1. What I Did on My Summer Vacation Olympus IX71 Characterization Substrate Comparison Substrate Cleaning Technique Comparison By: Amelia McCready

2. Limit of Detection on Olympus Microscope at 10x 200ms 100lamp fluorescein in PDMS channels Limit of detection is above 100nM for 10x

3. Limit of detection on Olympus Microscope at 40x 200ms 100lamp fluorescein in PDMS channels Limit of detection is between 10nM and 100nM for 40x.

4. Gain Series on Olympus Microscope 10e-9M Bodipy on 1.5 glass coverslide 100ms, 100x, 100%lamp The signal/noise increases as the gain increases and is good enough to detect single molecules

5. Gain series on SMT microscope 10e-9M Bodipy dried on 1.5coverglass, 50ms 100x 4.5pa 6em laser37 The signal/noise on the SMT is superior to that of the IX71 but the signal/noise does not increase as the gain increases

6. Single Molecule Image on SMT and Olympus Microscopes 10e-9MBodipy dried on 1.5coverglass 50ms, 100x cgain3, avgain3700 100%lamp 10e-9MBodipy 50ms 100x cgain4.5 avgain50 37laser Single molecules can be viewed on the IX71 with a 100x objective but considerable spherical aberration occurs because the objective is an uncorrected Zeiss and the tube lens on the IX71 is also uncorrected.

7. Dark Charge M-multiplication gain N-normal In the first ten images, multiplication gain decreases and normal mode increases. Also, multiplication gain shows a higher intensity but less fluctuation than normal mode 1uM fluorescein in PDMS channels, 100images, 12%lamp 0ms

8. Stddev of dark count intensity with increasing gain Noise increases as gain increases. 1uM fluorescein in PDMS channels, 100images, 12%lamp 0ms

9. Photobleaching of Fluorescein at 100% Lamp Power 100% causes fairly severe photobleaching. 10x, 3gain, 1uM fluorescein, 1000images, 4min 21sec

10. Photobleaching of Fluorescein at 50% Lamp Power Photobleaching is still considerable at 50% lamp power. 10x, 3gain, 1uM fluorescein, 1000images, 4min 21sec

11. Photobleaching of Fluorescein at 25% Lamp Power Photobleaching occurs even at 25%. 10x, 3gain, 1uM fluorescein, 1000images, 4min 21sec

12. Photobleaching of Fluorescein at 12% Lamp Power 10x, 3gain, 1uM fluorescein, 1000images, 4min 21sec 12% lamp power is the only one that does not demonstrate significant photobleaching.

13. Photobleaching of Fluorescein at various lamp powers For each power: 10x, 3gain, 1uM fluorescein, 1000images, 4min 21sec

14. Substrate Comparison 50ms exp, 100laser, 100em, 4.5pa, 100x, 10 frames, Glass and fused silica cleaned 10min in piranha rinsed with water and methanol and put in Argon plasma for 14 min at 60umHg The two substrates are very similar.

15. Further Comparison of Substrates substrateaverage mean of signalaverage stddev fused silica1406.4153.3 glass1429.0152.8 Magnitude of% difference1.582%0.347% Fused silica is slightly superior in mean and slightly inferior in stddev, but the difference is insignificant.

16. Figure1: 1.5glass coverslip, piranha 1 hour cgain3, avgain 3500, 100% lamp, 100ms 100x Figure2: 1.5glass coverslip, oxygen plasma 30 seconds 60umHg (same microscope conditions as Figure1) Comparison of cleaning techniques: Piranha vs. Oxygen Plasma Oxygen plasma is a superior cleaning method to piranha.

17. Salt Crystals on Oxygen Plasma Slide 1.5glass coverslip, oxygen plasma 30seconds 60umHg cgain3, avgain3500, 100% lamp, 100ms 100x Salt crystals occur on some plasma cleaned coverslips, making it a flawed method.

18. Comparison of Cleaning Techniques: Piranha then Plasma vs. Plasma then Piranha Figure1: 1hour piranha then 30sec oxygen plasma 60umHg 1.5glass coverslip,cgain3, avgain 3500, 100% lamp, 100ms 100x Figure2: 30sec oxygen plasma 60umHg then 1hour piranha on 1.5glass coverslip (same microscope conditions as Figure 1) Piranha followed by oxygen plasma solves the problem of salt crystals and results in spotless slides. The reverse method does not have the same result. It is slightly dirtier.

19. Varying Durations of Argon Plasma Firgure1: 30sec Ar plasma 60umHg (virtually ineffective) 1.5glass coverslip, Cgain3, avgain 3500, 100% lamp, 100ms 100x Figure2:10min Ar plasma 60umHg 1.5glass coverslip, (same microscope conditions as Figure1) Argon plasma requires a longer duration than Oxygen plasma, but 10 min is equal to piranha then plasma.