Time-lapse microscopy

Summary

Time-lapse microscopy is time-lapse photography applied to microscopy. Microscope image sequences are recorded and then viewed at a greater speed to give an accelerated view of the microscopic process. Before the introduction of the video tape recorder in the 1960s, time-lapse microscopy recordings were made on photographic film. During this period, time-lapse microscopy was referred to as microcinematography. With the increasing use of video recorders, the term time-lapse video microscopy was gradually adopted. Today, the term video is increasingly dropped, reflecting that a digital still camera is used to record the individual image frames, instead of a video recorder. Applications Time-lapse microscopy can be used to observe any microscopic object over time. However, its main use is within cell biology to observe artificially cultured cells. Depending on the cell culture, different microscopy techniques can be applied to enhance characteristics of the cells as most cells

Official source

https://en.wikipedia.org/wiki/Time-lapse_microscopy

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Uropathogenic Escherichia coli (UPEC) are the most common bacterial pathogens causing urinary tract infections (UTIs). Driven by the development of antibiotic resistant UPEC strains, UTIs have become a major public health issue and generate substantial healthcare costs. Even in the absence of antibiotic resistance, UTIs are notoriously difficult to treat and have high rates of recurrence, mostly due to: 1. the ability of UPEC to adhere to and invade bladder epithelial cells, which protects them from the action of antimicrobial agents and the immune system 2. the presence in antibiotic-susceptible UPEC populations of a small fraction of so-called persister cells, which are killed at a slower rate than the bulk of the population and can therefore survive prolonged antibiotic exposureThese two features allow some bacteria to remain viable in a protected niche for the whole duration of the antibiotic treatment, potentially leading to infection relapse when the treatment is terminated.High-content screening (HCS), which uses microscopy as a screening tool, allows the visualization of bacterial phenotypes at the single-cell level and can therefore provide a powerful and unbiased tool to understand the genetic basis of these phenotypes. The present thesis describes two complementary high-content screening approaches aimed at identifying UPEC mutants with altered adhesion to bladder cells and with altered antibiotic persistence respectively.In a first chapter, we report preparatory steps that led to the construction of a transposon insertion library of UPEC mutants that is suitable for high-content imaging, using the CFT073 clinical UTI isolate as background strain.In the second chapter, we performed fluorescence microscopy-based high-content screening to identify mutants with defects in early adhesion to bladder epithelial cells. We recovered 82 mutants with decreased adhesion and 54 mutants with increased adhesion. Unexpectedly, nine low-adhesion ÂÂhitsÂÂ mapped to the two P pili operons encoded by CFT073, which are thought to mediate adhesion to kidney cells rather than bladder cells. Additionally, six high-adhesion hits mapped to the operon coding for F1C pili, and we show that this phenotype is linked to increased P pili synthesis. These results therefore reveal a critical role for P pili in UPEC adhesion to bladder epithelial cells, which may inform the development of novel anti-adhesion therapies to prevent UTI recurrence.In the third chapter, we combined microfluidics with time-lapse microscopy to identify mutants with altered persistence to fosfomycin (FOS) âÂÂ a cell-wall inhibitor used as first-line agent in the treatment of UTIs âÂÂ in synthetic human urine (SHU), which mimics the physiology of UPEC in real urine. We recovered four mutants with decreased persistence, three of which had defects in lipopolysaccharide (LPS) synthesis. These results identify the outer membrane of UPEC as a key component for survival to FOS treatment, thus opening up new avenues in the fight against antibiotic persistence.

