3D Imaging of Single Cells in Bacterial Biofilms using Lattice Light-Sheet Microscopy

Cancer clone evolution takes place within tissue ecosystem habitats. But, how exactly tumors arise from a few malignant cells within an intact epithelium is a central, yet unanswered question. This is mainly due to the inaccessibility of this process to longitudinal imaging together with a lack of systems that model the progression of a fraction of transformed cells within a tissue. Here, we developed a new methodology based on primary mouse mammary epithelial acini, where oncogenes can be switched on in single cells within an otherwise normal epithelial cell layer. We combine this stochastic breast tumor induction model with inverted light-sheet imaging to study single-cell behavior for up to four days and analyze cell fates utilizing a newly developed image-data analysis workflow. The power of this integrated approach is illustrated by us finding that small local clusters of transformed cells form tumors while isolated transformed cells do not.

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Rapid high resolution 3D imaging of expanded biological specimens with lattice light sheet microscopy

Methods ◽

10.1016/j.ymeth.2019.04.006 ◽

2020 ◽

Vol 174 ◽

pp. 11-19 ◽

Cited By ~ 5

Author(s):

Yun-Chi Tsai ◽

Wei-Chun Tang ◽

Christine Siok Lan Low ◽

Yen-Ting Liu ◽

Jyun-Sian Wu ◽

...

Keyword(s):

High Resolution ◽

3D Imaging ◽

Light Sheet ◽

Light Sheet Microscopy

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3D imaging of cleared human skin biopsies using light-sheet microscopy: A new way to visualize in-depth skin structure

Skin Research and Technology ◽

10.1111/srt.12429 ◽

2018 ◽

Vol 24 (2) ◽

pp. 294-303 ◽

Cited By ~ 17

Author(s):

S. Abadie ◽

C. Jardet ◽

J. Colombelli ◽

B. Chaput ◽

A. David ◽

...

Keyword(s):

Human Skin ◽

3D Imaging ◽

Light Sheet ◽

Skin Structure ◽

Light Sheet Microscopy ◽

Skin Biopsies

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3D Imaging of BABB Cleared Whole-Mount Organs Using Light Sheet Microscopy

Microscopy and Microanalysis ◽

10.1017/s1431927618007432 ◽

2018 ◽

Vol 24 (S1) ◽

pp. 1390-1391

Author(s):

Kingsley A. Boateng ◽

Austin Cyphersmith ◽

Glenn A. Fried ◽

Barghav S. Sivaguru ◽

Xiaochen Lu ◽

...

Keyword(s):

3D Imaging ◽

Light Sheet ◽

Light Sheet Microscopy ◽

Whole Mount

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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 ◽

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.

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Label-free and Multimodal Second Harmonic Generation Light Sheet Microscopy

10.1101/2020.09.07.284703 ◽

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.

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Membrane dynamics of dividing cells imaged by lattice light-sheet microscopy

Molecular Biology of the Cell ◽

10.1091/mbc.e16-03-0164 ◽

2016 ◽

Vol 27 (22) ◽

pp. 3418-3435 ◽

Cited By ~ 58

Author(s):

François Aguet ◽

Srigokul Upadhyayula ◽

Raphaël Gaudin ◽

Yi-ying Chou ◽

Emanuele Cocucci ◽

...

Keyword(s):

Cell Surface ◽

Cell Imaging ◽

Single Cells ◽

Light Sheet ◽

Membrane Dynamics ◽

Bottom Surface ◽

Light Sheet Microscopy ◽

Dividing Cells ◽

Entire Cell ◽

Membrane Bound

Membrane remodeling is an essential part of transferring components to and from the cell surface and membrane-bound organelles and for changes in cell shape, which are particularly critical during cell division. Earlier analyses, based on classical optical live-cell imaging and mostly restricted by technical necessity to the attached bottom surface, showed persistent formation of endocytic clathrin pits and vesicles during mitosis. Taking advantage of the resolution, speed, and noninvasive illumination of the newly developed lattice light-sheet fluorescence microscope, we reexamined their assembly dynamics over the entire cell surface and found that clathrin pits form at a lower rate during late mitosis. Full-cell imaging measurements of cell surface area and volume throughout the cell cycle of single cells in culture and in zebrafish embryos showed that the total surface increased rapidly during the transition from telophase to cytokinesis, whereas cell volume increased slightly in metaphase and was relatively constant during cytokinesis. These applications demonstrate the advantage of lattice light-sheet microscopy and enable a new standard for imaging membrane dynamics in single cells and multicellular assemblies.

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