High throughput imaging cytometer with acoustic focussing

Abstract

We demonstrate an imaging flow cytometer that uses acoustic levitation to assemble cells and other particles into a sheet structure. This technique enables a high resolution, low noise CMOS camera to capture images of thousands of cells with each frame. While ultrasonic focussing has previously been demonstrated for 1D cytometry systems, extending the technology to a planar, much higher throughput format and integrating imaging is non-trivial, and represents a significant jump forward in capability, leading to diagnostic possibilities not achievable with current systems. A galvo mirror is used to track the images of the moving cells permitting exposure times of 10 ms at frame rates of 50 fps with motion blur of only a few pixels. At 80 fps, we demonstrate a throughput of 208 000 beads per second. We investigate the factors affecting motion blur and throughput, and demonstrate the system with fluorescent beads, leukaemia cells and a chondrocyte cell line. Cells require more time to reach the acoustic focus than beads, resulting in lower throughputs; however a longer device would remove this constraint.

Fig. 1. System schematic. A piezoelectric transducer (T) is driven by a sinusoidal waveform, inducing an ultrasonic standing wave in a rectangular channel of a glass microfluidic chip. Flowing particles are delivered from the syringe pump (S), and are acoustically focussed towards the pressure node plane which is located at the half-height of the device. This plane coincides with the focal plane of the imaging objective (O). Optical components include tube lens (L1), illumination optics (L3, 4), white light source (LS), and a fluorescence filter cube set (TFC). A programmable function generator (PFG) controls the angle of the rotating galvo mirror (GM), and the trigger signal for the camera (C1).

Fig. 2. Modelled flow profile in the channel. Normalised flow velocity profiles in the channel, top view across the width (a), and the depth (b), of the channel along the flow direction. Red dotted line indicates the position of the acoustic pressure nodes in the standing wave created by half wave acoustic resonance from piezoelectric transducer.

Fig. 3. System assembly and setup. Symbols describing the components are the same as for Fig. 1 (inset: the flow cell removed from the system). The imaging region is illuminated by the blue spot.

Fig. 4. Fluorescent 10 μm beads flowing in the channel with acoustic focussing deactivated (a), and activated (b). These images are taken at low flow rates, and no galvo mirror tracking to illustrate the requirement for acoustic focussing. A movie of this can be found in ESI. Scale bars 100 μm.

Fig. 5. (a) Moving the position of the field of view along the flow direction by rotating the galvo mirror in the infinity space of the objective. (b) Mirror control waveform and associated TTL camera trigger signals controlled by the signal generator's marker tool.

Fig. 6. Predicted deviation from a linear mapping between galvo mirror angle and displaced field of view seen by the camera.

Fig. 7. Displacement vector field of 10 μm fluorescent beads during a mirror cycle over the full field of view (1 Hz mirror cycle frequency). The vectors are scaled, with the average vector approximately 2 pixels long. The vectors highlight both mean motion from imperfect device alignment, and spatial variation resulting from acoustic causes. The flow direction is along the y-axis.

Fig. 8. Displacement vector fields at 20 Hz mirror cycle frequency. (a) Shows the displacement between the beginning and end of a single cycle. (b) The same data with median subtracted to highlight acoustic distortions. The main axes show position in pixels; vectors' lengths are over-scaled for viewing purpose according to the scale bar. The flow direction is along the y-axis.

Fig. 9. (right) Analysis of movements of bead images during the course of a single mirror cycle. Any movement results in blurring during normal operation. 14 frames are collected at a flow rate of 100 ml h^–1, corresponding to approximately a 20 Hz mirror frequency and 26 mm s^–1 particle velocity. (a and b) X-component of the displacement field, perpendicular to the flow, and (c and d) Y-component aligned along the flow. The boxes show the 25th and 75th percentiles, red crosses are outliers. The red bars show the median value. By subtracting the median in (b and c) we highlight non-uniform effects resulting from non-uniform flow and imperfect alignment. The sub-pixel deviations of (c and d) show that with accurate mirror synchronisation low levels of blurring are possible.

Fig. 10. Images of 10 μm fluorescent beads. (a and b) Static, sedimented beads compared to (c and d) an image aquired in flow at 104 mm s^–1 by synchronising the galvo mirror and camera frame rate to 80 fps (exposure time 2.5 ms). (e and f) show acquisition at 50 fps (exposure time 10 ms). The dimmer beads are from a photo-bleached sub-population. Images are 2048 × 2048 pixels (field of view 1.3 × 1.3 mm); zoomed sections are 256 × 256 pixels; and the dotted squares within them (30 × 30 pixels) are shown to give comparable scale. Scale bar 200 μm.

Fig. 11. Fluorescent beads (10 μm) recorded at 104 mm s^–1, 80 fps. Average throughput of 208 800 beads per second. (a–c) Represent a sample of three consecutive frames, the dotted regions highlight an overlap between frames of 140 pixels which can be adjusted by changing the amplitude of the signal driving the galvo mirror. Image size 2048 × 2048, field of view 1.3 × 1.3 mm. Scale bar 200 μm.

Fig. 12. (a) ATDC5 chondrocyte cells stained with cell tracker green, recorded at 104 mm s^–1, 80 fps. Average frame count 755 cells, throughput 60 400 cells per second. (b) CLL cells stained with SYTO-9 dye, recorded at 32.5 mm s^–1, 25 fps. Average frame count 2094 cells, throughput 52 350 cells per second. Image sizes 2048 × 2048. Scale bar 200 μm.