second, fluorophores are excited only in the focal plane, leading to strongly reduced photobleaching [4,5] and third, widefield detection enables high speed imaging by exploiting the high framerates of modern cameras [6]. Whereas these characteristics allow excellent imaging of nearly transparent objects like zebrafish [7], other objects like Drosophila melanogaster [8] or Arabidopsis root tips [9] make high-quality imaging deep inside the object more difficult, since scattering and absorption reduce the penetration depth and illumination homogeneity.

The effects of scattering and absorption could be reduced by the following 6 techniques: i) Two side illumination [10] does not correct artefacts, but doubles the maximum object size that can be imaged. ii) It is known that interference of scattered and unscattered laser light leads to inhomogeneous illumination often resulting in dark stripes [11]. By pivoting light-sheets these interference patterns are averaged out and the image quality is improved [10]. iii) Scanned light-sheets [12] are superior to static light-sheets, because interference artefacts can be strongly reduced. iv) Line-confocal detection [13,14] and structured illumination [15,16] reduce the influence of multiply scattered photons and thereby increase the image contrast. v) Adaptive optics have been applied to compensate for object induced aberrations [17–21]. vi) Bessel beams offer an increased penetration depth into scattering samples [22] and enhance the homogeneity of the illumination [23]. In addition, the resolution in detection direction can be improved since illuminating Bessel beams feature a higher numerical aperture (NA) at the same depth of field compared to Gaussian beams [24].

It is known that the main maximum of a Bessel beam is surrounded by a ring system, which carries a significant part of the total beam power and which illuminates out of focus regions, thus reducing image contrast. This detrimental effect is increased for higher NAs,

Vol. 10, No. 2 | 1 Feb 2019 | BIOMEDICAL OPTICS EXPRESS 670

#338517 https://doi.org/10.1364/BOE.10.000670 Journal © 2019 Received 10 Jul 2018; revised 6 Dec 2018; accepted 6 Dec 2018; published 18 Jan 2019

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filter, usually blocking the laser light. The coherent image Fig. 2(a) requires very low illumination power, so that the object is not stressed by the additional illumination and bleaching can be neglected.

In conventional LSM, the object point closest to the illumination objective z1 and the point furthest away from the objective z2 defines length and focal position of the ideal beam Fig. 2(b). The shape of the beam is not changed throughout the scan. That way the standard rectangular light-sheet forms, which we call reference light-sheet Fig. 2(c). In tailored LSM, the shape of the ideal light-sheet is reproduced by updating the beam forming hologram after a certain scan distance Fig. 2(d)).

A conical phase 2 20( , ) NAx y k x yφ = ⋅ ⋅ + is required in order to form a Bessel beam,

where the phase slope k0·NA defines the NA of the beam (at wave number k0 = 2π/λ0 with vacuum wavelength λ0). Remarkably, the phase of the hologram is independent from the axial beam profile, i.e. the phase and the NA are not changed during the measurement. To adapt the beam length Δz(x) and axial beam position z0(x), only an amplitude modulation is required. Figure 3(a) shows how amplitude and phase of the hologram displayed on the SLM define the beam shape.

We assume the typical case that the distance z0 between axial beam center position and hologram position is larger than the beam length Δz. Then the transmission function T(x,y) and the amplitude A(x,y) of the hologram have an annular shape [30,31]:

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φΔ Δ

= ⋅ ⋅

= − + − − − ⋅ ⋅ ⋅ (2)

With 2 2r x y= + and circ(r-r0) = 1 for r ≤ r0 and = 0 otherwise. The center diameter R0

of the ring defines the axial beam position according to 0 0 NAz n R= ⋅ with n being the

refractive index. The beam length is given by the width ΔR of the ring according to NAz n RΔ = ⋅ Δ . The magnification of the illumination system has been set to Mill = 1 in

order to keep things simple. To avoid oscillations of the axial beam profile a low-pass filtering of the amplitude A(x,y) is required.

