Raman spectroscopy - University of California, Irvinedmitryf/manuals/Fundamentals/Raman...

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Raman spectroscopy Information from Raman Spectroscopy characteristic Raman frequencies composition of material e.g. MoS 2 , MoO 3 changes in frequency of Raman peak stress/strai n state e.g. Si 10 cm -1 shift per % strain polarisation of Raman peak crystal symmetry and orientation e.g. orientation of CVD diamond grains width of Raman peak quality of crystal e.g. amount of plastic deformation parallel perpendicular intensity of Raman peak amount of material e.g. thickness of transparent coating

Transcript of Raman spectroscopy - University of California, Irvinedmitryf/manuals/Fundamentals/Raman...

Page 1: Raman spectroscopy - University of California, Irvinedmitryf/manuals/Fundamentals/Raman microsc… · Raman spectroscopy Information from Raman Spectroscopy characteristic Raman frequencies

Raman spectroscopy

Information from Raman Spectroscopy

characteristicRaman frequencies

composition ofmaterial

e.g. MoS2,MoO3

changes infrequency ofRaman peak

stress/strainstate

e.g. Si 10 cm-1 shift per% strain

polarisation ofRaman peak

crystal symmetry andorientation

e.g. orientation of CVDdiamond grains

width of Ramanpeak

quality ofcrystal

e.g. amount of plasticdeformation

parallel

perpendicular

intensity ofRaman peak

amount ofmaterial

e.g. thickness oftransparent coating

Page 2: Raman spectroscopy - University of California, Irvinedmitryf/manuals/Fundamentals/Raman microsc… · Raman spectroscopy Information from Raman Spectroscopy characteristic Raman frequencies

The coupling of a Raman spectrometer withan optical microscope provides a number ofadvantages:

1) Confocal Light collection

2) High lateral spatial resolution

3) Excellent depth resolution

4) Large solid collection angle for theRaman light

Collecting the light

Page 3: Raman spectroscopy - University of California, Irvinedmitryf/manuals/Fundamentals/Raman microsc… · Raman spectroscopy Information from Raman Spectroscopy characteristic Raman frequencies

The basic function of a Raman system

• Deliver the laser to the sampling point– With low power loss through the system

– Illuminating an area consistent with sampling dimensions

– Provide a selection/choice of laser wavelengths

• Collect the Raman scatter– High aperture

– High efficiency optics

– High level of rejection of the scattered laser light

• Disperse the scattered light– Short wavelength excitation requires high dispersion spectrometers

• Detect the scattered light

• Graphically / mathematically present the spectral data

Page 4: Raman spectroscopy - University of California, Irvinedmitryf/manuals/Fundamentals/Raman microsc… · Raman spectroscopy Information from Raman Spectroscopy characteristic Raman frequencies

Laser wavelength selection concerns for classical Raman

As the laser wavelength gets shorter

Raman scattering efficiency increases

The risk of fluorescence increases (except deep UV)

The risk of sample damage / heating increases

The cost of the spectrometer increases

Page 5: Raman spectroscopy - University of California, Irvinedmitryf/manuals/Fundamentals/Raman microsc… · Raman spectroscopy Information from Raman Spectroscopy characteristic Raman frequencies

Raman light source

System basics: lasers

1) UV lasers

2) visible lasers

3) NIR lasers

Common excitation wavelengths

244 nm- biological, catalysts(Resonance Raman)

325 nm- wide bandgap semiconductors

488 nm & 514 nm- semiconductor, catalysts,biological, polymers, minerals & generalpurpose

633 nm- corrosion & general purpose

785 nm - polymers, biological & generalpurpose

830 nm- biological

Page 6: Raman spectroscopy - University of California, Irvinedmitryf/manuals/Fundamentals/Raman microsc… · Raman spectroscopy Information from Raman Spectroscopy characteristic Raman frequencies

• Illuminate a Sample with an Intense Single Frequency Light Source

• Measure the relative frequency shift of the inelastically scattered light

Generic Raman system flow diagram

detector

laser sample

diffractiongrating

Page 7: Raman spectroscopy - University of California, Irvinedmitryf/manuals/Fundamentals/Raman microsc… · Raman spectroscopy Information from Raman Spectroscopy characteristic Raman frequencies

Raman microscopy: Dispersive instrument basics

Multi-channelDetector

sampleHNF

laser

slitGrating

100

200

300

Cou

nts

System basics:

1) laser

2) Rayleigh rejection filter

3) grating (resolution)

4) CCD detector

Page 8: Raman spectroscopy - University of California, Irvinedmitryf/manuals/Fundamentals/Raman microsc… · Raman spectroscopy Information from Raman Spectroscopy characteristic Raman frequencies

18 8 -2 -12

14

4

-6

Dispersion

0 200

Intensity

The Renishaw Raman spectrometer is an imagingspectrograph

on-axis stigmatic design with a -70 oC Peltier cooledCCD detector. Advanced inverted mode, deep depletionand UV optimized detectors are available as options.

We can easily demonstrate the high quality imaging andsystem performance advantages as seen in the imageof the Si 520 cm-1 Raman band on the CCD detector.

