Light Sheet Thickness Research Articles

Extended Depth of Focus Two-Photon Light-Sheet Microscopy for In Vivo Fluorescence Imaging of Large Multicellular Organisms at Cellular Resolution.

Two-photon excitation in light-sheet microscopy advances applications to live imaging of multicellular organisms. In a previous study, we developed a two-photon Bessel beam light-sheet microscope with a nearly 1-mm field of view and less than 4-μm axial resolution, using a low magnification (10×), middle numerical aperture (NA 0.5) detection objective. In this study, we aimed to construct a light-sheet microscope with higher resolution imaging while maintaining the large field of view, using low magnification (16×) with a high NA 0.8 objective. To address potential illumination and detection mismatch, we investigated the use of a depth of focus (DOF) extension method. Specifically, we used a stair-step device composed of five-layer annular zones that extended DOF two-fold, enough to cover the light-sheet thickness. Resolution measurements using fluorescent beads showed that the reduction in resolutions was small. We then applied this system to in vivo imaging of medaka fish and found that image quality degradation at the distal site of the beam injection could be compensated. This demonstrates that the extended DOF system combined with wide-field two-photon light-sheet microscopy offers a simple and easy setup for live imaging application of large multicellular organism specimens with sub-cellular resolution.

Pulsed LED line light for large-scale PIV—development and use in wave load measurements

In this paper the development of a high-power pulsed LED line light and its use to apply particle image velocimetry (PIV) during wave impact measurements are described. An electrical circuit that generates high-current pulses is designed and built, which is used to overdrive a number of commercially available LEDs. The limit for this overdrive-capacity is determined as function of pulse duration for various commercial available LEDs. Two systems of cylindrical convex lenses are designed to act as a collimator and reduce divergence of the LED bundle and the resulting light sheet properties (maximum light intensity and sheet thickness) are investigated. An array of LEDs of 60 cm length (referred to as the LED line light) is designed and manufactured. For the two lens systems, the LED line light provides proper light sheet conditions to illuminate measurement regions in the order of either 0.3 × 0.3 m2, or 1 × 1 m2, at a sufficiently constant light sheet thickness of 5 mm. The application of the LED line light is demonstrated by quantifying the instantaneous flow field of a wave impacting on a blunt object in a wave flume. PIV measurements are conducted at an acquisition rate of 25 frame pairs per second, quantifying maximum flow velocities in the order of 1.0 m s−1 at a LED pulse width of 200 μs. The system, consisting of the LED line light, a CMOS camera and open source PIV processing software provides the possibility to perform 2D planar PIV measurements for a fraction of the costs of a commercially available laser based PIV system.

Investigation of the extended focusing capability of the spherical aberration to enlarge the field of view in light-sheet fluorescence microscopy.

In light-sheet fluorescence microscopy (LSFM), using Gaussian beams for light-sheet generation results in a trade-off between the thickness and the field of view (FOV). Here we present a theoretical analysis of using spherical aberration to enlarge the FOV while keeping the light-sheet thickness small. Such spherical aberration can arise when focusing beams through an interface between materials of mismatched refractive indices. The depth-of-focus extension of the Gaussian beam is achieved when using air objectives to focus light into the samples dipped in the immersion medium with a higher refractive index. By scanning this elongated beam, a thin light sheet with a wide FOV can be used for LSFM imaging. Meanwhile, the accompanied sidelobes with the spherical aberrated light sheet, which are mainly distributed in the rear part of the light sheet, are also discussed. Simulation results show that an extended FOV of 64.4µm is possible for an objective lens of NA=0.3, which is about 5 times that of the unaberrated case. For such an extended FOV, a comparatively thin thickness of 1.38µm as well as the first sidelobe about 11.1% of the peak intensity in the center are also demonstrated.

Scattering-Based Light-Sheet Microscopy for Rapid Cellular Imaging of Fresh Tissue.

