High spatiotemporal resolution and low photo-toxicity fluorescence imaging in live cells and in vivo

2019 ◽  
Vol 47 (6) ◽  
pp. 1635-1650 ◽  
Author(s):  
Xiaohong Peng ◽  
Xiaoshuai Huang ◽  
Ke Du ◽  
Huisheng Liu ◽  
Liangyi Chen
Keyword(s):  
Fluorescence Microscopy ◽  
Spatial Resolution ◽  
Cell Biology ◽  
Critical Role ◽  
Super Resolution ◽  
Light Sheet ◽  
Live Cells ◽  
Spatiotemporal Resolution ◽  
And Function

Taking advantage of high contrast and molecular specificity, fluorescence microscopy has played a critical role in the visualization of subcellular structures and function, enabling unprecedented exploration from cell biology to neuroscience in living animals. To record and quantitatively analyse complex and dynamic biological processes in real time, fluorescence microscopes must be capable of rapid, targeted access deep within samples at high spatial resolutions, using techniques including super-resolution fluorescence microscopy, light sheet fluorescence microscopy, and multiple photon microscopy. In recent years, tremendous breakthroughs have improved the performance of these fluorescence microscopies in spatial resolution, imaging speed, and penetration. Here, we will review recent advancements of these microscopies in terms of the trade-off among spatial resolution, sampling speed and penetration depth and provide a view of their possible applications.

scholarly journals 3D Interferometric Lattice Light-Sheet Imaging

2020 ◽  
Author(s):  
Bin Cao ◽  
Guanshi Wang ◽  
Jieru Li ◽  
Alexandros Pertsinidis
Keyword(s):  
Single Molecule ◽  
Super Resolution ◽  
Light Sheet ◽  
Detection Sensitivity ◽  
Live Cells ◽  
Background Suppression ◽  
Axial Resolution ◽  
Volumetric Imaging ◽  
Spatio Temporal ◽  
And Function

Understanding cellular structure and function requires live-cell imaging with high spatio-temporal resolution and high detection sensitivity. Direct visualization of molecular processes using single-molecule/super-resolution techniques has thus been transformative. However, extracting the highest-resolution 4D information possible from weak and dynamic fluorescence signals in live cells remains challenging. For example, some of the highest spatial resolution methods, e.g. interferometric (4Pi) approaches1-6 can be slow, require high peak excitation intensities that accelerate photobleaching or suffer from increased out-of-focus background. Selective-plane illumination (SPIM)7-12 reduces background, but most implementations typically feature modest spatial, especially axial, resolution. Here we develop 3D interferometric lattice light-sheet (3D-iLLS) imaging, a technique that overcomes many of these limitations. 3D-iLLS provides, by virtue of SPIM, low light levels and photobleaching, while providing increased background suppression and significantly improved volumetric imaging/sectioning capabilities through 4Pi interferometry. We demonstrate 3D-iLLS with axial resolution and single-particle localization precision down to <100nm (FWHM) and <10nm (1σ) respectively. 3D-iLLS paves the way for a fuller elucidation of sub-cellular phenomena by enhanced 4D resolution and SNR live imaging.

scholarly journals Recent Progress in Light Sheet Microscopy for Biological Applications

2018 ◽  
Vol 72 (8) ◽  
pp. 1137-1169 ◽  
Author(s):  
Krishnendu Chatterjee ◽  
Feby Wijaya Pratiwi ◽  
Frances Camille M. Wu ◽  
Peilin Chen ◽  
Bi-Chang Chen
Keyword(s):  
Developmental Biology ◽  
Fluorescence Microscopy ◽  
Single Cell ◽  
Single Molecule ◽  
Super Resolution ◽  
Light Sheet ◽  
High Signal ◽  
Spatiotemporal Resolution ◽  
Light Sheet Microscopy ◽  
Resolution Imaging

The introduction of light sheet fluorescence microscopy (LSFM) has overcome the challenges in conventional optical microscopy. Among the recent breakthroughs in fluorescence microscopy, LSFM had been proven to provide a high three-dimensional spatial resolution, high signal-to-noise ratio, fast imaging acquisition rate, and minuscule levels of phototoxic and photodamage effects. The aforementioned auspicious properties are crucial in the biomedical and clinical research fields, covering a broad range of applications: from the super-resolution imaging of intracellular dynamics in a single cell to the high spatiotemporal resolution imaging of developmental dynamics in an entirely large organism. In this review, we provided a systematic outline of the historical development of LSFM, detailed discussion on the variants and improvements of LSFM, and delineation on the most recent technological advancements of LSFM and its potential applications in single molecule/particle detection, single-molecule super-resolution imaging, imaging intracellular dynamics of a single cell, multicellular imaging: cell–cell and cell–matrix interactions, plant developmental biology, and brain imaging and developmental biology.

