scholarly journals Development of single-cell-level microfluidic technology for long-term growth visualization of living cultures of Mycobacterium smegmatis

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Han Wang ◽  
Gloria M. Conover ◽  
Song-I Han ◽  
James C. Sacchettini ◽  
Arum Han
Keyword(s):  
Drug Treatment ◽  
Single Cell ◽  
Mycobacterium Smegmatis ◽  
Culture Media ◽  
Low Cost ◽  
Bacterial Pathogen ◽  
Growth Dynamics ◽  
Time Lapse ◽  
Cell Phenotype

AbstractAnalysis of growth and death kinetics at single-cell resolution is a key step in understanding the complexity of the nonreplicating growth phenotype of the bacterial pathogen Mycobacterium tuberculosis. Here, we developed a single-cell-resolution microfluidic mycobacterial culture device that allows time-lapse microscopy-based long-term phenotypic visualization of the live replication dynamics of mycobacteria. This technology was successfully applied to monitor the real-time growth dynamics of the fast-growing model strain Mycobacterium smegmatis (M. smegmatis) while subjected to drug treatment regimens during continuous culture for 48 h inside the microfluidic device. A clear morphological change leading to significant swelling at the poles of the bacterial membrane was observed during drug treatment. In addition, a small subpopulation of cells surviving treatment by frontline antibiotics was observed to recover and achieve robust replicative growth once regular culture media was provided, suggesting the possibility of identifying and isolating nonreplicative mycobacteria. This device is a simple, easy-to-use, and low-cost solution for studying the single-cell phenotype and growth dynamics of mycobacteria, especially during drug treatment.

scholarly journals Cell-to-cell heterogeneity in Escherichia coli stress response originates from pulsatile expression and growth

2020 ◽  
Author(s):  
Nadia M. V. Sampaio ◽  
Caroline M. Blassick ◽  
Jean-Baptiste Lugagne ◽  
Mary J. Dunlop
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Stress Response ◽  
Single Cell ◽  
Phenotypic Variability ◽  
Growth Dynamics ◽  
Time Lapse ◽  
Cell Heterogeneity ◽  
Temporal Expression ◽  
Functional Consequences

AbstractCell-to-cell heterogeneity in gene expression and growth can have critical functional consequences, such as determining whether individual bacteria survive or die following stress. Although phenotypic variability is well documented, the dynamics that underlie it are often unknown. This information is critical because dramatically different outcomes can arise from gradual versus rapid changes in expression and growth. Using single-cell time-lapse microscopy, we measured the temporal expression of a suite of stress response reporters in Escherichia coli, while simultaneously monitoring growth rate. In conditions without stress, we found widespread examples of pulsatile expression. Single-cell growth rates were often anti-correlated with gene expression, with changes in growth preceding changes in expression. These pulsatile dynamics have functional consequences, which we demonstrate by measuring survival after challenging cells with the antibiotic ciprofloxacin. Our results suggest that pulsatile expression and growth dynamics are common in stress response networks and can have direct consequences for survival.

scholarly journals Incubator-independent cell-culture perfusion platform for continuous long-term microelectrode array electrophysiology and time-lapse imaging

2015 ◽  
Vol 2 (6) ◽  
pp. 150031 ◽  
Author(s):  
Dirk Saalfrank ◽  
Anil Krishna Konduri ◽  
Shahrzad Latifi ◽  
Rouhollah Habibey ◽  
Asiyeh Golabchi ◽  
...  
Keyword(s):  
Cell Culture ◽  
Microelectrode Array ◽  
Low Cost ◽  
Time Lapse ◽  
Free Cell ◽  
Network Architectures ◽  
Hippocampal Networks ◽  
Time Lapse Imaging