Image of Si 520 cm-1

band

pixel number

pixe

l num

ber

Research Grade MicroRaman Spectrometer

Page 9: Raman spectroscopy - University of California, Irvinedmitryf/manuals/Fundamentals/Raman microsc… · Raman spectroscopy Information from Raman Spectroscopy characteristic Raman frequencies

Delivering the light

90 and 180 degree scattering

Porto notation

90 degree scattering x(z,z)y and

180 degree scattering x(z,z)x’

excitation direction (excitation polarization,

scattered polarization) scattering direction

The actual excitation and collection directions are the range of angles0 to γ

γ

mag N.A 2*γ(deg)

x5 0.12 11.5

x20 0.4 29

x50 0.75 97.2

x100 0.9 128.3

x

x’

y

γ

Delivering the light

Page 10: Raman spectroscopy - University of California, Irvinedmitryf/manuals/Fundamentals/Raman microsc… · Raman spectroscopy Information from Raman Spectroscopy characteristic Raman frequencies

Delivering the light (180 degree backscattering)

excitation

Raman

> 90% efficient

Holographic notch or edge filter

2

1

Delivering the light

Raman microscope systems typicallyoperate in with the excitation direction andcollected Raman scattering directionseparated 1800. This mode of collectionand excitation is referred to as “back-scattering”.

Typically back-scattered Raman collectionnecessitate special optics that operateboth as a Rayleigh filter and as a lasermirror. Holographic notch filters andspecial dielectric mirrors are often theoptics of choice, since they minimize laserintensity loss and Raman scattering lossesthat would otherwise occur when utilizing apartial reflector.

Relative laser excitation efficiency andRaman transmission efficiencies can beeasily calculated for most configurations

Page 11: Raman spectroscopy - University of California, Irvinedmitryf/manuals/Fundamentals/Raman microsc… · Raman spectroscopy Information from Raman Spectroscopy characteristic Raman frequencies

The minimum laser focus isdetermined by:

1. the focusing optic N.A.2. laser wavefront (distortionor M2)3. How the back aperture ofthe objective is filled

Laser focused spot size

Delivering the light

Raman spectroscopy utilizing a microscopefor laser excitation and Raman lightcollection offers that highest Raman lightcollection efficiencies.

When properly designed, Ramanmicroscopes allow Raman spectroscopywith very high lateral spatial resolution,minimal depth of field and the highestpossible laser energy density for a givenlaser power.

It is important to note that the laserminimum focused spot size is not typicallythe same size as the coupled Ramanscattered spot size.

The minimum laser focused spot size isoften compromised by improperly matchingthe laser size to the back aperture of anobjective and by wavefront errors inherentto the laser and introduced by the laserdelivery optics.

Page 12: Raman spectroscopy - University of California, Irvinedmitryf/manuals/Fundamentals/Raman microsc… · Raman spectroscopy Information from Raman Spectroscopy characteristic Raman frequencies

Without consideration of the laser mode quality and wavefront, orsource size the minimum laser focused spot for any optic isdescribed by equation 1:

Minimum laser focus

1) Excitation wavelength: λ

2) effective numerical aperture : N.A.

3) dl is determined by twice the Rayleigh criteria of the adjacent distancerequired to spatially resolve the presence of an identical size spots

Laser focused spot size

..

*22.1

ANdl

λ=

Delivering the light

N.A. 514.5 7850.12 2.72 4.150.25 1.31 1.990.4 0.82 1.250.75 0.44 0.660.9 0.36 0.55

dl

Page 13: Raman spectroscopy - University of California, Irvinedmitryf/manuals/Fundamentals/Raman microsc… · Raman spectroscopy Information from Raman Spectroscopy characteristic Raman frequencies

The laser focused spotsize does not necessarilydefine the lateral spatialresolution of the Ramansystem. The lateralspatial resolution, is oftendiscussed in terms of theRayleigh criteria for thecollected Raman light.The Rayleigh criteriarequires that the distancebetween two pointssources of light of equalintensity be greater thanthe distance from thepeak to the first airy diskminimum. Completediscrimination of twoadjacent materials occursat twice the Rayleighcriteria

Laser focused spot size

Delivering the light

Airy disk pattern

0

0.2

0.4

0.6

0.8

1

1.2

-2.5 -1.5 -0.5 0.5 1.5 2.5

Distance/microns

Rel

ativ

e In

ten

sity

objective N.A.: 0.75

Excitation wavelength/nm: 514.5

Separation distance 0.44 um

Page 14: Raman spectroscopy - University of California, Irvinedmitryf/manuals/Fundamentals/Raman microsc… · Raman spectroscopy Information from Raman Spectroscopy characteristic Raman frequencies

Diffration limited focus

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

-4 -3 -2 -1 0 1 2 3 4

distance/microns

rela

tiv

e e

ne

rgy

de

ns

ity

x50 (0.75) x20 (0.40) x5 (0.10)

It’s important toremember that theobjective used todeliver the laser lightaffects the laserenergy density.

The relative energydensity and peakpower for the X5,X20 and X50objectives areshown relative tothe X50 objective.

