Quantifying the volume of single cells continuously using a microfluidic pressure-driven trap with media exchange     

Jason Riordon  Michael Nash  Wenyang Jing  Michel Godin 《Biomicrofluidics》2014,8(1)

We demonstrate a microfluidic device capable of tracking the volume of individual cells by integrating an on-chip volume sensor with pressure-activated cell trapping capabilities. The device creates a dynamic trap by operating in feedback; a cell is periodically redirected back and forth through a microfluidic volume sensor (Coulter principle). Sieve valves are positioned on both ends of the sensing channel, creating a physical barrier which enables media to be quickly exchanged while keeping a cell firmly in place. The volume of individual Saccharomyces cerevisiae cells was tracked over entire growth cycles, and the ability to quickly exchange media was demonstrated.Measuring cell growth is of primary interest to researchers who seek to study the effects of drugs, nutrients, disease, and environmental stress. This has traditionally been accomplished by monitoring the optical transmittance of large ensembles of cells and applying the Beer-Lambert Law.^1,2 Such population-scale measurements provide important culture statistics, but averaging obscures the behaviour of individual cells. In addition, these techniques often require cell synchronicity in order to correlate growth with specific points in the cell cycle, but synchronicity typically decays rapidly in many cell lines including Saccharomyces cerevisiae (yeast) cultures.³ Researchers have thus adopted methods that study the growth of individual cells. Quantifying cellular growth is especially challenging since proliferating cells such as yeast or Escherichia coli are irregularly shaped, and will only increase in size by a factor of two.⁴ Growth will affect the mass, volume, and density of the cell; having access to each of these characteristics is important in obtaining a complete picture of this process. Time-lapse fluorescence microscopy can provide valuable information as to the cell cycle progression of individual cells,⁵ but 2D optics requires geometric assumptions, and, thus, can provide an incomplete picture of growth.^6,7Microfluidic lab-on-chip devices with integrated sensors can provide high-resolution growth tracking of individual cells, either through mass, volume, or density monitoring.^4,7,8 Recently, a microfluidic mass sensor was used to track the buoyant mass of individual cells using a suspended microchannel resonator (SMR).^4,9 Monitoring growth can also be accomplished by tracking volume using microfluidic volume sensors⁷ operating on the Coulter principle.¹⁰ Trapping can be achieved by either (1) cycling the target back and forth through the sensor (pressure-driven⁴ and electrokinetic⁷) or (2) holding a cell in place (posts,¹¹ chevron structure,¹² and E-Field¹³). The former, dynamic approach, allows a single cell to be sampled periodically by reversing flow directions after a cell is detected. Simple in its implementation, this technique also has the ability to compensate for a drifting baseline current resulting from parasitic ionic changes within the sensing channel or other sources of noise. On the other hand, static traps allow cells to be held in place while the buffer is rapidly exchanged.¹² The ability to dynamically change cellular growth conditions during an experiment can lead to significant insight into the behaviour of cells in environments of varying salinity,¹⁴ oxidative,^15,16 or osmotic conditions,¹⁷ as well as the effect of nutrients¹⁸ and drugs.¹⁹In this work, we propose a device capable of tracking growth using high-resolution volume measurements, combining the best attributes of both types of measurement systems; continuous baseline correction and the ability to rapidly exchange cell media. This is accomplished by using a pressure-driven, feedback-based dynamic trap, whereby a cell is cycled back and forth through the sensor within a microfluidic channel. On-chip sieve valves positioned at both ends of the sensing channel are able to selectively capture a cell while the solution is being replaced. As proof of principle, the volume of several individual yeast cells was monitored over the course of their respective growth cycles, and the ability to quantify growth response to media exchange was demonstrated.Devices were fabricated using multilayered soft lithography with polydimethylsiloxane (PDMS) molding.²⁰ The completed device is pictured in Figure 1(a); full fabrication protocols are presented as supplementary material.²¹ To maximize measurement sensitivity, it is optimal to choose a channel width and height slightly larger than the dimensions of the target cell.