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Article ; Online: Spinning Disk Confocal Microscopy for Optimized and Quantified Live Imaging of 3D Mitochondrial Network.

Ahmadian, Somaieh / Lindsey, Patrick J / Smeets, Hubert J M / van Tienen, Florence H J / van Zandvoort, Marc A M J

International journal of molecular sciences

2024 Volume 25, Issue 9

Abstract: Mitochondria are the energy factories of a cell, and depending on the metabolic requirements, the mitochondrial morphology, quantity, and membrane potential in a cell change. These changes are frequently assessed using commercially available probes. In ... ...

Abstract	Mitochondria are the energy factories of a cell, and depending on the metabolic requirements, the mitochondrial morphology, quantity, and membrane potential in a cell change. These changes are frequently assessed using commercially available probes. In this study, we tested the suitability of three commercially available probes-namely 5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolo-carbocyanine iodide (JC-1), MitoTracker Red CMX Rox (CMXRos), and tetramethylrhodamine methyl ester (TMRM)-for assessing the mitochondrial quantity, morphology, and membrane potential in living human mesoangioblasts in 3D with confocal laser scanning microscope (CLSM) and scanning disk confocal microscope (SDCM). Using CLSM, JC-1, and CMXRos-but not TMRM-uncovered considerable background and variation. Using SDCM, the background signal only remained apparent for the JC-1 monomer. Repetitive imaging of CMXRos and JC-1-but not TMRM-demonstrated a 1.5-2-fold variation in signal intensity between cells using CLSM. The use of SDCM drastically reduced this variation. The slope of the relative signal intensity upon repetitive imaging using CLSM was lowest for TMRM (-0.03) and highest for CMXRos (0.16). Upon repetitive imaging using SDCM, the slope varied from 0 (CMXRos) to a maximum of -0.27 (JC-1 C1). Conclusively, our data show that TMRM staining outperformed JC-1 and CMXRos dyes in a (repetitive) 3D analysis of the entire mitochondrial quantity, morphology, and membrane potential in living cells.
MeSH term(s)	Humans ; Mitochondria/metabolism ; Microscopy, Confocal/methods ; Imaging, Three-Dimensional/methods ; Fluorescent Dyes/chemistry ; Membrane Potential, Mitochondrial ; Carbocyanines/chemistry ; Rhodamines/chemistry
Chemical Substances	Fluorescent Dyes ; Carbocyanines ; Rhodamines
Language	English
Publishing date	2024-04-28
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2019364-6
ISSN	1422-0067 ; 1422-0067 ; 1661-6596
ISSN (online)	1422-0067
ISSN	1422-0067 ; 1661-6596
DOI	10.3390/ijms25094819
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Article: Molecular Imaging in Oncology: Advanced Microscopy Techniques.

Kapsokalyvas, Dimitrios / van Zandvoort, Marc A M J

Recent results in cancer research. Fortschritte der Krebsforschung. Progres dans les recherches sur le cancer

2020 Volume 216, Page(s) 533–561

Abstract: Preclinical studies usually require high levels of morphological, functional, and biochemical information at subcellular resolution. This type of information cannot be obtained from clinical imaging techniques, such as MRI, PET/CT, or US. Luckily, many ... ...

Abstract	Preclinical studies usually require high levels of morphological, functional, and biochemical information at subcellular resolution. This type of information cannot be obtained from clinical imaging techniques, such as MRI, PET/CT, or US. Luckily, many microscopy techniques exist that can offer this information, also for malignant tissues and therapeutic approaches. In this overview, we discuss the various advanced optical microscopy techniques and their applications in oncological research. After a short introduction in Sect. 16.1, we continue in Sect. 16.2 with a discussion on fluorescent labelling strategies, followed in Sect. 16.3 by an in-depth description of confocal, light-sheet, two-photon, and super-resolution microscopy. We end in Sect. 16.4 with a focus on the applications, specifically in oncology.
MeSH term(s)	Humans ; Medical Oncology ; Microscopy/methods ; Molecular Imaging ; Neoplasms/pathology
Language	English
Publishing date	2020-06-27
Publishing country	Germany
Document type	Journal Article ; Review
ISSN	0080-0015
ISSN	0080-0015
DOI	10.1007/978-3-030-42618-7_16
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Article ; Online: Multiphoton Imaging of Maturation in Tissue Engineering.

