Download New approaches for live cell imaging

New approaches for live cell imaging
Invitrogen Corporation, Eugene OR.
REAL POWER IS IN
LIVE CELL IMAGING
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Considerations for Live Cell Labeling
• Cell Loading Methods.
How to get stuff into cells.
• Challenges to contrast.
Sources of Background.
Autofluorescence.
• Multicolor Labeling.
More labels, more information.
• Photobleaching.
The eternal challenge.
• Phototoxicity.
Killing with light.
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Cell Loading Methods
• Chemical Permeability Characteristics
– Direct permeability
– Membrane permeant esters (AM esters)
– Loading assistance
ƒ NEW PowerLoad™ solution (P10020)
ƒ Pulronic F127 10% in DMSO (P6866)
• Active Cellular Uptake
– ATP-gated cation channels
– Endocytic uptake
– Peptide-mediated uptake (poly Arg, TAT).
• Mechanical permeabilization
– Microinjection
– Whole-cell patch pipet delivery
– Electroporation
– Osmotic permeabilization
– Ballistic microprojectile (“gene gun”) delivery
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Direct Permeability Dyes
LIVE/DEAD® BacLight™ Bacterial Viability Assay
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Permeable AM Ester Uptake
O
O
O
H N
2
O
N(CH COCH OCCH )
2
2
32
CH
O
2
OCCH
3
(CH COCH OCCH ) N
3
2
22
O
CH
2
CH CO
O
3
calcein AM
nonpolar
nonfluorescent
O
+
( OCCH ) NH
22
CH
2
O
O
esterase
O
O
O
+
NH(CH CO )
2
2
CH
2
O
calcein
polar
green
C O fluorescent
O
NH
2
H N
2
NH
2
+N
N+
+
+
(CH ) NH CH CH NH (CH )
23 2 2 2 2 23
ethidium homodimer-1(EthD-1)
membrane impermeant
red fluorescence enhanced on DNA binding
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Calcein AM/EthD-1 staining of tissue
3-D rendition of
rat lung stained
with calcein AM
(live cells) and
EthD-1 (dead
cells). Lance
Rodenkirch,
University of
Wisconsin
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Cell Loading Caveats
• Label the specimen to the minimum extent
required to obtain the biological
information that is sought.
• The following deleterious effects are all
positively correlated with increased label
concentration:
– phototoxicity,
– cytotoxicity,
– nonspecific localization,
– physiological or structural perturbation.
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Challenges to Contrast.
Sources of Background.
• Instrument
– Stray light, detector noise.
• Reagent
– Unbound or nonspecifically bound probes
– Leaking probe.
• Sample
– Solute and solvent autofluorescence,
– scattered excitation light (particle size and
wavelength dependent)
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Multicolor Image Considerations
• At the specimen level, the composition of
the image is primarily determined by four
factors:
1. Relative abundance of the targets
2. Fluorescence output per dye (extinction
coefficient, quantum yield, photobleaching
rate)
3. Spectral separation of dyes (minimum
overspill)
4. Specificity of dye localization
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Photobleaching and phototoxicity.
• Photobleaching: Irreversible destruction of excited
fluorophore by free radicals generated during
excitation.
• Phototoxicity: Cellular damage caused by the
action of light on biomolecules or indirectly by
radicals generated by fluorescent dyes.
• Proportional to time-integrated excitation intensity.
• Avoidance:
– Minimize excitation,
– Maximize detection efficiency,
– Use inherently stable dyes,
– Antifade reagents.
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Photobleaching in Action
HeLa cells labeled with MitoTracker Red CMXRos: 40X followed by 10X
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Phototoxicity
• Most dyes can
induce
phototoxicity as a
byproduct of the
photobleaching
process.
• Phototoxicity
induced in BPAE
cells loaded with
JC-1.
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Antifade Reagents for Live Cells
6–hydroxy–2,5,7,8–tetramethylchroman–2–carboxylic acid
(aka Trolox®, registered trademark of Hoffman–La Roche)
HO
HC
3
CH
3
O
O
CH
3
C OH
CH
3
•
•
•
Antifade compatible with
live specimens
Water-soluble
Working concentration
~100 µM
• Various other antioxidant or biocompatible
reducing agents maybe effective
• Vitamins E, B, C
• Reducing sugars, Glucose
• Typically require very high concentrations
to be effective.
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Guidelines for Live Cell Imaging
1. Ensure the cells/tissues are incubated
under ideal conditions.
