Thin Light Sheet Research Articles

Image post-processing for SILMAS: structured illumination light sheet microscopy with axial sweeping.

In this article, we propose a post-processing scheme for the novel volumetric microscopy technique SILMAS. We demonstrate this scheme on data from an alpha-synuclein transgenic mouse brain. By combining structured illumination and axial sweeping, a SILMAS measurement provides a prerequisite for quantitative data extraction through improved contrast and optical sectioning. However, due to the technique's efficient removal ofb multiple scattered light, image artifacts such as illumination inhomogeneity, shadowing stripes, and signal attenuation, are highlighted in the recorded volumes. To suppress these artifacts, we rely on the strengths of the imaging method. The SILMAS data, together with the Beer-Lambert law, allow for an approximation of real light extinction, which can be used to compensate for light attenuation in a near-quantitative way. Shadowing stripes can be suppressed efficiently using a computational strategy thanks to the large numerical aperture of an axially swept light sheet. Here, we build upon prior research that employed wavelet-Fourier filtering by incorporating an extra bandpass step. This allows us to filter high-contrast light sheet microscopy data without introducing new artifacts and with minimal distortion of the data. The combined technique is suitable for imaging cleared tissue samples of up to a centimeter scale with an isotropic resolution of a few microns. The combination of a thin and uniform light sheet, scattered light suppression, light attenuation compensation, and shadowing suppression produces volumetric data that is seamless and highly uniform.

Dynamic Wall Shear Stress Measurement Using Event-Based 3D Particle Tracking

We describe the implementation of a 3d Lagrangian particle tracking (LPT) system based on event-based vision (EBV) sensors and demonstrate its application for the near-wall characterization of a turbulent boundary layer (TBL) in air. The viscous sublayer of the TBL is illuminated by a thin light sheet that grazes the surface of a glass window inserted into the flat wall of the wind tunnel wall. The data simultaneously captured by three synchronized EBV-cameras is used to reconstruct the 3d particle tracks within 0.4 mm of the wall on a field of view of 12.0 mm by 7.5 mm. The velocity and position of particles within the viscous sub-layer permit the estimation of the local, unsteady wall shear stress vector under the assumption of linearity between particle velocity and wall shear stress. Thereby, 2d distributions and higher order statistics of the unsteady wall shear stress are obtained. The systematic underestimation of the spanwise shear stress fluctuation can be explained on the basis of DNS data; a methodology for its mitigation is proposed. The employed EBV hardware coupled with suited LPT algorithms provide data quality on par with currently used, considerably more expensive, high-speed framing cameras.

Converting a symmetrical Gaussian beam into a thin tunable light sheet

Converting a symmetrical Gaussian beam into a thin tunable light sheet

A metasurface-on-fiber light-sheet generator for biological imaging

Light sheet has been widely used for bioimaging. However, it is a big challenge to build a miniature light-sheet imaging system due to geometric constraints of bulky refractive and diffractive elements. Metasurface-based devices composed of subwavelength unit cells, with compact size and the ability to effectively manipulate optical fields, have been considered as potential planar analogs of conventional components to realize various functionalities. Integrating metasurfaces onto an optical fiber facet is sparking new ideas and emerging applications. Here, we propose a metasurface-on-fiber light-sheet generator, which can convert a fiber mode to a thin light sheet. By adopting a multi-core fiber and a dielectric metasurface on its facet, a light sheet with a thickness of 2 μm and a length of 83.0 μm is obtained. The generator can greatly simplify the illumination of light sheet imaging systems and be flexibly used for advanced biological imaging applications.

Thin light sheets image cells at organelle level

Thin light sheets image cells at organelle level

Construction and characterization of a versatile multimodal single-molecule super-resolution microscope for 3D whole cell studies.

The cellular environment is dense and complex, and imaging with 3D single-molecule super-resolution microscopy requires careful consideration of appropriate illumination methods to use in relation to the goals of the study. There is, however, typically a tradeoff between performance and simplicity when selecting illumination schemes. Epi-illumination is a common and simple technique, but it generates high unwanted background fluorescence and causes photodamage and photobleaching throughout the imaged cell. Total internal reflection fluorescence (TIRF) is another technique which provides excellent contrast, but it is limited to imaging a thin volume at the bottom of a sample and is thus incompatible with whole-cell imaging. Light sheet illumination is an alternative, whole-cell compatible approach where a thin sheet of light is directed orthogonally to the detection axis to optically section a cell and reduce the fluorescence background, photobleaching, and photodamage. In this work we demonstrate a versatile microscopy platform which integrates the sectioning and background reducing capability of light sheet illumination with homogeneous, flat-field epi- and TIRF illumination. A flat-field intensity profile improves the control of fluorophore blinking kinetics and the resolution across the entire field of view when compared to a standard Gaussian intensity profile. Additionally, this microscope is designed to allow very fast switching between illumination modalities and laser wavelengths, using primarily commercially available parts. When combined with point spread function engineering, our system conveniently enables 3D single-molecule super-resolution imaging and molecular counting throughout whole cells with optimized and adjustable illumination modalities.