Light-sheet microscopy (LSM) is a novel imaging technology that has been used for imaging fluorescence contrast in basic life science research. In this paper, we have developed a scattering-based LSM (sLSM) for rapidly imaging the cellular morphology of fresh tissues without any exogenous fluorescent dyes. In the sLSM device, a thin light sheet with the central wavelength of 834 nm was incident on the tissue obliquely, 45° relative to the tissue surface. The detection optics was configured to map the light sheet-illuminated area onto a two-dimensional imaging sensor. The illumination numerical aperture (NA) was set as 0.0625, and the detection NA 0.3. The sLSM device achieved a light sheet thickness of less than 6.7 µm over 284 µm along the illumination optical axis. The detection optics of the sLSM device had a resolution of 1.8 µm. The sLSM images of the swine kidney ex vivo visualized tubules with similar sizes and shapes to those observed in histopathologic images. The swine duodenum sLSM images revealed cell nuclei and villi architecture in superficial lesions and glands in deeper regions. The preliminary results suggest that sLSM may have the potential for rapidly examining the freshly-excised tissue ex vivo or intact tissue in vivo at microscopic resolution. Further optimization and performance evaluation of the sLSM technology will be needed in the future. Lasers Surg. Med. © 2020 Wiley Periodicals LLC.

Simulation analysis and comparison of the effects of aberrations on multiphoton fluorescent light sheets

Multi-photon scanning light-sheet microscope, which combines the advantages of light-sheet illumination and multi-photon excitation, has high axial resolution, low photobleaching, and a large field of view. However, as the light sheet goes deeper into the biological tissue, biological optical aberrations caused by the uneven refractive index inside the organism become non-negligible factors that dramatically increase the thickness and decrease the light intensity of the light sheet. This paper theoretically deduces and simulates the influence of aberrations on the intensity and thickness of the two-, three-, and four-photon fluorescent light sheet. A clear general trend is obvious. With the increasing amplitude of aberration, the effect of aberration on the light sheet thickness and intensity increases in each multiphoton light sheet. In terms of thickness, the impact of high-order aberrations diminishes from the two-photon to the four-photon light sheet. However, in terms of intensity, the impact of high-order aberrations increases from the two-photon to the four-photon light sheet. The number of items to be corrected for each multiphoton light sheet is given and compared under random aberrations with different amplitudes. The results provide important guidance for the design and evaluation of adaptive optics in multi-photon light sheet microscopes.

A shape-switch-block method for confocal light-sheet microscopy with sectioned Bessel beams and stimulated emission depletion

Microscopy seeks to simultaneously maximize optical resolution, contrast, speed, volume size, and probe tolerability, which is possible by combining different complementary imaging techniques with their specific strengths. Here, we show how to combine three physical concepts to increase resolution and contrast in light-sheet microscopy by making the effective light-sheet thinner through phase shaping, fluorophores-switching, and dynamic blocking of fluorescence. This shape-switch-block principle is realized by illumination with two holographically shaped, sectioned Bessel beams. Second, by switching off fluorophores in the proximity of the excitation center using continuous-wave stimulated emission depletion (STED). And third, by blocking fluorescence outside the switching region by confocal line detection. Thereby, we reduce the light-sheet thickness by 35%, achieving an isotropic resolution with beads in a 300 × 70 × 50 µm³ volume. Without STED, we obtain 0.37 µm resolution in cell clusters at improved sectioning and penetration depth. The shape-switch-block concept promises high potential, also for other microscopy techniques.

Thinner and Longer Working Distance Light Sheet Illumination and Microscopic Imaging

Light sheet fluorescence microscopy has become a basic tool in biology and medical research with its fast imaging speed, low phototoxicity and high spatiotemporal resolution. Here, we report a novel method to generate multiplane parallel light sheets with a micro structure named isosceles triangular array. The thickness of light sheet in each plane can approach 3.12 μm along with the working distance of 152.20 μm directly generated with the isosceles triangular array. We experimentally recorded the multiplane parallel light sheets illumination, which is consistent with the corresponding simulation by using ray-tracing method. To evaluate the imaging quality, we compared the light field generated with and without projected phase mask onto the spatial light modulator by using polystyrene fluorescent microspheres nanoparticles and transgenic zebrafish. This study provides a potential method for the building of volumetric imaging light sheet fluorescence microscopy with high resolution and low phototoxicity.

