scholarly journals Reflective imaging improves resolution, speed, and collection efficiency in light sheet microscopy

2017 ◽  
Author(s):  
Yicong Wu ◽  
Abhishek Kumar ◽  
Corey Smith ◽  
Evan Ardiel ◽  
Panagiotis Chandris ◽  
...  
Keyword(s):  
High Resolution ◽  
High Speed ◽  
Collection Efficiency ◽  
Single Cells ◽  
Numerical Aperture ◽  
Light Sheet ◽  
Three Dimensions ◽  
Spatiotemporal Resolution ◽  
Light Sheet Microscopy ◽  
Simultaneous Acquisition

AbstractLight-sheet fluorescence microscopy (LSFM) enables high-speed, high-resolution, gentle imaging of live biological specimens over extended periods. Here we describe a technique that improves the spatiotemporal resolution and collection efficiency of LSFM without modifying the underlying microscope. By imaging samples on reflective coverslips, we enable simultaneous collection of multiple views, obtaining 4 complementary views in 250 ms, half the period it would otherwise take to collect only two views in symmetric dual-view selective plane illumination microscopy (diSPIM). We also report a modified deconvolution algorithm that removes the associated epifluorescence contamination and fuses all views for resolution recovery. Furthermore, we enhance spatial resolution (to < 300 nm in all three dimensions) by applying our method to a new asymmetric diSPIM, permitting simultaneous acquisition of two high-resolution views otherwise difficult to obtain due to steric constraints at high numerical aperture (NA). We demonstrate the broad applicability of our method in a variety of samples of moderate (< 50 μm) thickness, studying mitochondrial, membrane, Golgi, and microtubule dynamics in single cells and calcium activity in nematode embryos.

scholarly journals Digital Spindle: A New Way to Explore Mitotic Functions by Whole Cell Data Collection and a Computational Approach

Cells ◽  
2020 ◽  
Vol 9 (5) ◽  
pp. 1255
Author(s):  
Norio Yamashita ◽  
Masahiko Morita ◽  
Hideo Yokota ◽  
Yuko Mimori-Kiyosue
Keyword(s):  
Cell Biology ◽  
Information Science ◽  
Three Dimensional ◽  
Light Sheet ◽  
Live Imaging ◽  
Three Dimensions ◽  
Complex Data ◽  
Spatiotemporal Resolution ◽  
Whole Cell ◽  
Light Sheet Microscopy

From cells to organisms, every living system is three-dimensional (3D), but the performance of fluorescence microscopy has been largely limited when attempting to obtain an overview of systems’ dynamic processes in three dimensions. Recently, advanced light-sheet illumination technologies, allowing drastic improvement in spatial discrimination, volumetric imaging times, and phototoxicity/photobleaching, have been making live imaging to collect precise and reliable 3D information increasingly feasible. In particular, lattice light-sheet microscopy (LLSM), using an ultrathin light-sheet, enables whole-cell 3D live imaging of cellular processes, including mitosis, at unprecedented spatiotemporal resolution for extended periods of time. This technology produces immense and complex data, including a significant amount of information, raising new challenges for big image data analysis and new possibilities for data utilization. Once the data are digitally archived in a computer, the data can be reused for various purposes by anyone at any time. Such an information science approach has the potential to revolutionize the use of bioimage data, and provides an alternative method for cell biology research in a data-driven manner. In this article, we introduce examples of analyzing digital mitotic spindles and discuss future perspectives in cell biology.

scholarly journals Observing the Cell in Its Native State: Imaging Subcellular Dynamics in Multicellular Organisms

2018 ◽  
Author(s):  
Tsung-Li Liu ◽  
Srigokul Upadhyayula ◽  
Daniel E. Milkie ◽  
Ved Singh ◽  
Kai Wang ◽  
...  
Keyword(s):  
Adaptive Optics ◽  
High Speed ◽  
Developmental Stages ◽  
Phenotypic Diversity ◽  
Metastatic Cancer ◽  
Light Sheet ◽  
Intrinsic Variability ◽  
Spatiotemporal Resolution ◽  
Light Sheet Microscopy

