SACNAS '13 poster
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` ` Conclusions and Future Directions Acoustic focusing allows for a vast increase in the throughput of dilute oceanic water through the submersible flow cytometer while keeping tight focusing without increasing the linear velocity of the sample. It was shown that algae can be focused in a silicon microfluididc chip. Particles of similar characteristics can be acoustically focused at high speeds in the flow cell Future directions will include testing the algae in the flow cell and automating the system. Acknowledgements: Funding was provided by NIGMS Introduction Microbes account for most of the primary productivity and biomass in the ocean, and the structure of the microbial community determines in large part that of higher trophic levels. A fundamental understanding of the factors that regulate community structure requires detailed and sustained observations of the plankton. A fully submersible hydrodynamic flow focusing based flow cytometer has been developed to analyze large phytoplankton and microzooplankton in the ocean. Ocean phytoplankton are continuously monitored for about six months via fluorescence, scattering, and high resolution images of phytoplankton acquired from the flow cytometer. However the main drawback of the current setup is its limited throughput. Signal and image quality are diminished with the increases in flow rates of sheath fluid and sample fluid in order to enhance throughput. In addition, the maximum flow rate for hydrodynamic focusing is limited to 125 μL/min. Thus, it is important to investigate alternative flow focusing techniques with enhanced throughput capabilities. The purpose of current research is to develop a flow focusing cell that utilizes standing acoustic waves and investigate its capability to focus and concentrate plankton at very high flow rates prior to analysis. Here we report design and fabrication of acoustic flow cells and our initial finding on focusing of phytoplankton. Gian Maestas 1 , Dan Kalb 1 Robert Olson 2 , Heidi M. Sosik 2 , Menake E. Piyasena 1 , Steven W. Graves 1 1 Center for Biomedical Engineering, Department of Chemical and Nuclear Engineering, University of New Mexico, 87131. 2 Biology Department, Woods Hole Oceanographic Institute, 02543. Fabrication of Acoustic Focusing Flow Cells for High Throughput Flow Cytometry Analysis of Plankton Acoustics A standing acoustic wave field produced by an acoustic transducer creates a force that will either move a particle to a pressure node or anti-node. This force is dependant on the relative compressibility and density of the particle and its surrounding medium. The acoustic force equation below describes the magnitude and direction of the force acting on the particle. = Radiation force = Radius of sphere = Wave number = Avg. acoustic energy density = Contrast factor = Distance from the pressure node Results The images to the left are fluorescent images of live dunaliella algae (emits at 488 nm) flowing in an etched silicon microfluidic device 300 μm wide that mimics the flow cell inside the flow cytometer. Image a) shows the non-focused algae flowing in the device The algae is not focused because the acoustic transducer is not given power. Image b) shows the algae being focused down the middle of the device. The algae is focused when a voltage of 100 mV is applied to the force transducer which equates to a power of 2.9 Mhz. These images show that the dunaliella species of algae can be acoustically focused. Current Setup The current flow cytometer used in practice is strong, fully automatic, and very efficient but it has limitations in space and power due to the requirement of being water proof. Our testing uses the internal flow cell of the current flow cytometer but differs in that it operates on the same acoustic focusing principle as previously described. As of now, the system is manual and is built using a microscope objective, a camera, a laser, and other components (as shown). Top Left : Image of current first and second generation submersible flow cytometer. Top Right : A schematic of the acoustic focusing system at the flow cell. The first transducer is used to create the standing wave while the second transducer maintains resonance. http://arch.ced.berkeley.edu/hiddenecologies/?p=62 Plankton Plankton is any floating organism in the ocean, large or microscopic. Microscopic plankton are called microbes. The microbial algae we are concerned with is the dunaliella species which is a genus of algae commonly found in marine waters. They are unicellular and range from rod to ovoid shaped. Microbes are important to the ocean because they are the basis of the oceanographic food web. An understanding of plankton is key if we want to gain knowledge about how the ocean works. Flow Cytometry Flow Cytometers are used in cell biology to analyze microscopic particles or cells. Inside the flow cell (pictured left),hydrodynamic focusing is used to focus particles into a single stream. Downstream, laser optics are used to shine light on the particle stream. Sensors read the differences in light when passing through and/or bending around the particles. This difference in Internal Flow Cell http://crl.berkeley.edu/ flow_cytometry_basic.html Sensor The images to the right are fluorescent images of polystyrene particles being acoustically focused in a flow cell setup. Image c) shows focusing at twice the speed of standard hydrodynamic focusing. Image d) is focusing at eight times faster than standard hydrodynamic focusing. The streak in image d) is not focused as well as image c) but this is to be expected at such high speeds. However, it is still sufficient enough for the laser optics in the flow cytometer. 150 μm 150 μm a) Un-focused algae a) Acoustically focused algae 100 μm 100 μm d) Polystyrene particles focused at eight times the speed of hydrodynamic focusing c) Polystyrene particles focused at twice the speed of hydrodynamic focusing F ac r k E ac A x ) 2 sin( 3 4 3 kx A kE r F ac ac
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Transcript of SACNAS '13 poster
