scholarly journals The changing face of live-cell imaging: From phase contrast to single photon

2004 ◽  
Vol 26 (3) ◽  
pp. 30-34
Author(s):  
Mark Jepson ◽  
Darran Clements
Keyword(s):  
Live Cell Imaging ◽  
Phase Contrast ◽  
Cell Imaging ◽  
Cell Function ◽  
Molecular Level ◽  
Single Photon ◽  
Living Cells ◽  
Live Cell ◽  
Live Cells ◽  
The Past

The imaging of live cells using light microscopy has come a long way from the early days of phase contrast. There have been many exciting developments in technology that now deliver live-cell images that previously would not have been thought possible. The study of dynamic processes right down to the molecular level as they happen in living cells is now common practice in the drive to understand cell function. So what has happened over the past 50 years to make live-cell imaging so much more accessible today?

scholarly journals Deep learning pipeline for cell edge segmentation of time-lapse live cell images

2017 ◽  
Author(s):  
Chuangqi Wang ◽  
Xitong Zhang ◽  
Hee June Choi ◽  
Bolun Lin ◽  
Yudong Yu ◽  
...  
Keyword(s):  
Neural Networks ◽  
Deep Learning ◽  
Live Cell Imaging ◽  
Phase Contrast ◽  
Deep Neural Networks ◽  
Cell Imaging ◽  
Time Lapse ◽  
Live Cell ◽  
Live Cells ◽  
Quantitative Analyses

AbstractQuantitative live cell imaging has been widely used to study various dynamical processes in cell biology. Phase contrast microscopy is a popular imaging modality for live cell imaging since it does not require labeling and cause any phototoxicity to live cells. However, phase contrast images have posed significant challenges for accurate image segmentation due to complex image features. Fluorescence live cell imaging has also been used to monitor the dynamics of specific molecules in live cells. But unlike immunofluorescence imaging, fluorescence live cell images are highly prone to noise, low contrast, and uneven illumination. These often lead to erroneous cell segmentation, hindering quantitative analyses of dynamical cellular processes. Although deep learning has been successfully applied in image segmentation by automatically learning hierarchical features directly from raw data, it typically requires large datasets and high computational cost to train deep neural networks. These make it challenging to apply deep learning in routine laboratory settings. In this paper, we evaluate a deep learning-based segmentation pipeline for time-lapse live cell movies, which uses small efforts to prepare the training set by leveraging the temporal coherence of time-lapse image sequences. We train deep neural networks using a small portion of images in the movies, and then predict cell edges for the entire image frames of the same movies. To further increase segmentation accuracy using small numbers of training frames, we integrate VGG16 pretrained model with the U-Net structure (VGG16-U-Net) for neural network training. Using live cell movies from phase contrast, Total Internal Reflection Fluorescence (TIRF), and spinning disk confocal microscopes, we demonstrate that the labeling of cell edges in small portions (5∼10%) can provide enough training data for the deep learning segmentation. Particularly, VGG16-U-Net produces significantly more accurate segmentation than U-Net by increasing the recall performance. We expect that our deep learning segmentation pipeline will facilitate quantitative analyses of challenging high-resolution live cell movies.

Autofocusing in digital holographic phase contrast microscopy on pure phase objects for live cell imaging

2008 ◽  
Vol 47 (19) ◽  
pp. D176 ◽  
Author(s):  
Patrik Langehanenberg ◽  
Björn Kemper ◽  
Dieter Dirksen ◽  
Gert von Bally
Keyword(s):  
Live Cell Imaging ◽  
Phase Contrast ◽  
Cell Imaging ◽  
Live Cell ◽  
Phase Contrast Microscopy ◽  
Pure Phase ◽  
Contrast Microscopy

scholarly journals tdLanYFP, a yellow, bright, photostable and pH insensitive fluorescent protein for live cell imaging and FRET-based sensing strategies

2021 ◽  
Author(s):  
Y. Bousmah ◽  
H. Valenta ◽  
G. Bertolin ◽  
U. Singh ◽  
V. Nicolas ◽  
...  
Keyword(s):  
Single Molecule ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Yellow Fluorescent Protein ◽  
Resonance Energy ◽  
Live Cell ◽  
Live Cells

