Article pubs.acs.org/acssensors
Characterization of Single Yeast Cell Phenotypes Using Microfluidic Impedance Cytometry and Optical Imaging Niels Haandbæk,*,† Sebastian C. Bürgel,† Fabian Rudolf,† Flavio Heer,‡ and Andreas Hierlemann† †
Department of Biosystems Science and Engineering, ETH Zurich, 4058, Basel, Switzerland Zurich Instruments AG, Technoparkstrasse 1, 8005, Zurich, Switzerland
‡
S Supporting Information *
ABSTRACT: Single-cell impedance cytometry is a noninvasive method for characterizing the dielectric properties of individual cells. The method has been used for counting and sizing of cells and particles, as well as for discriminating between different cell types in a label-free manner. However, finding a relationship between a specific cell phenotype and the corresponding measured impedance remains a challenging problem. This paper reports on a platform that combines a microfluidic impedance cytometer with a high-speed camera in order to allow for characterizing the physical morphology of single cells in parallel with the dielectric properties. This enables the extraction of signatures in the impedance data that can be associated with specific morphological phenotypes. We demonstrate the functionality of the platform by developing such signatures for discriminating between single and budding yeast cells. KEYWORDS: single-cell impedance cytometry, high-speed imaging, impedance spectroscopy, S. cerevisiae, morphology, PCA, QDA, classification
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channel. The channel features electrodes patterned either in an opposing configuration at the channel top and bottom, or in a coplanar configuration with electrodes only at the bottom. An AC voltage is applied to the electrodes, which causes a current to flow between them. The current change upon passage of a cell or particle between the electrodes is differentially measured and then analyzed to determine the particle dielectric properties. So far, single-cell impedance cytometry has mostly been used for discrimination between cells with relatively large differences in morphology. For example, Cheung et al. demonstrated the ability to discriminate between red blood cells and red blood cells that have been fixed by using glutaraldehyde.15 The investigation of the relationship between specific features in the impedance spectrum of cells of the same type and their morphologies and orientation relative to the detection electrodes has until now received little attention. A device suitable for such an investigation would need to acquire information related to cell morphology independently from, and in parallel with, the impedance data. Spencer et al. have demonstrated a microcytometer that allows for simultaneous recording of impedance and fluorescence data on a single-cell basis.18 The main limitation of their approach is that it is not label-free in the sense that it requires the generation of fluorescent markers, which target the specific cell morphologies
mpedance spectroscopy is a nondestructive experimental method for characterizing the electric and dielectric properties of materials or samples by measuring impedance values over a range of frequencies. The use of impedance spectroscopy for characterization of biological systems was pioneered by Höber, Fricke, Curtis, and Cole.1−3 Pethig, Foster, and Schwan have reviewed the theory and main contributions throughout the history of the field extensively.4−6 More recently, impedance spectroscopy has been used in diverse applications, such as evaluating the viability of tissue,7 monitoring cardiac function,8 and detecting breast cancer.9 In these applications the dielectric properties of larger samples of biological tissue are measured, which depend, among other things, on the density of cells, their membrane capacitances, and the electric conductivity of the intracellular medium. It has become increasingly important to develop analysis techniques with single-cell resolution in order to investigate details about the heterogeneity of individual cells in larger populations.10 Such information, which is difficult to acquire using traditional bulk assays,11 could assist in the development of mathematical descriptions and models of cellular behavior in the context of systems biology.12 The integration of impedance spectroscopy with microfluidic technology has enabled the creation of lab-on-chip devices, capable of label-free characterization of single cells.13 Many authors have reported on such devices with a similar general operating principle:14−17 Cells or particles are dispersed in an electrolyte, such as phosphatebuffered saline (PBS), and pumped through a microfluidic © XXXX American Chemical Society
Received: April 26, 2016 Accepted: July 13, 2016
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Figure 1. Schematic of the overall microfluidic cytometer setup. The lock-in amplifier drives the electrodes with an alternating current (AC) signal with four frequency components. The impedance is measured by the lock-in amplifier in the form of complex signals with real (X) and imaginary (Y) parts, which are buffered in a first-in-first-out (FIFO) queue and transmitted to a host PC for analysis. The high-speed camera monitors the cells after passage through the impedance electrodes. The camera features a digital output signal that indicates when images are being acquired, which was connected to one of the auxiliary analog-to-digital converters (ADC) of the lock-in amplifier and recorded together with the impedance data.
