published by aaas b. n. g. giepmans et al., science 312, 217 -224 (2006) fig. 3. parallel...
TRANSCRIPT
![Page 1: Published by AAAS B. N. G. Giepmans et al., Science 312, 217 -224 (2006) Fig. 3. Parallel application of targeting methods and fluorophores](https://reader036.vdocuments.us/reader036/viewer/2022070410/56649ed15503460f94be063f/html5/thumbnails/1.jpg)
Published by AAAS
B. N. G. Giepmans et al., Science 312, 217 -224 (2006)
Fig. 3. Parallel application of targeting methods and fluorophores
![Page 2: Published by AAAS B. N. G. Giepmans et al., Science 312, 217 -224 (2006) Fig. 3. Parallel application of targeting methods and fluorophores](https://reader036.vdocuments.us/reader036/viewer/2022070410/56649ed15503460f94be063f/html5/thumbnails/2.jpg)
Types of fluorochromes
1. organic molecules (polyphenolic)
natural & synthetic
2. metal chelates (lanthanides)
3. semiconductor crystals (Q-dots)
4. fluorescent proteins
natural & engineered
5. expressible affinity reagents
![Page 3: Published by AAAS B. N. G. Giepmans et al., Science 312, 217 -224 (2006) Fig. 3. Parallel application of targeting methods and fluorophores](https://reader036.vdocuments.us/reader036/viewer/2022070410/56649ed15503460f94be063f/html5/thumbnails/3.jpg)
QuickTime™ and aTIFF (Uncompressed) decompressor
are needed to see this picture.
![Page 4: Published by AAAS B. N. G. Giepmans et al., Science 312, 217 -224 (2006) Fig. 3. Parallel application of targeting methods and fluorophores](https://reader036.vdocuments.us/reader036/viewer/2022070410/56649ed15503460f94be063f/html5/thumbnails/4.jpg)
BODIPY fluorophores
4,4-difluoro-4-bora-3a,4a-diaza-s-indacene
![Page 5: Published by AAAS B. N. G. Giepmans et al., Science 312, 217 -224 (2006) Fig. 3. Parallel application of targeting methods and fluorophores](https://reader036.vdocuments.us/reader036/viewer/2022070410/56649ed15503460f94be063f/html5/thumbnails/5.jpg)
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
![Page 6: Published by AAAS B. N. G. Giepmans et al., Science 312, 217 -224 (2006) Fig. 3. Parallel application of targeting methods and fluorophores](https://reader036.vdocuments.us/reader036/viewer/2022070410/56649ed15503460f94be063f/html5/thumbnails/6.jpg)
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
![Page 7: Published by AAAS B. N. G. Giepmans et al., Science 312, 217 -224 (2006) Fig. 3. Parallel application of targeting methods and fluorophores](https://reader036.vdocuments.us/reader036/viewer/2022070410/56649ed15503460f94be063f/html5/thumbnails/7.jpg)
DiI images
![Page 8: Published by AAAS B. N. G. Giepmans et al., Science 312, 217 -224 (2006) Fig. 3. Parallel application of targeting methods and fluorophores](https://reader036.vdocuments.us/reader036/viewer/2022070410/56649ed15503460f94be063f/html5/thumbnails/8.jpg)
Fluorescence Spectra for DiXs
![Page 9: Published by AAAS B. N. G. Giepmans et al., Science 312, 217 -224 (2006) Fig. 3. Parallel application of targeting methods and fluorophores](https://reader036.vdocuments.us/reader036/viewer/2022070410/56649ed15503460f94be063f/html5/thumbnails/9.jpg)
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
