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Cells
Special Issues
Recent Advances in Intravital and Live Cell Imaging
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Recent Advances in Intravital and Live Cell Imaging

A special issue of Cells (ISSN 2073-4409). This special issue belongs to the section "Cell Methods".

Deadline for manuscript submissions: closed (5 February 2024) | Viewed by 17735

Special Issue Editors

Dr. Gregor Drummen

E-Mail Website
Guest Editor
Cellular Stress and Ageing Program, Bionanoscience and Bioimaging Program, BNS, 33647 Bielefeld, Germany
Interests: quantum dots; bionanotechnology; two-photon fluorescence imaging; cellular imaging; fluorescence microscopy; cancer; cell signaling; oxidative stress; lipids and biomembranes; lipid peroxidation; antioxidants; renal pathobiology; extracellular vesicles; Super-resolution microscopy
Special Issues, Collections and Topics in MDPI journals
Dr. Hellen Ishikawa-Ankerhold

E-Mail Website
Guest Editor
Walter Brendel Centre of Experimental Medicine, Department of Cardiology, Ludwig Maximilian University of Munich, Marchioninistraße 27, D-81377 München, Germany
Interests: multiphoton microscopy; intravital microscopy; confocal and super-resolution microscopy; fluorescent probes; leukocyte trafficking; atherosclerosis; inflammation; hematopoiesis and cytoskeleton proteins
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

Ever since the invention of lenses, man has not only looked into the skies, but also into the realm of the “invisible” microscopic world of tissues, cells, and micro-organisms. The use of fluorescent labels and the development of various forms of optical microscopy, particularly confocal laser scanning microscopy (CLSM), super-resolution microscopy (SRM), and intravital microscopy (IVM), have significantly advanced our knowledge about the basic mechanisms underpinning biology and the pathophysiological processes that lead to disease.

This Special Issue focuses exclusively on intravital, super-resolution, and life cell microscopy techniques which are developed to study cellular and subcellular events in health and diseases.

Previously unpublished experimental, theoretical, prospective, and review papers (with the exception of preprint servers) are solicited on the following and related topics:

  • Intravital imaging of animal model tissues to study cellular dynamics in acute or chronic set up (longitudinal imaging);
  • Generation of novel animal models to study cellular and subcellular events;
  • Recent advances in intravital imaging technology to study cellular dynamics behavior in vivo for instance through an imaging window or optical fibers;
  • Advances in super-resolution microscopy technologies;
  • New developments in life cell imaging in general;
  • Advances in fluorescence microscopy techniques, such as FRET, FLIM, FRAP, FLIP et al., with respect to life cell imaging;
  • Correlative microscopy;
  • Fundamental physicochemical properties, synthesis, and modification of novel fluorophores for imaging.

Dr. Gregor Drummen
Dr. Hellen Ishikawa-Ankerhold
Guest Editors

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Cells is an international peer-reviewed open access semimonthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 2700 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

  • fluorescence probe
  • fluorophore
  • fluorescence microscopy
  • super-resolution microscopy
  • nanoscopy
  • confocal laser scanning microscopy
  • two-photon microscopy
  • FRET
  • FLIM
  • FRAP
  • live cell imaging
  • intravital microscopy
  • longitudinal imaging techniques, imaging window
  • animal models for intravital imaging
  • multimodal imaging

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Further information on MDPI's Special Issue policies can be found here.

Published Papers (6 papers)

