Integrated Array Tomography

Integrated Array Tomography: Development and Applications of a Workflow for 3D Correlative Light and Electron Microscopy

Lane, R. (TU Delft ImPhys/Microscopy Instrumentation & Techniques)

Hoogenboom, J.P. (promotor)
Carroll, E.C.M. (copromotor)

Delft University of Technology

2022-10-04

Multi-modal imaging techniques have become essential for better understanding fundamental questions in cell biology such as disease progression. While individual microscopy methods have rapidly advanced in recent years, the information content of any one imaging technique is limited to the type of contrast that particular technique is sensitive to. By tagging particular biomolecules with a fluorescent protein, fluorescence microscopy (FM), for example, can relay dynamic information about the distribution of these biomolecules in their cellular environment. It struggles, however, to convey information regarding the structure of the organelles that might contain these biomolecules or the surroundings of their cellular environment. Electron microscopy (EM), on the other hand, can provide detailed layouts of cellular structure by staining membranes with heavy metals. Thus, by correlating these modalities (correlative light and electron microscopy, CLEM), a more holistic understanding of the relationship between structure and function at the (sub-)cellular level can be achieved. Array tomography (AT) is a technique combining FM and EM for volumetric imaging, first introduced in 2007 for studying brain tissue. The technique has since expanded, but the approach has largely remained the same. Biological material is cut into a series of ultrathin (∼100 nm) sections (an array) and prepared for sequential FM and EM imaging by applying a series of immunofluorescence and heavy metal stains. Correlative images of the serial sections are then computationally aligned to reconstruct the 3D structure (tomography). Compared to other volumetric imaging techniques in the life sciences, AT offers the ability to correlate structure and function at high resolution across large fields of view. Moreover, it enables high axial resolutionfor both EM and FM as determined by the section thickness...

correlative light and electron microscopy
volume electron microscopy
array tomography

https://doi.org/10.4233/uuid:951afaeb-1f03-45cb-8a86-cacbf9ca0f64

978-94-6366-603-9

Institutional Repository

doctoral thesis

© 2022 R. Lane