²² However, yeast cells are asymmetrically shaped and tend to tumble as they traverse the sensor. Preliminary testing suggested this effect could be mitigated by having cells flow along trajectories far from the electrodes (through buoyancy), where electric field is more uniform. Thus, a channel height of 20 μm was chosen as a compromise. Channel height increases to 28 μm in the wider part of the central and bypass channels, a result of using a mold made out of reflowed photoresist.²³ Channel width was set at 25 μm through the sensor, and widens to 80 μm at the sieve valves to facilitate valve actuation, which requires a high width to height ratio.²⁰ The fluidic layer is integrated in a 35 μm thick PDMS spin-coated layer, above which sits a 50 μm tall valve channel in a 4 mm PDMS layer. Tubing connects I1 and I2 to a common inlet vial, V1 and V2 to vials filled with deionised water and O1 and O2 connect to empty vials (not pictured). Inlet pressures I1 and I2, and valve pressures V1 and V2 are controlled with manual regulators (SMC IR2000-N02-R and SMC IR2010-N02-R); outlet pressures are computer-controlled (SMC ITV-1011). This pressure scheme is detailed elsewhere.²⁴ Current pulses caused by transiting particles/cells (Figure 1(d)) were acquired by applying a 50 kHz, 220 mV AC voltage between a pair of electrodes and measuring the drawn current. This frequency is sufficiently elevated to avoid the electrical double layer capacitance at the electrode-electrolyte interface,²⁵ but low enough to avoid sensitivity to cell impedance or substrate.²⁶ The electrical setup used for these experiments has been described previously.^24,27 A temperature controller maintains the device at 30 °C.Open in a separate windowFIG. 1.(a) Micrograph of the microfluidic device. Two parallel bypass channels are connected by a sensing channel with sensing electrodes. Pressure is applied at inlets (I1, I2) and outlets (O1, O2) to control flow conditions. Valves (V1, V2) are positioned over each end of the sensing channel. Food coloring is used to highlight the valve (red) and fluidic layers (blue). (b) Flow mode: valves are unpressurized, and cells flow freely through the device. (c) Trapping mode: valves are pressurized to capture a cell within the central channel. Pressure-driven flow cycles the cell back and forth across the sensor. (d)Typical current pulses measured for a yeast cell.The cell capture, media exchange, and detection process occurs as follows. A cell suspension is loaded into the bypass channel and made to flow through the central sensing channel by imposing a pressure gradient (Figure 1(b)). Cells flowing through the sensor are observed optically; once a cell of interest is observed (a cell without a bud), valves are sealed (V1 = V2 = 35 psi). This stops all flow through the sensor, and enables bypass channels to be flushed and replaced with fresh media. After 2 min, valve channels are pressurized to 24 psi where they compress the channel to a sufficient height to physically restrict the passage of yeast cells, while allowing the media to flow through the central channel (Figure 1(c)). The pressure gradient between bypasses causes the media in the central channel to be flushed out, while the target cell is physically trapped. Replacing the media in the central channel takes 2 min. At this stage, a pressure-driven feedback-based dynamic trap can be initiated. In this dynamic trap mode, the pressure settings at O1 and O2 are adjusted to redirect the cell back and forth through the sensor, based on current pulses measured from cells transiting through the sensor. Through custom LabView® software, these outlet pressure settings are feedback-adjusted to maintain a speed of 250 μm/s in both directions at a detection frequency of 30 cells/min (Figure 1(d)). To minimize the effects of channel stretching/shrinking, the sum of pressures at O1 and O2 is held constant. This precaution was taken since the sensing channel structured within the flexible PDMS polymer will alter its geometry based on internal pressure.²⁸ The short central channel ensures steady nutrient replenishment from the bypasses. For example, a glucose molecule takes ∼4 min to diffuse from the bypass to the electrodes. In practice, Taylor-Aris dispersion will reduce this replenishment time considerably. Based on video analysis, 25% of the central channel''s media is replenished every pressure reversal (video presented as supplementary material²¹). Polystyrene microspheres of 3.9 ± 0.3 μm, 5.6 ± 0.2 μm, and 8.3 ± 0.7 μm (NIST size standards) were used to calibrate the sensor, and obtain the current pulse-to-volume calibration for every solution (supplementary material²¹). The validity of this calibration method is discussed elsewhere.