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Live Cell Imaging Applications in Confocal Microscopy
“Introduction to Confocal Microscopy and Image Analysis”
UPDATED April 2003
Reference: J.Paul Robinson, Pawley “Introduction to Confocal Microscopy”,
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Applications
• Organelle Structure
• Probe ratioing
• Conjugated antibodies
• DNA/RNA
• Cytochemical Identification
• Oxidative Metabolism
• Exotic Applications
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Step 1: Cell Culture
Step 2: Cell Wash
Lab-Tek
1 2
3 4
5 6
7 8
top view
side view
170 µM coverslip
Step 3: Transfer to Lab-Tek plates
confocal microscopeoilimmersionobjective
37o heatedstage
stimulant/inhibitoradded
Step 4: Addition of DCFH-DA, Indo-1, or HE
Below: the culture dishes for live cell imaging using a confocal microscope and high NA objectives.
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Fluorescence Microscope image of Hoechst stained cells (plus DIC)Image collected with a 470T Optronics cooled camera
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• Use for DNA content and cell viability– 33342 for viability
• Less needed to stain for DNA content than for viability– decrease nonspecific fluorescence
• Low laser power decreases CVs
Measurement of DNA
G0-G1S
G2-M
Fluorescence Intensity
# of
Eve
nts
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Calcium Flux
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0 50 100 150 200
Rat
io: i
nten
sity
of 4
60nm
/ 40
5nm
sig
nals
Time (seconds)Time (Seconds)0 36 72 108 144 180
RA
TIO
[sho
rt/lo
ng]
0 2
00 4
00 6
00 8
0010
00
StimulationStimulation
Flow Cytometry Image Cytometry
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Oxidative Reactions
• Superoxide Hydroethidine
• Hydrogen Peroxide Dichlorofluorescein
• Glutathione levels Monobromobimane
• Nitric Oxide Dichlorofluorescein
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Exotic Applications of Confocal Microscopy
• FRAP (Fluorescence Recovery After Photobleaching)
• Release of “Caged” compounds
• Lipid Peroxidation (Parinaric Acid) Difficult to do with confocal, but possible with 2P (excitation is 325 nm)
• Membrane Fluidity (DPH)
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“Caged” Photoactivatable Probes
• Ca++: Nitr-5
• Ca++ - buffering: Diazo-2
• IP3
• cAMP
• cGMP
• ATP
• ATP-γ-S
Available Probes
Principle: Nitrophenyl blocking groups e.g. nitrophenyl ethyl ester undergoes photolysis upon exposure to UV light at 340-350 nm
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Release of “Caged” Compounds
UV Beam
Release of “Cage”
Culture dish
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18.2 Calcium Regulation
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Time (seconds) after UV FLASH
Release of Caged Nitric Oxide inAttached PMN
0
50
100
150
200
250
0 20 40 60 80 100 120 140 160Fluo
resc
ence
Em
issi
on a
t 515
nm
Release of Caged Compounds
CDUV excited
Control Region
Time (seconds) CONTROL
0
50
100
150
200
250 CONTROL STUDY
Flu
ores
cenc
e E
mis
sion
at 5
15 n
m
0 100 200 300 400
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