Figure 2. Schematic diagram of the microfluidic device. (A) Schematic diagram of the microfluidic device. (B) Schematic diagram of the inlet and filter. (C) Schematic top and front view of the focusing region, not drawn to scale, illustrating the tapered shape of the electrodes. The front view also shows how the focusing electrodes are electrically connected to the signal generator. The focusing electrodes have a width of 40 μm at the start of the focusing region, where they are aligned with the channel wall, and a width of 65 μm at the end of the focusing region.
throughput in terms of the number of cells that can be characterized within a given time frame is lower in comparison to what is possible with flowthrough devices. In this work we report on a platform that combines a microfluidic impedance cytometer with a high-speed camera in order to allow the physical morphology of single yeast cells of the S. cerevisiae species to be assessed in parallel with their dielectric properties. The goal was to develop a method for describing the cell cycle state based on the measured
to be investigated. Shaker et al. have demonstrated a device capable of measuring the impedance of single cells in multiple directions in order to assess cell morphology.19 Another method has been demonstrated by Zhu et al.,20 in the form of a device capable of immobilizing single yeast cells, and then monitoring their impedance over time. The device also allows for acquisition of optical images of the trapped cells. A limitation of that method is the need for manually trapping and releasing cells to be investigated. As a consequence, the B
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diluted with deionized water in order to decrease the conductivity to 0.1 S/m. The conductivity of the final medium was verified to be 0.12 S/m using a Mycom CLM141 conductivity transmitter (Endress +Hauser, Reinach, Switzerland), combined with a JUMO Blackline conductivity probe (JUMO, Stäfa, Switzerland). The cells were centrifuged for 5 min at 6000 rpm and then resuspended in the prepared measurement medium. The cell concentration in the solution was adjusted to approximately 200 × 103 cells/mL by further dilution with the measurement medium. A solution of monodisperse 3 μm polystyrene beads (Life Technologies Ltd., UK) was prepared in a similar fashion. The bead concentration in the suspension was adjusted to approximately 100 × 103 beads/mL. Cells and beads were mixed in equal proportions, loaded into a glass syringe and driven through the channel in the microfluidic device by means of a neMESYS syringe pump (Cetoni GmbH, Korbußen, Germany) at a flow rate of 0.5 μL/ min. Impedance Acquisition and Analysis. The impedance of the suspension was analyzed at four frequencies of f1 = 0.55 MHz, f 2 = 1.15 MHz, f 3 = 9.09 MHz, and f4 = 20.81 MHz. The frequency range covers cell size, measured at 0.55 MHz, as well as membrane capacitance, measured at up to around 20 MHz.27 The specific frequencies were chosen based on the dielectric spectra of yeast cells simulated by Asami et al.,28 in order to enable discrimination between single and budding cells. The stimulation signal was generated as a superposition of sine waves at the four analysis frequencies. The amplitude of the 0.55 MHz signal was 90 mV. The amplitude of each of the other frequency components was 45 mV. The time-domain impedance signals from the impedance spectroscope were recorded at a sampling rate of 55 kSamples/s with a lock-in filter bandwidth of 2 kHz. The signals were then analyzed using MATLAB (MathWorks, Inc., USA) to identify peaks corresponding to cells and particles passing the electrodes. The velocity was also calculated based on the temporal position of the two peaks in the impedance signal in combination with the known center-to-center spacing between the two electrode pairs of 36 μm. The combined information acquired in this way for each cell or particle is denoted as an impedance event. The impedance events were further processed using principal component analysis (PCA) in order to reduce the number of variables to be considered when identifying signatures associated with the different cell phenotypes.29 Details of the used PCA method can be found in the Supporting Information. Image Acquisition and Analysis. The high-speed camera acquired gray scale images in parallel with the impedance data at a rate of 1000 frames/s with an exposure time of 20 μs per frame. Cells and particles were detected in the frame sequence by means of the