Loss of SMAD4 from colorectal cancer cells causes BMP signaling to switch from tumor suppressive to metastasis promoting. Concurrent loss of SMAD4 and normal expression of BMP receptors in colorectal tumors was associated with reduced survival times of patients. Reagents that interfere with SMAD4-independent BMP signaling, such as ROCK inhibitors, might be developed as therapeutics for CRC.

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.

Summary We present an integrated light‐electron microscope in which an inverted high‐NA objective lens is positioned inside a scanning electron microscope (SEM). The SEM objective lens and the light objective lens have a common axis and focal plane, allowing high‐resolution optical microscopy and scanning electron microscopy on the same area of a sample simultaneously. Components for light illumination and detection can be mounted outside the vacuum, enabling flexibility in the construction of the light microscope. The light objective lens can be positioned underneath the SEM objective lens during operation for sub‐10 μm alignment of the fields of view of the light and electron microscopes. We demonstrate in situ epifluorescence microscopy in the SEM with a numerical aperture of 1.4 using vacuum‐compatible immersion oil. For a 40‐nm‐diameter fluorescent polymer nanoparticle, an intensity profile with a FWHM of 380 nm is measured whereas the SEM performance is uncompromised. The integrated instrument may offer new possibilities for correlative light and electron microscopy in the life sciences as well as in physics and chemistry.

In the biological sciences, data from fluorescence and electron microscopy is correlated to allow fluorescence biomolecule identification within the cellular ultrastructure and/or ultrastructural analysis following live-cell imaging. High-accuracy (sub-100 nm) image overlay requires the addition of fiducial markers, which makes overlay accuracy dependent on the number of fiducials present in the region of interest. Here, we report an automated method for light-electron image overlay at high accuracy, i.e. below 5 nm. Our method relies on direct visualization of the electron beam position in the fluorescence detection channel using cathodoluminescence pointers. We show that image overlay using cathodoluminescence pointers corrects for image distortions, is independent of user interpretation, and does not require fiducials, allowing image correlation with molecular precision anywhere on a sample.

Cathodoluminescence (CL) microscopy is an emerging analysis technique in the fields of biology and photonics, where it is used for the characterization of nanometer sized structures. For these applications, the use of transparent substrates might be highly preferred, but the detection of CL from nanostructures on glass is challenging because of the strong background generated in these substrates and the relatively weak CL signal from the nanostructures. We present an imaging system for highly efficient CL detection through the substrate using a high numerical aperture objective lens. This system allows for detection of individual nanophosphors down to thirty nanometer in size as well as the up to ninth order plasmon resonance modes of a gold nanowire on ITO coated glass. We analyze the CL signal-to-background dependence on the primary electron beam energy and discuss different approaches to minimize its influence on the measurement.

Electron beam lithography (EBL) requires conducting substrates to ensure pattern fidelity. However, there is an increasing interest in performing EBL on less well-conducting surfaces or even insulators, usually resulting in seriously distorted pattern formation. To understand the underlying charging phenomena, the authors use Monte Carlo simulations that include models for substrate charging, electron beam-induced current, and electric breakdown. Simulations of electron beam exposure of glass wafers are presented, exposing regular patterns which become distorted due to charge-induced beam deflection. The resulting displacements within the patterns are mapped and compared to experimental displacement maps obtained from patterns in PMMA resist on glass substrates. Displacements up to several hundreds of nanometers were observed at a primary beam energy of 50 keV. Also, various scan strategies were used to write the patterns, in the simulations as well as the experiments, revealing their strong effect on pattern distortion, in shape and in magnitude. A qualitative, in some cases even quantitative, good agreement was found between the simulations and the experiments, providing enough confidence in Monte Carlo simulations to predict charge-induced pattern displacement and shape distortion and to find smart scan strategies to minimize the effects of charging.

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There is a growing interest for patterning on curved or tilted surfaces using electron beam lithography. Computational proximity correction techniques are well established for flat surfaces and perpendicular exposure, but for curved and tilted surfaces adjustments are needed as the dose distribution is no longer cylindrically symmetric with respect to the surface normal. A graphical processing unitaccelerated 3D Monte Carlo simulation, based on first-principle scattering models, is used to simulate the asymmetric dose distribution. Based on that, an approximate adjustment is made to an existing high-performance proximity effect correction (PEC) algorithm aimed at the correct exposure of a pattern of nanowires on a 17°tilted surface. It was experimentally verified that using the adjusted PEC indeed leads to a more uniform exposure on tilted surfaces.