Multicolor 4D Fluorescence Microscopy using Ultrathin Bessel Light Sheets

Zhao, Teng; Lau, Sze Cheung; Wang, Ying; Su, Yumian; Wang, Hao; Cheng, Aifang; Herrup, Karl; Ip, Nancy Y.; Du, Shengwang; Loy, M. M. T.

doi:10.1038/srep26159

Cited by 44 publications

(40 citation statements)

References 15 publications

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“…In many previous publications a missing distinction between light sheet thickness and thickness of the main lobe could cause confusion, e.g. when referring to "ultrathin planar illumination produced by scanned Bessel beams" or "Ultrathin Bessel Light Sheets" when actually only the main lobe is thinner than a conventional Gaussian light-sheet of equal length [19,25]. For the most frequently used light sheet with a Gaussian profile there is only one sheet and the measure of main lobe thickness and light sheet thickness are strictly proportional.…”

Section: How Thick Is a Light Sheet? Main Lobe Thickness Vs Optical Smentioning

confidence: 99%

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

Remacha

Friedrich²,

Vermot

et al. 2019

Preprint

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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 lightsheet 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, by using simulation data we introduce a set of intuitive measures that helps to define the relationship between light sheet thickness and axial resolution. 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.

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Section: How Thick Is a Light Sheet? Main Lobe Thickness Vs Optical Smentioning

confidence: 99%

“…Another way to understand the dithered lattice light sheet is based on the Field Synthesis Theorem [27]. The dithered light-sheet can also be understood as an incoherent superposition of three light sheets: a line Bessel sheet [19] and two Focused flat-top light sheets with poor confinement along the detection axis.…”

Section: Comparative Analysismentioning

confidence: 99%

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

Remacha

Friedrich²,

Vermot

et al. 2019

Preprint

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“…As such it is of interest to look into nondiffracting, propagation‐invariant beams. Various reports have emerged over the past few years evaluating Bessel beams (Andilla et al, ; Fahrbach, Gurchenkov, Alessandri, Nassoy, & Rohrbach, ; Fahrbach & Rohrbach, ; Gao, Shao, Chen, & Betzig, ; Gohn‐Kreuz & Rohrbach, ; Zhang, Phipps, Goodwin, & Werner, ; Zhao et al, ) and Airy beams (Vettenburg et al, ) for LSFM, shown in Figure . Bessel beams are the most used as they are fairly easy to implement.…”

Section: Recent Technological Developmentsmentioning

confidence: 99%

“…Reportedly, a major advantage is the acquisition speed, that ranges between 200 to 1,000 planes/s in dithered mode, which is one order of magnitude more than with classic Bessel beams. The technique is undoubtedly very effective, but the set‐up is quite complex and the use of a SLM reduces the temporal resolution when multicolor imaging is required since the SLM must be rapidly switched and synchronized with every color change (Zhao et al, ). The solution proposed by Zhao and coworkers (Zhao et al, ) is to use a combination of “nonelectronic” optical components to create ultrathin Bessel sheets of the same thickness regardless of the input laser light, as long as it is in the visible range.…”

Section: Recent Technological Developmentsmentioning

confidence: 99%

“…The technique is undoubtedly very effective, but the set‐up is quite complex and the use of a SLM reduces the temporal resolution when multicolor imaging is required since the SLM must be rapidly switched and synchronized with every color change (Zhao et al, ). The solution proposed by Zhao and coworkers (Zhao et al, ) is to use a combination of “nonelectronic” optical components to create ultrathin Bessel sheets of the same thickness regardless of the input laser light, as long as it is in the visible range. The excitation light passes first through a slit to give it a line‐shape, then through an annulus imaged to the back focal plane of the illumination objective.…”

Section: Recent Technological Developmentsmentioning

confidence: 99%

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Technical implementations of light sheet microscopy

Zagato

Brans

Smedt

et al. 2018

Microscopy Res & Technique

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Fluorescence-based microscopy is among the most successful methods in biological studies. It played a critical role in the visualization of subcellular structures and in the analysis of complex cellular processes, and it is nowadays commonly employed in genetic and drug screenings. Among the fluorescence-based microscopy techniques, light sheet fluorescence microscopy (LSFM) has shown a quite interesting set of benefits. The technique combines the speed of epi-fluorescence acquisition with the optical sectioning capability typical of confocal microscopes. Its unique configuration allows the excitation of only a thin plane of the sample, thus fast, high resolution imaging deep inside tissues is nowadays achievable. The low peak intensity with which the sample is illuminated diminishes phototoxic effects and decreases photobleaching of fluorophores, ensuring data collection for days with minimal adverse consequences on the sample. It is no surprise that LSFM applications have raised in just few years and the technique has been applied to study a wide variety of samples, from whole organism, to tissues, to cell clusters, and single cells. As a consequence, in recent years numerous set-ups have been developed, each one optimized for the type of sample in use and the requirements of the question at hand. Hereby, we aim to review the most advanced LSFM implementations to assist new LSFM users in the choice of the LSFM set-up that suits their needs best. We also focus on new commercial microscopes and "do-it-yourself" strategies; likewise we review recent designs that allow a swift integration of LSFM on existing microscopes.

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High‐Throughput 3D Imaging Flow Cytometry of Suspended Adherent 3D Cell Cultures

Yamashita,

Tamamitsu,

Kirisako

et al. 2023

Small Methods

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Abstract3D cell cultures are indispensable in recapitulating in vivo environments. Among the many 3D culture methods, culturing adherent cells on hydrogel beads to form spheroid‐like structures is a powerful strategy for maintaining high cell viability and functions in the adherent states. However, high‐throughput, scalable technologies for 3D imaging of individual cells cultured on the hydrogel scaffolds are lacking. This study reports the development of a high throughput, scalable 3D imaging flow cytometry platform for analyzing spheroid models. This platform is realized by integrating a single objective fluorescence light‐sheet microscopy with a microfluidic device that combines hydrodynamic and acoustofluidic focusing techniques. This integration enabled unprecedentedly high‐throughput and scalable optofluidic 3D imaging, processing 1310 spheroids consisting of 28 117 cells min−1. The large dataset obtained enables precise quantification and comparison of the nuclear morphology of adhering and suspended cells, revealing that the adhering cells have smaller nuclei with less rounded surfaces. This platform's high throughput, robustness, and precision for analyzing the morphology of subcellular structures in 3D culture models hold promising potential for various biomedical analyses, including image‐based phenotypic screening of drugs with spheroids or organoids.

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Multicolor 4D Fluorescence Microscopy using Ultrathin Bessel Light Sheets

Cited by 44 publications

References 15 publications

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

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

Technical implementations of light sheet microscopy

High‐Throughput 3D Imaging Flow Cytometry of Suspended Adherent 3D Cell Cultures

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