Graduate Student Dane Santa

"Observing Disease Mechanisms at the Nanoscale Using Stimulated Emission Depletion Microscopy"

Light microscopy is highly influential in biomedical research due to minimal invasiveness, high biomarker specificity via fluorescent labelling, and live cell imaging compatibility. Ultimately limited by diffraction, the maximum resolution possible using conventional light microscopy is around 200 nm. This limitation prevents the imaging of subcellular disease mechanisms that occur at the nanoscale. Stimulated emission depletion (STED) is a fluorescence-based technique that selectively deactivates fluorophores by an excitation beam [1]. This increases resolution by minimizing the focal point area of illumination. STED can therefore impact the diagnostics and treatment of various diseases by elucidating subcellular mechanisms.

Querol-Vilaseca et al. used STED in tandem with array tomography to construct 3D images of soluble amyloid-β structures implicated in the pathogenesis of Alzheimer’s disease [2]. Previously, a lack of nanoscale resolution optical techniques prevented the study of these structures in intact human brain samples. This method uncovered differences in Amyloid-β between sporadic and autosomal dominant disease states, which traditional immunohistochemistry could not discern.

Using STED, Bergstrand et al. elucidated how tumor cells influence selective protein redistribution in platelets during cancer development [3]. The adhesion protein P-selectin was found to redistribute into distinct nanoscale patterns when incubated with various cancer cells. Images captured via STED were then used to develop a classification for both activated and inactivated platelets incubated in various environments. Ideally, STED can inform the development of platelet-based cancer diagnostic methods.

Baharom et al. established a novel method for visualizing early influenza A virus particle trafficking in human dendritic cells using three-color STED [4]. This technique improves upon the high cost, expert analysis, and laborious sample preparation required by electron microscopy. Dendritic cells act as a primary line of defense in early pathogenesis, but they are susceptible to viral entry via classical endocytic pathways. Visualizing where viral particles are located during endosomal uptake increases the understanding of immune cell impairment during infection.

(1) Blom, H. and Widengren, J. Stimulated Emission Depletion Microscopy. Chem. Rev. 2017, 117, 7377-7427. DOI: 10.1021/acs.chemrev.6b00653
(2) Querol-Vilaseca et al. Nanoscale structure of amyloid-β plaques in Alzheimer’s disease. Sci. Rep. 2018, 9, 5181. DOI: 10.1038/s41598-019-41443-3
(3) Bergstrand, J. et al. Super-resolution microscopy can identify specific protein distribution patterns in platelets incubated with cancer cells. Nanoscale. 2019. 11, 10023. DOI: 10.1039/c9nr01967g
(4) Baharom, F. et al. Visualization of early influenza A virus trafficking in human dendritic cells using STED microscopy. PLoS One. 2017. 12 (6), e0177920. DOI: 10.1371/journal.pone.0177920