![Page 10: Published by AAAS B. N. G. Giepmans et al., Science 312, 217 -224 (2006) Fig. 3. Parallel application of targeting methods and fluorophores](https://reader036.vdocuments.us/reader036/viewer/2022070410/56649ed15503460f94be063f/html5/thumbnails/10.jpg)
DiA
4-(4-(dihexadecylamino)styryl) -N-methylpyridinium iodide (DiA; 4-Di-16-ASP)
![Page 11: Published by AAAS B. N. G. Giepmans et al., Science 312, 217 -224 (2006) Fig. 3. Parallel application of targeting methods and fluorophores](https://reader036.vdocuments.us/reader036/viewer/2022070410/56649ed15503460f94be063f/html5/thumbnails/11.jpg)
DiA in mouse retina
3 days after optic nerve transection
10 days after optic nerve transection
![Page 12: Published by AAAS B. N. G. Giepmans et al., Science 312, 217 -224 (2006) Fig. 3. Parallel application of targeting methods and fluorophores](https://reader036.vdocuments.us/reader036/viewer/2022070410/56649ed15503460f94be063f/html5/thumbnails/12.jpg)
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
![Page 13: Published by AAAS B. N. G. Giepmans et al., Science 312, 217 -224 (2006) Fig. 3. Parallel application of targeting methods and fluorophores](https://reader036.vdocuments.us/reader036/viewer/2022070410/56649ed15503460f94be063f/html5/thumbnails/13.jpg)
Fluoro-Ruby
Fish spinal cord
![Page 14: Published by AAAS B. N. G. Giepmans et al., Science 312, 217 -224 (2006) Fig. 3. Parallel application of targeting methods and fluorophores](https://reader036.vdocuments.us/reader036/viewer/2022070410/56649ed15503460f94be063f/html5/thumbnails/14.jpg)
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
![Page 15: Published by AAAS B. N. G. Giepmans et al., Science 312, 217 -224 (2006) Fig. 3. Parallel application of targeting methods and fluorophores](https://reader036.vdocuments.us/reader036/viewer/2022070410/56649ed15503460f94be063f/html5/thumbnails/15.jpg)
Fluorescent Microspheres
polystyrene microspheres 0.02 – 15 m diameter
![Page 16: Published by AAAS B. N. G. Giepmans et al., Science 312, 217 -224 (2006) Fig. 3. Parallel application of targeting methods and fluorophores](https://reader036.vdocuments.us/reader036/viewer/2022070410/56649ed15503460f94be063f/html5/thumbnails/16.jpg)
Lucifer Yellow
![Page 17: Published by AAAS B. N. G. Giepmans et al., Science 312, 217 -224 (2006) Fig. 3. Parallel application of targeting methods and fluorophores](https://reader036.vdocuments.us/reader036/viewer/2022070410/56649ed15503460f94be063f/html5/thumbnails/17.jpg)
Lucifer Yellow
Lucifer Yellow
Texas Red
![Page 18: Published by AAAS B. N. G. Giepmans et al., Science 312, 217 -224 (2006) Fig. 3. Parallel application of targeting methods and fluorophores](https://reader036.vdocuments.us/reader036/viewer/2022070410/56649ed15503460f94be063f/html5/thumbnails/18.jpg)
End of tract tracers
![Page 19: Published by AAAS B. N. G. Giepmans et al., Science 312, 217 -224 (2006) Fig. 3. Parallel application of targeting methods and fluorophores](https://reader036.vdocuments.us/reader036/viewer/2022070410/56649ed15503460f94be063f/html5/thumbnails/19.jpg)
Types of fluorochromes
1. organic molecules (polyphenolic)
natural & synthetic
2. metal chelates (lanthanides)
3. semiconductor crystals (Q-dots)
4. fluorescent proteins
natural & engineered
5. expressible affinity reagents