scholarly journals A guide to super-resolution fluorescence microscopy

2010 ◽  
Vol 190 (2) ◽  
pp. 165-175 ◽  
Author(s):  
Lothar Schermelleh ◽  
Rainer Heintzmann ◽  
Heinrich Leonhardt
Keyword(s):  
Fluorescence Microscopy ◽  
Light Microscopy ◽  
Cell Biology ◽  
New Technologies ◽  
Optical Resolution ◽  
Super Resolution ◽  
Structure And Function ◽  
Precise Localization ◽  
New Approaches ◽  
And Function

For centuries, cell biology has been based on light microscopy and at the same time been limited by its optical resolution. However, several new technologies have been developed recently that bypass this limit. These new super-resolution technologies are either based on tailored illumination, nonlinear fluorophore responses, or the precise localization of single molecules. Overall, these new approaches have created unprecedented new possibilities to investigate the structure and function of cells.

scholarly journals Meeting experiments at the diffraction barrier: an in-silico widefield fluorescence microscopy

2021 ◽  
Author(s):  
Subhamoy Mahajan ◽  
Tian Tang
Keyword(s):  
Fluorescence Microscopy ◽  
In Silico ◽  
Super Resolution ◽  
Light Sheet ◽  
Atomic Resolution ◽  
Live Cells ◽  
Two Photon ◽  
Color Combinations ◽  
Super Resolution Microscopy ◽  
Diffraction Barrier

AbstractFluorescence microscopy allows the visualization of live cells and their components, but even with advances in super- resolution microscopy, atomic resolution remains unattainable. On the other hand, molecular simulations (MS) can easily access atomic resolution, but comparison with experimental microscopy images has not been possible. In this work, a novel in-silico widefield fluorescence microscopy is proposed, which reduces the resolution of MS to generate images comparable to experiments. This technique will allow cross-validation and compound the knowledge gained from experiments and MS. We demonstrate that in-silico images can be produced with different optical axis, object focal planes, exposure time, color combinations, resolution, brightness and amount of out-of-focus fluorescence. This allows the generation of images that resemble those obtained from widefield, confocal, light-sheet, two-photon and super-resolution microscopy. This technique not only can be used as a standalone visualization tool for MS, but also lays the foundation for other in-silico microscopy methods.

scholarly journals CD4+ regulatory T cells require CTLA-4 for the maintenance of systemic tolerance

2009 ◽  
Vol 206 (2) ◽  
pp. 421-434 ◽  
Author(s):  
Randall H. Friedline ◽  
David S. Brown ◽  
Hai Nguyen ◽  
Hardy Kornfeld ◽  
JinHee Lee ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Cell Function ◽  
Cytotoxic T Lymphocyte ◽  
Critical Role ◽  
Lymphoproliferative Disease ◽  
In Trans ◽  
And Function

Cytotoxic T lymphocyte antigen-4 (CTLA-4) plays a critical role in negatively regulating T cell responses and has also been implicated in the development and function of natural FOXP3+ regulatory T cells. CTLA-4–deficient mice develop fatal, early onset lymphoproliferative disease. However, chimeric mice containing both CTLA-4–deficient and –sufficient bone marrow (BM)–derived cells do not develop disease, indicating that CTLA-4 can act in trans to maintain T cell self-tolerance. Using genetically mixed blastocyst and BM chimaeras as well as in vivo T cell transfer systems, we demonstrate that in vivo regulation of Ctla4−/− T cells in trans by CTLA-4–sufficient T cells is a reversible process that requires the persistent presence of FOXP3+ regulatory T cells with a diverse TCR repertoire. Based on gene expression studies, the regulatory T cells do not appear to act directly on T cells, suggesting they may instead modulate the stimulatory activities of antigen-presenting cells. These results demonstrate that CTLA-4 is absolutely required for FOXP3+ regulatory T cell function in vivo.

scholarly journals Reading and Function of a Histone Code Involved in Targeting Corepressor Complexes for Repression

2005 ◽  
Vol 25 (1) ◽  
pp. 324-335 ◽  
Author(s):  
Ho-Geun Yoon ◽  
Youngsok Choi ◽  
Philip A. Cole ◽  
Jiemin Wong
Keyword(s):  
Transcriptional Activation ◽  
Transcriptional Repression ◽  
Hormone Receptors ◽  
Critical Role ◽  
Pericentric Heterochromatin ◽  
Histone Code ◽  
Stable Association ◽  
And Function

