Simultaneous Correlative Scanning Electron and High-NA Fluorescence Microscopy

Correlative light and electron microscopy (CLEM) is a unique method for investigating biological structure-function relations. With CLEM protein distributions visualized in fluorescence can be mapped onto the cellular ultrastructure measured with electron microscopy. Widespread application of correlative microscopy is hampered by elaborate experimental procedures related foremost to retrieving regions of interest in both modalities and/or compromises in integrated approaches. We present a novel approach to correlative microscopy, in which a high numerical aperture epifluorescence microscope and a scanning electron microscope illuminate the same area of a sample at the same time. This removes the need for retrieval of regions of interest leading to a drastic reduction of inspection times and the possibility for quantitative investigations of large areas and datasets with correlative microscopy. We demonstrate Simultaneous CLEM (SCLEM) analyzing cell-cell connections and membrane protrusions in whole uncoated colon adenocarcinoma cell line cells stained for actin and cortactin with AlexaFluor488. SCLEM imaging of coverglass-mounted tissue sections with both electron-dense and fluorescence staining is also shown.IST/Imaging Science and TechnologyApplied Science

Single-cell biological lasers

Since their invention some 50 years ago(1), lasers have made a tremendous impact on modern science and technology. Nevertheless, lasing has so far relied on artificial or engineered optical gain materials, such as doped crystals, semiconductors, synthetic dyes and purified gases(2,3). Here, we show that fluorescent proteins(4,5) in cells are a viable gain medium for optical amplification, and report the first successful realization of biological cell lasers based on green fluorescent protein (GFP). We demonstrate in vitro protein lasers using recombinant GFP solutions and introduce a laser based on single live cells expressing GFP. On optical pumping with nanojoule/nanosecond pulses, individual cells in a high-Q microcavity produce bright, directional and narrowband laser emission, with characteristic longitudinal and transverse modes. Lasing cells remained alive even after prolonged lasing action. Light amplification and lasing from and within biological systems pave the way to new forms of intracellular sensing, cytometry and imaging.</p

Correlated light and electron microscopy:ultrastructure lights up!

Microscopy has gone hand in hand with the study of living systems since van Leeuwenhoek observed living microorganisms and cells in 1674 using his light microscope. A spectrum of dyes and probes now enable the localization of molecules of interest within living cells by fluorescence microscopy. With electron microscopy (EM), cellular ultrastructure has been revealed. Bridging these two modalities, correlated light microscopy and EM (CLEM) opens new avenues. Studies of protein dynamics with fluorescent proteins (FPs), which leave the investigator 'in the dark' concerning cellular context, can be followed by EM examination. Rare events can be preselected at the light microscopy level before EM analysis. Ongoing development-including of dedicated probes, integrated microscopes, large-scale and three-dimensional EM and super-resolution fluorescence microscopy-now paves the way for broad CLEM implementation in biology

Inducing fluorescence of uranyl acetate as a dual-purpose contrast agent for correlative light-electron microscopy with nanometre precision

Abstract Correlative light-electron microscopy (CLEM) combines the high spatial resolution of transmission electron microscopy (TEM) with the capability of fluorescence light microscopy (FLM) to locate rare or transient cellular events within a large field of view. CLEM is therefore a powerful technique to study cellular processes. Aligning images derived from both imaging modalities is a prerequisite to correlate the two microscopy data sets, and poor alignment can limit interpretability of the data. Here, we describe how uranyl acetate, a commonly-used contrast agent for TEM, can be induced to fluoresce brightly at cryogenic temperatures (−195 °C) and imaged by cryoFLM using standard filter sets. This dual-purpose contrast agent can be used as a general tool for CLEM, whereby the equivalent staining allows direct correlation between fluorescence and TEM images. We demonstrate the potential of this approach by performing multi-colour CLEM of cells containing equine arteritis virus proteins tagged with either green- or red-fluorescent protein, and achieve high-precision localization of virus-induced intracellular membrane modifications. Using uranyl acetate as a dual-purpose contrast agent, we achieve an image alignment precision of ~30 nm, twice as accurate as when using fiducial beads, which will be essential for combining TEM with the evolving field of super-resolution light microscopy