Live Cell Imaging Applications in Confocal Microscopy

Introduction to Confocal Microscopy and Image Analysis

Reference: J.Paul Robinson, Pawley Introduction to Confocal Microscopy,

UPDATED April 2003

Slide 1 t:/classes/BMS524/lectures2000/524lec11.ppt

Applications
Organelle Structure Probe ratioing Conjugated antibodies DNA/RNA Cytochemical Identification Oxidative Metabolism Exotic Applications

Slide 2 t:/classes/BMS524/lectures2000/524lec11.ppt

Step 1: Cell
Culture

top view
1 2 4 6 8
Below: the culture dishes for live cell imaging using a confocal microscope and high NA objectives.
Lab-Tek

Step 2: Cell
Wash

3 5

Step 3: Transfer to LabTek plates

7
170 M coverslip

side view Step 4: Addition of DCFHDA, Indo-1, or HE

stimulant/inhibitor added 37o heated stage

oil immersion objective

confocal microscope

Slide 3 t:/classes/BMS524/lectures2000/524lec11.ppt

Fluorescence Microscope image of Hoechst stained cells (plus DIC) Image collected with a 470T Optronics cooled camera
Slide 4 t:/classes/BMS524/lectures2000/524lec11.ppt

Measurement of DNA
G0-G1 # of Events S G2-M

Use for DNA content and cell viability

33342 for viability

Fluorescence Intensity

Less needed to stain for DNA content than for viability

decrease nonspecific fluorescence

Low laser power decreases CVs

Slide 5 t:/classes/BMS524/lectures2000/524lec11.ppt

Calcium Flux
Flow Cytometry
Ratio: intensity of 460nm / 405nm signals
0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 0 50
1000

Image Cytometry

RATIO [short/long]

200

400

600

800

Stimulation
0

36

72

Time (Seconds)

108

144

180

Time (seconds)
100 150 200

Slide 6 t:/classes/BMS524/lectures2000/524lec11.ppt

Oxidative Reactions
Superoxide Hydrogen Peroxide Glutathione levels Nitric Oxide Hydroethidine Dichlorofluorescein Monobromobimane Dichlorofluorescein

Slide 7 t:/classes/BMS524/lectures2000/524lec11.ppt

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)

Slide 8 t:/classes/BMS524/lectures2000/524lec11.ppt

Caged Photoactivatable Probes

Principle: Nitrophenyl blocking groups e.g. nitrophenyl ethyl ester undergoes photolysis upon exposure to UV light at 340-350 nm

Available Probes
Ca++: Nitr-5 Ca++ - buffering: Diazo-2 IP3 cAMP cGMP ATP ATP--S

Slide 9 t:/classes/BMS524/lectures2000/524lec11.ppt

Release of Caged Compounds

UV Beam

Culture dish Release of Cage

Slide 10 t:/classes/BMS524/lectures2000/524lec11.ppt

18.2 Calcium Regulation

Slide 11 t:/classes/BMS524/lectures2000/524lec11.ppt

Slide 12 t:/classes/BMS524/lectures2000/524lec11.ppt

Slide 13 t:/classes/BMS524/lectures2000/524lec11.ppt

Slide 14 t:/classes/BMS524/lectures2000/524lec11.ppt

Release of Caged Compounds

UV excited

D
250 200 150 100 50 0 CONTROL STUDY

C
Control Region
Fluorescence Emission at 515 nm Release of Caged Nitric Oxide in Attached PMN 250 200 150 100 50 0
0 20 40 60 80 100 120 140 160

Fluorescence Emission at 515 nm

100 200 300

400

Time (seconds) CONTROL

Time (seconds) after UV FLASH

Slide 15 t:/classes/BMS524/lectures2000/524lec11.ppt

Slide 16 t:/classes/BMS524/lectures2000/524lec11.ppt

Slide 17 t:/classes/BMS524/lectures2000/524lec11.ppt

Slide 18 t:/classes/BMS524/lectures2000/524lec11.ppt

Slide 19 t:/classes/BMS524/lectures2000/524lec11.ppt