2013-­‐09-­‐23	  OutlineInfrared Spectroscopy
withSynchrotron Sources

September 30, 2013
Cheiron 2013 School
SPring-8
Hyogo, Japan

Ferenc Borondics
Mid-IR beamline
Canadian Light Source
Saskatoon, Canada SK• IR spectroscopy – basic theory• IR instrumentation• Some IR beamlines (CLS, ALS, NSLS, SRC…)• Exotic techniques – recent developments• Practical things• Examples

Cheiron 2013

Something to read…

IR beamlines around the worldFive	
  volumes	  Source: http://infrared.als.lbl.gov

1	  2013-­‐09-­‐23	
  

Sir William Herschel (1738-1822)
Composer Feb. 11, 1800
Astronomer
UranusOberonTitania
Mimas
Enceladus

W. Herschel, Phil. Trans. R. Soc. London, Vol 90, 284-292, (1800)

With numbers…
Near-IR
Mid-IR
Far-IR

12500 - 4000 cm-1
4000 - 500 cm-1
500 - 5 cm-1

Processes
0.8 - 2.5 µm
2.5 - 20 µm
20 - 2000 µm

What can we look at?
•••

Broad band electronic excitations
RotationsVibrations

1.55 - 0.5 eV
0.5 - 0.06 eV
60 - 0.6 meV

I

TR

Sample• Transmission• Absorption• Reflection2	  2013-­‐09-­‐23	  An infrared spectrum

Basic optical functions

Transmittance

Goal: determine ε(ω), the dielectric function
Information about the electronic structure

Measure: reflectivity, r(ω); transmittance, T(ω)

JAll of them are related

EnergyBasic optical functions
Goal: determine ε(ω), the dielectric function
Information about the electronic structure

Measure: reflectivity, r(ω); transmittance, T(ω)

LJAll complex functions
Kramers Kronig transformation

Studies on low energy gaps
conduction bande-h+valence band
Generalsemiconductor

Graphene3	
  2013-­‐09-­‐23	
  Energy gaps of semiconductors

Models to understand εDrude model

conduction band
e1.0

h

-2

-1

0

Energy / eV

valence band
Generalsemiconductor

Nanotubes

1

2

Reflectivity

0.8

+

0.6

0.4

0.2

0.0

0

1

2

3

4

E / eV

Models to understand ε

Molecular vibrations
Diatomic molecule, harmonic oscillator

Drude – Lorentz model

1.0

Reflectivity

0.8

0.6

m1

0.4

0.2

0.0

0

1

2

E / eV3

4m2

BH 2400 CH 3000 NH 3400 OH 3600 FH 4000
2.04
2.55
3.04
3.44
3.98
OH 3600 OD 26004	
  

2013-­‐09-­‐23	
  Molecular vibrations

Diatomic molecule, realistic case (solution)

Intensity

Intensity

Diatomic molecule, ideal case (alone)

Molecular vibrations

Frequency
ocw.mit.edu – 5.61 – Physical Chemistry

Frequency
ocw.mit.edu – 5.61 – Physical Chemistry

Molecular vibrations

Molecular vibrations
Quantized oscillator
Energy levels

Intensity

Diatomic molecule, realistic case (solution)

Frequency
ocw.mit.edu – 5.61 – Physical Chemistry5	
  2013-­‐09-­‐23	  Anharmonicity

Anharmonicity

Diatomic molecule, anharmonic

Diatomic molecule, anharmonic
0->1

Intensity

Intensity

0->1

0->2

1->2

1->2

2->3

2->3

1->3
2->4

Frequency

Frequency
ocw.mit.edu – 5.61 – Physical Chemistryocw.mit.edu – 5.61 – Physical ChemistryMore atoms# of molecular vibrations

Polyatomic molecule, ideal case, harmonic
ω1

ω3

ω4

ω2

3N-6 degrees of freedomor
3N-5 for linear molecules

Intensity

ω5

H2O ➙ 3x3-6=3
CO2 ➙ 3x3-5=4

3.0

H2O

Absorbance

2.5
2.0
1.5
1.0
0.5

Frequency

0.0

500

1000

1500

2000

2500

3000

3500

-1

Wavenumber / cm
ocw.mit.edu – 5.61 – Physical Chemistry6	
  2013-­‐09-­‐23	  More atoms

Larger molecules?

Polyatomic molecule, real case, anharmonic
ω1

ω5

ω2+ω4−ω1Intensity

ω2

ω4

ω5−ω1

ω3

2∗ω1

Chromophores

Frequency

Holt et al. Biochem J. 314, 1035-1039 (1996)

ocw.mit.edu – 5.61 – Physical Chemistry

IR active vibrations
Selection Rules IR:

Simplifying factors

Raman:

Group	  theory	
  Example: H2O• Chromophore groups• Selection rules• Symmetry

If there is an inversion symmetry IR≠Raman7	
  2013-­‐09-­‐23	
  Characteristic frequencies

