In the past decades, advances in microscopy have made it possible to study the dynamics of individual biomolecules in vitro and resolve intramolecular kinetics that would otherwise be hidden in ...ensemble averages. More recently, single-molecule methods have been used to image, localize, and track individually labeled macromolecules in the cytoplasm of living cells, allowing investigations of intermolecular kinetics under physiologically relevant conditions. In this review, we illuminate the particular advantages of single-molecule techniques when studying kinetics in living cells and discuss solutions to specific challenges associated with these methods.
Mapping a genetic perturbation to a change in phenotype is at the core of biological research. Advances in microscopy have transformed these studies, but they have largely been confined to examining ...a few strains or cell lines at a time. In parallel, there has been a revolution in creating synthetic libraries of genetically altered cells with relative ease. Here we describe methods that combine these powerful tools to perform live-cell imaging of pool-generated strain libraries for improved biological discovery.
The rise of antibiotic-resistant bacterial infections poses a global threat. Antibiotic resistance development is generally studied in batch cultures which conceals the heterogeneity in cellular ...responses. Using single-cell imaging, we studied the growth response of
to sub-inhibitory and inhibitory concentrations of nine antibiotics. We found that the heterogeneity in growth increases more than what is expected from growth rate reduction for three out of the nine antibiotics tested. For two antibiotics (rifampicin and nitrofurantoin), we found that sub-populations were able to maintain growth at lethal antibiotic concentrations for up to 10 generations. This perseverance of growth increased the population size and led to an up to 40-fold increase in the frequency of antibiotic resistance mutations in gram-negative and gram-positive species. We conclude that antibiotic perseverance is a common phenomenon that has the potential to impact antibiotic resistance development across pathogenic bacteria.
The emergence and spread of antibiotic-resistant bacteria are aggravated by incorrect prescription and use of antibiotics. A core problem is that there is no sufficiently fast diagnostic test to ...guide correct antibiotic prescription at the point of care. Here, we investigate if it is possible to develop a point-of-care susceptibility test for urinary tract infection, a disease that 100 million women suffer from annually and that exhibits widespread antibiotic resistance. We capture bacterial cells directly from samples with low bacterial counts (10⁴ cfu/mL) using a custom-designed microfluidic chip and monitor their individual growth rates using microscopy. By averaging the growth rate response to an antibiotic over many individual cells, we can push the detection time to the biological response time of the bacteria. We find that it is possible to detect changes in growth rate in response to each of nine antibiotics that are used to treat urinary tract infections in minutes. In a test of 49 clinical uropathogenic Escherichia coli (UPEC) isolates, all were correctly classified as susceptible or resistant to ciprofloxacin in less than 10 min. The total time for antibiotic susceptibility testing, from loading of sample to diagnostic readout, is less than 30 min, which allows the development of a point-of-care test that can guide correct treatment of urinary tract infection.
Isogenic E. coli cells growing in a constant environment display significant variability in growth rates, division sizes, and generation times. The guiding principle appears to be that each cell, ...during one generation, adds a size increment that is uncorrelated to its birth size. Here, we investigate the mechanisms underlying this “adder” behavior by mapping the chromosome replication cycle to the division cycle of individual cells using fluorescence microscopy. We have found that initiation of chromosome replication is triggered at a fixed volume per chromosome independent of a cell’s birth volume and growth rate. Each initiation event is coupled to a division event after a growth-rate-dependent time. We formalize our findings in a model showing that cell-to-cell variation in division timing and cell size is mainly driven by variations in growth rate. The model also explains why fast-growing cells display adder behavior and correctly predict deviations from the adder behavior at slow growth.
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•Replication initiates at a nearly fixed volume per chromosome for all growth rates•The time from initiation to division depends on the individual cell’s growth rate•Variation in growth rate sets the variation in generation time and division size•E. coli appears as a “sizer” at slow growth and an “adder” at fast growth
Cell-to-cell variation in division timing and cell size in E. coli is due to differences in growth rate, whereas the timing of replication is triggered at an invariant fixed volume per chromosome.
We provide an analytical tool based on a variational Bayesian treatment of hidden Markov models to combine the information from thousands of short single-molecule trajectories of intracellularly ...diffusing proteins. The method identifies the number of diffusive states and the state transition rates. Using this method we have created an objective interaction map for Hfq, a protein that mediates interactions between small regulatory RNAs and their mRNA targets.
Antimicrobial resistance is an increasing problem on a global scale. Rapid antibiotic susceptibility testing (AST) is urgently needed in the clinic to enable personalized prescriptions in ...high-resistance environments and to limit the use of broad-spectrum drugs. Current rapid phenotypic AST methods do not include species identification (ID), leaving time-consuming plating or culturing as the only available option when ID is needed to make the sensitivity call. Here we describe a method to perform phenotypic AST at the single-cell level in a microfluidic chip that allows subsequent genotyping by in situ FISH. By stratifying the phenotypic AST response on the species of individual cells, it is possible to determine the susceptibility profile for each species in a mixed sample in 2 h. In this proof-of-principle study, we demonstrate the operation with four antibiotics and mixed samples with combinations of seven species.
Single-particle tracking offers a noninvasive high-resolution probe of biomolecular reactions inside living cells. However, efficient data analysis methods that correctly account for various noise ...sources are needed to realize the full quantitative potential of the method. We report algorithms for hidden Markov-based analysis of single-particle tracking data, which incorporate most sources of experimental noise, including heterogeneous localization errors and missing positions. Compared to previous implementations, the algorithms offer significant speedups, support for a wider range of inference methods, and a simple user interface. This will enable more advanced and exploratory quantitative analysis of single-particle tracking data.
Summary
With the realization that bacteria display phenotypic variability among cells and exhibit complex subcellular organization critical for cellular function and behavior, microscopy has ...re‐emerged as a primary tool in bacterial research during the last decade. However, the bottleneck in today's single‐cell studies is quantitative image analysis of cells and fluorescent signals. Here, we address current limitations through the development of Oufti, a stand‐alone, open‐source software package for automated measurements of microbial cells and fluorescence signals from microscopy images. Oufti provides computational solutions for tracking touching cells in confluent samples, handles various cell morphologies, offers algorithms for quantitative analysis of both diffraction and non‐diffraction‐limited fluorescence signals and is scalable for high‐throughput analysis of massive datasets, all with subpixel precision. All functionalities are integrated in a single package. The graphical user interface, which includes interactive modules for segmentation, image analysis and post‐processing analysis, makes the software broadly accessible to users irrespective of their computational skills.
Oufti is an interactive, open source software package built for high throughput quantification of microscopy images. Oufti performs cell detection, localization of diffraction‐limited spots as well as boundary determination of image objects beyond the diffraction limit. Due to parallel processing, it is able to perform these tasks on large (>1Gb) datasets, which can be further expanded by high performance computing centers. Oufti also provides post‐processing tools for the statistical characterization of data following image analysis.
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.
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