algorithm outlined in the Supporting Information. Objects were then manually assigned the class of bead, single cell, or budding cell. Further information, such as the cell cross-sectional area, was also extracted from the image sequence. The internal memory of the camera limited the acquisition time to approximately 9 s per recording. The impedance signals were acquired for 18 s per recording. The camera issues an “acquisition active” signal when it starts recording, which was connected to the impedance spectroscope and saved as part of the impedance data. The “acquisition active” signal was then used to determine the temporal alignment between the optical and impedance data. In a further step, the impedance events were labeled with the corresponding optically determined class. Note that, due to the shorter recording times of the camera compared to the impedance spectroscope, only half of the impedance events could be labeled based on the optical information.
impedance signal. The platform records impedance values, as well as images of the cells as they pass through the microfluidic channel. The image sequence is analyzed in order to detect and track the cells. Moreover, the orientation of the cells upon passing the detectors is assessed. Each detected cell is further categorized according to its optically detected morphology, either as “single cell” or “budding cell”, which is taken as an indication of its state in the cell cycle. The impedance data of the cells are then labeled with the optical information. The two major principal-component scores of the labeled impedance data are then used to train a statistical classification algorithm based on quadratic discriminant analysis (QDA). The resulting algorithm can classify single yeast cells according to cell cycle state based solely on the measured impedance signature. The algorithm can additionally classify the budding cells according to the spatial orientation in which they passed the measurement electrodes. Budding cells that pass the electrodes in a vertical orientation produce a different impedance response as compared to those that pass the electrodes in a horizontal orientation even though the respective cell morphology is similar. The classification enables physical parameters, such as cell volume, to be reliably determined from the impedance data regardless of the cell orientation.
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MATERIALS AND METHODS
System Overview. Figure 1 shows a schematic of the cytometer. It combines a UHFLI lock-in amplifier (Zurich Instruments AG, Zurich, Switzerland) with a microfluidic device. The lock-in amplifier was interfaced with the microfluidic device by means of a power amplifier (PA) and a current-to-voltage converter (C2V) mounted on a frontend printed circuit board (PCB). These components have both been described in detail in our previous work.21 The lock-in amplifier contains multiple demodulators, which allow for several independent frequencies to be generated and analyzed in parallel. The microfluidic device was designed with a set of parallel facing measurement electrodes, as well as electrodes for dielectrophoretic focusing of the cells and particles.22 The front-end PCB was mounted in an inverted microscope (Leica DMI6000B, Leica Microsystems GmbH, Germany), which was focused on an area right after the measurement electrodes. Optical information was recorded by means of a high-speed camera (Phantom Miro eX 4, Vision Research Inc., Wayne, NJ, USA), which was mounted in one of the microscope camera ports. Microfluidic Device. The microfluidic device has been fabricated through a process based on bonding of SU-8 to glass.23 It consists of two glass plates with 200-nm-thick platinum electrodes that have been embedded into the glass after a short ion-beam etching step. The two plates were bonded face-to-face using a 10-μm-thick lithographically structured layer of SU-8 3005 as a spacer that defined the channel dimensions. Figure 2A shows a schematic view of the device. The 18.9mm-long microfluidic channel is divided into a focusing and a measurement region. Micrographs of these regions are shown in Figure S-1. The device includes filtering regions around the inlets and outlets as shown in Figure 2B. The circular structure of SU-8 pillars prevents large particles from entering the microfluidic channel where they could cause the channel to become blocked. The dielectrophoretic focusing scheme shown in Figure 2C uses an electrode structure similar to that presented by Holmes et al.24 The focusing electrodes were driven by a 7 V peak-to-peak sine wave at 123 kHz. The produced electric field has a strength of 3.5 kV/cm, which is significantly below the lysis threshold of yeast cells of 20 kV/cm.25 Cell and Bead Preparation. Yeast cells (S. cerevisiae, Euroscarf) of the B4741 wild-type strain were cultured at 30 °C in a complete synthetic medium, made of 0.17% Yeast Nitrogen Base (Becton Dickinson AG, Switzerland), 0.5% ammonium sulfate, and 2% glucose sulfate (both Sigma-Aldrich Co. LLC, USA), by using standard methods.26 A measurement medium was prepared based on 0.01 M PBS (Sigma-Aldrich Co. LLC, St. Louis, USA), which was further