Influencing factors
Shifts• Atomic masses• Bond order (single, double, triple)• Electronegativity• Symmetry – molecular or crystal• Solvent effects, hydrogen bonding• Conformational changes

www.brukeroptics.com/downloads.html

RotationsRotational spectrum example
Nitrous Oxide

R-BRANCH

P-BRANCH

Q-BRANCH

Criterion:	
  molecule	
  must	  have	  permanent	
  dipole	
  moment	
  

	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
34 31 29 26 24 21 19 17 14 12 09 07 05 02 00 97 95 93 90 88 85 83 80 78 76 73 71 68 66 64 61 59 56
2,8 2,8 2,8 2,8 2,8 2,8 2,8 2,8 2,8 2,8 2,8 2,8 2,8 2,8 2,8 2,7 2,7 2,7 2,7 2,7 2,7 2,7 2,7 2,7 2,7 2,7 2,7 2,7 2,7 2,7 2,7 2,7 2,7Δj=-­‐1	
   Δj=0	
   Δj=+1	
  Frequency / cm-1
Note: spectral range è high resolution8	
  2013-­‐09-­‐23	
  Spectrometer designs

Spectrometer designs
Fourier Transform
Michaelson interferometer

Dispersive

3.0
2.5

Slow, but simple
Resolution, throughput, etc…

2.0
1.5
1.0
0.5
0.0

500

1000

1500

2000

2500

3000

3500

-1

Wavenumber / cm

FT spectroscopy advantages••••

A spectrometer layout

Connes advantage: internal calibration He/Ne
Jacquinot advantage: interferometer is bright
Fellgett advantage: all n in the same time
No stray light∑: FT is 2000x more sensitive than dispersive

A – source
B – beamsplitterC – sample
D - detector9	
  2013-­‐09-­‐23	
  

FT-IR

Resolution

Michaelson interferometer

Wavelenth	
  

FT:	
  
discrete	
  

3.0
2.5

Nyquist:	  2.0

Resolution!

1.5

FT	
  

1.0
0.5
0.0

500

1000

1500

2000

2500

3000

3500

-1

Wavenumber / cm

Size matters

SoluNons:	
  

Zero	
  ﬁlling	
  –	
  looks	
  nicer,	
  but	
  useless	
  Pathlength	
  –	
  real	  informaNon	
  Conventional Light Sources

Bruker Alpha

Bruker 125hr

A4 paper size

Moving mirror - meters
http://infrared.als.lbl.gov

www.brukeroptics.com/downloads.html

10	
  2013-­‐09-­‐23	  Detectors

www.brukeroptics.com/downloads.html

Beamsplitterswww.brukeroptics.com/downloads.html

Producing IR light

IR Sources- a comparison -Black body radiation
GLOBAR – SiC rod, resistive heating11	
  2013-­‐09-­‐23	  Producing IR light

Producing IR light

Using moving charges
Dipole radiation

http://ocw.mit.edu
D. Attwood, UC Berkeley, http://ast.coe.berkeley.edu/srms/2007/Intro2007.pdf

Producing IR light

D. Attwood, UC Berkeley, http://ast.coe.berkeley.edu/srms/2007/Intro2007.pdf

Insertion devices

Moving frame of referenceLaboratory frame of reference

Synchrotron radiation
D. Attwood, UC Berkeley, http://ast.coe.berkeley.edu/srms/2007/Intro2007.pdf

D. Attwood, UC Berkeley, http://ast.coe.berkeley.edu/srms/2007/Intro2007.pdf

12	
  2013-­‐09-­‐23	  Producing IR light
Bending magnet radiation

Which one is better?

Bendingmagnete- beam
Problem: source size, focusing

Globar wins

GlobarSR

Synchrotron wins

Total flux
(CLS)

Brightness
(CLS)

13	
  

2013-­‐09-­‐23	  Pathlength and resolution

High-resolution
spectroscopy

Source size (pinhole)

SR advantage – “low” res…
50	
  
45	
  
40	
  
35	
  

S/N	
  

30	
  
25	
  
20	
  
15	
  
10	
  

Globar

SR

0	
  

35	
  
43	
  
51	
  
58	
  
66	
  
74	
  
82	
  
90	
  
97	
  
105	
  
113	
  
121	
  
129	
  
136	
  
144	
  
152	
  
160	
  
168	
  
175	
  
183	
  
191	
  
199	
  
207	
  
214	
  
222	
  
230	
  
238	
  
246	
  
253	
  
261	
  
269	
  
277	
  
285	
  
292	
  
300	
  
308	
  
316	
  
324	
  
331	
  
339	
  
347	
  

5	
  

Frequency	
  (cm-­‐1)	
  35-350 cm-1 | Resolution: 0.002 cm-1 | Aperture: 3.15 mm | Velocity: 30 KHz / 5KHz