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Research

13 pages, 10354 KiB  
Article
Click Chemistry with Cell-Permeable Fluorophores Expands the Choice of Bioorthogonal Markers for Two-Color Live-Cell STED Nanoscopy
by Carola Gregor, Florian Grimm, Jasmin Rehman, Christian A. Wurm and Alexander Egner
Cells 2024, 13(8), 683; https://doi.org/10.3390/cells13080683 - 15 Apr 2024
Cited by 2 | Viewed by 2206
Abstract
STED nanoscopy allows for the direct observation of dynamic processes in living cells and tissues with diffraction-unlimited resolution. Although fluorescent proteins can be used for STED imaging, these labels are often outperformed in photostability by organic fluorescent dyes. This feature is especially crucial [...] Read more.
STED nanoscopy allows for the direct observation of dynamic processes in living cells and tissues with diffraction-unlimited resolution. Although fluorescent proteins can be used for STED imaging, these labels are often outperformed in photostability by organic fluorescent dyes. This feature is especially crucial for time-lapse imaging. Unlike fluorescent proteins, organic fluorophores cannot be genetically fused to a target protein but require different labeling strategies. To achieve simultaneous imaging of more than one protein in the interior of the cell with organic fluorophores, bioorthogonal labeling techniques and cell-permeable dyes are needed. In addition, the fluorophores should preferentially emit in the red spectral range to reduce the potential phototoxic effects that can be induced by the STED light, which further restricts the choice of suitable markers. In this work, we selected five different cell-permeable organic dyes that fulfill all of the above requirements and applied them for SPIEDAC click labeling inside living cells. By combining click-chemistry-based protein labeling with other orthogonal and highly specific labeling methods, we demonstrate two-color STED imaging of different target structures in living specimens using different dye pairs. The excellent photostability of the dyes enables STED imaging for up to 60 frames, allowing the observation of dynamic processes in living cells over extended time periods at super-resolution. Full article
(This article belongs to the Special Issue Recent Advances in Intravital and Live Cell Imaging)
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27 pages, 18416 KiB  
Article
High-Resolution Microscopic Characterization of Tunneling Nanotubes in Living U87 MG and LN229 Glioblastoma Cells
by Nicole Matejka, Asieh Amarlou, Jessica Neubauer, Sarah Rudigkeit and Judith Reindl
Cells 2024, 13(5), 464; https://doi.org/10.3390/cells13050464 - 6 Mar 2024
Cited by 3 | Viewed by 2538
Abstract
Tunneling nanotubes (TNTs) are fine, nanometer-sized membrane connections between distant cells that provide an efficient communication tool for cellular organization. TNTs are thought to play a critical role in cellular behavior, particularly in cancer cells. The treatment of aggressive cancers such as glioblastoma [...] Read more.
Tunneling nanotubes (TNTs) are fine, nanometer-sized membrane connections between distant cells that provide an efficient communication tool for cellular organization. TNTs are thought to play a critical role in cellular behavior, particularly in cancer cells. The treatment of aggressive cancers such as glioblastoma remains challenging due to their high potential for developing therapy resistance, high infiltration rates, uncontrolled cell growth, and other aggressive features. A better understanding of the cellular organization via cellular communication through TNTs could help to find new therapeutic approaches. In this study, we investigate the properties of TNTs in two glioblastoma cell lines, U87 MG and LN229, including measurements of their diameter by high-resolution live-cell stimulated emission depletion (STED) microscopy and an analysis of their length, morphology, lifetime, and formation by live-cell confocal microscopy. In addition, we discuss how these fine compounds can ideally be studied microscopically. In particular, we show which membrane-labeling method is suitable for studying TNTs in glioblastoma cells and demonstrate that live-cell studies should be preferred to explore the role of TNTs in cellular behavior. Our observations on TNT formation in glioblastoma cells suggest that TNTs could be involved in cell migration and serve as guidance. Full article
(This article belongs to the Special Issue Recent Advances in Intravital and Live Cell Imaging)
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19 pages, 6394 KiB  
Article
Real-Time Multiphoton Intravital Microscopy of Drug Extravasation in Tumours during Acoustic Cluster Therapy
