A micro-scale Fluorescent Activated Cell Sorter (microFACS) for astrobiology applications is under development. This device is designed to have a footprint of 7 cm x 7 cm x 4 cm and allow live-dead counts and sorting of cells that have fluorescent characteristics from staining. The FACS system takes advantage of microfluidics to create a cell sorter that can fit in the palm of the hand. A micron-scale channel allows cells to pass by a blue diode which causes emission of marker-expressed cells which are detected by a filtered photodetector. A small microcontroller then counts cells and operates high speed valves to select which chamber the cell is collected in (a collection chamber or a waste chamber). Cells with the expressed characteristic will be collected in the collection chamber. This system has been built and is currently being tested. We are also designing a system with integrated MEMS-based pumps and valves for a small and compact unit to fly on small satellite-based biology experiments

Hoover, Richard B.

Platt, Donald W.

NASA Technical Reports Server

A micro fluorescent activated cell sorter for astrobiology applications
Donald W. Platt*', Richard B. Hooverb
'Micro Aerospace Solutions, Inc., 2280 Pineapple Ave, Melbourne, FL 32935;
bNASA/NSSTC, SD-50, 320 Sparkman Dr., Huntsville, AL 35805, USA
ABSTRACT
A micro-scale Fluorescent Activated Cell Sorter (µ&ACS) for astrobiology applications is under development. This
device is designed to have a footprint of 7 cm x 7 cm x 4 cm and allow live-dead counts and sorting of cells that have
fluorescent characteristics from staining. The µFACS system takes advantage of microfludics to create a cell sorter that
can fit in the palm of the hand. A micron-scale channel allows cells to pass by a blue diode which causes emission of
marker-expressed cells which are detected by a filtered photodetector. A small microcontroller then counts cells and
operates high speed valves to select which chamber the cell is collected in (a collection chamber or a waste chamber).
Cells with the expressed characteristic will be collected in the collection chamber. This system has been built and is
currently being tested. We are also designing a system with integrated 1\/EMS-based pumps and valves for a small and
compact unit to fly on small satellite-based biology experiments.
Keywords: Microfluidics, cell sorting, cell counting, live-dead staining, fluorescence
1. INTRODUCTION
A novel miniature instrument to assess in-situ cell viability as a function of exposure to microgravity and the space
environment is under development The instrument is a microfluidics-based fluorescent activated cell sorter (µFACS)
that is small enough to be flown aboard a free-flying CubeSat or micro-satellite to carry out viability studies of frozen
anaerobic and aerobic bacterial cells.
This device is designed to have a footprint of 7 cm x 7 cm x 4 cm and to allow live-dead bacterial counts and sorting of
cells that have characteristics that respond to BacLight TMLive/Dead bacterial green fluorescent stain (e.g., SYTO 9 for
live cells or SYTOX for dead cells) or molecular probes such as the BacLightTM Bacterial Membrane Potential probe.
Since these molecular probes use nucleic acid stains, they can distinguish live or dead biological cells from abiotic
particles. The µFACS system takes advantage of microfluidics to create a miniaturized cell sorter that can fit in the palm
of the hand. This staining system has been previously demonstrated with extermophilic bacteria.'
The detection of living Pleistocene bacteria in the Fox Tunnel and Antarctic Ice 2 suggests that ice encased bacteria might
survive for long periods in microgravity and the space environment. This enhances the feasibility of the transfer of
microorganisms by impact ejection phenomena. If cross-contamination of Solar System bodies might have resulted from
natural processes, perhaps some of the Planetary Protection Protocols that must be implemented for future spacecraft
could be relaxed at additional cost savings to NASA. This device can be used to determine the viability of this
hypothesis in a small nanosatellite or CubeSat system by carrying samples of bacteria in ice exposed to space.
The test µFACS System could also be used to determine if living or dead bacteria are present in the upper crust (2-5
meters) of Europa. This would be much cheaper than developing a cryobot to penetrate the 3-4 km thick ice to reach the
liquid water oceans.
