Single-shot, shadowless total internal reflection fluorescence microscopy via annular fiber bundle.

We demonstrate a method of generating instantaneous and uniform total internal reflection fluorescence (TIRF) excitation by using an annular fiber bundle and spatially incoherent light sources. We show the flexibility of our method in that it can generate TIRF excitation with either a laser light source or an LED of different wavelengths, and facilitate switching between TIRF and epi illumination. In this report we detail the design of the fiber bundle, then demonstrate the performance via single-molecule imaging in the presence of high background and high throughput, and uniform TIRF imaging of cells over a large field of view. Our versatile method will enable quantitative shadowless TIRF imaging.

Recent advancement of light-based single-molecule approaches for studying biomolecules.

Recent advances in single-molecule techniques have led to new discoveries in analytical chemistry, biophysics, and medicine. Understanding the structure and behavior of single biomolecules provides a wealth of information compared to studying large ensembles. However, developing single-molecule techniques is challenging and requires advances in optics, engineering, biology, and chemistry. In this paper, we will review the state of the art in single-molecule applications with a focus over the last few years of development. The advancements covered will mainly include light-based in vitro methods, and we will discuss the fundamentals of each with a focus on the platforms themselves. We will also summarize their limitations and current and future applications to the wider biological and chemical fields. This article is categorized under: Laboratory Methods and Technologies > Imaging Laboratory Methods and Technologies > Macromolecular Interactions, Methods Analytical and Computational Methods > Analytical Methods.

Facile single-molecule pull-down assay for analysis of endogenous proteins.

The single-molecule pull-down (SiMPull) assay analyzes molecular complexes in physiological conditions from cell or tissue lysates. Currently the approach requires a lengthy sample preparation process, which has largely prevented the widespread adoption of this technique in bioanalysis. Here, we present a simplified SiMPull assay based upon dichlorodimethylsilane-Tween-20 passivation and F(ab) fragment labeling. Our passivation is a much shorter process than the standard polyethylene glycol passivation used in most single-molecule studies. The use of F(ab) fragments for indirect fluorescent labeling rather than divalent F(ab')2 or whole IgG antibodies allows for the pre-incubation of the detection antibodies, reducing the sample preparation time for single-molecule immunoprecipitation samples. We examine the applicability of our approach to recombinant proteins and endogenous proteins from mammalian cell lysates.

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.

Endogenous Alpha-Synuclein Protein Analysis from Human Brain Tissues Using Single-Molecule Pull-Down Assay.

Alpha-synuclein (α-SYN) is a central molecule in Parkinson's disease pathogenesis. Despite several studies, the molecular nature of endogenous α-SYN especially in human brain samples is still not well understood due to the lack of reliable methods and the limited amount of biospecimens. Here, we introduce α-SYN single-molecule pull-down (α-SYN SiMPull) assay combined with in vivo protein crosslinking to count individual α-SYN protein and assess its native oligomerization states from biological samples including human postmortem brains. This powerful single-molecule assay can be highly useful in diagnostic applications using various specimens for neurodegenerative diseases including Alzheimer's disease and Parkinson's disease.