by Jessica Lage Fernandez, Sofie Snipstad, Astrid Bjørkøy and Catharina de Lange Davies
Cells 2024, 13(4), 349; https://doi.org/10.3390/cells13040349 - 16 Feb 2024
Cited by 1 | Viewed by 2199
Abstract
Optimising drug delivery to tumours remains an obstacle to effective cancer treatment. A prerequisite for successful chemotherapy is that the drugs reach all tumour cells. The vascular network of tumours, extravasation across the capillary wall and penetration throughout the extracellular matrix limit the [...] Read more.
Optimising drug delivery to tumours remains an obstacle to effective cancer treatment. A prerequisite for successful chemotherapy is that the drugs reach all tumour cells. The vascular network of tumours, extravasation across the capillary wall and penetration throughout the extracellular matrix limit the delivery of drugs. Ultrasound combined with microbubbles has been shown to improve the therapeutic response in preclinical and clinical studies. Most studies apply microbubbles designed as ultrasound contrast agents. Acoustic Cluster Therapy (ACT®) is a novel approach based on ultrasound-activated microbubbles, which have a diameter 5–10 times larger than regular contrast agent microbubbles. An advantage of using such large microbubbles is that they are in contact with a larger part of the capillary wall, and the oscillating microbubbles exert more effective biomechanical effects on the vessel wall. In accordance with this, ACT® has shown promising therapeutic results in combination with various drugs and drug-loaded nanoparticles. Knowledge of the mechanism and behaviour of drugs and microbubbles is needed to optimise ACT®. Real-time intravital microscopy (IVM) is a useful tool for such studies. This paper presents the experimental setup design for visualising ACT® microbubbles within the vasculature of tumours implanted in dorsal window (DW) chambers. It presents ultrasound setups, the integration and alignment of the ultrasound field with the optical system in live animal experiments, and the methodologies for visualisation and analysing the recordings. Dextran was used as a fluorescent marker to visualise the blood vessels and to trace drug extravasation and penetration into the extracellular matrix. The results reveal that the experimental setup successfully recorded the kinetics of extravasation and penetration distances into the extracellular matrix, offering a deeper understanding of ACT’s mechanisms and potential in localised drug delivery. Full article
(This article belongs to the Special Issue Recent Advances in Intravital and Live Cell Imaging)
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16 pages, 5819 KiB  
Article
Multiphoton In Vivo Microscopy of Embryonic Thrombopoiesis Reveals the Generation of Platelets through Budding
by Huan Liu, Hellen Ishikawa-Ankerhold, Julia Winterhalter, Michael Lorenz, Mykhailo Vladymyrov, Steffen Massberg, Christian Schulz and Mathias Orban
Cells 2023, 12(19), 2411; https://doi.org/10.3390/cells12192411 - 6 Oct 2023
Cited by 3 | Viewed by 1977
Abstract
Platelets are generated by specialized cells called megakaryocytes (MKs). However, MK’s origin and platelet release mode have remained incompletely understood. Here, we established direct visualization of embryonic thrombopoiesis in vivo by combining multiphoton intravital microscopy (MP-IVM) with a fluorescence switch reporter mouse model [...] Read more.
Platelets are generated by specialized cells called megakaryocytes (MKs). However, MK’s origin and platelet release mode have remained incompletely understood. Here, we established direct visualization of embryonic thrombopoiesis in vivo by combining multiphoton intravital microscopy (MP-IVM) with a fluorescence switch reporter mouse model under control of the platelet factor 4 promoter (Pf4CreRosa26mTmG). Using this microscopy tool, we discovered that fetal liver MKs provide higher thrombopoietic activity than yolk sac MKs. Mechanistically, fetal platelets were released from MKs either by membrane buds or the formation of proplatelets, with the former constituting the key process. In E14.5 c-Myb-deficient embryos that lack definitive hematopoiesis, MK and platelet numbers were similar to wild-type embryos, indicating the independence of embryonic thrombopoiesis from definitive hematopoiesis at this stage of development. In summary, our novel MP-IVM protocol allows the characterization of thrombopoiesis with high spatio-temporal resolution in the mouse embryo and has identified membrane budding as the main mechanism of fetal platelet production. Full article
(This article belongs to the Special Issue Recent Advances in Intravital and Live Cell Imaging)
