Correlative and integrated light and electron microscopy of in-resin GFP fluorescence, used to localise diacylglycerol in mammalian cells

Peddie, Christopher J.; Blight, Ken; Wilson, Emma R.; Melia, Charlotte E.; Marrison, Joanne; Carzaniga, Raffaella; Domart, Marie-Charlotte; O’Toole, Peter; Larijani, Banafshé; Collinson, Lucy M.

doi:10.1016/j.ultramic.2014.02.001

Cited by 117 publications

(144 citation statements)

References 35 publications

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“…Maintenance of a small amount of water in the final resin block is key to preserving FP activity. Indeed, our work indicates that a quick freeze substitution (QFS) protocol [13] that minimises dehydration time leads to stable, active FPs in resin-embedded cells [10,14].…”

Section: Introductionmentioning

confidence: 89%

Standard fluorescent proteins as dual-modality probes for correlative experiments in an integrated light and electron microscope

et al. 2015

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Integrated light and electron microscopes (ILEMs) will enable a new generation of high-precision correlative imaging experiments. To fully exploit these systems, samples must contain dual-modality probes that highlight the position of macromolecules in the context of cell ultrastructure. We demonstrate that the fluorescent proteins (FPs) GFP (green), YFP (yellow) and mCherry can be used as dualmodality probes for ILEM when preserved using the inresin fluorescence (IRF) technique, which delivers stable active fluorophores in lightly stained, resin-embedded cells and tissues. However, we found that vacuum pressure in the ILEM affects the photophysics of FPs in IRF sections. Here, we show that reducing the vacuum pressure reduces fluorescence intensity of GFP and YFP, which is a consequence of water extraction from the sample and is reversible on recreation of partial pressure with water vapour (but not oxygen or nitrogen gas). We also find that, although fluorescence intensity is reduced at a partial pressure of 200 Pa (created using water vapour), the FP intensity is remarkably stable over time in vacuum and resistant to photobleaching during imaging. We are thus able to define imaging strategies for standard FPs acting as dual-modality probes in a single 'multi-colour' integrated microscope system.

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Section: Introductionmentioning

confidence: 89%

Standard fluorescent proteins as dual-modality probes for correlative experiments in an integrated light and electron microscope

et al. 2015

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“…Recently, a correlative light and electron microscope (CLEM) was introduced to combine the advantages of EM and LM. [1][2][3] In this article,…”

Section: Development Of a Correlative Light And Electronmentioning

confidence: 99%

Development of a Correlative Light and Electron Microscope for Multi-scale Nano-bioimaging

Cho¹

2018

Phys. High Technol

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“…Immediate disadvantages of this approach are the inability to orient the samples and the time needed for the relatively long preparation procedure. The recently developed fast HPF-FS procedure (23)(24)(25) demonstrated that shorter overall exposure to substitution solutions increases the preservation success of genetically encoded fluorescent proteins after HPF fixation and resin embedding (26). It thus inspired us to modify the hybrid technique.…”

Section: Fast-hybrid Preparationmentioning

confidence: 99%

Adaptation of Cryo‐Sectioning for IEM Labeling of Asymmetric Samples: A Study Using Caenorhabditis elegans

Nicolle

Burel

Griffiths

et al. 2015

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Cryo-sectioning procedures, initially developed by Tokuyasu, have been successfully improved for tissues and cultured cells, enabling efficient protein localization on the ultrastructural level. Without a standard procedure applicable to any sample, currently existing protocols must be individually modified for each model organism or asymmetric sample.Here, we describe our method that enables reproducible cryo-sectioning of Caenorhabditis elegans larvae/adults and embryos. We have estab- Modern cell and developmental biology requires sophisticated high-resolution imaging. Be it light or electron microscopy, recent progress in technology has made it possible to apply a wide variety of methods to study processes such as molecular dynamics, protein localization and interactions between cellular components. While live imaging light microscopy (LM) allows visualization of the dynamics of molecular and cellular processes, it lacks high resolution of the structural context, even using the recently developed super-resolution LM techniques. Transmission electron microscopy (TEM), though static, gives access to high-resolution ultrastructure within the cellular context. Immuno-labeling of TEM samples is a powerful approach to localize antigens precisely at the ultrastructural level. The major disadvantage of immuno-electron microscopy (IEM) resides in the fact that it is performed on thin slices of specimens -that represent only a tiny fragment of the entire antigen-containing structures. As a result of sample dehydration and masking of active sites by resins, techniques that use resin embedding for IEM frequently lead to poor immuno-labeling signal. In contrast, the cryo-sectioning method developed by Tokuyasu www.traffic.dk 893

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Correlative and integrated light and electron microscopy of in-resin GFP fluorescence, used to localise diacylglycerol in mammalian cells

Cited by 117 publications

References 35 publications

Standard fluorescent proteins as dual-modality probes for correlative experiments in an integrated light and electron microscope

Standard fluorescent proteins as dual-modality probes for correlative experiments in an integrated light and electron microscope

Development of a Correlative Light and Electron Microscope for Multi-scale Nano-bioimaging

Adaptation of Cryo‐Sectioning for IEM Labeling of Asymmetric Samples: A Study Using Caenorhabditis elegans

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