Development of a Cellular Isolation System for Real-Time Single Cell Oxygen
Consumption Monitoring
Joe DragavonMarch 21, 2007
•Measure multiple parameters in individual living cells in real-time to correlate cellular events with genomic information.
•Develop modular, affordable microsystems for analyzing complex cellular processes.
•Application: To monitor and increase the understanding of programmed cell death, apoptosis (ap-a-tow’-sis)
• This is done by determining the cellular oxygen consumption rate before and after a stress is applied (LPS)
• Too much apoptosis = cell-loss disorders• Too little apoptosis = uncontrolled cell growth (cancer)
Highly interdisciplinary undertaking!!
Microscale Life Sciences Center (MLSC)
Comprehensive Understanding ofComplex Cellular Processes
www.nst.co.il/frontiers/apoptosis.htm
MajorContributors
My Objective
• To develop a Cell Isolation System (CIS) with which we can monitor oxygen consumption rates at the single cell level– Must be user friendly– Must be adaptable for performing stimulus
response experiments– Cells must be kept viable and in a “low-stress”
environment– Hopefully inexpensive (relatively)
Why Oxygen
• History of O2 sensors at the UW– Professor Callis: pressure sensitive paints
• Direct indicator of cell viability and metabolic activity
• Difficult analyte to exclusively monitor– If we can do this, we should be able to do other
analytes in a similar manner
Why Single Cell
• Heterogeneous subpopulations
1. Cell death due to inflammatory pathology (pyroptosis)
2. Malignancy due to unregulated cell growth
3. Susceptibility and resistance to cell death leading to clones with aberrant survival
electronic ground state
T1
T2
S0
S1
S2
Sn
IC
ISC ICA F
P
Ene
rgy
electronic ground state
T1
T2
S0
S1
S2
Sn
IC
ISC ICA F
P
Ene
rgy
electronic ground state
T1
T2
S0
S1
S2
Sn
IC
ISC ICA F
P
Ene
rgy 0% 20%
Dy, E. S.; Kasai, H. e-Journal of Surface Science and Nanotechnology 2005, 3, 473-475.
McGraw, C. M., Callis, J. B. Experiments in Fluids 2006, 40, 2, 203-211.
Porphyrin O2 Sensor
Clark Electrode Response
y = -2E-05x + 0.0006R2 = 0.9997
0.0000
0.0001
0.0002
0.0003
0.0004
0.0005
0.0006
4 6 8 10 12 14 16
Time (min)
Mol
O/L
Bead response
y = -2E-05x + 0.0006R2 = 0.955
0
0.0001
0.0002
0.0003
0.0004
0.0005
0.0006
4 6 8 10 12 14 16
Time (min)
Mol
O/L
Clark Electrode Response
y = -2E-05x + 0.0006R2 = 0.9997
0.0000
0.0001
0.0002
0.0003
0.0004
0.0005
0.0006
4 6 8 10 12 14 16
Time (min)
Mol
O/L
Clark Electrode Response
y = -2E-05x + 0.0006R2 = 0.9997
0.0000
0.0001
0.0002
0.0003
0.0004
0.0005
0.0006
4 6 8 10 12 14 16
Time (min)
Mol
O/L
Bead response
y = -2E-05x + 0.0006R2 = 0.955
0
0.0001
0.0002
0.0003
0.0004
0.0005
0.0006
4 6 8 10 12 14 16
Time (min)
Mol
O/L
Bead response
y = -2E-05x + 0.0006R2 = 0.955
0
0.0001
0.0002
0.0003
0.0004
0.0005
0.0006
4 6 8 10 12 14 16
Time (min)
Mol
O/L
microspheres Clark Electrode
Strovas, T.; Dragavon, J.; et al. Appl. Environ. Microbiol. 2006, 72 (2), 1692-1695.
Sensor Deposition2-Photon photo polymerization
Laser
Substrate
63x 1.4 NAobjective
Nano-cube
Dichroic mirror
Shutter
Camera
YoungDragavon
• Good for individual sensor deposition• Low throughput• Porphyrin embedded into polymer
100µm
Young, A. C., Dragavon, J. et al. Two-Photon Lithography of Platinum-Porphyrin Oxygen Sensors. IEEE Sensors. Accepted
Big Idea: Integrated ParallelCell Fluorescence System
NutrientSupply
CCDOnly those pixels above the tube are excited. Collects fluorescence data and allows feedback via computer to LED layer.