AbstractTrue physiological imaging of subcellular dynamics requires studying cells within their parent organisms, where all the environmental cues that drive gene expression, and hence the phenotypes we actually observe, are present. A complete understanding also requires volumetric imaging of the cell and its surroundings at high spatiotemporal resolution without inducing undue stress on either. We combined lattice light sheet microscopy with two-channel adaptive optics to achieve, across large multicellular volumes, noninvasive aberration-free imaging of subcellular processes, including endocytosis, organelle remodeling during mitosis, and the migration of axons, immune cells, and metastatic cancer cells in vivo. The technology reveals the phenotypic diversity within cells across different organisms and developmental stages, and may offer insights into how cells harness their intrinsic variability to adapt to different physiological environments.One Sentence SummaryCombining lattice light sheet microscopy with adaptive optics enables high speed, high resolution in vivo 3D imaging of dynamic processes inside cells under physiological conditions within their parent organisms.

scholarly journals Imaging mitotic processes in three dimensions with lattice light-sheet microscopy

2021 ◽  
Author(s):  
Yuko Mimori-Kiyosue
Keyword(s):  
Dimensional Space ◽  
Three Dimensional ◽  
Image Data ◽  
Light Sheet ◽  
Three Dimensions ◽  
Cell Level ◽  
Spatiotemporal Resolution ◽  
New Era ◽  
Light Sheet Microscopy ◽  
Three Dimensional Space

AbstractThere are few technologies that can capture mitotic processes occurring in three-dimensional space with the desired spatiotemporal resolution. Due to such technical limitations, our understanding of mitosis, which has been studied since the early 1880s, is still incomplete with regard to mitotic processes and their regulatory mechanisms at a molecular level. A recently developed high-resolution type of light-sheet microscopy, lattice light-sheet microscopy (LLSM), has achieved unprecedented spatiotemporal resolution scans of intracellular spaces at the whole-cell level. This technology enables experiments that were not possible before (e.g., tracking of growth of every spindle microtubule end and discrimination of individual chromosomes in living cells), thus providing a new avenue for the analysis of mitotic processes. Herein, principles of LLSM technology are introduced, as well as experimental techniques that became possible with LLSM. In addition, issues remaining to be solved for use of this technology in mitosis research, big image data problems, are presented to help guide mitosis research into a new era.

scholarly journals Multi-color 4D superresolution light-sheet microscopy reveals organelle interactions at isotropic 100-nm resolution and sub-second timescales

2021 ◽  
Author(s):  
Peng Fei
Keyword(s):  
Dimensional Space ◽  
Three Dimensional ◽  
Light Sheet ◽  
Three Dimensions ◽  
Live Cells ◽  
Spatiotemporal Resolution ◽  
Volumetric Imaging ◽  
Light Sheet Microscopy ◽  
Intracellular Organelles ◽  
Microscopy Techniques

Long-term visualization of the dynamic organelle-organelle or protein-organelle interactions throughout the three-dimensional space of whole live cells is essential to better understand their functions, but this task remains challenging due to the limitations of existing three-dimensional fluorescence microscopy techniques, such as an insufficient axial resolution, low volumetric imaging rate, and photobleaching. Here, we present the combination of a progressive deep-learning superresolution strategy with a dual-ring-modulated SPIM design capable of visualizing the dynamics of intracellular organelles in live cells for hours at an isotropic spatial resolution of ~100 nm in three dimensions and a temporal resolution up to ~17 Hz. With a compelling spatiotemporal resolution, we substantially reveal the complex spatial relationships and interactions between the endoplasmic reticulum (ER) and mitochondria throughout live cells, providing new insights into ER-mediated mitochondrial division. We also localized the motion of Drp1 oligomers in three dimensions and observed Drp1-mediated mitochondrial branching for the first time.

scholarly journals LITE microscopy: Tilted light-sheet excitation of model organisms offers high resolution and low photobleaching

2018 ◽  
Vol 217 (5) ◽  
pp. 1869-1882 ◽  
Author(s):  
Tanner C. Fadero ◽  
Therese M. Gerbich ◽  
Kishan Rana ◽  
Aussie Suzuki ◽  
Matthew DiSalvo ◽  
...  
Keyword(s):  
High Resolution ◽  
Fluorescence Microscopy ◽  
Focal Plane ◽  
Numerical Aperture ◽  
Light Sheet ◽  
Model Organisms ◽  
Spatiotemporal Resolution ◽  
High Spatiotemporal Resolution ◽  
Light Gathering ◽  
Microscopy Techniques