Werner, Maximilian P / Kučikas, Vytautas / Voß, Kirsten / Abel, Dirk / Jockenhoevel, Stefan / van Zandvoort, Marc A M J / Schmitz-Rode, Thomas

Tissue engineering. Part C, Methods

2023 Volume 30, Issue 1, Page(s) 38–48

Abstract: Donor cell-specific tissue-engineered (TE) implants are a promising therapy for personalized treatment of cardiovascular diseases, but current development protocols lack a stable longitudinal assessment of tissue development at subcellular resolution. As ...

Abstract	Donor cell-specific tissue-engineered (TE) implants are a promising therapy for personalized treatment of cardiovascular diseases, but current development protocols lack a stable longitudinal assessment of tissue development at subcellular resolution. As a first step toward such an assessment approach, in this study we establish a generalized labeling and imaging protocol to obtain quantified maturation parameters of TE constructs in three dimensions (3D) without the need of histological slicing, thus leaving the tissue intact. Focusing on intracellular matrix (ICM) and extracellular matrix (ECM) networks, multiphoton laser scanning microscopy (MPLSM) was used to investigate TE patches of different conditioning durations of up to 21 days. We show here that with a straightforward labeling procedure of whole-mount samples (so without slicing into thin histological sections), followed by an easy-to-use multiphoton imaging process, we obtained high-quality images of the tissue in 3D at various time points during development. The stacks of images could then be further analyzed to visualize and quantify the volume of cell coverage as well as the volume fraction and network of structural proteins. We showed that collagen and alpha-smooth muscle actin (α-SMA) volume fractions increased as normalized to full tissue volume and proportional to the cell count, with a converging trend to the final density of (4.0% ± 0.6%) and (7.6% ± 0.7%), respectively. The image analysis of ICM and ECM revealed a developing and widely branched interconnected matrix. We are currently working on the second step, that is, to integrate MPLSM endoscopy into a dynamic bioreactor system to monitor the maturation of intact TE constructs over time, thus without the need to take them out.
MeSH term(s)	Tissue Engineering/methods ; Extracellular Matrix/chemistry ; Collagen/metabolism ; Microscopy, Fluorescence, Multiphoton/methods
Chemical Substances	Collagen (9007-34-5)
Language	English
Publishing date	2023-12-20
Publishing country	United States
Document type	Journal Article
ZDB-ID	2420585-0
ISSN	1937-3392 ; 1937-3384
ISSN (online)	1937-3392
ISSN	1937-3384
DOI	10.1089/ten.TEC.2023.0141
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Article ; Online: A time window for rescuing dying retinal ganglion cells.

You, Wenting / Knoops, Kèvin / Boesten, Iris / Berendschot, Tos T J M / van Zandvoort, Marc A M J / Benedikter, Birke J / Webers, Carroll A B / Reutelingsperger, Chris P M / Gorgels, Theo G M F

Cell communication and signaling : CCS

2024 Volume 22, Issue 1, Page(s) 88

Abstract: Background: Retinal ganglion cell (RGC) degeneration and death cause vision loss in patients with glaucoma. Regulated cell death, once initiated, is generally considered to be an irreversible process. Recently, we showed that, by timely removing the ... ...

Abstract	Background: Retinal ganglion cell (RGC) degeneration and death cause vision loss in patients with glaucoma. Regulated cell death, once initiated, is generally considered to be an irreversible process. Recently, we showed that, by timely removing the cell death stimulus, stressed neuronal PC12 cells can recover from phosphatidylserine (PS) exposure, nuclear shrinkage, DNA damage, mitochondrial fragmentation, mitochondrial membrane potential loss, and retraction of neurites, all hallmarks of an activated cell death program. Whether the cell death process can be reversed in neurons of the central nervous system, like RGCs, is still unknown. Here, we studied reversibility of the activated cell death program in primary rat RGCs (prRGCs). Methods: prRGCs were exposed to ethanol (5%, vol/vol) to induce cell death. At different stages of the cell death process, ethanol was removed by washing and injured prRGCs were further cultured in fresh medium to see whether they recovered. The dynamics of single cells were monitored by high-resolution live-cell spinning disk microscopy. PS exposure, mitochondrial structure, membrane potential, and intracellular Ca Results: Analysis of temporal relationships between mitochondrial changes and PS exposure showed that fragmentation of the mitochondrial network and loss of mitochondrial membrane potential occurred before PS exposure. Mitochondrial changes proceeded caspase-independently, while PS exposure was caspase dependent. Interestingly, prRGCs recovered quickly from these mitochondrial changes but not from PS exposure at the plasma membrane. Correlative light and electron microscopy showed that stress-induced decrease in mitochondrial area, length and cristae number was reversible. Intracellular Ca Conclusions: Our study demonstrates that RGCs with impaired mitochondrial structure and function can fully recover if there is no mitochondrial cytochrome c release yet, and no PS is exposed at the plasma membrane. This finding indicates that there is a time window for rescuing dying or injured RGCs, by simply removing the cell death stimulus. Video Abstract.
MeSH term(s)	Animals ; Rats ; Apoptosis ; Caspases/metabolism ; Cytochromes c/metabolism ; Ethanol ; Retinal Ganglion Cells/metabolism
Chemical Substances	Caspases (EC 3.4.22.-) ; Cytochromes c (9007-43-6) ; Ethanol (3K9958V90M)
Language	English
Publishing date	2024-01-31
Publishing country	England
Document type	Video-Audio Media ; Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2126315-2
ISSN	1478-811X ; 1478-811X
ISSN (online)	1478-811X
ISSN	1478-811X
DOI	10.1186/s12964-023-01427-3
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Article ; Online: Two-Photon Endoscopy: State of the Art and Perspectives.

