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In this article, we report the number of cyclin B1 proteins tagged with enhanced green fluorescent protein (eGFP) in fixed U-2 OS cells across the cell cycle. We use a quantitative analysis of epifluorescence to determine the number of eGFP molecules in a nondestructive way, and integrated over the cell we find 104 to 105 molecules. Based on the measured number of eGFP tagged cyclin B1 proteins, knowledge of cyclin B1 dynamics through the cell cycle, and the cell morphology, we identify the stages of cells in the cell cycle. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals LLC. on behalf of International Society for Advancement of Cytometry.

Polysialylation of neural cell adhesion molecule (NCAM) in small-cell lung cancer (SCLC) is thought to regulate NCAM-mediated cell–surface interactions, imparting antiadhesive properties to cells. However, SCLC cells in culture demonstrate anchorage-independent growth and spontaneously generate adherent forms. Here, the ability of polySia-NCAM to influence cell proliferation and adherence is unclear. We analyzed live SCLC cell polySia-NCAM expression by flow cytometry, using the novel combination of a polySia antibody-mimetic eGFP-tagged endosialidase and the viability dye DRAQ7. Enrichment for adherence ( 150 population doublings) resolved cells with stable re-expression of polySia and increased growth rates both in vitro and in vivo. Endoneuraminidase removal of polySia from re-expressing cells showed that rapid adherence to extracellular matrix components was functionally independent of polySia. PolySia expression was not altered when isolated adherent forms underwent enforced cell–cell contact in three-dimensional culture. Coculture of polySia expression variants modulated overall polySia expression profiles indicating an influence of SCLC microcommunity composition independent of substrate adherence potential. We conclude that an obligatory linkage between substrate adherence potential and polySia expression is rejected for SCLC cells. We suggest that a degree of homeostasis operates to regulate polysialylation within heterogeneous cell populations. The findings suggest a new model for SCLC progression while the application of live cell profiling of polysialylation could be used to assess polySia-NCAM-targeted therapies.

We outline a simple approach involving instrument setup and calibration for the analysis of Hoechst dye 33342-loading in human cell lines for exploring heterogeneity in dye efflux efficiency and the status of side population (SP) A549 lung cancer cells. Dual excitation 488 nm/multiline UV (351–364 nm) flow cytometry was used to confirm ABCG2-specific inhibition of dye efflux using Fumitremorgin C. Transporter gene expression, assayed by qRT-PCR, confirmed higher expression of ABCG2 versus ABCB1, reiterated in a cloned subline. Coexpression of aldehyde dehydrogenase genes ranked as aldehyde dehydrogenase class 1A1 (ALDH1A1) > ALDH3A1 > ALDH3, relative expression of all genes was again reiterated in a cloned subline. Permeabilized cells were used to create red:violet (660:405 nm Em wavelengths) ratiometric references for mapping temporal changes in Hoechst 33342–DNA fluorescence in live cells. A live cell “kinetic SP gate” tracked progressive dye loading of the whole population and coapplication of the far red (>695 nm wavelength) fluorescing dye DRAQ7 enabled viable cell gating. Kinetic gating revealed a continuum for dye accumulation suggesting that SP enumeration is critically dependent upon the nonlinear relationship of the spectral shift with progressive dye–DNA binding and thus requires accurate definition. To this end, permeabilized cell reference samples permit reproducible instrument setup, guide gate boundaries for SP and compromised cells, and offer a simple means of comparing SP enumeration across laboratory sites/platforms. Our approach reports the dynamic range for the spectral shift, revealing noninformative staining conditions and explaining a source of variability for SP enumeration. We suggest that live cell kinetic sorting of all cells with the same dye:DNA load but with differences in efflux capacity can be used to explore drug resistance capability without prejudice. The SP phenotype should be regarded as a kinetic parameter and not a fixed characteristic—critical for functional assay design and the interpretation of heterogeneity.

