Date of Award

Spring 2022

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biomedical Engineering (ENAS)

First Advisor

Bewersdorf, Joerg

Abstract

Fluorescence light microscopy is an essential tool in biomedical research. In immunofluorescence, fluorophore-conjugated antibodies are used to detect specific proteins of interest in a fixed biological sample. With recently developed nanoscopy techniques, whole cells can be imaged at an isotropic spatial resolution of ~10 nm, revealing accurate protein distributions on the nanoscale. However, most localized proteins are imaged against a dark background, which forbids seeing the overall subcellular compartments (ultrastructural context) that encompass them. Electron microscopy (EM), on the other hand, offers a complete cellular overview on the scale of a few nanometers. However, EM fails to reliably detect specific molecules of interest. To this end, correlated light and electron microscopy (CLEM) techniques have emerged to combine the high molecular contrast of fluorescence microscopy with the ultrastructural imaging capabilities of EM. Despite the merits of CLEM, sample preparation and image alignment are extremely laborious, limiting this correlative approach to only proof-of-concept biological experiments. This thesis poses this specific question: why is light microscopy alone incapable of resolving the ultrastructural context of cells, despite extraordinary improvements in spatial resolution? We argue that the limitation stems from the physical properties of fluorescent dyes: dyes are ~1 nm in diameter, a size comparable to the distance between proteins in the densely crowded cell. If labeled in bulk, fluorescent dyes would sterically hinder and self-quench via electron transfer and dipole-dipole interactions, which would limit the achievable staining density and thereby the sampling necessary to resolve the crowded cellular interior. This thesis made the conceptual realization that if the sample protein content is isotropically expanded up to 20-fold in all three dimensions, the relative size of fluorescent dyes would shrink by the same factor. Here, the relative radius of a fluorescent dye would approach ~50 pm, which is comparable to the size of an osmium atom (~200 pm) used in heavy metal EM staining. Bulk fluorescence staining of the decrowded cell will therefore no longer be limited by sampling and quenching restrictions, and ultrastructural details, previously accessible with only EM, can now be revealed on a standard light microscope. We call the underlying sample preparation technique pan-Expansion Microscopy (pan-ExM). pan-ExM combines the philosophy of bulk- (pan-) staining of the total protein content with a newly developed Expansion Microscopy (ExM) protocol capable of 20-fold linear sample expansion and protein retention. We first develop pan-ExM in cultured cells as a proof-of-concept demonstration. We then develop the technique in dissociated neuronal cultures and in thick (~70 μm) mouse brain tissue sections to establish its applicability in neurobiological research. Finally, in a method we call panception, we demonstrate that the conceptual advance of pan-staining is also applicable to transmitted light microscopy. Using polymers of varying refractive indices and light-scattering analogs of fluorescent dyes, we show that sample ultrastructure can be imaged with brightfield microscopy, and that sample microstructure can be revealed with the un-aided eye.

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