Simultaneous Correlative Scanning Electron and High-NA Fluorescence Microscopy

Abstract

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 epi-fluorescence 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.

Cathodoluminescence Microscopy of nanostructures on glass substrates

“…A general approach to reduce the background contribution is therefore to lower the acceleration voltage: for a sufficiently small energy the interaction range can be restricted to the structure of interest. However, reducing the acceleration voltage decreases the CL intensity: coherent CL emission, as the one coming from SPPs and transition radiation, increases with the acceleration voltage (above 1kV) [15]. This is also valid for CL rising from inelastic electron-electron collisions, where the CL signal is proportional to the current and the inelastic cross section (σ): CL ∝ I × σ.…”

“…1(b)). In this system light microscopy and scanning electron microscopy can be performed simultaneously on the same area of a sample [15]. The alignment of the light and electron axes guarantees a direct spatial correlation of the different imaging modes, which is particularly useful to perform CL detection through the substrate.…”

Multicolour correlative imaging using phosphor probes

“…While the need for multiphoton light microscopy is a relatively expensive option for fluorescence imaging, further developments of phosphor probes will increase their suitability and sensitivity for standard light microscopy [7]. New combinations of electron and optical microscopes are being introduced [15], but in all cases, there is a clear need for fiducial markers. The photo-and electron stability of the probes compared to standard organic dyes also makes these highly desirable for future studies and development.…”

Integrated Microscopy: Highly Accurate Light-Electron Image Correlation Anywhere on a Sample

“…By correlating data from both techniques [4], molecules can be localized within the context of cells and tissue and with reference to their live dynamics, but throughput and quantification are hindered by elaborate, expert procedures involving separate microscopes. We have developed an integrated approach with high-numerical aperture LM inside an SEM, such that the electron beam can be positioned anywhere within the fluorescence field of view [5,6]. Here, we will show that this approach allows for automated light-electron overlay, i.e.…”

“…Samples containing fluorescence can be prepared either via standard chemical fixation procedures followed by post-embedding immuno-labelling, or using an adapted sample preparation protocol for maintaining genetic expressed fluorescence during EM sample preparation [4]. In all cases, integrated microscopy allows for rapid identification of regions of interest for SEM based on fluorescence expression and seamless exchange between both fluorescence and electron microscopy acquisition [6]. However, both modalities are still separated imaging systems, each with their own field distortions.…”