Parallel Imaging Microfluidic Cytometer
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Parallel Imaging Microfluidic Cytometer
Methods Cell Biol
By adding an additional degree of freedom from multichannel flow, the parallel microfluidic cytometer (PMC) combines some of the best features of fluorescence-activated flow cytometry (FCM) and microscope-based high-content screening (HCS). The PMC (i) lends itself to fast processing of large numbers of samples, (ii) adds a 1D imaging capability for intracellular localization assays (HCS), (iii) has a high rare-cell sensitivity, and (iv) has an unusual capability for time-synchronized sampling. An inability to practically handle large sample numbers has restricted applications of conventional flow cytometers and microscopes in combinatorial cell assays, network biology, and drug discovery. The PMC promises to relieve a bottleneck in these previously constrained applications. The PMC may also be a powerful tool for finding rare primary cells in the clinic. The multichannel architecture of current PMC prototypes allows 384 unique samples for a cell-based screen to be read out in ∼6-10 min, about 30 times the speed of most current FCM systems. In 1D intracellular imaging, the PMC can obtain protein localization using HCS marker strategies at many times for the sample throughput of charge-coupled device (CCD)-based microscopes or CCD-based single-channel flow cytometers. The PMC also permits the signal integration time to be varied over a larger range than is practical in conventional flow cytometers. The signal-to-noise advantages are useful, for example, in counting rare positive cells in the most difficult early stages of genome-wide screening. We review the status of parallel microfluidic cytometry and discuss some of the directions the new technology may take.
Copyright © 2011 Elsevier Inc. All rights reserved.
(a) Parallel microfluidic cytometer (PMC) for cell-based assays. The system is designed for automated fluorescence measurements on 384-channel microfluidic plates and comprises up to two temperature-controlled microfluidic “chips” (16 to 384 channels each), a scanning detector, and automated pipettor/sample elevator for automated maintenance of cell suspensions/cultures. Cell suspensions are pulled by vacuum suction from injection wells using a positive-displacement syringe pump. Multicolor detection is via a scanned confocal detector that oscillates below the microfluidics. ((Modified from (El-Difrawy 2005) with permission. Copyright 2005, American Institute of Physics.))
(a) Plan-view detail of the PMC showing microfluidics, sample and wash plates and the optical scanner located beneath the fluidics; (b) A 384-channel microfluidic plate and (c) a segment of data collected from several channels. A short time sequence from one of four photomultipliers is shown with each pixel representing 35 μm in the (horizontal) scan direction. Data is collected at a rate of 3 scans/s, (0.33 s vertical displacement of each row of pixels in the data image); 240 s of data shown. Signal amplitude is shown in RGB color scale with blue representing low signal and red high signal. [From (McKenna et al. 2009). Reproduced with permission of the Royal Society of Chemistry.]
(a/b) optical diagram of the laser-induced fluorescence (LIF) detector including detail of rotary scanner that introduces the 488-nm laser beam and returns fluorescence onto four photomultipliers (PMTs, other configurations shown below in Fig. 4). The scanner uses a 3-inch radius of rotation, a DC rotary motor, and optical encoder; (c) detail of a typical condensed-time data scans for a single microfluidic channel [(c), left and center] and reduced-difference scan to identify positives [(c)), right]. See text. [From (McKenna et al. 2009). Reproduced with permission of the Royal Society of Chemistry.]
Two configurations of the optical detector to match cell assays used for the PTHR screen (Fig. 4(a)), and for the dilution study on primary leukemia cells (Fig. 4(b)). The scattered forward light sensor (Fig. 4(b)) is a fiber optic (910-μm dia., 0.22 NA) on a rotatable mount that can be adjusted in the range from 20 degrees to 70 degrees off the forward direction.
A plate of 32-channel PMC microdevices at the lithography stage of fabrication. Five devices are fabricated simultaneously on a 250x250mm alumina silicate glass plate. There are economies of scale from batch fabrication -particularly yield improvements at bonding stage. As a last step individual devices are separated by diamond sawing.
