scholarly journals Microscopy quantification of microbial birth and death dynamics

2018 ◽  
Author(s):  
Samuel F. M. Hart ◽  
David Skelding ◽  
Adam J. Waite ◽  
Justin Burton ◽  
Li Xie ◽  
...  
Keyword(s):  
Fluorescent Proteins ◽  
Time Lapse ◽  
Microbial Evolution ◽  
Cell Counting ◽  
Live Cells ◽  
Nutrient Concentrations ◽  
Death Rates ◽  
Time Lapse Microscopy ◽  
Wide Range ◽  
Total Fluorescence

AbstractMicrobes live in dynamic environments where nutrient concentrations fluctuate. Quantifying fitness (birth and death) in a wide range of environments is critical for understanding microbial evolution as well as ecological interactions where one species alters the fitness of another. Here, using high-throughput time-lapse microscopy, we have quantified howSaccharomyces cerevisiaemutants incapable of synthesizing an essential metabolite grow or die in various concentrations of the required metabolite. We establish that cells normally expressing fluorescent proteins lose fluorescence upon death and that the total fluorescence in an imaging frame is proportional to the number of live cells even when cells form multiple layers. We validate our microscopy approach of measuring birth and death rates using flow cytometry, cell counting, and chemostat culturing. For lysine-requiring cells, very low concentrations of lysine are not detectably consumed and do not support cell birth, but delay the onset of death phase and reduce the death rate. In contrast, in low hypoxanthine, hypoxanthine-requiring cells can produce new cells, yet also die faster than in the absence of hypoxanthine. For both strains, birth rates under various metabolite concentrations are better described by the sigmoidal-shaped Moser model than the well-known Monod model, while death rates depend on the metabolite concentration and can vary with time. Our work reveals how time-lapse microscopy can be used to discover non-intuitive microbial dynamics and to quantify growth rates in many environments.

scholarly journals Fibronectin-Based Nanomechanical Biosensors to Map 3D Strains in Live Cells and Tissues

2020 ◽  
Author(s):  
Daniel J. Shiwarski ◽  
Joshua W. Tashman ◽  
Alkiviadis Tsamis ◽  
Jacqueline M. Bliley ◽  
Malachi A. Blundon ◽  
...  
Keyword(s):  
Spatial Resolution ◽  
Single Cells ◽  
Time Lapse ◽  
Confocal Imaging ◽  
Square Lattice ◽  
Live Cells ◽  
3D Analysis ◽  
Wide Range ◽  
And Function ◽  
Analysis Platform

AbstractMechanical forces are integral to a wide range of cellular processes including migration, differentiation and tissue morphogenesis; however, it has proved challenging to directly measure strain at high spatial resolution and with minimal tissue perturbation. Here, we fabricated, calibrated, and tested a fibronectin (FN)-based nanomechanical biosensor (NMBS) that can be applied to cells and tissues to measure the magnitude, direction, and dynamics of strain from subcellular to tissue length-scales. The NMBS is a fluorescently-labeled, ultrathin square lattice FN mesh with spatial resolution tailored by adjusting the width and spacing of the lattice fibers from 2-100 µm. Time-lapse 3D confocal imaging of the NMBS demonstrated strain tracking in 2D and 3D following mechanical deformation of known materials and was validated with finite element modeling. Imaging and 3D analysis of the NMBS applied to single cells, cell monolayers, and Drosophila ovarioles demonstrated the ability to dynamically track microscopic tensile and compressive strains in various biological applications with minimal tissue perturbation. This fabrication and analysis platform serves as a novel tool for studying cells, tissues, and more complex systems where forces guide structure and function.

scholarly journals ACDC: Automated Cell Detection and Counting for Time-Lapse Fluorescence Microscopy

2020 ◽  
Vol 10 (18) ◽  
pp. 6187
Author(s):  
Leonardo Rundo ◽  
Andrea Tangherloni ◽  
Darren R. Tyson ◽  
Riccardo Betta ◽  
Carmelo Militello ◽  
...  
Keyword(s):  
Large Scale ◽  
Time Lapse ◽  
Cell Counting ◽  
Cell Detection ◽  
Cell Nuclei ◽  
Similarity Coefficients ◽  
Laboratory Practice ◽  
Microscopy Imaging ◽  
Time Lapse Microscopy ◽  
Detection And Counting

