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Live Cell Imaging
Toxicity
Phototoxicity
Compartmentalization
Compatibility
Interference (buffer / sink)
Temporal Constraints
Permeability
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Permeability
1. Endocytosis2. Injection / infusion3. Esterification
acetateacetoxymethyl (AM)
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Ion Selective Dyes
Calcium / Magnesium / Zinc ions
Protons (pH)
Sodium & Potassium ions
Chloride / Halide ions
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Ion Selective Dyes
5-carboxy-2’,7’-chlorofluorescein
Calcium Green (BAPTA)
Magnesium Green (APTRA)
Sodium Green (diaza crown ether)
1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid
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Ion Selective Dyes
Spectral compatibility
Stoichiometry
Specificity
Affinity (KD)
Dynamic range
Permeability
Optical response
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Optical Response
1. Change (+/-) in Quantum Yield with little change in spectra
e.g., fluo-3, Calcium Green, SPQ
2. Blue shift of absorption (excitation) spectrum with little change in maximum emission
e.g., fura-2, Fura Red, SBFI, PBFI, BCECF
3. Blue shift in both absorption and emission spectra
e.g., indo-1, SNARF & SNAFL
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Calcium Dyes - Issues
Qualitative vs quantitative (calibration)
KD for calcium
Compartmentalization
pH effects
Interaction with other ions
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Calcium Green
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Calcium Green
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Calcium Green
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Dynamic Range
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Calcium Green
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Fluo Dyes
Fluo 4
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Fluo-3
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Fluo Calcium Indicators
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Optical Response
1. Change (+/-) in Quantum Yield with little change in spectra
e.g., fluo-3, Calcium Green, SPQ
2. Blue shift of absorption (excitation) spectrum with little change in maximum emission
e.g., fura-2, Fura Red, SBFI, PBFI, BCECF
3. Blue shift in both absorption and emission spectra
e.g., indo-1, SNARF & SNAFL
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Fura-2 & Indo-1
Fura-2 Indo-1
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Fura-2
Isosbestic point
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Published by AAAS
H. Ma et al., Science 287, 1647 -1651 (2000)
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Published by AAAS
M. A. Takasu et al., Science 295, 491 -495 (2002)
No Caption Found
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Optical Response
1. Change (+/-) in Quantum Yield with little change in spectra
e.g., fluo-3, Calcium Green, SPQ
2. Blue shift of absorption (excitation) spectrum with little change in maximum emission
e.g., fura-2, Fura Red, SBFI, PBFI, BCECF
3. Blue shift in both absorption and emission spectra
e.g., indo-1, SNARF & SNAFL
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Fura-2 & Indo-1
Fura-2 Indo-1
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Calcium indicators - affinity
Table 20.8 Comparison of in vitro and in situ Kd values for fluo-3, fura-2 and indo-1.
Indicator Kd in vitro * Kd in situ Cell/Tissue Type
fluo-3 390 nM 2570 nM Frog skeletal muscle
fura-2 145 nM 371 nM U373-MG astrocytoma cell
fura-2 145 nM 350 nM Rabbit gastric gland
indo-1 230 nM 844 nM Rabbit cardiac myocyte
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Photo-activated “Caged” compounds
GABA
ATP
uncaging requires violet - UV irradiation
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Red calcium indicators
Rhod-2
X-rhod-1
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Rhod-2 & X-Rhod-1
X-Rhod-1 Rhod-2
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Red calcium indicators
Fura Red Calcium Orange
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Fura Red
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Fura-2
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More Furas
fura-4F fura-5F
fura-6F fura-FF
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Fura-2 C18
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Indo-1
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Other Ratiometric Dyes
Quin-2AM
benzothiaza-1
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Ion Selective Dyes
Calcium / Magnesium / Zinc ions
Protons (pH)
Sodium & Potassium ions
Chloride / Halide ions
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pH Sensitive DyesFluoroscein Diacetate (FDA)
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Protonation of FDA
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Fluorescein Diacetate
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Permeability of pH-sensitive dyes
1) calcein AM
2) BCECF AM
3) TFFDA
4) FDA
5) HFFDA
6) CFDA
7) CMFDA
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BCECF-AM
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BCECF-AM Fluorescence Spectrum
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BCECF-AM Spectra
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SNARF & SNAFL
SemiNAphthoRhodaFluors & SemiNAphthoFLuoresceins
Carboxy-SNAFL-1 AM
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Indo / SNARF-1
Fluorescence from the dual-emission Ca2+ indicator, indo-1 AM, is shown at 405 and 475 nm (left panels). Fluorescence from the dual-emission pH indicator, SNARF-1 AM, is shown at 575 and 640 nm (right panels)
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Applications of pH selective dyes
•Cl–/HCO3– exchange
•K+/H+ exchange
•Lactate transport and metabolism
•Na+/H+ exchange
•Na+/Ca2+ exchange
•NH4+ transport
•Apoptosis
•Cytotoxicity
•Multidrug resistance
•Cell volume changes
•Cytosolic pH regulation in osteoblasts and osteoclasts
•pH in lateral intercellular spaces of epithelial cell monolayers and interstitial spaces of normal and neoplastic tissue
•Phagocytosis
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Reactive Dyes
Chloromethyl-SNARF-1
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Reactive Dyes
Chloromethylfluorescein Diacetate
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“Cell Tracker” Dyes
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Ion Selective Dyes
Calcium / Magnesium / Zinc ions
Protons (pH)
Sodium & Potassium ions
Chloride / Halide ions
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Sodium & Potassium Selective Dyes
SBFIPBFI
benzofuranyl fluorophores
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SBFI Excitation Spectra
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SBFI Fluorescence Spectrum
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Sodium Green
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Ion Selective Dyes
Calcium / Magnesium / Zinc ions
Protons (pH)
Sodium & Potassium ions
Chloride / Halide ions
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Halide Sensitive Dyes
N-(ethoxycarbonylmethyl)-6- methoxyquinolinium bromide (MQAE)
6-methoxy-N-(3-sulfopropyl) quinolinium, inner salt (SPQ)
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Stern–Volmer constant
Stern–Volmer constant (KSV)
the reciprocal of the ion concentration that produces 50% of maximum quenching.