Improved optical slicing by stimulated emission depletion light sheet microscopy.

Three-dimensional microscopy is mandatory for biological investigation. We describe a stimulated emission depletion selective plane illumination microscope (STED-SPIM) that provides both ease of implementation and an efficient optical slicing. This self-aligned system is based on a single diode-pumped solid-state laser and phase masks made of simple cover glass. A three-fold reduction of the light sheet thickness is achieved together with an enhancement of the sheet uniformity. This method is validated by using fluorescent microspheres and thick slices of fixed and clarified mouse brain to provide an enhanced imaging of Alzheimer's disease models.

How to define and optimize axial resolution in light-sheet microscopy: a simulation-based approach.

"How thick is your light sheet?" is a question that has been asked frequently after talks showing impressive renderings of 3D data acquired by a light-sheet microscope. This question is motivated by the fact that most of the time the thickness of the light-sheet is uniquely associated to the axial resolution of the microscope. However, the link between light-sheet thickness and axial resolution has never been systematically assessed and it is still unclear how both are connected. The question is not trivial because commonly employed measures cannot readily be applied or do not lead to easily interpretable results for the many different types of light sheet. Here, we introduce a set of intuitive measures that helps to define the relationship between light sheet thickness and axial resolution by using simulation data. Unexpectedly, our analysis revealed a trade-off between better axial resolution and thinner light-sheet thickness. Our results are surprising because thicker light-sheets that provide lower image contrast have previously not been associated with better axial resolution. We conclude that classical Gaussian illumination beams should be used when image contrast is most important, and more advanced types of illumination represent a way to optimize axial resolution at the expense of image contrast.

Generation of a subdiffraction-limit light sheet with micrometer-order thickness using a computer-generated hologram

We present the generation of a subdiffraction-limit light sheet using a computer-generated hologram (CGH) with a lens system, comprising a cylindrical lens and objective lens. The algorithm for designing a CGH generating an array of subdiffraction-limit spots (Ogura et al. in Opt Express 22:25196–25207, 2014) is modified to design a CGH generating a subdiffraction-limit light sheet. The modulation of CGHs is limited to one dimension to avoid the unnecessary fluctuation of intensity along the height direction of the light sheet. In design, we can obtain an isolated light sheet with 80% thickness of the diffraction-limit light sheet. In addition, a subdiffraction-limit triplet light sheet can be generated owing to design flexibility. Analysis of the three-dimensional structure of light sheets suggests that our method can be used to reduce the thickness of a light sheet, while suppressing a reduction in depth. Experimental results demonstrate that the designed CGH generates a subdiffraction-limit light sheet with a micrometer-order thickness.

Flexible Light-Sheet Generation by Field Synthesis

Light-sheet microscopy has revolutionized biological imaging by affording rapid and efficient volumetric imaging while minimizing the applied light-dosage and thus phototoxicity. However, due to beam divergence, there is a trade-off between the light-sheet thickness and the useful field-of-view for imaging: the confocal parameter, which approximates the propagation length of a light-sheet, shrinks nonlinearly with the size of the beam waist, and consequently, modest axial resolution is achieved when imaging large specimens. To overcome this trade-off, propagation invariant beams have been employed in light-sheet generation, such as Bessel or Airy beams. These beams maintain their cross-sectional profile over extended distances and overcome the beam divergence of Gaussian beams. To generate a light-sheet, a propagation invariant beam is scanned laterally to create a time averaged sheet of light. Such setups require dedicated optical trains, which increase the cost and complexity of a light-sheet microscope. We have recently discovered and proven the ‘Field Synthesis’ theorem, which describes a universal mechanism for light-sheet generation. Field synthesis predicts that any scanned light-sheet can be synthesized by an incoherent superposition of 1D intensity patterns. We demonstrate that in practice, this results in far less complex optical systems and much higher light-efficiency compared to previous setups using propagation invariant beams. Through careful comparisons, we show that through Field Synthesis we can re-create the time averaged intensity of Bessel and Lattice light-sheets. However, in contrast to scanned Bessel beams, we achieve 100% spatial duty cycle, which results in drastically reduced photobleaching. Compared to lattice light-sheet microscopy, Field Synthesis is achromatic and can hence perform simultaneous multicolor imaging. We present careful comparisons of each illumination mode and demonstrate biological imaging applications of light-sheets produced by Field Synthesis.