Most in vitro electrophysiology studies extract information and draw conclusions from representative, temporally limited snapshot experiments. This approach bears the risk of missing decisive moments that may make a difference in our understanding of physiological events. This feasibility study presents a simple benchtop cell-culture perfusion system adapted to commercial microelectrode arrays (MEAs), multichannel electrophysiology equipment and common inverted microscopy stages for simultaneous and uninterrupted extracellular electrophysiology and time-lapse imaging at ambient CO 2 levels. The concept relies on a transparent, replica-casted polydimethylsiloxane perfusion cap, gravity- or syringe-pump-driven perfusion and preconditioning of pH-buffered serum-free cell-culture medium to ambient CO 2 levels at physiological temperatures. The low-cost microfluidic in vitro enabling platform, which allows us to image cultures immediately after cell plating, is easy to reproduce and is adaptable to the geometries of different cell-culture containers. It permits the continuous and simultaneous multimodal long-term acquisition or manipulation of optical and electrophysiological parameter sets, thereby considerably widening the range of experimental possibilities. Two exemplary proof-of-concept long-term MEA studies on hippocampal networks illustrate system performance. Continuous extracellular recordings over a period of up to 70 days revealed details on both sudden and gradual neural activity changes in maturing cell ensembles with large intra-day fluctuations. Correlated time-lapse imaging unveiled rather static macroscopic network architectures with previously unreported local morphological oscillations on the timescale of minutes.

Long-Term Single-Cell Culture of Human Pluripotent Stem Cells on Precoated rLaminin-521 Cultureware in Multiple Culture Media

Cytotherapy ◽  
2016 ◽  
Vol 18 (6) ◽  
pp. S130
Author(s):  
H. Nandivada ◽  
J. Montoya ◽  
P. Flaherty ◽  
M. Albouy ◽  
B. Onteniente ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Culture ◽  
Single Cell ◽  
Pluripotent Stem Cells ◽  
Culture Media ◽  
Human Pluripotent Stem Cells ◽  
Single Cell Culture

scholarly journals Development of an Inverted Epifluorescence Microscope for Long-Term Monitoring of Bacteria in Multiplexed Microfluidic Devices

Sensors ◽  
2020 ◽  
Vol 20 (15) ◽  
pp. 4140
Author(s):  
Amaro Torres-Simón ◽  
María Henar Marino ◽  
Clara Gómez-Cruz ◽  
Marina Cañadas ◽  
Miguel Marco ◽  
...  
Keyword(s):  
Single Cell ◽  
Bacterial Resistance ◽  
Single Cell Analysis ◽  
Low Cost ◽  
Microfluidic Devices ◽  
Cell Analysis ◽  
Antibiotic Development ◽  
Long Term Monitoring ◽  
Term Monitoring

Developing more efficient methods for antibiotic susceptibility testing is a pressing issue in novel drug development as bacterial resistance to antibiotics becomes increasingly common. Microfluidic devices have been demonstrated to be powerful platforms that allow researchers to perform multiplexed antibiotic testing. However, the level of multiplexing within microdevices is limited, evidencing the need of creating simple, low-cost and high-resolution imaging systems that can be integrated in antibiotic development pipelines. This paper describes the design and development of an epifluorescence inverted microscope that enables long-term monitoring of bacteria inside multiplexed microfluidic devices. The goal of this work is to provide a simple microscope powerful enough to allow single-cell analysis of bacteria at a reduced cost. This facilitates increasing the number of microscopes that are simultaneously used for antibiotic testing. We prove that the designed system is able to accurately detect fluorescent beads of 100 nm, demonstrating comparable features to high-end commercial microscopes and effectively achieving the resolution required for single-cell analysis of bacteria. The proposed microscope could thus increase the efficiency in antibiotic testing while reducing cost, size, weight, and power requirements, contributing to the successful development of new antibiotic drugs.

