Proceedings of Meetings on Acoustics

Transcription

1 Proceedings of Meetings on Acoustics Volume 19, ICA 2013 Montreal Montreal, Canada 2-7 June 2013 Physical Acoustics Session 1pPAb: Acoustics in Microfluidics and for Particle Separation III: Biological Applications 1pPAb2. Application of acoustic radiation pressure to align cells in a commercial flow cytometer Gregory Kaduchak* and Michael D. Ward *Corresponding author's address: Life Technologies, Eugene, OR 97402, greg.kaduchak@lifetech.com Forces derived from acoustic radiation pressure can be used to replace or partly replace hydrodynamic forces to align cells and particles in flow cytometry. The ability to focus cells into a tight line without relying on hydrodynamic forces allows many new possibilities for sample delivery. Dilute samples can be processed quickly. Flow velocities can be varied allowing control of particle delivery parameters such as laser interrogation time and volumetric sample input rates. Recently, a commercial flow cytometer that directs particles into the laser interrogation region using acoustic radiation pressure in a 200 micron flow cell has been developed. In this talk, the application of acoustic radiation pressure in flow cytometry systems from fundamental principles to implementation details will be presented. Data will be shown for both the operational implementation of the acoustic focusing device as well as demonstrating its ability to perform for complex biological assays. Published by the Acoustical Society of America through the American Institute of Physics 2013 Acoustical Society of America [DOI: / ] Received 22 Jan 2013; published 2 Jun 2013 Proceedings of Meetings on Acoustics, Vol. 19, (2013) Page 1

2 ACOUSTIC CYTOMETRY FIGURE 1. Acoustic focusing capillary driven by a PZT line source. Particles enter at the top and experience a drift force that transports them to the axis of the capillary as they flow in the downward direction in the capillary. The force field is two dimensional in the cross section of the capillary, forcing the particles into a single-file line. The acoustic focusing capillary is a particle focusing device that applies acoustic radiation pressure to align particles in sample streams to replace or partially replace hydrodynamic focusing of the sample. 1 The device focuses cells or particles into a single line using an elastic capillary coupled to a piezoelectric transducer as shown in Fig. 1. 2,3,4 Particles experience an acoustic radiation pressure based drift force that transports them to the central axis of the capillary. Figure 2(a) displays an image at the exit of a capillary with a sample containing 3 m fluorescent beads in phosphate buffered saline flowing at 1 ml/min. In Fig 2(b), the acoustic field is activated and the particles are confined to the axial region. (a) (b) FIGURE 2. Images of 3 m fluorescent microspheres exiting an acoustic focusing capillary. The internal diameter of the capillary is ~340 m. (a) Piezoelectric element is not activated and the particles are distributed throughout the cross section of the capillary. (b) Piezoelectric element is activated and the particles are focused to the central axis of the capillary. The flow rate is 1 ml/min. Proceedings of Meetings on Acoustics, Vol. 19, (2013) Page 2