The peak energydensity decreases by~50% for the X20and 87% for the X5objective

Airy disk calculation for X5, X20 and X50 objective calculated for 514.5 nm l

Page 15: Raman spectroscopy - University of California, Irvinedmitryf/manuals/Fundamentals/Raman microsc… · Raman spectroscopy Information from Raman Spectroscopy characteristic Raman frequencies

2

*53.2

=

ll D

fh λ

The system laser focus depth (hl) is determined by:

1) Excitation wavelength: λ

2) Microscope objective focal length : f

3) Effective laser beam diameter at the the objective backaperture: Dl

Laser focus and depth of field

Delivering the light

DO NOT CONFUSE LASER FOCUS DEPTH WITH CONFOCAL COLLECTION DEPTH

Page 16: Raman spectroscopy - University of California, Irvinedmitryf/manuals/Fundamentals/Raman microsc… · Raman spectroscopy Information from Raman Spectroscopy characteristic Raman frequencies

4

3*21.3

=

ll D

fλτ

The system laser focus volume (τl) is determined by:

1) Excitation wavelength: λ

2) Microscope objective focal length : f

3) Effective laser beam diameter at the the objective backaperture: Dl

Laser focus and illuminated volume

Delivering the light

DO NOT CONFUSE LASER FOCUS VOLUME WITH CONFOCAL COLLECTION VOLUME

Page 17: Raman spectroscopy - University of California, Irvinedmitryf/manuals/Fundamentals/Raman microsc… · Raman spectroscopy Information from Raman Spectroscopy characteristic Raman frequencies

N.A. vs. Intensity

0

0.5

1

1.5

2

2.5

0 0.5 1 1.5

numerical aperture

Si

Ra

ma

n i

nte

ns

ity

Opaque sample

Objective N.A rel σ5x 0.12 0.0210X 0.25 0.0820X 0.4 0.250Xulwd 0.55 0.3750X 0.75 0.69100X 0.9 1100X oil 1.2 1.78

Solid collection angle is proportional to (N.A.)2 not 1/(f/#)^2

Measured vs. calculated

σ = 4/π *(N.A.)2

Collecting the light

Oil immersion objectiveincrease is likely due toreduced reflection losses

Page 18: Raman spectroscopy - University of California, Irvinedmitryf/manuals/Fundamentals/Raman microsc… · Raman spectroscopy Information from Raman Spectroscopy characteristic Raman frequencies

Relative collection volume

0

1

2

3

4

5

6

400 500 600 700 800 900

Wavelength (nm)

Re

lati

ve

vo

lum

e

Macro-sampling isimproved with longerwavelength excitation

Collecting the light

4

3*21.3

=

ll D

fλτ

The system laser focus volume (τl)

Page 19: Raman spectroscopy - University of California, Irvinedmitryf/manuals/Fundamentals/Raman microsc… · Raman spectroscopy Information from Raman Spectroscopy characteristic Raman frequencies

Extended scanning(Renishaw patent EP 0638788)

From the Renishaw Raman software the user can select:

• a fixed grating measurement with a spectrum 'window'of 400 cm-1 to 1000 cm-1 (configuration dependent)

• a unique 'extended scanning' facility allowing the userto choose any Raman shift range up to about 10000 cm-1

(configuration dependent). Essential for extended rangescanning for Raman and photoluminescence

Extended scanning is implemented by moving the grating andthe charge generated on the CCD camera synchronously.

This feature is NOT available on any other instrument and isKEY to system performance

Page 20: Raman spectroscopy - University of California, Irvinedmitryf/manuals/Fundamentals/Raman microsc… · Raman spectroscopy Information from Raman Spectroscopy characteristic Raman frequencies

CCD Basics

Page 21: Raman spectroscopy - University of California, Irvinedmitryf/manuals/Fundamentals/Raman microsc… · Raman spectroscopy Information from Raman Spectroscopy characteristic Raman frequencies

Extended scanning: how it works

Page 22: Raman spectroscopy - University of California, Irvinedmitryf/manuals/Fundamentals/Raman microsc… · Raman spectroscopy Information from Raman Spectroscopy characteristic Raman frequencies

Extended scanning vs stitched scanning

Advantages of extended scanninguse a single grating no stitching required and no “discontinuities” at joinsflexible wavenumber coverage (up to 10000 cm-1 )pixel-to-pixel variation is averaged out - enhancing noise reductionno compromise on resolution across the scanned rangesimple to use

Page 23: Raman spectroscopy - University of California, Irvinedmitryf/manuals/Fundamentals/Raman microsc… · Raman spectroscopy Information from Raman Spectroscopy characteristic Raman frequencies

Adequate S/N

To acquire useful Raman spectra all you need is:

Sufficient spectral and spatial resolution and coverage

Repeatability

The ability to collect and detect enough photons to distinguish theirelectronic signal above system generated noise before the sample changesor dies.

The ability to separate spectral peaks narrower than the narrowestanticipated spectral features of your sample

The ability to collect all of the spectral data required for the analysis

The ability to optically restrict the data collection to an area / volumesmall enough to eliminate acquisition of unwanted spectral data ofnearby substances

The ability to consistently get the same right or wrong values

Page 24: Raman spectroscopy - University of California, Irvinedmitryf/manuals/Fundamentals/Raman microsc… · Raman spectroscopy Information from Raman Spectroscopy characteristic Raman frequencies

Confocal Raman microscopy without pinhole optics

The use of a stigmatic spectrograph and stigmatic microscope-spectrometercoupling optics creates two additional conjugate image planes at the slit andCCD eliminating the need for pinhole optics!

Conjugate image planes - Square pinhole

Collection

optic

Preslit

focusing lens

Slit

grating

CC

D

CCD CCD

Slit

Slit

Spatial filtering

Confocal Raman collection

Page 25: Raman spectroscopy - University of California, Irvinedmitryf/manuals/Fundamentals/Raman microsc… · Raman spectroscopy Information from Raman Spectroscopy characteristic Raman frequencies

0

500

1000

Cou

nts

400 600 800 1000 1200 1400 1600 1800 2000 Raman Shift (cm-1)

Confocal 100X

Laser: 15802.78cm-1White Light Correction:

3: CONFO~15

αβχδη

50X

100X

100X oil

Si polymer2 um polymer film

Silicon Wafer

Higher numericalaperture objectiveseffectively eliminate theRaman spectrum ofunderlying layers!