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RESULTS AND DISCUSSION Parallel Acquisition of Optical Images and Impedance. First, the morphology of each cell was classified as either “single cell” or “budding cell” based on the analyzed imagesequence from the high-speed camera. Asami et al. have demonstrated that the impedance of yeast cells, measured in between two parallel electrodes, depends not only on their state in the cell cycle, but also on the physical orientation of the C
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Figure 3. Optical images and impedance signals of single cells and particles. (A) Example images of cells and particles as recorded by the high-speed camera. The red arrows in the images of the budding cells indicate the position of the bud. The labeling is “B” for beads, “SC” for single, “HBC” for horizontally budding, and “VBC” for vertically budding cells with reference to the configuration in which they passed the electrodes. Larger groupings of cells are labeled “AC”. (B) The top two panels show the real (green, solid) and imaginary (magenta, dashed) part of the raw impedance signal recorded at 0.55 and 9.08 MHz, as multiple cells and a single bead pass the measurement electrodes. The lowest panel shows the area of the cells as extracted from the image sequence, which was recorded in parallel with the impedance data. Each vertical line shows the area computed from a single image frame.
cells.28 For example, as shown in Figure S-3, the impedance of a budding cell passing the electrodes in the horizontal configuration will be virtually indistinguishable from that of a single cell with a correspondingly larger volume. In contrast, when a budding cell passes in the vertical configuration, the electric field can interrogate both the neck opening separating mother and daughter as well as the coupling between the membranes of the two cells.30 As a consequence, budding cells passing in the vertical configuration are expected to show a substantially different impedance response in comparison to single cells. For this reason, the budding cells were further divided according to the configuration in which they passed the measurement electrodes, either horizontally or vertically. It has been shown by other authors31 that asymmetrical cells that are subjected to a dielectrophoretic force (as is the case here due to the use of dielectrophoretic focusing) tend to align with their longest axis parallel to the direction of the electrical field lines. The electric field in the presented device is mainly oriented in two distinct directions due to the geometry of the focusing electrodes. In the regions between the upper and lower focusing electrodes, the field is oriented in the vertical direction. In the center of the channel and in the regions between two electrodes in the same plane, the field is oriented in the horizontal direction. Therefore, the budding cells will predominantly be oriented horizontally or vertically, depending on how well they are focused toward the center of the channel. Figure 2A shows example images of the cells and particles after classification. The intensity of the images has been adjusted in order to make the objects stand out from the background. As can be seen from the images, the beads can be clearly discriminated from the cells due to their uniform shape and darker coloring. An example of two beads that stick together is also shown. The figure also shows examples of atypical cells where more than two cells are found to be sticking together. Such configurations were classified in a separate group in order to then evaluate their impedance characteristics in comparison to those of budding cells. Next, particles and cells were detected in the raw time-domain impedance signals recorded by the impedance spectroscope. Figure 3B shows an example of the real and imaginary part of this signal, as a bead and several cells pass the electrodes. The figure shows the
