Application Of Single-molecule Imaging Research Articles

Deep learning in single-molecule imaging and analysis: recent advances and prospects.

Single-molecule microscopy is advantageous in characterizing heterogeneous dynamics at the molecular level. However, there are several challenges that currently hinder the wide application of single molecule imaging in bio-chemical studies, including how to perform single-molecule measurements efficiently with minimal run-to-run variations, how to analyze weak single-molecule signals efficiently and accurately without the influence of human bias, and how to extract complete information about dynamics of interest from single-molecule data. As a new class of computer algorithms that simulate the human brain to extract data features, deep learning networks excel in task parallelism and model generalization, and are well-suited for handling nonlinear functions and extracting weak features, which provide a promising approach for single-molecule experiment automation and data processing. In this perspective, we will highlight recent advances in the application of deep learning to single-molecule studies, discuss how deep learning has been used to address the challenges in the field as well as the pitfalls of existing applications, and outline the directions for future development.

Single-Molecule Imaging in Living Plant Cells: A Methodological Review.

Single-molecule imaging is emerging as a revolutionary approach to studying fundamental questions in plants. However, compared with its use in animals, the application of single-molecule imaging in plants is still underexplored. Here, we review the applications, advantages, and challenges of single-molecule fluorescence imaging in plant systems from the perspective of methodology. Firstly, we provide a general overview of single-molecule imaging methods and their principles. Next, we summarize the unprecedented quantitative details that can be obtained using single-molecule techniques compared to bulk assays. Finally, we discuss the main problems encountered at this stage and provide possible solutions.

Rational Design of Bright Long Fluorescence Lifetime Dyad Fluorophores for Single Molecule Imaging and Detection.

Increasing demand for detecting single molecules in challenging environments has raised the bar for the fluorophores used. To achieve better resolution and/or contrast in fluorescence microscopy, it is now essential to use bright and stable dyes with tailored photophysical properties. While long fluorescence lifetime fluorophores offer many advantages in time-resolved imaging, their inherently lower molar absorption coefficient has limited applications in single molecule imaging. Here we propose a generic approach to prepare bright, long fluorescence lifetime dyad fluorophores comprising an absorbing antenna chromophore with high absorption coefficient linked to an acceptor emitter with a long fluorescence lifetime. We introduce a dyad consisting of a perylene antenna and a triangulenium emitter with 100% energy transfer from donor to acceptor. The dyad retained the long fluorescence lifetime (∼17 ns) and high quantum yield (75%) of the triangulenium emitter, while the perylene antenna increased the molar absorption coefficient (up to 5 times) in comparison to the free triangulenium dye. These triangulenium based dyads with significantly improved brightness can now be detected at the single molecule level and easily discriminated from bright autofluorescence by time-gated and other lifetime-based detection schemes.

Support Recovery for Sparse Signals With Unknown Non-Stationary Modulation

The problem of estimating a sparse signal from low dimensional noisy observations arises in many applications, including super resolution, signal deconvolution, and radar imaging. In this paper, we consider a sparse signal model with non-stationary modulations, in which each dictionary atom contributing to the observations undergoes an unknown, distinct modulation. By applying the lifting technique, under the assumption that the modulating signals live in a common subspace, we recast this sparse recovery and non-stationary blind demodulation problem as the recovery of a column-wise sparse matrix from structured linear observations, and propose to solve it via block $\ell_{1}$-norm regularized quadratic minimization. Due to observation noise, the sparse signal and modulation process cannot be recovered exactly. Instead, we aim to recover the sparse support of the ground truth signal and bound the recovery errors of the signal's non-zero components and the modulation process. In particular, we derive sufficient conditions on the sample complexity and regularization parameter for exact support recovery and bound the recovery error on the support. Numerical simulations verify and support our theoretical findings, and we demonstrate the effectiveness of our model in the application of single molecule imaging.

Proteome-wide Quantification of Labeling Homogeneity at the Single Molecule Level.

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a fluorescent dye and are spotted by a photodetector following their separation. Single molecule fluorescence imaging can provide ultrasensitive protein detection with its ability for visualizing individual fluorescent molecules. However, the application of this powerful imaging method to electrophoresis assays is hampered by the lack of ways to characterize the homogeneity of fluorescent labeling of each protein species across the proteome. Here, we developed a method to evaluate the labeling homogeneity across the proteome based on a single molecule fluorescence imaging assay. In our measurement using a HeLa cell sample, the proportion of proteins labeled with at least one dye, which we termed 'labeling occupancy' (LO), was determined to range from 50% to 90%, supporting the high potential of the application of single molecule imaging to sensitive and precise proteome analysis.

Fluorogen Activating Peptides that Enhance Photostability through Encapsulation of the Fluorogen

Biological imaging has profited from the development of a variety of fluorescent probes such as organic dyes, quantum dots and fluorescent proteins. More recently, Fluorogen Activating Peptides (FAP) have been developed through directed evolution. These FAPs bind non-covalently to an otherwise non-fluorescent molecule (fluorogen) to form fluorescent complexes. FAPs have been successfully employed in various live-cell imaging applications in the past two years. For live-cell and single molecule imaging applications, photostability is a key requirement for any fluorescent probe. FAPs have been shown to exhibit high photostability in cell-imaging applications. An understanding of the mechanism underlying the photostability of these FAPs will help us in future to develop FAP clones with higher photostability. In this study, we have characterized the photobleaching behavior of two Malachite green (MG) binding FAP clones: dL5NP138 and HL4. These studies have been performed at a single molecule level. These studies have shown FAPs to be more photostable compared to a widely used single molecule dye, Cy5, at a single molecule condition. Additionally we have exposed FAPs and the Green fluorescent protein to vigorous oxidizing conditions to assess the encapsulation of the chromophore in the protein environment.Our studies show that FAPs protect MG (the fluorogen) from external environments that are rich in reactive oxygen species. The same behavior was observed by GFP compared to a synthetic soluble GFP chromophore. Using single molecule approaches in the presence of excess dye, we have demonstrated that these MG-binding FAPs are photostable through encapsulation of the fluorogen, and not by exchange of the bleached fluorogen. This mechanism provides MG-FAP complexs that can be used for single molecule detection over periods greater than Cy5 labels, yet with the convenience of genetically encoded reporters such as GFP.

Single Molecule Genotyping by TIRF Microscopy

As part of a programme to develop a metrological framework for single molecule measurements in biology, we have investigated the applications of single molecule imaging to genomics. Specifically, we have developed a technique for measuring the frequencies of single nucleotide polymorphisms (SNPs) in complex or pooled samples of DNA. We believe that this technique has applications to statistical genotyping-the identification of correlations between SNP frequencies and particular phenotypes-and other areas where it is desirable to track the frequencies of SNPs in complex DNA populations.

Single-molecule signal enhancement using a high-impedance ground plane substrate

We introduce a “high-impedance ground plane” (HIGP) substrate for use in single-molecule imaging applications. The HIGP is a quarter-wave-thick layer of dielectric material is deposited over a regular metallic mirror. Comparison of confocal fluorescence images of single DiI molecules on HIGP substrates and on glass substrates indicates a dramatic increase in the number of collected photons for the HIGP substrate. Signal enhancement ranges from a factor of 8 at low excitation powers to a lifetime-limited factor of 2 at higher powers.