*dplatt@micro-a.net-, phone 1 321243-4633-, fax 1 928 396-3256; micro-amet
https://ntrs.nasa.gov/search.jsp?R=20090042980 2019-08-30T08:27:41+00:00Z
2. SYSTEM DESIGN
A micron-scale claxuuel allows study of cells stained with a mixture of SYTO 9 green fluoresc era (excitationlemission
maxima of 48Of5OO ruts) aryl the red pwpidium iodide (excitationlemission maxima of 4901635) nucleic acid stairs. The
3YTO 9TM stain penetrates through both intact ce11 membrane (live cells) and damaged ce11 membranes (dead cells)
whereas the propidiurn iodide reacts only with the damaged cell membrane and enters into the dead cells resulting into
red fluorescent cells.
Since it does not react with the irdact membrane, live cells fluoresce green. The stained cells enter the tricrofluidics
capillaries and pass by the blue light emitting diode (LE.D ^ which causes the stained live cells to fluoresce green and the
dead cells to fluoresce red. The fluorescence emitted by cells is detected by a filtered photodetector. A small
microcontroller then operates high speed valves to divert the cell to the collection chamber (live cells) or the waste
chamber (dead cells). We take advantage of prior experience with micro -scale valve technology to create a system with
integrated WE M-based pumps and valves for a small and compact unit to fly on small satellite leased biology
experiments. Figure 1 shows a schematic of current system and Figure 2 slows the integrated prototype electronti ,cs aryl
microfluidics assembly
Photo(hode vkith filters (2, 51-1 iurn :iml 615 iurn)
4lit filter (2)
LED	 I%IifiTOfl_kuxh(s -CItU E-1
( Side F
LED
Fig. 1. Mi,crofhiidi,c s Chunu 1Pe sir, Showing Row Charuiel, CeUsEriLa on RightOptic sand Split Gha=lfor GeI Sorting
(L MM ead Sorg
A basic microfluidics channel and electroncs control system has been developed and is un ergoivg testing. The next
phase of this project will take the microfluidics channel and integrate valving aril controls to allow cells to be
automatic ally fed into the system and then sent to one of the two chambers (one for live cells and one for dead cells as
indicatedby staining).
Photodiodes with appropriate wavelength filters observe the flaw channel and associated electronics count the cells and
sort them using a microcontroller-based circuit board. A breadboard design has already been built and tested} but the
current system is manually fed and only counts cells and pro-ides live-dead ratios. We are currently evaluating 1ow we
can integrate other micromechanical components into the microfluidics system design For instance micravalles,
6microlenses and micropumps could be incorporated into the µFACS system design to make the system more compact
and efficient. A variety of microoptics systems are also under evaluation.
Fig. 2. Protype System (Electronics, left and Microfluidics, right).
The end goal will be to create a complete astrobiology lab-on-a-chip system to sample bacteria, count the live-dead ratio
overtime and then sort the bacteria for further analysis as seen in Figure 3. This system integrates microfluidics, optics,
control electronics and valves in a package that can easily fit into a 3U CubeSat with multiple systems to study different
types of bacteria and/or different bacteria exposures. The system can also be adaptable to different excitation LEDs and
filter wavelengths for detection.
Overall system mass is 100 grams within the footprint of 7 cm x 7 cm x 4 cm. It uses under 100 mA of power to process
a bacterial sample. The LED has emission centered at 485 nm. The optics system consists of micro lenses with filters
optimized for green and red emission for SYTO 9 and red propidium iodide, respectively. A photodiode measures
emission intensity. Simple green/red ratios can be measured using the two channels to determine percent live bacteria or
more sophisticated analysis can take place and bacteria samples separated based upon viability. Figure 3 shows the
final system design.
Control
Valve	 Waste ('hamber
CoRec tiOn Chamber	 Inlet
Fig. 3 System Design Schematic Show the Microfluidcs Flow Channel with Sheathing Buffer Inlet and Cell Inlet, Blue Illumination
LED (for emission) and redfgree filtered photodetector output. A microcontroller counts cells and controls valves for collection.
A sheathing flow is used to hydrodynamically focus the cells. Wolff et al. implemented a "smoking chimney" focusing
technique that is used in the system described in this paper. 3 An important factor will be the required relative flow rate
of cell-suspended fluid compared to the buffer fluid flow. Increasing the ratio of sheath flow to sample flow increases
the focusing of the system. The transverse injection of the cell-suspension fluid should provide the proper level of
mixing even in a microgravity environment. With microcontroller-based control of sheathing flow and sample flow, the
system can be optimized for various conditions and focusing can be done "on the fly". This focusing flow is illustrated
in Figure 4.