signals at two of the four measurement frequencies, namely, 0.55 and 9.08 MHz. The overall shape of the impedance signals can be qualitatively explained as follows: The real part of the impedance signal is proportional to the conductivity between the measurement electrodes, whereas the imaginary part is proportional to the capacitance. As can be seen in the top panel of Figure 3B, the first peak of the real part of the impedance signal at 0.55 MHz is positive and the second peak is negative for all events regardless of object class. Because of the differential measurement principle, the implication is that both cells and particles cause a decrease in conductivity of the measurement region. The particles cause a decrease in conductivity because they are composed of a nonconductive material, which displaces the conductive suspending medium, when they pass between the measurement electrodes. The cells also cause a decrease in conductivity because their conductive core, in the form of the cytoplasm, is isolated from the suspending medium by the nonconductive cell membrane. In addition, the imaginary part of the signal at 0.55 MHz of the budding cell, labeled 1, has a polarity opposite to that of the bead, labeled 2. The explanation for this effect is that the budding cells cause an increase in channel capacitance, whereas the beads cause a decrease in channel capacitance. The decrease in capacitance caused by the beads is a consequence of the dielectric constant of the polystyrene material of 2.6, which is significantly lower than that of the suspending medium of 80. In short, the magnitude of the impedance at 0.55 MHz will depend on object volume, because the current is forced to flow around the cells and particles.15 The following panel shows the signal at 9.08 MHz, where the polarity of the peaks associated with the cells can be seen to be opposite to those at the lower frequency. The explanation is that the measurement current can penetrate through the capacitive cell membrane at the higher frequency. As a consequence, the cells will cause an increase in conductivity of the region between the measurement electrodes, because the conductivity of the cytoplasm is larger than that of the suspending medium. The presented findings are consistent with those of Asami et al.28 Cell Classification. The opacity, defined as the ratio of high-frequency (9.08 MHz) to low-frequency (0.55 MHz) impedance, is an overall measure of how the dielectric D
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Figure 4. Impedance data classified according to optically determined cell morphology. (A) Scatter plot of the particle opacity, computed as the ratio of impedance measured at 9.08 MHz to impedance measured at 0.55 MHz, against the magnitude of the impedance measured at 0.55 MHz. The data points are labeled according to the image-based classification of the detected cells and particles. The labeling follows the nomenclature of Figure 3B. The panel on the right shows outlines of histograms of the opacity values. (B) Scatter plot of the first two principal components of the impedance data. The background coloring of the plot shows the decision regions extracted after training of the classification algorithm with the principal component values of the cells and particles.
points belonging to each of the four classes are clustered in different regions of the plot. The beads now form a distinct cluster in the left part of the figure, which is a consequence of their dielectric properties being vastly different from those of the cells. The large difference only becomes apparent when the impedance at all measurement frequencies is considered. The single cells and the horizontally budding cells are roughly clustered along the line labeled as “L1” for illustration purposes. The linear relationship is an indication that the dielectric properties of the two morphologies are very similar. Discrimination between these two morphologies will, therefore, have to be based mainly on the detected volume of the cells. A small overlap between the two clusters can be observed, which is expected, because some SCs, typically older cells,33 may have volumes comparable to those of smaller budding cells.34 The ACs are mainly larger groupings of cells. Their dielectric properties are also similar to those of the SCs and HBCs as indicated by them being mostly clustered along the same line. Their main distinctive