The monitoring of cells labeled with quantum dot endosome-targeted markers in a highly proliferative population provides a quantitative approach to determine the redistribution of quantum dot signal as cells divide over generations. We demonstrate that the use of time-series flow cytometry in conjunction with a stochastic numerical simulation to provide a means to describe the proliferative features and quantum dot inheritance over multiple generations of a human tumor population. However, the core challenge for long-term tracking where the original quantum dot fluorescence signal over time becomes redistributed across a greater cell number requires accountability of background fluorescence in the simulation. By including an autofluorescence component, we are able to continue even when this signal predominates (i.e., >80% of the total signal) and obtain valid readouts of the proliferative system. We determine the robustness of the technique by tracking a human osteosarcoma cell population over 8 days and discuss the accuracy and certainty of the model parameters obtained. This systems biology approach provides insight into both cell heterogeneity and division dynamics within the population and furthermore informs on the lineage history of its members.

Single cell encoding with quantum dots as live cell optical tracers for deriving proliferation parameters has been developed using modelling to investigate cell cycle and proliferative outputs of human osteosarcoma cells undergoing mitotic bypass and endocycle routing. A computer-based simulation of the evolving cell population provides information on the dilution and segregation of nanoparticle dose cell by cell division and allows quantitative assessment of patterns of division, at both single cell and including whole population level cell cycle routing, with no a-priori knowledge of the population proliferation potential. The output therefore provides a unique mitotic distribution function that represents a convolution of cell cycle kinetics (cell division) and the partitioning coefficient for the labelled cell compartment (daughter-daughter inheritance or lineage asymmetry). The current study has shown that the cellular quantum dot fluorescence reduced over time as the particles were diluted by the process of cell division and had the properties of a non-random highly asymmetric event. Asymmetric nanoparticle segregation in the endosomal compartment has major implications on cell-fate determining signaling pathways and could lead to an understanding of the origins of unique proliferation and drug-resistance characteristics within a tumour cell lineage.

The maintenance of genomic stability in mitotic and meiotic cycles through mismatch repair (MMR) demands the coordination of MMR functions with multiple processes including cell cycle traverse, linked changes in microtubule dynamics, protein translocation at chromatin sites and checkpoint activation. We have studied changes in the intracellular location of the MMR protein Msh2 in response to mitosis, microtubule disruption by colcemid and DNA damage induction by cis-platin in mouse embryonic fibroblasts (MEFs). Image analysis indicated that MEFs have a normally high nuclear retention of Msh2 during interphase with a precipitous dispersal of protein from chromatin sites into the cytoplasm at mitosis. Dispersal was also observed in cisplatin- and colcemid-treated interphase MEFs without any change in the overall Msh2 levels throughout the cell cycle. There was no evidence of co-localization of the punctate cytoplasmic Msh2 foci with any microtubule structures and knockout of Msh2 altered neither the extent of microtubule disruption nor the functional activation of the spindle assembly checkpoint by colcemid. Critically, extra-nuclear relocation of protein did not alter the ability to mount an Msh2-dependent G2 checkpoint delay in response to cisplatin-induced DNA damage. Depletion of the nuclear pool of Msh2 protein in cells undergoing dispersal was found to involve a rapid relocation of protein from AT-rich chromatin sites as defined by coassociation studies exploiting a newly-characterized base-pair preference of the fluorescent DNA binding probe DRAQ5. The study reveals the unexpected mobility of MMR protein pools during the MEF cell cycle and in response to different stress-inducing agents. The results link for the first time microtubule-integrity with intra-nuclear Msh2 protein dynamics. The high nuclear retention of Msh2 in interphase MEFs is in contrast to human tumor cells while the observations on protein dispersal suggest that only low levels of nuclear-located Msh2 are needed for G2 checkpoint activation by DNA damage.

Dysfunction of cell-cycle checkpoints in DNA mismatch repair (MMR)-deficient cells in response to DNA damage has implications for anticancer therapy and genetic instability. We have studied the cell-cycle effects of MMR deficiency (Msh2(-/-)) in primary mouse embryonic fibroblasts (MEFs) exposed to cisplatin (10 microM x 1 h) using time-lapse microscopy. Kinetic responses of MEFs from different embryos and passage ages varied, but we report a consistent drug-induced inhibition of mitotic entry (approx. 50%). There was a loss of an early-acting (5 h) delay in G2 to M transition in Msh2(-/-) cells, although a later-acting G2 arrest was apparently normal. This suggests that Msh2 primarily acts to delay mitotic entry of cells already in G2, that is, DNA damage incurred during G2 does not influence the cell once committed to mitotic traverse. Irrespective of Msh2 status, cisplatin treatment and the incurred DNA damage did not effect mitotic traverse or show any evidence for early (within 24 h) cell death. The results indicate that Msh2(-/-) status can result in the premature commitment to mitosis of a cell subpopulation, determined by the fraction residing in G2 at the time of damage induction. The findings suggest a new route to MMR-driven genetic instability that does not rely primarily on the integrity of the late-acting checkpoint.