Multi-target 3D single-molecule super resolution imaging throughout mammalian cells with a single-objective tilted light sheet.

Single-molecule based super-resolution fluorescence microscopy offers an approach to sub-diffraction-limited imaging of biological samples. While these approaches have proved significant to understanding subcellular structures, numerous super-resolution systems suffer from drawbacks such as decreased localization precision due to high background fluorescence, lack of control of the extracellular environment, decreased multi-target accuracy due to chromatic offsets in multicolor imaging, and the inability to image structures in three dimensions (3D). Light sheet fluorescence microscopy, which involves illuminating the sample with a thin sheet of light, offers a gentle illumination scheme that greatly reduces photobleaching, photodamage, and out of focus background fluorescence, thereby improving the localization precision. Here, we present a single-objective tilted light sheet super-resolution setup with five added innovations: (i) a microfluidic chip with reflective sidewalls and transparent top, (ii) sequential DNA Point Accumulation for Imaging in Nanoscale Topography (DNA-PAINT) known as Exchange-PAINT, (iii) deep learning for high density single emitter localization, (iv) double helix point spread functions for 3D imaging, and (iv) galvanometric dithering of the illumination beam to reduce scattering artifacts and provide homogenous sample illumination. By utilizing light sheet illumination in conjunction with Exchange-PAINT and deep learning, this setup allows for the use of high concentrations of imager strands given that background fluorescence is drastically reduced and sparse emitters are no longer necessary for accurate localization. Furthermore, sequential washes of imager strands are enabled through the use of microfluidics, which allows for accurate, multi-target reconstructions that are not subject to chromatic offsets. Thus, we demonstrate a setup that offers improved localization precision, extracellular environment control, multi-target information, and 3D imaging that can easily be implemented for fast, accurate, and precise multi-target super-resolution whole cell imaging.

Linearly modulated multi-focal diffractive lens for multi-sheet excitation of flow driven samples in a light-sheet fluorescence microscope

Light sheet fluorescence microscope with single light sheet illumination enables rapid 3D imaging of living cells. In this paper we show the design, fabrication and characterization of a diffractive optical element producing several light sheets along a 45° inclined tube. The element, which is based on a multi-focal diffractive lens and a linear grating, generates five thin light sheets with equal intensities when combined with a refractive cylindrical lens. The generated uniform light sheets can be applied for the scanning of samples in tubes enabling flow-driven 3-dimensional imaging.

Thermocapillary-driven coherent structures by low-Stokes-number particles and their morphology in high-aspect-ratio liquid bridges

Thermocapillary-driven coherent structures by low-Stokes-number particles are experimentally investigated. Time-dependent convection is induced in a tall or high-aspect-ratio half-zone liquid bridge. We successfully realize coherent structures, which seem rigidly rotating by following the traveling-wave-type convection of mHTW=1 in azimuthal wave number due to so-called hydrothermal-wave instability. The Poincaré sections at different heights of a single particle forming the coherent structure are obtained in the rotating frame of reference by preparing a thin light sheet. Projected distribution of the multiple particles forming the coherent structure are also depicted to examine the correlation with the Poincaré points. The stability of the coherent structures is discussed by tracking the particles exhibiting turnover motion in the liquid bridge.

Quasi-non-diffracting static light sheets generated by holistically optimized pupil masks

Thin and wide light sheets are crucial for light sheet microscopy with wide field of views and near diffraction-limited resolutions. Non-diffracting light sheets feature wide span ranges but often come with strong sidelobes or increased thicknesses. We develop a computational optimization method for creating light sheets that balance the trade-offs between thickness, non-diffracting range and sidelobes. We show in theory and experiment that our optimized mask extends the static light sheet's non-diffracting range by 50 % while keeping its sidelobes below the 20 % level.

Dual-sided illumination light-sheet fluorescence microscopy with virtual single-pixel imaging deconvolution

In light-sheet fluorescence microscopy (LSFM) a thin light sheet is used to excite the specimen from the side and imaging is performed in the direction perpendicular to the light-sheet. It has the advantages of fast imaging speed, high optical sectioning capability and low photobleaching and phototoxicity to samples. Therefore, it is suitable for high-quality, long-term three-dimensional dynamic observation of large living biological samples. However, the traditional Gaussian light sheet illumination microscopy technology has the problems of small imaging field of view and low spatial resolution. Based on the existing dual-sided illumination LSFM, a large field of view and high resolution LSFM combined with virtual single-pixel imaging deconvolution is presented in this paper, which improves the field of view and resolution of LSFM simultaneously. The relevant microscope is designed and built, and three-dimensional optical sectioning imaging experiments on fluorescent beads and transgenic zebrafish standard samples are carried out. The experimental results prove the three-dimensional high resolution imaging capability of the microscope, which is of great significance in developing the large field of view and high resolution LSFM.