The SLM used in the current setup is a pure phase shifting element, although we used an algorithm to modulate the amplitude as well by transforming the continuous amplitude modulation into a binary pattern [30]. Therefore, in order to modulate beam length and axial beam position, a dynamic binary amplitude modulation in combination with a static phase modulation can replace the often slow SLMs. Hence, an axicon can be used as static phase element and a digital micromirror device (DMD) for dynamic binary amplitude modulation. The framerate of the currently used SLM is limited to 60 Hz, which is not enough to enable real time light-sheet adaptation. In contrast, DMDs feature framerates of more than 10 kHz [32–34]. Thus, for a field of view of 500 µm and a framerate of 50 Hz, totally 200 beams can be adapted laterally in steps of 2.5 µm within only 20 ms. This step size is small enough to sample all relevant object shapes. Also binary phase SLMs have been used for fast Bessel beam position control [35]. In this study, beams were rapidly shifted axially to enabled tiled light-sheets of rectangular shape, hence, light-sheets were not adapted to the shape of the object.

Figure 1 shows a confocal detection based on the rolling shutter technique, which requires a minimum scanning speed (minimum speed of rolling shutter). For the proof of principle measurements presented in this work, a DMD has not been available, so that beam adaptation could not be realized at the scanning speed required for rolling shutter based confocal detection. To circumvent this problem, individual images were captured automated at each lateral scan position. The final image was calculated by multiplying the individual images with a mask representing the confocal slit and subsequently summing up all images [13]. To


enable a fair influenced byrecorded at eworkaround pnovel beam shpoint out that detection is psame format adaption withbe used as a li

Fig. 3formathe beis ideBessemaximbackgconfo

As explainbeam length. of the image method sectioinfinite slit w

shows that the= 0.2 - is procontain usefulength, if the

comparison ofy sample drifteach scan posperformed wellhaping principreal time light

possible by impand pixel size

h more than 10ibrary for diffe

3. Bessel beam lenation. Only the ameam. The conical pentical for all 3 bel rings at the imamum, qn the n-th rground ratio (SBRcal (ds = 0.1 µm) d

ned above, theFigure 3(b) illin dependency

on for more inwidth sd → ∞

e proportion ofoportional to 1ul image informe useful signal

f imaging witht or bleachingsition before tl for our proof-

ple, but is mucht-sheet tailoringplementing a D

e as the used S0 kHz. In additerent beam form

ngth, ring energy amplitude of the holophase φ(r) (depicteeams. b) Calculat

age formation for ring in the ring sy

R) decreases with detection.

e power in the ustrates the infy of the confonformation abis identical to

f photons in th/Δz. Assuming

mation, the totl level should

h tailored and u, an image wthe beam was-of-principle exh too slow for g in combinatioDMD as fast 2SLM are commtion, DMDs prming amplitude

and signal-to-backogram defines the ed by the white linted relative energ4 different beam

ystem. ds is the cothe beam length

ring system ofluence qi of th

ocal slit width bout the calculo the power dis

he main maximg that only phtal beam poweremain consta

untailored lighwith and witho

s moved to thxperiments, clemost application with rolling2D amplitude mercially avairovide onboarde patterns.

kground ratio. A) length Δz and cen

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lengths Δz. q0 reonfocal slit width. Δz for convention

of the Bessel bhe individual rds and the be

lation of qi.) Tstribution in th

mum - with FWhotons emitted er must be proant. However,

ht-sheets, withoout beam adaphe next positiearly demonstrions. Again, wg shutter based

mask. DMDsilable and enabd memories, w

Principle of beamnter position z0 ofA of the beam and

of the individualepresents the main

c) The signal–to-nal (ds → ∞) and

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WHM of 0.88 µin the main m

oportional to t, with increasi

out being ption was ion. This rating the

we want to d confocal with the ble beam

which can

m f d l n -d

s with the formation (See the

on for an bar chart

µm at NA maximum the beam ing beam


length the total beam power and therefore the amount of bleaching increase. In other words, a reduction in beam length is proportional to a reduction in bleaching.