` `
Conclusions and Future Directions
Acoustic focusing allows for a vast increase in the throughput of dilute oceanic water through the submersible flow cytometer while keeping tight focusing without increasing the linear velocity of the sample. It was shown that algae can be focused in a silicon microfluididc chip. Particles of similar characteristics can be acoustically focused at high speeds in the flow cell Future directions will include testing the algae in the flow cell and automating the system.Acknowledgements: Funding was provided by NIGMS grant NIGMS #GM060201 and NSF grant NSF 1131134
Introduction
Microbes account for most of the primary productivity and biomass in the ocean, and the structure of the microbial community determines in large part that of higher trophic levels. A fundamental understanding of the factors that regulate community structure requires detailed and sustained observations of the plankton. A fully submersible hydrodynamic flow focusing based flow cytometer has been developed to analyze large phytoplankton and microzooplankton in the ocean. Ocean phytoplankton are continuously monitored for about six months via fluorescence, scattering, and high resolution images of phytoplankton acquired from the flow cytometer. However the main drawback of the current setup is its limited throughput. Signal and image quality are diminished with the increases in flow rates of sheath fluid and sample fluid in order to enhance throughput. In addition, the maximum flow rate for hydrodynamic focusing is limited to 125 μL/min. Thus, it is important to investigate alternative flow focusing techniques with enhanced throughput capabilities. The purpose of current research is to develop a flow focusing cell that utilizes standing acoustic waves and investigate its capability to focus and concentrate plankton at very high flow rates prior to analysis. Here we report design and fabrication of acoustic flow cells and our initial finding on focusing of phytoplankton.
Gian Maestas1, Dan Kalb1 Robert Olson2, Heidi M. Sosik2, Menake E. Piyasena1, Steven W. Graves11Center for Biomedical Engineering, Department of Chemical and Nuclear Engineering, University of New Mexico, 87131.
2Biology Department, Woods Hole Oceanographic Institute, 02543.
Fabrication of Acoustic Focusing Flow Cells for High Throughput Flow Cytometry Analysis of Plankton
AcousticsA standing acoustic wave field produced by an acoustic transducer creates a force that will either move a particle to a pressure node or anti-node. This force is dependant on the relative compressibility and density of the particle and its surrounding medium. The acoustic force equation below describes the magnitude and direction of the force acting on the particle.
FacrkEacAx
= Radiation force= Radius of sphere= Wave number= Avg. acoustic energy density= Contrast factor = Distance from the pressure node
)2sin(3
4 3 kxAkErF acac
Results
The images to the left are fluorescent images of live dunaliella algae (emits at 488 nm) flowing in an etched silicon microfluidic device 300 μm wide that mimics the flow cell inside the flow cytometer. Image a) shows the non-focused algae flowing in the device The algae is not focused because the acoustic transducer is not given power. Image b) shows the algae being focused down the middle of the device. The algae is focused when a voltage of 100 mV is applied to the force transducer which equates to a power of 2.9 Mhz. These images show that the dunaliella species of algae can be acoustically focused.Current Setup
The current flow cytometer used in practice is strong, fully automatic, and very efficient but it has limitations in space and power due to the requirement of being water proof. Our testing uses the internal flow cell of the current flow cytometer but differs in that it operates on the same acoustic focusing principle as previously described. As of now, the system is manual and is built using a microscope objective, a camera, a laser, and other components (as shown).
Top Left: Image of current first and second generation submersible flow cytometer.
Top Right: A schematic of the acoustic focusing system at the flow cell. The first transducer is used to create the standing wave while the second transducer maintains resonance.
Bottom left: Labeled image of our current setup
http://arch.ced.berkeley.edu/hiddenecologies/?p=62
PlanktonPlankton is any floating organismin the ocean, large ormicroscopic. Microscopicplankton are called microbes. The microbial algae we are concerned with is the dunaliella species which is a genus of algaecommonly found in marine waters. They are unicellular andrange from rod to ovoid shaped. Microbes are important to the ocean because they are the basisof the oceanographic food web. An understanding of plankton is key if we want to gain knowledge about how the ocean works.
Flow CytometryFlow Cytometers are used in cell biology to analyze microscopic particles or cells. Inside the flow cell (pictured left),hydrodynamic focusing is used to focus particles into a single stream. Downstream, laser optics are used to shine light on the particle stream. Sensors read the differences in light when passing through and/or bending around the particles. This difference in light can be translated into different properties.
Internal Flow Cell
http://crl.berkeley.edu/flow_cytometry_basic.html
Sensor
The images to the right are fluorescent images of polystyrene particles being acoustically focused in a flow cell setup. Image c) shows focusing at twice the speed of standard hydrodynamic focusing. Image d) is focusing at eight times faster than standard hydrodynamic focusing. The streak in image d) is not focused as well as image c) but this is to be expected at such high speeds. However, it is still sufficient enough for the laser optics in the flow cytometer.
150 μm
150 μm a) Un-focused algae
a) Acoustically focused algae
100 μm
100 μm
d) Polystyrene particles focused at eighttimes the speed of hydrodynamic focusing
c) Polystyrene particles focused at twice the speed of hydrodynamic focusing