Tubular Cage Layer3D microfabricated polymer pressed into the layer below to trap individual cells. The tubes are perforated to allow nutrients to pass through. The inner surface of the tubes is functionalized to act as an external sensor.
Specimen LayerFluorescent bacteria cells on a flat transparent slide. Cells are sustained at temperature by a continuous flow of nutirents.
LED Excitation LayerMulticolored LED with microfabricated half-spherical lens array shown here to be uniformly illuminating blue. The intensity and color can either be static or dynamic to meet experiment needs.
NutrientSupply
CCDOnly those pixels above the tube are excited. Collects fluorescence data and allows feedback via computer to LED layer.
Tubular Cage Layer3D microfabricated polymer pressed into the layer below to trap individual cells. The tubes are perforated to allow nutrients to pass through. The inner surface of the tubes is functionalized to act as an external sensor.
Specimen LayerFluorescent bacteria cells on a flat transparent slide. Cells are sustained at temperature by a continuous flow of nutirents.
LED Excitation LayerMulticolored LED with microfabricated half-spherical lens array shown here to be uniformly illuminating blue. The intensity and color can either be static or dynamic to meet experiment needs.
NutrientSupply
CCDOnly those pixels above the tube are excited. Collects fluorescence data and allows feedback via computer to LED layer.
Tubular Cage Layer3D microfabricated polymer pressed into the layer below to trap individual cells. The tubes are perforated to allow nutrients to pass through. The inner surface of the tubes is functionalized to act as an external sensor.
Specimen LayerFluorescent bacteria cells on a flat transparent slide. Cells are sustained at temperature by a continuous flow of nutirents.
LED Excitation LayerMulticolored LED with microfabricated half-spherical lens array shown here to be uniformly illuminating blue. The intensity and color can either be static or dynamic to meet experiment needs.
JenYoung
Microwell Array
• High throughput• Approaching the requirements for
commercial implementation• Decent uniformity
1 2 3
4 5 6
7 8 9
• All microwells seeded simultaneously (random seeding)
• Can sequentially perform oxygen consumption measurements on multiple locations
Sensors
1 2 3
4 5 6
7 8 9
20 40 60 80 100
10
20
30
40
50
60
70
80
90
100
1000
2000
3000
4000
5000
6000
7000
8000
9000
Phase Modulation Data Processing
• System calibrated using a PMT, MLE, and the OOFD
• Improved fit by going to higher order polynomial
• Multiple phosphorescent lifetimes being detected (non-linear Stern-Volmer)
DragavonMolter
]O[1 2SV0 K
Ψ
Ψ
mKΨ
Ψ]O[1 2
0
])O[1/(])O[1/(
1
2SV2022SV101
0
KfKfΨ
Ψ
1)
2)
3)
Model
HollDragavonMolter
ActuatorSystem
• System acts as a manual linear actuator
• Load cell allows for accurate monitoring of the exerted force
• Rigid structure allows for a significant amount of force to be applied
• V-groove system ensures proper piston alignment
Living Cell Array
• Increased support– Aluminum plate
– 1/8” quartz window
• One macrowell format– ~5mL volume
• Reusable seal!– VERY IMPORTANT!!
• The glass lid is also made of borosilicate, just like the chips
• The thickness of the PDMS layer is ≈ the same as the lid
• Adhesive not optimized
Plunger(stainless steel)
PDMS
Glass
LaserScanningModule
Lid Actuator
Heat Source
405nmExcitation
TemperatureControl
PressureControl
QA/QC
bad seal
unsealed sealed (visual confirmation!) cleaned
PDMS delaminationdense array sealand other flaws
Cell Test 6/26/06
-0.05
0
0.05
0.1
0.15
0.2
0.25
0 1 2 3
# of cells
delta
O2
(ppm
/min
)de
lta O
2 (p
pm/m
in)
O
2 (
pp
m/m
in)
• 2 Locations (3X3)• 1 chip (81 wells)• 4 sequential
repetitions• Mouse macrophage
– very fragile!
