Development of a water refractive index-matched microneedle integrated into a light sheet microscopy system for continuous embryonic cell imaging

Lab on a Chip ◽  
2022 ◽  
Author(s):  
Yu-Jui Fan ◽  
Han-Yun Hsieh ◽  
Yen-Ru Huang ◽  
Chieh Tsao ◽  
Chia-Ming Lee ◽  
...  
Keyword(s):  
Refractive Index ◽  
Cell Imaging ◽  
Light Sheet ◽  
Live Imaging ◽  
Embryonic Cell ◽  
Imaging Technology ◽  
Light Sheet Microscopy

Leveraging advances in microfluidics and light sheet imaging technology. We developed a water refractive index-matched microneedle to catch embryos for live imaging.

scholarly journals High-throughput live imaging using Light Sheet Microscopy

2020 ◽  
Author(s):  
Emilio J. Gualda ◽  
Matteo Bernardello ◽  
Maria Marsal ◽  
Pablo Loza Alvarez
Keyword(s):  
High Throughput ◽  
Light Sheet ◽  
Live Imaging ◽  
Light Sheet Microscopy

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 Comparison of Transparency and Shrinkage During Clearing of Insect Brains Using Media With Tunable Refractive Index

2020 ◽  
Vol 14 ◽  
Author(s):  
Bo M. B. Bekkouche ◽  
Helena K. M. Fritz ◽  
Elisa Rigosi ◽  
David C. O'Carroll
Keyword(s):  
Refractive Index ◽  
Rapid Development ◽  
Vacuum Treatment ◽  
Building Blocks ◽  
Light Sheet ◽  
Tracheal System ◽  
Light Sheet Microscopy ◽  
Tissue Shrinkage ◽  
Tunable Refractive Index ◽  
The Brain

Improvement of imaging quality has the potential to visualize previously unseen building blocks of the brain and is therefore one of the great challenges in neuroscience. Rapid development of new tissue clearing techniques in recent years have attempted to solve imaging compromises in thick brain samples, particularly for high resolution optical microscopy, where the clearing medium needs to match the high refractive index of the objective immersion medium. These problems are exacerbated in insect tissue, where numerous (initially air-filled) tracheal tubes branching throughout the brain increase the scattering of light. To date, surprisingly few studies have systematically quantified the benefits of such clearing methods using objective transparency and tissue shrinkage measurements. In this study we compare a traditional and widely used insect clearing medium, methyl salicylate combined with permanent mounting in Permount (“MS/P”) with several more recently applied clearing media that offer tunable refractive index (n): 2,2′-thiodiethanol (TDE), “SeeDB2” (in variants SeeDB2S and SeeDB2G matched to oil and glycerol immersion, n = 1.52 and 1.47, respectively) and Rapiclear (also with n = 1.52 and 1.47). We measured transparency and tissue shrinkage by comparing freshly dissected brains with cleared brains from dipteran flies, with or without addition of vacuum or ethanol pre-treatments (dehydration and rehydration) to evacuate air from the tracheal system. The results show that ethanol pre-treatment is very effective for improving transparency, regardless of the subsequent clearing medium, while vacuum treatment offers little measurable benefit. Ethanol pre-treated SeeDB2G and Rapiclear brains show much less shrinkage than using the traditional MS/P method. Furthermore, at lower refractive index, closer to that of glycerol immersion, these recently developed media offer outstanding transparency compared to TDE and MS/P. Rapiclear protocols were less laborious compared to SeeDB2, but both offer sufficient transparency and refractive index tunability to permit super-resolution imaging of local volumes in whole mount brains from large insects, and even light-sheet microscopy. Although long-term permanency of Rapiclear stored samples remains to be established, our samples still showed good preservation of fluorescence after storage for more than a year at room temperature.

scholarly journals Automatic and adaptive heterogeneous refractive index compensation for light-sheet microscopy

