Vitrification of Tokuyasu-style immuno-labelled sections for correlative cryo light microscopy and cryo electron tomography

Bos, Erik; Hussaarts, Leonie; Weering, Jan R.T. van; Ellisman, Mark H.; Wit, Heidi de; Koster, Abraham J.

doi:10.1016/j.jsb.2014.03.021

Cited by 31 publications

(27 citation statements)

References 47 publications

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“…Compared with EM, simplicity in sample preparation is an immediate advantage of the developed SMLM methodology, as serial sections from routine FFPE blocks can be prepared for SMLM with only minor adjustments to the IF staining protocol, primarily in the concentration and class of utilized fluorophores. SMLM also provides more specific and generally higher efficiency labeling than either the Tokuyasu technique or other post-embedding immunostaining methods used for EM34. Additionally, samples prepared for SMLM imaging can be imaged using conventional microscopy without additional staining or sample preparation steps, enabling convenient association between high resolution SMLM structures to those visualized in histopathology (Fig.…”

Section: Discussionmentioning

confidence: 99%

Superresolution Imaging of Clinical Formalin Fixed Paraffin Embedded Breast Cancer with Single Molecule Localization Microscopy

Creech

Wang

Nan

et al. 2017

Sci Rep

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Millions of archived formalin-fixed, paraffin-embedded (FFPE) specimens contain valuable molecular insight into healthy and diseased states persevered in their native ultrastructure. To diagnose and treat diseases in tissue on the nanoscopic scale, pathology traditionally employs electron microscopy (EM), but this platform has significant limitations including cost and painstaking sample preparation. The invention of single molecule localization microscopy (SMLM) optically overcame the diffraction limit of light to resolve fluorescently labeled molecules on the nanoscale, leading to many exciting biological discoveries. However, applications of SMLM in preserved tissues has been limited. Through adaptation of the immunofluorescence workflow on FFPE sections milled at histological thickness, cellular architecture can now be visualized on the nanoscale using SMLM including individual mitochondria, undulations in the nuclear lamina, and the HER2 receptor on membrane protrusions in human breast cancer specimens. Using astigmatism imaging, these structures can also be resolved in three dimensions to a depth of ~800 nm. These results demonstrate the utility of SMLM in efficiently uncovering ultrastructural information of archived clinical samples, which may offer molecular insights into the physiopathology of tissues to assist in disease diagnosis and treatment using conventional sample preparation methods.

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Section: Discussionmentioning

confidence: 99%

Superresolution Imaging of Clinical Formalin Fixed Paraffin Embedded Breast Cancer with Single Molecule Localization Microscopy

Creech

Wang

Nan

et al. 2017

Sci Rep

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“…Tokuyasu (1973Tokuyasu ( , 1986Tokuyasu ( , 1989) applied a high-molar sucrose solution or a mixture of polyvinylpyrrolidone (PVP) and high molar sucrose solution to prevent ice-crystal formation within specimens, and saw good preservation of both the ultrastructure and antigenicity on the ultrathin cryosections of the specimens. Many studies have since reported using ultrathin cryosections prepared by the Tokuyasu method (van Donselaar et al 2007;Koike et al 2013;Bos et al 2014), but this method has not been applied to SEM.…”

Section: Research-article2015mentioning

confidence: 99%

Correlative Light and Scanning Electron Microscopy for Observing the Three-Dimensional Ultrastructure of Membranous Cell Organelles in Relation to Their Molecular Components

Koga

Kusumi

Bochimoto

et al. 2015

J Histochem Cytochem.

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SummaryAlthough the osmium maceration method has been used to observe three-dimensional (3D) structures of membranous cell organelles with scanning electron microscopy (SEM), the use of osmium tetroxide for membrane fixation and the removal of cytosolic soluble proteins largely impairs the antigenicity of molecules in the specimens. In the present study, we developed a novel method to combine cryosectioning with the maceration method for correlative immunocytochemical analysis. We first immunocytochemically stained a semi-thin cryosection cut from a pituitary tissue block with a cryoultramicrotome, according to the Tokuyasu method, before preparing an osmium-macerated specimen from the remaining tissue block. Correlative microscopy was performed by observing the same area between the immunostained section and the adjacent face of the tissue block. Using this correlative method, we could accurately identify the gonadotropes of pituitary glands in various experimental conditions with SEM. At 4 weeks after castration, dilated cisternae of rough endoplasmic reticulum (RER) were distributed throughout the cytoplasm. On the other hand, an extremely dilated cisterna of the RER occupied the large region of the cytoplasm at 12 weeks after castration. This novel method has the potential to analyze the relationship between the distribution of functional molecules and the 3D ultrastructure in different composite tissues. (J Histochem Cytochem 63:968-979, 2015) Keywords correlative light and electron microscopy, gonadotrope, immunofluorescence microscopy, osmium maceration method, pituitary gland, scanning electron microscopy, Tokuyasu method

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“…CLEM studies that involve fluorescence microscopy may benefit from fluorescent markers that can be attached to molecules of interest to allow their identification and localization. To date, most readily this has been done by fluorescent fusion proteins, by fluorescent antibody labelling or by the chemical modification of a protein with a fluorescent detection group [1][2][3]. As well as these fluorescent detection moieties, structures must be present in the CLEM sample that are both EM and LM detectable in order to correlate (overlay) the LM image with the EM image.…”

Section: Introductionmentioning

confidence: 99%

“…stained nuclei) or fluorescently labelled electron-dense particles (e.g. fluorescent microspheres) [1][2][3][4].…”

Section: Introductionmentioning

confidence: 99%

“…It is also possible to use photoactivatable chemical groups for socalled photoclick-reactions. Nitrile imine mediated [1,3]-dipolar cycloaddition reaction and has been employed to selectively functionalize an alkene genetically encoded in a protein inside E. coli cells [28]. The reaction procedure was reported to be simple, straightforward and non-toxic to E. coli cells (Fig.…”

Section: Introductionmentioning

confidence: 99%

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The potential of bioorthogonal chemistry for correlative light and electron microscopy: a call to arms

et al. 2015

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With correlative light and electron microscopy (CLEM), the ultrastructural cellular location of a biomolecule of interest can be determined using a combination of light microscopy (LM) and electron microscopy (EM). In many cases, the application of CLEM requires the use of markers that need to be attached to a biomolecule of interest to allow its identification and localization. Here, we review the potential of bioorthogonal chemistry to introduce such markers for CLEM.

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Vitrification of Tokuyasu-style immuno-labelled sections for correlative cryo light microscopy and cryo electron tomography

Cited by 31 publications

References 47 publications

Superresolution Imaging of Clinical Formalin Fixed Paraffin Embedded Breast Cancer with Single Molecule Localization Microscopy

Superresolution Imaging of Clinical Formalin Fixed Paraffin Embedded Breast Cancer with Single Molecule Localization Microscopy

Correlative Light and Scanning Electron Microscopy for Observing the Three-Dimensional Ultrastructure of Membranous Cell Organelles in Relation to Their Molecular Components

The potential of bioorthogonal chemistry for correlative light and electron microscopy: a call to arms

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