Ron R. Lin

Development of new photon-counting detectors for single-molecule fluorescence microscopy

Two optical configurations are commonly used in single-molecule fluorescence microscopy: point-like excitation and detection to study freely diffusing molecules, and wide field illumination and detection to study surface immobilized or slowly diffusing molecules. Both approaches have common features, but also differ in significant aspects. In particular, they use different detectors, which share some requirements but also have major technical differences. Currently, two types of detectors best fulfil the needs of each approach: single-photon-counting avalanche diodes (SPADs) for point-like detection, and electron-multiplying charge-coupled devices (EMCCDs) for wide field detection. However, there is room for improvements in both cases. The first configuration suffers from low throughput owing to the analysis of data from a single location. The second, on the other hand, is limited to relatively low frame rates and loses the benefit of single-photon-counting approaches. During the past few years, new developments in point-like and wide field detectors have started addressing some of these issues. Here, we describe our recent progresses towards increasing the throughput of single-molecule fluorescence spectroscopy in solution using parallel arrays of SPADs. We also discuss our development of large area photon-counting cameras achieving subnanosecond resolution for fluorescence lifetime imaging applications at the single-molecule level.

High-throughput single-molecule optofluidic analysis

We describe a high-throughput, automated single-molecule measurement system, equipped with microfluidics. The microfluidic mixing device has integrated valves and pumps to accurately accomplish titration of biomolecules with picoliter resolution. We demonstrate that the approach enabled rapid sampling of biomolecule conformational landscape and of enzymatic activity, in the form of transcription by Escherichia coli RNA polymerase, as a function of the chemical environment.

IDOL Stimulates Clathrin-Independent Endocytosis and Multivesicular Body-Mediated Lysosomal Degradation of the Low-Density Lipoprotein Receptor

ABSTRACT The low-density lipoprotein receptor (LDLR) is a critical determinant of plasma cholesterol levels that internalizes lipoprotein cargo via clathrin-mediated endocytosis. Here, we show that the E3 ubiquitin ligase IDOL stimulates a previously unrecognized, clathrin-independent pathway for LDLR internalization. Real-time single-particle tracking and electron microscopy reveal that IDOL is recruited to the plasma membrane by LDLR, promotes LDLR internalization in the absence of clathrin or caveolae, and facilitates LDLR degradation by shuttling it into the multivesicular body (MVB) protein-sorting pathway. The IDOL-dependent degradation pathway is distinct from that mediated by PCSK9 as only IDOL employs ESCRT (endosomal-sorting complex required for transport) complexes to recognize and traffic LDLR to lysosomes. Small interfering RNA (siRNA)-mediated knockdown of ESCRT-0 (HGS) or ESCRT-I (TSG101) components prevents IDOL-mediated LDLR degradation. We further show that USP8 acts downstream of IDOL to deubiquitinate LDLR and that USP8 is required for LDLR entry into the MVB pathway. These results provide key mechanistic insights into an evolutionarily conserved pathway for the control of lipoprotein receptor expression and cellular lipid uptake.

Enzymatically Incorporated Genomic Tags for Optical Mapping of DNA-Binding Proteins

Affordable DNA sequencing is revolutionizing genetic research and is enabling multiple novel biomedical applications. Among the inherent properties of today’s high-throughput sequencing technologies is the fact that it compiles long-range sequences from the assembly of numerous short-read data.[1] This leads to two fundamental limitations: loss of long-range contextual information on the single-genome level and difficulties coping with repetitive or variable genomic regions. Optical mapping and its variants[2–10] rely on the visualization of individual, long (50 kb–1000 kb) DNA molecules and extraction of genomic information by fluorescent labeling of the DNA. These techniques lack the resolution of sequencing but offer genomic context and therefore are attractive both in combination with sequencing to aid in sequence assembly[11–13] and for investigation of genomic structural variations on the individual chromosome level.[14, 15] Such variations include deletions, duplications, copy-number variants (CNVs), insertions, inversions, and translocations, all of which have a major impact on the phenotypic variations within a population (or somatic mutations, important in cancer progression). In addition, the available information content of the genome extends beyond the sequence, and the long-range data offered by optical mapping may provide crucial information regarding the distribution of DNA-binding proteins such as transcription factors and histones along the genome.