Confocal Raman collection

Page 26: Raman spectroscopy - University of California, Irvinedmitryf/manuals/Fundamentals/Raman microsc… · Raman spectroscopy Information from Raman Spectroscopy characteristic Raman frequencies

Spectral resolution and coverage are controlled by focal length and groove density

Page 27: Raman spectroscopy - University of California, Irvinedmitryf/manuals/Fundamentals/Raman microsc… · Raman spectroscopy Information from Raman Spectroscopy characteristic Raman frequencies

Spectrometer issues associated with different excitations

Shorter wavelength excitation requires higher dispersion spectrometers andproduce higher levels of stray light in the system.

1 nm is equivalent to:

160 cm-1 @ 250 nm excitation 38 cm-1 @ 514 nm excitation 94 cm-1 @ 325 nm excitation 16 cm-1 @ 785 nm excitation

Page 28: Raman spectroscopy - University of California, Irvinedmitryf/manuals/Fundamentals/Raman microsc… · Raman spectroscopy Information from Raman Spectroscopy characteristic Raman frequencies

System parameters that affect spectral resolution and coverage

• The dispersion of the spectrometer– Focal length– Grating groove density

•Multiple gratings• for resolution/coverage trade-off•For using multiple excitation wavelength

– Grating rotation– Optical aberrations in the spectrometer– Mutichannel Detector

•Pixel size•Width (under certain circumstances)

– Laser•Wavelength stability•Wavelength choice

Page 29: Raman spectroscopy - University of California, Irvinedmitryf/manuals/Fundamentals/Raman microsc… · Raman spectroscopy Information from Raman Spectroscopy characteristic Raman frequencies

Raman microscopy: spectral resolution

Spectrometer resolution

The slit-width determined resolution of the spectrometer isdetermined by the convolution of the entrance slit with theCCD pixel.

ccd

Spectral lines

spectrum

Page 30: Raman spectroscopy - University of California, Irvinedmitryf/manuals/Fundamentals/Raman microsc… · Raman spectroscopy Information from Raman Spectroscopy characteristic Raman frequencies

10000

15000

20000

25000

30000

35000

40000

Co

un

ts

60 70 80 90 100 110 Raman Shift (cm-1)

Time: 300secs

File # 1 : BBF

Air 15 um slit Laser: 15803cm-1

The air spectrum shows thatsystem resolution is limited to~0.7 cm-1 FWHM utilizing 633 nmexcitation.

O2 & N2

Spectrometer resolution is best determined by measuring the air spectrum

Raman microscopy: spectral resolution

Page 31: Raman spectroscopy - University of California, Irvinedmitryf/manuals/Fundamentals/Raman microsc… · Raman spectroscopy Information from Raman Spectroscopy characteristic Raman frequencies

50000

100000

150000

200000

Co

un

ts

40 60 80 100 120 140 160 180 200 220 Raman Shift (cm-1)

Time: 300secs

File # 3 : BBJ

Air 100 um slit Laser: 15803cm-1

100 um

30 um15 um

Raman spectrum of air30 min, 7 mW, 633 nm.

Resolution 3.4 cm-1

vs.Resolution 0.70 cm-1

When the spectrometer determines measured spectral linewidths

Increasing the entrance slit increases light throughput but decreases resolution

Page 32: Raman spectroscopy - University of California, Irvinedmitryf/manuals/Fundamentals/Raman microsc… · Raman spectroscopy Information from Raman Spectroscopy characteristic Raman frequencies

0

100000

200000

300000

400000

Co

un

ts

380 400 420 440 460 480 500 520 Raman Shift (cm-1)

Time: 1secs

File # 3 : BBK

CCl4 100 um Laser: 15803cm-1

Trading spectral resolution for throughput

Raman spectrum ofCCl4, 10 - 1 secaccumulations withdifferent slit setting

Intensity increases 7fold, resolutiondecreases 5 fold

100 um

30 um

15 um

Increasing the entrance slit increases throughput but decreases resolution

2400 l/mm grating

Page 33: Raman spectroscopy - University of California, Irvinedmitryf/manuals/Fundamentals/Raman microsc… · Raman spectroscopy Information from Raman Spectroscopy characteristic Raman frequencies

0

.2

.4

.6

.8

1

1.2

1.4

Co

un

ts

400 600 800 1000 1200 1400 1600 1800 2000 Raman Shift (cm-1)

Time: 10secs

File # 1 : BBO

Caff 100 um match step and repeat Laser: 15803cm-1

Sample determines measured spectral linewidths

The caffeine Ramanspectrumidentical laser powers

Resolution 7 cm-1

(600 l/mm)

Resolution 3.5 cm-1

(2400 l/mm)

Single static scan with 600 l/mm, 10 stitched static scans with 2400 l/mm

10 sec

~2 min

Page 34: Raman spectroscopy - University of California, Irvinedmitryf/manuals/Fundamentals/Raman microsc… · Raman spectroscopy Information from Raman Spectroscopy characteristic Raman frequencies

0

.1

.2

.3

.4

.5

.6

.7

Co

un

ts

1050 1100 1150 1200 1250 1300 1350 1400 1450 Raman Shift (cm-1)

Time: 10secs

File # 1 : BBO

Caff 100 um match step and repeat Laser: 15803cm-1

600 l/mm gratingDecreases measurement timean order of magnitude,

Increases S/N an > order ofmagnitude.

