scholarly journals In vivo NIR-II structured-illumination light-sheet microscopy

2021 ◽  
Vol 118 (6) ◽  
pp. e2023888118
Author(s):  
Feifei Wang ◽  
Zhuoran Ma ◽  
Yeteng Zhong ◽  
Felix Salazar ◽  
Chun Xu ◽  
...  
Keyword(s):  
Light Scattering ◽  
Molecular Imaging ◽  
Single Cell ◽  
Light Sheet ◽  
Single Cell Level ◽  
Structured Illumination ◽  
Cell Level ◽  
Spatiotemporal Resolution ◽  
Light Sheet Microscopy

Noninvasive optical imaging with deep tissue penetration depth and high spatiotemporal resolution is important to longitudinally studying the biology at the single-cell level in live mammals, but has been challenging due to light scattering. Here, we developed near-infrared II (NIR-II) (1,000 to 1,700 nm) structured-illumination light-sheet microscopy (NIR-II SIM) with ultralong excitation and emission wavelengths up to ∼1,540 and ∼1,700 nm, respectively, suppressing light scattering to afford large volumetric three-dimensional (3D) imaging of tissues with deep-axial penetration depths. Integrating structured illumination into NIR-II light-sheet microscopy further diminished background and improved spatial resolution by approximately twofold. In vivo oblique NIR-II SIM was performed noninvasively for 3D volumetric multiplexed molecular imaging of the CT26 tumor microenvironment in mice, longitudinally mapping out CD4, CD8, and OX40 at the single-cell level in response to immunotherapy by cytosine-phosphate-guanine (CpG), a Toll-like receptor 9 (TLR-9) agonist combined with OX40 antibody treatment. NIR-II SIM affords an additional tool for noninvasive volumetric molecular imaging of immune cells in live mammals.

Deep-learning on-chip light-sheet microscopy enabling video-rate volumetric imaging of dynamic biological specimens

Lab on a Chip ◽  
2021 ◽  
Author(s):  
Xiaopeng Chen ◽  
Junyu Ping ◽  
Yixuan Sun ◽  
Chengqiang Yi ◽  
Sijian Liu ◽  
...  
Keyword(s):  
Deep Learning ◽  
Light Scattering ◽  
Light Sheet ◽  
High Contrast ◽  
Spatiotemporal Resolution ◽  
Volumetric Imaging ◽  
Light Sheet Microscopy ◽  
High Spatiotemporal Resolution ◽  
On Chip

Volumetric imaging of dynamic signals in a large, moving, and light-scattering specimen is extremely challenging, owing to the requirement on high spatiotemporal resolution and difficulty in obtaining high-contrast signals. Here...

scholarly journals High-resolution label-free 3D mapping of extracellular pH of single living cells

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Yanjun Zhang ◽  
Yasufumi Takahashi ◽  
Sung Pil Hong ◽  
Fengjie Liu ◽  
Joanna Bednarska ◽  
...  
Keyword(s):  
Single Cell ◽  
Cancer Cells ◽  
Extracellular Ph ◽  
Single Cell Level ◽  
Ph Sensing ◽  
Label Free ◽  
Cell Level ◽  
Spatiotemporal Resolution ◽  
Ion Conductance ◽  
Scanning Ion Conductance Microscopy

AbstractDynamic mapping of extracellular pH (pHe) at the single-cell level is critical for understanding the role of H+ in cellular and subcellular processes, with particular importance in cancer. While several pHe sensing techniques have been developed, accessing this information at the single-cell level requires improvement in sensitivity, spatial and temporal resolution. We report on a zwitterionic label-free pH nanoprobe that addresses these long-standing challenges. The probe has a sensitivity > 0.01 units, 2 ms response time, and 50 nm spatial resolution. The platform was integrated into a double-barrel nanoprobe combining pH sensing with feedback-controlled distance dependance via Scanning Ion Conductance Microscopy. This allows for the simultaneous 3D topographical imaging and pHe monitoring of living cancer cells. These classes of nanoprobes were used for real-time high spatiotemporal resolution pHe mapping at the subcellular level and revealed tumour heterogeneity of the peri-cellular environments of melanoma and breast cancer cells.

scholarly journals Quantification of Unintegrated HIV-1 DNA at the Single Cell Level In Vivo

PLoS ONE ◽  
2012 ◽  
Vol 7 (5) ◽  
pp. e36246 ◽  
Author(s):  
Rodolphe Suspène ◽  
Andreas Meyerhans
Keyword(s):  
Single Cell ◽  
Single Cell Level ◽  
Cell Level ◽  
Hiv 1

scholarly journals A New Gene Set Identifies Senescent Cells and Predicts Senescence-Associated Pathways Across Tissues

2021 ◽  
Author(s):  
Sundeep Khosla ◽  
Dominik Saul ◽  
Robyn Laura Kosinsky ◽  
Elizabeth Atkinson ◽  
Madison Doolittle ◽  
...  
Keyword(s):  
Single Cell ◽  
Mesenchymal Cells ◽  
Senescent Cell ◽  
Single Cell Level ◽  
Cell Level ◽  
Gene Set ◽  
Age Related ◽  
Cell Clearance ◽  
Bone Biopsies