feature, in comparison to the other morphologies, is their substantially larger volume. The VBCs form a cluster separate from those of the SCs and HBCs along the line labeled “L2”. The line L2 is almost perpendicular to L1. The implication is that VBCs can be discriminated from SCs and HBCs based mostly on the value of the second principal component. The shaded regions in the background of Figure 4B shows the result of training the QDA classifier with the labeled data. The performance of the classifier was verified by means of an independent data set, which had also been labeled with optical information. The classifier assigned data points that fall within these decision regions to the corresponding class indicated by the labels. The classifier achieved a relative classification accuracy of 86% in comparison to the manual classification of the independent data set. The trained QDA classifier was also used on a larger data set originating from a complete measurement, including particles for which no optical information was available. The corresponding results are shown in Figure S-8. This larger data set was also used for the analysis in the next section. Analysis of Cell Parameters from Impedance Data. The classification as described above enables the independent
properties change with frequency in relation to those of the suspending medium. A plot of the opacity against the magnitude of the low-frequency impedance was used by Han et al. to differentiate between different types of leukocytes.32 Figure 4A shows recorded data of a single measurement, for which cells and particles could be assigned an optically detected class using a similar plot as that of Han et al. In total, 94% of the impedance events could be assigned to one of the optically determined classes. As can be seen from the figure, the cells give rise to a larger low frequency impedance signal than the beads (B), which indicates that they have a greater volume. The horizontally budding cells (HBC) and vertically budding cells (VBC) can also be observed to be generally larger than the single cells (SC), as expected. The beads are composed of a uniform material with dielectric properties that are constant within the frequency range used for the experiment. Therefore, the opacity is expected to remain at a value of unity as the frequency is increased. Indeed, the histogram in the right panel of Figure 4A shows a Gaussian distribution of the opacity of the beads with a mean value of 1.02 ± 0.13, which confirms that the impedance is constant with frequency. In contrast, the SCs, HBCs, and VBCs have different dielectric properties at the higher frequency, as indicated by their opacity values of 0.77 ± 0.15, 0.85 ± 0.12, and 1.24 ± 0.16, respectively. The change in dielectric properties at the higher frequency is a consequence of the multilayered structure of the cell. As shown in Figure 3B, at the frequency of 9.08 MHz the measurement current can pass through the shorted cell membrane and interrogate the conductive cytoplasm of the cell, thereby causing a change in measured opacity. The data as displayed in Figure 4A show considerable overlap between the points belonging to the different classes, which makes automatic classification based on this plot alone difficult. It is, however, possible to improve the separation between the classes by taking the impedance information, measured at all applied frequencies at the same time, into account. Therefore, the impedance values at the four measurement frequencies were analyzed along with the particle velocity by using PCA. The first two of the resulting principal components are shown in Figure 4B. The corresponding weights are shown in Table S-2. In contrast to Figure 4A, data E
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Figure 5. Cell parameters extracted from impedance data. (A) Histograms of the volumes of the different classes of cells detected in a complete measurement. The top panel shows a histogram of the volumes of the entire cell population. The three following panels show the histograms of the individual classes. The classes are labeled according to Figure 3A. The histograms have been fitted with Gaussian functions the key parameters of which are indicated in the figure. The single cells (SC) have further been divided into daughter (SC,D) and mother cells (SC,M). (B) Conductivity (σ) and relative permittivity (εr) of the different classes of cells at the four measurement frequencies have been computed by fitting a model to the measured impedance data.
