scholarly journals Open-Top Light-Sheet Microscopy Image Atlas of Prostate Core Needle Biopsies

2019 ◽  
Vol 143 (9) ◽  
pp. 1069-1075 ◽  
Author(s):  
Nicholas P. Reder ◽  
Adam K. Glaser ◽  
Erin F. McCarty ◽  
Ye Chen ◽  
Lawrence D. True ◽  
...  
Keyword(s):  
Immunohistochemical Staining ◽  
Structural Information ◽  
Ex Vivo ◽  
Three Dimensional ◽  
Light Sheet ◽  
Fixed Tissue ◽  
Core Needle ◽  
Light Sheet Microscopy ◽  
Wide Range ◽  
Hematoxylin Eosin

Context.— Ex vivo microscopy encompasses a range of techniques to examine fresh or fixed tissue with microscopic resolution, eliminating the need to embed the tissue in paraffin or produce a glass slide. One such technique is light-sheet microscopy, which enables rapid 3D imaging. Our pathology-engineering collaboration has resulted in an open-top light-sheet (OTLS) microscope that is specifically tailored to the needs of pathology practice. Objective.— To present an image atlas of OTLS images of prostate core needle biopsies. Design.— Core needle biopsies (N = 9) were obtained from fresh radical prostatectomy specimens. Each biopsy was fixed in formalin, dehydrated in ethanol, stained with TO-PRO3 and eosin, optically cleared, and imaged using OTLS microscopy. The biopsies were then processed, paraffin embedded, and sectioned. Hematoxylin-eosin and immunohistochemical staining for cytokeratin 5 and cytokeratin 8 was performed. Results.— Benign and neoplastic histologic structures showed high fidelity between OTLS and traditional light microscopy. OTLS microscopy had no discernible effect on hematoxylin-eosin or immunohistochemical staining in this pilot study. The 3D histology information obtained using OTLS microscopy enabled new structural insights, including the observation of cribriform and well-formed gland morphologies within the same contiguous glandular structures, as well as the continuity of poorly formed glands with well-formed glands. Conclusions.— Three-dimensional OTLS microscopy images have a similar appearance to traditional hematoxylin-eosin histology images, with the added benefit of useful 3D structural information. Further studies are needed to continue to document the OTLS appearance of a wide range of tissues and to better understand 3D histologic structures.

scholarly journals Three-dimensional, isotropic imaging of mouse brain using multi-view deconvolution light sheet microscopy

2017 ◽  
Vol 10 (05) ◽  
pp. 1743006 ◽  
Author(s):  
Sa Liu ◽  
Jun Nie ◽  
Yusha Li ◽  
Tingting Yu ◽  
Dan Zhu ◽  
...  
Keyword(s):  
Mouse Brain ◽  
Pyramidal Neurons ◽  
Structural Information ◽  
Fluorescent Microscopy ◽  
Three Dimensional ◽  
Light Sheet ◽  
Nerve Fibers ◽  
Single View ◽  
Light Sheet Microscopy ◽  
The Brain

We present a three-dimensional (3D) isotropic imaging of mouse brain using light-sheet fluorescent microscopy (LSFM) in conjunction with a multi-view imaging computation. Unlike common single view LSFM is used for mouse brain imaging, the brain tissue is 3D imaged under eight views in our study, by a home-built selective plane illumination microscopy (SPIM). An output image containing complete structural information as well as significantly improved resolution ([Formula: see text]4 times) are then computed based on these eight views of data, using a bead-guided multi-view registration and deconvolution. With superior imaging quality, the astrocyte and pyramidal neurons together with their subcellular nerve fibers can be clearly visualized and segmented. With further including other computational methods, this study can be potentially scaled up to map the connectome of whole mouse brain with a simple light-sheet microscope.

scholarly journals Three-dimensional quantification of twisting in the Arabidopsis petiole

2021 ◽  
Author(s):  
Yuta Otsuka ◽  
Hirokazu Tsukaya
Keyword(s):  
Cross Sections ◽  
Three Dimensional ◽  
Light Sheet ◽  
Underlying Mechanism ◽  
Two Dimensions ◽  
Observation Angle ◽  
Differential Growth ◽  
Light Sheet Microscopy ◽  
Definition Of ◽  
Twisting Angle

AbstractOrganisms have a variety of three-dimensional (3D) structures that change over time. These changes include twisting, which is 3D deformation that cannot happen in two dimensions. Twisting is linked to important adaptive functions of organs, such as adjusting the orientation of leaves and flowers in plants to align with environmental stimuli (e.g. light, gravity). Despite its importance, the underlying mechanism for twisting remains to be determined, partly because there is no rigorous method for quantifying the twisting of plant organs. Conventional studies have relied on approximate measurements of the twisting angle in 2D, with arbitrary choices of observation angle. Here, we present the first rigorous quantification of the 3D twisting angles of Arabidopsis petioles based on light sheet microscopy. Mathematical separation of bending and twisting with strict definition of petiole cross-sections were implemented; differences in the spatial distribution of bending and twisting were detected via the quantification of angles along the petiole. Based on the measured values, we discuss that minute degrees of differential growth can result in pronounced twisting in petioles.

scholarly journals Three-Dimensional Cross-Sectional Light-Sheet Microscopy Imaging of the Inflamed Mouse Gut

