What I Did on My Summer Vacation

• Olympus IX71 Characterization

• Substrate Comparison

• Substrate Cleaning Technique Comparison

By: Amelia McCready

Limit of Detection on Olympus Microscope at 10x

gain (3,3500) mag 10x channel

-60000

-40000

-20000

0

20000

40000

60000

80000

100000

0 20 40 60 80 100 120

concentration (nM)

inte

nsity

(cou

nts)

200ms 100lamp fluorescein in PDMS channels

Limit of detection is above 100nM for 10x

10x

0

5

10

15

20

25

0 20 40 60 80 100 120

concentration(nM)si

ng

al/n

ois

e 3, 35003, 30003,02,01,0

Limit of detection on Olympus Microscope at 40x

gain (3,3500) 40x channel

01000020000300004000050000600007000080000

0 20 40 60 80 100 120

concentration (nM)

inte

ntsi

ty (c

ount

s)

200ms 100lamp fluorescein in PDMS channels

Limit of detection is between 10nM and 100nM for 40x.

40x

0

2

4

6

8

10

12

0 20 40 60 80 100 120

concentration(nM)

sign

al/n

oise

3, 35003, 30003,02,01,0

Gain Series on Olympus MicroscopeOlympus microscope

0

2

4

6

8

10

12

14

3000 3100 3200 3300 3400 3500 3600 3700 3800 3900 4000

gain

sig

na

l/n

ois

e

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

Gain series on SMT microscope

SMT Microscope

0

5

10

15

20

25

30

35

0 20 40 60 80 100 120

gain

sig

na

l/n

ois

e r

ati

o

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

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.

Dark Charge

1742

1741

120100806040200frame

2260

2250

2499

2498

2497

3440343034203410

395139503949

intensity, cnts

6000

5950

4230

4220

4150

4140

4130

4070

4060

gain 3 4095 M gain 3 3600 M gain 3 3000 M gain 3 0000 M gain 3 0000 N gain 2 0000 M gain 2 0000 N gain 1 0000 M gain 1 0000 N

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

Stddev of dark count intensity with increasing gain

50

40

30

20

10

0

stde

v of

dar

k co

nt in

tens

ity, c

nts

gain

3 4

095

M

gain

3 3

600

M

gain

3 3

000

M

gain

3 0

000

M

gain

3 0

000

N

gain

2 0

000

M

gain

2 0

000

N

gain

1 0

000

M

gain

1 0

000

N

Noise increases as gain increases.

1uM fluorescein in PDMS channels, 100images, 12%lamp 0ms

Photobleaching of Fluorescein at 100% Lamp Power

4400

4200

4000

Inte

nstiy

, cnt

s

10008006004002000frame

3850

3825

3800

Lamp: 100%

100% causes fairly severe photobleaching.

10x, 3gain, 1uM fluorescein, 1000images, 4min 21sec

Photobleaching of Fluorescein at 50% Lamp Power

4200

4000

3800

Inte

nstiy

, cnt

s

8006004002000frame

3675

3670

3665

3660

Lamp: 50%

Photobleaching is still considerable at 50% lamp power.

10x, 3gain, 1uM fluorescein, 1000images, 4min 21sec

Photobleaching of Fluorescein at 25% Lamp Power

4200

4100

4000

3900

Inte

nstiy

, cnt

s

10008006004002000frame

3596

3592

3588

3584

Lamp: 25%

Photobleaching occurs even at 25%.

10x, 3gain, 1uM fluorescein, 1000images, 4min 21sec

Photobleaching of Fluorescein at 12% Lamp Power

3940

3920

3900

3880

3860

Inte

nstiy

, cn

ts

10008006004002000frame

3564

3560

3556

3552

Lamp: 12%

10x, 3gain, 1uM fluorescein, 1000images, 4min 21sec

12% lamp power is the only one that does not demonstrate significant photobleaching.

Photobleaching of Fluorescein at various lamp powers

For each power: 10x, 3gain, 1uM fluorescein, 1000images, 4min 21sec

4400

4200

4000

3800

3600

Inte

nsi

ty

10008006004002000 Image

lamp12%_signal lamp12%_background lamp25%_signal lamp25%_background lamp50%_signal lamp50%_background lamp100%_signal lamp100%_background

Substrate Comparison

1420

1400

1380

1360

1340

1320

1300

1280

86420

'Mean fused silica' 'Mean glass'

154

152

150

148

146

144

142

140

86420

'StdDev fused silica' 'StdDev glass'

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.

Further Comparison of Substrates

substrate average mean of signal average stddev

fused silica 1406.4 153.3

glass 1429.0 152.8

Magnitude of% difference 1.582% 0.347%

Fused silica is slightly superior in mean and slightly inferior in stddev, but the difference is insignificant.

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.

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.

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.

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.