![Page 20: Published by AAAS B. N. G. Giepmans et al., Science 312, 217 -224 (2006) Fig. 3. Parallel application of targeting methods and fluorophores](https://reader036.vdocuments.us/reader036/viewer/2022070410/56649ed15503460f94be063f/html5/thumbnails/20.jpg)
![Page 21: Published by AAAS B. N. G. Giepmans et al., Science 312, 217 -224 (2006) Fig. 3. Parallel application of targeting methods and fluorophores](https://reader036.vdocuments.us/reader036/viewer/2022070410/56649ed15503460f94be063f/html5/thumbnails/21.jpg)
Published by AAAS
G. Gaietta et al., Science 296, 503 -507 (2002)
No Caption Found
![Page 22: Published by AAAS B. N. G. Giepmans et al., Science 312, 217 -224 (2006) Fig. 3. Parallel application of targeting methods and fluorophores](https://reader036.vdocuments.us/reader036/viewer/2022070410/56649ed15503460f94be063f/html5/thumbnails/22.jpg)
Published by AAAS
G. Gaietta et al., Science 296, 503 -507 (2002)
No Caption Found
![Page 23: Published by AAAS B. N. G. Giepmans et al., Science 312, 217 -224 (2006) Fig. 3. Parallel application of targeting methods and fluorophores](https://reader036.vdocuments.us/reader036/viewer/2022070410/56649ed15503460f94be063f/html5/thumbnails/23.jpg)
Published by AAAS
G. Gaietta et al., Science 296, 503 -507 (2002)
No Caption Found
![Page 24: Published by AAAS B. N. G. Giepmans et al., Science 312, 217 -224 (2006) Fig. 3. Parallel application of targeting methods and fluorophores](https://reader036.vdocuments.us/reader036/viewer/2022070410/56649ed15503460f94be063f/html5/thumbnails/24.jpg)
Published by AAAS
J. Lippincott-Schwartz et al., Science 300, 87 -91 (2003)
FRAP FLIP &PhRAP
![Page 25: Published by AAAS B. N. G. Giepmans et al., Science 312, 217 -224 (2006) Fig. 3. Parallel application of targeting methods and fluorophores](https://reader036.vdocuments.us/reader036/viewer/2022070410/56649ed15503460f94be063f/html5/thumbnails/25.jpg)
Published by AAAS
T. Misteli Science 291, 843 -847 (2001)
Fluorescence Recovery After Photobleaching (FRAP)
![Page 26: Published by AAAS B. N. G. Giepmans et al., Science 312, 217 -224 (2006) Fig. 3. Parallel application of targeting methods and fluorophores](https://reader036.vdocuments.us/reader036/viewer/2022070410/56649ed15503460f94be063f/html5/thumbnails/26.jpg)
Published by AAAS
A. B. Houtsmuller et al., Science 284, 958 -961 (1999)
Fluorescence Recovery After Photobleaching (FRAP)
![Page 27: Published by AAAS B. N. G. Giepmans et al., Science 312, 217 -224 (2006) Fig. 3. Parallel application of targeting methods and fluorophores](https://reader036.vdocuments.us/reader036/viewer/2022070410/56649ed15503460f94be063f/html5/thumbnails/27.jpg)
Published by AAAS
J. Lippincott-Schwartz et al., Science 300, 87 -91 (2003)
No Caption Found
![Page 28: Published by AAAS B. N. G. Giepmans et al., Science 312, 217 -224 (2006) Fig. 3. Parallel application of targeting methods and fluorophores](https://reader036.vdocuments.us/reader036/viewer/2022070410/56649ed15503460f94be063f/html5/thumbnails/28.jpg)
Published by AAAS
R. Ando et al., Science 306, 1370 -1373 (2004)
Fig. 3. Monitoring the nuclear import and export of importin-{beta}-Dronpa in COS7 cells
