Miriam S. Lucas

Last Name

Lucas

First Name

Miriam S.

ORCID

0000-0001-9998-6372

Organisational unit

02891 - ScopeM / ScopeM

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Publications 1 - 10 of 25

Clot-entrapped blood cells in synergy with human mesenchymal stem cells create a pro-angiogenic healing response
Burkhardt, Melanie A.; Gerber, Isabel; Moshfegh, Cameron; et al. (2017)
Biomaterials Science
Correlative 3D Imaging: CLSM and FIB-SEM Tomography Using High-Pressure Frozen, Freeze-Substituted Biological Samples
Lucas, Miriam S.; Guenthert, Maja; Gasser, Philippe; et al. (2014)
Methods in Molecular Biology ~ Electron Microscopy: Methods and Protocols
One for All, All for One: A Close Look at In-Resin Fluorescence Protocols for CLEM
Heiligenstein, Xavier; Lucas, Miriam S. (2022)
Frontiers in Cell and Developmental Biology
Sample preparation is the novel bottleneck for high throughput correlative light and electron microscopy (CLEM). Protocols suitable for both imaging methods must therefore balance the requirements of each technique. For fluorescence light microscopy, a structure of interest can be targeted using: 1) staining, which is often structure or tissue specific rather than protein specific, 2) dye-coupled proteins or antibodies, or 3) genetically encoded fluorescent proteins. Each of these three methods has its own advantages. For ultrastructural investigation by electron microscopy (EM) resin embedding remains a significant sample preparation approach, as it stabilizes the sample such that it withstands the vacuum conditions of the EM, and enables long-term storage. Traditionally, samples are treated with heavy metal salts prior to resin embedding, in order to increase imaging contrast for EM. This is particularly important for volume EM (vEM) techniques. Yet, commonly used contrasting agents (e.g., osmium tetroxide, uranyl acetate) tend to impair fluorescence. The discovery that fluorescence can be preserved in resin-embedded specimens after mild heavy metal staining was a game changer for CLEM. These so-called in-resin fluorescence protocols present a significant leap forward for CLEM approaches towards high precision localization of a fluorescent signal in (volume) EM data. Integrated microscopy approaches, combining LM and EM detection into a single instrument certainly require such an "all in one" sample preparation. Preserving, or adding, dedicated fluorescence prior to resin embedding requires a compromise, which often comes at the expense of EM imaging contrast and membrane visibility. Especially vEM can be strongly hampered by a lack of heavy metal contrasting. This review critically reflects upon the fundamental aspects of resin embedding with regard to 1) specimen fixation and the physics and chemistry underlying the preservation of protein structure with respect to fluorescence and antigenicity, 2) optimization of EM contrast for transmission or scanning EM, and 3) the choice of embedding resin. On this basis, various existing workflows employing in-resin fluorescence are described, highlighting their common features, discussing advantages and disadvantages of the respective approach, and finally concluding with promising future developments for in-resin CLEM.
Regulation of mammalian pexophagy by oxygen-regulated hypoxia-inducible factors alpha
Schönenberger, M.; Walter, K.M.; Horn, M.; et al. (2014)
Molecular Biology of the Cell
Noninvasive measurement of cell volume changes by negative staining
Lucas, Miriam S.; Biel, Stefan S.; Terstegen, Lara; et al. (2005)
Journal of Biomedical Optics
Correlative 3D microscopy: CLSM and FIB/SEM tomography
Lucas, Miriam S.; Gasser, Philippe; Günthert, Maja; et al. (2008)
Imaging & Microscopy
A Compartmentalized Neuronal Cell-Culture Platform Compatible With Cryo-Fixation by High-Pressure Freezing for Ultrastructural Imaging
Tran, Hung Tri; Lucas, Miriam S.; Ishikawa, Takashi; et al. (2021)
Frontiers in Neuroscience
The human brain contains a wide array of billions of neurons and interconnections, which are often simplified for analysis in vitro using compartmentalized microfluidic devices for neuronal cell culturing, to better understand neuronal development and disease. However, such devices are traditionally incompatible for high-pressure freezing and high-resolution nanoscale imaging and analysis of their sub-cellular processes by methods including electron microscopy. Here we develop a novel compartmentalized neuronal co-culture platform allowing reconstruction of neuronal networks with high variable spatial control, which is uniquely compatible for high-pressure freezing. This cryo-fixation method is well-established to enable high-fidelity preservation of the reconstructed neuronal networks and their sub-cellular processes in a near-native vitreous state without requiring chemical fixatives. To direct the outgrowth of neurites originating from two distinct groups of neurons growing in the two different compartments, polymer microstructures akin to microchannels are fabricated atop of sapphire disks. Two populations of neurons expressing either enhanced green fluorescent protein (EGFP) or mCherry were grown in either compartment, facilitating the analysis of the specific interactions between the two separate groups of cells. Neuronally differentiated PC12 cells, murine hippocampal and striatal neurons were successfully used in this context. The design of this device permits direct observation of entire neuritic processes within microchannels by optical microscopy with high spatial and temporal resolution, prior to processing for high-pressure freezing and electron microscopy. Following freeze substitution, we demonstrate that it is possible to process the neuronal networks for ultrastructural imaging by electron microscopy. Several key features of the embedded neuronal networks, including mitochondria, synaptic vesicles, axonal terminals, microtubules, with well-preserved ultrastructures were observed at high resolution using focused ion beam – scanning electron microscopy (FIB-SEM) and serial sectioning – transmission electron microscopy (TEM). These results demonstrate the compatibility of the platform with optical microscopy, high-pressure freezing and electron microscopy. The platform can be extended to neuronal models of brain disease or development in future studies, enabling the investigation of subcellular processes at the nanoscale within two distinct groups of neurons in a functional neuronal pathway, as well as pharmacological testing and drug screening.
Bridging Microscopes: 3D Correlative Light and Scanning Electron Microscopy of Complex Biological Structures
Lucas, Miriam S.; Günthert, Maja; Gasser, Philippe; et al. (2012)
Methods in Cell Biology ~ Correlative light and electron microscopy
Phase tomography from x-ray coherent diffractive imaging projections
Guizar-Sicairos, Manuel; Diaz, Ana; Holler, Mirko; et al. (2011)
Optics Express
Simultaneous Correlative Scanning Electron and High-NA Fluorescence Microscopy
Liv, Nalan; Zonnevylle, A. Christiaan; Narvaez, Angela C.; et al. (2013)
PLoS ONE
Correlative light and electron microscopy (CLEM) is a unique method for investigating biological structure-function relations. With CLEM protein distributions visualized in fluorescence can be mapped onto the cellular ultrastructure measured with electron microscopy. Widespread application of correlative microscopy is hampered by elaborate experimental procedures related foremost to retrieving regions of interest in both modalities and/or compromises in integrated approaches. We present a novel approach to correlative microscopy, in which a high numerical aperture epi-fluorescence microscope and a scanning electron microscope illuminate the same area of a sample at the same time. This removes the need for retrieval of regions of interest leading to a drastic reduction of inspection times and the possibility for quantitative investigations of large areas and datasets with correlative microscopy. We demonstrate Simultaneous CLEM (SCLEM) analyzing cell-cell connections and membrane protrusions in whole uncoated colon adenocarcinoma cell line cells stained for actin and cortactin with AlexaFluor488. SCLEM imaging of coverglass-mounted tissue sections with both electron-dense and fluorescence staining is also shown.

Publications 1 - 10 of 25

Miriam S. Lucas

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