For SPQ, KSV is reported to be 118 M-1 in aqueous
solution and 12 M-1 inside cells.
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MQAE Spectra
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Potentiometric Dyes
ANEP Dyes (AminoNaphthylEthenylPyridinium)
di-4-ANEPPS
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di-4-ANEPPS
depol
ex
475
em em
560 620
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Rina Hildesheim (RH) Family of Potentiometric Dyes
Dialkylaminophenylpolyenylpyridinium
Oxonol dyes with phenylsulfonate substituents
RH414
RH155
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RH414
Cultured olfactory bulb neuron stained with RH 414 and (-)-DM-BODIPY dihydropyridine Left: image at >580 nm; Middle: image at 510–580 nm; Right: ratio of the middle image divided by the left image. Images were acquired with a Leica confocal laser scanning microscope
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Potentiometric Dyes: RH155
RH 155 is nonfluorescent; its potentiometric response is detected using absorption changes at approximately 720 nm.
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Photodiode Array Recording
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RH155 A fast potentiometric dye
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PDA Recording Setup
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SCN Slice Preparation
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PDA Recording of SCN Activity
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PDA Recording of SCN Activity
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PDA Recording of SCN Activity
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EAA Pharmacology of Evoked Responses
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Rat Enteric Plexus
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Obaid et al., 1999
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Obade et al., 1999
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Obade et al., 1999
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Cell Signaling - NO
DAF-FM diacetate
SNAP (S-nitroso-N-acetylpenicillamine)
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Cell Signaling - NO
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Coumarin Fluorophores
6-((7-amino-4-methylcoumarin- 3-acetyl)amino)hexanoic acid
AMCA-X
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Coumarin Fluorophores
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Coumarin Fluorophores
Marina Blue Pacific Blue
*351
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Infrared Fluorophores
Laser Pro IR 790
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BODIPY fluorophores
4,4-difluoro-4-bora-3a,4a-diaza-s-indacene
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BODIPY fluorophores
Normalized fluorescence emission spectra of1) BODIPY FL 2) BODIPY R6 3) BODIPY TMR 4) BODIPY 581/5915) BODIPY TR 6) BODIPY 630/650 7) BODIPY 650/665
fluorophores in methanol
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Green Fluorescent Protein
imidazolidinone ring
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Green Fluorescent Protein
The excitation spectrum of native GFP from A. victoria (blue) has two excitation maxima at 395 nm and at 470 nm. The fluorescence emission spectrum (green) has a peak at 509 nm and a shoulder at 540 nm.
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DiI Family
1,1'-dioctadecyl-3,3,3',3'- tetramethylindocarbocyanine perchlorate ('DiI'; DiIC18(3))
Tract tracing in fixed tissue
Remarkably stable – up to 2 years in vitro
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DiI images
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Fluorescence Spectra for DiXs
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DiI Family
3,3'-dioctadecyloxacarbocyanine perchlorate ('DiO'; DiOC18(3))
1,1'-dioctadecyl-3,3,3',3'-
tetramethylindodicarbocyanine perchlorate ('DiD' oil; DiIC18(5) oil)
DiO DiD
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1,1'-dioctadecyl-3,3,3',3'- tetramethylindotricarbocyanine iodide ('DiR'; DiIC18(7))
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Fast DiO
3,3'-dilinoleyloxacarbocyanine perchlorate (FAST DiO solid; DiO 9,12-C18(3), ClO4)
Diunsaturated linoleyl (C18:2) tails in place of the saturated
octadecyl tails (C18:0) of DiI and DiO increase rate of transport
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Sulfonate forms
1,1'-dioctadecyl-6,6'-di(4- sulfophenyl)-3,3,3',3'-
tetramethylindocarbocyanine (SP-DiIC18(3))
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DiA
4-(4-(dihexadecylamino)styryl) -N-methylpyridinium iodide (DiA; 4-Di-16-ASP)
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DiA in mouse retina
3 days after optic nerve transection
10 days after optic nerve transection
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Dextrans
Substituted dextran polymers
Poly-( -D-1,6-glucose) linkages, which render them resistant to cleavage by most endogenous cellular glycosidases
Wide molecular weight range (3000 – 300K)
Multiple fluorophores
Biotin
Lysine
Fluoro-Ruby
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Fluoro-Ruby
Fish spinal cord
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Cell lineage with dextrans
The image on the left shows a 13 µm-thick section of a stage 6 (32-cell) Xenopus embryo fixed right after injection; this section exhibits significant autofluorescence due to the presence of residual yolk. The image on the right is a stage 10 (early gastrula) embryo
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Fluorescent Microspheres
polystyrene microspheres 0.02 – 15 m diameter
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Lucifer Yellow
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Lucifer Yellow
Lucifer Yellow
Texas Red
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End of tract tracers