Extension of the span and optimization of the optical "magic carpet": generation of a wide quasi-nondiffracting light sheet.

Light sheet illumination is the basis in developing light sheet microscopy (LSM), a technique with significant advantages compared with other classical techniques. Most proposed optical systems to generate light sheets for LSM use many optical elements, which require extensive adjustments and are costly; moreover, they generate a nonuniform or semiuniform light sheet or have a short depth of field (DOF). A simple scheme using a pair of double slits and a cylindrical lens for generating a quasi-nondiffracting 2D light sheet was reported in Opt. Lett.40, 5121 (2015)OPLEDP0146-959210.1364/OL.40.005121. In the present investigation, we elaborate on the optimization of the mask used. As the separation between the two slits increases, the light sheet becomes thinner and the DOF smaller and vice versa. The slits' width does not affect the light sheet thickness, but it does affect the intensity of the side lobes. For convergence angles of the inner slits from 0.75° to 8°, an optimum ratio of the slits' separation/width of 2.182 is recommended. The obtained light sheet is quasi-diffraction-free, namely, while its DOF is comparable with that of a Gaussian beam, its diffraction broadening is substantially smaller. We also add to the previously developed configuration a Powell lens in order to expand the beam in the spanwise direction while keeping nearly constant intensity in this dimension. We perform scalar diffraction theory calculations and conduct measurements showing the nearly constant intensity in the significantly broadened span of the light sheet. Potential applications for the augmented width include imaging of certain large embryos, laser micromachining, and microparticle image velocimetry.

Microfabricated devices for single objective single plane illumination microscopy (SoSPIM).

Light sheet microscopy is a relatively new form of fluorescence microscopy that has been receiving a lot of attention recently. The strong points of the technique, such as high signal to noise ratio and its reduced photodamage of fluorescently labelled samples, come from its unique feature to illuminate only a thin plane in the sample that coincides with the focal plane of the detection lens. Typically this requires two closely positioned perpendicular objective lenses, one for detection and one for illumination. Apart from the fact that this special configuration of objective lenses is incompatible with standard microscope bodies, it is particularly problematic for high-resolution lenses which typically have a short working distance. To address these issues we developed sample holders with an integrated micromirror to perform single lens light sheet microscopy, also known as single objective single plane illumination microscopy (SoSPIM). The first design is based on a wet-etched silicon substrate, the second on a microfabricated polished polymer plug. We achieved an on-chip light sheet thickness of 2.3 μm (FWHM) at 638 nm with the polymer micromirror and of 1.7 μm (FWHM) at 638 nm with the silicon micromirror, comparable to reported light sheet thicknesses obtained on dedicated light sheet microscopes. A marked contrast improvement was obtained with both sample holders as compared to classic epi-fluorescence microscopy. In order to evaluate whether this technology could be made available on a larger scale, in a next step we evaluated the optical quality of inexpensive replicas from both types of master molds. We found that replicas from the polished polymer based mold have an optical quality close to that of the master component, while replicas from the silicon based mold were of slightly lower but still acceptable quality. The suitability of the replicated polymer based sample holder for single-lens light sheet microscopy was finally demonstrated by imaging breast cancer spheroids.

Selectable light-sheet uniformity using tuned axial scanning.

