Live Cell Imaging Applications in Confocal
Microscopy
BMS 524 - “Introduction to Confocal Microscopy and Image Analysis”
1Credit course offered by Purdue University Department of Basic Medical Sciences, School of
Veterinary Medicine
J.Paul Robinson, Ph.D.
Professor of Immunopharmacology
Director, Purdue University Cytometry Laboratories
These slides are intended for use in a lecture series. Copies of the graphics are distributed and students encouraged to take their notes on these
graphics. The intent is to have the student NOT try to reproduce the figures, but to LISTEN and UNDERSTAND the material. All material
copyright J.Paul Robinson unless otherwise stated, however, the material may be freely used for lectures, tutorials and workshops. It may not
be used for any commercial purpose.
The text for this course is Pawley “Introduction to Confocal Microscopy”, Plenum Press, 2nd Ed. A number of
the ideas and figures in these lecture notes are taken from this text.
© 1993-2004 J. Paul Robinson, Purdue University Cytometry UPDATED
Laboratories
April 2003
Slide 1 t:/classes/BMS524/lectures2000/524lec11.ppt
Applications
• Organelle Structure & Function
– Mitochondria (Rhodamine 123)
– Golgi (C6-NBD-Ceramide)
– Actin (NBD-Phaloidin)
– Lipid (DPH)
© 1993-2004 J. Paul Robinson, Purdue University Cytometry Laboratories
Slide 3 t:/classes/BMS524/lectures2000/524lec11.ppt
top view
Step 1: Cell
Culture
Step 2: Cell
Wash
Step 3: Transfer to LabTek plates
side
view
Step 4: Addition of DCFHDA, Indo-1, or HE
1
2
3
4
5
6
7
8
Below: the culture
dishes for live cell
imaging using a
confocal
microscope and
high NA
objectives.
170 M coverslip
stimulant/inhibitor
added 37o heated
stage
oil
immersion
objective
confocal microscope
© 1993-2004 J. Paul Robinson, Purdue University Cytometry Laboratories
Slide 4 t:/classes/BMS524/lectures2000/524lec11.ppt
Confocal System
Culture System
Photos taken in Purdue University Cytometry Labs
Photo taken from Nikon promotion material
© 1993-2004 J. Paul Robinson, Purdue University Cytometry Laboratories
Slide 5 t:/classes/BMS524/lectures2000/524lec11.ppt
Example of DIC and Fluorescnece
Giardia (DIC image)
(no fluorescence)
Human cheek epithelial cells (from JPR!) stained
with Hoechst 33342 - wet prep, 20 x objective, 3 x
zoom (Bio-Rad 1024 MRC)
© 1993-2004 J. Paul Robinson, Purdue University Cytometry Laboratories
(photo taken from a 35 mm
slide and scanned - cells were
live when photographed)
Slide 6 t:/classes/BMS524/lectures2000/524lec11.ppt
Fluorescence Microscope image of Hoechst stained cells (plus DIC)
Image collected with a 470T Optronics cooled camera
© 1993-2004 J. Paul Robinson, Purdue University Cytometry Laboratories
Slide 7 t:/classes/BMS524/lectures2000/524lec11.ppt
Measurement of DNA
# of Events
G0-G1
G2-M
S
Fluorescence Intensity
• 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
© 1993-2004 J. Paul Robinson, Purdue University Cytometry Laboratories
Slide 8 t:/classes/BMS524/lectures2000/524lec11.ppt
PI - Cell Viability
How the assay works:
• PI cannot normally cross the cell membrane
• If the PI penetrates the cell membrane, it is assumed to be damaged
• Cells that are brightly fluorescent with the PI are damaged or dead
Viable Cell
Damaged Cell
PI
PI
PI
PI
PI
PI
PI
PI
PI
PI
PI
PI
PI
© 1993-2004 J. Paul Robinson, Purdue University Cytometry Laboratories
PI
Slide 9 t:/classes/BMS524/lectures2000/524lec11.ppt
104
Fluorescence
Transmission
102
oocysts
Flow cytometric scatter plot of
gamma irradiated C.
parvum oocysts. The oocysts region is clearly
distinguished from ghosts and debris. Images on the right
show Sytox green fluorescence and transmission images of
these regions. Note ghosts do not take up Sytox green dye.