The anticancer agent topotecan is considered to be S-phase specific. This implies that cancer cells that are not actively replicating DNA could resist the effects of the drug. The cycle specificity of topotecan action was investigated in MCF-7 cells, using time-lapse microscopy to link the initial cell cycle position during acute exposures to topotecan with the antiproliferative consequences for individual cells. The bioactive dose range (0.5-10 microM) for 1-h topotecan exposures was defined by rapid drug delivery and topoisomerase I trapping. Topotecan caused pan-cycle induction and activation of p53. Lineage analysis of the time-lapse sequences identified cells initially in S-phase and G2, and defined the time to mitosis for cells originating from G2, S-phase and G1. Topotecan prevented all mitoses from S-phase cells and G1 cells (half-maximal effects at 0.14 microM and 0.96 microM, respectively). No dose of topotecan completely prevented mitosis among G2 cells, and at saturating doses of topotecan about half the cells of G2 origin continued dividing (the half-maximal effects was at 0.31 microM). Overall, topotecan differentially targeted G1-, S- and G2-phase cells, but many G2 cells were resistant to topotecan, presenting a clear route for cell cycle-mediated drug resistance.

Time-lapse microscopy can be described as the repeated collection of an image (in n-dimensions; x, y, z, λ) or field of view from a microscope at discrete time intervals. The duration of the time interval defines the temporal resolution, which in turn characterizes the type of event detected. This unit describes the implementation of time-lapse microscopy to link initial cell cycle position during acute exposures to anti-cancer agents with anti-proliferative consequences for individual cells. The approach incorporates fundamental concepts arising from the ability to capture simple video sequences of cells from which it is possible to extract kinetic descriptors that reflect the interplay of mitosis and cell death in the growth of an unsynchronized tumor population. Utilizing a multi-well format enables the user to screen different drug derivatives, multiple dose ranges, or cell cultures with unique genetic backgrounds. The objective of this unit is to present the basic methodology for capturing time-lapse sequences and touch upon subsequent mining of the data for deriving event curves and possible cell lineage maps.

An automated technique for the identification, tracking and analysis of biological cells is presented. It is based on the use of nanoparticles, enclosed within intra-cellular vesicles, to produce clusters of discrete, point-like fluorescent, light sources within the cells. Computational analysis of these light ensembles in successive time frames of a movie sequence, using k-means clustering and particle tracking algorithms, provides robust and automated discrimination of live cells and their motion and a quantitative measure of their proliferation. This approach is a cytometric version of the moving light display technique which is widely used for analyzing the biological motion of humans and animals. We use the endocytosis of CdTe/ZnS, core-shell quantum dots to produce the light displays within an A549, epithelial, lung cancer cell line, using time-lapse imaging with frame acquisition every 5 minutes over a 40 hour time period. The nanoparticle moving light displays provide simultaneous collection of cell motility data, resolution of mitotic traversal dynamics and identification of familial relationships allowing construction of multi-parameter lineage trees.

The delivery of nanoparticles into cells is important in therapeutic applications and in nanotoxicology. Nanoparticles are generally targeted to receptors on the surfaces of cells and internalized into endosomes by endocytosis, but the kinetics of the process and the way in which cell division redistributes the particles remain unclear. Here we show that the chance of success or failure of nanoparticle uptake and inheritance is random. Statistical analysis of nanoparticle-loaded endosomes indicates that particle capture is described by an over-dispersed Poisson probability distribution that is consistent with heterogeneous adsorption and internalization. Partitioning of nanoparticles in cell division is random and asymmetric, following a binomial distribution with mean probability of 0.52-0.72. These results show that cellular targeting of nanoparticles is inherently imprecise due to the randomness of nature at the molecular scale, and the statistical framework offers a way to predict nanoparticle dosage for therapy and for the study of nanotoxins.

