Cathodoluminescence (CL), the excitation of light by an electron beam, has gained attention as an analysis tool for investigating the optical response of a structure, at a resolution that approaches that in electron microscopy, in the nanometer range. However, the application possibilities are limited because the use of transparent substrates, one of the most common sample substrates for optical characterizations in multiple research fields, is normally avoided in CL microscopy, since these materials generate a strong signal that contributes as a background to the measurement. The main goal in this thesis is to achieve cathodoluminescence detection of nanostructures on glass-based substrates. For that purpose, a CL system with enhanced collection efficiency and confocal detection of the signal was developed, built and tested. The design is based on an integrated Scanning Electron and Optical Microscope, a setup that offers simultaneous correlated acquisition of the electron and light signals. Besides cathodoluminescence, other interesting applications derive from the combination of these techniques, but they are out of the scope of this thesis. Chapter 1 intends to give general introduction to cathodoluminescence as a microscopy analysis tool. First we discuss its generation principle: considering the excitation and emission mechanisms, electron-hole recombination, transition radiation and surface plasmon polariton radiative outcoupling are identified as the main CL sources in the structures investigated in this thesis. In bulk samples, the emission is not restricted to the nanometer size spot where the incoming electron beam is focused, but it extends to a region that spreads below it, where electrons scatter and interact with the host material. Cathodoluminescence is potentially generated throughout this volume, the size of which increases dramatically with the electron beam energy. Therefore it should be considered as an extended excitation, although its size can be modulated by a spatially confined generation yield. Most of the CL setups are incorporated in the vacuum chamber of an electron microscope, where the light collector is a parabolic mirror placed on top of the sample. The advantages, challenges and improvement examples of these standard setups are discussed. An overview of applications in cell and molecular biology, geosciences and nanophotonics emphasizes the increasing interest on applying the technique at the nanometer regime. The chapter ends by summarizing the main challenges that cathodoluminescence microscopy encounters for successful imaging of nanostructures on glass, which define the design criteria for our setup. The system details are presented in chapter 2: a brief description of the integrated electron light microscope functionalities and the implementation of the confocal detection path are presented. Explanation of the available acquisition modes, alignment procedures and typical imaging examples serve to establish an operation routine. The effect of the pinhole can be observed by comparing unfiltered and confocal CL images on the same region of a sample. Additionally, the filtering is evaluated without using the electron beam: a laser excitation path included in the setup allows acquiring confocal fluorescence images of a sample with luminescent beads on a glass substrate, for different sizes of the pinhole diameter. Besides efficient CL detection, potential applications of the setup could include: (i) emission localization for excitations with long propagation length, (ii) simultaneous light and electron excitation, (iii) monitoring the effect of electron excitation with subsequent light microscopy, and (iv) the incorporation of light or electron pulses for time-resolved characterization. The use of low energies for the electron excitation probe is proposed in chapter 3 as a strategy to reduce the background CL contribution. This is further investigated with Monte Carlo simulations that show the dependence of the electron interaction volume on the electron beam acceleration voltage. We observe however, that to detect nanostructures with a weak cathodoluminescence signal it is necessary to increase the electron current, which in the low acceleration voltage regime may compromise the spatial resolution. With the low energy approach, individual 30nm phosphor particles are resolved and the high order resonant modes of a gold nanowire on an indium tin oxide (ITO) covered glass microscope slide are detected. For high electron energies, the substrate cathodoluminescence is too strong and overwhelms the signal. Chapter 4 demonstrates confocal filtering as an effective tool for background rejection at high acceleration voltages. The filtering achieved for a given pinhole size is estimated with simulations of the electron interaction volume and measurements of the axial intensity distribution of a phosphor nanoparticle, which acts as a point source. As an illustrative example, a series of CL confocal sections of a gold nanowire on a transparent substrate shows a contrast inversion at the plane where the nanowire is in focus. Here, the highest CL intensity is detected at the position of the structure. The need of a high resolution electron probe is evidenced by acquiring the CL spectral distribution of a gold triangle nano plate, which shows a strong sensitivity to the excitation probe position. Both of the strategies presented in this thesis, the use of low energy excitation and confocal filtering are applicable not only for transparent substrates but for any highly cathodoluminescent material. Chapter 5 explores the use of quantum dots as cathodoluminescent biological markers. In cellular biology, investigation of cellular interactions requires imaging the specific functional proteins on top of the organelles ultrastructure. Therefore, direct correlation between electron and light optical information is a key element for understanding cell function at a molecular level. Among other potential cathodoluminescent markers, quantum dots have the additional advantage that they are already routinely incorporated as bio-labels in fluorescence and consequently, many different bio functionalization possibilities are currently available. Here, we report on the cathodoluminescence detection of bio-functionalized quantum-dots embedded in cells. A high similarity between the fluorescence and cathodoluminescence signals is observed, but the cathodoluminescence signal originates from a smaller sample volume defined by the electron penetration depth. We observe a bleaching of the quantum dots emission under high electron irradiation dose, which so far prevents high magnification imaging. However, recording the fluorescence emission after incremental low dose electron irradiation reveals a complicated dependence of the emission intensity on electron dose, featuring even a regime wherein intensity slightly increases. The origin of this behavior is discussed as a charging mechanism, building on existing models that are also used to explain photo blinking, -bleaching and -brightening of fluorescence from quantum dots. The results presented support the use of cathodoluminescence as a high resolution imaging technique for optical characterization of biological systems. Finally, the main findings on the cathodoluminescence emitted from ITO-covered glass slides, the substrate through this work, are summarized in chapter 6. A dynamic behavior of the intensity and spectral distribution of the emission is observed. Cathodoluminescence measurements at different electron doses reveal a faster cathodoluminescence bleaching with increasing dose, but also the appearance and growth of a new intensity peak at a different position in the spectra. Secondary electron images of the irradiated areas suggest that deposition may be involved in this process. Additionally, experiments with different thicknesses for the ITO conductive layer point to glass as the main responsible for the background emission in our measurements. The results reinforce the importance of sample pre-exposure and confocal filtering for CL characterization at high electron energies.