Spectral resolution isdetermined by the sample

Sample determines measured spectral linewidths

Matching the spectrometer resolution and the and CCD pixel resolution to the natural linewidthsof the sample optimizes S/N

Page 35: Raman spectroscopy - University of California, Irvinedmitryf/manuals/Fundamentals/Raman microsc… · Raman spectroscopy Information from Raman Spectroscopy characteristic Raman frequencies

System parameters that affect S/N

• Laser– Power– Wavelength– Modality– Stability– Delivery optics

• Collection Optics– Aperture– Focus

•Diffraction limited spot size

– Transmission– Robustness

Page 36: Raman spectroscopy - University of California, Irvinedmitryf/manuals/Fundamentals/Raman microsc… · Raman spectroscopy Information from Raman Spectroscopy characteristic Raman frequencies

Factors affecting S/N

Page 37: Raman spectroscopy - University of California, Irvinedmitryf/manuals/Fundamentals/Raman microsc… · Raman spectroscopy Information from Raman Spectroscopy characteristic Raman frequencies

Select a CCD for best Raman performance

What limits the CCD performance?

1. Read noise: How many photon generated electrons are required to achievea signal level greater than the read noise?

Raman systems that require off chip binning increase read noise to thesquare root of the number of pixels binned.

2. Dark Charge rate: How long can you integrate before the binned CCDpixels generate a charge equivalent to the read noise?

At the integration time that the dark charge signal contributes to the noiseeither through shot noise or uniformity of response, it must be subtracted.

3. Uniformity of response: How many photon generated electrons can bemeasured before the shot noise is exceeded by the non-uniformity ofresponse?

At the point uniformity of response noise exceeds shot noise the pixels mustbe read out individually (without binning) for response correction.

Page 38: Raman spectroscopy - University of California, Irvinedmitryf/manuals/Fundamentals/Raman microsc… · Raman spectroscopy Information from Raman Spectroscopy characteristic Raman frequencies

Optimal CCD operating temperature

The best CCD temperature operation is determined by the CCD darkcharge rate and the requirements for operation near the detectorlimit of ~1050 nm

• Low temperatures decrease the CCD dark charge rate. The CCD darkcharge rate decreases ~50% for each 6-9 degree decrease in operatingtemperature.

e*T*122 QdQd T

-64003

0

=

Dark Charge e/p/s

0.001

0.01

0.1

1

10

-80 -60 -40 -20 0

Temperature C

Dar

k C

harg

e ra

te e

/p/s

Qd dark charge rate (e/p/s) atoperating temperature T

Qdo - dark charge rate at reference

temperature (typically 23-25 oC)

A1A-CCD02-06 Deep Depletion Sensor Issue 3, January2000

Page 39: Raman spectroscopy - University of California, Irvinedmitryf/manuals/Fundamentals/Raman microsc… · Raman spectroscopy Information from Raman Spectroscopy characteristic Raman frequencies

Select response uniformity rather than QE

Renishaw CCDtypicalresponsecurve. Thepeak QE is~50%, butthe responseuniformity isan order ofmagnitudebetter thanwith higher QECCD chips

Select a CCD for best Raman performance

Page 40: Raman spectroscopy - University of California, Irvinedmitryf/manuals/Fundamentals/Raman microsc… · Raman spectroscopy Information from Raman Spectroscopy characteristic Raman frequencies

StreamLine™

• StreamLine™ technology– Unique Renishaw technology

(patent pending)

– Combination ofhardware and software

– Enables very fastRaman imaging of samples

• Application areas– Pharmaceuticals

– Materials science

– Semiconductors

– Polymers

– Biosciences

– etc.

Page 41: Raman spectroscopy - University of California, Irvinedmitryf/manuals/Fundamentals/Raman microsc… · Raman spectroscopy Information from Raman Spectroscopy characteristic Raman frequencies

Spectral imaging

• Acquire data from different pointson the sample.

• Generate maps based onparameters of resulting spectra.Examples:

– Univariate: intensity of band

– Multivariate: chemometrics:

• Component analysis based onreference samples

• Principal component analysis (noreferences)

Page 42: Raman spectroscopy - University of California, Irvinedmitryf/manuals/Fundamentals/Raman microsc… · Raman spectroscopy Information from Raman Spectroscopy characteristic Raman frequencies

• Measure the Raman spectrum of ~1 µm Siparticle with 1 µm laser spot (backlash of 10 µmenabled)

• Move away from then return to the particle torepeat the Raman measurement (32 times eachdirection)

• Compare performance of the motorized mappingstage with the 0.1 µm encoders on to theperformance with the encoders off

Specification:

unencoded 2 µm

encoded 0.3 µm

Mapping stage repeatability

Page 43: Raman spectroscopy - University of California, Irvinedmitryf/manuals/Fundamentals/Raman microsc… · Raman spectroscopy Information from Raman Spectroscopy characteristic Raman frequencies

The Si particle

Page 44: Raman spectroscopy - University of California, Irvinedmitryf/manuals/Fundamentals/Raman microsc… · Raman spectroscopy Information from Raman Spectroscopy characteristic Raman frequencies

Raman line mapping

• Method– Generate laser line on sample

– Simultaneously acquire spectrafrom positions along the line

– Move line over sample,perpendicular to its length

• Advantages– Larger area illuminated by line

• Disadvantages– Stop/start movement overhead

– Artefacts…line uniformity

Page 45: Raman spectroscopy - University of California, Irvinedmitryf/manuals/Fundamentals/Raman microsc… · Raman spectroscopy Information from Raman Spectroscopy characteristic Raman frequencies

StreamLine™

• Move line the other way!

