With the growing popularity of cryogenic correlative light and electron microscopy, it is becoming increasingly important to bridge the resolution gap between these two modalities. At cryogenic temperatures, the photon yield of fluorophores is a few orders of magnitude higher than at room temperature, enabling localization precisions on the Ångström scale. The current challenge is to induce sparsity at cryogenic temperatures such that individual fluorescent molecules can be localized. In this paper, we demonstrate the progress of using polarized stimulated-emission depletion (STED) to induce sparsity at cryogenic temperatures and in vacuum. We generate linear polarization of arbitrary in-plane orientations to achieve polarized STED with a sparsity of 3.3:1. Furthermore, we have probed the dark-state lifetime of ATTO 647N at cryogenic temperatures and in vacuum at room temperature. This dark state in vacuum is long-lived (τ=38 ms) and could be the cause for reduced photostability of fluorophores under STED illumination in vacuum. The experiments were done on an in-house designed and built liquid nitrogen cryostat, enabling 30 hours of stable cryogenic fluorescence microscopy.

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Total internal reflection fluorescence (TIRF) microscopy is an important imaging tool for the investigation of biological structures, especially the study on cellular events near the plasma membrane. Imaging at cryogenic temperatures not only enables observing structures in a near-native and fixed state but also suppresses irreversible photo-bleaching rates, resulting in increased photo-stability of fluorophores. Traditional TIRF microscopes produce an evanescent field based on high numerical aperture immersion objective lenses with high magnification, which results in a limited field of view and is incompatible with cryogenic conditions. Here, we present a waveguide-based TIRF microscope, which is able to generate a uniform evanescent field using high refractive index waveguides on photonic chips and to obtain cellular observation at cryogenic temperatures. Our method provides an inexpensive way to achieve total-internal-reflection fluorescence imaging under cryogenic conditions.

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Crucial for the field of ultrafast electron microscopy is the creation of sub-picosecond, high brightness electron pulses. The use of a blanker to chop the beam that originates from a high brightness Schottky source may provide an attractive alternative to direct pulsed laser illumination of the source. We have recently presented the concept of a laser-triggered ultrafast beam blanker and argued that generation of 100 fs pulses could be possible [Weppelman et al., Ultramicroscopy 184, 8-17 (2017)]. However, a detailed analysis of the influence of a deflection field changing sign on sub-picoseconds time scale on the quality of the resulting electron pulses has so far been lacking. Here, we present such an analysis using time-dependent, three-dimensional numerical simulations to evaluate the time-evolution of deflection fields in and around a micrometers-scale deflector connected to a photo-conductive switch. Further particle tracing through the time-dependent fields allows us to evaluate beam quality parameters such as energy spread and temporal broadening. We show that with a shielded, "tunnel-type" design of the beam blanker limiting the spatial extent of fringe fields outside the blanker, the blanker-induced energy spread can be limited to 0.5 eV. Moreover, our results confirm that it could be possible to bring laser-triggered 100 fs focused electron pulses on the sample using a miniaturized ultrafast beam blanker. This would enable us to resolve ultrafast dynamics using focused electron pulses in an SEM or STEM.

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In article number 1700323, a sparsity‐inducing scheme based on widefield stimulated emission depletion, which requires stringent control of the polarization state of both the excitation and depletion laser, is introduced by Bernd Rieger and co‐workers for superresolution fluorescence microscopy. The ideal state involves excitation and depletion lasers with linear polarizations, orthogonally oriented in the sample plane. This state is obtained from linearly polarized light from the laser, which is made elliptically polarized before reflecting off the dichroic mirror. @en

We present a new method to create ultrashort electron pulses by integrating a photoconductive switch with an electrostatic deflector. This paper discusses the feasibility of such a system by analytical and numerical calculations. We argue that ultrafast electron pulses can be achieved for micrometer scale dimensions of the blanker, which are feasible with MEMS-based fabrication technology. According to basic models, the design presented in this paper is capable of generating 100 fs electron pulses with spatial resolutions of less than 10 nm. Our concept for an ultrafast beam blanker (UFB) may provide an attractive alternative to perform ultrafast electron microscopy, as it does not require modification of the microscope nor realignment between DC and pulsed mode of operation. Moreover, only low laser pulse energies are required. Due to its small dimensions the UFB can be inserted in the beam line of a commercial microscope via standard entry ports for blankers or variable apertures. The use of a photoconductive switch ensures minimal jitter between laser and electron pulses.