2. Use the minimum amount of reagent to
achieve staining.
3. Use the lowest level of excitation light
possible.
4. Don’t unnecessarily expose cells to
excitation light, particularly once loaded
with dyes.
5. Maximize detector sensitivity before
increasing excitation light.
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Cell Structure Labeling
• Organelle Lights™
• Cellular Lights™
• Classic Organelle Stains
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New probes for cutting edge imaging
• Organelle Lights™ and Cellular Lights ™ Targeted FP’s for dissecting cell biology.
• Premo™ line of genetically encoded biosensors.
• Intracellular Ion Indicators.
• pHrodo ™ tools for endocytosis/ phagoctyosis.
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Baculovirus mediated gene delivery……
Targeted fluorescent proteins + BacMam
Organelle LightsTM
Cellular LightsTM
PremoTM sensors
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Organelle Lights™
Organelle Lights™
CFP
GFP
YFP
OFP
RFP
(440/480)
(488/510)
(514/528)
(548/565)
(555/584)
Cytoplasm
O36227
Endoplasmic
Reticulum
O36212
Endosomes
O10104
Golgi
O36215
O36223
O36224
Lysosomes
O10100
Mitochondria
O36210
Nuclear Envelope
O36213
Nucleus
O36218
Peroxisome
Plasma Membrane
Synaptophysin
Null virus (control)
O10098
O36209
O36222
O36219
O36211
O36216
O36214
O36220
O10099
O36225
O36217
O36226
C10080
C10130
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Organelle Lights™ Organelle Markers
BacMam virus delivered expression of organelle targeted FPs
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Cellular Lights™
Cellular Lights™
CFP
GFP
YFP
OFP
RFP
(440/480)
(488/510)
(514/528)
(548/565)
(555/584)
Actin
C10126
CFS1r
C10078
Exo1
C10079
Histones
C10128
MAP4
C10105
Tubulin
C10106
Null virus (control)
C10127
C10129
C10112
C10130
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Imaging cell division with Cellular Lights™
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Membrane Trafficking
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Real time imaging of cytoskeleton disruption
Delta Vision core imaging system.
Image acquisition every 20s for 30 minutes
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A snapshot of our intracellular targets…….
SV40 nuclear localization
sequence (N- or C- terminus)
Nesprin 1alpha C-terminal
transmembrane domain (aa 923-982)
Nuclear envelope
Histones
H2B
Tubulin monomer
MAP4
human Golgi-resident enzyme
N-acetylgalactosaminyltransferase-2
LAMP
Exocyst
Exoc1 (Sec3p homologue)
Signal sequence of Calreticulin
KDEL
Synapse
Synaptophysin
leader sequence of E1alpha
pyruvate dehydrogenase
Nuclear export sequence
(N- or C- terminus)
Actin monomer
Peroxisomal C-terminal
targeting sequence
Cell surface
CSF1r (colony stimulating factor
1 receptor, cell surface marker)
Endosome
Rab5
Myristoylation/palmitoylation
sequence from Lck tyrosine kinase
Organelle lights ™ and Cellular Lights™
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Organelles Stains and Dyes
• There are a wide variety of fluorescent dyes that stain specific
intracellular structures in living cells based on their chemical
nature.
• Examples include…
– Mitotrackers for mitochondria.
– Nucleic acid stains for nuclei.
– Lysotrackers and Lysosensors for acidic compartments.
– Lipophilic stains for membranes.
– ER Tracker for Endoplasmic reticulum.
– Ceramide conjugates for Golgi apparatus.
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Mitochondrial Stains
• MitoTracker (and MitoFluor)
– M7514 MitoTracker® Green FM
– M7511 MitoTracker® Orange CM-H2TMRos
– M7510 MitoTracker® Orange CMTMRos
– M22425 MitoTracker® Red 580
– M7513 MitoTracker® Red CM-H2XRos
– M7512 MitoTracker® Red CMXRos
– M22426 MitoTracker® Deep Red 633
– MitoTracker® Red CMXRos
ƒ Ex/Em = 579/599 nm
ƒ Potential dependent
ƒ Aldehyde fixable
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Live Cell Organelle Labeling
Live BPAE cells incubated simultaneously with MitoTracker® Deep Red FM,
LysoTracker® Green DND-26, and Hoechst 33342
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Endoplasmic Reticulum
• E34251 ER-Tracker™ Green
• E34250 ER-Tracker™ Red
– Conjugates of drug
Gilbencalmide
– Withstand fixation but not
heavy permeabilization
• D273 DiOC6(3)
– Used in live and
glutaraldehyde fixed
– Not all cell types
(concentration-dependent
staining)
– Very phototoxic.
