Three dimensional live-cell STED microscopy at increased depth using a water immersion objective

2018 ◽  
Vol 89 (5) ◽  
pp. 053701 ◽  
Author(s):  
Jörn Heine ◽  
Christian A. Wurm ◽  
Jan Keller-Findeisen ◽  
Andreas Schönle ◽  
Benjamin Harke ◽  
...  
Keyword(s):  
Water Immersion ◽  
Three Dimensional ◽  
Live Cell ◽  
Sted Microscopy ◽  
Water Immersion Objective

Cost-Effective High-Speed, Three-Dimensional Live-Cell Imaging of HIV-1 Transfer at the T Cell Virological Synapse

2021 ◽  
Author(s):  
Alice Sandmeyer ◽  
Lili Wang ◽  
Wolfgang Hübner ◽  
Marcel Müller ◽  
Benjamin Chen ◽  
...  
Keyword(s):  
T Cell ◽  
Live Cell Imaging ◽  
High Speed ◽  
Cell Imaging ◽  
Three Dimensional ◽  
Cost Effective ◽  
Live Cell ◽  
Virological Synapse ◽  
Hiv 1

scholarly journals CRISPR-mediated Multiplexed Live Cell Imaging of Nonrepetitive Genomic Loci

2020 ◽  
Author(s):  
Patricia A. Clow ◽  
Nathaniel Jillette ◽  
Jacqueline J. Zhu ◽  
Albert W. Cheng
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Repetitive Sequences ◽  
Binary Sequence ◽  
Three Dimensional ◽  
Live Cell ◽  
Live Cells ◽  
Imaging Method ◽  
Genomic Locus ◽  
Time Dynamics

AbstractThree-dimensional (3D) structures of the genome are dynamic, heterogeneous and functionally important. Live cell imaging has become the leading method for chromatin dynamics tracking. However, existing CRISPR- and TALE-based genomic labeling techniques have been hampered by laborious protocols and low signal-to-noise ratios (SNRs), and are thus mostly applicable to repetitive sequences. Here, we report a versatile CRISPR/Casilio-based imaging method, with an enhanced SNR, that allows for one nonrepetitive genomic locus to be labeled using a single sgRNA. We constructed Casilio dual-color probes to visualize the dynamic interactions of cohesin-bound elements in single live cells. By forming a binary sequence of multiple Casilio probes (PISCES) across a continuous stretch of DNA, we track the dynamic 3D folding of a 74kb genomic region over time. This method offers unprecedented resolution and scalability for delineating the dynamic 4D nucleome.One Sentence SummaryCasilio enables multiplexed live cell imaging of nonrepetitive DNA loci for illuminating the real-time dynamics of genome structures.

scholarly journals High-precision 3D inkjet technology for live cell bioprinting

2019 ◽  
Vol 5 (2) ◽  
pp. 27 ◽  
Author(s):  
Daisuke Takagi ◽  
Waka Lin ◽  
Takahiko Matsumoto ◽  
Hidekazu Yaginuma ◽  
Natsuko Hemmi ◽  
...  
Keyword(s):  
Three Dimensional ◽  
Cell Types ◽  
Cell Number ◽  
Live Cell ◽  
Drop On Demand ◽  
Long Time ◽  
Spatial Positioning ◽  
Different Cell Types ◽  
Membrane Vibrations

In recent years, bioprinting has emerged as a promising technology for the construction of three-dimensional (3D) tissues to be used in regenerative medicine or in vitro screening applications. In the present study, we present the development of an inkjet-based bioprinting system to arrange multiple cells and materials precisely into structurally organized constructs. A novel inkjet printhead has been specially designed for live cell ejection. Droplet formation is powered by piezoelectric membrane vibrations coupled with mixing movements to prevent cell sedimentation at the nozzle. Stable drop-on-demand dispensing and cell viability were validated over an adequately long time to allow the fabrication of 3D tissues. Reliable control of cell number and spatial positioning was demonstrated using two separate suspensions with different cell types printed sequentially. Finally, a process for constructing stratified Mille-Feuille-like 3D structures is proposed by alternately superimposing cell suspensions and hydrogel layers with a controlled vertical resolution. The results show that inkjet technology is effective for both two-dimensional patterning and 3D multilayering and has the potential to facilitate the achievement of live cell bioprinting with an unprecedented level of precision.