• 10x objective
Sequential Drawdowns
Cell Drawdown Histogram
0
2
4
6
8
10
12
14
0 0.02 0.04 0.06 0.07 0.09 0.11 0.13 0.15
Bin
Freq
uenc
y
Δ O2 (ppm/min)
Fre
qu
en
cy
HistogramSingle Cell Drawdown
# of cells
Cell Test 6/26/06 1
-0.05
0
0.05
0.1
0.15
0.2
1 2 3 4 5 6 7 8 9
w ell #
de
lta
O2
(p
pm
/min
)
1
2
3
4
Average
2
1
0
2
1
0 00 0
del
ta O
2(p
pm
/min
)
Location 1Cell Test 6/26/06 1
-0.05
0
0.05
0.1
0.15
0.2
1 2 3 4 5 6 7 8 9
w ell #
de
lta
O2
(p
pm
/min
)
1
2
3
4
Average
2
1
0
2
1
0 00 0
del
ta O
2(p
pm
/min
)
Location 1 Cell Test 6/26/06 2
-0.05
0
0.05
0.1
0.15
0.2
0.25
1 2 3 4 5 6 7 8 9
w ell #
de
lta
O2
(p
pm
/min
)
1
2
3
4
Average
2
1
0
1 1 1
0 0 0
Location 2
del
ta O
2(p
pm
/min
)
Cell Test 6/26/06 2
-0.05
0
0.05
0.1
0.15
0.2
0.25
1 2 3 4 5 6 7 8 9
w ell #
de
lta
O2
(p
pm
/min
)
1
2
3
4
Average
2
1
0
1 1 1
0 0 0
Location 2
del
ta O
2(p
pm
/min
)
• General linearity• Not centered around 0• Superimposed biological
and system variance
paper in progress
0
100000
200000
300000
400000
500000
600000
700000
800000
0 5 10 15 20 25 30 35
step
int 20%
0%inte
nsi
ty
Power Spectrum
0
5E+11
1E+12
1.5E+12
2E+12
2.5E+12
3E+12
0 5 10 15 20
frequency
Fo
uri
er c
oef
fici
entMethod
# of data points
# of laser shots
time (sec)
Original 42 210 42
Current 21 105 21
In Progress 31 31 6.2*
TBD (Nyquist) 6 6 1.2*
For a Single Oxygen Determination
* Camera Limited
Final Work:New Data Collection
DragavonYoungBurgess
Φ = tan-1(I/R)
My Objective
• To develop a Cell Isolation System (CIS) with which we can monitor oxygen consumption rates at the single cell level– Must be user friendly– Must be adaptable for performing stimulus
response experiments– Cells must be kept viable and in a “low-stress”
environment– Hopefully inexpensive (relatively)
(working on this still)
HIBISCUS
• Hybrid Integraged BIophotonic Sensors Created
by Ultrafast laser Systems
• 7 universities and companies
• 6 different countries
• Aiming to develop a single production machine
for biophotonic chips
• Use a high-power femtosecond laser to write
waveguides and etch the channels
Proposed Synthesis
• Gramicidin S, a cyclic pentapeptide antibiotic
ONH
NH
O
O
(9H -f luoren-9-yl)methyl 3-(benzylamino)-3-oxopropylcarbamate
Chemical Formula: C25H24N2O3Exact Mass: 400.1787
Molecular Weight: 400.4697m/z: 400.18 (100.0%), 401.18 (28.2%), 402.19 (3.6%)
Elemental Analysis: C, 74.98; H, 6.04; N, 7.00; O, 11.99
ONH
O
O
O
F
F
F
F
F
perf luorophenyl 3-(((9H -f luoren-9-yl)methoxy)carbonylamino)propanoate
Chemical Formula: C24H16F5NO4Exact Mass: 477.0999
Molecular Weight: 477.3802m/z: 477.10 (100.0%), 478.10 (26.5%), 479.11 (3.3%)
Elemental Analysis: C, 60.38; H, 3.38; F, 19.90; N, 2.93; O, 13.41
H2N
phenylmethanamine
Chemical Formula: C7H9NExact Mass: 107.0735
Molecular Weight: 107.1531m/z: 107.07 (100.0%), 108.08 (7.7%)
Elemental Analysis: C, 78.46; H, 8.47; N, 13.07
• Representative reaction to be used to test various analytical methods