A finished PMC microdevice similar to those in Fig. 4 (slightly different design) but after attachment of G-10 fiberglass pumping block and fluid reservoirs. The suction port and wash port are threaded to receive standard 10–32 HPLC fittings. The 32 open sample ports are 2-mm diameter and 10-mm deep, on 9 mm centers (other designs use 4.5mm centers), and are compatible with a standard multi-tip pipettors.
A 16-channel PMC microdevice with 3-sided hydrodynamic focusing. This design can be fabricated with one microlithographically-defined fluidic level and captures the three (out of four) directions for flow focusing, The glue-on fiberglass block (see e.g., Fig. 5 above) is machined to combine the three “blue” buffer flows into a single manifold and reservoir. Other flow focusing designs are provided in Section 3.4.
A 384-channel PMC microdevice plate at mask stage, finished device shown in Fig. 2(b). The flow channels fan out on the “loading” (top) end to allow room for the sample-well array that must match the 4.5-mm spacing of the robotic pipettor. At the “scan” end the flow channels converge to a maximum density allowed by the bonding process, 5 channels per mm. The channel cross-section is hemispherical, 60-μm radius. This channel structure is etched into the glass plate (flat-panel display glass), the access holes are laser drilled, conical shape terminating with a 80-μm diameter at the etched channel, then the plate is sealed by high-temperature fusion bonding.
(left side) A simple crossing junction used as a design element in software and imaging calibrations; two inlet flows from PA and PB. single outlet flow from PD. No flow allowed through PC (wall boundary condition). The analysis channel is on top. The sheath channel is on bottom. Percentages of flow from PA and PB are in reference to PD, the total flow after the junction; (right side) illustrates the four-level compensated vertical focusing device modeled in Figs. 10 and 11. Additive sheath (symmetric sheath inputs S1 and S3) and additive analysis (symmetric S2) are combined upstream of a correction flow (symmetric S4). The device is driven by suction from a port at the right end. Adjustable flow resistances on the channels S1–S4 are used to tune the device. [(Reprinted from (Lin et al. 2009) with permission. Copyright 2009, American Institute of Physics.)]
Plan view layout of the device designed to test vertical flow focusing and subtractive compensation. Eight variations of the 4-sheath configuration shown in Fig. 9(b), labeled “A”– “H”(right side of die), are included on the single test die. The two layers of etched channels are indicated as red (top plate) and black (bottom plate) respectively. A single laser-drilled hole is provided for each input or output (S1–S4, Fig. 9(b)) and for a common suction port (common to configurations A–H, right side of die). The full die size is 3x7 cm. ((Reprinted with permission from (Lin et al. 2009). Copyright 2009, American Institute of Physics.))
Simulations of four-layer focused flow, (a) before and, (b) after, the channel S4 junction and subtractive correction flow (plane V4). As the traces pass beyond the channel S4 junction (Fig. 9) they are preferentially pulled downward and outward. The flow interface indicated by the arrows is most strongly altered by the subtractive flow. ((Reprinted with permission from (Lin et al. 2009). Copyright 2009, American Institute of Physics.))
Raw data. Two sample types showing raw signal on all four photomultipliers (PMT’s ) using the detector layout of Fig. 4(b). The x-axis is time, designated as scan number, 12 scans per second. The labeled and unlabeled cells show up as events on the scatter detector, while GFP cells appear as fluorescent spikes on the P1–P3 color PMT’s. Weak autofluorescence is occasionally observable on P3 (unlabeled cells). For the GFP sample, the signal ratios vary significantly, e.g., P2 (GFP channel) compared to P3 (Yellow channel).
Unreduced image data: Raw data (e.g., Fig. 12) is plotted as an image of the microfluidic channel cross-section (vertical axis labeled spinner position) versus time, raw data is collected at 300 pixels per 16-channel (or 384-channel) scan and 12 scans per second.
Distinguishing positives from autofluorescence. A scatter-plot comparison of the ratio of the GFP channel to yellow channel for objects with a sufficient maximum GFP value (threshold) in positive and negative samples. The low ratio in the negative sample shows how auto-fluorescence cells can be rejected as negatives. By determining the mean and standard deviation for cells in the negative sample it is possible to calculate an outlier threshold (> mean + 4 sd).