Advances in microscopy imaging technologies have enabled the visualization of live-cell dynamic processes using time-lapse microscopy imaging. However, modern methods exhibit several limitations related to the training phases and to time constraints, hindering their application in the laboratory practice. In this work, we present a novel method, named Automated Cell Detection and Counting (ACDC), designed for activity detection of fluorescent labeled cell nuclei in time-lapse microscopy. ACDC overcomes the limitations of the literature methods, by first applying bilateral filtering on the original image to smooth the input cell images while preserving edge sharpness, and then by exploiting the watershed transform and morphological filtering. Moreover, ACDC represents a feasible solution for the laboratory practice, as it can leverage multi-core architectures in computer clusters to efficiently handle large-scale imaging datasets. Indeed, our Parent-Workers implementation of ACDC allows to obtain up to a 3.7× speed-up compared to the sequential counterpart. ACDC was tested on two distinct cell imaging datasets to assess its accuracy and effectiveness on images with different characteristics. We achieved an accurate cell-count and nuclei segmentation without relying on large-scale annotated datasets, a result confirmed by the average Dice Similarity Coefficients of 76.84 and 88.64 and the Pearson coefficients of 0.99 and 0.96, calculated against the manual cell counting, on the two tested datasets.

scholarly journals Preventing Photomorbidity in Long-Term Multi-color Fluorescence Imaging of Saccharomyces cerevisiae and S. pombe

2020 ◽  
Vol 10 (12) ◽  
pp. 4373-4385
Author(s):  
Gregor W. Schmidt ◽  
Andreas P. Cuny ◽  
Fabian Rudolf
Keyword(s):  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Single Cells ◽  
Time Lapse ◽  
Light Dose ◽  
Live Cells ◽  
Excitation Light ◽  
Minimal Impact ◽  
Imaging Conditions

Time-lapse imaging of live cells using multiple fluorescent reporters is an essential tool to study molecular processes in single cells. However, exposure to even moderate doses of visible excitation light can disturb cellular physiology and alter the quantitative behavior of the cells under study. Here, we set out to develop guidelines to avoid the confounding effects of excitation light in multi-color long-term imaging. We use widefield fluorescence microscopy to measure the effect of the administered excitation light on growth rate (here called photomorbidity) in yeast. We find that photomorbidity is determined by the cumulative light dose at each wavelength, but independent of the way excitation light is applied. Importantly, photomorbidity possesses a threshold light dose below which no effect is detectable (NOEL). We found, that the suitability of fluorescent proteins for live-cell imaging at the respective excitation light NOEL is equally determined by the cellular autofluorescence and the fluorescent protein brightness. Last, we show that photomorbidity of multiple wavelengths is additive and imaging conditions absent of photomorbidity can be predicted. Our findings enable researchers to find imaging conditions with minimal impact on physiology and can provide framework for how to approach photomorbidity in other organisms.

scholarly journals ACDC: Automated Cell Detection and Counting for Time-lapse Fluorescence Microscopy

2020 ◽  
Author(s):  
Leonardo Rundo ◽  
Andrea Tangherloni ◽  
Darren R. Tyson ◽  
Riccardo Betta ◽  
Carmelo Militello ◽  
...  
Keyword(s):  
Large Scale ◽  
Time Lapse ◽  
Cell Counting ◽  
Cell Detection ◽  
Cell Nuclei ◽  
Similarity Coefficients ◽  
Laboratory Practice ◽  
Microscopy Imaging ◽  
Time Lapse Microscopy ◽  
Detection And Counting

AbstractAdvances in microscopy imaging technologies have enabled the visualization of live-cell dynamic processes using time-lapse microscopy imaging. However, modern methods exhibit several limitations related to the training phases and to time constraints, hindering their application in the laboratory practice. In this work, we present a novel method, named Automated Cell Detection and Counting (ACDC), designed for activity detection of fluorescent labeled cell nuclei in time-lapse microscopy. ACDC overcomes the limitations of the literature methods, by first applying bilateral filtering on the original image to smooth the input cell images while preserving edge sharpness, and then by exploiting the watershed transform and morphological filtering. Moreover, ACDC represents a feasible solution for the laboratory practice, as it can leverage multi-core architectures in computer clusters to efficiently handle large-scale imaging datasets. Indeed, our Parent-Workers implementation of ACDC allows to obtain up to a 3.7× speed-up compared to the sequential counterpart. ACDC was tested on two distinct cell imaging datasets to assess its accuracy and effectiveness on images with different characteristics. We achieved an accurate cell-count and nuclei segmentation without relying on large-scale annotated datasets, a result confirmed by the average Dice Similarity Coefficients of 76.84 and 88.64 and the Pearson coefficients of 0.99 and 0.96, calculated against the manual cell counting, on the two tested datasets.