Other focusing techniques require microfabrication of electrodes on both the bottom and top of the channel, precision
alignment of these electrodes, electronics for generating electric AC field, and conductivity of the focusing liquid. These
additional complications are eliminated in this design allowinga smaller, more compact design ideal for small satellite
systems.
Fell F1cnN-
Buffer Fl(ny
Side V. iew
Top View
Fig. 4 Microchannel sheathing (buffer) flow and focusing of the introduced bacterial fluid. Arrows indicate flow direction.
Figure 5 shows BacLight TM Live/Dead stain of Spirochaeta americana pure culture from Owens Lake, CA showing live
cells (green) and dead cells (red). This illustrates the ease in distinguishing between viable and dead bacteria with this
staining technique. New high intensity, yet miniature LEDs with emissions around 470 to 490 nm allows this
technology to be implemented in a microfluidics system.
Fig. 5 BacLightT"' Live/Dead stain of Spirochaeta americana pure culture from Owens Lake, CA showing live cells (green) and dead
cells (red).
3. APPLICATIONS
This system relies on technology based on Live/Dead Molecular probes and microfluidics capillary to permit in-situ
bioanalytical assessment of the viability of selected anaerobic bacteria and archaea thawed out after different periods of
exposure to the space environment. This work has drwan on expertise in microfluidics-based system combined with
recent discoveries of survivability of microbial extremophiles entrapped in the ancient ice from the Fox Tunnel of
Alaska, Deep Vostok Ice Cores as well as more modern ice from the Anuchin Glacier, Ice Sculptures and Ice-Bubbles
studied in-situ  during the 2008 Antarctic Expeditions.
This instrumentation can have valuable commercial applications and permit significant cost reductions to future Space
Missions. A miniature, lightweight, low-power instrument that can distinguish live bacteria from dead bacteria and
bacterial cells in ice from abiotic particles could be incorporated into future SMD Planetary Science Missions.
Verification that microbial life entrained in ice can remain viable suggests that trans-planetary transfer of intact
microbiota may have occurred during great cosmic impacts.
Recently there was a discovery of living Pleistocene microorganisms, (Camobacterium pleistocenium, sp. nov.) that had
been frozen in the ice of the Fox Tunnel of Alaska for 32,000 years and was still alive and expressed a motility
immediately as the ice thawed. This finding implies that ancient living bacteria may be crypopreserved in frozen water
ice glaciers at the Polar Caps of Mars. It also means that it may not be necessary to drill through several kilometers of ice
to penetrate the crust of icy moons such as Europa or Enceladus to search for life. Living microbes may be present
within the first few centimeters or meters of the ice crust, if they are protected from the UV and radiation environment
by the ice sheet. These icy moons have become of great interest to Astrobiology due to indications of liquid water oceans
beneath their icy crusts. However, viable microorganisms have been discovered in the permafrost and frozen thermokarst
ponds of Siberia and Alaska (Carnobacterium pleistocenium). During Antarctic Expeditions, living and physiologically
active microorganisms were observed in abundance in freshly thawed ice from the Anuchin glacier and ice bubbles of
Lake Untersee and the frozen ice sheets covering the Schirmacher Oasis lakes (unpublished data). These facts expand
the possibility of surviving of microbial extremophiles on the Polar Ice Caps of Mars, and the crusts of other frozen
bodies of our Solar Systems. Understanding the nature and distribution of microbial life in the glaciers and perennially
ice covered lakes of Antarctica should provide meaningful and valuable data required to develop and evaluate
instruments and mission operational techniques to search for and recognize evidence of extant or extinct life during
future Mars Missions or during explorations of other Solar System bodies. This compact device can be used to analyze
bacteria in-situ in these environments. Combining this mFACS with a compact imaging instrument would create a
complete field laboratory that is transportable in a backpack.
The possibility that viable microorganisms entrapped in ice may survive exposure to microgravity and the space
environment enhances the feasibility of transfer of microorganisms via impact ejection phenomena. If ice-entrapped
microorganisms remain viable in the space environment and can be revived on melting then it is feasible to search for
living bacteria in the uppermost 2-5 meters of the crust of Europa or Enceladus using the pFACS system. This is much
less expensive than developing robotic systems to penetrate a 3-4 km thick ice shell to reach liquid water. Ice is an
excellent absorber of high energy particles — only 20 cm of ice reduces the flux of 100 MeV protons to Ile.