http://www.newport.com/images/web600w-EN/images/2407.gif

14	
  

0	
  

500	
  
512	
  
524	
  
535	
  
547	
  
559	
  
571	
  
583	
  
594	
  
606	
  
618	
  
630	
  
642	
  
653	
  
665	
  
677	
  
689	
  
701	
  
712	
  
724	
  
736	
  
748	
  
760	
  
771	
  
783	
  
795	
  
807	
  
819	
  
830	
  
842	
  
854	
  
866	
  
878	
  
889	
  
901	
  
913	
  
925	
  
937	
  
948	
  
960	
  
972	
  
984	
  
996	
  

S/N	
  
0	
  

35	
  
44	
  
53	
  
62	
  
71	
  
80	
  
89	
  
98	
  
107	
  
116	
  
125	
  
134	
  
143	
  
152	
  
161	
  
170	
  
179	
  
188	
  
197	
  
206	
  
215	
  
224	
  
233	
  
242	
  
251	
  
260	
  
269	
  
278	
  
287	
  
296	
  
305	
  
314	
  
323	
  
332	
  
341	
  
350	
  
359	
  
368	
  
377	
  
386	
  
395	
  

S/N	
  

SR advantage
25	
  

20	
  

15	
  

10	
  

5	
  

35-350 cm-1 | Resolution: 0.00096 cm-1 | Aperture: 2 mm | Velocity: 30 KHz / 5KHz

SR advantage

30	
  

25	
  

20	
  

15	
  

10	
  

5	
  

Frequency	
  (cm-­‐1)	
  
0	
  

335	
  
340	
  
345	
  
350	
  
355	
  
360	
  
365	
  
370	
  
375	
  
380	
  
385	
  
390	
  
395	
  
400	
  
405	
  
410	
  
415	
  
420	
  
425	
  
430	
  
435	
  
440	
  
445	
  
450	
  
455	
  
460	
  
465	
  
470	
  
475	
  
480	
  
485	
  
490	
  
495	
  
500	
  
505	
  
510	
  
515	
  
520	
  
525	
  
530	
  

S/N	
  

2013-­‐09-­‐23	  SR advantage
80	
  

70	
  

60	
  

50	
  

40	
  

30	
  

20	
  

10	
  

Frequency	
  (cm-­‐1)	
  335-535 cm-1 | Resolution: 0.001 cm-1 | Aperture: 1.5 mm | Velocity: 40 KHz

Frequency	
  (cm-­‐1)	
  Applications35	
  High resolution FTIR on gases– Important for Astronomy(interstellar gases, small molecules)– Atmospheric sciences(ozone, pollution, radicals)– Basic physical chemistry(molecular dynamics, radicals, etc…)

500-1000 cm-1 | Resolution: 0.001 cm-1 | Aperture: 1.15 mm | Velocity: 40 KHz15	
  2013-­‐09-­‐23	
  Sample preparation
Gases

Gas cells

IR transparent
windows

Multi-pass cells

GAS	
  

• 30 cm White Cell
• Ambient Temperature• Paths up to 12 m

Gas cells (discharge)• 2 m White Cell
• Coolable (down to -80 °C)• Paths up to 80 m

The CLS high resolution IR BL• 1 m White Cell
• AdjustablePower• 4 m Path
Bruker IFS 125 HR
Maximum Resolution: 0.00096 cm-1
~1.2x10-7 eVBeamsplitter
Mylar 6 µm
Mylar 75 µm
Ge/KBrCaF2
Detectors
MCT N
MCT B
DTGS
DTGS PESi Bolometer
Ge:Cu
Internal Sources
Globar
Hg – Lamp
Tungsten Lamp

Spectral Range
30-630 cm-1
12-35 cm-1
400-4800 cm-1
1850-20000 cm-1
600-10000 cm-1
450-10000 cm-1
100-3000 cm-1
15-700 cm-1
10-370 cm-1
300-1850 cm-1
10 – 13000 cm-1
10 – 1000 cm-1
1000-25000 cm-1

16	
  

2013-­‐09-­‐23	  More information
The CLS Far IR website: http://goo.gl/idUP

Microscopy- spectromicroscopy -Brant Billinghurst

Tim May

Resolution
Fraunhofer diffraction

Rayleigh criterion

W2
Occurs in the “far field” λ << L ,Two objects are resolved if:

where W is the size of theDIFFRACTION
entrance3.23aperture, λ is the wavelengthand L is the distance from W

S! 2J ( x ) $I (θ ) = I 0 # 1 &
" x %

S'

L

The	
  ﬁrst	  object’s	
  Airy	  disk’s	
  maximum	
  coincides	
  with	  the	  second’s	
  ﬁrs	  minimum	
  

2

Airy	
  disk	  A
FIGURE 18 Fraunhofer diffraction of an aperture A , with a converging
spherical wave. S : point source. L: converging lens. S !: image.