ABSTRACT A central question in histone code theory is how various codes are recognized and utilized in vivo. Here we show that TBL1 and TBLR1, two WD-40 repeat proteins in the corepressor SMRT/N-CoR complexes, are functionally redundant and essential for transcriptional repression by unliganded thyroid hormone receptors (TR) but not essential for transcriptional activation by liganded TR. TBL1 and TBLR1 bind preferentially to hypoacetylated histones H2B and H4 in vitro and have a critical role in targeting the corepressor complexes to chromatin in vivo. We show that targeting SMRT/N-CoR complexes to the deiodinase 1 gene (D1) requires at least two interactions, one between unliganded TR and SMRT/N-CoR and the other between TBL1/TBLR1 and hypoacetylated histones. Neither interaction alone is sufficient for the stable association of the corepressor complexes with the D1 promoter. Our data support a feed-forward working model in which deacetylation exerted by initial unstable recruitment of SMRT/N-CoR complexes via their interaction with unliganded TR generates a histone code that serves to stabilize their own recruitment. Similarly, we find that targeting of the Sin3 complex to pericentric heterochromatin may also follow this model. Our studies provide an in vivo example that a histone code is not read independently but is recognized in the context of other interactions.

scholarly journals Hydroxychloroquine Synergizes With The PI3K Inhibitor BKM120 to Exhibit Antitumor Efficacy Independent of Autophagy

2021 ◽  
Author(s):  
Xin Peng ◽  
Shaolu Zhang ◽  
Wenhui Jiao ◽  
Zhenxing Zhong ◽  
Yuqi Yang ◽  
...  
Keyword(s):  
Tumor Cells ◽  
Cell Biology ◽  
Molecular Mechanisms ◽  
Critical Role ◽  
Single Agent ◽  
Antitumor Efficacy ◽  
Antitumor Effects ◽  
Autophagy Inhibitor

Abstract Background: The critical role of phosphoinositide 3-kinase (PI3K) activation in tumor cell biology has prompted massive efforts to develop PI3K inhibitors (PI3Kis) for cancer therapy. However, recent results from clinical trials have shown only a modest therapeutic efficacy of single-agent PI3Kis in solid tumors. Targeting autophagy has controversial context-dependent effects in cancer treatment. As a FDA-approved lysosomotropic agent, hydroxychloroquine (HCQ) has been well tested as an autophagy inhibitor in preclinical models. Here, we elucidated the novel mechanism of HCQ alone or in combination with PI3Ki BKM120 in the treatment of cancer.Methods: The antitumor effects of HCQ and BKM120 on three different types of tumor cells were assessed by in vitro PrestoBlue assay, colony formation assay and in vivo zebrafish and nude mouse xenograft models. The involved molecular mechanisms were investigated by MDC staining, LC3 puncta formation assay, immunofluorescent assay, flow cytometric analysis of apoptosis and ROS, qRT-PCR, Western blot, comet assay, homologous recombination (HR) assay and immunohistochemical staining. Results: HCQ significantly sensitized cancer cells to BKM120 in vitro and in vivo. Interestingly, the sensitization mediated by HCQ could not be phenocopied by treatment with other autophagy inhibitors (Spautin-1, 3-MA and bafilomycin A1) or knockdown of the essential autophagy genes Atg5/Atg7, suggesting that the sensitizing effect might be mediated independent of autophagy status. Mechanistically, HCQ induced ROS production and activated the transcription factor NRF2. In contrast, BKM120 prevented the elimination of ROS by inactivation of NRF2, leading to accumulation of DNA damage. In addition, HCQ activated ATM to enhance HR repair, a high-fidelity repair for DNA double-strand breaks (DSBs) in cells, while BKM120 inhibited HR repair by blocking the phosphorylation of ATM and the expression of BRCA1/2 and Rad51. Conclusions: Our study revealed that HCQ and BKM120 synergistically increased DSBs in tumor cells and therefore augmented apoptosis, resulting in enhanced antitumor efficacy. Our findings provide a new insight into how HCQ exhibits antitumor efficacy and synergizes with PI3Ki BKM120, and warn that one should consider the “off target” effects of HCQ when used as autophagy inhibitor in the clinical treatment of cancer.