Light-sheet fluorescence microscopy (LSFM) is an optical sectioning technique capable of rapid three-dimensional (3D) imaging of a wide range of specimens with reduced phototoxicity and superior background rejection. However, traditional light-sheet generation approaches based on elliptical or circular Gaussian beams suffer an inherent trade-off between light-sheet thickness and area over which this thickness is preserved. Recently, an increase in light-sheet uniformity was demonstrated using rapid biaxial Gaussian beam scanning along the lateral and beam propagation directions. Here we apply a similar scanning concept to an elliptical beam generated by a cylindrical lens. In this case, only z-scanning of the elliptical beam is required and hence experimental implementation of the setup can be simplified. We introduce a simple dimensionless uniformity statistic to better characterize scanned light-sheets and experimentally demonstrate custom tailored uniformities up to a factor of 5 higher than those of unscanned elliptical beams. This technique offers a straightforward way to generate and characterize a custom illumination profile that provides enhanced utilization of the detector dynamic range and field of view, opening the door to faster and more efficient 2D and 3D imaging.

Uncertainty quantification of three-dimensional velocimetry techniques for small measurement depths

In this paper, the multi-camera techniques tomographic PTV and 3D-PTV as well as the single-camera defocusing PTV approach are assessed for flow measurements with a small measurement depth in conjunction with a high resolution along the optical axis. This includes the measurement of flows with strong velocity gradients in z direction and flow features, which have smaller scales than the actual light sheet thickness. Furthermore, in fields like turbomachinery, the measurement of flows in domains with small depth dimensions is of great interest. Typically, these domains have dimensions on the order of 100 mm in z direction and of 101 mm in x and y direction. For small domain depths, employing a 3D flow velocimetry technique is inevitable, since the measurement depths lie in the range of the light sheet thickness. To resolve strong velocity gradients and small-scale flow features along the z axis, the accuracy and spatial resolution of the 3D technique are very important. For the comparison of the different measurement methods, a planar Poiseuille flow is investigated. Quantitative uncertainty analyses reveal the excellent suitability of all three methods for the measurement of flows in domains with small measurement depths. Naturally, the multi-camera approaches tomographic PTV and 3D-PTV yield lower uncertainties, since they image the measurement volume from different angles. Other criteria, such as optical access requirements, hardware costs, and setup complexity, clearly favor defocusing PTV over the more complex multi-camera techniques.

Active focus stabilization for upright selective plane illumination microscopy.

Due to its sectioning capability, large field of view, and minimal light exposure, selective plane illumination microscopy has become the preferred choice for 3D time lapse imaging. Single cells in a dish can be conveniently imaged using an upright/inverted configuration. However, for measurements on long time scales (hours to days), mechanical drift is a problem; especially for studies of mammalian cells that typically require heating to 37°C which causes a thermal gradient across the instrument. Since the light sheet diverges towards the edges of the field of view, such a drift leads to a decrease in axial resolution over time. Or, even worse, the specimen could move out of the imaging volume. Here, we present a simple, cost-effective way to stabilize the axial position using the microscope camera to track the sample position. Thereby, sample loss is prevented and an optimal axial resolution is maintained by keeping the sample at the position where the light sheet is at its thinnest. We demonstrate the virtue of our approach by measurements of the light sheet thickness and 3D time lapse imaging of a cell monolayer at physiological conditions.

STED-SPIM made simple.

We report the development of a stimulated emission depletion (STED) selective plane illumination (SPIM) microscope based on a single diode-pumped solid state (DPSS) laser that simultaneously delivers nanosecond-pulses at two wavelengths. The two wavelengths, 355 nm and 532 nm, are generated by harmonic conversion and they are used to induce respectively excitation and stimulated emission depletion. This source should allow a low-cost, compact, very efficient and simplified STED scheme since the two beams are intrinsically aligned and synchronized. Using a chromatic beam shaping device which leaves the excitation beam unaffected and produces a donut-shaped STED beam, we demonstrate a 300% reduction of the light sheet thickness, together with an enhancement of the sheet uniformity over larger field of view, at low STED power, in Coumarin dye solution.