We present a new approach to the handling and interrogating of large flow cytometry data where cell status and function can be described, at the population level, by global descriptors such as distribution mean or co-efficient of variation experimental data. Here we link the “real” data to initialise a computer simulation of the cell cycle that mimics the evolution of individual cells within a larger population and simulates the associated changes in fluorescence intensity of functional reporters. The model is based on stochastic formulations of cell cycle progression and cell division and uses evolutionary algorithms, allied to further experimental data sets, to optimise the system variables. At the population level, the in-silico cells provide the same statistical distributions of fluorescence as their real counterparts; in addition the model maintains information at the single cell level. The cell model is demonstrated in the analysis of cell cycle perturbation in human osteosarcoma tumour cells, using the topoisomerase II inhibitor, ICRF-193. The simulation gives a continuous temporal description of the pharmacodynamics between discrete experimental analysis points with a 24 hour interval; providing quantitative assessment of inter-mitotic time variation, drug interaction time constants and sub-population fractions within normal and polyploid cell cycles. Repeated simulations indicate a model accuracy of ±5%. The development of a simulated cell model, initialized and calibrated by reference to experimental data, provides an analysis tool in which biological knowledge can be obtained directly via interrogation of the in-silico cell population. It is envisaged that this approach to the study of cell biology by simulating a virtual cell population pertinent to the data available can be applied to “generic” cell-based outputs including experimental data from imaging platforms., Author Summary One of the key challenges facing cell biologists today is understanding the influence of molecular controls in shaping and controlling cell growth and proliferation. There is growing recognition that abnormal progression through the cell cycle and the associated effects on the growth of cell populations has a major impact on a wide range of biological and clinical problems, including: tumour growth, developmental control, origins of chromosomal instability and drug resistance. Multiparameter flow cytometry is frequently used to assess proliferation dynamics of cellular populations using fluorescent reporters generating large data sets that can inform simulation models. We have developed stochastic computing approaches allied to evolutionary algorithms to produce simulated cell populations—providing a new approach to the analysis of real multi-variate data sets obtained by flow cytometry. The methodology delivers new insight on biological processes in delivering a continuous simulation of the dynamic evolution of a cellular system between fixed sampling points, hence, converting static data into dynamic data revealing the effective traverse of the cell cycle, restriction points and commitment gateways. The approach also permits the visualisation of the variation between individual cells reflecting biological heterogeneity and potentially Darwinian fitness, given that the simulation delivers a report on population dynamics in which each and every cell can be tracked.

Cells cycle checkpoints guard against the inapproriate commitment to critical cell events such as mitosis. The bisdioxxopiperazzine ICRF-193, a catalytic inhibitor of DNA topoisomerase II causes a reversible stalling of the exit of cells from G(2) at the decatenation checkpoint (DC) and can generate tetraploidy via the compromising of chromosome segregation and mitotic failure. We have addressed an alternative origin-endocycle entry-for the tetraploidisation step in ICRF-193 exposed cells. Here we show that DC-proficient p53-functional tumor cells can undergo a transition to tetraploidy and subbsequent aneuploidy via an initial bypass of mitosis and the mitotic spindle checkpoint. DC-deficient SV4-tranformed cells move exclusively through mitosis to tetraploidy. In p53-functional tumor cells, escape through mitosis is enhanced by dominant negative p53 co-expression. The mitotic bypass transition phase (termed G(2)(endo)) disconnects cyclin B1 degradation from nuclear envelope breakdown and allows cells to evade the action of Taxol. G(2)(endo) constitutes a novel and alternative cell cycle phase-lasting some 8 h-with distinct molecular motifs at its boundaries for G(2) exit and subsequent entry into a delayed G(1) tetraploid state. The result challenge the paradigm that checkpoint breaching leads directly to abnormal ploidy states via mitosis alone. We further propose that the induction of bypass could: facilitate the covert development of tetraploidy in p53 functional cancers, lead to a misinterpretation of phase allocation during cell cycle arrest and contribbute to tumor cell drug resistance.

We present details of the development of a optical biochip, with integrated on-chip laser excitation, for fluorescence intensity cell based assays. The biochip incorporates an "active surface" for the control and manipulation of fluorescent species placed directly on the device. The active elements of the biochip are one-dimensional periodic sub-wavelength corrugations fabricated on a thin gold film. We have made fluorescence intensity measurements of both an organic dye (Cy5), and immobilized and fluorescently labeled (with 705 nm emitting quantum dots), mammalian tumor cells in contact with the active surface. Here we show that the presence of the periodic grating can be used to control both the excitation and fluorescence generation process itself. We demonstrate that the gratings convert evanescent surface optical modes into well-defined beams of radiation in the far-field and at the surface of the device this produces highly contrasting regions of fluorescence excitation providing regions of high spatial selectivity.

