raman scattering -why raman scattering? all molecules scatter light chemical selectivity no labeling...
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Raman scattering
-Why Raman scattering?
All molecules scatter lightChemical selectivityNo labeling needed!
-Possible to combine with microscopy-3D images and deep penetration possible.
K.U.LEUVEN
LEUVEN 2005
CARS microscopie(Coherent anti-Stokes Raman Scattering microscopie)
-No labeling needed!-3D images and deep penetration possible.
Fibroblast cells, C-H streching vibration
Energy scheme CARS mircroscopy
K.U.LEUVEN
LEUVEN 2005
CARS microscopie
K.U.LEUVEN
LEUVEN 2005
SERS spectroscopy
Dramatic enhancement of Raman signal occurs
when the analyte molecule is adsorbed to metal
particles of subwavelength dimension.
Käll et al. Phys. Rev. E 62, 2000
Mechanism
• Enhancement of the electromagnetic fields close to the surface through interaction with the plasmon excitation (EM enhancement).
• Chemical bonding and the subsequent formation of a charge transfer state (chemical enhancement).
• Particle size, the presence of anions etc.
SERRS cross-section : ~10-15 cm2 per molecule
Heterogeneous and localized active sites
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LEUVEN 2005
Single molecule SERRS
AgNO3 aq
sodium citrate
refluxSilver colloids
Silver colloids
Molecules (R6G, EGFP)
+1 hour The molecules adsorbed on the colloidal particles
(NaCl = 0.75 mM).
The colloidal particles were immobilized on polylysine coated glass surface. After evaporating the solvent, cover slip samples were rinsed with ultra pure water, and dried with nitrogen flow.
K.U.LEUVEN
LEUVEN 2005
Single molecule SERRS
Stage scan confocal microscope(1 image / 2.5 min)
SM-SERRS images show on-off blinking in the order of second to minute.
K.U.LEUVEN
LEUVEN 2005
Single molecule SERRS
1800 1600 1400 1200 1000 800 6000
50
100
150
200
250
300
62077711201183
1305
136015121579
Inte
nsity
Raman Shift / cm-1
1659
Nie et al. Science 275, 1997
614 :
773 :
1127 :
1363 :
1509 :
1575 :
1650 :
C-C-C ring in plane bend
C-H out of plane bend
C-H in plane bend
aromatic C-C stretch
aromatic C-C stretch
aromatic C-C stretch
aromatic C-C stretch
C-C-C ring in plane bend
Wavenumber / cm-1 Assignment
Wavenumber / cm-1 Assignment
K.U.LEUVEN
LEUVEN 2005
Single molecule SERRS
Hemoglobin
The non-covalently bound porphyrin group might diffuse out of the protein to be directly on the silver particles.
GFPs
The chromophore is part of the protein and is kept in place in the 3-dimensional barrel structure by a complex hydrogen bonding network.
K.U.LEUVEN
LEUVEN 2005
Single molecule SERRS
Wild-type GFP
HO
NN
O
N
OH
NH N
H2N
NH3+
NHNH2
O
O
HO
Serine65
Arginine96
Glutamine94
Tyrosine66
Glu222
Glycine67
Phenylalanine64
Histidine148
K.U.LEUVEN
LEUVEN 2005
Fluorescent proteins
K.U.LEUVEN
LEUVEN 2005
Fluorescent proteins
•monitoring proteins, organelles, cells in living tissue.
•protein-protein interaction using double labeling and FRET.
•membrane traffic studies.
•pH sensor.
•Ca2+ sensor.
•……….
Applications :K.U.LEUVEN
LEUVEN 2005
Fluorescent proteins
O
H
N
N
O
R
H
HO
OOHO
R
O
H
Glu222
Ser205
W12O
HH
O
RR
W22
Thr203
O HThr203
N
N
H
O
RR
Asn146
His148
His148
Asn146
Thr203O H
Thr203
W22W12
Ser205
Glu222
H
O
R
O
RR
N
N
O
R
H
HO
OOHO
H
N
N
H
O
O
H
H
O
RR
(A , A * states)
( I * state) (B *, B states)
N
N
O
R
H
HO
OO
H
O
RR
R
O
H
Gl u222
Ser205
W12W22
Thr203
Thr203
Asn146
His148
O H
OH
N
N
H
O
O
H
H
O
RR
ES
PT
SL O W RE L A X A TI O N
A
A*I*
I B
B*
400
nm
450
nm
490
nm
508
nm, 3
.3 n
s
470
nm
508
nm, 2
.8 n
s
SLOWRELAX
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LEUVEN 2005
Fluorescent proteins
Fluorescent proteins
New trends in GFP-research
• Optical marking (following intracellular dynamics) or kindling
Patterson, G. H. & Lippincott-Schwartz, J. Science 2002, 297, 1873.