In an imaging situation (see Fig. 18), a diverging spherical wave is brought to a focus in
the image plane. This is also an example of Fraunhofer diffraction pattern of the light
distribution in the aperture A by a converging spherical wave. To realize Fraunhofer
diffraction, a similar situation is obtained when a narrow diffracting aperture is held next tothe eye when it is focused on a distant point source. The diffraction pattern is observed in
the plane of the source.
An optical processing setup is shown in Fig. 19 where collimated or parallel light isincident normally on plane 1. In this arrangement an inverted image of plane 1 is formedin plane 3 (see Chap. 1). The imaging process may be thought of as a Fourier transform(Fraunhofer diffraction) of the light distribution in plane 1 onto plane 2, followed byanother Fourier transform of the light distribution in plane 2 onto plane 3.
Recall our earlier discussion in relation to Eqs. (33) and (34). When the quadratic phasefactor, exp ["iπ (x 2s " y 2s ) / # r ] , may be approximated by unity, we are in the domain of
Fraunhofer diffraction. From the point of view of Fresnel zone construction, the far-field
condition, z  d 2 / # , means that for these distances z the first Fresnel zone overfills theaperture. The entire aperture contributes to the disturbance at any point in the Fraunhofer
pattern. In Fresnel diffraction only relatively small portions of the aperture contribute to
any one point in the pattern.
In this context the term Fresnel number is frequently used. It is defined in terms of the
product of two ratios. The radius r of the aperture to the wavelength # times the radius of
the aperture to the distance b measured from the aperture to the plane of observation:

In case of a circular aperturer r 1 d2
Fresnel number ! N $ % $# b 4b #(35)

Thus, Fresnel number can also be expressed as the ratio of the far-field distance, d 2 / # , tohXp://goo.gl/cp6WGT	
  

17	
  

2013-­‐09-­‐23	  In practice1.22 λNAcond + NAobj

NA = n ⋅ sin (θ )26.3%	
  dip	
  Rayleigh resolution limit: R =Beyond Rayleighn	
  –	
  refracNve	
  index	
  of	
  the	
  medium	  	
  the	
  objecNve	
  is	
  looking	
  into	  But…

General IR microscope Layout

Σ:	  Higher	  resoluNon	
  possible	
  Image	
  processing	  algorithms,	
  2D	
  deconvoluNon	
  techniques	
  
	
  	
  	
  with	  PSF	  model	
  
	
  	
  	
  or	
  Bayesian	
  (model	
  free)	
  	
  Oversampling!	
  The Schwarzschild objective18	
  2013-­‐09-­‐23	
  Why use a synchrotron?

Globar Light Spot in Planeof Confocal Aperture

GlobarBeam spot in the CLS

Synchrotron Light Spot inPlane of Confocal
ApertureSR

Beam spot in the CLS19	
  2013-­‐09-­‐23	
  Globar vs. Synchrotron

Globar vs. synchrotron

Globar vs. synchrotron

2x2 mm pinhole

5x5 mm pinhole

3x3 mm pinhole

10x10 mm pinhole

Spatial resolution

Absorbance	  Absorbance	  6x6 µm aperture (single cell)	
  3500	
  	
  	
  

	
  3000	
  	
  	
  

	
  2500	
  	
  	
  

	
  2000	  	  	
  Wavenumbers	
  (cm-­‐1)	
  	
  1500	
  	
  	
  

	
  1000	
  	
  	
  

	
  3500	
  	
  	
  

	
  3000	
  	
  	
  

	
  2500	
  	
  	
  

	
  2000	
  	
  	
  

	
  1500	
  	
  	
  

	
  1000	  	  	
  Wavenumbers	
  (cm-­‐1)	
  

20	
  

2013-­‐09-­‐23	  Spatial resolution…
Visible	  …diffraction limited

Synchrotron	  RadiaBon	  	
  Globar	
  

2.46	
  μm	
  
3000	
  cm-­‐1	
  2000	
  cm-­‐1	
  1500	
  cm-­‐1	
  2000	
  cm-­‐1	
  Diffraction limited resolution
can be achieved only by SR

Canadian Light Source Mid-IR

-40

-50

)mm( y

-20

-60

-40

M6	
  

0

-20

0

20

x (mm)

root:Mirr7G_y_0 vs. Mirr7G_x_0

400x (mm)

root:Mirr2G_y_1 vs. Mirr2G_x_1
Good:14904 σx: 14.725mm Avg:23.075
σy: 53.038mm Avg:3.1469

50

UHV
Window
Focus

20
σy: 23.105mm Avg:-4.6752
Good:14904 σx: 6.9572mm Avg:8.3454

0

x (mm)

-6

-4

-2

0

2

4

4

8

6

root:Mirr1G_y_0 vs. Mirr1G_x_0

8

6
4
2

20
10

y (mm)

-8

-4

-6

0

-2

y (mm)2-2

0x (mm)

root:Star3G_x_1 vs. Star3G_z_1-5x (mm)

4

60

Good:14904 σx: 20.126mm Avg:-1.5859
σy: 11.053mm Avg:0.066009

Good:14904 σx: 1.7001mm Avg:-1.0273
σy: 3.3622mm Avg:1.5963

root:Star6G_x_0 vs. Star6G_z_0

-8

-10
0

root:Star8G_x_0 vs. Star8G_z_0

0

-5

-15

-10

Good:14904 σx: 3.2889mm Avg:1.4637
σy: 1.7268mm Avg:-1.0759

40

M2

0

M5

2

y (mm)RV Target
Focus

20
x (mm)

-2

M7

0

-50

y (mm)FTIR

5

Good:14904 σx: 4.7954mm Avg:-1.5743
σy: 2.8756mm Avg:-5.8612

Infrared beamlines- some examples -

0

-40

30

0

20

y (mm)y (mm)10y (mm)