Demonstrating the feasibility of cryo-panspermia could have implications to the NASA Origins Program concerning
distribution and Evolution of Life in the Cosmos and the Origin of Life on Earth as well as the NASA Planetary
Protection Program. If cross-contamination of Solar System bodies can feasibly occur by natural impact ejection transfer
of ice containing viable microbes, it may be possible to relax Planetary Protection Protocols at significant cost savings to
NASA.
Potential commercial applications of the device include lab-on-a-chip systems, field-based medical instruments and
astronaut health monitoring systems to measure blood counts or pathogen activity on long duration space missions.
Since the system can be tailored to counting other cell-types and particles; these devices may be used for counter-
bioterrorism efforts by detecting potentially deadly microbiological agents in their intact states. Very small, portable
medical research devices may be used for pathogen diagnostics in field settings in the rural US and developing countries.
Their low cost, ease of use, small footprint and low power consumption could bring state of the art medical care to areas
of the world which have not previously had such services. A main issue previously has been the large size, complex
support equipment, and cost associated with many FAGS systems.
4. FUTURE WORD
The newly developing field of C ub e Sat-bas ed science missions is theperfect setting to test the µFACS system and the
viability of bacteria submerged in ice in the space environment. NASA has previously flown G enesat and Pharmasat 3U
Cub eSats with total spacecraft masses under 5 kilograms. This system can be flown on a similar Cub eSat platform for
astrobiology research into bacterial viability in space. A simple 3U CubeSat concept is shown in Figure 6
An exposure section can be configured on the bottom and top surfaces of the spacecraft (the circle in Figure 6). This
exposure area would then interface to a valve manifold sy stem to all ow bacterial samples to be made at desired intervals
during the mission. For instance, each week ormonth a sample could be taken and bacterial viability analyzed using the
p.FACS device.
A series of ground tests will also be conducted with various extremophile bacteria to gain a system baseline and for
calibration purposes. The system can also be tested with bacteria in extreme Earth environments such as the polar
regions and radiation chambers.
Figure 6 A 3U CubeSat with anExposure Platform (bottom circle) for anice-bacteria sample
5. CONCLUSION
The mFA CS system offers a low co st and responsive solution for astrobiology missions to analyze bacterial viability in
space as well as detect life on other solar system bodies and analyze bacteria in remote locations on Earth. The design is
based upon the fundamental principles that a microfluidic-µFA CS system in a CubeSat format that would function in
conjunction with a well established commercially available fluorescent dye-based microbial detection technology that
allows specific identification of the live cells from dead. This overall system makes a low-cost, low-mass and low-
p ower a strobi of o gy lab -o n-a- chip.
We are working towards the development of a flight instrument prototype that could form the basis for future flight
instruments to address the most fundamental question of Astrobiology: Is life restricted to Planet Earth or is it distributed
throughout the Cosmos?
REFERENCES
[1] Leuko, S., Legat, A., Fendrihan, S., and Stan-Lotter, H., "Evaluation of the LIVE/DEAD BacLight Kit for Detection
of Extremophilic Archaea and Visualization of Microorganisms in Environmental Hypersaline Samples", AEM, 70 (11),
6884-6886(2004).
[2] Pikuta, E., Marsic, D., Bej, A., Tang, J., Krader, P., and Hoover, R. B., "Carnobacterium pleistocenium sp. nov., a
novel psychrotolerant, facultative anaerobe isolated from permafrost of the Fox Tunnel in Alaska", IIJS, 55, 473-478
(2005).
[3] Wolff, A., Perch-Nielsen, I. R., Larsen, U.D., Friis, P., Goranovic, G., Poulsen, C. R., Kutter, J. P., and Telleman, P.,
"Integrating advanced functionality in a microfabricated high-throughput fluorescent-activated cell sorter", Lab Chip, 3,
22-27 (2003).
[4]Parra, M., McGinnis, M.R., Ricco, A.J., Yost, B., and Hines, J.W., "Studying space effects on microorganisms
autonomously: genesat, pharmasat and the future of bio-nanosatellites", Gravitational and Space Biology 21(2), 9-17
(June 2008).


A Micro Fluorescent Activated Cell Sorter for Astrobiology Applications

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