Correlative Stochastic Optical Reconstruction Microscopy and Electron Microscopy

Abstract

Correlative fluorescence light microscopy and electron microscopy allows the imaging of spatial distributions of specific biomolecules in the context of cellular ultrastructure. Recent development of super-resolution fluorescence microscopy allows the location of molecules to be determined with nanometer-scale spatial resolution. However, correlative super-resolution fluorescence microscopy and electron microscopy (EM) still remains challenging because the optimal specimen preparation and imaging conditions for super-resolution fluorescence microscopy and EM are often not compatible. Here, we have developed several experiment protocols for correlative stochastic optical reconstruction microscopy (STORM) and EM methods, both for un-embedded samples by applying EM-specific sample preparations after STORM imaging and for embedded and sectioned samples by optimizing the fluorescence under EM fixation, staining and embedding conditions. We demonstrated these methods using a variety of cellular targets.

Correlated confocal and super-resolution imaging by VividSTORM

“…For the correlative application of superresolution microscopy with other imaging methods, several approaches have been published. STORM has been used in combination with EM 33,45 , with confocal microscopy 46 and with live-cell imaging followed by automated immunostaining in microfluidic devices 34 . The main goal of the aforementioned approaches is to observe the same signal at different scales, and at different time points, with increased resolution.…”

Plastic embedding immunolabeled large-volume samples for three-dimensional high-resolution imaging

“…In the embedded tissues, the fluorescent intensity of fluorophores further depends on the resin types and embedding modes [19][20][21][22][23]. In this study, we labeled the brain slices with the same type of primary antibody (anti-TH) and the same type of secondary antibodies with commonly used fluorescent dyes, and embedded the slices in GMA/Lowicryl HM20/LR White resins.…”

“…In this case, whether the whole-mount immunolabelling method is compatible with plastic embedding should be examined first, as plastic embedding of the sample is a pre-requisite to enable precise and ultra-thin slicing. In fact, plastic embedding has been applied to immunolabelled thin samples like cells and tissues [19][20][21][22], and could improve the axial resolution of immunolabelled sections [23]. However, procedures and resins for immunostaining and embedding the thin sample are unsuitable for large-volume sample.…”

Correlating 3D light to 3D electron microscopy for systems biology

“…Rather than performing LM prior to fixation and embedding for EM, the fluorescence of LM probes is either preserved during preparation for imaging in EM (see [8] for references), or sections are immunolabelled with a fluorescent moiety [53], so that both imaging modalities are performed on the exact same sample. Serial sections with both electron dense stains and fluorescent labels may be imaged sequentially in separate fluorescence and electron microscopes [54][55][56], or in situ inside integrated light and electron microscopes [57,58]. These techniques have not yet been applied routinely to larger tissue samples, and compromises tend to be made on both fluorescence intensity and electron contrast in the sample, but the technique has the potential to produce high (super) resolution macromolecular localisations within easily-aligned [58] 3D image stacks from both imaging modalities.…”