Instrument Performance --Tests

• Field illumination (plastic slides, beads)

• Axial resolution (mirror)

• Chromatic aberration (1 u beads, mirror)

• Laser power (meter, test sample)

• Laser stability (plastic slide, transmission detector)

• Beam alignment (focal check,mirror,bead)

UV Laser Stability

100.0

120.0

140.0

160.0

180.0

200.0

0:00 0:15 0:30 0:45 1:00 1:15 1:30 1:45 2:00 2:15

time (hr:min)

inte

nsity

60s intervals, 121 sections

32 min15 min

Deviation + 6%

WARM-UP

UV Laser Stability Thermal Effects

140.0

145.0

150.0

155.0

160.0

165.0

0:00 0:15 0:30 0:45 1:00 1:15 1:30 1:45 2:00

time (hr:min)

inte

nsi

ty

30s intervals, 225 Deviation + 5%

UV Laser Stability

125.0

130.0

135.0

140.0

145.0

150.0

155.0

160.0

0:00 0:15 0:30 0:45 1:00 1:15 1:30 1:45 2:00 2:15

time (hr:min)

inte

nsit

y

Deviation + 8%

UV Laser Stability

100.0

105.0

110.0

115.0

120.0

0:00 0:15 0:30 0:45

time (hr:min)

inte

nsity

10s intervals, 225 sections Deviation + 4%

UV Laser Stability

100.0

120.0

140.0

160.0

180.0

200.0

0:00 0:15 0:30 0:45 1:00 1:15 1:30 1:45

time (hr:min)

inte

nsity

60s intervals, 100 sectionsDeviation + 20%

Laser Stability (568nm)

100.0

120.0

140.0

160.0

180.0

200.0

0:00 0:15 0:30 0:45 1:00 1:15 1:30 1:45 2:00 2:15 2:30

time (hr:min)

inte

ns

ity

30s intervals, 225 sections Deviation + 17%

Laser Stability (Visible)

120.0

140.0

160.0

180.0

200.0

220.0

0:00 0:15 0:30 0:45 1:00 1:15 1:30 1:45 2:00 2:15 2:30

time (hr:min)

inte

ns

ity

488 nm

568 nm

647 nm

647

568

488

Deviation = +10%, +17%, +13%

UV Field Illumination

100X Objective (1.4 NA)

12.5% intervals

INTENSITY=230

123

47

5X Objective

(.25 NA)

12.5% intervals

20X Objective

(.7 NA)

12.5% intervals

Axial Registration

Mirror Test63X objective (1.2NA)

(Chromatic Aberration)

Axial Registration

0

50

100

150

200

250

0 1 2 3 4

x-z resolution (mm)

inte

nsi

ty

488 nm

568 nm

647 nm

63x ( 1.2NA) objective

647

488

568

Axial Resolution40X Objective (1.0 NA)

0

50

100

150

200

250

0 2 4 6 8x-z resolution (mm)

inte

nsi

ty

488 nm

568 nm

647 nm647568

488

Bad Galvanometer

568 nm

488 nm + 568nm

488 nm + 568nm

568 nm

Test Samples

• Beads: Spherotech 10 micron, MP focal check, 1 micron, submicron

• Front silvered mirror

• Silicon chip

• Biological samples-Diatoms, Pollen, MP Fluo slide, User defined specimen

• Fluorescence Plastic Slide

A B C

D E F

C

• Lower NA’s --Less resolution

• Longer working distance

• Cleaner working environment

Dry Objectives

Bleaching

0.0

50.0

100.0

150.0

200.0

250.0

0 50 100 150 200

number of scans

inte

ns

ity

Transmitted

Fluorescence - TRITC

180.0

190.0

200.0

210.0

0:00 0:15 0:30 0:45

time (hr:min)

inte

nsi

tyThick Fluorescent Slide

488

568