2017 ◽  
Vol 8 (1) ◽  
Author(s):  
Duncan P. Ryan ◽  
Elizabeth A. Gould ◽  
Gregory J. Seedorf ◽  
Omid Masihzadeh ◽  
Steven H. Abman ◽  
...  
Keyword(s):  
Refractive Index ◽  
Light Sheet ◽  
Light Sheet Microscopy

scholarly journals Light sheet microscopy and live imaging of plants

2016 ◽  
Vol 263 (2) ◽  
pp. 158-164 ◽  
Author(s):  
BÉATRICE BERTHET ◽  
ALEXIS MAIZEL
Keyword(s):  
Light Sheet ◽  
Live Imaging ◽  
Light Sheet Microscopy

scholarly journals Rapid Whole Cell Imaging Reveals A Calcium-APPL1-Dynein Nexus That Regulates Cohort Trafficking of Stimulated EGF Receptors

2018 ◽  
Author(s):  
H M York ◽  
A Patil ◽  
U K Moorthi ◽  
A Kaur ◽  
A Bhowmik ◽  
...  
Keyword(s):  
Signal Processing ◽  
Cell Imaging ◽  
Light Sheet ◽  
Endosomal Trafficking ◽  
Egf Receptors ◽  
Whole Cell ◽  
Cellular Processes ◽  
Light Sheet Microscopy ◽  
Signaling Receptors ◽  
Cell Surface Signaling

ABSTRACTMulticellular life processes such as proliferation and differentiation depend on cell surface signaling receptors that bind ligands generally referred to as growth factors. Recently, it has emerged that the endosomal system provides rich signal processing capabilities for responses elicited by these factors [1-3]. At the single cell level, endosomal trafficking becomes a critical component of signal processing, as exemplified by the epidermal growth factor (EGF) receptors of the receptor tyrosine kinase family. EGFRs, once activated by EGF, are robustly trafficked to the phosphatase-enriched peri-nuclear region (PNR), where they are dephosphorylated [4-8]. However, the details of the mechanisms regulating the movements of stimulated EGFR in time and space, i.e., towards the PNR, are not known. What endosomal regulators provide specificity to EGFR? Do modifications to the receptor upon stimulation regulate its trafficking? To understand the events leading to EGFR translocation, and especially the early endosomal dynamics that immediately follow EGFR internalization, requires the real-time, long-term, whole-cell imaging of multiple elements. Here, exploiting the advantages of lattice light-sheet microscopy [9], we show that the binding of EGF by its receptor, EGFR, triggers a transient calcium increase that peaks by 30 s, causing the desorption of APPL1 from pre-existing endosomes within one minute, the rebinding of liberated APPL1 to EGFR within three minutes, and the dynein-dependent translocation of APPL1-EGF-bearing endosomes to the PNR within five minutes. The novel, cell spanning, fast acting network that we reveal integrates a cascade of events dedicated to the cohort movement of activated EGFR receptors. Our findings support the intriguing proposal that certain endosomal pathways have shed some of the stochastic strategies of traditional trafficking, and have evolved behaviors whose predictability is better suited to signaling [10, 11]. Work presented here demonstrates that our whole cell imaging approach can be a powerful tool in revealing critical transient interactions in key cellular processes such as receptor trafficking.

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

2016 ◽  
Vol 27 (22) ◽  
pp. 3418-3435 ◽  
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.

Live Imaging of Influenza Viral Ribonucleoproteins Using Light-Sheet Microscopy

2018 ◽  
pp. 303-327 ◽  
Author(s):  
Amar R. Bhagwat ◽  
Valerie Le Sage ◽  
Seema S. Lakdawala
Keyword(s):  
Light Sheet ◽  
Live Imaging ◽  
Light Sheet Microscopy

scholarly journals Erratum: Live imaging and quantitative analysis of gastrulation in mouse embryos using light-sheet microscopy and 3D tracking tools

2014 ◽  
Vol 9 (10) ◽  
pp. 2513-2513 ◽  
Author(s):  
Takehiko Ichikawa ◽  
Kenichi Nakazato ◽  
Philipp J Keller ◽  
Hiroko Kajiura-Kobayashi ◽  
Ernst H K Stelzer ◽  
...  
Keyword(s):  
Quantitative Analysis ◽  
Light Sheet ◽  
Live Imaging ◽  
Mouse Embryos ◽  
3D Tracking ◽  
Light Sheet Microscopy
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