scholarly journals Digital Microfluidics for Single Bacteria Capture and Selective Retrieval Using Optical Tweezers

Micromachines ◽  
2020 ◽  
Vol 11 (3) ◽  
pp. 308 ◽  
Author(s):  
Phalguni Tewari Kumar ◽  
Deborah Decrop ◽  
Saba Safdar ◽  
Ioannis Passaris ◽  
Tadej Kokalj ◽  
...  
Keyword(s):  
Optical Tweezers ◽  
Cell Sorting ◽  
Magnetic Beads ◽  
Single Cells ◽  
Digital Microfluidics ◽  
Cell Behavior ◽  
Bacterial Cells ◽  
Microfluidic Platforms ◽  
Dynamic Cell ◽  
Downstream Analysis

When screening microbial populations or consortia for interesting cells, their selective retrieval for further study can be of great interest. To this end, traditional fluorescence activated cell sorting (FACS) and optical tweezers (OT) enabled methods have typically been used. However, the former, although allowing cell sorting, fails to track dynamic cell behavior, while the latter has been limited to complex channel-based microfluidic platforms. In this study, digital microfluidics (DMF) was integrated with OT for selective trapping, relocation, and further proliferation of single bacterial cells, while offering continuous imaging of cells to evaluate dynamic cell behavior. To enable this, magnetic beads coated with Salmonella Typhimurium-targeting antibodies were seeded in the microwell array of the DMF platform, and used to capture single cells of a fluorescent S. Typhimurium population. Next, OT were used to select a bead with a bacterium of interest, based on its fluorescent expression, and to relocate this bead to a different microwell on the same or different array. Using an agar patch affixed on top, the relocated bacterium was subsequently allowed to proliferate. Our OT-integrated DMF platform thus successfully enabled selective trapping, retrieval, relocation, and proliferation of bacteria of interest at single-cell level, thereby enabling their downstream analysis.

scholarly journals Mechanosensitive channels and bacterial cell wall integrity: does life end with a bang or a whimper?

2014 ◽  
Vol 11 (91) ◽  
pp. 20130850 ◽  
Author(s):  
Marcel Reuter ◽  
Nicholas J. Hayward ◽  
Susan S. Black ◽  
Samantha Miller ◽  
David T. F. Dryden ◽  
...  
Keyword(s):  
Optical Tweezers ◽  
Cell Sorting ◽  
Cell Shape ◽  
Fluorescent Protein ◽  
Osmotic Shock ◽  
Mechanosensitive Channels ◽  
Bacterial Cells ◽  
Water Influx ◽  
Cell Turgor ◽  
Green Fluorescent

Mechanogated channels are fundamental components of bacterial cells that enable retention of physical integrity during extreme increases in cell turgor. Optical tweezers combined with microfluidics have been used to study the fate of individual Escherichia coli cells lacking such channels when subjected to a bursting stress caused by increased turgor. Fluorescence-activated cell sorting and electron microscopy complement these studies. These analyses show that lysis occurs with a high probability, but the precise path differs between individual cells. By monitoring the loss of cytoplasmic green fluorescent protein, we have determined that some cells release this protein but remain phase dark (granular) consistent with the retention of the majority of large proteins. By contrast, most cells suffer cataclysmic wall failure leading to loss of granularity but with the retention of DNA and overall cell shape (protein-depleted ghosts). The time span of these events induced by hypo-osmotic shock varies but is of the order of milliseconds. The data are interpreted in terms of the timing of mechanosensitive channel gating relative to osmotically induced water influx.

scholarly journals The nucleus measures shape changes for cellular proprioception to control dynamic cell behavior

Science ◽  
2020 ◽  
Vol 370 (6514) ◽  
pp. eaba2644 ◽  
Author(s):  
Valeria Venturini ◽  
Fabio Pezzano ◽  
Frederic Català Castro ◽  
Hanna-Maria Häkkinen ◽  
Senda Jiménez-Delgado ◽  
...  
Keyword(s):  
Functional Module ◽  
Single Cells ◽  
Physical Space ◽  
Cell Behavior ◽  
Geometrical Shape ◽  
Zebrafish Model ◽  
Calcium Dependent ◽  
Dynamic Cell ◽  
And Migration ◽  
Shape Changes

The physical microenvironment regulates cell behavior during tissue development and homeostasis. How single cells decode information about their geometrical shape under mechanical stress and physical space constraints within tissues remains largely unknown. Here, using a zebrafish model, we show that the nucleus, the biggest cellular organelle, functions as an elastic deformation gauge that enables cells to measure cell shape deformations. Inner nuclear membrane unfolding upon nucleus stretching provides physical information on cellular shape changes and adaptively activates a calcium-dependent mechanotransduction pathway, controlling actomyosin contractility and migration plasticity. Our data support that the nucleus establishes a functional module for cellular proprioception that enables cells to sense shape variations for adapting cellular behavior to their microenvironment.

