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

New Era ◽

Light Sheet Microscopy ◽

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.

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

10.1101/2021.05.09.443230 ◽

2021 ◽

Author(s):

Peng Fei

Keyword(s):

Dimensional Space ◽

Three Dimensional ◽

Light Sheet ◽

Three Dimensions ◽

Live Cells ◽

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.

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

Cells ◽

10.3390/cells9051255 ◽

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 ◽

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.

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

Micromachines ◽

10.3390/mi10090599 ◽

2019 ◽

Vol 10 (9) ◽

pp. 599 ◽

Cited By ~ 2

Author(s):

Chen ◽

Liu ◽

Lu ◽

Lee ◽

Tsai ◽

...

Keyword(s):

Cell Culture ◽

Hippocampal Neurons ◽

Three Dimensional ◽

Light Sheet ◽

Three Dimensions ◽

Laser Exposure ◽

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.

Extension of the Spatial PDI Model to Three Dimensions

Perceptual and Motor Skills ◽

10.2466/pms.1997.84.1.176 ◽

1997 ◽

Vol 84 (1) ◽

pp. 176-178

Author(s):

Frank O'Brien

Keyword(s):

Population Density ◽

Dimensional Space ◽

Finite Lattice ◽

Three Dimensional ◽

Upper Bounds ◽

Three Dimensions ◽

Lower And Upper Bounds ◽

Density Index ◽

The author's population density index ( PDI) model is extended to three-dimensional distributions. A derived formula is presented that allows for the calculation of the lower and upper bounds of density in three-dimensional space for any finite lattice.

Actin-based protrusions of migrating neutrophils are intrinsically lamellar and facilitate direction changes

eLife ◽

10.7554/elife.26990 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 46

Author(s):

Lillian K Fritz-Laylin ◽

Megan Riel-Mehan ◽

Bi-Chang Chen ◽

Samuel J Lord ◽

Thomas D Goddard ◽

...

Keyword(s):

Three Dimensional ◽

Light Sheet ◽

Time Lapse ◽

Three Dimensions ◽

Cortical Actin ◽

Linear Arrangement ◽

Collagen Matrices ◽

Flat Surfaces ◽

Actin Networks ◽

Light Sheet Microscopy

Leukocytes and other amoeboid cells change shape as they move, forming highly dynamic, actin-filled pseudopods. Although we understand much about the architecture and dynamics of thin lamellipodia made by slow-moving cells on flat surfaces, conventional light microscopy lacks the spatial and temporal resolution required to track complex pseudopods of cells moving in three dimensions. We therefore employed lattice light sheet microscopy to perform three-dimensional, time-lapse imaging of neutrophil-like HL-60 cells crawling through collagen matrices. To analyze three-dimensional pseudopods we: (i) developed fluorescent probe combinations that distinguish cortical actin from dynamic, pseudopod-forming actin networks, and (ii) adapted molecular visualization tools from structural biology to render and analyze complex cell surfaces. Surprisingly, three-dimensional pseudopods turn out to be composed of thin (<0.75 µm), flat sheets that sometimes interleave to form rosettes. Their laminar nature is not templated by an external surface, but likely reflects a linear arrangement of regulatory molecules. Although we find that Arp2/3-dependent pseudopods are dispensable for three-dimensional locomotion, their elimination dramatically decreases the frequency of cell turning, and pseudopod dynamics increase when cells change direction, highlighting the important role pseudopods play in pathfinding.

Proceedings of the Royal Society of London. Series A, Containing Papers of a Mathematical and Physical Character ◽

On a set of quartic surfaces in space of four dimensions; and a certain involutory transformation

10.1098/rspa.1926.0042 ◽

1926 ◽

Vol 110 (756) ◽

pp. 700-708

Keyword(s):

Present Note ◽

Dimensional Space ◽

Three Dimensional ◽

Three Dimensions ◽

Quartic Surface ◽

Four Dimensions ◽

Cubic Surfaces ◽

Mutual Relations ◽

Quartic Surfaces ◽

There exists in space of four dimensions an interesting figure of 15 lines and 15 points, first considered by Stéphanos (‘Compt. Rendus,’ vol. 93, 1881), though suggested very clearly by Cremona’s discussion of cubic surfaces in three-dimensional space. In connection with the figure of 15 lines there arises a quartic surface, the intersection of two quadrics, which is of importance as giving rise by projection to the Cyclides, as Segre has shown in detail (‘Math. Ann.,’ vol. 24, 1884). The symmetry of the figure suggests, howrever, the consideration of 15 such quartic surfaces; and it is natural to inquire as to the mutual relations of these surfaces, in particular as to their intersections. In general, two surfaces in space of four dimensions meet in a finite number of points. It appears that in this case any two of these 15 surfaces have a curve in common; it is the purpose of the present note to determine the complete intersection of any two of these 15 surfaces. Similar results may be obtained for a system of cubic surfaces in three dimensions, corresponding to those here given for this system of quartic surfaces in four dimensions, since the surfaces have one point in common, which may be taken as the centre of a projection.

