Correlative Fluorescence and Electron Microscopy on Ultrathin Cryosections: Bridging the Resolution Gap

Here we show that ultrathin cryosections of placental tissue can be used as a substrate in immunofluorescence experiments. A high degree of spatial resolution can be achieved in these preparations because there is essentially no out-of-focus fluorescence. Therefore, immunofluorescence microscopy using ultrathin cryosections provides a very useful method for determining the precise subcellular localization of antigens in tissues. In addition, ultrathin cryosections of placenta also serve as a substrate for correlative immunofluorescence and immunoelectron microscopy using FluoroNanogold as the detection system. In correlative microscopy, the exact same structures in the same ultrathin section were observed by both fluorescence and electron microscopy. Using a particle counting procedure and electron microscopy, we compared the labeling obtained with colloidal gold and FluoroNanogold and found a higher number of particles with silver-enhanced FluoroNanogold than with colloidal gold.

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FluoroNanogold Is a Bifunctional Immunoprobe for Correlative Fluorescence and Electron Microscopy

Journal of Histochemistry & Cytochemistry ◽

10.1177/002215540004800405 ◽

2000 ◽

Vol 48 (4) ◽

pp. 481-485 ◽

Cited By ~ 40

Author(s):

Toshihiro Takizawa ◽

John M. Robinson

Keyword(s):

Electron Microscopy ◽

Fluorescence Microscopy ◽

Gold Particle ◽

High Spatial Resolution ◽

Human Neutrophils ◽

Secondary Antibody ◽

Marker Protein ◽

Cytoplasmic Granules ◽

Fluorescence And Electron Microscopy ◽

Ultrathin Cryosections

We applied a fluorescent ultrasmall immunogold probe, FluoroNanogold (FNG), to immunocytochemistry on ultrathin cryosections. FNG has the properties of both a fluorescent dye-conjugated antibody for fluorescence microscopy and a gold particle-conjugated antibody for electron microscopy. Therefore, this bifunctional immunoprobe permits correlative microscopic observation of the same cell profiles labeled in a single labeling procedure by these two imaging methods. We demonstrate the utility of FNG as a secondary antibody for immunocytochemical labeling of myeloperoxidase (a marker protein for azurophilic granules) in ultrathin cryosectioned human neutrophils. Its detection requires high spatial resolution because neutrophils contain many cytoplasmic granules. There was a one-to-one relationship between fluorescent structures labeled with FNG and organelle profiles labeled with the same silver-enhanced FNG in ultrathin cryosections. Use of FNG immunocytochemistry on ultrathin cryosections is an ideal methodology for highresolution correlative fluorescence and electron microscopy and can provide unique information that may be difficult to obtain with a single imaging regimen.

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Correlative Microscopy Using FluoroNanogold on Ultrathin Cryosections: Proof of Principle

Journal of Histochemistry & Cytochemistry ◽

10.1177/002215549804601001 ◽

1998 ◽

Vol 46 (10) ◽

pp. 1097-1102 ◽

Cited By ~ 57

Author(s):

Toshihiro Takizawa ◽

Kouki Suzuki ◽

John M. Robinson

Keyword(s):

Electron Microscopy ◽

Imaging Techniques ◽

Human Neutrophils ◽

Fluorescence Signal ◽

Correlative Microscopy ◽

Reporter System ◽

Fluorescence And Electron Microscopy ◽

Specific Granules ◽

Ultrathin Cryosections ◽

Proof Of Principle

We demonstrate a fluorescent ultrasmall immunogold probe, FluoroNanogold (FNG), to be a versatile reporter system for immunocytochemical labeling of ultrathin cryosections. FNG-labeled molecules in the same ultrathin cryosections can be resolved by two imaging techniques (i.e., fluorescence and electron microscopy). Lactoferrin, a marker protein for the specific granules in human neutrophils, was employed as the target for FNG immunolabeling. The spatial resolution of the fluorescence signal from FNG-labeled specific granules was compatible with that of silver-enhanced gold signal from the same granules in electron microscopy. Our results confirm that FNG can be used as a probe for highresolution correlation between immunofluorescence and electron microscopy.

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Faculty Opinions recommendation of A Cyclometalated Iridium (III) Complex as a Microtubule Probe for Correlative Super-Resolution Fluorescence and Electron Microscopy.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.738533948.793580586 ◽

2020 ◽

Author(s):

Ke Xu

Keyword(s):

Electron Microscopy ◽

Super Resolution ◽

Fluorescence And Electron Microscopy

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Photooxidation Microscopy: Bridging the Gap Between Fluorescence and Electron Microscopy

Neuromethods - Super-Resolution Microscopy Techniques in the Neurosciences ◽

10.1007/978-1-62703-983-3_13 ◽

2014 ◽

pp. 325-341

Author(s):

Annette Denker ◽

Silvio O. Rizzoli

Keyword(s):

Electron Microscopy ◽

Fluorescence And Electron Microscopy

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A Novel Flat-embedding Method to Prepare Ultrathin Cryosections from Cultured Cells in Their In Situ Orientation

Journal of Histochemistry & Cytochemistry ◽

10.1177/002215540205000809 ◽

2002 ◽

Vol 50 (8) ◽

pp. 1067-1080 ◽

Cited By ~ 42

Author(s):

Viola Oorschot ◽

Heidi de Wit ◽

Wim G. Annaert ◽

Judith Klumperman

Keyword(s):

Electron Microscopy ◽

Quantitative Method ◽

Cultured Cells ◽

Petri Dish ◽

Ultrastructural Level ◽

Immunogold Labeling ◽

Polarized Cells ◽

Ultrathin Cryosections ◽

Flat Embedding

Immunogold labeling of ultrathin cryosections provides a sensitive and quantitative method to localize proteins at the ultrastructural level. An obligatory step in the routine preparation of cryosections from cultured cells is the detachment of cells from their substrate and subsequent pelleting. This procedure precludes visualization of cells in their in situ orientation and hampers the study of polarized cells. Here we describe a method to sample cultured cells from a petri dish or coverslip by embedding them in a 12% gelatin slab. Subsequently, sections can be prepared in parallel or perpendicular to the plane of growth. Our method extends the cryosectioning technique to applications in studying polarized cells and correlative light–electron microscopy.

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