050

M3

root:Mirr4G_y_0 vs. Mirr4G_x_0

M450x (mm)
-10

100

100

Good:14904 σx: 33.412mm Avg:-4.4553
σy: 32.184mm Avg:44.646
150

root:Mirr5G_y_1 vs. Mirr5G_x_1
Good:14904 σx: 16.976mm Avg:-23.986
σy: 59.41mm Avg:7.509

0
-10
-20
-20

0

20

x (mm)SHADOW Beam shapeson mirror surfaces for 25
micron wavelength.

n
ro
ot
hr
c
n

M1

Sy

21	
  

2013-­‐09-­‐23	  Canadian Light Source Mid-IR

Advanced Light Source, Bl 5.4~10 m+ Beamline 1.4

NSLS VUV-IR ringU2A	  Geophysics,	
  materials	
  at	
  extreme	
  pressures	
  and	  microspectroscopy	
  

U2B	
  

Mid-­‐IR	
  microspectroscopy	
  IRENI beamline, SRC

U4IR	
   InstrumentaNon	
  development	
  U10A	
   Far-­‐IR	
  microspectroscopy	
  U10B	
   Mid-­‐IR	
  microspectroscopy	
  U12IR	
   Far-­‐IR,	
  THz	
  and	
  millimeter	
  wave	  spectroscopy.	
  Materials	
  physics	
  Shutting down soon because of NSLS II22	
  2013-­‐09-­‐23	
  

IRENI beamline, SRC
Shugng	  down	  in	
  November	
  2013	
  IR spectroscopy- samples and measurement
techniques -Focal Plane Array
Absorbance (a.u.)

(a)

a

b

Thermal source
Synchrotron source
ATR linear array (1.56 m) Point mapping (10 m)cdThermal source
74× FPA (0.54 m)

(b)
(c)

e

(d)

Multi-beam synchr. source 3,800 3,400 3,000 2,600
Wavenumber (cm-1)
74× FPA (0.54 m)

Nasse et al., Nature Methods 2011, DOI:10.1038/nmeth.1585

Supplementary Figure 1 | Additional chemical images from various FTIR systems. Panels a!d show the same
unstained cancerous prostate tissue section as in Figure 2, but here using the peak height at 3,300 cm-1
(Amide A band, N-H stretch). (a) Attenuated total reflection (ATR) image acquired with a PerkinElmer Spotlight
FTIR system using a ZnSe crystal (1.56  1.56 m2 pixel size). Even though ATR can provide relatively highspatial resolution, it is not really a far-field technique and requires close, homogeneous contact of a crystal withthe sample surface. This requirement restricts the number of samples that can be studied with ATR and canpotentially lead to sample damage and artifacts, for example due to sample parts sticking to the crystal andbeing dragged along during the acquisition (e.g., see the !line" in the lower left part of the image a, indicated by
the green arrow). (b) A dual-aperture microscope (Thermo Nicolet Continum) with a conventional single-beamsynchrotron source. Such a setup can in principle yield diffraction-limited resolution at all wavelengths, but thesmall aperture (3  3 m2) and step size (0.8 m) required to achieve this make it practically impossible. For
this sample, in particular, ~135,000 pixels would need to be mapped, which at ~7 s (16 scans) per spectrum
would require 11 days of uninterrupted mapping. Thus, sample size and acquisition time (here ~5 hours) canmake larger apertures and step sizes necessary (here 10  10 m2), leading to loss of resolution. In sharp
contrast, we recorded the fully diffraction-limited image using the system presented here (d) with ~295,000
pixels in ~30 minutes (16 scans). (c) A thermal-source-equipped system (Bruker Hyperion 3000 using a 64 
64 pixel FPA) with pixel size 0.54  0.54 m2 loses spectral (see e) and image quality, making the approach
impractical. (d) The multi-beam synchrotron image (pixel size 0.54  0.54 m2) demonstrates farfield/contactless high spatial resolution and high spectral quality (see e). Both c and d have been acquired on
the same instrument with identical imaging conditions. The crosshairs in a!d indicate the positions of thecorresponding, unprocessed spectra (NH/OH and CH stretch region) shown in panel e. Yellow scale bar in a:
100 m.

Sample forms

Gases

IR transparent
windows

Sample forms
Liquids

SampleSpacerMulti-pass cells

Nature Methods: doi.10.1038/nmeth.1585

GAS	
  

IR transparent
windows

23	
  2013-­‐09-­‐23	
  Sample forms

Reflection setups

SolidsRegularConvenient
SampleTransmission

Reflection

SampleSubstrate
Reflection-absorption
Transflecion

Self supporting
Lying on substrate
Suspended in carrier
KBr
Nujol

DRIFTS
Diffuse reflection
Integrating sphere

Very thin samples
Grazing angle incidence

Special microscope objectives
Increases optical pathlength
Monolayers
Sub-monolayers

SampleSubstrate

Reflection setups
We are happy

SampleSubstrate

Reflection setups
Not so lucky

Application:- Powders- Catalysis!!!