2017 ◽  
Vol 153 (4) ◽  
pp. 898-900 ◽  
Author(s):  
Sebastian Zundler ◽  
Anika Klingberg ◽  
Daniela Schillinger ◽  
Sarah Fischer ◽  
Clemens Neufert ◽  
...  
Keyword(s):  
Three Dimensional ◽  
Light Sheet ◽  
Cross Sectional ◽  
Microscopy Imaging ◽  
Light Sheet Microscopy

scholarly journals Three Dimensional Fluorescence Imaging Using Multiple Light-Sheet Microscopy

PLoS ONE ◽  
2014 ◽  
Vol 9 (6) ◽  
pp. e96551 ◽  
Author(s):  
Kavya Mohan ◽  
Subhajit B. Purnapatra ◽  
Partha Pratim Mondal
Keyword(s):  
Fluorescence Imaging ◽  
Three Dimensional ◽  
Light Sheet ◽  
Light Sheet Microscopy

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

eLife ◽  
2017 ◽  
Vol 6 ◽  
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.

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 Label-free and Multimodal Second Harmonic Generation Light Sheet Microscopy

2020 ◽  
Author(s):  
Niall Hanrahan ◽  
Simon I. R. Lane ◽  
Peter Johnson ◽  
Konstantinos Bourdakos ◽  
Christopher Brereton ◽  
...  
Keyword(s):  
Second Harmonic Generation ◽  
Harmonic Generation ◽  
Biological Samples ◽  
3D Imaging ◽  
Second Harmonic ◽  
Three Dimensional ◽  
Light Sheet ◽  
Optical Methods ◽  
Label Free ◽  
Light Sheet Microscopy

AbstractLight sheet microscopy (LSM) has emerged as one of most profound three dimensional (3D) imaging tools in the life sciences over the last decade. However, LSM is currently performed with fluorescence detection on one- or multi-photon excitation. Label-free LSM imaging approaches have been rather limited. Second Harmonic Generation (SHG) imaging is a label-free technique that has enabled detailed investigation of collagenous structures, including its distribution and remodelling in cancers and respiratory tissue, and how these link to disease. SHG is generally regarded as having only forward- and back-scattering components, apparently precluding the orthogonal detection geometry used in Light Sheet Microscopy. In this work we demonstrate SHG imaging on a light sheet microscope (SHG-LSM) using a rotated Airy beam configuration that demonstrates a powerful new approach to direct, without any further processing or deconvolution, 3D imaging of harmonophores such as collagen in biological samples. We provide unambiguous identification of SHG signals on the LSM through its wavelength and polarisation sensitivity. In a multimodal LSM setup we demonstrate that SHG and two-photon signals can be acquired on multiple types of different biological samples. We further show that SHG-LSM is sensitive to changes in collagen synthesis within lung fibroblast 3D cell cultures. This work expands on the existing optical methods available for use with light sheet microscopy, adding a further label-free imaging technique which can be combined with other detection modalities to realise a powerful multi-modal microscope for 3D bioimaging.

scholarly journals Single-molecule light-sheet microscopy with local nanopipette delivery

2020 ◽  
Author(s):  
B. Li ◽  
A. Ponjavic ◽  
W. H. Chen ◽  
L. Hopkins ◽  
C. Hughes ◽  
...  
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Single Molecule ◽  
Single Molecules ◽  
Light Sheet ◽  
Local Delivery ◽  
Live Cells ◽  
Biological Processes ◽  
Light Sheet Microscopy ◽  
Wide Range

AbstractDetection of single molecules in biological systems has rapidly increased in resolution over the past decade. However, delivery of single molecules has remained a challenge. Currently there is no effective method that can both introduce a precise amount of molecules onto or into a single cell at a defined position, and then image the cellular response. Here we have combined light sheet microscopy with local delivery, using a nanopipette, to accurately deliver individual proteins to a defined position. We call this method local delivery selective plane illumination microscopy (ldSPIM). ldSPIM uses a nanopipette and the ionic feedback current at the nanopipette tip to control the position from which molecules are delivered. The number of proteins delivered can be controlled by varying the voltage applied. For single-molecule detection, we implemented single-objective SPIM using a reflective atomic force microscopy cantilever to create a 2µm thin sheet. Using this setup, we demonstrate that ldSPIM can deliver single fluorescently-labeled proteins onto the plasma membrane of HK293 cells or into the cytoplasm. Next, we deposited aggregates of amyloid-β, which causes proteotoxicity relevant to Alzheimer’s disease, onto a single macrophage stably expressing a MyDD88-eGFP fusion construct. Whole-cell imaging in 3D mode enables live detection of MyDD88 accumulation and formation of MyDDosome signaling complexes, as a result of aggregate-induced triggering of toll-like receptor 4. Overall, we demonstrate a novel multifunctional imaging system capable of precise delivery of single proteins to a specific location on the cell surface or inside the cytoplasm and high-speed 3D detection at single-molecule resolution within live cells.Statement of SignificanceThis paper describes and validates a new method to study biological processes based on the controlled local delivery of molecules onto or into the cell, combined with single molecule imaging using light sheet microscopy. we not only demonstrate the instrument’s capability of delivering controlled numbers of molecules to a defined position, down to the level of single molecules, but also its potential in study of the triggering of the innate immune response by protein aggregates, a key process in the development of neurodegenerative diseases such as Alzheimer’s disease. The same approach could be applied to a wide range of other important biological processes allowing them to be followed in live cells in real-time, hence it will be of great interest to the biophysical community.

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