3 In a conventional flow cytometer particles in a sample stream are aligned for optical interrogation by hydrodynamic forces. The alignment is done to allow the use of tightly focused laser beams (typically 10 m x 70 m) to increase photon flux at the target and reduce illumination variations experienced by the individual particles. In hydrodynamic focusing, the sample stream is wrapped with a high velocity sheath to decrease the sample stream diameter in the analysis region as shown in Fig. 3(a). These systems have a volumetric sheath to sample flow ratio in the range of 100 to 1000 resulting in sheath usage that can reach > 1 L/hour. This generates an equivalent amount of biological waste that must be handled carefully when operating with hazardous biological samples. Hydrodynamic particle delivery has been the historical means of particle delivery in flow cytometers for decades. Recently, a particle delivery system based upon acoustic radiation pressure has been implemented in a commercial flow cytometer. The implementation of the acoustically focused particle stream is shown in Fig. 3(b) where a sample stream is initially acoustically focused to the central axis of a capillary. It is then injected into a sheath flow. The sheath flow is necessary to keep the optical interfaces clean. The ratio of volumetric sheath flow to the volumetric sample flow ranges from 1 to 100 thereby reducing sheath consumption and the generation of biological waste. FIGURE 3. (a) Hydrodynamic focused sample stream. A sample is injected into a fast flowing sheath wrapper that hydrodynamcially pinches the diameter of the sample stream thereby focusing the particles for interrogation. (b) Acoustic focusing of particles prior to weak hydrodynamic focus. Particles are focused with acoustic radiation pressure prior to entering a sheath flow. The pre-focusing allows for much greater volumetric sample throughput without sacrificing particle alignment in the interrogation region. Systems that employ an acoustic focusing particle stream are different from a conventional hydrodynamically sheath focused system in that the particles or cells in the acoustic capillary are focused toward a tight central line along the axis of the capillary whether fluid is flowing or not. In these systems, fluid flow can be controlled at any rate using appropriate pumps. A large dynamic range of sample input volumes can be achieved allowing for dilute samples to be run at conventional analysis rates due to an inherent concentration effect that occurs within the focusing system. These fundamental differences between acoustic and hydrodynamic particle focusing systems allows for many potential benefits from an acoustically focused system, but the ramifications of these differences must be fully understood in order to realize them. Transit Time In a flow cytometer increased particle transit times within the optical interrogation region can be used to increase system sensitivity. Figure 4 is data taken on an acoustic particle focusing flow cytometer using Sphereotech s 3. 0 µm diameter, 8 peak rainbow bead set (Spherotech RCP-30-5A). The bead set contains eight populations of microspheres each carrying a different concentration of fluorophores. Mult-level beads are standards used for Proceedings of Meetings on Acoustics, Vol. 19, (2013) Page 3

4 determining the dynamic range and sensitivity of flow cytometers. In this figure the sample flow rate is 100 ul/min. In (a), only six of the populations are resolved. By slowing down the transit rate of the sample in the laser interrogation zone and increasing the residence time in the laser by 4x, the data in (b) is generated. The increased residence time of the particles in the laser increases the resolution for the dimmer populations making the distinction of all eight populations possible. Assuming measurements to be limited by photon statistics, increasing transit time increases resolution and sensitivity relative to the square root of the increase. 5 Slowing the flow to get more photons is by no means unique to acoustic cytometry, but the tight focus achievable with little regard to flow rates or sheath gives acoustic cytometry an edge over previous slow flow systems, at least for particles large enough to be focused. A slow flow hydrodynamic system with millisecond transit times and single molecule sensitivity for phycoerythrin and DNA fragments has been demonstrated. 6 The key to this system is its small sample delivery dimensions. As mentioned previously, tight focus in a hydrodynamic system requires a large sheath to sample ratio. Tight focus and slow flow therefore requires small fluidic dimensions which are suitable for pure preparations of DNA, protein or virus but not for cells which might clog the system. Additionally, without the benefit of the acoustic concentration effect, hydrodynamic slow flow systems must process highly concentrated samples to achieve a reasonable throughput. This in turn makes them even more subject to clogging. Other researchers have experimented with slowing flow using a conventional flow cytometer by using flow resistors but found it very difficult to control precise fluid delivery and focus in this manner. 7 (a) (b) Fluorescence Intensity Fluorescence Intensity FIGURE 4. (a) Eight peak calibration bead set run at 100 ul/min on acoustic focusing cytometer at standard transit time. Only six peaks are resolved. (b) Slowing down the flow rate and increasing the transit time 4x allows for resolution of all eight populations. The x-axis is proportional to fluorescence intensity. Data is collected with excitation from a 488nm laser using a 675nm long-pass filter. Optimization of Event Rate In a conventional cytometer, a low sample input rate of approximately µl/minute is generally used to achieve higher precision analysis that results from a tighter hydrodynamic focus of the particle stream. At 10µl /min, 6 x 10 7 cells/ml are required to achieve a 10,000 particles/s analysis rate. For many assays, this requires a centrifugation step to concentrate cells and for some assays, this concentration can cause problematic cell sticking and the selective loss of rare sample types. 8 For a more conventional sample concentration of 6 x 10 6 cells/ml, the user is limited to a maximum of 1,000 particles/s at the high precision sample input rate. A medium concentration sample of 6 x 10 5 cells/ml would have a maximum rate of just 100 particles/s at this input rate. If sample throughput is more important than sensitivity and resolution, a conventional flow user can increase the cell particle analysis rate by an order of magnitude by using the standard maximum sample input of 120µl/min. For the medium concentration example of 6 x 10 5 cells/ml, the event rate is still just 1,000 cells/s. Table 1 displays particle event rate as a function of sample concentration for different sample input flow rates. This table is independent of the type of focusing used as the event rate is only dependent upon the concentration of the input sample and the volumetric sample input rate. The values in red represent sample input rates that to date have only been accessible by an acoustically focused sample stream. It is seen that the particle event rate can be greatly increased and the total analysis time of a sample decreased for dilute samples e.g. concentration 1 x 10 5 part/ml and lower. Proceedings of Meetings on Acoustics, Vol. 19, (2013) Page 4

