Optical properties of semiconducting pyrite deposited by aerosol
07_05_2015
1. Ca2+ Signaling Imaging Analysis
Center of Molecular Imaging and Therapeutics (University of Pittsburgh
Medical Center)
Darwin Kwok – July 7, 2015 – Supervisor: Francois Yu
2. Introduction
Background Theory Results Discussion Conclusion
Figure 1: In vivo experimental set-up under fluorescence
Goal (In Vivo):
To investigate methods that improve
visualization of Rhod 2 fluorescence in
cremaster
To observe calcium transients across
cremaster tissue caused by positive
variables and potentially microbubble (MB)
experimental variables
Goal (In Vivo):
To analyze and quantify the relationship
between Rhod 2-AM
concentration/exposure time to that of
fluorescence.
To compare and contrast the effectiveness
of Rhod 2-AM concentration to that of
exposure time in increasing magnitudes
3. Experimental Materials: Rhod 2
Rhod 2 is a calcium indicator with an excitation and emission maxima of 557 nm and 581 nm,
respectively.
Purified Rhod-2’s fluorescence increases 80-100 times with the introduction of Ca2+ to create a Rhod-2 Ca
complex
Carboxylic acid functional groups creates a pocket with high affinity for Ca2+
Figure 5: Functional properties of Rhod 2 in presence of calcium ionsP8
Background Theory Results Discussion Conclusion
4. Experimental Materials: Rhod 2-AM
Extracellular calcium ion concentration is not of high priority in Ca2+ signaling
Rhod 2-AM, an esterified form of Rhod 2, allows membrane permeability into cell
Esterase in cytosol cleaves ester group from Rhod 2-AM and replaces the R group with an alcohol group
Rhod 2 Ca2+ complex lacks ester functional groups increases polarity decreases membrane permeability
Figure 6: Hydrolysis of Rhod 2-AM into its functional form through presence of esteraseP9
Background Theory Results Discussion Conclusion
5. In Vitro Experimental Set-Up (6/30-7/5)
Nine HUVEC plates (in 3 x 3) are cultured and prepared
Each row of plate is stained with Rhod-2 AM dye of increasing concentration
Row A = 2 uM
Row B = 10 uM
Row C = 50 uM
Each column of plate is exposed to dye for increasing time intervals
Column 1 = 24 seconds
Column 2 = 2 minutes
Column 3 = 10 minutes
Wells are washed with PBS three times
Plates are incubated in media (250 uL) for 10 minutes before analysis under scope
Image is captured in BF, Fluo-baseline, and Fluo after addition of ionomycin
Background Theory Results Discussion Conclusion
6. Important Considerations In Vitro (6/22)
Sheer cell in vitro number
cannot be representative to
that in vivo
Cell confluence decreases
upon the completion of
experimental procedure.
This can be due to a change in
media to DMEM in the first trial
(which is not the optimal media
for HUVEC)
Further experimentation can
be done with proper media
Exposure to ionomycin
(dissolved in DMSO)
negatively affects cells
Background Theory Results Discussion Conclusion
8. (2 uM)
(10 uM)
(50 uM)
(24 s) (2 min) (10 min)
Background Theory Results Discussion Conclusion
HUVEC (6/22/15)
In DMEM
200 Gain
0.0100 Exposure
2 Binning
9. (2 uM)
(10 uM)
(50 uM)
(24 s) (2 min) (10 min)
Background Theory Results Discussion Conclusion
HUVEC (6/30/15)
In ETS-2 (P9)
200 Gain
0.0200 Exposure
2 Binning
10. (2 uM)
(10 uM)
(50 uM)
(24 s) (2 min) (10 min)
Background Theory Results Discussion Conclusion
HUVEC (7/5/15)
In ETS-2 (P10-1)
200 Gain
0.0200 Exposure
2 Binning
11. (2 uM)
(10 uM)
(50 uM)
(24 s) (2 min) (10 min)
Background Theory Results Discussion Conclusion
HUVEC (7/5/15)
In ETS-2 (P10-2)
200 Gain
0.0200 Exposure
2 Binning
12. Contrast between Concentration and Exposure Time
Background Theory Results Discussion Conclusion
0
1000
2000
3000
4000
5000
6000
7000
8000
9000
10000
0 100 200 300 400 500 600 700
Average Intensity
Time (s)
Concentration/Time Dependence vs. Fluorescence
(Passage 9)
2 uM
10 uM
50 uM
Figure 2: Passage 9 HUVEC’s in EDM-2 fluorescence with increasing magnitudes and concentration of Rhod 2-AM
13. Contrast between Concentration and Exposure Time
Background Theory Results Discussion Conclusion
Figure 3: Passage 10 HUVEC’s in EDM-2 fluorescence with increasing magnitudes and concentration of Rhod 2-AM
0
1000
2000
3000
4000
5000
6000
0 100 200 300 400 500 600 700
Average Intensity
Time (s)
Concentration/Time Dependence vs. Fluorescence (Passage 10)
2 uM (1)
10 uM (1)
50 uM (1)
2 uM (2)
10 uM (2)
50 uM (2)
14. In Vivo Experimental Set-Up (7/2)
Small (160 g) rat is prepared prior to experimentation
Rat is placed on a movable, inverted dock setup allowing ultrasound (US) signalling to
pass through the underside
A small catheter is inserted into the left femoral artery, allowing external injection
Hamamatsu camera is adjusted accordingly through course of experimentation:
250 uL of saline is initially injected to verify flow and its direction 0 gain, 0.01 exp, 4 binning (BF)
50 uL of 1 mM Rhod-2 is then injected with needle 255 gain, 0.01 exp, 4 binning (Fluo)
50 uL of MB in 200 uL saline is then injected in increasing frequencies 255 gain, 0.01 exp, 4
binning (Fluo)
50 uL of 0.1 mM ionomycin in 200 uL saline is then injected 255 gain, 0.01 exp, 4 binning (Fluo)
250 uL of saline is finally injected to confirm flow and its direction to the initial injection 0 gain,
0.01 exp, 4 binning (BF)
Background Theory Results Discussion Conclusion
15. In Vivo Results (7/2)
Background Theory Results Discussion Conclusion
16. Conclusions
Background Theory Results Discussion Conclusion
In Vitro:
As noted in the discussion, it can be
concluded that due to its staggering
effects (relative to one another),
concentration of exposed Rhod 2-AM
cells allows greater fluorescence in the
presence of Ca2+ than the duration of
exposure, itself.
In Vivo:
No full conclusion can be drawn from
the conducted experiment. Though
results from the experimental variable
was obtained, results from the positive
control was unobtainable due to
movement of the injection line during
experimentation. Further
experimentation and repetitions are
needed to solidify any findings.