scholarly journals Initiation of chromosome replication controls both division and replication cycles in E. coli through a double-adder mechanism

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Guillaume Witz ◽  
Erik van Nimwegen ◽  
Thomas Julou
Keyword(s):  
Cell Cycle ◽  
Time Lapse ◽  
Growth Conditions ◽  
Replication Initiation ◽  
Stochastic Simulations ◽  
E Coli ◽  
Time Lapse Microscopy ◽  
Statistical Framework ◽  
Wide Range ◽  
Cell Cycle Models

Living cells proliferate by completing and coordinating two cycles, a division cycle controlling cell size and a DNA replication cycle controlling the number of chromosomal copies. It remains unclear how bacteria such as Escherichia coli tightly coordinate those two cycles across a wide range of growth conditions. Here, we used time-lapse microscopy in combination with microfluidics to measure growth, division and replication in single E. coli cells in both slow and fast growth conditions. To compare different phenomenological cell cycle models, we introduce a statistical framework assessing their ability to capture the correlation structure observed in the data. In combination with stochastic simulations, our data indicate that the cell cycle is driven from one initiation event to the next rather than from birth to division and is controlled by two adder mechanisms: the added volume since the last initiation event determines the timing of both the next division and replication initiation events.

scholarly journals Maturing autophagosomes are transported towards the cell periphery

2021 ◽  
Author(s):  
Anna Hilverling ◽  
Eva M. Szegö ◽  
Elisabeth Dinter ◽  
Diana Cozma ◽  
Theodora Saridaki ◽  
...  
Keyword(s):  
Subcellular Distribution ◽  
Intracellular Trafficking ◽  
Fluorescent Proteins ◽  
Time Lapse ◽  
Vesicle Transport ◽  
Cell Periphery ◽  
Time Lapse Microscopy ◽  
Acidic Vesicles ◽  
Luminal Ph ◽  
Autophagosome Maturation

Abstract Autophagosome maturation comprises fusion with lysosomes and acidification. It is a critical step in the degradation of cytosolic protein aggregates that characterize many neurodegenerative diseases. In order to better understand this process, we studied intracellular trafficking of autophagosomes and aggregates of α-synuclein, which characterize Parkinson’s disease and other synucleinopathies. The autophagosomal marker LC3 and the aggregation prone A53T mutant of α-synuclein were tagged by fluorescent proteins and expressed in HEK293T cells and primary astrocytes. The subcellular distribution and movement of these vesicle populations were analyzed by (time-lapse) microscopy. Fusion with lysosomes was assayed using the lysosomal marker LAMP1; vesicles with neutral and acidic luminal pH were discriminated using the RFP-GFP “tandem fluorescence” tag. With respect to vesicle pH, we observed that neutral autophagosomes, marked by LC3 or synuclein, were located more frequently in the cell center, and acidic autophagosomes were observed more frequently in the cell periphery. Acidic autophagosomes were transported towards the cell periphery more often, indicating that acidification occurs in the cell center before transport to the periphery. With respect to autolysosomal fusion, we found that lysosomes preferentially moved towards the cell center whereas autolysosomes moved towards the cell periphery, suggesting a cycle where lysosomes are generated in the periphery and fuse to autophagosomes in the cell center. Unexpectedly, many acidic autophagosomes were negative for LAMP1, indicating that acidification does not require fusion to lysosomes. Moreover, we found both neutral and acidic vesicles positive for LAMP1, consistent with delayed acidification of the autolysosome lumen. Individual steps of aggregate clearance thus occur in dedicated cellular regions. During aggregate clearance, autophagosomes and autolysosomes form in the center and are transported towards the periphery during maturation. In this process, luminal pH could regulate the direction of vesicle transport.

