Diverse protocols for correlative super-resolution fluorescence imaging and electron microscopy of chemically fixed samples

Abstract

Our groups have recently developed related approaches for sample preparation for super-resolution imaging within endogenous cellular environments using correlative light and electron microscopy (CLEM). Four distinct techniques for preparing and acquiring super-resolution CLEM datasets on aldehyde-fixed specimens are provided, including Tokuyasu cryosectioning, whole-cell mount, cell unroofing and platinum replication, and resin embedding and sectioning. Choice of the best protocol for a given application depends on a number of criteria that are discussed in detail. Tokuyasu cryosectioning is relatively rapid but is limited to small, delicate specimens. Whole-cell mount has the simplest sample preparation but is restricted to surface structures. Cell unroofing and platinum replica creates high-contrast, 3-dimensional images of the cytoplasmic surface of the plasma membrane, but is more challenging than whole-cell mount. Resin embedding permits serial sectioning of large samples, but is limited to osmium-resistant probes, and is technically difficult. Expected results from these protocols include super-resolution localization (~10–50 nm) of fluorescent targets within the context of electron microscopy ultrastructure, which can help address cell biological questions. These protocols can be completed in 2–7 days, are compatible with a number of super-resolution imaging protocols, and are broadly applicable across biology.

Ultrastructural Imaging of Salmonella–Host Interactions Using Super‐resolution Correlative Light‐Electron Microscopy of Bioorthogonal Pathogens

“…By lowering OsO 4 concentrations during post‐fixing and optimizing resin embedding, fluorophore quenching could be partially prevented. This sample preparation technique was reported with both PALM (photoactivated localization microscopy) and STED (stimulated emission depletion) microscopy in combination with TEM and scanning electron microscopy (SEM) . Of the various super‐resolution imaging techniques, STORM offers higher spatial resolution and sensitivity at the cost of longer acquisition times.…”

Structurally distinct endocytic pathways for B cell receptors in B lymphocytes

“…Large BCR clusters are associated with distinct endocytic structures at the plasma membrane Correlative light and electron microscopy (CLEM) provides a unique nanoscale view of the cellular environment of proteins localized by fluorescence microscopy (Kopek et al, 2017;Sochacki et al, 2014). Figure 2a shows correlated super-resolution fluorescence and platinum replica EM image (CLEM image) of the inner plasma membrane of an unstimulated DG-75 B cell plasma membrane.…”

Correlating Catalyst Structure and Activity at the Nanoscale

“…In correlative light and electron microscopy (CLEM) (Figure a), FM and EM are performed consecutively by employing dedicated microscopes. This allows full flexibility over the FM configuration, even enabling SRFM, but requires sample transfer which could lead to contamination and complicates image registration ,. Fiducial markers, visible in both FM and EM, are commonly mixed into the sample to facilitate the image registration process.…”