![Page 29: Published by AAAS B. N. G. Giepmans et al., Science 312, 217 -224 (2006) Fig. 3. Parallel application of targeting methods and fluorophores](https://reader036.vdocuments.us/reader036/viewer/2022070410/56649ed15503460f94be063f/html5/thumbnails/29.jpg)
Published by AAAS
R. Ando et al., Science 306, 1370 -1373 (2004)
Fig. 2. Monitoring the nuclear import and export of ERK1-Dronpa in COS7 cells during stimulation with 100 ng/ml EGF
![Page 30: Published by AAAS B. N. G. Giepmans et al., Science 312, 217 -224 (2006) Fig. 3. Parallel application of targeting methods and fluorophores](https://reader036.vdocuments.us/reader036/viewer/2022070410/56649ed15503460f94be063f/html5/thumbnails/30.jpg)
Foerster Resonance Energy Transfer
dipole - dipole interaction
![Page 31: Published by AAAS B. N. G. Giepmans et al., Science 312, 217 -224 (2006) Fig. 3. Parallel application of targeting methods and fluorophores](https://reader036.vdocuments.us/reader036/viewer/2022070410/56649ed15503460f94be063f/html5/thumbnails/31.jpg)
Foerster Resonance Energy Transfer
1. Distance between fluors
2. Spectral overlap
3. Orientation of dipoles
![Page 32: Published by AAAS B. N. G. Giepmans et al., Science 312, 217 -224 (2006) Fig. 3. Parallel application of targeting methods and fluorophores](https://reader036.vdocuments.us/reader036/viewer/2022070410/56649ed15503460f94be063f/html5/thumbnails/32.jpg)
Foerster Resonance Energy Transfer
E = 1
1+ (r/R0)6
“donor” “acceptor”
![Page 33: Published by AAAS B. N. G. Giepmans et al., Science 312, 217 -224 (2006) Fig. 3. Parallel application of targeting methods and fluorophores](https://reader036.vdocuments.us/reader036/viewer/2022070410/56649ed15503460f94be063f/html5/thumbnails/33.jpg)
Foerster Resonance Energy Transfer
E = 1
1+ (r/R0)6
“donor” “acceptor”
efficiency Foerster radius
![Page 34: Published by AAAS B. N. G. Giepmans et al., Science 312, 217 -224 (2006) Fig. 3. Parallel application of targeting methods and fluorophores](https://reader036.vdocuments.us/reader036/viewer/2022070410/56649ed15503460f94be063f/html5/thumbnails/34.jpg)
Foerster Resonance Energy Transfer
r < 50 A
![Page 35: Published by AAAS B. N. G. Giepmans et al., Science 312, 217 -224 (2006) Fig. 3. Parallel application of targeting methods and fluorophores](https://reader036.vdocuments.us/reader036/viewer/2022070410/56649ed15503460f94be063f/html5/thumbnails/35.jpg)
Foerster Resonance Energy Transfer (FRET)
R06 = 8.8 x 10-28 2 n-4
Q0 J
E = 1
1+ (r/R0)6
![Page 36: Published by AAAS B. N. G. Giepmans et al., Science 312, 217 -224 (2006) Fig. 3. Parallel application of targeting methods and fluorophores](https://reader036.vdocuments.us/reader036/viewer/2022070410/56649ed15503460f94be063f/html5/thumbnails/36.jpg)
Foerster Resonance Energy Transfer (FRET)
R06 = 8.8 x 10-28 2
n-4 Q0 J
E = 1
1+ (r/R0)6
dipole orientation factor
2 ~ 2/3
![Page 37: Published by AAAS B. N. G. Giepmans et al., Science 312, 217 -224 (2006) Fig. 3. Parallel application of targeting methods and fluorophores](https://reader036.vdocuments.us/reader036/viewer/2022070410/56649ed15503460f94be063f/html5/thumbnails/37.jpg)
Foerster Resonance Energy Transfer (FRET)
R06 = 8.8 x 10-28 2
n-4 Q0 J
E = 1
1+ (r/R0)6
refractive index
quantum yield of donor
spectral overlap integral