scholarly journals Isolating live cells after high-throughput, long-term, time-lapse microscopy

2019 ◽  
Vol 17 (1) ◽  
pp. 93-100 ◽  
Author(s):  
Scott Luro ◽  
Laurent Potvin-Trottier ◽  
Burak Okumus ◽  
Johan Paulsson
Keyword(s):  
High Throughput ◽  
Time Lapse ◽  
Live Cells ◽  
Time Lapse Microscopy

scholarly journals Maturing Autophagosomes are Transported Towards the Cell Periphery

2021 ◽  
Author(s):  
Anna Hilverling ◽  
Eva M. Szegö ◽  
Elisabeth Dinter ◽  
Diana Cozma ◽  
Theodora Saridaki ◽  
...  
Keyword(s):  
Intracellular Trafficking ◽  
Fluorescent Proteins ◽  
Time Lapse ◽  
Cell Periphery ◽  
Microtubule Organizing Center ◽  
Time Lapse Microscopy ◽  
Organizing Center ◽  
Acidic Vesicles ◽  
Luminal Ph ◽  
Autophagosome Maturation

AbstractAutophagosome maturation comprises fusion with lysosomes and acidification. It is a critical step in the degradation of cytosolic protein aggregates that characterize many neurodegenerative diseases. In order to better understand this process, we studied intracellular trafficking of autophagosomes and aggregates of α-synuclein, which characterize Parkinson’s disease and other synucleinopathies. The autophagosomal marker LC3 and the aggregation prone A53T mutant of α-synuclein were tagged by fluorescent proteins and expressed in HEK293T cells and primary astrocytes. The subcellular distribution and movement of these vesicle populations were analyzed by (time-lapse) microscopy. Fusion with lysosomes was assayed using the lysosomal marker LAMP1; vesicles with neutral and acidic luminal pH were discriminated using the RFP-GFP “tandem-fluorescence” tag. With respect to vesicle pH, we observed that neutral autophagosomes, marked by LC3 or synuclein, were located more frequently in the cell center, and acidic autophagosomes were observed more frequently in the cell periphery. Acidic autophagosomes were transported towards the cell periphery more often, indicating that acidification occurs in the cell center before transport to the periphery. With respect to autolysosomal fusion, we found that lysosomes preferentially moved towards the cell center, whereas autolysosomes moved towards the cell periphery, suggesting a cycle where lysosomes are generated in the periphery and fuse to autophagosomes in the cell center. Unexpectedly, many acidic autophagosomes were negative for LAMP1, indicating that acidification does not require fusion to lysosomes. Moreover, we found both neutral and acidic vesicles positive for LAMP1, consistent with delayed acidification of the autolysosome lumen. Individual steps of aggregate clearance thus occur in dedicated cellular regions. During aggregate clearance, autophagosomes and autolysosomes form in the center and are transported towards the periphery during maturation. In this process, luminal pH could regulate the direction of vesicle transport. Graphic Abstract (1) Transport and location of autophagosomes depend on luminal pH: Acidic autophagosomes are preferentially transported to the cell periphery, causing more acidic autophagosomes in the cell periphery and more neutral autophagosomes at the microtubule organizing center (MTOC). (2) Autolysosomes are transported to the cell periphery and lysosomes to the MTOC, suggesting spatial segregation of lysosome reformation and autolysosome fusion. (3) Synuclein aggregates are preferentially located at the MTOC and synuclein-containing vesicles in the cell periphery, consistent with transport of aggregates to the MTOC for autophagy.

scholarly journals Sample preparation and imaging conditions affect mEos3.2 photophysics in fission yeast cells

2020 ◽  
Author(s):  
Mengyuan Sun ◽  
Kevin Hu ◽  
Joerg Bewersdorf ◽  
Thomas D. Pollard
Keyword(s):  
Sample Preparation ◽  
Yeast Cell ◽  
Single Molecule ◽  
Fluorescent Proteins ◽  
Yeast Cells ◽  
Live Cells ◽  
Fluorescence Signal ◽  
Wide Range ◽  
Imaging Conditions ◽  
In Cells