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Light microscopy, allowing sub-diffraction-limited resolution, has been among the fastest developing techniques at the interface of biology, chemistry, and physics. Intriguingly no theoretical limit exists on how far the underlying measurement uncertainty can be lowered. In particular data fusion of large amounts of images can reduce the measurement error to match the reso-lution of structural methods like cryo-electron microscopy. Fluorescence, although reliant on a reporter molecule and therefore not the first choice to obtain ultraresolution structures, brings highly specific labeling of molecules in a large assembly to the table and inherently allows the detection of multiple colors, which enables the interrogation of multiple molecular species at the same time in the same sample. Here, the problems to be solved in the coming years, with the aim of higher resolution, are discussed, and what polarization depletion of fluorescence at cryogenic temperatures can contribute for fluorescence imaging of biological samples, like whole cells, is described.@en

Many applications in (quantum) nanophotonics rely on controlling light-matter interaction through strong, nanoscale modification of the local density of states (LDOS). All-optical techniques probing emission dynamics in active media are commonly used to measure the LDOS and benchmark experimental performance against theoretical predictions. However, metal coatings needed to obtain strong LDOS modifications in, for instance, nanocavities, are incompatible with all-optical characterization. So far, no reliable method exists to validate theoretical predictions. Here, we use sub-nanosecond pulses of focused electrons to penetrate the metal and excite a buried active medium at precisely-defined locations inside sub-wavelength resonant nanocavities. We reveal the spatial layout of the spontaneous-emission decay dynamics inside the cavities with deep-subwavelength detail, directly mapping the LDOS. We show that emission enhancement converts to inhibition despite an increased number of modes, emphasizing the critical role of optimal emitter location. Our approach yields fundamental insight in dynamics at deep-subwavelength scales for a wide range of nano-optical systems.

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Nanomaterials can be identified in high-resolution electron microscopy images using spectrally-selective cathodoluminescence. Capabilities for multiplex detection can however be limited, e.g., due to spectral overlap or availability of filters. Also, the available photon flux may be limited due to degradation under electron irradiation. Here, we demonstrate single-pass cathodoluminescence-lifetime based discrimination of different nanoparticles, using a pulsed electron beam. We also show that cathodoluminescence lifetime is a robust parameter even when the nanoparticle cathodoluminescence intensity decays over an order of magnitude. We create lifetime maps, where the lifetime of the cathodoluminescence emission is correlated with the emission intensity and secondary-electron images. The consistency of lifetime-based discrimination is verified by also correlating the emission wavelength and the lifetime of nanoparticles. Our results show how cathodoluminescence lifetime provides an additional channel of information in electron microscopy.

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We show cathodoluminescence-based time-resolved electron beam spectroscopy in order to directly probe the spontaneous emission decay rate that is modified by the local density of states in a nanoscale environment. In contrast to dedicated laser-triggered electron-microscopy setups, we use commercial hardware in a standard SEM, which allows us to easily switch from pulsed to continuous operation of the SEM. Electron pulses of 80–90 ps duration are generated by conjugate blanking of a high-brightness electron beam, which allows probing emitters within a large range of decay rates. Moreover, we simultaneously attain a resolution better than λ/10, which ensures details at deep-subwavelength scales can be retrieved. As a proof-of-principle, we employ the pulsed electron beam to spatially measure excited-state lifetime modifications in a phosphor material across the edge of an aluminum half-plane, coated on top of the phosphor. The measured emission dynamics can be directly related to the structure of the sample by recording photon arrival histograms together with the secondary-electron signal. Our results show that time-resolved electron cathodoluminescence spectroscopy is a powerful tool of choice for nanophotonics, within reach of a large audience.@en