![Page 38: Published by AAAS B. N. G. Giepmans et al., Science 312, 217 -224 (2006) Fig. 3. Parallel application of targeting methods and fluorophores](https://reader036.vdocuments.us/reader036/viewer/2022070410/56649ed15503460f94be063f/html5/thumbnails/38.jpg)
Foerster Resonance Energy Transfer (FRET)
J = fD() A() 4 d
![Page 39: Published by AAAS B. N. G. Giepmans et al., Science 312, 217 -224 (2006) Fig. 3. Parallel application of targeting methods and fluorophores](https://reader036.vdocuments.us/reader036/viewer/2022070410/56649ed15503460f94be063f/html5/thumbnails/39.jpg)
Foerster Resonance Energy Transfer (FRET)
J = fD() A() 4 d normalized
donor spectrum
molar extinctioncoef of acceptor
wavelength
![Page 40: Published by AAAS B. N. G. Giepmans et al., Science 312, 217 -224 (2006) Fig. 3. Parallel application of targeting methods and fluorophores](https://reader036.vdocuments.us/reader036/viewer/2022070410/56649ed15503460f94be063f/html5/thumbnails/40.jpg)
Foerster Resonance Energy Transfer (FRET)
R06 = 8.8 x 10-28 2
n-4 Q0 J
E = 1
1+ (r/R0)6
J = fD() A() 4 d
![Page 41: Published by AAAS B. N. G. Giepmans et al., Science 312, 217 -224 (2006) Fig. 3. Parallel application of targeting methods and fluorophores](https://reader036.vdocuments.us/reader036/viewer/2022070410/56649ed15503460f94be063f/html5/thumbnails/41.jpg)
Foerster Resonance Energy Transfer
![Page 42: Published by AAAS B. N. G. Giepmans et al., Science 312, 217 -224 (2006) Fig. 3. Parallel application of targeting methods and fluorophores](https://reader036.vdocuments.us/reader036/viewer/2022070410/56649ed15503460f94be063f/html5/thumbnails/42.jpg)
Foerster Resonance Energy Transfer
![Page 43: Published by AAAS B. N. G. Giepmans et al., Science 312, 217 -224 (2006) Fig. 3. Parallel application of targeting methods and fluorophores](https://reader036.vdocuments.us/reader036/viewer/2022070410/56649ed15503460f94be063f/html5/thumbnails/43.jpg)
CFP CFPYFP YFP
FRET433 nm476 nm433 nm 527 nm
Ca2+
CaM
M13
![Page 44: Published by AAAS B. N. G. Giepmans et al., Science 312, 217 -224 (2006) Fig. 3. Parallel application of targeting methods and fluorophores](https://reader036.vdocuments.us/reader036/viewer/2022070410/56649ed15503460f94be063f/html5/thumbnails/44.jpg)
Published by AAAS
P. Niethammer et al., Science 303, 1862 -1866 (2004)
Fig. 2. Phosphorylation-dependent patterns of stathmintubulin interaction in Xenopus A6 cells
![Page 45: Published by AAAS B. N. G. Giepmans et al., Science 312, 217 -224 (2006) Fig. 3. Parallel application of targeting methods and fluorophores](https://reader036.vdocuments.us/reader036/viewer/2022070410/56649ed15503460f94be063f/html5/thumbnails/45.jpg)
Published by AAAS
P. J. Verveer et al., Science 290, 1567 -1570 (2000)
Fluorescence Lifetime Imaging
![Page 46: Published by AAAS B. N. G. Giepmans et al., Science 312, 217 -224 (2006) Fig. 3. Parallel application of targeting methods and fluorophores](https://reader036.vdocuments.us/reader036/viewer/2022070410/56649ed15503460f94be063f/html5/thumbnails/46.jpg)
Foerster Resonance Energy Transfer
![Page 47: Published by AAAS B. N. G. Giepmans et al., Science 312, 217 -224 (2006) Fig. 3. Parallel application of targeting methods and fluorophores](https://reader036.vdocuments.us/reader036/viewer/2022070410/56649ed15503460f94be063f/html5/thumbnails/47.jpg)