Photo-Switchable Fluorescent Protein Dronpa
• Dronpa is a monomeric GFP-like fluorescent protein from coral Echinophyllia sp.
• Dronpa shows reversible photoswitching on irradiation with a 488 nm and 405 nm light.
On
Off
Inte
nsi
ty
488 nm
405 nm
Time
Steady-State Spectra of Dronpa
pH = 7.4 pH = 5.0
O
NN
O
NN
O
OH
• Deprotonated form (B form); fluorescent state, fl488 = 0.85, fl = 3.6 ns
• Protonated form (A1 form); dim state, fl390 = 0.02, fl = 14 ps
300 400 500 600
Flu
ores
cenc
e In
tens
ity
Abs
orba
nce
Wavelength / nm300 400 500 600
Flu
ores
cenc
e In
tens
ity
A
bsor
banc
e
Wavelength / nm
300 400 500 6000.00
0.05
0.10
0.15
Abs
orba
nce
Wavelength / nm
Photoswitching of Dronpa at the Ensemble Level
488 nm
405 nm
300 400 500 6000.00
0.05
0.10
0.15
Abs
orba
nce
Wavelength / nm
0.00
0.05
0.10
0.15
0 300 600 900 12000.00
0.02
0.04
Ab
sorb
an
ceTime / sec
k = 9.0 x 10-3 s-1
Ab
ao
rba
nce
k = 9.6 x 10-3 s-1
0.00
0.05
0.10
0.15
0 20 40 60 800.00
0.02
0.04
Ab
sorb
an
ce
Time / min
k = 6.9 x 10-4 s-1
Ab
ao
rba
nce
k = 6.7 x 10-4 s-1pH = 7.4
pH = 7.4
300 400 500 6000.00
0.02
0.04
0.06
0.08
0.10
Ab
sorb
an
ce
Wavelength / nm
Photoswitched Protonated (A2) Form
488 nm
405 nm
pH = 5.0
pH = 5.0
300 400 500 6000.00
0.02
0.04
0.06
0.08
0.10
Ab
sorb
an
ce
Wavelength / nm
0.0
0.5
1.0
0 20 40 60 80 1000.0
0.5
1.0
1.5
[CA
2] / 1
0-6 M
Time / min
k = 5.6 x 10-4 s-1
[CB] /
10
-6 M
k = 5.1 x 10-4 s-1
0.0
0.5
1.0
0 300 600 900 12000.0
0.5
1.0
1.5
[CA
2] / 1
0-6 M
Time / sec
k = 9.1 x 10-3 s-1
[CB] /
10
-6 M
k = 1.0 x 10-2 s-1
Scheme of the Photoswitching
On
Off
Inte
nsi
ty
488 nm
405 nm
Time
Photoswitched protonated form Non-fluorescent
intermediate
S0
S1
Fluorescent deprotonated form
= 3.2 ×10-4
= 0.37
3.6 ns
Protonated form
14 ps
New trends in GFP-research
• Diffraction-unlimited microscopy in far field
Hell, S. W. Curr. Opin. Neurobiol. 2004, 14, 599.
Time (s)
Co
un
ts/m
s
Co
un
ts/m
s
Time (s)
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LEUVEN 2005
Fluorescent proteins
0 5 10 15 200
10
20
30
40
50
60
Cou
nts
/ m
s
Time / sec
Stage scan confocal microscope(1 image / 2.5 min)
SM-SERRS images show on-off blinking in the order of second to minute.
K.U.LEUVEN
LEUVEN 2005
SERRS of fluorescent proteins
1560 delocalized imidazolinone/exocyclic C=C stretching mode
Wavenumber / cm-1 Assignment
Protonated form Deprotonated form
1537
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LEUVEN 2005
SERRS of fluorescent proteins
Most of the peaks in the spectrum are in agreement with the ensemble Raman spectrum.