Total internal reflection< micron

Sample24	  2013-­‐09-­‐23	
  Reflection setups
Attenuated total internal reflection

Single reflection
Surface sensitive
Microscopy

Reflection setups
Attenuated total internal reflection

Multiple reflection
Very sensitive
Number of reflections:

Artifacts?
Samples are not ideal…thickness/roughness

Possible problems

Life gets very complicated25	
  2013-­‐09-­‐23	
  Artifacts?

Artifacts?

Samples are not ideal…

Samples are not ideal…scattering.
Mie Scattering

Mohlenhoﬀ	
  et	
  al.,	
  Biophysical	
  Journal	
  88,	
  3635-­‐3640,	
  (2005)	
  Mohlenhoﬀ	
  et	
  al.,	
  Biophysical	
  Journal	
  88,	
  3635-­‐3640,	
  (2005)	
  Artifacts? – solutions…

Artifacts? – RMieSc• Second derivatives• EMSC (extended multiplicative scattering correction)• Mie Scattering corr., Resonant Mie Scattering corr.

How to differentiate spectra?
• Statistical analysis– Cluster analysis– Principal Component Analysis– Partial Least Squares regression

Cytospec
R, Matlab,
Eigenvetcor
The Unscrambler

Samples are not ideal…
resonant scattering.
Amide I – 1641 cm-1
b-sheet, random

Amide I – 1650 cm-1
random, turns

Amide I – 1654 cm-1
a-helix
Bassan et al., The Analyst 135, 268-277, (2010)26	
  2013-­‐09-­‐23	
  Artifacts? – RMieSc…
Samples are not ideal…resonant scattering.

Artifacts? – RMieSc…
Samples are not ideal…resonant scattering.

Uncorrected	
  Bassan et al., Analyst 135, 268-277 (2010)Mie	
  scaXering	  	
  corrected	
  Resonant	  Mie	
  scaXering	  	
  corrected	
  Bassan et al., Analyst 135, 268-277 (2010)

Artifacts?
Does the synchrotron light heat the sample?
Synchrotron	
  source:	  	
  Measured	
  1	
  mW	
  focused	
  onto	  ~10	  X	  10	
  μm2	
  

DPPC	
  =	
  dipalmitoylphosphaNdylcholine,	
  	
  
	
  
	
  a	
  phospholipid	
  bilayer	  	  Undergoes	
  a	
  gel	  to	
  liquid-­‐crystalline	
  phase	  transiNon	
  at	
  T=	
  316	  K	  SR IR spectroscopy- applications -Observed < 0.5˚ C temp rise on sample
Important: No Evidence for Cytotoxic Effects on Living Cells
M.C. Martin et al., Applied Spectroscopy 55, 111 (2001)27	
  2013-­‐09-­‐23	  Courtesy of H. A. Bechtel, ALS LBNLIR SNOM

Courtesy of H. A. Bechtel, ALS LBNL

Background suppression in s-SNOM

Scattering type SNOM

Scanning Near-field Optical Microscopy

Nanometer spatial resolution
Wavelength independent
Soft and hard matter
Amplitude and phase of optical field

Courtesy of H. A. Bechtel, ALS LBNL

Experimental setup

Courtesy of H. A. Bechtel, ALS LBNL

Experimental setup
Lock-­‐in	
  ampliﬁer	
  

AFM	
  
sample	
  

ALS	
  

28	
  

2013-­‐09-­‐23	  Courtesy of H. A. Bechtel, ALS LBNL

Approach curves

Courtesy of H. A. Bechtel, ALS LBNL

Broadband nano-imaging

Courtesy of H. A. Bechtel, ALS LBNL

Broadband nano-spectroscopy:
interferograms

Courtesy of H. A. Bechtel, ALS LBNL

Broadband nano-spectroscopy29	
  2013-­‐09-­‐23	  Courtesy of H. A. Bechtel, ALS LBNL

Courtesy of H. A. Bechtel, ALS LBNL

Linescans

Biological Samples

SPring-8, BL43B

Commercial, non SR system

Three IR endstations- spectromicroscopy- magneto-optical spectroscopy- high-pressure studies- near field microscopy

NeaSNOM
from
NeaSpec
www.neaspec.com
Uses QCL sources
Possible with Globar30	
  2013-­‐09-­‐23	  Kinetics: problem RC time constant of the electrodesolution: decrease size

e-chem cell	

Small electrode size ~ 10 µm
Need for SR

Apply Estep	

rest	

ref	

Flow Through Holes	

CE	

Ecell/I	DAQ
Computer	potentiostat	

WE	

3.8
3.6

Signal (V)

1.0
0.15

0.8
i / µA0.10

q / µC

0.6

0.00

FTIR
Microscope	

-0.05
-0.10
-0.4

-0.2

0.0

0.2

0.4

0.6

E vs Ag wire

0.4

Let’s roll!

0.05

1.0
0.5
0.0

Get ready…

-0.5

0.6

0.2

E / V vs Ag

0.4

0.00

0.0

0.0

0

30

32

34

36

Spectra38

time / s

0

0.05

0.2

-0.2

1

2

3

4

time / s

5

6

7

0.10

0.15

time / s

Direct detector signal	

8RE	

Rosendahl SM; Borondics F; May T; Pedersen T; Burgess I, Anal. Chem. 83 (10), 3632-3639, 2011.

