Preservation of Fluorescence Signal and Imaging Optimization for Integrated Light and Electron Microscopy

Baatsen, Pieter; Gabarre, Sergio; Vandael, Dorien; Wouters, Rosanne; Goodchild, Rose; Munck, Sebastian; Gounko, Natalia V.

doi:10.3389/fcell.2021.737621

Cited by 6 publications

(3 citation statements)

References 39 publications

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“…[57] provided a detailed comparison of the advantages of the three immune‐CLEM approaches, which allowed the identification of tissue stem cells in whole zebrafish brain. More recently, a protocol was published for increasing the in‐resin fluorescence of GFP [58]. This protocol refined previously established workflows using freeze substitution and Lowicryl HM20 embedding [37].…”

Section: Discussionmentioning

confidence: 99%

Fluorescence CLEM in biology: historic developments and current super‐resolution applications

2022

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Correlative light and electron microscopy (CLEM) is a powerful imaging approach that allows the direct correlation of information obtained on a light and an electron microscope. There is a growing interest in the application of CLEM in biology, mainly attributable to technical advances in field of fluorescence microscopy in the past two decades. In this review, we summarize the important developments in CLEM for biological applications, focusing on the combination of fluorescence microscopy and electron microscopy. We first provide a brief overview of the early days of fluorescence CLEM usage starting with the initial rise in the late 1970s and the subsequent optimization of CLEM workflows during the following two decades. Next, we describe how the engineering of fluorescent proteins and the development of super‐resolution fluorescence microscopy have significantly renewed the interest in CLEM resulting in the present application of fluorescence CLEM in many different areas of cellular and molecular biology. Lastly, we present the promises and challenges for the future of fluorescence CLEM discussing novel workflows, probe development and quantification possibilities.

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Section: Discussionmentioning

confidence: 99%

Fluorescence CLEM in biology: historic developments and current super‐resolution applications

2022

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“…Regions of interest in the CA1-stratum radiatum (SR) were punched out from these sections and frozen in 3 mm carriers with a high-pressure freezer (HPF ICE, Leica Microsystems). Frozen samples were quick freeze-substituted and embedded according to our protocol [ 23 ] using EM AFS2 apparatus (Leica Microsystems). Subsequently, 70 nm sections were cut with an ultramicrotome (Ultracut S, Leica Microsystems).…”

Section: Methodsmentioning

confidence: 99%

Cdk5-dependent rapid formation and stabilization of dendritic spines by corticotropin-releasing factor

Vandael,

Vints,

Baatsen

et al. 2024

Transl Psychiatry

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The neuropeptide corticotropin-releasing factor (CRF) exerts a pivotal role in modulating neuronal activity in the mammalian brain. The effects of CRF exhibit notable variations, depending on factors such as duration of exposure, concentration, and anatomical location. In the CA1 region of the hippocampus, the impact of CRF is dichotomous: chronic exposure to CRF impairs synapse formation and dendritic integrity, whereas brief exposure enhances synapse formation and plasticity. In the current study, we demonstrate long-term effects of acute CRF on the density and stability of mature mushroom spines ex vivo. We establish that both CRF receptors are present in this hippocampal region, and we pinpoint their precise subcellular localization within synapses by electron microscopy. Furthermore, both in vivo and ex vivo data collectively demonstrate that a transient surge of CRF in the CA1 activates the cyclin-dependent kinase 5 (Cdk5)-pathway. This activation leads to a notable augmentation in CRF-dependent spine formation. Overall, these data suggest that upon acute release of CRF in the CA1-SR synapse, both CRF-Rs can be activated and promote synaptic plasticity via activating different downstream signaling pathways, such as the Cdk5-pathway.

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“…This reinforces the primary fixation, but concomitantly reduces a proteins’ ability to fluoresce ( Berryman and Rodewald, 1990 ; Nixon et al, 2009 ). Hence, consensus has grown around the need to minimize fixatives as well as contrasting agents in order to preserve fluorescence after resin embedding ( Biel et al, 2003 ; Nixon et al, 2009 ; Kukulski et al, 2011 ; Lucas et al, 2012 ; Peddie et al, 2014 ; Delevoye et al, 2016 ; Baatsen et al, 2021 ). For TEM applications, sensitive detection systems (EMCCD, CMOS and direct electron detectors) enable minimal use of uranyl acetate, at concentrations below 1% in FS protocols, yielding impressive image quality ( Hawes et al, 2007 ).…”

Section: Contrast Enhancement: a Necessary Evil?mentioning

confidence: 99%

One for All, All for One: A Close Look at In-Resin Fluorescence Protocols for CLEM

Heiligenstein¹,

Lucas

2022

Front. Cell Dev. Biol.

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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.

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Preservation of Fluorescence Signal and Imaging Optimization for Integrated Light and Electron Microscopy

Cited by 6 publications

References 39 publications

Fluorescence CLEM in biology: historic developments and current super‐resolution applications

Fluorescence CLEM in biology: historic developments and current super‐resolution applications

Cdk5-dependent rapid formation and stabilization of dendritic spines by corticotropin-releasing factor

One for All, All for One: A Close Look at In-Resin Fluorescence Protocols for CLEM

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