Croop, B. | Search bioRxiv

bioRxiv2018

Flat-field illumination for quantitative fluorescence imaging

The uneven illumination of a Gaussian profile makes quantitative analysis highly challenging in laser-based wide-field fluorescence microscopy. Here we present flat-field illumination (FFI) where the Gaussian beam is reshaped into a uniform flat-top profile using a high-precision refractive optical component. The long working distance and high spatial coherence of FFI allows us to accomplish uniform epi and TIRF illumination for multi-color single-molecule imaging. In addition, high-throughput borderless imaging is demonstrated with minimal image overlap.

biophysics↗

bioRxiv2017

Endogenous α-SYN protein analysis on human brain tissues using single-molecule pull-down assay

Alpha-synuclein (-SYN) is a central molecule in Parkinsons disease pathogenesis. Despite several studies, the molecular nature of endogenous -SYN especially in human brain samples is still not well understood due to the lack of reliable methods and the limited amount of bio-specimens. Here, we introduce -SYN single-molecule pull-down (-SYN SiMPull) assay combined with in vivo protein crosslinking to count individual -SYN protein and assess its native oligomerization states from biological samples including human postmortem brains. A SiMPull assay enables us to count the number of immuno-precipitated proteins, and reveal the stoichiometry of protein complexes by single-molecule fluorescence imaging. This powerful assay can be highly useful in diagnostic applications using various specimens for neurodegenerative diseases including Alzheimers disease and Parkinsons disease.

biophysics↗