(atypical cells) because their dielectric properties are highly similar to those of the HBCs. Second, a more detailed analysis of the structure of the budding cells revealed that the mother cells that grow buds are approximately 30% larger than single mother cells. Figure S-7 shows the details of this analysis. These two factors cause an increase in the actual volume of the HBCs as compared to the expected value. The mean volume of the VBCs is 85 ± 22 μm3, as shown in the bottom panel, which, at a first glance, matches well the expected value of 86 μm3. However, as demonstrated by Asami et al.,28 the electrically measured volume of budding cells in the vertical configuration will appear appreciably smaller than the true physical value. The reason for the discrepancy between horizontally and vertically budding cells can be qualitatively understood by the fact that the current in the channel between the electrodes experiences less obstruction through the budding cells in the vertical configuration as compared to the horizontal configuration. The measured current is governed by the total volume of the measured object, but also by its projection onto the electrodes along the current direction. A numerical analysis of the presented system, illustrated in Figure S-3, has shown that the electrically measured volume of a VBC will be approximately 75% of the actual physical value. Once this factor has been compensated for, the measured volume of the VBCs (114 μm3) can be seen to be quite comparable to that of the HBCs (115 μm3). The problem of accurately sizing nonspherical objects is well-known from using more traditional methods, like the Coulter counter.37 Finally, it should be noted that the electrically measured volumes presented here show similar trends as the values that have been extracted from the images of the cells reported in Table S-1. The classification of the cells as either single or budding also gives a rough indication of their state in the cell cycle:38 The SCs, which account for 48% of the cells, are in the G1 phase, whereas the HBCs and VBCs are in one of the phases of S, G2, or M. We next examined whether more detailed information about the cell cycle could be derived from an impedance analysis of the cells. For example, as shown by other
extraction of quantitative information for each of the detected classes. Figure 5A shows the resulting volume distribution of the complete measurement set computed from the measured impedance signal and the conversion formula of equation S-4.8. The top panel of Figure 5A shows a histogram of the volumes of the entire population of cells, which represents the information that is available prior to classification. As can be seen from the figure, it is not possible to extract details about the different cell classes from this histogram alone. Still, for illustrative purposes, the figure has been fitted with two Gaussian functions representing SCs and budding cells (BC) as shown by the lines in the figure. Based on this “uninformed” classification 17% of the SCs would be wrongly classified as BCs. With the classification method based on combined impedance and optical data it is, however, possible to produce detailed histograms of the detected cell classes as shown in the lower panels. The histogram of the SCs contains two peaks, which are associated with so-called daughter and mother cells.35 The daughters are new cells, typically smaller in size, which have yet to go through mitosis. In contrast, the mother cells have gone through the complete cell cycle once or several times and are typically larger. In this measurement, the daughters have a mean volume of 29 ± 7 μm3, which is comparable to the range of 24 to 36 μm3 reported by Ferrezuelo et al.36 for daughter cells in the state between birth and the start of the first budding process. The mother cells have a mean volume of 57 ± 17 μm3. The mean volume of the HBCs as indicated in the third panel of Figure 5A is 115 ± 21 μm3, which is larger than the value of 86 μm3 that would be expected, based on the volumes of the mother and daughter cells. The discrepancy between expected and measured volume of the HBCs is caused by two factors. First, a fraction of the budding cells was found to have started the budding process anew. Figure S-11 shows two examples of cells where this is the case. The extra bud often only becomes visible as the cell rotates during its movement through the channel. According to the optical images, this is the case for approximately 15% of the budded cells. The classifier is not able to categorize such cells as belonging to the AC group F
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authors,28,30 the relative permittivity of the budding cells is expected to be reduced by almost 50%, as the cells complete the last stage of mitosis (M phase), where the neck opening between mother and daughter cell is closed (the relative permittivity is defined as the ratio of the capacitance of a capacitor filled with dielectric material as compared to an identical capacitor that has vacuum inside). We, therefore, determined the dielectric properties of the cells by fitting a multishelled model to the experimental data of each cell individually. The model is similar to that employed by Asami et al.39 in that it considers the cell to be a multishelled object suspended in a medium with uniform dielectric properties. The cell consists of an inner sphere of cytoplasm enclosed by a thin cell membrane, and a thicker shell representing the cell wall. The dielectric properties of such an object can be computed in a recursive manner using Maxwells mixture theory.40 The main feature of the used model is that it allows for determining