Fluorescence microscopy is a powerful approach for studying subcellular dynamics at high spatiotemporal resolution; however, conventional fluorescence microscopy techniques are light-intensive and introduce unnecessary photodamage. Light-sheet fluorescence microscopy (LSFM) mitigates these problems by selectively illuminating the focal plane of the detection objective by using orthogonal excitation. Orthogonal excitation requires geometries that physically limit the detection objective numerical aperture (NA), thereby limiting both light-gathering efficiency (brightness) and native spatial resolution. We present a novel live-cell LSFM method, lateral interference tilted excitation (LITE), in which a tilted light sheet illuminates the detection objective focal plane without a sterically limiting illumination scheme. LITE is thus compatible with any detection objective, including oil immersion, without an upper NA limit. LITE combines the low photodamage of LSFM with high resolution, high brightness, and coverslip-based objectives. We demonstrate the utility of LITE for imaging animal, fungal, and plant model organisms over many hours at high spatiotemporal resolution.

scholarly journals Light Sheet Theta Microscopy for High-resolution Quantitative Imaging of Large Biological Systems

2017 ◽  
Author(s):  
Bianca Migliori ◽  
Malika S. Datta ◽  
Christophe Dupre ◽  
Mehmet C. Apak ◽  
Shoh Asano ◽  
...  
Keyword(s):  
High Resolution ◽  
High Speed ◽  
Quantitative Imaging ◽  
Biological Systems ◽  
Light Sheet ◽  
Planar Imaging ◽  
Light Sheet Microscopy ◽  
Significant Step ◽  
Resolution Imaging ◽  
And Function

AbstractAdvances in tissue clearing and molecular labelling methods are enabling unprecedented optical access to large intact biological systems. These advances fuel the need for high-speed microscopy approaches to image large samples quantitatively and at high resolution. While Light Sheet Microscopy (LSM), with its high planar imaging speed and low photo-bleaching, can be effective, scaling up to larger imaging volumes has been hindered by the use of orthogonal light-sheet illumination. To address this fundamental limitation, we have developed Light Sheet Theta Microscopy (LSTM), which uniformly illuminates samples from same side as the detection objective, thereby eliminating limits on lateral dimensions without sacrificing imaging resolution, depth and speed. We present detailed characterization of LSTM, and show that this approach achieves rapid high-resolution imaging of large intact samples with superior uniform high-resolution than LSM. LSTM is a significant step in high-resolution quantitative mapping of structure and function of large intact biological systems.

scholarly journals Converting lateral scanning into axial focusing to speed up three-dimensional microscopy

2020 ◽  
Vol 9 (1) ◽  
Author(s):  
Tonmoy Chakraborty ◽  
Bingying Chen ◽  
Stephan Daetwyler ◽  
Bo-Jui Chang ◽  
Oliver Vanderpoorten ◽  
...  
Keyword(s):  
High Resolution ◽  
Spherical Aberration ◽  
Optical Design ◽  
Light Sheet ◽  
Three Dimensions ◽  
High Resolution Imaging ◽  
Light Sheet Microscopy ◽  
Scanning Rate ◽  
Resolution Imaging ◽  
Axial Scanning

Abstract In optical microscopy, the slow axial scanning rate of the objective or the sample has traditionally limited the speed of volumetric imaging. Recently, by conjugating either a movable mirror to the image plane in a remote-focusing geometry or an electrically tuneable lens (ETL) to the back focal plane, rapid axial scanning has been achieved. However, mechanical actuation of a mirror limits the axial scanning rate (usually only 10–100 Hz for piezoelectric or voice coil-based actuators), while ETLs introduce spherical and higher-order aberrations that prevent high-resolution imaging. In an effort to overcome these limitations, we introduce a novel optical design that transforms a lateral-scan motion into a spherical aberration-free axial scan that can be used for high-resolution imaging. Using a galvanometric mirror, we scan a laser beam laterally in a remote-focusing arm, which is then back-reflected from different heights of a mirror in the image space. We characterize the optical performance of this remote-focusing technique and use it to accelerate axially swept light-sheet microscopy by an order of magnitude, allowing the quantification of rapid vesicular dynamics in three dimensions. We also demonstrate resonant remote focusing at 12 kHz with a two-photon raster-scanning microscope, which allows rapid imaging of brain tissues and zebrafish cardiac dynamics with diffraction-limited resolution.