Toward Single-Molecule Optical Mapping of the Epigenome

The past decade has seen an explosive growth in the utilization of single-molecule techniques for the study of complex systems. The ability to resolve phenomena otherwise masked by ensemble averaging has made these approaches especially attractive for the study of biological systems, where stochastic events lead to inherent inhomogeneity at the population level. The complex composition of the genome has made it an ideal system to study at the single-molecule level, and methods aimed at resolving genetic information from long, individual, genomic DNA molecules have been in use for the last 30 years. These methods, and particularly optical-based mapping of DNA, have been instrumental in highlighting genomic variation and contributed significantly to the assembly of many genomes including the human genome. Nanotechnology and nanoscopy have been a strong driving force for advancing genomic mapping approaches, allowing both better manipulation of DNA on the nanoscale and enhanced optical resolving power for analysis of genomic information. During the past few years, these developments have been adopted also for epigenetic studies. The common principle for these studies is the use of advanced optical microscopy for the detection of fluorescently labeled epigenetic marks on long, extended DNA molecules. Here we will discuss recent single-molecule studies for the mapping of chromatin composition and epigenetic DNA modifications, such as DNA methylation.

Single-molecule FRET experiments with a red-enhanced custom technology SPAD

Single-molecule fluorescence spectroscopy of freely diffusing molecules in solution is a powerful tool used to investigate the properties of individual molecules. Single-Photon Avalanche Diodes (SPADs) are the detectors of choice for these applications. Recently a new type of SPAD detector was introduced, dubbed red-enhanced SPAD (RE-SPAD), with good sensitivity throughout the visible spectrum and with excellent timing performance. We report a characterization of this new detector for single-molecule fluorescence resonant energy transfer (smFRET) studies on freely diffusing molecules in a confocal geometry and alternating laser excitation (ALEX) scheme. We use a series of doubly-labeled DNA molecules with donor-to-acceptor distances covering the whole range of useful FRET values. Both intensity-based (μs-ALEX) and lifetime-based (ns-ALEX) measurements are presented and compared to identical measurements performed with standard thick SPADs. Our results demonstrate the great potential of this new detector for smFRET measurements and beyond.

Parallel multispot smFRET analysis using an 8-pixel SPAD array

Single-molecule Förster resonance energy transfer (smFRET) is a powerful tool for extracting distance information between two fluorophores (a donor and acceptor dye) on a nanometer scale. This method is commonly used to monitor binding interactions or intra- and intermolecular conformations in biomolecules freely diffusing through a focal volume or immobilized on a surface. The diffusing geometry has the advantage to not interfere with the molecules and to give access to fast time scales. However, separating photon bursts from individual molecules requires low sample concentrations. This results in long acquisition time (several minutes to an hour) to obtain sufficient statistics. It also prevents studying dynamic phenomena happening on time scales larger than the burst duration and smaller than the acquisition time. Parallelization of acquisition overcomes this limit by increasing the acquisition rate using the same low concentrations required for individual molecule burst identification. In this work we present a new two-color smFRET approach using multispot excitation and detection. The donor excitation pattern is composed of 4 spots arranged in a linear pattern. The fluorescent emission of donor and acceptor dyes is then collected and refocused on two separate areas of a custom 8-pixel SPAD array. We report smFRET measurements performed on various DNA samples synthesized with various distances between the donor and acceptor fluorophores. We demonstrate that our approach provides identical FRET efficiency values to a conventional single-spot acquisition approach, but with a reduced acquisition time. Our work thus opens the way to high-throughput smFRET analysis on freely diffusing molecules.

Parallel multispot smFRET analysis using an 8-pixel SPAD array.

Single-molecule Förster resonance energy transfer (smFRET) is a powerful tool for extracting distance information between two fluorophores (a donor and acceptor dye) on a nanometer scale. This method is commonly used to monitor binding interactions or intra- and intermolecular conformations in biomolecules freely diffusing through a focal volume or immobilized on a surface. The diffusing geometry has the advantage to not interfere with the molecules and to give access to fast time scales. However, separating photon bursts from individual molecules requires low sample concentrations. This results in long acquisition time (several minutes to an hour) to obtain sufficient statistics. It also prevents studying dynamic phenomena happening on time scales larger than the burst duration and smaller than the acquisition time. Parallelization of acquisition overcomes this limit by increasing the acquisition rate using the same low concentrations required for individual molecule burst identification. In this work we present a new two-color smFRET approach using multispot excitation and detection. The donor excitation pattern is composed of 4 spots arranged in a linear pattern. The fluorescent emission of donor and acceptor dyes is then collected and refocused on two separate areas of a custom 8-pixel SPAD array. We report smFRET measurements performed on various DNA samples synthesized with various distances between the donor and acceptor fluorophores. We demonstrate that our approach provides identical FRET efficiency values to a conventional single-spot acquisition approach, but with a reduced acquisition time. Our work thus opens the way to high-throughput smFRET analysis on freely diffusing molecules.