²⁹ Care was taken to limit trajectory-based variations in signal: the device is positioned with electrodes at the top of the sensing channel, and with the negatively buoyant cells/particles flowing along the bottom. Based on previous experimental and theory work, we found that signal amplitude can vary as much as 3.5 fold for different heights.²⁷ The effect of trajectory on current pulse amplitude has also been reported elsewhere.^30,31 In this work, buoyancy is used to ensure that the cell flows along a trajectory at the same distance from the electrodes for every measurement.Saccharomyces cerevisiae (BY4743 Mat a/alpha, genotype: his3Δ1/his3Δ1 leu2Δ0/leu2Δ0 LYS2/lys2Δ0 met15Δ0/MET15 ura3Δ0/ura3Δ0 ade2::LEU2/ade2::URA3) was cultured to exponential phase at 30 °C in an incubator/shaker in yeast bacto-peptone (YPD) with 2% w/v glucose, supplemented with 0.2 M NaCl, 0.05% bovine serum albumin (BSA) and 42 mg/l adenine. Sodium chloride was added to enable the current pulse measurement, at a concentration where cells are viable;³² BSA was used to prevent cell agglomeration; adenine was supplemented since this particular yeast mutant does not produce its own supply. A cell suspension was introduced into the device, from which a cell at the early stages of its cell cycle was captured, and dynamically trapped for 100 min. Three typical cell growth results are shown in Figure 2(a). Since the culture was not synchronized, this leads to variability between “initial” cell volumes: there is a 27% difference in initial volume between the cells identified by red squares and green triangles. This is caused by (1) optical limits, whereby cells chosen for study are not all at the exact same cell cycle stage and (2) differences in the age of the mother cell: the more buds a mother cell has produced, the larger it becomes.³³ On average, captured yeast cell demonstrated a doubling time consistent with growth rates under ideal incubator/shaker conditions; nutrient depletion, electric field, and shear stresses are not affecting growth. Optical inspection of budding cells confirms that most growth is occurring at the daughter cell, as expected.³³ An elevated signal-to-noise ratio allows for high resolution volumetric measurements (4 μm³); cell asymmetry⁷ and trajectory variability^27,30,31 lead to a relative standard deviation of 6% for cells and 4% for microspheres of similar size. While mass or protein synthesis methods have indicated linear³⁴ or exponential^4,6,35,36 growth curves, volume-based methods have suggested sigmoidal patterns.^7,37 Prior to daughter cell emergence, and later in the cycle as the daughter cell emerges, volumetric growth rate declines.³⁸ In this work, it is difficult to ascertain with mathematical rigor the shape of the growth profile; however, for each cell, volume increases steadily throughout the growth cycle before declining near the end of the cycle.Open in a separate windowFIG. 2.(a) Growth curves for 3 cells trapped in succession. Simultaneous optical and electrical measurements allow cell cycle stage to be correlated with volume. Pictures of cell corresponding to the red squares are presented in 15 min increments. A cell is cycled through the sensor every 2 s. For clarity, each data point for yeast volume represents the average of data points over a period of 5 min, with standard deviation. (b) Demonstration of an interrupted growth cycle, where YPD + 0.2 M NaCl was replaced with 0.2 M NaCl at 40 min, and then again returned to YPD + 0.2 M NaCl at 80 min. The media exchange process takes 4 min.To demonstrate our ability to easily exchange media while maintaining a trap, the solution was exchanged 40 min into a yeast growth cycle; culture media was replaced with a pure saline solution 0.2 M NaCl + 0.05% BSA, and then replaced again with culture media at 80 min (Figure 2(b)). Cell growth is halted temporarily while in saline solution, before resuming normal growth thereafter. The cell cycle time is extended by this period. The cell volume drifts downward after the initial solution change at 40 min. Though this drift lies within our uncertainty bounds, cellular responses to osmotic shock on similar timescales have been documented elsewhere.³⁹ This result demonstrates an ability to quickly exchange cell media, and observe cellular response.In conclusion, we have demonstrated a microfluidic device capable of maintaining a dynamic, pressure-driven cell trap, which can monitor cellular volume over the cell cycle. Concurrent optical microscopy allows for real-time visual inspection of the cells. In addition, sieve valve integration provides for the exchange of media or the addition of drugs. Such a platform could also be key in cancer cell cytotoxicity assays,⁴⁰ where growth response to anticancer drugs could be monitored.  相似文献   