• Synchronise the stage and thedetector

• Advantages– Smooth fast continuous movement

– Artefacts eliminated

– Large area illuminated by line

Page 46: Raman spectroscopy - University of California, Irvinedmitryf/manuals/Fundamentals/Raman microsc… · Raman spectroscopy Information from Raman Spectroscopy characteristic Raman frequencies

Features of StreamLine™ imaging

• StreamLine™ offers:

– Power density up to 100x less than point laser configurations

– No joining or uniformity artefacts

– Macro (whole tablet) and micro (<1 µm) sampling capabilities

– Zero dead time between sequential spectral acquisitions

– Confocal information maintained

– High spectral resolution options available

– Multi-wavelength capabilities from the UV to NIR

– High speed with unparalleled data quality

Page 47: Raman spectroscopy - University of California, Irvinedmitryf/manuals/Fundamentals/Raman microsc… · Raman spectroscopy Information from Raman Spectroscopy characteristic Raman frequencies

StreamLine™

• Technology for very fast Ramanimaging

• Unique Renishaw technology(patent pending)

• Innovative hardware, uniquesoftware

• Collect excellent quality data wherepoint by point would damagesample

• Parallel data readout, synchronisedwith sample movement

100µm

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StreamLine™ imaging: how it works?

– Each sample point passes beneath each part of the laser line

– The charge is stepped synchronously with the stage movement

– Parallel data readout

Page 49: Raman spectroscopy - University of California, Irvinedmitryf/manuals/Fundamentals/Raman microsc… · Raman spectroscopy Information from Raman Spectroscopy characteristic Raman frequencies

White-light montage

• Used for detailed sample survey

• 4_4 white-light montagegenerated with 50_ objective

• Montage can then be used todefine multiple imagingexperiments

• Multiple experiments may bequeued

• White-light image saved withspectral data

Page 50: Raman spectroscopy - University of California, Irvinedmitryf/manuals/Fundamentals/Raman microsc… · Raman spectroscopy Information from Raman Spectroscopy characteristic Raman frequencies

StreamLine™ in action

• Example StreamLine™ video

• Silicon target with metallisedstructure, imaged with 532nm laser

• 4 x 4 montage white-light imageused to define experimental area

• Imaged area bigger than 50xobjective field of view

• Live imaging of Si 520cm-1 band

• Bicubic interpolation applied toimage during acquisition

Page 51: Raman spectroscopy - University of California, Irvinedmitryf/manuals/Fundamentals/Raman microsc… · Raman spectroscopy Information from Raman Spectroscopy characteristic Raman frequencies

Metallised Si target analysis

• Line profile along the Y axis

• Feature size approximately 5 µm

Page 52: Raman spectroscopy - University of California, Irvinedmitryf/manuals/Fundamentals/Raman microsc… · Raman spectroscopy Information from Raman Spectroscopy characteristic Raman frequencies

Image quality comparison

Image quality comparison

StreamLine™ 1 minute, 40 seconds Point by point 1hour, 6 minutes

• Same area on tablet analysed using point by point and StreamLine™ techniques

• Image generated using direct classical least square (DCLS) multivariate method

• Image shows distribution of Aspirin, Paracetamol and Caffeine components

• Identical data quality

Page 53: Raman spectroscopy - University of California, Irvinedmitryf/manuals/Fundamentals/Raman microsc… · Raman spectroscopy Information from Raman Spectroscopy characteristic Raman frequencies

Image of complete tooth section

• First ever whole tooth Raman image

• Different regions clearly indentified

• Color coding:– Yellow: enamel– Green: dentine– Red: fluorescent areas

• Details– 9 mm x 16 mm area– 84,024 spectra– 20 minutes– 785 nm excitation

Page 54: Raman spectroscopy - University of California, Irvinedmitryf/manuals/Fundamentals/Raman microsc… · Raman spectroscopy Information from Raman Spectroscopy characteristic Raman frequencies

Spectrum from dentine region of tooth

Page 55: Raman spectroscopy - University of California, Irvinedmitryf/manuals/Fundamentals/Raman microsc… · Raman spectroscopy Information from Raman Spectroscopy characteristic Raman frequencies

Dental caries polarisation images

• Image is a ratio of two data sets:– Parallel-polarisation– Crossed-polarisation

• Color coding:– Red: strong polarisation dependence -

Sound enamel– Green: weak polarisation dependence -

Carious region

• Details– 1.5 mm x 3.4 mm area– 42,642 spectra– 27 minutes– 785 nm excitation

• StreamLine is compatible with the fullrange of spectral options available forthe inVia Raman microscope

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Dental caries curve-fit analysis image