LncRNA MALAT1 exhibits positive effects on nucleus pulposus cell biology in vivo and in vitro by sponging miR-503

2020 ◽  
Author(s):  
Hongyu Zheng ◽  
Tingting Wang ◽  
Xiangmin Li ◽  
Wei He ◽  
Zhiqiang Gong ◽  
...  
Keyword(s):  
Nucleus Pulposus ◽  
Disc Degeneration ◽  
Cell Biology ◽  
Mapk Pathway ◽  
Critical Role ◽  
Mapk Signaling ◽  
Positive Effects ◽  
Lncrna Malat1

Abstract Background: Intervertebral disc degeneration (IDD) is characterized by the loss of nucleus pulposus cells (NPCs) and phenotypic abnormalities. Accumulating evidence suggests that long noncoding RNAs (lncRNAs) are involved in the pathogenesis of IDD. In this study, we aimed to investigate the functional effects of lncRNA MALAT1 on NPCs in IDD and the possible mechanism governing these effects. Results: We validated the decreased expression of MALAT1 in the IDD tissues, which was associated with decreased Collagen II and Aggrecan expression. In vitro, overexpressed MALAT1 could attenuate the effect of IL-1β on NPC proliferation, apoptosis, and Aggrecan degradation. In vivo, MALAT1 overexpression attenuated the severity of disc degeneration in IDD model rats. Our molecular study further demonstrated that MALAT1 could sponge miR-503, modulate the expression of miR-503, and activate downstream MAPK signaling pathways. The effects of MALAT1 on NPCs were partially reversed/aggregated by miR-503 mimics/inhibitor treatment. Conclusion: Our data suggested that the MALAT1-miR-503-MAPK pathway plays a critical role in NPCs, which may be a potential strategy for alleviating IDD.

009. KALLIKREIN-RELATED PROTEASES AS NOVEL THERAPEUTIC TARGETS IN PROSTATE AND OVARIAN CANCER

2009 ◽  
Vol 21 (9) ◽  
pp. 6
Author(s):  
J. A. Clements ◽  
Y. Dong ◽  
D. Loessner ◽  
O. Tan ◽  
S. Sieh ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Cancer Progression ◽  
Cell Biology ◽  
Critical Role ◽  
Bone Matrix ◽  
Therapeutic Targets ◽  
Fibrillar Structure ◽  
Pilot Studies ◽  
Novel Therapeutic

The kallikrein-related (KLKs) peptidases are implicated in prostate and ovarian cancer invasion/metastasis via activation of growth factors, proteases and extracellular matrix degradation involved in. In our published work, we used cell biology approaches to show novel associations of KLK peptidases with processes indicative of metastasis and the potential of our novel sunflower trypsin inhibitor scaffold-engineered KLK4 inhibitor. Our current studies are directed towards discovering the precise KLK target proteins/substrates and the subsequent signalling pathways involved in these events in order to determine their therapeutic target potential. In this regard, we are using novel tissue engineered biomimetic 3D gel matrices to better mimic the in vivo micro-environment of prostate cancer cells especially in bone metastasis and peritoneal invasion in ovarian cancer. Pilot studies show that PC3 cells cultured on an osteoblast-derived bone matrix undergo an EMT-like change but remain dispersed on the cell surface. In contrast, LNCaP cells cluster aligning with the fibrillar structure as they invade into the bone matrix as typically seen in vivo. KLK4 proteolysis of the osteoblast-derived bone matrix has identified additional novel substrates. In addition, we are exploring the cell biology that underlies the reported high KLK4 or KLK7 levels associated with poorer outcome in women with epithelial ovarian cancer (EOC). Of note, KLK4 or KLK7 transfected SKOV3 EOC cells have increased chemoresistance to taxol and/or cisplastin suggesting a mechanism for this poor outcome. Furthermore, KLK7 transfected SKOV-3 cells form multicellular aggregates (MCA) in agarose suspension (a process indicative of peritoneal tumour cell spread seen in ascites fluid clinically) which can be reversed by a KLK7 blocking antibody indicating the critical role played by KLK7 in this event. These new paradigms are providing novel information on the role of KLK peptidases in prostate and ovarian cancer progression and their potential as novel therapeutic targets.

scholarly journals Canalicular domain structure and function in matrix-free hepatic spheroids

2020 ◽  
Vol 8 (1) ◽  
pp. 485-496 ◽  
Author(s):  
Vikas Raj Sharma ◽  
Ananya Shrivastava ◽  
Benoit Gallet ◽  
Elizaveta Karepina ◽  
Peggy Charbonnier ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Fluorescence Microscopy ◽  
Light Sheet ◽  
Structure And Function ◽  
Bile Canaliculi ◽  
Combined Use ◽  
3D Electron Microscopy ◽  
And Function ◽  
Matrix Free ◽  
Light Sheet Fluorescence Microscopy

The combined use of light sheet fluorescence microscopy and 3D electron microscopy enables to reveal the fine details of bile canaliculi structure and function in matrix-free hepatic spheroids.

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