scholarly journals The Applications of Lattice Light-sheet Microscopy for Functional Volumetric Imaging of Hippocampal Neurons in a Three-Dimensional Culture System

Micromachines ◽  
2019 ◽  
Vol 10 (9) ◽  
pp. 599 ◽  
Author(s):  
Chen ◽  
Liu ◽  
Lu ◽  
Lee ◽  
Tsai ◽  
...  
Keyword(s):  
Cell Culture ◽  
Hippocampal Neurons ◽  
Three Dimensional ◽  
Light Sheet ◽  
Three Dimensions ◽  
Laser Exposure ◽  
Spatiotemporal Resolution ◽  
Strong Light ◽  
Light Sheet Microscopy ◽  
Volumetric Images

The characterization of individual cells in three-dimensions (3D) with very high spatiotemporal resolution is crucial for the development of organs-on-chips, in which 3D cell cultures are integrated with microfluidic systems. In this study, we report the applications of lattice light-sheet microscopy (LLSM) for monitoring neuronal activity in three-dimensional cell culture. We first established a 3D environment for culturing primary hippocampal neurons by applying a scaffold-based 3D tissue engineering technique. Fully differentiated and mature hippocampal neurons were observed in our system. With LLSM, we were able to monitor the behavior of individual cells in a 3D cell culture, which was very difficult under a conventional microscope due to strong light scattering from thick samples. We demonstrated that our system could study the membrane voltage and intracellular calcium dynamics at subcellular resolution in 3D under both chemical and electrical stimulation. From the volumetric images, it was found that the voltage indicators mainly resided in the cytosol instead of the membrane, which cannot be distinguished using conventional microscopy. Neuronal volumetric images were sheet scanned along the axial direction and recorded at a laser exposure of 6 ms, which covered an area up to 4800 μm2, with an image pixel size of 0.102 μm. When we analyzed the time-lapse volumetric images, we could quantify the voltage responses in different neurites in 3D extensions.

scholarly journals A Versatile Oblique Plane Microscope for Large-Scale and High-Resolution Imaging of Subcellular Dynamics

2020 ◽  
Author(s):  
Etai Sapoznik ◽  
Bo-Jui Chang ◽  
Jaewon Huh ◽  
Robert J. Ju ◽  
Evgenia V. Azarova ◽  
...  
Keyword(s):  
High Resolution ◽  
Numerical Aperture ◽  
Light Sheet ◽  
Membrane Dynamics ◽  
Field Of View ◽  
Large Field ◽  
Endoplasmic Reticulum Membrane ◽  
High Resolution Imaging ◽  
Light Sheet Microscopy ◽  
Resolution Imaging

AbstractWe present an Oblique Plane Microscope that uses a bespoke glass-tipped tertiary objective to improve the resolution, field of view, and usability over previous variants. Owing to its high numerical aperture optics, this microscope achieves lateral and axial resolutions that are comparable to the square illumination mode of Lattice Light-Sheet Microscopy, but in a user friendly and versatile format. Given this performance, we demonstrate high-resolution imaging of clathrin-mediated endocytosis, vimentin, the endoplasmic reticulum, membrane dynamics, and Natural Killer-mediated cytotoxicity. Furthermore, we image biological phenomena that would be otherwise challenging or impossible to perform in a traditional light-sheet microscope geometry, including cell migration through confined spaces within a microfluidic device, subcellular photoactivation of Rac1, diffusion of cytoplasmic rheological tracers at a volumetric rate of 14 Hz, and large field of view imaging of neurons, developing embryos, and centimeter-scale tissue sections.

scholarly journals Reflective imaging improves spatiotemporal resolution and collection efficiency in light sheet microscopy

2017 ◽  
Vol 8 (1) ◽  
Author(s):  
Yicong Wu ◽  
Abhishek Kumar ◽  
Corey Smith ◽  
Evan Ardiel ◽  
Panagiotis Chandris ◽  
...  
Keyword(s):  
Collection Efficiency ◽  
Light Sheet ◽  
Spatiotemporal Resolution ◽  
Light Sheet Microscopy
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