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

10.1101/154807 ◽

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 ◽

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.

On the Interpretation of Vowel “Quality”: The Dimension of Rounding

Journal of the International Phonetic Association ◽

10.1017/s0025100300005880 ◽

1989 ◽

Vol 19 (1) ◽

pp. 24-30 ◽

Cited By ~ 3

Author(s):

Leigh Lisker

Keyword(s):

Vocal Tract ◽

Dimensional Space ◽

Three Dimensional ◽

Three Dimensions ◽

Vowel Space ◽

Tongue Position ◽

Two Measures ◽

The One ◽

Jaw Position ◽

The usual description of vowels in respect to their “phonetic quality” requires the linguist to locate them within a so-called “vowel space,” apparently articulatory in nature, and having three dimensions labeled high-low (or close-open), front-back, and unrounded-rounded. The first two are coordinates of tongue with associated jaw position, while the third specifies the posture of the lips. It is recognized that vowels can vary qualitatively in ways that this three-dimensional space does not account for. So, for example, vowels may differ in degree of nasalization, and they may be rhotacized or r-colored. Moreover, it is recognized that while this vowel space serves important functions within the community of linguists, both the two measures of tongue position and the one for the lips inadequately identify those aspects of vocal tract shapes that are primarily responsible for the distinctive phonetic qualities of vowels (Ladefoged 1971). With all this said, it remains true enough that almost any vowel pair of different qualities can be described as occupying different positions with the space. Someone hearing two vowels in sequence and detecting a quality difference will presumably also be able to diagnose the nature of the articulatory shift executed in going from one vowel to the other.

THREE-D SINGLETONS AND 2-D C.F.T.

International Journal of Modern Physics A ◽

10.1142/s0217751x92000971 ◽

1992 ◽

Vol 07 (10) ◽

pp. 2193-2206 ◽

Cited By ~ 9

Author(s):

A.M. HARUN AR-RASHID ◽

C. FRONSDAL ◽

M. FLATO

Keyword(s):

Dimensional Space ◽

Three Dimensional ◽

Three Dimensions ◽

Alternative Formulation ◽

Boundary Values ◽

Witten Theory ◽

Space Time Structure ◽

Dimensional Space Time ◽

Left And Right ◽

Two-dimensional Wess-Zumino-Novikov-Witten theory is extended to three dimensions, where it becomes a scalar gauge theory of the singleton type. The three-dimensional formulation involves a scalar field valued in a compact group G, a Nakanishi-Lautrup field valued in Lie (G) and Faddeev-Popov ghosts. The physical sector, characterized by the vanishing of the Nakanishi-Lautrup field, coincides with the WZNW theory of the group G. Three-dimensional space-time structure involves a generalized metric, but only its boundary values are of consequence. An alternative formulation in terms of left and right movers (in three dimensions!) is also possible.

A notation for vectors and tensors

Mathematical Proceedings of the Cambridge Philosophical Society ◽

10.1017/s0305004100030450 ◽

1955 ◽

Vol 51 (3) ◽

pp. 449-453

Author(s):

F. C. Powell

Keyword(s):

Scalar Product ◽

Dimensional Space ◽

Three Dimensional ◽

Three Dimensions ◽

Usual Form ◽

Three Dimensional Space ◽

Scalar Moment

The vector notation commonly employed in elementary physics cannot be applied in its usual form to spaces of other than three dimensions. In plane dynamics, for instance, it cannot be used to represent the velocity (– ωx2, ωx1) at the point (x1, x2) due to a rotation ω about the origin, or the (scalar) moment about the origin of the force (F1, F2) acting at (x1, x2). In relativity physics the symbol ⋅ is often used to denote the scalar product of two vectors, it is true, and the tensor aαbβ – aβbα is sometimes denoted by a × b, but there exists no body of rules for the manipulation of these symbols that enables one to dispense with the suffix notation as in the case of vectors in three-dimensional space.