Rosendahl SM; Borondics F; May T; Pedersen T; Burgess I, Anal. Chem. 83 (10), 3632-3639, 2011.

Results	

2
0

Fe(CN)64- = Fe(CN)63- + e-

ΔS/S

0.03

Δ(abs) x 1000

0.04

-2

Fe(CN)64- 	

loss	

-4
-6
-8
-10
-12
0

0.02

1

2

3

4

5

6

7

time / s
1.2

0.01

creation	Fe(CN)63- 	

-0.01
1900

2000

2100

2200

Wavenumber / cm

-1

2300

ΔConcentration (mM)

0.8

0.00

0.4
0.0
-0.4
-0.8
-1.2

0

1

2

3

time / s

Rosendahl SM; Borondics F; May T; Pedersen T; Burgess I, Anal. Chem. 83 (10), 3632-3639, 2011.

Rosendahl SM; Borondics F; May T; Pedersen T; Burgess I, Anal. Chem. 83 (10), 3632-3639, 2011.

31	
  2013-­‐09-­‐23	  Biology	Spectromicroscopy in Rod Cells	

Ideal size range for resolution
Cells, tissues, bacteria, fungi
globar source	Absorbance	Absorbance	

SR source	

0.8	

0.7	

0.6	

0.5	

0.4	

0.3	

0.2	

0.1	

4000 	

3500 	

3000 	

2500 	

2000 	

1500 	

1000 	

3500 	

3000 	

2500 	

2000 	

Wavenumbers (cm-1)	

1500 	

1000 	

Lipid distribution:
absorbance at 2925 cm-1

we derive hgi from the crystal structure of rhodopsin[14] [protein data bank (PDB) entry 1F88; see the Supporting Information], thereby obtaining a value of hgi = 248, which is in good
agreement with our spectroscopic results. The orientation ofrhodopsin a helices relative to the normal of the membrane
has been previously measured by means of FTIR VLD using reet al., ChemPhysChem
constituted disks or ensemblesQuaroni
of fractured
ROS. Values of h9g(10)i 1380-1382, 2008	

ranging between 38 and 518 were reported in these[12, 15]
works.
To date, our results—obtained in vivo—provide thebest agreement between the spectroscopic measurement of
the hgi value of rhodopsin a helices and the corresponding
crystallographic value.
The amide-II band overlaps with bands assigned to tyrosine
Courtesy	
  of	
  M.	
  J.	
  HackeX	
  residues in rhodopsin,[12] which prevents a quantitative assessment of polarization ratios by curve fitting. Nonetheless, theband clearly displays opposite dichroism to that of amide I, as
expected based on the orientation of its transition moment.
[14]
An approximate
value of hgi = 338 can be calculated for the
we derive hgi from the crystal structure of rhodopsin[proa helices,in fair agreement with the more accurate value obtein data bank (PDB) entry 1F88; see the Supporting
Informatainedtion], thereby obtaining a value of hgi = 248, whichis in fromgoodthe analysis of the amide-I absorption.
Dichroism
agreement with our spectroscopic results. The orientationof is also observed for the absorption band at!1
rhodopsin a helices relative to the normal of the1740–1720
membranecm , which is tentatively assigned to the acylic carbonylof thehas been previously measured by means of FTIR VLD
using
re- phospholipid headgroups. The transition moment
for thisofvibration
is taken to be aligned with the C=O group,
constituted disks or ensembles of fractured ROS. Values
hgi
ranging between 38 and 518 were reportedthusin providing
these an average orientation of hgi = 728 and hgi =678, relative
works.[12, 15] To date, our results—obtained in vivo—provide
the to the ROS axis, for non-hydrated and hydrated
carbonyl groups,
respectively.[16] This result is consistent withbest agreement between the spectroscopic measurementofmeasurements on model systems.[17] However, a different orithe hgi value of rhodopsin a helices and the corresponding
entation for the population of hydrated carbonyl groups has
crystallographic value.
been reported in the past. The protonated forms of asThe amide-II band overlaps with bands assignedneverto tyrosine
partic assessand glutamic acid residues of rhodopsin absorb in thisresidues in rhodopsin,[12] which prevents a quantitative
same region.
ment of polarization ratios by curve fitting. Nonetheless,the Although their contribution to the total absorbanceissmall, it is sufficient to account for the differband clearly displays opposite dichroism to that of amiderelatively
I, asencemoment.
in polarization of the two components of the carbonyl
expected
based
the absorption
orientationof with
its transition
Figure 1. a) ROS spectra
recordedin the on
carbonyl
regionlight polarized perpendicular(g) and parallel
the
ROScanaxis,be calculatedband,forthus
setting a limit to the accuracy of this measurement.
An approximate
value (c)of hgtoi =338theb) ROS spectra recorded in the methyl absorption region with light polarized
in the 2700–3100 cm!1 region also show remarkablea helices, in fair agreement with the more accurate Bands
value obperpendicular (g) and parallel (c) to the ROS axis.
polarization, in particular thetained from the analysis of the amide-I absorption.
methylene
52x	
  osymmetricbjecNve	
  handigh	
  antires	
  imaging.	
  RepresentaNve	
  Spectrum	
  from	  a	
  single	
  Dichroism is also observed for the absorption band atsymmetric
at1740–1720 cm!1, which is tentatively assigned to the acylic car0.77	
  stretchingum	
  pixel	  (bands
512	
  scans)	
  