AbstractPhotoconvertible fluorescent proteins (PCFPs) are widely used in super-resolution microscopy and studies of cellular dynamics. However, our understanding of their photophysics is still limited, hampering their quantitative application. For example, we do not know the optimal sample preparation methods or imaging conditions to count protein molecules fused to PCFPs by single-molecule localization microscopy in live and fixed cells. We also do not know how the behavior of PCFPs in live cells compares with fixed cells. Therefore, we investigated how formaldehyde fixation influences the photophysical properties of the popular green-to-red PCFP mEos3.2 in fission yeast cells under a wide range of imaging conditions. We estimated photophysical parameters by fitting a 3-state model of photoconversion and photobleaching to the time course of fluorescence signal per yeast cell expressing mEos3.2. We discovered that formaldehyde fixation makes the fluorescence signal, photoconversion rate and photobleaching rate of mEos3.2 sensitive to the buffer conditions by permeabilizing the yeast cell membrane. Under some imaging conditions, the time-integrated mEos3.2 signal per yeast cell is similar in live cells and fixed cells imaged in buffer at pH 8.5 with 1 mM DTT, indicating that light chemical fixation does not destroy mEos3.2 molecules. We also discovered that some red-state mEos3.2 molecules entered an intermediate dark state that is converted back to the red fluorescent state by 561-nm illumination. Our findings provide a guide to compare quantitatively conditions for imaging and counting of mEos3.2-tagged molecules in yeast cells. Our imaging assay and mathematical model are easy to implement and provide a simple quantitative approach to measure the time-integrated signal and the photoconversion and photobleaching rates of fluorescent proteins in cells.STATEMENT OF SIGNIFICANCEMaking quantitative measurements with single-molecule localization microscopy (SMLM) has been impeded by limited understanding of the photophysics of the fluorophores, which is very sensitive to the sample preparation and imaging conditions. We characterized the photophysics of the green-to-red photoconvertible fluorescent protein mEos3.2, which is widely used in SMLM. We combined quantitative fluorescence microscopy and mathematical modeling to measure the fluorescence signal and rate constants for photoconversion and photobleaching of mEos3.2 in live and fixed cells under a wide range of illumination intensities. Our findings provide a guide to compare conditions for imaging and counting mEos3.2-tagged proteins in cells. The presented approach is generally applicable to characterize other fluorescent proteins or dyes in cells.

scholarly journals Control of division and microtubule dynamics in Chlamydomonas by cyclin B/CDKB1 and the anaphase-promoting complex

2021 ◽  
Author(s):  
Frederick Cross ◽  
Kresti Pecani ◽  
Kristi Lieberman ◽  
Natsumi Tajima-Shirasaki ◽  
Masayuki Onishi
Keyword(s):  
Time Lapse ◽  
Microtubule Dynamics ◽  
Cyclin B ◽  
Live Cells ◽  
Cyclin Dependent Kinase ◽  
Anaphase Promoting Complex ◽  
Mitotic Progression ◽  
Time Lapse Microscopy ◽  
Spindle Poles ◽  
Multiple Fission

In yeast and animals, cyclin B binds and activates the cyclin-dependent kinase ('CDK') CDK1 to drive entry into mitosis. We show that CYCB1, the sole cyclin B in Chlamydomonas, activates the plant-specific CDKB1 rather than the CDK1 ortholog CDKA1. Time-lapse microscopy shows that CYCB1 is synthesized before each division in the multiple fission cycle, then is rapidly degraded 3-5 minutes before division occurs. CYCB1 degradation is dependent on the anaphase-promoting complex (APC). Like CYCB1, CDKB1 is not synthesized until late G1; however, CDKB1 is not degraded with each division within the multiple fission cycle. The microtubule plus-end-binding protein EB1 labeled with mNeonGreen (EB1-NG) allowed detection of mitotic events in live cells. The earliest detectable step in mitosis, splitting of polar EB1-NG signal into two foci, likely associated with future spindle poles, was dependent on CYCB1. CYCB1-GFP localized close to these foci immediately before spindle formation. Spindle breakdown, cleavage furrow formation and accumulation of EB1 in the furrow were dependent on the APC. In interphase, rapidly growing microtubules are marked by 'comets' of EB1; comets are absent in the absence of APC function. Thus CYCB1/CDKB1 and the APC mitosis modulate microtubule dynamics while regulating mitotic progression.

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