Total Internal Reflection Microscopy
![Page 48: Published by AAAS B. N. G. Giepmans et al., Science 312, 217 -224 (2006) Fig. 3. Parallel application of targeting methods and fluorophores](https://reader036.vdocuments.us/reader036/viewer/2022070410/56649ed15503460f94be063f/html5/thumbnails/48.jpg)
Published by AAAS
D. J. Stephens et al., Science 300, 82 -86 (2003)
Total Internal Reflection Microscopy
![Page 49: Published by AAAS B. N. G. Giepmans et al., Science 312, 217 -224 (2006) Fig. 3. Parallel application of targeting methods and fluorophores](https://reader036.vdocuments.us/reader036/viewer/2022070410/56649ed15503460f94be063f/html5/thumbnails/49.jpg)
![Page 50: Published by AAAS B. N. G. Giepmans et al., Science 312, 217 -224 (2006) Fig. 3. Parallel application of targeting methods and fluorophores](https://reader036.vdocuments.us/reader036/viewer/2022070410/56649ed15503460f94be063f/html5/thumbnails/50.jpg)
Synapto-pHluorin
Fusion protein comprised of VAMP-2/synaptobrevin - GFP (luminal domain)
Developed by Miesenbock et al (98)
Vesicular uptake is powered by V-type H+-ATPase2; pH ~ 5.5
GFP is pH sensitive (pKa = 7.1)
97% of GFP fluorescence is quenched in SV
Some forms permit ratiometric quantification
![Page 51: Published by AAAS B. N. G. Giepmans et al., Science 312, 217 -224 (2006) Fig. 3. Parallel application of targeting methods and fluorophores](https://reader036.vdocuments.us/reader036/viewer/2022070410/56649ed15503460f94be063f/html5/thumbnails/51.jpg)
Synapto-pHluorin
Hela Cells
![Page 52: Published by AAAS B. N. G. Giepmans et al., Science 312, 217 -224 (2006) Fig. 3. Parallel application of targeting methods and fluorophores](https://reader036.vdocuments.us/reader036/viewer/2022070410/56649ed15503460f94be063f/html5/thumbnails/52.jpg)
Synapto-pHluorin
Visualizing synaptic transmission in hippocampal neurons
Miesenbock et al Nature 394:192-195 (1998)
![Page 53: Published by AAAS B. N. G. Giepmans et al., Science 312, 217 -224 (2006) Fig. 3. Parallel application of targeting methods and fluorophores](https://reader036.vdocuments.us/reader036/viewer/2022070410/56649ed15503460f94be063f/html5/thumbnails/53.jpg)
Synapto-pHluorin
Visualizing exocytosis in RBL-2H3 cells
Miesenbock et al Nature 394:192-195 (1998)
![Page 54: Published by AAAS B. N. G. Giepmans et al., Science 312, 217 -224 (2006) Fig. 3. Parallel application of targeting methods and fluorophores](https://reader036.vdocuments.us/reader036/viewer/2022070410/56649ed15503460f94be063f/html5/thumbnails/54.jpg)
Copyright ©2006 Society for Neuroscience
Atluri, P. P. et al. J. Neurosci. 2006;26:2313-2320
Figure 1. Rapid acid quench of surface SpH fluorescence allows measurement of internal alkaline vesicle pool and postendocytic reacidification time course
![Page 55: Published by AAAS B. N. G. Giepmans et al., Science 312, 217 -224 (2006) Fig. 3. Parallel application of targeting methods and fluorophores](https://reader036.vdocuments.us/reader036/viewer/2022070410/56649ed15503460f94be063f/html5/thumbnails/55.jpg)
Copyright ©2005 by the National Academy of Sciences
Li, Zhiying et al. (2005) Proc. Natl. Acad. Sci. USA 102, 6131-6136
Fig. 1. Characterizing transgenic mice expressing spH