Single-view volumetric PIV via high-resolution scanning, isotropic voxel restructuring and 3D least-squares matching (3D-LSM)

Scanning PIV as introduced by Brücker (1995 Exp. Fluids 19 255–63, 1996a Appl. Sci. Res. 56 157–79) has been successfully applied in the last 20 years to different flow problems where the frame rate was sufficient to ensure a ‘frozen’ field condition. The limited number of parallel planes however leads typically to an under-sampling in the scan direction in depth; therefore, the spatial resolution in depth is typically considerably lower than the spatial resolution in the plane of the laser sheet (depth resolution = scan shift Δz ≫ pixel unit in object space). In addition, a partial volume averaging effect due to the thickness of the light sheet must be taken into account. Herein, the method is further developed using a high-resolution scanning in combination with a Gaussian regression technique to achieve an isotropic representation of the tracer particles in a voxel-based volume reconstruction with cuboidal voxels. This eliminates the partial volume averaging effect due to light sheet thickness and leads to comparable spatial resolution of the particle field reconstructions in x-, y- and z-axes. In addition, advantage of voxel-based processing with estimations of translation, rotation and shear/strain is taken by using a 3D least-squares matching method, well suited for reconstruction of grey-level pattern fields. The method is discussed in this paper and used to investigate the ring vortex instability at Re = 2500 within a measurement volume of roughly 75 × 75 × 50 mm3 with a spatial resolution of 100 µm/voxel (750 × 750 × 500 voxel elements). The volume has been scanned with a number of 100 light sheets and scan rates of 10 kHz. The results show the growth of the Tsai–Widnall azimuthal instabilities accompanied with a precession of the axis of the vortex ring. Prior to breakdown, secondary instabilities evolve along the core with streamwise oriented striations. The front stagnation point's streamwise distance to the core starts to decrease while the rear stagnation point distance remains constant which indicates that the front part of the ring is at first losing its mass during breakdown.

Scanning thin-sheet laser imaging microscopy (sTSLIM) with structured illumination and HiLo background rejection

We report replacement of one side of a static illumination, dual sided, thin-sheet laser imaging microscope (TSLIM) with an intensity modulated laser scanner in order to implement structured illumination (SI) and HiLo image demodulation techniques for background rejection. The new system is equipped with one static and one scanned light-sheet and is called a scanning thin-sheet laser imaging microscope (sTSLIM). It is an optimized version of a light-sheet fluorescent microscope that is designed to image large specimens (<15 mm in diameter). In this paper we describe the hardware and software modifications to TSLIM that allow for static and uniform light-sheet illumination with SI and HiLo image demodulation. The static light-sheet has a thickness of 3.2 µm; whereas, the scanned side has a light-sheet thickness of 4.2 µm. The scanned side images specimens with subcellular resolution (<1 µm lateral and <4 µm axial resolution) with a size up to 15 mm. SI and HiLo produce superior contrast compared to both the uniform static and scanned light-sheets. HiLo contrast was greater than SI and is faster and more robust than SI because as it produces images in two-thirds of the time and exhibits fewer intensity streaking artifacts.

Quantification of dispersed phase concentration using light sheet imaging methods

With the prevalence of particle image velocimetry (PIV) as a quantitative tool for fluid mechanics diagnostics, its application for analyzing complicated multiphase flows has been steadily increasing over the last several decades. While the primary issue in using PIV for multiphase flows is in separating the information of the phases for independent analysis with a minimum of spurious “cross-talk,” an equally crucial but often overlooked point is in the accurate quantitative measurement of the dispersed phase concentration. Accurate concentration measurement is important due to the fact that the dispersed phase is often heterogeneously distributed in both space and time, either due to a non-uniformity of the source of particulates (such as a spray nozzle or sediment boundary) or due to inertial migration of the particles even from originally homogeneous spatial distributions. In the current work, we examine the effects of light sheet profile distortion and attenuation by tracer seeding particles, as well as reflected light from local wall boundaries on the effective light sheet thickness. The effective thickness is critical for concentration measurements, as it dictates the dispersed phase detection volume. A direct calibration method is demonstrated to measure the effective light sheet thickness in a water/glass bead system, which shows that systematic bias errors on the order of 30% can result if the reflective bed condition is not accounted for, and the errors can be as high as 50% or more if a single-point measure of the sheet width is used.