A distributional analysis of the effect of physical exercise on a choice reaction time task     

Davranche K  Audiffren M  Denjean A 《Journal of sports sciences》2006,24(3):323-329

The aim of this study was to examine the facilitating effects of physical exercise on the reaction process. Eleven participants with specific expertise in decision-making sports performed a choice reaction time task during moderate sub-maximal exercise (90% of their ventilatory threshold power). Participants were tested at rest and while cycling. During exercise, the participants were faster, without being more variable. We suggest that the effect of exercise on cognitive performance was due to a major generalized improvement of the whole distribution of response time and, although the benefit effect was small, it was consistent throughout the entire range of reaction times.  相似文献   

Effective learning activities in observation taks when learning to write and read argumentative texts     

Martine A. H. Braaksma  Huub van den Bergh  Gert Rijlaarsdam  Michel Couzijn 《European Journal of Psychology of Education - EJPE》2001,16(1):33-48

On repeated occasions, observational learning has proved itself to be an effective instruction method. Experimental studies have shown to be effective for complex tasks such as reading and writing for both teachers and students as models. The problem when interpreting the results of such research is that, in observation tasks, several mental activities play a simultaneous role. In this study we therefore set out to identify the effective elements of observation tasks. We focused on two elements of the observation tasks, both aimed at stimulating monitoring activities: evaluation of the model’s performance and elaboration on this evaluation. We have also distinguished between elaboration on the observed products (the models’ written answers), and elaboration on the observed processes (the models’ verbalisations of their mental activities). The data were subjected to a LISREL analysis. First of all, it was observed that subjects who performed “evaluation” and “productelaboration” better, and “process-elaboration” more often in one lesson, also performed these activities better or more often in the subsequent lesson. Next, we observed an effect of aptitude on the learning activities: pre skill scores influence “evaluation” and “product-elaboration”. The most important finding is that “evaluation” and “product-elaboration” contribute positively to argumentative writing skills. It is discussed that these findings confirm the importance of the monitoring, evaluative and reflective activities when learning complex tasks as writing.  相似文献   

The Economic Functions of Schooling     

Michel Kostecki 《Compare》1985,15(1):5-19

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Etude en temps réel de la production eEcrite chez des enfants de sept et huit ans     

Jean-Noël Foulin  Michel Fayol 《European Journal of Psychology of Education - EJPE》1988,3(4):461-475

The aim of this research is to study on-line written production in children. Twelve seven year old children and twelve eight year old children were asked to compose two texts (a narrative and a report). They were filmed as they wrote in order to investigate the temporal caracteristics of their composition (pauses, rates…). The results show three main findings:

- the rates increases from seven to eight year old.

- pauses times varied according to the syntactic organisation of the next sentence.

- longer pause times are localized after punctuation marks and before connective.

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Le mouvement de réforme de la formation des enseignants en Grèce     

Michel Cassotakis 《欧洲师范教育杂志》1985,8(2):133-146

The article undertakes a close analysis of the perceived weaknesses of Secondary teacher training in Greece. The analysis is based upon the criticisms of other observers and of the Greek Federation of Secondary Teachers, and especially upon an enquiry conducted of 233 Secondary teachers in May 1983 (approximately 2% of the cadre). The teachers concerned were invited to report upon their own perceptions of the training which they had undergone, and of its relevance to their later professional careers. The overall picture is one of considerable dissatisfaction with a training which is labelled, at one point, ‘anachronistic'; the view is expressed that the education given does not even attain its declared aim of instilling an understanding of scientific methodology.