ghosts
10
1
Side Scatter
103
Flow Cytometry Dot Plot
10
0
debris
Forward Scatter
100
101
102
103
104
Green Fluorescence
© 1993-2004 J. Paul Robinson, Purdue University Cytometry Laboratories
Slide 10 t:/classes/BMS524/lectures2000/524lec11.ppt
Specific Organelle Probes
Probe
BODIPY
NBD
DPH
TMA-DPH
Rhodamine 123
DiO
diI-Cn-(5)
diO-Cn-(3)
Site
Excitation
Golgi
505
Golgi
488
Lipid
350
Lipid
350
Mitochondria 488
Lipid
488
Lipid
550
Lipid
488
Emission
511
525
420
420
525
500
565
500
BODIPY - borate-dipyrromethene complexes
NBD - nitrobenzoxadiazole
DPH - diphenylhexatriene
TMA - trimethylammoniumSlide 11 t:/classes/BMS524/lectures2000/524lec11.ppt
© 1993-2004 J. Paul Robinson, Purdue University Cytometry Laboratories
Organelle Function
•
•
•
•
Mitochondria
Endosomes
Golgi
Endoplasmic Reticulum
© 1993-2004 J. Paul Robinson, Purdue University Cytometry Laboratories
Rhodamine 123
Ceramides
BODIPY-Ceramide
DiOC6(3) Carbocyanine
Slide 12 t:/classes/BMS524/lectures2000/524lec11.ppt
Calcium Related Applications
• Probe Ratioing
– Calcium Flux (Indo-1)
– pH indicators (BCECF, SNARF)
Molecule-probe
Excitation
Emission
Calcium - Indo-1
Calcium- Fluo-3
Calcium - Fura-2
Calcium - Calcium Green
Magnesium - Mag-Indo-1
Phospholipase A- Acyl Pyrene
351 nm
488 nm
363 nm
488 nm
351 nm
351 nm
405, >460 nm
525 nm
>500 nm
515 nm
405, >460 nm
405, >460 nm
© 1993-2004 J. Paul Robinson, Purdue University Cytometry Laboratories
Slide 13 t:/classes/BMS524/lectures2000/524lec11.ppt
Probes for Ions
•
•
•
•
INDO-1
QUIN-2
Fluo-3
Fura -2
Ex350
Ex350
Ex488
Ex330/360
© 1993-2004 J. Paul Robinson, Purdue University Cytometry Laboratories
Em405/480
Em490
Em525
Em510
Slide 14 t:/classes/BMS524/lectures2000/524lec11.ppt
Ionic Flux Determinations
• Calcium
• Intracellular pH
Indo-1
BCECF
How the assay works:
• Fluorescent probes such as Indo-1 are able to bind to calcium in
a ratiometric manner
• The emission wavelength decreases as the probe binds available
calcium
600
400
200
RATIO [short/long]
800
1000
Ratio: intensity of 460nm / 405nm signals
0.8
0
Stimulation
0
36
72
108
Time (Seconds)
144
180
Flow Cytometry
0.7
0.6
0.5
0.4
0.3
0.2
0.1
Time (seconds)
0
0
Image Analysis
© 1993-2004 J. Paul Robinson, Purdue University Cytometry Laboratories
50
100
150
200
Slide 15 t:/classes/BMS524/lectures2000/524lec11.ppt
Calcium Flux
Flow Cytometry
Image Cytometry
Ratio: intensity of 460nm / 405nm signals
1000
0.8
0.7
800
0.6
600
0.5
400
0.4
0.3
200
0.2
0
Stimulation
0
36
72
108
Time (Seconds)
144
0.1
180
Time (seconds)
0
0
© 1993-2004 J. Paul Robinson, Purdue University Cytometry Laboratories
50
100
150
200
Slide 16 t:/classes/BMS524/lectures2000/524lec11.ppt
Oxidative Reactions
•
•
•
•
Superoxide
Hydrogen Peroxide
Glutathione levels
Nitric Oxide
© 1993-2004 J. Paul Robinson, Purdue University Cytometry Laboratories
Hydroethidine
Dichlorofluorescein
Monobromobimane
Dichlorofluorescein
Slide 17 t:/classes/BMS524/lectures2000/524lec11.ppt
DCFH-DA
DCFH
DCF
2’,7’-dichlorofluorescin diacetate
O
O
CH3-C-O
O
O-C-CH3
Cl
2’,7’-dichlorofluorescin
Cl
H
COOH
O
HO
Cellular Esterases
OH
Cl
Fluorescent
Cl
H
COOH
Hydrolysis
2’,7’-dichlorofluorescein
O
HO
O
H 2O 2
Cl
Oxidation
DCFH-DA
Cl
H
COOH
Neutrophils
DCFH-DA
8
0
Monocytes
H 2O 2
counts
DCFH
60
DCF
PMA-stimulated PMN
Control
40
20
Lymphocytes
0
.