scholarly journals In vivo imaging with a water immersion objective affects brain temperature, blood flow and oxygenation

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Morgane Roche ◽  
Emmanuelle Chaigneau ◽  
Ravi L Rungta ◽  
Davide Boido ◽  
Bruno Weber ◽  
...  
Keyword(s):  
Blood Flow ◽  
Brain Temperature ◽  
Water Immersion ◽  
Phosphorescence Lifetime ◽  
Cranial Window ◽  
Hemoglobin Saturation ◽  
Tissue Po2 ◽  
Physiological Values ◽  
Water Immersion Objective

Previously, we reported the first oxygen partial pressure (Po2) measurements in the brain of awake mice, by performing two-photon phosphorescence lifetime microscopy at micrometer resolution (Lyons et al., 2016). However, this study disregarded that imaging through a cranial window lowers brain temperature, an effect capable of affecting cerebral blood flow, the properties of the oxygen sensors and thus Po2 measurements. Here, we show that in awake mice chronically implanted with a glass window over a craniotomy or a thinned-skull surface, the postsurgical decrease of brain temperature recovers within a few days. However, upon imaging with a water immersion objective at room temperature, brain temperature decreases by ~2–3°C, causing drops in resting capillary blood flow, capillary Po2, hemoglobin saturation, and tissue Po2. These adverse effects are corrected by heating the immersion objective or avoided by imaging through a dry air objective, thereby revealing the physiological values of brain oxygenation.

Electrochemical Tip-Enhanced Raman Spectroscopy with Improved Sensitivity Enabled by a Water Immersion Objective

2019 ◽  
Vol 91 (17) ◽  
pp. 11092-11097 ◽  
Author(s):  
Sheng-Chao Huang ◽  
Jiu-Zheng Ye ◽  
Xiao-Ru Shen ◽  
Qing-Qing Zhao ◽  
Zhi-Cong Zeng ◽  
...  
Keyword(s):  
Raman Spectroscopy ◽  
Water Immersion ◽  
Tip Enhanced Raman Spectroscopy ◽  
Water Immersion Objective

scholarly journals Small-Molecule Labeling of Live Cell Surfaces for Three-Dimensional Super-Resolution Microscopy

2014 ◽  
Vol 136 (40) ◽  
pp. 14003-14006 ◽  
Author(s):  
Marissa K. Lee ◽  
Prabin Rai ◽  
Jarrod Williams ◽  
Robert J. Twieg ◽  
W. E. Moerner
Keyword(s):  
Small Molecule ◽  
Three Dimensional ◽  
Super Resolution ◽  
Live Cell ◽  
Cell Surfaces ◽  
Super Resolution Microscopy

scholarly journals Extending resolution of structured illumination microscopy with sparse deconvolution

2021 ◽  
Author(s):  
Weisong Zhao ◽  
Shiqun Zhao ◽  
Liuju Li ◽  
Xiaoshuai Huang ◽  
Shijia Xing ◽  
...  
Keyword(s):  
Signal To Noise Ratio ◽  
Three Dimensional ◽  
Super Resolution ◽  
Live Cell ◽  
Frame Rate ◽  
Live Cells ◽  
Photon Flux ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Spatiotemporal Resolution

Abstract The spatial resolutions of live-cell super-resolution microscopes are limited by the maximum collected photon flux. Taking advantage of a priori knowledge of the sparsity and continuity of biological structures, we develop a deconvolution algorithm that further extends the resolution of super-resolution microscopes under the same photon budgets by nearly twofold. As a result, sparse structured illumination microscopy (Sparse-SIM) achieves ~60 nm resolution at a 564 Hz frame rate, allowing it to resolve intricate structural intermediates, including small vesicular fusion pores, ring-shaped nuclear pores formed by different nucleoporins, and relative movements between the inner and outer membranes of mitochondria in live cells. Likewise, sparse deconvolution can be used to increase the three-dimensional resolution and contrast of spinning-disc confocal-based SIM (SD-SIM), and operates under conditions with the insufficient signal-to-noise-ratio, all of which allows routine four-color, three-dimensional, ~90 nm resolution live-cell super-resolution imaging. Overall, sparse deconvolution may be a general tool to push the spatiotemporal resolution limits of live-cell fluorescence microscopy.

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