AbstractYellow fluorescent proteins (YFP) are widely used as optical reporters in Förster Resonance Energy Transfer (FRET) based biosensors. Although great improvements have been done, the sensitivity of the biosensors is still limited by the low photostability and the poor fluorescence performances of YFPs at acidic pHs. In fact, today, there is no yellow variant derived from the EYFP with a pK1/2 below ∼5.5. Here, we characterize a new yellow fluorescent protein, tdLanYFP, derived from the tetrameric protein from the cephalochordate B. lanceolatum, LanYFP. With a quantum yield of 0.92 and an extinction coefficient of 133 000 mol−1.L.cm−1, it is, to our knowledge, the brightest dimeric fluorescent protein available, and brighter than most of the monomeric YFPs. Contrasting with EYFP and its derivatives, tdLanYFP has a very high photostability in vitro and preserves this property in live cells. As a consequence, tdLanYFP allows the imaging of cellular structures with sub-diffraction resolution with STED nanoscopy. We also demonstrate that the combination of high brightness and strong photostability is compatible with the use of spectro-microscopies in single molecule regimes. Its very low pK1/2 of 3.9 makes tdLanYFP an excellent tag even at acidic pHs. Finally, we show that tdLanYFP can be a FRET partner either as donor or acceptor in different biosensing modalities. Altogether, these assets make tdLanYFPa very attractive yellow fluorescent protein for long-term or single-molecule live-cell imaging that is also suitable for FRET experiment including at acidic pH.

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.

Use of rhodamine-allyl Schiff base in chemodosimetric processes for total palladium estimation and application in live cell imaging

2018 ◽  
Vol 42 (21) ◽  
pp. 17351-17358 ◽  
Author(s):  
Anup Kumar Bhanja ◽  
Snehasis Mishra ◽  
Ketaki Kar ◽  
Kaushik Naskar ◽  
Suvendu Maity ◽  
...  
Keyword(s):  
Schiff Base ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Living Cells ◽  
Live Cell ◽  
Raw 264.7 ◽  
Macrophage Cells ◽  
Raw 264.7 Macrophage ◽  
Raw 264.7 Macrophage Cells

An allyl-rhodamine Schiff base shows excellent palladium sensitivity (LOD, 95 nM) irrespective of Pd(0,ii,iv) and practical applicability is judged in living cells of RAW 264.7 (macrophage) cells.

scholarly journals Live Cell Imaging: 3-Dimensional Tracking of Non-blinking ‘Giant’ Quantum Dots in Live Cells (Adv. Funct. Mater. 30/2014)

2014 ◽  
Vol 24 (30) ◽  
pp. 4795-4795 ◽  
Author(s):  
Aaron M. Keller ◽  
Yagnaseni Ghosh ◽  
Matthew S. DeVore ◽  
Mary E. Phipps ◽  
Michael H. Stewart ◽  
...  
Keyword(s):  
Quantum Dots ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell ◽  
Live Cells ◽  
3 Dimensional

Synthesis of photoactivatable azido-acyl caged oxazine fluorophores for live-cell imaging

2016 ◽  
Vol 52 (60) ◽  
pp. 9442-9445 ◽  
Author(s):  
Andrew V. Anzalone ◽  
Zhixing Chen ◽  
Virginia W. Cornish
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Living Cells ◽  
Live Cell

A new cell-permeable caged oxazine fluorophore was synthesized for protein specific labeling and photoactivation in living cells.

scholarly journals Label-free and live cell imaging by interferometric scattering microscopy

2018 ◽  
Vol 9 (10) ◽  
pp. 2690-2697 ◽  
Author(s):  
Jin-Sung Park ◽  
Il-Buem Lee ◽  
Hyeon-Min Moon ◽  
Jong-Hyeon Joo ◽  
Kyoung-Hoon Kim ◽  
...  
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Complex Structure ◽  
Live Cell ◽  
Fluorescent Label ◽  
Live Cells ◽  
Label Free ◽  
Cytoplasmic Organelles ◽  
Microscopic Techniques