1
© 1993-2004 J. Paul Robinson, Purdue University Cytometry Laboratories
1
100
log FITC 10
Fluorescence
100
0
Slide 18 t:/classes/BMS524/lectures2000/524lec11.ppt
Hydroethidine
HE
EB
H2N
NH2
H
N
O2-
H2N
NH2
N + Br
CH2CH3
-
CH2CH3
Phagocytic Vacuole
NADPH Oxidase
NADPH
O2
HE
O2-
NADP
SOD
O2H2O2
DCF
H2O2
DCF
OH-
Example: Neutrophil Oxidative Burst
© 1993-2004 J. Paul Robinson, Purdue University Cytometry Laboratories
Slide 19 t:/classes/BMS524/lectures2000/524lec11.ppt
Macrovascular Endothelial Cells
in Culture
0
Time (minutes)
© 1993-2004 J. Paul Robinson, Purdue University Cytometry Laboratories
60
Slide 20 t:/classes/BMS524/lectures2000/524lec11.ppt
Hydrogen peroxide measurements with DCFH-DA
1
2
3
4
Change in fluorescence was measured
using Bio-Rad software and the data
exported to a spread sheet for analysis.
5
525 nm
Step 7B: Export data from Excel data
base to Delta Graph
% change (DCF fluorescence)
Step 6B: Export data from measured
regions to Microsoft Excel
2000
1800
1600
1400
1200
1000
800
600
400
200
0
cell 1
cell 2
cell 3
cell 4
cell 5
0
500
© 1993-2004 J. Paul Robinson, Purdue University Cytometry Laboratories
1000 1500 2000 2500 3000
Time in seconds
Slide 21 t:/classes/BMS524/lectures2000/524lec11.ppt
Superoxide measured with hydroethidine
cell 1
Change in fluorescence was measured
using Bio-Rad software and the data
exported to a spread sheet for analysis.
cell 3
cell 4
cell 2
Export data from measured
regions to Microsoft Excel
Export data from Excel data
base to Delta Graph
%change (DCF fluorescence)
cell 5
1800
1600
1400
1200
1000
800
600
400
cell 1
cell 2
cell 3
cell 4
200
0
cell 5
-200
200 400
600 800
1000 1200 1400 1600 1800
Time in seconds
© 1993-2004 J. Paul Robinson, Purdue University Cytometry Laboratories
Slide 22 t:/classes/BMS524/lectures2000/524lec11.ppt
H2O2 stimulation and DCF & EB loading in Rat
Pulmonary Artery Endothelial Cells
24 treatments - 5000 cells each
200
ENDO HBSS
ENDO HBSS TNFa
ENDO L-arg
ENDO/ L-arg TNFa
ENDO/ D-arg
ENDO/ D-arg TNFa
Endo + 200uM H2O2
Endo + 200uM H2O2
Endo + 200uM H2O2
Endo / TNFa + 200uM H2O2
Endo / TNFa + 200uM H2O2
Endo / TNFa + 200uM H2O2
Confocal System Fluorescence
Measurements
.
180
160
DCF Fluorescence
Mean EB Fluorescence
Endo / L-arg + 200uM H2O2
Endo / L-arg + 200uM H2O2
Endo / L-arg + 200uM H2O2
Endo / L-arg TNFa + 200uM H2O2
Endo / L-arg TNFa + 200uM H2O2
Endo / L-arg TNFa + 200uM H2O2
Endo / D-arg + 200uM H2O2
Endo / D-arg + 200uM H2O2
Endo / D-arg + 200uM H2O2
Endo / D-arg TNFa + 200uM H2O2
Endo / D-arg TNFa + 200uM H2O2
Endo / D-arg TNFa + 200uM H2O2
140
120
100
80
60
40
20
200uM
H2O2
added
200uM
H2O2
added
0
0
20
40
60
80
100
120
140
© 1993-2004 J. Paul Robinson, Purdue University Cytometry Laboratories
Time (minutes)
Time (seconds)
Slide 23 t:/classes/BMS524/lectures2000/524lec11.ppt
pH Sensitive Indicators
Probe
Excitation
Emission
• SNARF-1
488
575
• BCECF
488
440/488
525/620
525
[2’,7’-bis-(carboxyethyl)-5,6-carboxyfluorescein]
© 1993-2004 J. Paul Robinson, Purdue University Cytometry Laboratories
Slide 24 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)
© 1993-2004 J. Paul Robinson, Purdue University Cytometry Laboratories
Slide 25 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
© 1993-2004 J. Paul Robinson, Purdue University Cytometry Laboratories
Slide 26 t:/classes/BMS524/lectures2000/524lec11.ppt
Release of “Caged” Compounds
UV Beam
Culture dish
Release of “Cage”
© 1993-2004 J. Paul Robinson, Purdue University Cytometry Laboratories
Slide 27 t:/classes/BMS524/lectures2000/524lec11.ppt
Release of Caged Compounds
UV excited
CONTROL STUDY
Fluorescence Emission at 515 nm
250
C
Control Region
200
150
100
50
0
0
100 200 300
400
Time (seconds) CONTROL
© 1993-2004 J. Paul Robinson, Purdue University Cytometry Laboratories
Fluorescence Emission at 515 nm
D
Release of Caged Nitric Oxide in
Attached PMN
250
200
150
100
50
0
0
20 40
60 80 100 120 140 160
Time (seconds) after UV FLASH
Slide 28 t:/classes/BMS524/lectures2000/524lec11.ppt
Membrane Polarization
• Polarization/fluidity
Diphenylhexatriene
How the assay works: The DPH partitions into liphophilic portions of the cell and is
excited by a polarized UV light source. Polarized emissions are collected and changes
can be observed kinetically as cells are activated.