Magnetic Digital Microfluidics for Point-of-Care Testing: Where Are We Now?

2020 ◽  
Vol 27 ◽  
Author(s):  
Yi Zhang
Keyword(s):  
System Integration ◽  
Diagnostic Tests ◽  
Point Of Care ◽  
Digital Microfluidics ◽  
Controlled Environment ◽  
Point Of Care Testing ◽  
Microfluidic Platforms ◽  
Interface System ◽  
Technical Issues ◽  
Sample System

: Point-of-care (POC) testing decentralizes the diagnostic tests to the sites near the patient. Many POC tests rely microfluidic platforms for sample-to-answer analysis. Compared to other microfluidic systems, magnetic digital microfluidics demonstrate compelling advantages for POC diagnostics. In this review, we have examined the capability of magnetic digital microfluidics-based POC diagnostic platforms. More importantly, we have categorized POC settings into three classes based on “where is the point”, “who to care” and “how to test”, and evaluated the suitability of magnetic digital microfluidics in various POC settings. Furthermore, we have addressed other technical issues associated with POC testing such as controlled environment, sample-system interface, system integration and information connectivity. We hope this review would provide a guideline for the future development of magnetic digital microfluidics-based platforms for POC testing.

scholarly journals Stochasticity in Colonial Growth Dynamics of Individual Bacterial Cells

2013 ◽  
Vol 79 (7) ◽  
pp. 2294-2301 ◽  
Author(s):  
Konstantinos P. Koutsoumanis ◽  
Alexandra Lianou
Keyword(s):  
Kinetic Parameters ◽  
Single Cells ◽  
Growth Curves ◽  
Growth Dynamics ◽  
Time Lapse ◽  
Initial Population ◽  
Bacterial Cells ◽  
Stochastic Growth ◽  
Bacterial Populations ◽  
Content Type

ABSTRACTConventional bacterial growth studies rely on large bacterial populations without considering the individual cells. Individual cells, however, can exhibit marked behavioral heterogeneity. Here, we present experimental observations on the colonial growth of 220 individual cells ofSalmonella entericaserotype Typhimurium using time-lapse microscopy videos. We found a highly heterogeneous behavior. Some cells did not grow, showing filamentation or lysis before division. Cells that were able to grow and form microcolonies showed highly diverse growth dynamics. The quality of the videos allowed for counting the cells over time and estimating the kinetic parameters lag time (λ) and maximum specific growth rate (μmax) for each microcolony originating from a single cell. To interpret the observations, the variability of the kinetic parameters was characterized using appropriate probability distributions and introduced to a stochastic model that allows for taking into account heterogeneity using Monte Carlo simulation. The model provides stochastic growth curves demonstrating that growth of single cells or small microbial populations is a pool of events each one of which has its own probability to occur. Simulations of the model illustrated how the apparent variability in population growth gradually decreases with increasing initial population size (N0). For bacterial populations withN0of >100 cells, the variability is almost eliminated and the system seems to behave deterministically, even though the underlying law is stochastic. We also used the model to demonstrate the effect of the presence and extent of a nongrowing population fraction on the stochastic growth of bacterial populations.

A microfluidic device for reversible environmental changes around single cells using optical tweezers for cell selection and positioning

Lab on a Chip ◽  
2010 ◽  
Vol 10 (5) ◽  
pp. 617-625 ◽  
Author(s):  
Emma Eriksson ◽  
Kristin Sott ◽  
Fredrik Lundqvist ◽  
Martin Sveningsson ◽  
Jan Scrimgeour ◽  
...  
Keyword(s):  
Optical Tweezers ◽  
Microfluidic Device ◽  
Environmental Changes ◽  
Single Cells ◽  
Cell Selection

scholarly journals The Development of an Effective Bacterial Single-Cell Lysis Method Suitable for Whole Genome Amplification in Microfluidic Platforms

Micromachines ◽  
2018 ◽  
Vol 9 (8) ◽  
pp. 367 ◽  
Author(s):  
Yuguang Liu ◽  
Dirk Schulze-Makuch ◽  
Jean-Pierre de Vera ◽  
Charles Cockell ◽  
Thomas Leya ◽  
...  
Keyword(s):  
Single Cell ◽  
Single Cells ◽  
Bacterial Species ◽  
Cell Manipulation ◽  
Microfluidic Platforms ◽  
Gram Positive ◽  
Gram Negative ◽  
Genome Amplification ◽  
Single Cell Sequencing ◽  
Wide Range

