Waveguide-based total internal reflection fluorescence microscope enabling cellular imaging under cryogenic conditions.

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.

Nanoscale Imaging of Light-Matter Coupling Inside Metal-Coated Cavities with a Pulsed Electron Beam.

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 subnanosecond pulses of focused electrons to penetrate the metal and excite a buried active medium at precisely defined locations inside subwavelength 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.

Time-resolved cathodoluminescence microscopy with sub-nanosecond beam blanking for direct evaluation of the local density of states.

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.

Self-assembled silver nanoparticles in a bow-tie antenna configuration.

The self-assembly of silver nanoparticles into a bow-tie antenna configuration is achieved with the DNA origami method. Instead of complicated particle geometries, spherical silver nanoparticles are used. Formation of the structures in high yields is verified with transmission electron microscopy and agarose gel electrophoresis. According to finite-difference time-domain simulations, the antenna configuration could be used as a DNA sensor.

Shaping single emitter emission with metallic hole arrays: strong focusing of dipolar radiation.

Nanoscale plasmonic structures allow for control of the emission of single emitters, such as fluorescent molecules and quantum dots, enabling phenomena such as lifetime reduction, emission redirection and color sorting of photons. We present single emitter emission tailored with arrays of holes of heterogeneous size, perforated in a gold film. With spatial control of the local amplitude and phase of the electromagnetic field radiated by the emitter, a desired near- or far-field distribution of the electromagnetic waves can be obtained. This control is established by varying the aspect ratio of the individual holes and the periodicity of the array surrounding the emitter. As an example showing the versatility of the technique, we present the strong focusing of the radiation of a highly divergent dipole source, for both p- and s-polarized waves.

Cathodoluminescence Microscopy of nanostructures on glass substrates.

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 nano-phosphors 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.

Fluorescence Polarization Control for On-Off Switching of Single Molecules at Cryogenic Temperatures.

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 resolution 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.

Nanoparticle discrimination based on wavelength and lifetime-multiplexed cathodoluminescence microscopy.

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.

Reversible polarization control of single photon emission.

We present reversible and a-priori control of the polarization of a photon emitted by a single molecule by introducing a nanoscale metal object in its near field. It is experimentally shown that, with the metal close to the emitter, the polarization ratio of the emission can be varied by a factor of 2. The tunability of polarization decays, when the metal is displaced by typically 30 nm. Calculations based on the multiple multipole method agree well with our experiments and predict even further enhancement with a suitable nanoantenna design.

Lambda/4 resonance of an optical monopole antenna probed by single molecule fluorescence.

We present a resonant optical nanoantenna positioned at the end of a metal-coated glass fiber near-field probe. Antenna resonances, excitation conditions, and field localization are directly probed in the near field by single fluorescent molecules and compared to finite integration technique simulations. It is shown that the antenna is equivalent to its radio frequency analogue, the monopole antenna. For the right antenna length and local excitation conditions, antenna resonances occur that lead to an enhanced localized field near the antenna apex. Direct mapping of this field with single fluorescent molecules reveals a spatial localization of 25 nm, demonstrating the importance of such antennas for nanometer resolution optical microscopy.