5 TABLE 1. Particle event rate through the interrogation laser as a function of sample volumetric input rate and sample concentration. The values are given as events/s and the table is limited to a maximum event rate of 10,000 events/s. The values in red are made accessible by an acoustic focused sample stream. Concentration 12 l/min 60 l/min 100 l/min 200 l/min 500 l/min 1000 l/min 1x10 4 part/ml x10 4 part/ml x10 5 part/ml ,667 5x10 5 part/ml ,667 4,167 8,333 1x10 6 part/ml 200 1,000 1,667 3,333 8,333 5x10 6 part/ml 1,000 5,000 8,333 1x10 7 part/ml 2,000 10,000 SUMMARY Acoustic focusing is not likely to fully replace hydrodynamic focusing in flow cytometry but it will be used both independently and in conjunction with hydrodynamic focusing to enhance sample handling and analytical capabilities for a broad range of flow assays. The ability to tightly focus particles or cells over an extensive range of flow rates, with or without sheath puts a tremendous amount of power in the hands of the user by allowing him or her to optimize performance and throughput as is suited to each assay. The specific benefits that provide this power are: High throughput for dilute samples without resorting to centrifugation High precision focusing combined with high particle rates over a broad range of initial sample concentrations High sensitivity and resolution from slower flow velocity combined with tight focus Slower flow velocity can be used to increase performance, but it may also be used to reduce cost. Longer transit times translate into not only more photons but also more time to integrate noise. Integration of noise is a pathway to effective use of noisy lower cost lasers and detectors. Reducing cost is often more important to fueling discovery than increasing performance and acoustic focusing could make a large impact by placing the power of cytometry in a greater number of hands. REFERENCES 1. G. Kaduchak, G. Goddard, G. Salzman, D. Sinha, J. C. Martin, C. S. Kwiatkowski, S. W. Graves, Ultrasonic Particle Concentration and Application in Flow Cytometry, US Patent 7,340,957 (2008). 2. G. Goddard and G. Kaduchak, Ultrasonic particle concentration in a line-driven cylindrical tube, J. Acoust. Soc. Am. 117, (2005). 3. G. Goddard, J. C. Martin, S. W. Graves, and G. Kaduchak, Ultrasonic particle-concentration for sheathless focusing of particles for analysis in a flow cytometer, Cytometry 69A, (2006). 4. G. Goddard, C. K. Sanders, J. C. Martin, G. Kaduchak, S. W. Graves, Analytical performance of an ultrasonic particle focusing flow cytometer, Anal Chem 79(22), (2007). 5. H. B. Steen, Noise, sensitivity and resolution of flow cytometers, Cytometry 13, (1992). 6. R. C. Habbersett and J. J. Jett, An analytical system based on a compact cytometer for DNA fragment sizing and single molecule detection, Cytometry, 60A, (2004). 7. R. M. P. Doornbos, B. G. de Grooth, J. Greve, Experimental and Model Investigations of Bleaching and Saturation of Fluorescence in Flow Cytometry, Cytometry 29, (1997). 8. J. W. Gratama, P. Menendez, J. Kraan, and A. Orfao, Loss of CD34+ hematopoetic progenitor cells due to washing can be reduced by the use of a fixative-free erythrocyte lysing reagent, J. Immunol. Methods 239, (2000). Proceedings of Meetings on Acoustics, Vol. 19, (2013) Page 5