Making an unbiased library

Sequencing the genome of single cells gives insight into issues such as cell-to-cell heterogeneity and genome instability. Key to single-cell sequencing techniques are whole-genome amplification (WGA) methods that provide sufficient DNA for next-generation sequencing. Current WGA methods have been hampered by low accuracy and spatial resolution of gene copy numbers and by low amplification fidelity. Chen et al. report an improved single-cell WGA method, Linear Amplification via Transposon Insertion (LIANTI). The DNA is randomly fragmented by Tn5 transposition of a transposon that includes a T7 promoter, which allows linear amplification. The authors used the method to determine the spectrum of single-nucleotide variations in a single human cell after ultraviolet radiation.

Science, this issue p. 189

Imaging with molecular vibrations

The vibrational spectra of biomolecules could in principle image cells and tissue without added markers. Practically, several technical problems need to be overcome to achieve sufficient imaging depths, resolution, and data acquisition speed. Cheng and Xie review emerging bioimaging methods for use in the lab and the clinic.

Science, this issue p. 10.1126/science.aaa8870

Quantitative SRS microscopy can detect human brain tumor infiltration with high sensitivity and specificity, even in tissues appearing grossly normal.

Stimulated Raman scattering microscopy provides a rapid, label-free means of detecting tumor infiltration of brain tissue ex vivo and in vivo.

Abstract

Super-resolution imaging in live Escherichia coli reveals protein clusters that sequester DNA loci and organize the chromosome.

Skin-Deep Raman Spectroscopy

Raman spectroscopy allows for molecular identification via vibrational spectra at optical wavelengths. However, if the optical signal is scattered, as occurs when trying to image tissue, the signal becomes very weak, and it becomes difficult to image a sample with high time resolution. Saar et al. (p. 1368) now show that by improving the optics and electronics of the acquisition of the backscattered signal, stimulated Raman scattering spectroscopy can be performed at video rates on human skin, which should enable label-free studies of tissues and, for example, the tracking of the delivery of a drug.

Abstract

Abstract

Label-free chemical contrast is highly desirable in biomedical imaging. Spontaneous Raman microscopy provides specific vibrational signatures of chemical bonds, but is often hindered by low sensitivity. Here we report a three-dimensional multiphoton vibrational imaging technique based on stimulated Raman scattering (SRS). The sensitivity of SRS imaging is significantly greater than that of spontaneous Raman microscopy, which is achieved by implementing high-frequency (megahertz) phase-sensitive detection. SRS microscopy has a major advantage over previous coherent Raman techniques in that it offers background-free and readily interpretable chemical contrast. We show a variety of biomedical applications, such as differentiating distributions of omega-3 fatty acids and saturated lipids in living cells, imaging of brain and skin tissues based on intrinsic lipid contrast, and monitoring drug delivery through the epidermis.

Abstract

Abstract

By monitoring fluorescently labeled lactose permease with single-molecule sensitivity, we investigated the molecular mechanism of how an Escherichia coli cell with the lac operon switches from one phenotype to another. At intermediate inducer concentrations, a population of genetically identical cells exhibits two phenotypes: induced cells with highly fluorescent membranes and uninduced cells with a small number of membrane-bound permeases. We found that this basal-level expression results from partial dissociation of the tetrameric lactose repressor from one of its operators on looped DNA. In contrast, infrequent events of complete dissociation of the repressor from DNA result in large bursts of permease expression that trigger induction of the lac operon. Hence, a stochastic single-molecule event determines a cell's phenotype.

Abstract

Transcription factors regulate gene expression through their binding to DNA. In a living Escherichia coli cell, we directly observed specific binding of a lac repressor, labeled with a fluorescent protein, to a chromosomal lac operator. Using single-molecule detection techniques, we measured the kinetics of binding and dissociation of the repressor in response to metabolic signals. Furthermore, we characterized the nonspecific binding to DNA, one-dimensional (1D) diffusion along DNA segments, and 3D translocation among segments through cytoplasm at the single-molecule level. In searching for the operator, a lac repressor spends ∼90% of time nonspecifically bound to and diffusing along DNA with a residence time of <5 milliseconds. The methods and findings can be generalized to other nucleic acid binding proteins.

Abstract

The combination of specific probes and advanced optical microscopy now allows quantitative probing of biochemical reactions in living cells. On selected systems, one can detect and track a particular protein with single-molecule sensitivity, nanometer spatial precision, and millisecond time resolution. Metabolites, usually difficult to detect, can be imaged and monitored in living cells with coherent anti-Stokes Raman scattering microscopy. Here, we describe the application of these techniques in studying gene expression, active transport, and lipid metabolism.

Abstract

We directly observed real-time production of single protein molecules in individual Escherichia coli cells. A fusion protein of a fast-maturing yellow fluorescent protein (YFP) and a membrane-targeting peptide was expressed under a repressed condition. The membrane-localized YFP can be detected with single-molecule sensitivity. We found that the protein molecules are produced in bursts, with each burst originating from a stochastically transcribed single messenger RNA molecule, and that protein copy numbers in the bursts follow a geometric distribution. The quantitative study of low-level gene expression demonstrates the potential of single-molecule experiments in elucidating the workings of fundamental biological processes in living cells.

Imaging of giant vesicles: Coherent anti-Stokes Raman scattering (CARS) microscopy is used for imaging of giant vesicles of binary mixtures of lipids. The vibrational selectivity of the CARS microscope allows determination of molecules based on differences in the vibrational modes. The picture shows Raman and CARS spectra as well as CARS images of a deuterated giant unilamellar vesicle.

Abstract

Electron transfer is used as a probe for angstrom-scale structural changes in single protein molecules. In a flavin reductase, the fluorescence of flavin is quenched by a nearby tyrosine residue by means of photo-induced electron transfer. By probing the fluorescence lifetime of the single flavin on a photon-by-photon basis, we were able to observe the variation of flavin-tyrosine distance over time. We could then determine the potential of mean force between the flavin and the tyrosine, and a correlation analysis revealed conformational fluctuation at multiple time scales spanning from hundreds of microseconds to seconds. This phenomenon suggests the existence of multiple interconverting conformers related to the fluctuating catalytic reactivity.

Abstract

We used a multiplexed approach based on flow-stretched DNA to monitor the enzymatic digestion of λ-phage DNA by individual bacteriophage λ exonuclease molecules. Statistical analyses of multiple single-molecule trajectories observed simultaneously reveal that the catalytic rate is dependent on the local base content of the substrate DNA. By relating single-molecule kinetics to the free energies of hydrogen bonding and base stacking, we establish that the melting of a base from the DNA is the rate-limiting step in the catalytic cycle. The catalytic rate also exhibits large fluctuations independent of the sequence, which we attribute to conformational changes of the enzyme-DNA complex.