K.U.LEUVEN
LEUVEN 2005
1600 1400 1200 1000
Raman Shift / cm-1
Inte
nsi
ty
1617 15
60
1536
1493
1448
1364
1300 12
54
1166
1079
1001
Ensemble spectrum (pH = 7.4)
Inte
nsi
ty 1618 15
63
1450 13
73
1324 12
72
1230
1150
1084
1022
1528 Averaged SM spectrum
Inte
nsi
ty
1617
1598
1560
1493
1448
1364
1300
1254
1166
1079
1001
1536 Ensemble spectrum (pH = 5.0)
SERRS of fluorescent proteins
1800 1600 1400 1200 1000 800
4
1
5
2
3
1151
1282
1311
1558
1596
1634
1153
1281
131415
6215
9616
34
1259
136115
24
1143
116312
251263
1348
1530
1606
1652
1524
1143
116712
2312
59
1334
1376
1597
1620
1661
Inte
nsi
ty
Raman Shift / cm-1
Conversion from deprotonated to protonated form of the chromophore.
1800 1600 1400 1200 1000 8000
20
40
60
coun
ts
time
/ sec
Raman shift / cm-1
K.U.LEUVEN
LEUVEN 2005
SERRS of fluorescent proteins
• Reversible conversion takes place in less time than the accumulation time.
1800 1600 1400 1200 1000
Inte
nsity
Raman Shift / cm-1
1800 1600 1400 1200 1000
Raman Shift / cm-1
1800 1600 1400 1200 1000
Raman Shift / cm-1
K.U.LEUVEN
LEUVEN 2005
SERRS of fluorescent proteins
AgAgAgAg
NN
O
OH
NN
O
OH
NN
O
OH
+ H+
- H+
+ H+
- H+
1800 1600 1400 1200 1000 800 600
Inte
nsity
Raman Shift / cm-1
0 - 5 s
5 - 10 s
10 - 15 s
15 - 20 s
20 - 25 s
O
NN
O
K.U.LEUVEN
LEUVEN 2005
SERRS of fluorescent proteins
Habuchi, S.; Cotlet, M.; Gronheid, R.; Dirix, G.; Michiels, J.; Vanderleyden, J.; De Schryver, F. C.; Hofkens, J. J. Am. Chem. Soc. 2003, 125, 8446-8447.
Single Molecule DNA Sequencing
Human Genome Project
identify all the approximately 30,000 genes in human DNA, determine the sequences of the 3 billion chemical base pairs that make up human DNA, store this information in databases, improve tools for data analysis, transfer related technologies to the private sector, and address the ethical, legal, and social issues (ELSI) that may arise from the project.
Conventional methods involve manual intensive procedures that cannot be completely automated, are time consuming, suffer from low reproducibilityand accuracy and high cost.
Single molecule detection using laser-induced fluorescence might solve problems such as automation, low cost, high throughput (speed).
Conventional methods
DNA Sequencing
Single molecule DNA Sequencing
Microcapillary-basedSM DNA sequencing(M.Sauer et al. Heidelberg)
femtotip
Laser beam
3’-5’ exonuclease
Microcapillary-based SM DNA sequencing
Microcapillary-based SM DNA sequencing
Micro-capillary-basedSM DNA sequencing
Flow-cytometry based DNA sequencingP. Ambrose et al. LANL
micropipette
microsphere
exonuclease
Flow stream
control
cleavage
Experimental setup
Flow-cytometry based DNA sequencingA. Castro et al. LANL
Photon bursts detected from individual Rhodamine 6 G molecules
10 fM Rhodamine 6G
In aqueous solution
only aqueous solution
Single molecule DNA Sequencing
Double-label assay: PNA (peptide nucleic acids) are used instead of DNA probes because of their stronger binding to DNA targets
Single molecule DNA Sequencing
Two PNA probes labeled with different dyes bind to a DNA target, the new complex passes the laser focus and produces a signal in both detectors (coincidence). Targets hybridized to one probe will give non-coincident signals.