• Experimental parameters already determined by current Watts group members
+
Optical TechniquesMethod Information How
SPR RI, Binding Spreeta™ by TI
ATR RI, Binding Waveguides in glass
µMS RI, Diffusion PSD
Raman Compound Identification Microscope
Fluorescence Functional Groups, Protected vs. Unprotected Spectral Deconvolution
HPLC Product Separation, Standard Technique HPLC
Costin, c. D., Synovec, R. E. Anal. Chem. 2002, 74, 4558 – 4565
Multi-Channel Chip
• Promotes phase separation
• Diffusion properties can be used to improve analysisBen Wahab,
Watts group
Phase Separation
• Multiple channels will promote the separation/isolation of desired compounds
• Increases the effectiveness of the optical analysis
• Good for:– µMS– SPR– Fluorescence
Acknowledgements
University of Washington
MLSC-NIH
CPAC
Cody Young, Tim Molter,
Sarah McQuaide
Drs. Jen, Lidstrom, Meldrum, Holl, Chao, Lutz, Marquardt, Strovas
Lloyd Burgess, Gordon Mitchell
University of Hull
HIBISCUS
Paul Watts
and Watts group members
Phase Modulated Determination(OOFD)
Delay of the intensifier is established using the camera software
Laser period = Intensifier periodThe period is variable
ExternalTrigger
Laser
Camera
Intensifier
-180°
-90°
0°
90°
180°
1
2
3
4
5
StepDegree Shift
100μsec
Shonat, R., Kight, A. Annals of Biomedical Engineering, Vol. 31, pp. 1084–1096, 2003
Thermoelectric Cooler Effect
0
1
2
3
4
5
6
7
8
9
1 2 3 4 5 6 7 8 9
well #
ave
-20
-10
0
Offav
era
ge
pp
mAccumulation Comparison
0
1
2
3
4
5
6
7
8
9
1 2 3 4 5 6 7 8 9
well #
aver
age
ppm
1
2
3
4
5av
era
ge
pp
m
Gain Comparison
0
1
2
3
4
5
6
7
8
9
1 2 3 4 5 6 7 8 9
well #
aver
age
ppm 50
100
150
av
era
ge
pp
m
Focus Comparison
0
1
2
3
4
5
6
7
8
9
1 2 3 4 5 6 7 8 9
well #
aver
age
ppm
-75um
-25um
0um
25um
75umav
era
ge
pp
m
N = 30
OXYGEN SENSOR CALIBRATION AND REPRODUCIBILITYThermoelectric Cooler Effect
Gain Comparison Focus Comparison
Accumulation Comparison
well #
well #
well #
well #
Bin Comparison
0
1
2
3
4
5
6
7
8
9
1 2 3 4 5 6 7 8 9
well #
aver
age
ppm
4x4
6x6
10x10
12x12
20x20av
era
ge
pp
mShot Comparison
0
50000
100000
150000
200000
250000
1 2 3 4 5 6 7 8 9
well #
coun
tsa
ve
rag
e i
nte
gra
ted
in
ten
sit
y
Microwell Location Comparison
0
1
2
3
4
5
6
7
8
9
1 2 3 4 5 6 7 8 9
well #
Av single
wholeav
era
ge
pp
m
1 hour Stability
0
1
2
3
4
5
6
7
8
9
1 2 3 4 5 6 7 8 9
well #
ava
ve
rag
e p
pm
Long-term Stability
17,000 x 50sec exposures( >150 O2 determinations)
OXYGEN SENSOR CALIBRATION AND REPRODUCIBILITYShot Comparison
1800 x 50µsec exposures
Microwell Location Comparison
Bin Comparison
N = 30well #
well #
well #
well #
-0.1
-0.08
-0.06
-0.04
-0.02
0
0.02
0.04
0.06
0.08
0.1
1 2 3 4 5 6 7 8 9
well #
delta
O2 1
2
3
delta
O2
(ppm
/min
)
10 Acquisitions
-0.1
-0.08
-0.06
-0.04
-0.02
0
0.02
0.04
0.06
0.08
0.1
1 2 3 4 5 6 7 8 9
well #
delta
O2 1
2
3
delta
O2
(ppm
/min
)
10 Acquisitions
-0.1
-0.08
-0.06
-0.04
-0.02
0
0.02
0.04
0.06
0.08
0.1
1 2 3 4 5 6 7 8 9
