Combining light and electron microscopy in an integrated system allows for the combination of two different sorts of information in an automated fashion. This type of imaging, called integrated correlative light and electron microscopy (CLEM), is used for imaging of biological specimen and allows us to put the biomolecular context provided by fluorescence microscope into the specimen’s ultrastructural context provided by the electron microscope. However, while electron microscopy (EM) creates images at nanometre resolution, fluorescence microscopy (FM) is typically limited by the diffraction limit to hundreds of nanometers of resolution. This leads to a significant discrepancy in resolution when combining both image modalities. Thus, the application of CLEM is limited. This is especially the case for integrated CLEM, as the resolution of FM in non-integrated systems can be increased to tens of nanometres using conventional superresolution (SR) techniques. While there are a few reported cases of SR in integrated systems, overall, the techniques are limited due to a limited amount of space for complex excitation techniques, or stringent limits on the blinking of fluorescent molecules used for localization microscopy (LM) imposed by the vacuum of the EM. Finding new ways to make fluorescent molecules blink in a controlled fashion in the vacuum of an electron microscope would resolve all the issues mentioned above.
The goal of this thesis is to manipulate fluorescent molecules using low energy electrons for superresolution microscopy in the vacuum of a scanning electron microscope (SEM). By manipulating these molecules, and understanding the electron-induced effects, a versatile platform for LM could become available. Using low energy electrons of a few electronvolts, the different electron-induced mechanisms induced would become limited and thus more controlled. For all of this to work, a setup suitable for integrated CLEM, with electron energies available down to a few eV needs to be built. Furthermore, the electron-induced mechanisms for fluorescent molecules, and their effects on the fluorescence should then be understood, characterized, and verified as suitable for LM.
In chapter 2, we show how we modified a commercially available platform for integrated microscopy to achieve electron landing energies down to 0 eV, with 0.3 eV energy spread. For this we use a retarding field by applying a negative voltage to the setup’s stage. We show by reflecting the electron beam and detecting it with an in-column detector that we can determine the electron beam's landing energy and energy spread. In addition to this, we show that the setup improves the signal acquired for tissue sections optimized for simultaneous correlative microscopy. These tissue sections often have lower signals than samples optimized for one imaging modality only. For in-resin samples especially, this leads to poor EM signal for tissue sections of 100 nm thick or thinner. Using the negative stage bias we show that these in-resin CLEM samples can be imaged without extremely long dwell times or high beam currents even for ultrathin (50 nm) sections.
We use the setup presented in chapter 2 to study the effect of different electron landing energies down to a few eV on different fluorescent molecules in chapter 3. We find that fluorescent molecules can act as reporters for different electron-molecule reaction mechanisms. We show how electron irradiation of perylene-diimide (PDI), leads to a remarkable recovery in fluorescence after electron irradiation. We monitor this recovery continuously for different electron landing energies down to 0 eV and find based on the strength of the recovery component that electron-attachment to a transient anionic dark state is the main contributor to this process. This transient dark state can be manipulated by depositing the emitters on a conducting substrate, or by using a different dye of which the anionic dark state can be excited using a different excitation wavelength. With Rhodamine B ITC, we show an instantaneous recovery of the electron-induced dark state close to 0 eV landing energies using a short 405 nm excitation. Finally, we also demonstrate the versatility of low-energy electron irradiation by showing a dye that increases in fluorescence after electron irradiation.
Based on the electron-induced dynamics reported in chapter 3, we aim to determine what sort of strategy would be feasible for superresolution microscopy in the vacuum of a SEM. In chapter 4, we assess the resolution and quality of the reconstructed images of different molecular arrangements using simulations and different localization microscopy analysis techniques. We studied how extended photobleaching lifetimes in vacuum could improve easy-to-implement bleaching assisted localization techniques, or how low energy electron induced fluorescence fluctuations could be distinguished using Haar wavelet kernel filters and used to improve the resolution. We find that the latter approach results in both higher resolution and number of correct localizations, even if the photoswitching is switched off and only photobleaching occurs. We also propose new techniques relying on sparsity in each frame using instantaneous photoswitching of electron-induced dark states, or by temporarily switching emitters on with electrons. In general, we find that these approaches lead to a higher resolution of tens of nanometres, but that the current experimentally available photoswitching parameters are insufficient for resolving small molecular arrangements down to tens of nanometres.
The results presented in chapter 4 show promising prospects for superresolution microscopy in the vacuum of a SEM. However, with the setup presented in chapter 2, and the currently experimentally verified photoswitching parameters, resolutions down to tens of nanometeres are still unfeasible. In chapter 5, we show an integrated microscope modified by having a laser and easy to customize excitation and imaging path. The increased laser power should allow for higher accuracy localizations, but also allows for faster image acquisition. By then introducing a photomultiplier tube in the imaging path, we can monitor the electron induced dynamics down to sub-milliseconds timescales. Using the experimental approach presented in chapter 3, we quantify the fluorescence recovery timescales of perylene diimide for electron landing energies ranging from 1000 eV down to 2 eV. We find that the fluorescence recovery can be described with a double exponential behaviour characterized by time constants varying between 5-150 ms and 0.2–2s, respectively. For 2 eV electron landing energy, close to the resonance energy of electron attachment, we find a reduction in the slower exponential recovery term. Potential mechanisms responsible for these observed dynamics and follow-up experiments are then discussed.
With the results presented throughout this thesis we show how low energy electrons could be used to manipulate fluorescent molecules to achieve higher optical resolutions in an integrated light- and electron microscope. While the first steps have been made, considerable effort needs to be made to (i) understand the electron-induced dynamics and optimize fluorescent dyes, and (ii) to perform the electron-induced dynamics on biological specimen. In our outlook chapter, we discuss experimental approaches for these next steps, and other applications of low-energy electrons outside of superresolution microscopy in integrated microscopy.