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Using New 3D CLEM Imaging Technique to Investigate the Effects of Substrate Mechanics on Cellular Uptake of Nanoparticle

In the past years, there have been many new developments in biological imaging, ranging from synchrotron X-ray crystallography for protein structure to 3D maps of entire human body [1]. Confocal microscopy, one of the most rapidly growing areas, is commonly used to construct the 3D images of biological samples where the area of interest is stained with fluorescent dyes conjugated to specific targeting antibodies. However, the physical resolution limit of light microscopy (~0.2μm) does not permit visualization of features that are on the nanoscale. Transmission electron microscopy, TEM, is another popular technique where serial sectioning has permitted 3D reconstruction of biological elements. However, this technique is very difficult and requires extremely accurate positioning of the sections, which are each floated from the air/water interface. Lately, focused ion beam (FIB), has been introduced as a viable alternative, which can produce rapid and precise milling (sectioning) of the sample. In combination with scanning electron microscopy (SEM), it becomes a straightforward and powerful tool for 3D imaging of biological samples. Yet, despite its accuracy and high resolution, the EM techniques are not able to uniquely identify the proteins, which comprise the observed structures, or functional domains and receptors which determine the structure of the adhered cells. Hence immunohistochemical staining with fluorescently labeled antibodies has recently been used in combination with electron microsopy, otherwise known as, correlative light and electron microscopy (CLEM). It is a convenient technique which can be used to obtain comprehensive 3D images of tissues elucidating structure, composition, and cell lineage.