well #
delta
O2 1
2
3
delta
O2
(ppm
/min
)
42 Acquisitions
-0.1
-0.08
-0.06
-0.04
-0.02
0
0.02
0.04
0.06
0.08
0.1
1 2 3 4 5 6 7 8 9
well #
delta
O2 1
2
3
delta
O2
(ppm
/min
)
42 AcquisitionsΔ O2 (15 min, 42 Kinetic Series) Δ O2 (11 min, 10 Kinetic Series)
well # well #
Table 1. Integrated Intensities of One Microwell Over Multiple Kinetic Series
Kinetic Series Step #
Series # 1 2 … 20 21 Average StDev CV
1 252649 252012 … 254735 254643 252552.4 1397.15 0.553
10 250113 251660 … 248569 247600 249158 1707.178 0.685
20 246669 243976 … 248299 248096 246771.5 1650.655 0.669
30 244905 247621 … 246993 248892 247372 1530.93 0.619
Average 248584 248817.3 … 249649 249807.8
STDEV 3467.195 3792.816 … 3459.799 3267.134
CV 1.394778 1.524338 … 1.385865 1.307859
Integrated intensity at a fixed phase shift
Coefficient of Variance = CV = (StDev/Average)*100
* will return to this camera issue
Benzylamine
Pentafluorophenol
http://www.aist.go.jp/RIODB/SDBS/cgi-bin/direct_frame_top.cgi?lang=eng
Ongoing Work:External Stimulus
• External Stimulus Response– LPS Application
• Monitor O2 consumption prior to and after application
• Monitor O2 over time
• This would add the metabolic rate of the cell as another dimension to the data set
5 bead
1920 x 1200 pixels
Jesacher et. al., 23 August 2004 / Vol. 12, No. 17 / OPTICS EXPRESS 4129
Ongoing Work: SLM used to generate dynamic optical tweezers
Size exclusion optical trapping
BurgessYoung
High-throughput cell deposition
Ongoing Work:Sample Cassette
Holl
Technology from the current CIS is the foundation for the cassette
Ongoing Work: Multispectral Analysis
HollBurgessPowellKarlsgodt Cheaper than the current set-up!
Clark Electrode Response
y = -2E-05x + 0.0006R2 = 0.9997
0.0000
0.0001
0.0002
0.0003
0.0004
0.0005
0.0006
4 6 8 10 12 14 16
Time (min)
Mol
O/L
Bead response
y = -2E-05x + 0.0006R2 = 0.955
0
0.0001
0.0002
0.0003
0.0004
0.0005
0.0006
4 6 8 10 12 14 16
Time (min)
Mol
O/L
Clark Electrode Response
y = -2E-05x + 0.0006R2 = 0.9997
0.0000
0.0001
0.0002
0.0003
0.0004
0.0005
0.0006
4 6 8 10 12 14 16
Time (min)
Mol
O/L
Clark Electrode Response
y = -2E-05x + 0.0006R2 = 0.9997
0.0000
0.0001
0.0002
0.0003
0.0004
0.0005
0.0006
4 6 8 10 12 14 16
Time (min)
Mol
O/L
Bead response
y = -2E-05x + 0.0006R2 = 0.955
0
0.0001
0.0002
0.0003
0.0004
0.0005
0.0006
4 6 8 10 12 14 16
Time (min)
Mol
O/L
Bead response
y = -2E-05x + 0.0006R2 = 0.955
0
0.0001
0.0002
0.0003
0.0004
0.0005
0.0006
4 6 8 10 12 14 16
Time (min)
Mol
O/L
Bulk O2 Consumption and Biological Compatibility
y = 5160.2x + 1.0671R2 = 0.9978
0
0.5
1
1.5
2
2.5
3
3.5
4
0 0.0001 0.0002 0.0003 0.0004 0.0005
Mol O/L
To/
T
0
10
20
30
40
50
60
70
Life
time
s (
sec)
DragavonStrovas
Strovas, T.; Dragavon, J.; et al. “Measurement of Respiration Rates of Methylobacterium extorquens AM1 Cultures by Use of a Phosphorescence-Based Sensor”, Appl. Environ. Microbiol. 2006, 72 (2), 1692-1695.
microspheres Clark Electrode