The bar chart in Fig. 3(b) shows the fraction q0 of photons that form a useful light-sheet image i.e. photons excited in the main maximum. This fraction q0(Δz) depends on the beam length Δz and, in the case of confocal detection, on the slit width ds. For a slit width of ds = 1 µm, one finds q0 = 54% for Δz = 200 µm and q0 = 42% for Δz = 600 µm. Assuming that only the photons excited in the main maximum contribute to the useful signal, whereas all photons from the Bessel rings form the image background, the signal-to-background ratio is given by

0 0

01

( z) ( z)1 ( z)( z)

SBR( z)nn

q qqq>

Δ Δ− ΔΔΔ = =

(3)

Therefore, the SBR increases by 62% if the beam length is reduced from 600 µm to 200 µm. Figure 3(c) shows the decay of SBR(Δz) with beam length Δz for conventional and confocal detection mode. In conventional mode, the relative improvement achieved by keeping the illumination beam short is more pronounced than in the confocal case. Nevertheless, the absolute SBR is always higher if confocal detection is applied.

3. Results

3.1 Increasing contrast with adaptive light sheets

To demonstrate the contrast enhancement capabilities of tailored LSM, we imaged an Arabidopsis root tip with (LTi6b:eGFP) membrane labeling and a fixed spherical cell cluster of T47D breast cancer cells stained with propidium iodide DNA labeling. The ideal light-sheet and the reference light-sheet – without object adaptation – used for the Arabidopsis root are presented in Fig. 2(c),(d). The NA of the illumination beam has been set to 0.13, resulting in a FWHM of the first maximum of 1.36 µm. Figure 4 shows a comparison of the corresponding fluorescent images using the reference light-sheet and a tailored light-sheet (both using line-confocal detection). The magnified image sections A and B underline the improvement in image contrast. To determine the contrast enhancement quantitatively, the Fourier transformations ( ),ref rP k θ and ( ),tay rP k θ of the reference image pref(r,φ) and the

tailored image ptay(r,φ) were calculated and averaged over the polar angle:

( ) ( )2

0,xx r xx rP k P k d

πθ θ= . For better comparability, both spectra have been normalized to

the same noise level and an identical DC component. The ratios of the tailored and reference spectra, ( ) ( )tay r ref rP k P k are plotted separately for the upper left rectangular area (Region 1)

and lower right area (Region 2) of the root image. Because all spectra have been normalized to the same DC component, the ratio ( ) ( ) 1tay r ref rP k P k = for kr = 0. It can be seen that the

amount of spatial frequencies above DC are enhanced up to 50% by light-sheet tailoring, i.e.

( ) ( ) 1.5tay r ref rP k P k ≈ . This corresponds to a background reduction of 33%. The effect

reduces with increasing frequency because noise becomes more dominant in this range. The contrast improvement is more pronounced in the lower right part of the image. This can be explained by the shorter beam length which was applied in this area for tailored imaging. This effect is especially pronounced in the conventional detection mode. Although contrast improvement is better for the conventional detection, the pure image contrast with confocal detection is still much better than with conventional detection - with and without adaptive Bessel beams.

3.2 Adapting the beam length to the penetration depth

The line-confocal light-sheet fluorescence images of the cell cluster are shown in Fig. 5(a),(b). The cells had been in culture for 28 days, growing to a diameter of roughly 500 µm


of the cell cludue to scatteri

Fig. 4tailoribeam 1.36 µ(d) thillustrdiffereµm.

In such a cpenetration defor the investimprovement.

( ) (tay r refP k P

of the line scsome areas oreference lighmaximum intthe maximumideal-light she

uster. In conseing and absorp

4. Fluorescent imaing. The images wlength was adapt

µm). The NA of thhe normalized imarate the contrast iment spatial frequen

case, the ideal epth. The purptigated cell clu. This finding i

( )rk , which in

can is marked of the image thht-sheet, which tensity of the b

m intensity of teet is lower for

quence, the peption.

ages of an Arabiwere recorded withted to the object, he detection objectage spectra as a mprovement by lincies are shown al

light-sheet is nple lines outlinuster. Again, ais quantified in

ncreased by up

by the dashedhe intensity wis caused by th

beam profile atthe beam center the tailored li

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ith (b) light-sheetof ds = 1 µm. They 0.13 (FWHM =

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ct shape, but aland the ideal liest reveals theaveraged imagFig. 5(d)). The

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by the longer beamn a certain x-inte

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a spherical ceble range of srations based oeam length fro% and 70%. In70 µm, correstailored beam wood, but slightof the image f

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(4)

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n that the and 70%, ast (SBR) 3), which reference

ent of the d 150 µm, is can be ributions, on of the or shorter iction. ight-sheet f the root d thus the


Furthermore, most light-sheet microscopes allow a rotation only around the x-axis (perpendicular to both optical axes). By rotating the root by 90° around x, light-sheet tailoring becomes unnecessary, but at the same time the size of the object in detection direction would increase and thus also the number of frames per 3D-stack.