Kučikas, Vytautas / Werner, Maximilian P / Schmitz-Rode, Thomas / Louradour, Frédéric / van Zandvoort, Marc A M J

Molecular imaging and biology

2021 Volume 25, Issue 1, Page(s) 3–17

Abstract: In recent years, the demand for non-destructive deep-tissue imaging modalities has led to interest in multiphoton endoscopy. In contrast to bench top systems, multiphoton endoscopy enables subcellular resolution imaging in areas not reachable before. ... ...

Abstract	In recent years, the demand for non-destructive deep-tissue imaging modalities has led to interest in multiphoton endoscopy. In contrast to bench top systems, multiphoton endoscopy enables subcellular resolution imaging in areas not reachable before. Several groups have recently presented their development towards the goal of producing user friendly plug and play system, which could be used in biological research and, potentially, clinical applications. We first present the technological challenges, prerequisites, and solutions in two-photon endoscopic systems. Secondly, we focus on the applications already found in literature. These applications mostly serve as a quality check of the built system, but do not answer a specific biomedical research question. Therefore, in the last part, we will describe our vision on the enormous potential applicability of adult two-photon endoscopic systems in biological and clinical research. We will thus bring forward the concept that two-photon endoscopy is a sine qua non in bringing this technique to the forefront in clinical applications.
MeSH term(s)	Endoscopy/methods ; Diagnostic Imaging/methods ; Photons ; Biomedical Research
Language	English
Publishing date	2021-11-15
Publishing country	United States
Document type	Journal Article ; Review ; Research Support, Non-U.S. Gov't
ZDB-ID	2079160-4
ISSN	1860-2002 ; 1536-1632
ISSN (online)	1860-2002
ISSN	1536-1632
DOI	10.1007/s11307-021-01665-2
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Article ; Online: Structural and Mechanical Aberrations of the Nuclear Lamina in Disease.

Stiekema, Merel / van Zandvoort, Marc A M J / Ramaekers, Frans C S / Broers, Jos L V

Cells

2020 Volume 9, Issue 8

Abstract: The nuclear lamins are the major components of the nuclear lamina in the nuclear envelope. Lamins are involved in numerous functions, including a role in providing structural support to the cell and the mechanosensing of the cell. Mutations in the genes ... ...

Abstract	The nuclear lamins are the major components of the nuclear lamina in the nuclear envelope. Lamins are involved in numerous functions, including a role in providing structural support to the cell and the mechanosensing of the cell. Mutations in the genes encoding for lamins lead to the rare diseases termed laminopathies. However, not only laminopathies show alterations in the nuclear lamina. Deregulation of lamin expression is reported in multiple cancers and several viral infections lead to a disrupted nuclear lamina. The structural and mechanical effects of alterations in the nuclear lamina can partly explain the phenotypes seen in disease, such as muscular weakness in certain laminopathies and transmigration of cancer cells. However, a lot of answers to questions about the relation between changes in the nuclear lamina and disease development remain elusive. Here, we review the current understandings of the contribution of the nuclear lamina in the structural support and mechanosensing of healthy and diseased cells.
MeSH term(s)	Humans ; Lamins/metabolism ; Mechanoreceptors/metabolism ; Mutation/genetics ; Nuclear Lamina/metabolism
Chemical Substances	Lamins
Keywords	covid19
Language	English
Publishing date	2020-08-11
Publishing country	Switzerland
Document type	Journal Article ; Review
ZDB-ID	2661518-6
ISSN	2073-4409 ; 2073-4409
ISSN (online)	2073-4409
ISSN	2073-4409
DOI	10.3390/cells9081884
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Article ; Online: Stressed neuronal cells can recover from profound membrane blebbing, nuclear condensation and mitochondrial fragmentation, but not from cytochrome c release.