Encapsulated Petri dish system for single-cell drug delivery and long-term time lapse microscopy

2007 ◽  
Vol 371 (2) ◽  
pp. 208-214 ◽  
Author(s):  
Marcelo Salierno ◽  
Ricardo Cabrera ◽  
Oscar Filevich ◽  
Roberto Etchenique
Keyword(s):  
Drug Delivery ◽  
Single Cell ◽  
Petri Dish ◽  
Time Lapse ◽  
Time Lapse Microscopy

The use of a low cost, time-lapse camera for high frequency monitoring of intertidal beach morphology

2020 ◽  
Author(s):  
Emilia Guisado-Pintado ◽  
Derek W.T. Jackson
Keyword(s):  
Time Scales ◽  
High Frequency ◽  
Sandy Beach ◽  
Low Cost ◽  
Time Lapse ◽  
Airborne Lidar ◽  
Camera System ◽  
Monitoring Method ◽  
Coastal Systems

<p>Coastal monitoring of sandy beach areas requires data gathering at regular time scales to capture daily to weekly geomorphological changes that are modified through tidal and wave action. Regular GPS profiling surveys carried out at medium (weekly) to long-term (month/annual) frequency can lead to misinterpretation of beach changes as they are not able to pick up subtle gross changes and instead usually only capture net changes that have occurred. Recently, a new suite of monitoring devices such as Terrestrial Laser Scanners, airborne LiDAR or the use of Unnamed Aerial Vehicles have become more readily available and have in many cases replaced traditional monitoring methods (e.g. the use of Emery method and GPS) as they can capture contemporary morphological impacts faster and more conveniently. Monitoring coastal systems using video cameras is also an increasingly common monitoring method as it allows a continuous monitoring method through the image capture at different time-scales.</p><p>Here, we present the use of high-frequency imagery generated from a low-cost, fixed, time-lapse camera system as an effective method for quantifying intertidal bar migration patterns on a daily to annual time scale. The time-lapse camera system was deployed over-looking a beach-dune complex at Five Finger strand, NW Ireland. It was located on high ground (around 80 m) obliquely overlooking the study site, with a field of view of 59° and set to acquire images every 30 min. Images captured were calibrated using multiple ground truth Ground Control Points (GCPs), positioned at regular geo-located intervals along intertidal profile lengths. Further, average distance of each pixel on the ground was converted into real-world distance using a pre-calculated scaling factor.</p><p>The method successfully tracked the leading edge of an onshore migrating intertidal bar using a set of chronological captured images over a shoreward distance of 31.23 m in a 3-month period. The technique can also be used in the monitoring of wave run-up and dune toe encroachment events by waves during high energy events. The use of the camera over long time periods provided a rich dataset for examining both long-term intertidal beach dynamics of sites to help fully compare forcing and response phenomena in between forcing events. We believe that this easy-to-use and low-cost technique will enhance future monitoring of highly dynamic coastal systems enabling a more detailed spatial and temporal analysis of intertidal sandy beach areas.</p><p> </p>

scholarly journals Analytics and visualization tools to characterize single-cell stochasticity using bacterial single-cell movie cytometry data

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Athanasios D. Balomenos ◽  
Victoria Stefanou ◽  
Elias S. Manolakos
Keyword(s):  
Cell Division ◽  
Cell Growth ◽  
Single Cell ◽  
Bacterial Communities ◽  
Information Transfer ◽  
Community Organization ◽  
Growth Dynamics ◽  
Time Lapse ◽  
Bioimage Analysis