Linearly modulated multi-focal diffractive lens for multi-sheet excitation of flow-driven samples in a light-sheet fluorescence microscope

Light-sheet fluorescence microscopy (LSFM) with single light-sheet illumination enables rapid 3Dimaging of living cells. In this paper we show the design, fabrication and characterization of a diffractive optical element producing several light sheets along an inclined tube for applications in flow-driven imaging. The element, which is based on a multi-focal Fresnel zone plate and a linear grating, generates in combination with a refractive cylindrical lens five thin light sheets of equal intensity.

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.

An open-hardware sample mounting solution for inverted light-sheet microscopes with large detection objective lenses.

Implementations of light-sheet microscopes are often incompatible with standard methods of sample mounting. Light-sheet microscopy uses orthogonal illumination and detection to create a thin sheet of light which does not illuminate the sample outside of the depth of field of the detection axis. Typically, this configuration involves a pair of orthogonal objectives which constrains the positioning and length of cover slips in the range of the detection objective. Here, we present an open-hardware sample mounting system for light-sheet microscopes using large detection objectives, built using 3D printed components and demonstrate the chamber's efficacy on live biological samples in a custom light-sheet microscope. LAY DESCRIPTION: Implementations of light-sheet microscopes are often incompatible with standard methods of sample mounting. Light-sheet microscopy creates a thin sheet of light at a certain depth of field within a volumetric sample. Typically, this configuration involves a pair of orthogonal objectives which constrains the positioning of samples and sample-mounting apparatus in range of the detection objective. To overcome the limitations of this setup, we present an open-hardware sample mounting system for light-sheet microscopes using large detection objectives, built using 3D printed components and demonstrate the chamber's efficacy on live biological samples in a custom light-sheet microscope.

Light-sheet engineering using the Field Synthesis theorem

Recent advances in light-sheet microscopy have enabled sensitive imaging with high spatiotemporal resolution. However, the creation of thin light-sheets for high axial resolution is challenging, as the thickness of the sheet, field of view and confinement of the excitation need to be carefully balanced. Some of the thinnest light-sheets created so far have found little practical use as they excite too much out-of-focus fluorescence. In contrast, the most commonly used light-sheet for subcellular imaging, the square lattice, has excellent excitation confinement at the cost of lower axial resolving power. Here we leverage the recently discovered Field Synthesis theorem to create light-sheets where thickness and illumination confinement can be continuously tuned. Explicitly, we scan a line beam across a portion of an annulus mask on the back focal plane of the illumination objective, which we call C-light-sheets. We experimentally characterize these light-sheets and demonstrate their application on biological samples.

Calculation of diffractive optical elements for the formation of thin light sheet

In the work, the calculation and study of diffractive optical elements (DOE) for the formation of a diffraction-free beam in the form of a thin light sheet, which can be used in planar microscopy, were performed. The calculation of phase DOEs is made on the basis of an iterative algorithm, taking into account the quantization of the phase function.

Propagating-path uniformly scanned light sheet excitation microscopy for isotropic volumetric imaging of large specimens.

.We demonstrate a propagating-path uniformly scanned light sheet excitation (PULSE) microscopy based on the oscillation of voice coil motor that can rapidly drive a thin light sheet along its propagation direction. By synchronizing the rolling shutter of a camera with the motion of laser sheet, we can obtain a uniform plane-illuminated image far beyond the confocal range of Gaussian beam. A stable optical sectioning under a wide field of view (FOV) has been achieved for up to 20 Hz volumetric imaging of large biological specimens. PULSE method transforms the extent of plane illumination from one intrinsically limited by the short confocal range ( scale) to one defined by the motor oscillation range (mm scale). Compared to the conventional Gaussian light sheet imaging, our method greatly mitigates the compromise of axial resolution and successfully extends the FOV over 100 times. We demonstrate the applications of PULSE method by rapidly imaging cleared mouse spinal cord and live zebrafish larva at isotropic subcellular resolution.

DNA skybridge: 3D structure producing a light sheet for high-throughput single-molecule imaging.

Real-time visualization of single-proteins or -complexes on nucleic acid substrates is an essential tool for characterizing nucleic acid binding proteins. Here, we present a novel surface-condition independent and high-throughput single-molecule optical imaging platform called ‘DNA skybridge’. The DNA skybridge is constructed in a 3D structure with 4 μm-high thin quartz barriers in a quartz slide. Each DNA end is attached to the top of the adjacent barrier, resulting in the extension and immobilization of DNA. In this 3D structure, the bottom surface is out-of-focus when the target molecules on the DNA are imaged. Moreover, the DNA skybridge itself creates a thin Gaussian light sheet beam parallel to the immobilized DNA. This dual property allows for imaging a single probe-tagged molecule moving on DNA while effectively suppressing interference with the surface and background signals from the surface.

Light sheets with extended length

High resolution light-sheet optical microscopy requires illuminating a sample with a thin excitation light sheet with large area. Here, we describe the method of two-beam interference for producing light sheets with extended length, as compared to Gaussian-beam-type light sheets with the same thickness, without scanning or dithering the light beams.