You, Wenting / Zhou, Tao / Knoops, Kèvin / Berendschot, Tos T J M / van Zandvoort, Marc A M J / Germeraad, Wilfred T V / Benedikter, Birke / Webers, Carroll A B / Reutelingsperger, Chris P M / Gorgels, Theo G M F

Scientific reports

2023 Volume 13, Issue 1, Page(s) 11045

Abstract: Loss of neurons in chronic neurodegenerative diseases may occur over a period of many years. Once initiated, neuronal cell death is accompanied by distinct phenotypic changes including cell shrinkage, neurite retraction, mitochondrial fragmentation, ... ...

Abstract	Loss of neurons in chronic neurodegenerative diseases may occur over a period of many years. Once initiated, neuronal cell death is accompanied by distinct phenotypic changes including cell shrinkage, neurite retraction, mitochondrial fragmentation, nuclear condensation, membrane blebbing and phosphatidylserine (PS) exposure at the plasma membrane. It is still poorly understood which events mark the point of no return for dying neurons. Here we analyzed the neuronal cell line SH-SY5Y expressing cytochrome C (Cyto.C)-GFP. Cells were exposed temporarily to ethanol (EtOH) and tracked longitudinally in time by light and fluorescent microscopy. Exposure to EtOH induced elevation of intracellular Ca
MeSH term(s)	Humans ; Cytochromes c/metabolism ; Apoptosis/physiology ; Neuroblastoma/metabolism ; Neurons/metabolism ; Neurodegenerative Diseases/metabolism
Chemical Substances	Cytochromes c (9007-43-6)
Language	English
Publishing date	2023-07-08
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2615211-3
ISSN	2045-2322 ; 2045-2322
ISSN (online)	2045-2322
ISSN	2045-2322
DOI	10.1038/s41598-023-38210-w
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Article ; Online: Multiview deconvolution approximation multiphoton microscopy of tissues and zebrafish larvae.

Kapsokalyvas, Dimitrios / Rosas, Rodrigo / Janssen, Rob W A / Vanoevelen, Jo M / Nabben, Miranda / Strauch, Martin / Merhof, Dorit / van Zandvoort, Marc A M J

Scientific reports

2021 Volume 11, Issue 1, Page(s) 10160

Abstract: Imaging in three dimensions is necessary for thick tissues and small organisms. This is possible with tomographic optical microscopy techniques such as confocal, multiphoton and light sheet microscopy. All these techniques suffer from anisotropic ... ...

Abstract	Imaging in three dimensions is necessary for thick tissues and small organisms. This is possible with tomographic optical microscopy techniques such as confocal, multiphoton and light sheet microscopy. All these techniques suffer from anisotropic resolution and limited penetration depth. In the past, Multiview microscopy-imaging the sample from different angles followed by 3D image reconstruction-was developed to address this issue for light sheet microscopy based on fluorescence signal. In this study we applied this methodology to accomplish Multiview imaging with multiphoton microscopy based on fluorescence and additionally second harmonic signal from myosin and collagen. It was shown that isotropic resolution was achieved, the entirety of the sample was visualized, and interference artifacts were suppressed allowing clear visualization of collagen fibrils and myofibrils. This method can be applied to any scanning microscopy technique without microscope modifications. It can be used for imaging tissue and whole mount small organisms such as heart tissue, and zebrafish larva in 3D, label-free or stained, with at least threefold axial resolution improvement which can be significant for the accurate quantification of small 3D structures.
MeSH term(s)	Animals ; Image Processing, Computer-Assisted ; Imaging, Three-Dimensional ; Larva ; Microscopy, Confocal/methods ; Microscopy, Fluorescence, Multiphoton/instrumentation ; Microscopy, Fluorescence, Multiphoton/methods ; Zebrafish
Language	English
Publishing date	2021-05-12
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2615211-3
ISSN	2045-2322 ; 2045-2322
ISSN (online)	2045-2322
ISSN	2045-2322
DOI	10.1038/s41598-021-89566-w
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Article ; Online: Super-Resolution Imaging of the A- and B-Type Lamin Networks: A Comparative Study of Different Fluorescence Labeling Procedures.