Despite recent remarkable advances in microscopic techniques, it still remains very challenging to directly observe the complex structure of cytoplasmic organelles in live cells without a fluorescent label.

scholarly journals Illuminating the Sites of Enterovirus Replication in Living Cells by Using a Split-GFP-Tagged Viral Protein

mSphere ◽  
2016 ◽  
Vol 1 (4) ◽  
Author(s):  
H. M. van der Schaar ◽  
C. E. Melia ◽  
J. A. C. van Bruggen ◽  
J. R. P. M. Strating ◽  
M. E. D. van Geenen ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Lipid Composition ◽  
Viral Protein ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Living Cells ◽  
Live Cell ◽  
Genome Replication ◽  
Fluorescent Reporter ◽  
Unique Protein

ABSTRACT Enteroviruses induce the formation of membranous structures (replication organelles [ROs]) with a unique protein and lipid composition specialized for genome replication. Electron microscopy has revealed the morphology of enterovirus ROs, and immunofluorescence studies have been conducted to investigate their origin and formation. Yet, immunofluorescence analysis of fixed cells results in a rather static view of RO formation, and the results may be compromised by immunolabeling artifacts. While live-cell imaging of ROs would be preferred, enteroviruses encoding a membrane-anchored viral protein fused to a large fluorescent reporter have thus far not been described. Here, we tackled this constraint by introducing a small tag from a split-GFP system into an RO-resident enterovirus protein. This new tool bridges a methodological gap by circumventing the need for immunolabeling fixed cells and allows the study of the dynamics and formation of enterovirus ROs in living cells. Like all other positive-strand RNA viruses, enteroviruses generate new organelles (replication organelles [ROs]) with a unique protein and lipid composition on which they multiply their viral genome. Suitable tools for live-cell imaging of enterovirus ROs are currently unavailable, as recombinant enteroviruses that carry genes that encode RO-anchored viral proteins tagged with fluorescent reporters have not been reported thus far. To overcome this limitation, we used a split green fluorescent protein (split-GFP) system, comprising a large fragment [strands 1 to 10; GFP(S1-10)] and a small fragment [strand 11; GFP(S11)] of only 16 residues. The GFP(S11) (GFP with S11 fragment) fragment was inserted into the 3A protein of the enterovirus coxsackievirus B3 (CVB3), while the large fragment was supplied by transient or stable expression in cells. The introduction of GFP(S11) did not affect the known functions of 3A when expressed in isolation. Using correlative light electron microscopy (CLEM), we showed that GFP fluorescence was detected at ROs, whose morphologies are essentially identical to those previously observed for wild-type CVB3, indicating that GFP(S11)-tagged 3A proteins assemble with GFP(S1-10) to form GFP for illumination of bona fide ROs. It is well established that enterovirus infection leads to Golgi disintegration. Through live-cell imaging of infected cells expressing an mCherry-tagged Golgi marker, we monitored RO development and revealed the dynamics of Golgi disassembly in real time. Having demonstrated the suitability of this virus for imaging ROs, we constructed a CVB3 encoding GFP(S1-10) and GFP(S11)-tagged 3A to bypass the need to express GFP(S1-10) prior to infection. These tools will have multiple applications in future studies on the origin, location, and function of enterovirus ROs. IMPORTANCE Enteroviruses induce the formation of membranous structures (replication organelles [ROs]) with a unique protein and lipid composition specialized for genome replication. Electron microscopy has revealed the morphology of enterovirus ROs, and immunofluorescence studies have been conducted to investigate their origin and formation. Yet, immunofluorescence analysis of fixed cells results in a rather static view of RO formation, and the results may be compromised by immunolabeling artifacts. While live-cell imaging of ROs would be preferred, enteroviruses encoding a membrane-anchored viral protein fused to a large fluorescent reporter have thus far not been described. Here, we tackled this constraint by introducing a small tag from a split-GFP system into an RO-resident enterovirus protein. This new tool bridges a methodological gap by circumventing the need for immunolabeling fixed cells and allows the study of the dynamics and formation of enterovirus ROs in living cells.

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