Single-cell sequencing is a powerful technology that provides the capability of analyzing a single cell within a population. This technology is mostly coupled with microfluidic systems for controlled cell manipulation and precise fluid handling to shed light on the genomes of a wide range of cells. So far, single-cell sequencing has been focused mostly on human cells due to the ease of lysing the cells for genome amplification. The major challenges that bacterial species pose to genome amplification from single cells include the rigid bacterial cell walls and the need for an effective lysis protocol compatible with microfluidic platforms. In this work, we present a lysis protocol that can be used to extract genomic DNA from both gram-positive and gram-negative species without interfering with the amplification chemistry. Corynebacterium glutamicum was chosen as a typical gram-positive model and Nostoc sp. as a gram-negative model due to major challenges reported in previous studies. Our protocol is based on thermal and chemical lysis. We consider 80% of single-cell replicates that lead to >5 ng DNA after amplification as successful attempts. The protocol was directly applied to Gloeocapsa sp. and the single cells of the eukaryotic Sphaerocystis sp. and achieved a 100% success rate.

scholarly journals Microfluidic Platforms Designed for Morphological and Photosynthetic Investigations of Chlamydomonas reinhardtii on a Single-Cell Level

Cells ◽  
2022 ◽  
Vol 11 (2) ◽  
pp. 285
Author(s):  
Eszter Széles ◽  
Krisztina Nagy ◽  
Ágnes Ábrahám ◽  
Sándor Kovács ◽  
Anna Podmaniczki ◽  
...  
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Single Cell ◽  
Single Cells ◽  
Fluorescence Induction ◽  
Photochemical Quenching ◽  
Support Surface ◽  
Single Cell Level ◽  
Microfluidic Platforms ◽  
Cell Level ◽  
Non Photochemical Quenching

Chlamydomonas reinhardtii is a model organism of increasing biotechnological importance, yet, the evaluation of its life cycle processes and photosynthesis on a single-cell level is largely unresolved. To facilitate the study of the relationship between morphology and photochemistry, we established microfluidics in combination with chlorophyll a fluorescence induction measurements. We developed two types of microfluidic platforms for single-cell investigations: (i) The traps of the “Tulip” device are suitable for capturing and immobilizing single cells, enabling the assessment of their photosynthesis for several hours without binding to a solid support surface. Using this “Tulip” platform, we performed high-quality non-photochemical quenching measurements and confirmed our earlier results on bulk cultures that non-photochemical quenching is higher in ascorbate-deficient mutants (Crvtc2-1) than in the wild-type. (ii) The traps of the “Pot” device were designed for capturing single cells and allowing the growth of the daughter cells within the traps. Using our most performant “Pot” device, we could demonstrate that the FV/FM parameter, an indicator of photosynthetic efficiency, varies considerably during the cell cycle. Our microfluidic devices, therefore, represent versatile platforms for the simultaneous morphological and photosynthetic investigations of C. reinhardtii on a single-cell level.

scholarly journals scHiCSRS: A Self-Representation Smoothing Method with Gaussian Mixture Model for Imputing single-cell Hi-C Data

2021 ◽  
Author(s):  
Shili Lin ◽  
Qing Xie
Keyword(s):  
Single Cell ◽  
Gaussian Mixture Model ◽  
Mixture Model ◽  
Single Cells ◽  
High Sensitivity ◽  
Gaussian Mixture ◽  
Smoothing Method ◽  
Intermediate Step ◽  
Downstream Analysis ◽  
Structural Zeros

Motivation: Single-cell Hi-C techniques make it possible to study cell-to-cell variability in genomic features. However, excess zeros are commonly seen in single-cell Hi-C (scHi-C) data, making scHi-C matrices extremely sparse and bringing extra difficulties in downstream analysis. The observed zeros are a combination of two events: structural zeros for which the loci never inter- act due to underlying biological mechanisms, and dropouts or sampling zeros where the two loci interact but are not captured due to insufficient sequencing depth. Although quality improvement approaches have been proposed as an intermediate step for analyzing scHi-C data, little has been done to address these two types of zeros. We believe that differentiating between structural zeros and dropouts would benefit downstream analysis such as clustering. Results: We propose scHiCSRS, a self-representation smoothing method that improves the data quality, and a Gaussian mixture model that identifies structural zeros among observed zeros. scHiCSRS not only takes spatial dependencies of a scHi-C 2D data structure into account but also borrows information from similar single cells. Through an extensive set of simulation studies, we demonstrate the ability of scHiCSRS for identifying structural zeros with high sensitivity and for accurate imputation of dropout values in sampling zeros. Downstream analysis for three real datasets show that data improved from scHiCSRS yield more accurate clustering of cells than simply using observed data or improved data from several comparison methods.

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