Stiekema, Merel / Ramaekers, Frans C S / Kapsokalyvas, Dimitrios / van Zandvoort, Marc A M J / Veltrop, Rogier J A / Broers, Jos L V

International journal of molecular sciences

2021 Volume 22, Issue 19

Abstract: A- and B-type lamins are type V intermediate filament proteins. Mutations in the genes encoding these lamins cause rare diseases, collectively called laminopathies. A fraction of the cells obtained from laminopathy patients show aberrations in the ... ...

Abstract	A- and B-type lamins are type V intermediate filament proteins. Mutations in the genes encoding these lamins cause rare diseases, collectively called laminopathies. A fraction of the cells obtained from laminopathy patients show aberrations in the localization of each lamin subtype, which may represent only the minority of the lamina disorganization. To get a better insight into more delicate and more abundant lamina abnormalities, the lamin network can be studied using super-resolution microscopy. We compared confocal scanning laser microscopy and stimulated emission depletion (STED) microscopy in combination with different fluorescence labeling approaches for the study of the lamin network. We demonstrate the suitability of an immunofluorescence staining approach when using STED microscopy, by determining the lamin layer thickness and the degree of lamin A and B1 colocalization as detected in fixed fibroblasts (co-)stained with lamin antibodies or (co-)transfected with EGFP/YFP lamin constructs. This revealed that immunofluorescence staining of cells does not lead to consequent changes in the detected lamin layer thickness, nor does it influence the degree of colocalization of lamin A and B1, when compared to the transfection approach. Studying laminopathy patient dermal fibroblasts (
MeSH term(s)	3T3 Cells ; Animals ; Cell Line ; Fibroblasts/metabolism ; Fluorescent Antibody Technique ; Intermediate Filament Proteins/genetics ; Intermediate Filament Proteins/metabolism ; Lamin Type A/genetics ; Lamin Type A/metabolism ; Lamin Type B/genetics ; Lamin Type B/metabolism ; Laminopathies/genetics ; Laminopathies/pathology ; Mice ; Microscopy, Confocal ; Nuclear Envelope/metabolism
Chemical Substances	Intermediate Filament Proteins ; Lamin Type A ; Lamin Type B
Language	English
Publishing date	2021-09-22
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2019364-6
ISSN	1422-0067 ; 1422-0067 ; 1661-6596
ISSN (online)	1422-0067
ISSN	1422-0067 ; 1661-6596
DOI	10.3390/ijms221910194
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Article: The Role of Lamins in the Nucleoplasmic Reticulum, a Pleiomorphic Organelle That Enhances Nucleo-Cytoplasmic Interplay.

Stiekema, Merel / Houben, Frederik / Verheyen, Fons / Borgers, Marcel / Menzel, Julia / Meschkat, Martin / van Zandvoort, Marc A M J / Ramaekers, Frans C S / Broers, Jos L V

Frontiers in cell and developmental biology

2022 Volume 10, Page(s) 914286

Abstract: Invaginations of the nuclear membrane occur in different shapes, sizes, and compositions. Part of these pleiomorphic invaginations make up the nucleoplasmic reticulum (NR), while others are merely nuclear folds. We define the NR as tubular invaginations ... ...

Abstract	Invaginations of the nuclear membrane occur in different shapes, sizes, and compositions. Part of these pleiomorphic invaginations make up the nucleoplasmic reticulum (NR), while others are merely nuclear folds. We define the NR as tubular invaginations consisting of either both the inner and outer nuclear membrane, or only the inner nuclear membrane. Specifically, invaginations of both the inner and outer nuclear membrane are also called type II NR, while those of only the inner nuclear membrane are defined as type I NR. The formation and structure of the NR is determined by proteins associated to the nuclear membrane, which induce a high membrane curvature leading to tubular invaginations. Here we review and discuss the current knowledge of nuclear invaginations and the NR in particular. An increase in tubular invaginations of the nuclear envelope is associated with several pathologies, such as laminopathies, cancer, (reversible) heart failure, and Alzheimer's disease. Furthermore, viruses can induce both type I and II NR. In laminopathies, the amount of A-type lamins throughout the nucleus is generally decreased or the organization of lamins or lamin-associated proteins is disturbed. Also, lamin overexpression or modulation of lamin farnesylation status impacts NR formation, confirming the importance of lamin processing in NR formation. Virus infections reorganize the nuclear lamina
Language	English
Publishing date	2022-06-16
Publishing country	Switzerland
Document type	Journal Article ; Review
ZDB-ID	2737824-X
ISSN	2296-634X
ISSN	2296-634X
DOI	10.3389/fcell.2022.914286
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