3D integrated Correlative Light and Electron Microscopy via Confocal Laser Scanning Microscope and Focused Ion Beam / Scanning Electron Microscope

This thesis is about the integration of two 3D microscopes. One is light based – the Confocal Laser Scanning microscope (CLSM). The other is an Electron Microscope – Focused Ion Beam /Scanning Electron Microscope. The resulting microscope is called 3D integrated Correlative Light and Electron Microscope (CLEM), it allows to use the two modalities (light and electron) to acquire 3D-data from the same sample. The added value of is in the guidance of a very precise but slow FIB/SEM to the particular regions of interests (ROIs) by a less precise but faster CLSM. The thesis begins with the Introduction, where the challenge of microscopy is formulated and how CLEM methods allow to address it. The differences of Light and Electron Microscopy from the fundamental point of view are discussed. A brief review of the past and currently used CLEM geometries is given. In Chapter 1, the integrated CLEM system is described. The configuration allowed for the use of a high Numerical Aperture objective in the CLSM. The high performance of both LM and EM modalities is verified. The test of 3D CLEM is performed using a model bio-sample. The accuracy of CLSM and FIB/SEM registration is demonstrated. In Chapter 2 the integrated system is applied to a biological sample to study intracellular transport. It is demonstrated that with the use of fluorescent nano-particles (fiducial markers) that a ROI with a footprint as small as 2×2 μm^2 can be found. With the correlated data, the relationship between the cell ultrastructure and organelle movement is observed. In Chapter 3 the integrated CLEM is applied to a zeolite/bentonite catalyst. The accurate registration of SEM and integrated CLSM images is used. The relationship between the pore geometry with the reaction products is shown. A new modality of the setup is demonstrated – 3D CLSM/FIB, which generates 3D optical datasets from opaque samples. In Chapter 4 the 3D CLEM is applied to microscopic ink droplet stains on a (coated) photopaper. The relationship between the radius and the depth of the stains was studied by using FIB to cut through the paper coating and CLSM to image the stains. The distributions of ink were compared to the extensive numerical models of the collaborators. Again, the new modality of the setup demonstrated – the 3D distribution of ink inside the coating layer of paper is acquired. In Chapter 5 the optical challenges related to CLSM integration into the vacuum conditions of the FIB/SEM, and looking into samples of high refractive indexes are studied. In these conditions of refractive index mismatch (RIM) CLSM focal spots suffer both from RIM-induced aberrations and from the finite detector size. When the theories of RIM and detector size influence of the focal spots of CLSM are combined, they predict stronger focal-spots shape distortions than measured. We show that, two subtle phenomena are present. First, the role of the excitation beam shape becomes influential under RIM. Second, the small deviation from geometrical optics inside many CLSMs decreases the effective NA of the objective.