An image showing
DPH fluorescence in
cultured endothelial
cells.
© 1993-2004 J. Paul Robinson, Purdue University Cytometry Laboratories
Slide 29 t:/classes/BMS524/lectures2000/524lec11.ppt
Calcium ratios with Indo-1
1
1
2
2
3
3
460 nm
405/35 nm
Changes in the fluorescence were measured
using the Bio-Rad calcium ratioing software.
The same region in each wave length was
measured and the relative change in each region
was recorded and exported to a spread sheet for
analysis..
Export data from measured regions to
Microsoft Excel
Ratio: intensity1 (460nm) / intensity2 (405/35nm)
0.8
Export data from Excel data
base to Delta Graph
cell 1
cell 2
cell 3
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
0
© 1993-2004 J. Paul Robinson, Purdue University Cytometry Laboratories
50
100
150
200
Slide 30 t:/classes/BMS524/lectures2000/524lec11.ppt
FRAP
Intense laser Beam
Bleaches Fluorescence
%F
Time
Recovery of fluorescence
Zero time
10 seconds
© 1993-2004 J. Paul Robinson, Purdue University Cytometry Laboratories
30 seconds
Slide 31 t:/classes/BMS524/lectures2000/524lec11.ppt
4D confocal microscopy
• Time vs 3D sections
• Used when evaluating kinetic changes in
tissue or cells
• Requires fast 3D sectioning
• Difficult to evaluate
© 1993-2004 J. Paul Robinson, Purdue University Cytometry Laboratories
Slide 32 t:/classes/BMS524/lectures2000/524lec11.ppt
4D Imaging
Time
1
2
3
4
5
This could also be achieved using an X-Z scan on a point scanner.
© 1993-2004 J. Paul Robinson, Purdue University Cytometry Laboratories
Slide 33 t:/classes/BMS524/lectures2000/524lec11.ppt
Imaging 3D ECM structures
• Mainly collagen based materials
• Usually 40-120 microns thick
• Require both transmitted and fluorescent
signals
• Often require significant image processing
to extract information
© 1993-2004 J. Paul Robinson, Purdue University Cytometry Laboratories
Slide 34 t:/classes/BMS524/lectures2000/524lec11.ppt
© 1993-2004 J. Paul Robinson, Purdue University Cytometry Laboratories
Slide 35 t:/classes/BMS524/lectures2000/524lec11.ppt
© 1993-2004 J. Paul Robinson, Purdue University Cytometry Laboratories
Slide 36 t:/classes/BMS524/lectures2000/524lec11.ppt
Thick Tissue - Bone and
Cartilage
• Very difficult to image thick
specimens
• Can use live specimens if
appropriately stained
• Special preparation
techniques
© 1993-2004 J. Paul Robinson, Purdue University Cytometry Laboratories
Slide 37 t:/classes/BMS524/lectures2000/524lec11.ppt
Lecture Summary
• Live cell applications are relatively common using
confocal microscopy
• Correct use of fluorescent probes necessary
• Temperature and atmosphere control may be
required
• Thick specimens often require advanced image
processing
• Exotic applications are potentially useful
• A limited window of time is available to image
live cells before cells deteriorate
© 1993-2004 J. Paul Robinson, Purdue University Cytometry Laboratories
Slide 38 t:/classes/BMS524/lectures2000/524lec11.ppt