The epigenetic mark 5-hydroxymethylcytosine (5-hmC) is a distinct product of active DNA demethylation that is linked to gene regulation, development, and disease. In particular, 5-hmC levels dramatically decline in many cancers, potentially serving as an epigenetic biomarker. The noise associated with next-generation 5-hmC sequencing hinders reliable analysis of low 5-hmC containing tissues such as blood and malignant tumors. Additionally, genome-wide 5-hmC profiles generated by short-read sequencing are limited in providing long-range epigenetic information relevant to highly variable genomic regions, such as the 3.7 Mbp disease-related Human Leukocyte Antigen (HLA) region. We present a long-read, highly sensitive single-molecule mapping technology that generates hybrid genetic/epigenetic profiles of native chromosomal DNA. The genome-wide distribution of 5-hmC in human peripheral blood cells correlates well with 5-hmC DNA immunoprecipitation (hMeDIP) sequencing. However, the long single-molecule read-length of 100 kbp to 1 Mbp produces 5-hmC profiles across variable genomic regions that failed to show up in the sequencing data. In addition, optical 5-hmC mapping shows a strong correlation between the 5-hmC density in gene bodies and the corresponding level of gene expression. The single-molecule concept provides information on the distribution and coexistence of 5-hmC signals at multiple genomic loci on the same genomic DNA molecule, revealing long-range correlations and cell-to-cell epigenetic variation.

Optical genome mapping in nanochannels is a powerful genetic analysis method, complementary to deoxyribonucleic acid (DNA) sequencing. The method is based on detecting a pattern of fluorescent labels attached along individual DNA molecules. When such molecules are extended in nanochannels, the labels create a fluorescent genetic barcode that is used for mapping the DNA molecule to its genomic locus and identifying large-scale variation from the genome reference. Mapping resolution is currently limited by two main factors: the optical diffraction limit and the thermal fluctuations of DNA molecules suspended in the nanochannels. Here, we utilize single-molecule tracking and super-resolution localization in order to improve the mapping accuracy and resolving power of this genome mapping technique and achieve a 15-fold increase in resolving power compared to currently practiced methods. We took advantage of a naturally occurring genetic repeat array and labeled each repeat with custom-designed Trolox conjugated fluorophores for enhanced photostability. This model system allowed us to acquire extremely long image sequences of the equally spaced fluorescent markers along DNA molecules, enabling detailed characterization of nanoconfined DNA dynamics and quantitative comparison to the Odijk theory for confined polymer chains. We present a simple method to overcome the thermal fluctuations in the nanochannels and exploit single-step photobleaching to resolve subdiffraction spaced fluorescent markers along fluctuating DNA molecules with ∼100 bp resolution. In addition, we show how time-averaging over just ∼50 frames of 40 ms enhances mapping accuracy, improves mapping P-value scores by 3 orders of magnitude compared to nonaveraged alignment, and provides a significant advantage for analyzing structural variations between DNA molecules with similar sequence composition.

The human genome contains multiple layers of information that extend beyond the genetic sequence. In fact, identical genetics do not necessarily yield identical phenotypes as evident for the case of two different cell types in the human body. The great variation in structure and function displayed by cells with identical genetic background is attributed to additional genomic information content. This includes large-scale genetic aberrations, as well as diverse epigenetic patterns that are crucial for regulating specific cell functions. These genetic and epigenetic patterns operate in concert in order to maintain specific cellular functions in health and disease. Single-molecule optical genome mapping is a high-throughput genome analysis method that is based on imaging long chromosomal fragments stretched in nanochannel arrays. The access to long DNA molecules coupled with fluorescent tagging of various genomic information presents a unique opportunity to study genetic and epigenetic patterns in the genome at a single-molecule level over large genomic distances. Optical mapping entwines synergistically chemical, physical, and computational advancements, to uncover invaluable biological insights, inaccessible by sequencing technologies. Here we describe the method’s basic principles of operation, and review the various available mechanisms to fluorescently tag genomic information. We present some of the recent biological and clinical impact enabled by optical mapping and present recent approaches for increasing the method’s resolution and accuracy. Finally, we discuss how multiple layers of genomic information may be mapped simultaneously on the same DNA molecule, thus paving the way for characterizing multiple genomic observables on individual DNA molecules.

Fluorescent peptide nanodots (PNDs) are bioorganic nanoparticles self-assembled from peptide biomolecules of different origin and complexity. These recently discovered nanodots of biological origin are highly promising for biomedical imaging applications due to their biocompatibility, bright and tunable fluorescence over the entire visible range and photostability. Here we apply single-particle microscopy methods to study the photophysical properties of individual PNDs. We show that the fluorescence spectrum tunability, studied previously only for PND ensembles in solutions, origins at the single-particle level. Temporal dynamics measurements of the single particles reveal fluorescence lifetime in the range of nanoseconds and pronounced fluorescence blinking with continuous bright states of seconds. The latter provides a first evidence of quantum emitter transitions between two states (ON and OFF) in fluorescent PNDs. All these findings advance the understanding of the fluorescence mechanism of PNDs and provide strong motivation for using PNDs as fluorescent agents for various bioimaging and super-resolution techniques.

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