Abstract Background Time-lapse microscopy live-cell imaging is essential for studying the evolution of bacterial communities at single-cell resolution. It allows capturing detailed information about the morphology, gene expression, and spatial characteristics of individual cells at every time instance of the imaging experiment. The image analysis of bacterial "single-cell movies" (videos) generates big data in the form of multidimensional time series of measured bacterial attributes. If properly analyzed, these datasets can help us decipher the bacterial communities' growth dynamics and identify the sources and potential functional role of intra- and inter-subpopulation heterogeneity. Recent research has highlighted the importance of investigating the role of biological "noise" in gene regulation, cell growth, cell division, etc. Single-cell analytics of complex single-cell movie datasets, capturing the interaction of multiple micro-colonies with thousands of cells, can shed light on essential phenomena for human health, such as the competition of pathogens and benign microbiome cells, the emergence of dormant cells (“persisters”), the formation of biofilms under different stress conditions, etc. However, highly accurate and automated bacterial bioimage analysis and single-cell analytics methods remain elusive, even though they are required before we can routinely exploit the plethora of data that single-cell movies generate. Results We present visualization and single-cell analytics using R (ViSCAR), a set of methods and corresponding functions, to visually explore and correlate single-cell attributes generated from the image processing of complex bacterial single-cell movies. They can be used to model and visualize the spatiotemporal evolution of attributes at different levels of the microbial community organization (i.e., cell population, colony, generation, etc.), to discover possible epigenetic information transfer across cell generations, infer mathematical and statistical models describing various stochastic phenomena (e.g., cell growth, cell division), and even identify and auto-correct errors introduced unavoidably during the bioimage analysis of a dense movie with thousands of overcrowded cells in the microscope's field of view. Conclusions ViSCAR empowers researchers to capture and characterize the stochasticity, uncover the mechanisms leading to cellular phenotypes of interest, and decipher a large heterogeneous microbial communities' dynamic behavior. ViSCAR source code is available from GitLab at https://gitlab.com/ManolakosLab/viscar.

Fine Particle Mass Monitoring with Low-Cost Sensors: Corrections and Long-Term Performance Evaluation

2019 ◽  
Author(s):  
Carl Malings ◽  
Rebecca Tanzer ◽  
Aliaksei Hauryliuk ◽  
Provat K. Saha ◽  
Allen L. Robinson ◽  
...  
Keyword(s):  
Performance Evaluation ◽  
Fine Particle ◽  
Low Cost ◽  
Particle Mass ◽  
Long Term Performance ◽  
Term Performance

scholarly journals Surface Immobilization of Nano-Silver on Polymeric Medical Devices to Prevent Bacterial Biofilm Formation

Pathogens ◽  
2019 ◽  
Vol 8 (3) ◽  
pp. 93 ◽  
Author(s):  
Riau ◽  
Aung ◽  
Setiawan ◽  
Yang ◽  
Yam ◽  
...  
Keyword(s):  
Medical Devices ◽  
Biofilm Formation ◽  
Culture Media ◽  
Silver Ions ◽  
Bacterial Biofilm ◽  
Surface Immobilization ◽  
Nano Silver ◽  
Gram Positive Bacteria ◽  
Tissue Microenvironment

: Bacterial biofilm on medical devices is difficult to eradicate. Many have capitalized the anti-infective capability of silver ions (Ag+) by incorporating nano-silver (nAg) in a biodegradable coating, which is then laid on polymeric medical devices. However, such coating can be subjected to premature dissolution, particularly in harsh diseased tissue microenvironment, leading to rapid nAg clearance. It stands to reason that impregnating nAg directly onto the device, at the surface, is a more ideal solution. We tested this concept for a corneal prosthesis by immobilizing nAg and nano-hydroxyapatite (nHAp) on poly(methyl methacrylate), and tested its biocompatibility with human stromal cells and antimicrobial performance against biofilm-forming pathogens, Pseudomonas aeruginosa and Staphylococcus aureus. Three different dual-functionalized substrates—high Ag (referred to as 75:25 HAp:Ag); intermediate Ag (95:5 HAp:Ag); and low Ag (99:1 HAp:Ag) were studied. The 75:25 HAp:Ag was effective in inhibiting biofilm formation, but was cytotoxic. The 95:5 HAp:Ag showed the best selectivity among the three substrates; it prevented biofilm formation of both pathogens and had excellent biocompatibility. The coating was also effective in eliminating non-adherent bacteria in the culture media. However, a 28-day incubation in artificial tear fluid revealed a ~40% reduction in Ag+ release, compared to freshly-coated substrates. The reduction affected the inhibition of S. aureus growth, but not the P. aeruginosa. Our findings suggest that Ag+ released from surface-immobilized nAg diminishes over time and becomes less effective in suppressing biofilm formation of Gram-positive bacteria, such as S. aureus. This advocates the coating, more as a protection against perioperative and early postoperative infections, and less as a long-term preventive solution.

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