There exists a strong demand for a greater emphasis upon pedagogy, with the approach through psycho‐pedagogics being much favoured; the author enters the reservation that the scepticism which begins to be shown in some European countries is not yet evident in Greece. There is also a demand for greater emphasis on practical training, though there exists a debate over the question of consecutive versus concurrent training, with the majority favouring the former.

Explanations for the present ‘disastrous’ situation in Greek teacher training are varied, ranging from the view that forces of political conservatism are at work to the view that Greek educational thinking is over‐dependent upon ideas brought in from other European countries. Certainly the author sees the Greek system as imposing a high level of conformity upon the beginning teacher, who is ill equipped to offer challenging ideas to existing practice. The climate, however, is one of change, and the author is finally optimistic about the growing contribution of educational sciences to Greek teacher training.  相似文献   

Developing a quality curriculum in a technological era     

Twining  Peter  Butler  Deirdre  Fisser  Petra  Leahy  Margaret  Shelton  Chris  Forget-Dubois  Nadine  Lacasse  Michel 《Educational technology research and development : ETR & D》2021,69(4):2285-2308

Educational technology research and development - There is considerable rhetoric internationally around the need for national curricula to reflect the changes that are taking place in the world...  相似文献   

科克苏-穹库什太古生代构造-岩浆作用及其对西南天山造山时代的约束     

王博  舒良树Michel FAURE Dominique CLUZEL Jacques CHARVET 《中国科技期刊研究》2007,23(6):1354-1368

天山造山带是古生代多期碰撞增生作用的产物,其确切的造山时代是当前争议较多的热点问题.分布在西南天山的科克苏-穹库什太剖面经历了复杂的构造变形,最明显的两期变形事件分别为朝北的推覆作用和NE-SW韧性走滑作用.本剖面可分为伊犁岩浆岛弧、伊犁结晶基底、高压变质杂岩三个岩石-构造单元.野外可见黑云母花岗闪长岩侵入到绿片岩相变质岩中,岩脉切穿绿片岩中面理构造.通过锆石U-Pb LA-ICPMS测年,科克苏剖面钾长花岗岩的年龄为341±6Ma和338±8Ma,穹库什太黑云母花岗闪长岩的年龄为313±4Ma.其中,花岗闪长岩发生了黑云母定向排列,通过对黑云母进行^40 Ar/^39 Ar测年,获得坪年龄为263.4士0.6Ma,表明该花岗闪长岩受过后期热事件的干扰.地球化学分析表明,黑云母花岗闪长岩属于钙碱性系列,Nb和Ta含量低而Rb,Ba和TH含量很高,与俯冲作用有关的岛弧岩浆岩地球化学组成非常相似.结合前人对该地区高压变质岩、花岗岩和火山岩的研究成果,本文提出,西南天山俯冲-碰撞造山作用发生在晚石炭世之前,研究区后碰撞区域走滑作用标志着西南天山碰撞造山作用在二叠纪之前全部结束.  相似文献   

The development of inventive thinking skills in the upper secondary language classroom     

Alexander Sokol  David Oget  Michel Sonntag  Nikolai Khomenko 《Thinking Skills and Creativity》2008,3(1):34-46

The given paper presents the results of an empirical study into the efficacy of the Thinking Approach (TA) to language teaching and learning which is aimed at the development of students’ inventive thinking skills in the context of foreign language education, namely learning of English. The study was conducted among upper secondary students of two schools in Latvia and aimed to answer whether students working with the Thinking Approach demonstrate an increase in their inventive thinking skills. An inventive thinking test was employed as the research instrument. The results of the study suggest that students working with the TA demonstrate a significant increase in their inventive thinking skills in comparison with the control group (t = 3.32, p = 0.001). At the same time a number of limiting factors that appeared in the process of the study due to its naturalistic setting call for further research that could increase the reliability of the findings.  相似文献   

Three Generations of Complexity Theories: Nuances and ambiguities   总被引：1，自引：0，他引：1  

Michel  Alhadeff-Jones 《Educational Philosophy and Theory》2008,40(1):66-82

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