Electron‐Beam Induced Luminescence and Bleaching in Polymer Resins and Embedded Biomaterial

Abstract

Electron microscopy is crucial for imaging biological ultrastructure at nanometer resolution. However, electron irradiation also causes specimen damage, reflected in structural and chemical changes that can give rise to alternative signals. Here, luminescence induced by electron‐beam irradiation is reported across a range of materials widely used in biological electron microscopy. Electron‐induced luminescence is spectrally characterized in two epoxy (Epon, Durcupan) and one methacrylate resin (HM20) over a broad electron fluence range, from 10−4 to 103 mC cm−2, both with and without embedded biological samples. Electron‐induced luminescence is pervasive in polymer resins, embedded biomaterial, and occurs even in fixed, whole cells in the absence of resin. Across media, similar patterns of intensity rise, spectral red‐shifting, and bleaching upon increasing electron fluence are observed. Increased landing energies cause reduced scattering in the specimen shifting the luminescence profiles to higher fluences. Predictable and tunable electron‐induced luminescence in natural and synthetic polymer media is advantageous for turning many polymers into luminescent nanostructures or to fluorescently visualize (micro)plastics. Furthermore, these findings provide perspective to direct electron‐beam excitation approaches like cathodoluminescence that may be obscured by these nonspecific electron‐induced signals.

Cathodoluminescent and Characteristic X-ray-emissive Rare-Earth-doped Core/Shell Immunolabels for Spectromicroscopic Analysis of Cell Surface Receptors

“…Figure S7). [20] Moreover, EDX detectors are offered in many STEM systems, which allows more widespread access and use of EDX-based ColorEM compared to CL that additionally also requires cryo temperatures during data acquisition and often more data processing. Through lanthanide substitution, the library of the immunolabels presented here is expandable, allowing multiple colours with unique (X-ray fingerprints to be used for the elucidation of structure-function relationships at the nanometer scale.…”

“…In this SEM-based multi-color approach, spectral filters were introduced, simplifying the dual dopant recognition on the outer cell membrane, but veiling nonlocal excitations through secondary electrons were found and the obstacles associated with epoxy-embedding, required for intracellular ultrastructure assessment, were neglected. [19,20]…”

“…In this SEM-based multi-color approach, spectral filters were introduced, simplifying the dual dopant recognition on the outer cell membrane, but veiling nonlocal excitations through secondary electrons were found and the obstacles associated with epoxy-embedding, required for intracellular ultrastructure assessment, were neglected. [19,20] Alternatively to CL, characteristic X-ray emission may be leveraged with EDX-SM for label identification and characterization of the elemental distribution within samples. Work by Scotuzzi et al demonstrated its feasibility using quantum dots and gold-based immunolabels.…”

“…are used, which potentially veil or interfere with the label's CL and/or EDX-SM signal. [20,[64][65][66] Initally, phase-transferred (BF -4 -capped) particles deposited on a TEM grid investigated. In this set-up the interaction of the particles with their environment was kept to a minimum and non-radiative quenching effects reduced, highly favouring (CL)-emissive properties.…”

Integrated Array Tomography for 3D Correlative Light and Electron Microscopy

“…The traditional way to compensate for diminished contrast is to boost the EM signal by increasing the dwell time per pixel, but this comes at the expense of throughput. An additional complication in integrated CLEM is electron-beam-induced quenching of the fluorescence ( Srinivasa Raja et al, 2021 ). This imposes the constraint that the fluorescence in a given area must be acquired prior to exposure from the electron beam, which prohibits uniformly pre-irradiating the sample with the electron beam to enhance and stabilize contrast ( Kuipers et al, 2015 ).…”