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11 pages, 4723 KiB  
Article
Cell Surface Charge Mapping Using a Microelectrode Array on ITO Substrate
by Leixin Ouyang, Rubia Shaik, Ruiting Xu, Ge Zhang and Jiang Zhe
Cells 2023, 12(4), 518; https://doi.org/10.3390/cells12040518 - 4 Feb 2023
Cited by 2 | Viewed by 2682
Abstract
Many cellular functions are regulated by cell surface charges, such as intercellular signaling and metabolism. Noninvasive measurement of surface charge distribution of a single cell plays a vital role in understanding cellular functions via cell membranes. We report a method for cell surface [...] Read more.
Many cellular functions are regulated by cell surface charges, such as intercellular signaling and metabolism. Noninvasive measurement of surface charge distribution of a single cell plays a vital role in understanding cellular functions via cell membranes. We report a method for cell surface charge mapping via photoelectric interactions. A cell is placed on an array of microelectrodes fabricated on a transparent ITO (indium tin oxide) surface. An incident light irradiates the ITO surface from the backside. Because of the influence of the cell surface charge (or zeta potential), the photocurrent and the absorption of the incident light are changed, inducing a magnitude change of the reflected light. Hence, the cell surface charge distribution can be quantified by analyzing the reflected light intensity. This method does not need physical or chemical modification of the cell surface. We validated this method using charged microparticles (MPs) and two types of cells, i.e., human dermal fibroblast cells (HDFs) and human mesenchymal stem cells (hMSC). The measured average zeta potentials were in good agreement with the standard electrophoresis light scattering method. Full article
(This article belongs to the Special Issue Recent Advances in Intravital and Live Cell Imaging)
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18 pages, 3041 KiB  
Article
IL18 Receptor Signaling Inhibits Intratumoral CD8+ T-Cell Migration in a Murine Pancreatic Cancer Model
by Elena Nasiri, Malte Student, Katrin Roth, Nadya Siti Utami, Magdalena Huber, Malte Buchholz, Thomas M. Gress and Christian Bauer
Cells 2023, 12(3), 456; https://doi.org/10.3390/cells12030456 - 31 Jan 2023
Cited by 7 | Viewed by 4216
Abstract
In pancreatic ductal adenocarcinoma (PDAC), the infiltration of CD8+ cytotoxic T cells (CTLs) is an important factor in determining prognosis. The migration pattern and interaction behavior of intratumoral CTLs are pivotal to tumor rejection. NLRP3-dependent proinflammatory cytokines IL-1β and IL-18 play a [...] Read more.
In pancreatic ductal adenocarcinoma (PDAC), the infiltration of CD8+ cytotoxic T cells (CTLs) is an important factor in determining prognosis. The migration pattern and interaction behavior of intratumoral CTLs are pivotal to tumor rejection. NLRP3-dependent proinflammatory cytokines IL-1β and IL-18 play a prominent role for CTL induction and differentiation. Here, we investigate the effects of T-cellular IL-1R and IL-18R signaling for intratumoral T-cell motility. Murine adenocarcinoma cell line Panc02 was stably transfected with ovalbumin (OVA) and fluorophore H2B-Cerulean to generate PancOVA H2B-Cerulean tumor cells. Dorsal skinfold chambers (DSFC) were installed on wild-type mice, and PancOVA H2B-Cerulean tumor cells were implanted into the chambers. PancOVA spheroids were formed using the Corning® Matrigel®-based 3D cell culture technique. CTLs were generated from OT-1 mice, Il1r−/− OT-1 mice, or Il18r−/− OT-1 mice and were marked with fluorophores. This was followed by the adoptive transfer of CTLs into tumor-bearing mice or the application into tumor spheroids. After visualization with multiphoton microscopy (MPM), Imaris software was used to perform T-cell tracking. Imaris analysis indicates a significantly higher accumulation of Il18r−/− CTLs in PancOVA tumors and a significant reduction in tumor volume compared to wild-type CTLs. Il18r−/− CTLs covered a longer distance (track displacement length) in comparison to wild-type (WT) CTLs, and had a higher average speed (mean track speed). The analysis of instantaneous velocity suggests a higher percentage of arrested tracks (arrests: <4 μm/min) for Il18r−/− CTLs. Our data indicate the contribution of IL-18R signaling to T-cell effector strength, warranting further investigation on phenomena such as intratumoral T-cell exhaustion. Full article
(This article belongs to the Special Issue Recent Advances in Intravital and Live Cell Imaging)
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