EPFL2022

Single-cell division and killing dynamics of bacteria exposed to isocratic and time-dependent concentrations of antibiotic

Katrin Esther Schneider

Antibiotic resistance, defined as the ability of a bacteria to survive an antibiotic drug to which it was initially sensitive, has become increasingly worrying. While the discovery of antibiotics is considered to be one of the most important of all biomedical advances of the twentieth century, bacterial infections are still problematic, and the medical and financial costs that they entail are a great burden to society. In the last 30 years the number of new antibiotic approvals has been decreasing dramatically, leading to a lack of replacements for antibiotics whose effectiveness has been compromised. The drug discovery pipeline is very long and expensive with high rates of attrition at each stage of the process. Massive economic investments have attempted to address this problem but they will bear little fruit if methods are not improved to make the process faster, less expensive, and more efficient. One of the fundamental steps in the evaluation of any new compound is the characterization of the relationship between its pharmacokinetic (PK) and pharmacodynamic (PD) parameters, which will ultimately determine the optimal dosing regimen to be used in clinical trials. A more detailed understanding of these parameters can make a critical difference in early go/no-go decisions, thereby increasing the effectiveness of the drug discovery process. The use of novel assays with single-cell resolution has demonstrated how conventional measurements, taken at the population level, can hide important underlying dynamics of cellular behavior. Since none of the PK/PD models available today allow single-cell measurements, this thesis is focused on the development and implementation of new tools that address this need by enabling the study of essential parameters of antibiotic effectiveness at the single-cell level. We first assessed the effect of different isocratic concentrations of antibiotic by exposing Mycobacterium smegmatis to various concentrations of isoniazid. We find that the higher number of surviving bacteria at lower concentrations of antibiotic is entirely due to increased division rates, as killing rates remain constant. Second, in order to study more realistic pharmacokinetic profiles, we construct a novel microfluidic platform that uses computer-controlled microvalves to enable the administration of time-dependent antibiotic concentration gradients. These gradients serve as input to an array of 960 microchambers in which the cells are imaged by time-lapse microscopy. By studying uropathogenic Escherichia coli (UPEC) treated with ampicillin, we evaluated dosing regimens of intermittently dosed patients. As a proof-of-principle, we accurately predict the pharmacokinetic driver for dose-optimization. Finally, we employ quantitative phase microscopy to complement our single-cell analyses with dry-mass measurements, which give us deeper insights into fundamental processes. Interestingly, we find that this technique can be used as a more rigorous tool to identify dead cells by microscopy.

EPFL2018

Propagation of microorganisms is based on three fundamental processes: cell growth, DNA replication, and cell division. Although important for antibacterial drug development, these processes are poorly understood in Actinobacteria, a medically important phylum that includes Mycobacterium tuberculosis. Using microfluidic cultures and time-lapse microscopy, we studied single-cell growth, DNA replication, and cell division in the model organism Mycobacterium smegmatis.M. smegmatis is rod-shaped and grows by tip elongation in a biphasic manner due to a "new end take-off" (NETO) event. We find that pole elongation speed is increased and NETO occurs earlier in fast-growing cells than in slow-growing cells. As a consequence of variable timing of NETO, single-cell growth can be monophasic, biphasic, or triphasic. We propose that cells optimize pole growth speed and the timing of NETO to maximize their overall growth.We show that fast-growing cells initiate DNA replication earlier than slow-growing cells. We also find that single-cell growth speed is linked to cell-cycle progression, which is similar when comparing cells growing at the same speed under different conditions. We also report that multifork replication occurs when the time between DNA replication initiation events is shorter than the C period, which may occur even in slow-growing cells with interdivision times longer than the C period.Division site selection in many bacteria is governed by the nucleoid occlusion (Noc) and minicell (Min) systems. Mycobacteria do not encode homologs of these proteins and have no known mechanism for division site selection. We found that the DNA replisome and cell division ring colocalize and move together in a biphasic trajectory determined by the chromosome partitioning (Par) system. We propose a model in which Par-dependent movement of the replisome and division ring ultimately determines the site of cell division.Coordination of cell growth and division is required for cell size homeostasis. Three models of division control have been proposed: sizer (cells divide after reaching a critical size), timer (cells divide at a certain time after birth), and adder (cells add a specific amount of mass before dividing). We show that the single-cell growth model (exponential, linear, or bilinear) constrains the possible division control models. Thus, it is crucial to know the true model of single-cell growth in order to distinguish the true mechanism of cell size homeostasis.

EPFL2021

EPFL2022

Single-cell division and killing dynamics of bacteria exposed to isocratic and time-dependent concentrations of antibiotic

Katrin Esther Schneider

EPFL2018

EPFL2021