According to the bar chart shown in Fig. 3(b), it was expected that bleaching is proportional to the beam length. The experimental data presented in Fig. 6 shows that the percentage of bleached fluorophores increases by a factor of 2, if the beam length is scaled up by a factor of 2.5. This can be explained by the fact that the light-sheet images have been captured with line-confocal detection, where the influence of the bleached areas far away from the focal plane is reduced. This is more relevant for longer beams, which are significantly broader than the shorter beam. Nevertheless, we could demonstrate a tremendous effect of the beam length on fluorophore bleaching. Therefore, we expect that the possible acquisition time of biological specimen sensitive to fluorophore bleaching can be doubled by light-sheet tailoring. This nearly compensates the main disadvantage of Bessel beams relative to Gaussian beams, which is enhanced bleaching, but maintains the strong advantages of Bessel beam illumination such as higher axial resolution and more homogeneous illumination.

5. Conclusion and outlook

We presented a concept for object adapted light-sheet tailoring. Adapting the Bessel beams lengths and positions to the space variant extent of the biological object result in a significant improvement in contrast and reduced photo bleaching. The image improvement depends on the shape and orientation of the object, which was estimated by a pre-scan using low-power scattered laser light. Light-sheet tailoring can be added to any system using Bessel beams, which are generated by either a static phase axicon or spatial light modulators, and this without any drawbacks or compromises. Especially photobleaching is expected to be reduced by light-sheet tailoring, although this effect has to be validated with different specimen in long term imaging experiments in the future.

Due to the characteristics of Bessel beams, light-sheet tailoring requires only binary amplitude modulation, which is also possible by phase-only SLMs. Due to the low frame rate of conventional SLMs, the proof of principle measurements presented in this article have been captured with a very limited frame rate. However, because only binary amplitude modulation is required, the phase-only SLM can be replaced by a fast DMDs or binary SLM. These devices feature frame rates, which enable tailored light-sheet microscopy at the maximum imaging speed of modern sCMOS cameras. With pure amplitude modulation, it is not possible to adapt Gaussian beams, which require a phase modulation in order to optimize the focal position and the depth of field.

Beside the automated object adapted light-sheet tailoring, the shape of the light sheet can also be defined by the user. In this way, parts of the object can be excluded from illumination. It is also possible to create light-sheets with user defined intensity distributions. In scanned LSM this is easily achieved in scanning direction by modulating the illumination intensity. In addition, the presented technique allows adaptation along the illumination direction. This enables the compensation of a varying fluorophore density as well as the correction of the intensity drop due to absorption of the illumination beam.

In summary, the simple and straightforward automation of tailored light-sheets enables this concept promising to become a standard feature in LSM with Bessel beams.

6. Method

Simulating the contribution qi of the individual Bessel rings onto the image formation a) Calculate the 3D intensity distribution of Bessel beams with different lengths by the angular spectrum wave propagation method. b) Separate the beam into its rings. c) Separate convolution of the Beam rings with the 3D-Detection-PSF in order to get the images of the


individual beam rings for the case the object is a homogeneous distribution of fluorophores. d) Cut the focal plane out of the 3D images. e) Calculate the ratio qi of the power inside a slit of width ds for a certain ring i and the total power in the image of all rings.

Acknowledgments

The authors thank Prof. Wolfgang Driever, Dr. Olaf Tietz and Dr. Andreas Thomsen for providing the biological samples and Dr. Felix Juenger and Luis Koebele for helpful comments on the manuscript.

The article processing charge was funded by the German Research Foundation (DFG) and the University of Freiburg in the funding programme Open Access Publishing.

Disclosures

The authors declare that there are no conflicts of interest related to this article.

References

1. H. Siedentopf and R. Zsigmondy, “Uber Sichtbarmachung und Größenbestimmung ultramikoskopischer Teilchen, mit besonderer Anwendung auf Goldrubingläser,” Ann. Phys. 315(1), 1–39 (1902).