Single molecule DNA Sequencing
The Helicos approach
Based on work by the S. Quake group in CaltechBraslavsky et al PNAS Vol 100, 3960-3964, 2003PDF on webside
DNA-sequencing:Helicos approach
K.U.LEUVEN
LEUVEN 2005
DNA-sequencing: the Helicos-approach
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DNA-sequencing: the Helicos-approach
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DNA-sequencing: the Helicos-approach
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DNA-sequencing: the Helicos-approach
K.U.LEUVEN
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DNA-sequencing: the Helicos-approach
K.U.LEUVEN
LEUVEN 2005
DNA-sequencing: the Helicos-approach
K.U.LEUVEN
LEUVEN 2005
DNA-sequencing: the Helicos-approach
K.U.LEUVEN
LEUVEN 2005
DNA-sequencing: the Helicos-approach
K.U.LEUVEN
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DNA-sequencing: the Helicos-approach
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DNA-sequencing: the Helicos-approach
K.U.LEUVEN
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Simplicity
Helicos' technology has the inherent advantage of working directly from genomic DNA, thereby eliminating the need for DNA amplification (PCR). In addition to greatly simplifying the overall sample preparation process, this abolishes the introduction of amplification errors and bias, and ultimately reduces cost.
Density
By eliminating amplification, DNA molecules can be closely packed on the substrate, thereby providing the largest amount of sequence information from a given surface area. The entire human genome can be represented on a single, compact, glass substrate.
Throughput
Imaging a substrate densely packed with single molecules of DNA provides the largest amount of sequence information per image, thus per unit time, enabling the sequencing of entire genomes in a day as opposed to years.
DNA-sequencing: the Helicos-approach
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Low Cost
The advantages listed above translate directly into reductions in cost both in terms of sample preparation and sequencing chemistry. Reagent use is orders of magnitude lower than alternative amplification based technologies for the equivalent amount of data.
Detection of Rare Events
High-density and throughput allow the detection of rare mutations and transcripts in a biological sample, at levels never before possible. This approach allows reading the sequences of over a billion DNA strands in a very short time, dramatically increasing the chance of finding mutations present at very low frequencies.
DNA-sequencing: the Helicos-approach
K.U.LEUVEN
LEUVEN 2005
Cancer Research
Cancer is ultimately a disease of the genes. Identifying the entire gamut of genetic aberrations in all tumor types will elucidate the molecular mechanisms responsible for uncontrolled malignant cell growth and metastasis.
Examples Sequencing the DNA from thousands of tumor samples for the identification
of common genetic aberrations Transcriptional profiling of tumors, with the ability to enumerate mRNA
transcripts Combining mutational profiling with transcription profiling to obtain allele-
specific expression information
Genome-wide methylation studies
DNA can undergo a chemical modification known as methylation. The expression of many genes is controlled by their methylation state; highly methylated genes are suppressed, and can be “turned on” by de-methylation. Discovering which genes are methylated at which stage in normal tissue development or during cancer progression will reveal important pathways.
Examples Quantitative assessment of the methylation state of genomic DNA Correlation of the methylation status with transcription levels
DNA-sequencing: the Helicos-approachStem cell research
Stem cells, by definition, have the potential to differentiate into every type of tissue in the body. Each tissue type has a distinct expression profile and differentiation pathway, which involves selectively turning genes “on” or “off” in a particular sequence. Using single molecule sequencing, the RNA expression profile of stem cells can be tracked at various stages of development and differentiation. Equipped with this information, scientists will eventually harness stem cells to engineer tissues of their choice, for the purpose of replacing damaged, deficient or diseased tissues in patients (e.g. pancreatic islet cells for the production of insulin in diabetics).
Examples Transcription profiling of pluripotent cells prior to and post-differentiation
Systems biology
A human cell can be studied as an integrated system of many interacting components. Systems biology aims to compile large amounts of diverse experimental data (genomic, transcriptomic, proteomic, metabolomic, etc.) and to create mathematical models of how the components interact. Helicos' technology provides a single platform for the analysis of DNA and RNA, and can eventually serve as a platform for the analysis of proteins at the single molecule level.
Examples Integration of multiple “omics” technologies into one platformSequencing DNA and RNA from the same sample
High degree of parallelization: 12000000 templates in a 25 mm square
FRET approach gets rid of the no specific background
FRET only works over 5 nm (15 base pairs), so new donors have to be build in at regular intervals or donor on polymerase
97% confidence level for discrimination between sequences