Quantum dot fluorophores provide a photo and bio-stable optical marker signal well suited to the tracking of lineage within large cell populations over multiple generations. We have used a Monte Carlo algorithm to model the process of dot partitioning and dilution by cell mitosis. A Genetic Algorithm was used to compare simulated and experiment quantum dot distributions, which shows that the dot fluorescence is divided with a stochastic variation about an asymmetric mean split ratio.

We have developed a range of optical biochip devices for conducting live and fixed cell-based assays. The devices encompass the ability to process an entire assay including fluorescently labelling cells, a microfluidic system to transport and maintain cells to deliver them to an optical area of the device for measurement, with the possibility of a incorporating a sorting step in between. On-chip excitation provided by red emitting LED and lasers define the excitation wavelength of the fluorophore to be incorporated into the assay readout. The challenge for such an integrated microfluidic optical biochip has been to identify and characterise a longterm fluorescent label suitable for tracking cell proliferation status in living cells. Traditional organic fluorophores have inherent disadvantages when considering their use for an on-chip device requiring longterm cellular tracking. This has led us to utilise inorganic quantum dots (QDots) as fluorophores for on- chip assays. QDs have unique properties such as photostability, broad absorption and narrow emission spectra and are available in a range of emission wavelengths including far red. They also have much higher quantum efficiencies than traditional organic fluorophores thus increasing the possible dynamic range for on-chip detection. Some of the QDots used have the added advantage of labelling intact cells and being retained and distributed among daughter cells at division, allowing their detection for up to 6 generations. The use of these QDs off-chip has suggested that they are ideal for live cell, nonperturbing labelling of division events, whereby over time the QD signal becomes diluted with each generation. Here we describe the use of quantum dots as live cell tracers for proliferating populations and the potential applications in drug screening and optical biochip environments.

In this paper, we report on the development of a fully integrated biochip that uses surface plasmons to control all aspects of a fluorescence assay. This includes both excitation and emission characteristics and so in addition to the fluorescence enhancement/quenching also control of the spatial location, beam direction and polarization of the excitation light. These effects are investigated with both bare fluorescent molecules and quantum dot-labeled cells

Background: We report on the potential DNA binding modes and spectral characteristics of the cell-permeant far red fluorescent DNA dye, DRAQ5, in solution and bound within intact cells. Our aim was to determine the constraints for its use in flow cytometry and bioimaging. Methods: Solution characteristics and quantum yields were determined by spectroscopy. DRAQ5 binding to nuclear DNA was analyzed using fluorescence quenching of Hoechst 33342 dye, emission profiling by flow cytometry, and spectral confocal laser scanning microscopy of the complex DRAQ5 emission spectrum. Cell cycle profiling utilized an EGFP–cyclin B1 reporter as an independent marker of cell age. Molecular modeling was used to explore the modes of DNA binding. Results: DRAQ5 showed a low quantum yield in solution and a spectral shift upon DNA binding, but no significant fluorescence enhancement. DRAQ5 caused a reduction in the fluorescence intensity of Hoechst 33342 in live cells prelabeled with the UV excitable dye, consistent with molecular modeling that suggests AT preference and an engagement of the minor groove. In vivo spectral analysis of DRAQ5 demonstrated shifts to longer wavelengths upon binding with DNA. Analysis of spectral windows of the dual emission peaks at 681 and 707 nm in cells showed that cell cycle compartment recognition was independent of the far red–near IR emission wavelengths monitored. Conclusions: The study provides new clues to modes of DNA binding of the modified anthraquinone molecule in vivo, and its AT base-pair selectivity. The combination of low quantum yield but high DNA affinity explains the favorable signal-to-noise profile of DRAQ5-nuclear fluorescence. The robust nature of cell cycle reporting using DRAQ5, even when restricted spectral windows are selected, facilitates the analysis of encroaching spectral emissions from other fluorescent reporters, including GFP-tagged proteins. © 2006 International Society for Analytical Cytology