2. A. H. Voie, D. H. Burns, and F. A. Spelman, “Orthogonal-plane fluorescence optical sectioning: three-dimensional imaging of macroscopic biological specimens,” J. Microsc. 170(Pt 3), 229–236 (1993).

3. J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. Stelzer, “Optical sectioning deep inside live embryos by selective plane illumination microscopy,” Science 305(5686), 1007–1009 (2004).

4. E. H. Stelzer, “Light-sheet fluorescence microscopy for quantitative biology,” Nat. Methods 12(1), 23–26 (2015).

5. E. G. Reynaud, U. Kržič, K. Greger, and E. H. K. Stelzer, “Light sheet-based fluorescence microscopy: more dimensions, more photons, and less photodamage,” HFSP J. 2(5), 266–275 (2008).

6. F. O. Fahrbach, F. F. Voigt, B. Schmid, F. Helmchen, and J. Huisken, “Rapid 3D light-sheet microscopy with a tunable lens,” Opt. Express 21(18), 21010–21026 (2013).

7. M. B. Ahrens, M. B. Orger, D. N. Robson, J. M. Li, and P. J. Keller, “Whole-brain functional imaging at cellular resolution using light-sheet microscopy,” Nat. Methods 10(5), 413–420 (2013).

8. U. Krzic, S. Gunther, T. E. Saunders, S. J. Streichan, and L. Hufnagel, “Multiview light-sheet microscope for rapid in toto imaging,” Nat. Methods 9(7), 730–733 (2012).

9. A. Maizel, D. von Wangenheim, F. Federici, J. Haseloff, and E. H. Stelzer, “High-resolution live imaging of plant growth in near physiological bright conditions using light sheet fluorescence microscopy,” Plant J. 68(2), 377–385 (2011).

10. J. Huisken and D. Y. R. Stainier, “Even fluorescence excitation by multidirectional selective plane illumination microscopy (mSPIM),” Opt. Lett. 32(17), 2608–2610 (2007).

11. A. Rohrbach, “Artifacts resulting from imaging in scattering media: a theoretical prediction,” Opt. Lett. 34(19), 3041–3043 (2009).

12. P. J. Keller, A. D. Schmidt, J. Wittbrodt, and E. H. Stelzer, “Reconstruction of zebrafish early embryonic development by scanned light sheet microscopy,” Science 322(5904), 1065–1069 (2008).

13. F. O. Fahrbach and A. Rohrbach, “Propagation stability of self-reconstructing Bessel beams enables contrast-enhanced imaging in thick media,” Nat. Commun. 3(1), 632 (2012).

14. E. Baumgart and U. Kubitscheck, “Scanned light sheet microscopy with confocal slit detection,” Opt. Express 20(19), 21805–21814 (2012).

15. T. Breuninger, K. Greger, and E. H. K. Stelzer, “Lateral modulation boosts image quality in single plane illumination fluorescence microscopy,” Opt. Lett. 32(13), 1938–1940 (2007).

16. P. J. Keller, A. D. Schmidt, A. Santella, K. Khairy, Z. Bao, J. Wittbrodt, and E. H. K. Stelzer, “Fast, high-contrast imaging of animal development with scanned light sheet-based structured-illumination microscopy,” Nat. Methods 7(8), 637–642 (2010).

17. L. A. Royer, W. C. Lemon, R. K. Chhetri, Y. Wan, M. Coleman, E. W. Myers, and P. J. Keller, “Adaptive light-sheet microscopy for long-term, high-resolution imaging in living organisms,” Nat. Biotechnol. 34(12), 1267–1278 (2016).

18. T. Meinert, B. A. Gutwein, and A. Rohrbach, “Light-sheet microscopy in a glass capillary: feedback holographic control for illumination beam correction,” Opt. Lett. 42(2), 350–353 (2017).

19. R. Jorand, G. Le Corre, J. Andilla, A. Maandhui, C. Frongia, V. Lobjois, B. Ducommun, and C. Lorenzo, “Deep and clear optical imaging of thick inhomogeneous samples,” PLoS One 7(4), e35795 (2012).

20. C. Bourgenot, C. D. Saunter, J. M. Taylor, J. M. Girkin, and G. D. Love, “3D adaptive optics in a light sheet microscope,” Opt. Express 20(12), 13252–13261 (2012).