the dielectric properties in a volume-independent manner. Figure 5B shows the dielectric properties of the cells at the four measurement frequencies condensed into two values: conductivity and relative permittivity. These values represent an average of the properties of the individual shells of the model. The complete set of dielectric properties, as well as a more detailed description of the model can be found in the Supporting Information. Overall, both conductivity and relative permittivity are consistent with the results reported for suspensions of yeast cells.41 The conductivity is low at low frequencies due to the presence of the cell membrane. It then increases with frequency, as the capacitance of the cell membrane is gradually short-circuited. In the frequency range between 10 and 20 MHz, the conductivity is determined by the properties of the cell wall and the cytoplasm. At frequencies higher than have been used here, the conductivity will approach that of the cytoplasm alone. The relative permittivity shows a decrease with frequency mostly caused by the cell membrane, which loses capacitor characteristics at frequencies above 1 MHz. Looking at the values of the individual classes in Figure 5B, we note a small but statistically significant (p < 0.001) difference in both conductivity and relative permittivity between daughter (SC,D) and mother cells (SC,M) as confirmed by using an unpaired two-sample t-test. For example, the conductivity of the daughter cells was found to be 0.12 ± 0.04 S m−1 and the conductivity of the mother cells to be 0.14 ± 0.04 S m−1 at 9.08 MHz. As shown by Uchida et al.,42 daughter cells have a much lower organelle-volume-to-cell-volume ratio in comparison to mother cells. The lack of higher-conductivity organelles and associated membrane structures could explain why the conductivity and relative permittivity of the daughter cells are lower than that of the mother cells. Both conductivity and relative permittivity of the mother cells and the HBCs are similar at all frequencies, as already indicated by the data in Figure 4. Based on this finding it seems unlikely that further information regarding the specific cell phase of the HBCs can be extracted from the impedance data. Finally, the VBCs show markedly different dielectric properties in comparison to both SCs and HBCs at all frequencies. The higher conductivity and relative permittivity in comparison to the HBCs is a consequence of the current needing to pass through both mother and daughter cell in series in the vertical configuration. The simple multishell model used in this work does not include this current path, which causes the conductivity and permittivity to be reported higher than their true values.
CONCLUSION One of the challenges of developing new label-free experimental protocols based on microfluidic impedance cytometry is to find clear correlations between the physical attributes to be studied, and the measured cell impedance. Here we have reported on a platform that solves part of this problem by allowing the optical assessment of the physical morphology and the orientation of single cells relative to the detection electrodes in parallel with their impedance. The work has demonstrated a method for identifying signatures in the impedance data alone that can be associated with specific cell morphologies and orientations based on the combined recording of optical images and impedance at a single-cell level. The method was used for classifying cells of the S. cerevisiae species as either single or budding cells, which was taken as an indication of their state in the cell cycle. As a result, it was possible to extract information about cell volume and dielectric properties as a function of the cell cycle state. The resulting data demonstrated a lower conductivity and relative permittivity of newborn daughter cells in comparison to mother cells, although their physical morphology is largely similar. The differences were attributed to the organelles of the daughter cells being less developed in comparison to those in the mother cells. It was also found that the dielectric properties of mother cells and budding cells are generally similar, even though their physical morphology may be substantially different. However, it also became evident that the spatial orientation of budding cells, as they flow through the measurement region, plays a significant role. The impedance characteristics of the vertically budding cells are so markedly different from those of single and horizontally budding cells that it is important to detect them and treat them differently in the analysis. A remaining question concerns whether information about later stages of the yeast cell cycle can be extracted from the impedance data, in addition to the basic classification as single or budded cell. Other authors have demonstrated a change in impedance of budding cells, measured in the vertical configuration, as the neck closes between mother and daughter cell at the end of cytokinesis.30 A possible future direction would be to improve the dielectrophoretic focusing principle in order to orient a larger fraction of the budding cells in the vertical direction. It could then become possible to investigate the effect of cytokinesis on the impedance of the cells.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssensors.6b00286. Detailed description of data analysis protocols, supporting figures and a summary table (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS Financial support through the ERC Advanced Grant 267351 “NeuroCMOS” is acknowledged. G
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DOI: 10.1021/acssensors.6b00286 ACS Sens. XXXX, XXX, XXX−XXX