Calibration for dilution study on primary splenocytes. Ratio of GFP/yellow channels as a plot of objects and as histogram for a positive sample (right) and a (mostly) negative sample (left). From the histogram we conclude that cells with a PMT ratio greater than 0.8 would (a) definitely be GFP cells and (b) these cells would represent about half of the cell number that was contained in the GFP source sample.
Calibration of dilution study on primary leukocytes. For all the objects identified by the scatter detector we plot the maximum GFP channel value vs. the yellow channel value. Note that most objects in the negative sample have lower fluorescence then the positive sample, a more sensitive measure is made by comparing the ratio of the two PMTs.
Results for dilution study on primary splenocytes. Measured percentage and expected percentage of GFP labeled cells for all samples (ordered by expected percentage) shows a clear distinction between negative samples and positive samples down to dilutions of 0.01%. There is observable saturation of the count at high abundance (likely due to multiple-cell counts).
Figure 18. Results for a 384-channel run (clonal osteocytes)
(a) histogram of dsRed-cell counts for a cell dilution curve (dsRed-expressing cells diluted serially with GFP-expressing cells). The histogram is organized by well placement on the PMC fluidics. Counts for all 384 microfluidic channels are shown. Sample dilutions are run redundantly in 2-ea. columns of 8-well rows (layout on the microfluidic device), i.e., 24 channels for each dilution. (b) Total counts are summed for each sample and used to generate the serial dilution curve (log vertical scale) which shows slight saturation at the highest concentration of positives (100% positives, right side of the figure). [From (McKenna et al. 2009). Reproduced with permission of the Royal Society of Chemistry.]
Schematic representation of a cDNA expression cloning study that identified a new target for the CPTHR receptor. The most difficult first two stages were completed on the PMC using the rare-cell detection advantages of the variable integration detector.
Fig. 20. Cartoon of typical 1-D images that are encountered in a protein localization assay
The left column shows 2-D (microscope) images with the marker (green) and cytoplasm (pink). Three positives are shown top; three negatives on bottom. The confocal slit in our detector discriminates strongly against out-of-focus images. The right column shows the several principal 1-D image types that are generated depending on how the laser scanner traverses the cell. The dashed arrow shows the location of the single line scan that is taken per cell. Some of the most diagnostic signatures are surprising.
1-D images of 6-μm beads (one of four PMTs). On left 350 pixels in line scan (X) vs. scan number (Y) with fluorescence intensity values shown as growing from blue to green to red. On right two sections are magnified, showing image of 6-μm bead moving at a higher speed through the detector (top) and slower at bottom. Slower speed yields a more 2-D “picture-like” image.
Fig. 22. Results showing 1-D HCS data using a 3.5-μm laser spot to scan αSyn-GFP expression patterns in S. cerevisiae
The first version of the detector is (just) able to distinguish the localization patterns. Top: raw scans for whole-cell (red) and αSyn-GFP (green). Left (a) negative cells, right (b) positives showing αSyn aggregates. Below: Filtered data using a modified “roundness” parameter distinguishes positive (induced) sample from a negative with baseline αSyn expression.
Figure 23. Proof of principle for NT assay
The FWHM for the green and orange channels are compared in a scatter plot. For the “un-stimulated” CDFE sample cells, the wider green line scan skews the sample above the center diagonal line (proving marker in the cytoplasm). For the “stimulated Sytox Green sample cells the distribution centers along the diagonal (marker confined to the nucleus).
Figure 24. Proof of principle for nuclear translation (NT) assay
(a) Representative line scans from each sample show green fluorescent signal difference between whole cell marker and nuclear stain when compared to orange nuclear stain (FWHM point for normalized scans marked by blue line). In (b) an algorithm classifies objects by first eliminating all green signal outside of green FWHM and inside of orange FWHM, then measuring the remaining green signal. This value is significantly larger for “positives” (stimulated cells) in the NT assay. 38
Figure 25. Box plots of the two samples that simulate nuclear translocation
The green signal outside the nuclear area (nuclear area defined by the Orange PMT channel) is plotted (vertical axis in the plot). As a group, the “un-stimulated” cell type (left column) shows more green signal outside the nucleus then the “stimulated” cell sample (right column).
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