• Curve fit data analysis onP-O symmetric stretchband

– Peak width– Peak position

• Details– 1.5 mm x 3.4 mm area– 42,642 spectra– 27 minutes– 785 nm excitation

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Whole ink character imaging

• 43,400 spectra

• 29 minutes

• 514 nm

• 20x objective

• 30 µm spatial resolution

• Image created usingcomponent method

White light montage

Raman ink image

First ever whole ink character Raman image

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Whole ink character imaging

High quality ink spectrum from image (noise filtered)

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Whole ink character imaging

Image can also reveal pen contact on paper

Blue – paper

Red – paper with pen contactaway from ink

Useful information provided onpen angle during stroke

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Crossed ink example 1: dual crossed ink lines

• Details– 1040 µm (X) by 2607 µm (Y)

– 90,440 spectra

– 90 minutes total time

– 514 nm excitation

– 20x objective

Double crossed line exampleusing two black ball point pens

Image collected of all lines and paper inone experiment

First ever Raman crossed lineimage

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Crossed ink example 1: dual crossed ink lines

Component Raman image ofink 1 (green)

Shows smearing of top horizontal line

Also the image provides information onthe direction of the crossing

This suggests that the top ink 1 line isbeneath a further line crossing it.

Directionof

smearing

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Crossed ink example 1: dual crossed ink lines

Component Raman image overlayfrom WiRE 3 – confirms order ofdeposition

Top cross – Ink 1 (green) under ink 2

Bottom cross – Ink 2 (red) under ink 1

Ink 1 (green)Ink 2 (red)Paper (blue)

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Crossed ink example 2: single crossed ink lines

• Details– 990 µm (X) by 1430 µm (Y)

– 47,422 spectra

– 33 minutes total time

– 514 nm laser wavelength

– 20x objective

Single crossed line example using two black ball point pens

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Crossed ink example 2: single crossed ink lines

Component Raman imageoverlay from WiRE 3superimposed on white lightimage

Cross – Black ink 1 (green) over blackink 2 (red)

Black ink 1 (green)Black ink 2 (red)

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Crossed ink example 2: spectra from image

Black ink 1 (green)

Black ink 2 (red)

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Raman and PL imaging of polycrystalline CVD diamond film

• CVD diamond sample grownonto silicon substrate usingchemical vapour deposition(CVD) method

• Rough growth surface issubsequently polishedoptically flat

• Raman andphotoluminescencemeasured in sequentialexperiments

• Curve-fit analysis used forimage generation

• 18,000 spectra collected in 6minutes

Diamond peak position

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Raman and PL imaging of polycrystalline CVD diamond film

1332 cm-1

Diamond

1.68 eV [Si-V]0

(intensity capped)

1.95 eV[N-V]-

1.77 eV

Raman range

Photoluminescence range

• Standard spectralresolutionconfiguration usedfor Raman bands,lower resolutionused forphotoluminescencefeatures

Details of spectral analysis

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Raman and PL imaging of polycrystalline CVD diamond film

• 50,000 spectra collected in 15 minutes with 0.5 micrometer resolution

• Sequence derived from 2 imaging experiments (Raman and Photoluminescence)

• A and B highlight two areas of highly twinned crystal facets exhibiting five-foldsymmetry

Position of 1332 cm-1 Raman bandWidth of 1332 cm-1 Raman bandArea of 1332 cm-1 Raman bandIntensity of 1.68 eV [Si-V]0 PL band

A B

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Diamond nucleation region

• Diamond film– nucleation side of a free-standing

diamond film

• Map shows:– Top image: Diamond-rich area

• 1332cm-1 diamond band component

– Bottom image: Graphitic-rich region• G and D bands graphite components

• Details– 110 µm _ 110 µm area

– 10,000 spectra

– 10 minutes

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Si-Ge cross hatch

• Semiconductor sample– SiGe layer deposited onto substrate– Graded layer with increasing

germanium content towards layersurface

– Crosshatch structure is notengineered

– Pattern is generated a mechanismfor strain relief

• Map shows:– Variation in Si-Si 510 cm-1 band

position (~ 0.2 cm-1 positional bandshift)

• Details– 55,000 spectra– 13 minutes– 0.5 micrometer resolution– 532 nm excitation

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Carbon nanotube lattice structures

SWNT Raman spectrum

MWT

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If the wavelength of the laser is close to an excited electronic state of a bond in molecule, i.e. where it is strongly absorbed or fluoresces, the signal enhancement can be increased by a factor between 100 and 10,000.

Resonance Raman

Advantage: You can select a wavelength to enhance the sensitivity toa particular type of bond or vibrations.

Potential disadvantages: increased fluorescence increase absorption/heating

In the study of carbon nanotubes multiple laserwavelengths are used to increase sensitivity tospecific vibrational modes within a molecule.

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0

.2

.4

.6

.8

1

Cou

nts

500 1000 1500 2000 Raman Shift (cm-1)Time: 10secs

File # 1 : RICE(8)C

Solid Film Laser: 12834.5cm-1

244 nm and 780 nm excited Raman spectra of nanotubes

244 nm

RBMC-C

Resonance Raman spectroscopy of SWNT

Since the Raman spectral measurment for nano-tubes is typically a resonance Raman measurementthe excitation wavelength can dramatically affect the spectral feature intensity and shift.