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Golgi Apparatus
•
N1154 NBD-ceramide
•
D3521 BODIPY® FL ceramide
•
D7540 BODIPY® TR ceramide
– Live and aldehyde fixed
cells
– Not retained through
fixation and
permeabilization
BODIPY® FL Ceramide
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Lysosome
• LysoTracker
– L7525
– L12490
– L7526
– L7528
– L12491
LysoTracker®
LysoTracker®
LysoTracker®
LysoTracker®
LysoTracker®
Blue DND-22
Blue-White DPX
Green DND-26
Red DND-99
Yellow HCK-123
– Live cell; not fixable
• LysoSensor
– Live cell; not fixable
– pH indicators
L7528 LysoTracker® Red DND-99
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Lysotracker Red in Live Cells
Lysotracker® Red DND-99 and Hoechst 33342
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Nucleic Acid Stains
• Nucleic acid stains are not specific for only one type of nucleic
acid. Depending on the staining conditions used some
cytoplasmic staining may be observed.
• There are a wide range of nucleic acid stains but only a select
few can enter live cells due to the plasma membrane
polarization.
• Examples
are…
D1306 DAPI
A1301 Acridine orange
H1399 Hoechst 33342
S7575 SYTO® 13
S7578 SYTO® 16
Structures not disclosed.
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Live Cell Nucleic Acid Labeling
SYTO 13 Live-Cell Nucleic Acid Stain
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Membrane Stains
•
Dialkylcarbocyanine and
dialkylaminostyryl probes
– D282 DiI,
– D275 DiO,
– D307 DiD
– T3163 FM® 1-43
– F35355 FM® 1-43 Fixable
– T3168 FM® 4-64
– F34653 FM® 4-64 Fixable
•
Fatty Acid analogs, phospholipids,
sphingolipids, etc
– Fatty acid analogs (label in the
carbon chain)
– Phospholipids labeled on the head
group.
FM 1-43
DiI
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Studying internalization using pHrodo™ indicators
• pHrodo™-labeled S. aureus or E.coli Bioparticles® conjugates for
researchers studying phagocytosis
• pHrodo™ SE – the amine-reactive form, lets the users label the
biomolecule of interest (microorganism, antibody, protein)
MMM Cells: pHrodo™
MMM Cells: Calcein AM & pHrodo™
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Phagocytosis Assays using pHrodo™ Particles.
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pHrodo™ particles for phagocytosis
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Fluorescent Ion Indicators
• Calcium
– NEW – Rhod-3 red calcium indicator.
– Fura-2, Indo-1, Fluo-4, Fluo-3, Calcium Green-1, Rhod-2
• Magnesium
– Mag-fura-2, Mag-indo-1, Magnesium Green
• Sodium
– SBFI, CoroNa Green, CoroNa Red, Sodium Green
• Potassium
– PBFI
• Chloride
– MEQ, SPQ
• H+ (pH)
– BCECF, Fluorescein, SNARF-1, Oregon Green 488
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How ion Indicators Work
Ion-dependent
spectral shift
Examples
•Fura-2
•Indo-1
•Premo Cameleon
Ion-dependent
fluorescence increase,
no spectral shift
Dye
Dye
Ca2+
Ca2+
Examples
•Fluo-3/Fluo-4
•Rhod-2
•Calcium Green
•Oregon Green
488 BAPTA
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What’s a Biosensor?
• A biological element
(protein) that has been
rendered sensitive to an
external signal
– Naturally occurring (e.g.
light-sensitive pigments in
the eye)
– Constructed (e.g. blood
glucose biosensor)
• Can be targeted to cell
type or organelle
• Based on naturally
occurring fluorescent
signal proteins (e.g. GFP)
and “detector” proteins
(e.g. calmodulin)
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Biosensors: Premo™ Cameleon calcium indicators
Adult stem cell from left atrial appendage of pig heart stimulated with 20 uM ATP
Image obtained in collaboration with Drs. Michael Rutten and Kenton Gregory, Oregon Medical Laser
Center Bioimaging Suite, Providence St. Vincent Hospital, Portland, Oregon
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THANK YOU!
Questions?
[email protected]
800.955.6288
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