Raman scattering -Why Raman scattering? All molecules scatter light Chemical selectivity No labeling...

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

K.U.LEUVEN

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


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


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




C-C-C ring in plane bend

Wavenumber / cm-1 Assignment


K.U.LEUVEN

LEUVEN 2005


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


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


•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


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

K.U.LEUVEN

LEUVEN 2005


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)

K.U.LEUVEN

LEUVEN 2005


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


Protonated form Deprotonated form

1537

K.U.LEUVEN

LEUVEN 2005


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)


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


• 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


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


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

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


Double-label assay: PNA (peptide nucleic acids) are used instead of DNA probes because of their stronger binding to DNA targets


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.


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

K.U.LEUVEN

LEUVEN 2005


K.U.LEUVEN

LEUVEN 2005

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.


K.U.LEUVEN

LEUVEN 2005

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.


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

Raman scattering -Why Raman scattering? All molecules scatter light Chemical selectivity No labeling...

Documents

Transcript of Raman scattering -Why Raman scattering? All molecules scatter light Chemical selectivity No labeling...