21. D. Wilding, P. Pozzi, O. Soloviev, G. Vdovin, and M. Verhaegen, “Adaptive illumination based on direct wavefront sensing in a light-sheet fluorescence microscope,” Opt. Express 24(22), 24896–24906 (2016).


22. F. O. Fahrbach, P. Simon, and A. Rohrbach, “Microscopy with self-reconstructing beams,” Nat. Photonics 4(11), 780–785 (2010).

23. T. Meinert, O. Tietz, K. J. Palme, and A. Rohrbach, “Separation of ballistic and diffusive fluorescence photons in confocal Light-Sheet Microscopy of Arabidopsis roots,” Sci. Rep. 6(1), 30378 (2016).

24. T. A. Planchon, L. Gao, D. E. Milkie, M. W. Davidson, J. A. Galbraith, C. G. Galbraith, and E. Betzig, “Rapid three-dimensional isotropic imaging of living cells using Bessel beam plane illumination,” Nat. Methods 8(5), 417–423 (2011).

25. F. O. Fahrbach, V. Gurchenkov, K. Alessandri, P. Nassoy, and A. Rohrbach, “Light-sheet microscopy in thick media using scanned Bessel beams and two-photon fluorescence excitation,” Opt. Express 21(11), 13824–13839 (2013).

26. B. C. Chen, W. R. Legant, K. Wang, L. Shao, D. E. Milkie, M. W. Davidson, C. Janetopoulos, X. S. Wu, J. A. Hammer 3rd, Z. Liu, B. P. English, Y. Mimori-Kiyosue, D. P. Romero, A. T. Ritter, J. Lippincott-Schwartz, L. Fritz-Laylin, R. D. Mullins, D. M. Mitchell, J. N. Bembenek, A. C. Reymann, R. Böhme, S. W. Grill, J. T. Wang, G. Seydoux, U. S. Tulu, D. P. Kiehart, and E. Betzig, “Lattice light-sheet microscopy: imaging molecules to embryos at high spatiotemporal resolution,” Science 346(6208), 1257998 (2014).

27. L. Gao, L. Shao, C. D. Higgins, J. S. Poulton, M. Peifer, M. W. Davidson, X. Wu, B. Goldstein, and E. Betzig, “Noninvasive imaging beyond the diffraction limit of 3D dynamics in thickly fluorescent specimens,” Cell 151(6), 1370–1385 (2012).

28. A. K. Chmielewski, A. Kyrsting, P. Mahou, M. T. Wayland, L. Muresan, J. F. Evers, and C. F. Kaminski, “Fast imaging of live organisms with sculpted light sheets,” Sci. Rep. 5, 9385 (2015).

29. K. M. Dean and R. Fiolka, “Uniform and scalable light-sheets generated by extended focusing,” Opt. Express 22(21), 26141–26152 (2014).

30. F. O. Fahrbach and A. Rohrbach, “A line scanned light-sheet microscope with phase shaped self-reconstructing beams,” Opt. Express 18(23), 24229–24244 (2010).

31. A. Müller, M. C. Wapler, and U. Wallrabe, “Segmented Bessel beams,” Opt. Express 25(19), 22640–22647 (2017).

32. P. Zhang, M. E. Phipps, P. M. Goodwin, and J. H. Werner, “Light-sheet microscopy by confocal line scanning of dual-Bessel beams,” J. Biomed. Opt. 21(10), 100502 (2016).

33. K. Szulzycki, V. Savaryn, and I. Grulkowski, “Generation of dynamic Bessel beams and dynamic bottle beams using acousto-optic effect,” Opt. Express 24(21), 23977–23991 (2016).

34. A. S. Rao and G. K. Samanta, “On-axis intensity modulation-free, segmented, zero-order Bessel beams with tunable ranges,” Opt. Lett. 43(13), 3029–3032 (2018).

35. Q. Fu, B. L. Martin, D. Q. Matus, and L. Gao, “Imaging multicellular specimens with real-time optimized tiling light-sheet selective plane illumination microscopy,” Nat. Commun. 7, 11088 (2016).


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Light-sheet microscopy with length-adaptive Bessel beams