785 nm

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Resonance Raman of SWINTs

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500 1000 1500 2000 2500 3000 3500

Raman shift / cm-1

0

5000

10000

15000

20000

25000

Cou

nts

514 nm excited and 488 nm excited Raman spectra MWNT material

Raman spectroscopy of MWNT

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Raman and Fraud

Lewis, I. R.; Edwards, H. G. M., Lewis, I. R.; Edwards, H. G. M., Handbook of Raman Spectroscopy: From the Research Laboratory to theHandbook of Raman Spectroscopy: From the Research Laboratory to theProcess Line, Process Line, Marcel Dekker, New York: 2001.0Marcel Dekker, New York: 2001.0

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Ivory or Plastic?

Lewis, I. R.; Edwards, H. G. M., Lewis, I. R.; Edwards, H. G. M., Handbook of Raman Spectroscopy: From the ResearchHandbook of Raman Spectroscopy: From the ResearchLaboratory to the Process Line, Laboratory to the Process Line, Marcel Dekker, New York: 2001.Marcel Dekker, New York: 2001.

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The Vinland Map: Genuine or Forged?

Brown, K. L.; Clark, J. H. R., Brown, K. L.; Clark, J. H. R., Anal. Chem. Anal. Chem. 20022002, 74,, 74,3658.3658.

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The Vinland Map: Forged!

Brown, K. L.; Clark, J. H. R., Brown, K. L.; Clark, J. H. R., Anal. Chem. Anal. Chem. 20022002, 74,, 74,3658.3658.

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Surface-Enhanced Raman Scattering (SERS)

Haynes, McFarland, and Van Duyne, Anal. Chem.,77, 338A-346A (2005).

SERS: Surface Enhanced Raman Scattering

Discovered in 1977, Jeanmire et al. & Albrecht et al.

--Strongly increased Raman signals from molecules attachedto metal nanostructures

--SERS active substrates: metallic structures with sizeabout 10--100 nm (e.g. colloidal Ag, Au, roughened surfaces)

General contributions:

1)Electromagnetic field enhancement

2) Chemical ‘first layer’ effect

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SERS Enhancement Mechanisms

Chemical Mechanism:Laser excites (a) new electronic states arising fromchemisorption or (b) shifted or broadened adsorbateelectronic states yielding a resonance condition.

• Short range (1-5 Å)• No roughness requirement• Contributes EF ~ 102 – 104

Electromagnetic Mechanism:LSPR induces large electromagnetic fields at roughenedmetal surface where molecules are adsorbed.

• Long range (2-4 nm)• Affected by all factors determining LSPR• Contributes EF > 104

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Localized Surface Plasmon Resonance

The resonance results in (1) wavelength-selective extinction and (2) enhanced EMfields at the surface.

Spectral location of the LSPR is dependent upon particle size, shape, composition,and dielectric environment.

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Localized Surface Plasmon Resonance

Non-resonant Resonant

1) Resonant λ is absorbed2) EM fields localized at nanoparticle surface

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Nanostructured Substrates

http://pubs.acs.org/cgi-bin/article.cgi/ancham-a/0000/77/i17/pdf/905feature_vanduyne.pdfhttp://pubs.acs.org/cgi-bin/article.cgi/ancham-a/0000/77/i17/pdf/905feature_vanduyne.pdf

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Commercial SERS Substrates

D3 produces the Klarite range of substrates for SurfaceEnhanced Raman Spectroscopy. Klarite substrates enable faster,higher accuracy detection of biological and chemical samples atlower detection limits for a wide range of applications inhomeland security, forensics, medical diagnostics andpharmaceutical drug discovery. Manufactured using techniquesfrom semiconductor processing Klarite substrates offer highlevels of enhancement and reliability.

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References

Raman Microscopy: Developments and ApplicationsRaman Microscopy: Developments and Applications, G., G. Turrell Turrell, J. Corset,, J. Corset, eds eds. (Elsevier Academic Press,. (Elsevier Academic Press,1996)1996)

Introductory Raman SpectroscopyIntroductory Raman Spectroscopy, J.R. Ferraro, K., J.R. Ferraro, K. Nakamoto Nakamoto, C.W. Brown, Academic Press, 2003., C.W. Brown, Academic Press, 2003.

Raman Spectroscopy for Chemical AnalysisRaman Spectroscopy for Chemical Analysis, R.L., R.L. McCreery McCreery (Wiley (Wiley Interscience Interscience, 2000)., 2000).

Handbook of Raman SpectroscopyHandbook of Raman Spectroscopy, I.R. Lewis, H.G.M. Edwards,, I.R. Lewis, H.G.M. Edwards, eds eds. (Marcel. (Marcel Dekker Dekker, 2001), 2001)

Raman Technology for TodayRaman Technology for Today’’ss Spectroscopists Spectroscopists, 2004 Technology primer, Supplement to Spectroscopy, 2004 Technology primer, Supplement to Spectroscopymagazine.magazine.

FT Raman spectroscopy, P. Hendra et al., Ellis Horwood.

Raman and IR spectroscopy in biology and chemistry, J. Twardowski and P. Anzenbacher, Ellis Horwood.

Ch 18 in Skoog, Holler, Nieman, Principles of Instrumental Analysis, Saunders.

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Raman Websites and On-Line Databases

www.spectroscopynow.com/coi/cda/landing.cda?chId=6&type=Education(many links including,An Introduction to Raman Spectroscopy: Introduction and Basic Principles, by J. Javier Laserna,)

Database:http://wwwobs.univ-bpclermont.fr/sfmc/ramandb2/index.html

Vendors:http://www.renishaw.comhttp://www.optics.bruker.com/http://www.nicolet.com/http://www.jobinyvon.com