Abstract Although cellular senescence is increasingly recognized as driving multiple age-related co-morbidities through the senescence-associated secretory phenotype (SASP), in vivo senescent cell identification, particularly in bulk or single cell RNA-sequencing (scRNA-seq) data remains challenging. Here, we generated a novel gene set (SenMayo) and first validated its enrichment in bone biopsies from two aged human cohorts. SenMayo also identified senescent cells in aged murine brain tissue, demonstrating applicability across tissues and species. For direct validation, we demonstrated significant reductions in SenMayo in bone following genetic clearance of senescent cells in mice, with similar findings in adipose tissue from humans in a pilot study of pharmacological senescent cell clearance. In direct comparisons, SenMayo outperformed all six existing senescence/SASP gene sets in identifying senescent cells across tissues and in demonstrating responses to senescent cell clearance. We next used SenMayo to identify senescent hematopoietic or mesenchymal cells at the single cell level from publicly available human and murine bone marrow/bone scRNA-seq data and identified monocytic and osteolineage cells, respectively, as showing the highest levels of senescence/SASP genes. Using pseudotime and cellular communication patterns, we found senescent hematopoietic and mesenchymal cells communicated with other cells through common pathways, including the Macrophage Migration Inhibitory Factor (MIF) pathway, which has been implicated not only in inflammation but also in immune evasion, an important property of senescent cells. Thus, SenMayo identifies senescent cells across tissues and species with high fidelity. Moreover, using this senescence panel, we were able to characterize senescent cells at the single cell level and identify key intercellular signaling pathways associated with these cells, which may be particularly useful for evolving efforts to map senescent cells (e.g., SenNet). In addition, SenMayo represents a potentially clinically applicable panel for monitoring senescent cell burden with aging and other conditions as well as in studies of senolytic drugs.

scholarly journals Observing the Cell in Its Native State: Imaging Subcellular Dynamics in Multicellular Organisms

2018 ◽  
Author(s):  
Tsung-Li Liu ◽  
Srigokul Upadhyayula ◽  
Daniel E. Milkie ◽  
Ved Singh ◽  
Kai Wang ◽  
...  
Keyword(s):  
Adaptive Optics ◽  
High Speed ◽  
Developmental Stages ◽  
Phenotypic Diversity ◽  
Metastatic Cancer ◽  
Light Sheet ◽  
Intrinsic Variability ◽  
Spatiotemporal Resolution ◽  
Light Sheet Microscopy

AbstractTrue physiological imaging of subcellular dynamics requires studying cells within their parent organisms, where all the environmental cues that drive gene expression, and hence the phenotypes we actually observe, are present. A complete understanding also requires volumetric imaging of the cell and its surroundings at high spatiotemporal resolution without inducing undue stress on either. We combined lattice light sheet microscopy with two-channel adaptive optics to achieve, across large multicellular volumes, noninvasive aberration-free imaging of subcellular processes, including endocytosis, organelle remodeling during mitosis, and the migration of axons, immune cells, and metastatic cancer cells in vivo. The technology reveals the phenotypic diversity within cells across different organisms and developmental stages, and may offer insights into how cells harness their intrinsic variability to adapt to different physiological environments.One Sentence SummaryCombining lattice light sheet microscopy with adaptive optics enables high speed, high resolution in vivo 3D imaging of dynamic processes inside cells under physiological conditions within their parent organisms.

scholarly journals The iSplit GFP assay detects intracellular recombinant proteins in Bacillus subtilis

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Patrick Lenz ◽  
Fabienne Hilgers ◽  
Alina Burmeister ◽  
Leonie Zimmermann ◽  
Kristina Volkenborn ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Recombinant Protein ◽  
Single Cell ◽  
Recombinant Protein Production ◽  
Protein Production ◽  
Online Monitoring ◽  
Protein Detection ◽  
Single Cell Level ◽  
Cell Level

Abstract Background Bacillus subtilis is one of the most important microorganisms for recombinant protein production. It possesses the GRAS (generally recognized as safe) status and a potent protein secretion capacity. Secretory protein production greatly facilitates downstream processing and thus significantly reduces costs. However, not all heterologous proteins are secreted and intracellular production poses difficulties for quantification. To tackle this problem, we have established a so-called intracellular split GFP (iSplit GFP) assay in B. subtilis as a tool for the in vivo protein detection during expression in batch cultures and at a single-cell level. For the iSplit GFP assay, the eleventh β-sheet of sfGFP is fused to a target protein and can complement a detector protein consisting of the respective truncated sfGFP (GFP1-10) to form fluorescent holo-GFP. Results As proof of concept, the GFP11-tag was fused C-terminally to the E. coli β-glucuronidase GUS, resulting in fusion protein GUS11. Variable GUS and GUS11 production levels in B. subtilis were achieved by varying the ribosome binding site via spacers of increasing lengths (4–12 nucleotides) for the GUS-encoding gene. Differences in intracellular enzyme accumulation were determined by measuring the GUS11 enzymatic activity and subsequently by adding the detector protein to respective cell extracts. Moreover, the detector protein was co-produced with the GUS11 using a two-plasmid system, which enabled the in vivo detection and online monitoring of glucuronidase production. Using this system in combination with flow cytometry and microfluidics, we were able to monitor protein production at a single-cell level thus yielding information about intracellular protein distribution and culture heterogeneity. Conclusion Our results demonstrate that the iSplit GFP assay is suitable for the detection, quantification and online monitoring of recombinant protein production in B. subtilis during cultivation as well as for analyzing production heterogeneity and intracellular localization at a single-cell level. Graphic abstract

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