In this paper we report on the development of an optical biochip to control both the excitation and resultant fluorescence using grating coupled surface plasmons. Electron beam lithography is used to fabricate line gratings in thin layers of gold on the surface of 150μm thick coverslips. Laser diodes operating at 630nm are close coupled to the coverslip resulting in the excitation of surface plasmons. In the region of the grating light can radiate into the far-field, and both the angle of emission and beam divergence can be controlled by the grating pitch and the number of lines included in the pattern. A model is presented which treats the grating as an optical antenna array which shows how these characteristics can be explained in terms of the wave vector matching between the surface plasmons and the grating. Fluorescence has also been excited in standard organic dyes on-chip. When placed in close proximity to the surface of the sample strong quenching of the fluorescence is seen in the region of the grating. In contrast an enhancement of the signal is seen when the fluorophores are placed on a 200nm thick spacer layer.

Time-lapse microscopy can be described as the repeated collection of a field of view from a microscope at discrete time intervals. The duration of the time interval defines the temporal resolution, which in turn characterizes the type of event detected. This unit describes the implementation of time-lapse microscopy to link initial cell cycle position during acute exposures to anti-cancer agents with anti-proliferative consequences for individual cells. The approach incorporates fundamental concepts arising from the ability to capture simple video sequences of cells from which it is possible to extract kinetic descriptors that reflect the interplay of mitosis and cell death in the growth of an unsynchronized tumor population. Utilizing a multi-well format enables the user to test different drug derivatives, multiple dose ranges, or cell cultures with unique genetic backgrounds. The objective of this unit is to present a generic methodology for capturing time-lapse sequences and subsequently mining the data for comprehensive event analysis. Keywords: video microscopy; time-lapse; data mining; cell cycle dynamics

Dynamic physical interactions between proteins underpin all key cellular processes and are a highly attractive area for the development of research tools and medicines. Protein-protein interactions frequently involve α-helical structures, but peptides matching the sequences of these structures usually do not fold correctly in isolation. Therefore, much research has focused on the creation of small peptides that adopt stable α-helical structures even in the absence of their intended protein targets. We show that short peptides alkylated with azobenzene crosslinkers can be used to photo-stimulate mitochondrial membrane depolarization and cytochrome c release in permeabilised cells, the initial events of the intrinsic apoptosis pathway.

Background The inheritance of cellular material between parent and daughter cells during mitosis is highly influential in defining the properties of the cell and therefore the population lineage. This is of particular relevance when studying cell population evolution to assess the impact of a disease or the perturbation due to a drug treatment. The usual technique to investigate inheritance is to use time-lapse microscopy with an appropriate biological marker, however, this is time consuming and the number of inheritance events captured are too low to be statistically meaningful. Results Here we demonstrate the use of a high throughput fluorescence measurement technique e.g. flow cytometry, to measure the fluorescence from quantum dot markers which can be used to target particular cellular sites. By relating, the fluorescence intensity measured between two time intervals to a transfer function we are able to deconvolve the inheritance of cellular material during mitosis. To demonstrate our methodology we use CdTe/ZnS quantum dots to measure the ratio of endosomes inherited by the two daughter cells during mitosis in the U2-OS, human osteoscarcoma cell line. The ratio determined is in excellent agreement with results obtained previously using a more complex and computational intensive bespoke stochastic model. Conclusions The use of a transfer function approach allows us to utilise high throughput measurement of large cell populations to derive statistically relevant measurements of the inheritance of cellular material. This approach can be used to measure the inheritance of organelles, proteins etc. and also particles introduced to cells for drug delivery.

We report on experimental observations of highly collimated beams of radiation generated when a periodic sub-wavelength grating interacts with surface bound plasmon-polariton modes of a thin gold film. We find that the radiation process can be fully described in terms of interference of emission from a dipole antenna array and modeling the structure in this way enables the far-field radiation pattern to be predicted. The directionality, multiplicity and divergence of the beams can be completely described within this framework. Essential to the process are the surface plasmon excitations: these are the driving mechanism behind the beam formation, phase-coupling radiation from the periodic surface structure and thus imposing a spatial coherence. Detailed fitting of the experimental and modeled data indicates the presence of scattering events involving the interaction of two surface plasmon polariton modes.