=	
  10	
  minute	
  image	
  Nme	  (20	  minutes	
  with	  BG).	
  !1Table 1. Band frequencies for selected ROS spectral bands measured with polarized light and corresponding, respective2852 andbonyl of the phospholipid headgroups. The transition moment180	
  2924
mA	
  cm
beam	
  
current	  polarization ratio (R), average transition moment orientation (hqi), and average structural element orientation
ly,
and
the
stretchingvibration(hgi) relative to thefor
ROSthisaxis.vibration is taken to be aligned with the C=O group,of C!H bonds on trigonal carthus providing an average orientation of hgi = 728 and hgi =Assignment
ratio
hqi
hgi
Frequency of the absorption
bandto thebons at 3012 cm!1. The values of678,
relative
ROS axis, for non-hydratedPolarizationand hydrated
R = A j j /A ?
[8][8][cm!1] w̃ k ;w̃ ?
[16]
the average angle of the transicarbonyl groups, respectively. This result is consistent with
1651; 1651
amide-I
contribution
30
tion moment to the ROS axis,
measurements on
modelsystems.[17] However, 1.94
a different ori- 45
from a helices
hqi, for these groups are shownentation for the population of hydrated carbonyl groups has" 1545amide-II contribution" 0.5" 63 " 33in Table 1, together with correnever been reported
the past. The protonated forms of asfrom ainhelices
sponding values of hgi. Contri1742; 1741
non-H
(or D)-bonded
acyl car- 0.78
72
partic and glutamic
acid
residues lipidof rhodopsin
absorb in this 72
bonylgroupsbutions to the absorption in thissame region. Although their contribution to the total absorb1724; 1725
H (or D)-bonded lipid acyl
0.67
67
67
spectral region arise predomiance is relatively small, it is sufficient to account for the differcarbonyl groups
nantly from lipid acyl chains. A
ence in polarization
Figure 1. a) ROS spectra recorded in the carbonyl absorption region
2853;with
2852
nCH2,S of the two components of
0.57the carbonyl
62
35[a]contribution from C!H vibralight polarized perpendicular (g) and parallel (c) to the ROS
axis,2922
2926;
nCH2,AS57
band, thus setting
a limit to the accuracy of this0.72
measurement.
!1b) ROS spectra recorded in the methyl absorption region with light
0.61
61
61
3013;polarized
3012
n
=C!H
tions of amino-acid side chains
Bands in the 2700–3100 cm region also show remarkable
Wavenumbers (cm-1)	

Results – polarization studies	

Measurement of molecular orientation ina subcellular compartment bysynchrotron infrared spectromicroscopy	

FPA imaging of brain samples
for stroke research

The measurement confirmed previous	

results obtained by crystallography	

on model systems.	

32	
  2013-­‐09-­‐23	  Courtesy	
  of	
  M.	
  J.	
  HackeX	
  Courtesy	
  of	
  M.	
  J.	
  HackeX	
  Imaging Total Protein in
Cerebellum Purkinje Neurons
Histochemistry provided evidence that Purkinje neurons have low protein content (relative toother neurons, and surrounding tissue), however, results were could have been misleading due
to removal of proteins from cell during fixation/staining protocol. FTIR imaging at the cellularlevel revealed that the Purkinje neuron is rich in Protein. (A) Bright Field image of unstained
tissue. (B) Bright field image of cresyl violet stained tissue showing neuron body (stained after
FTIR). (C) FTIR image protein (amide I band, 1690 – 1610 cm-1)

HackeX	
  et	
  al.	
  ACS	
  Chemical	
  Neuroscience	
  2013	  Courtesy	
  of	
  M.	
  J.	
  HackeX	
  Subcellular imaging with SR/FPA

Something to remember
Synchrotron = high brightness
Diffraction limited resolution
Small samples!
High spectral resolution
Data processing…pay attention!

33	
  2013-­‐09-­‐23	
  Resources
•
•
•
•
•
•
•

CLS Mid-IR webpage http://midir.lightsource.ca
ALS Infrared beamlines http://infrared.als.lbl.gov
SPring-8 publications website https://user.spring8.or.jp/uisearch/publication2/
William Reusch: Virtual Textbook of Organic Chemistry http://goo.gl/NEUWDavid Attwood SR course (EE290f) at UC Berkeley (http://www.youtube.com/watch?v=1WWnxA6odso)
Wolfram Demonstrations Project(http://demonstrations.wolfram.com)
Synchrotron Simulator
T. Shintake, Nuclear Instruments and Methods in Physics, 507 (1-2), 89-92 (2003)(http://www-xfel.spring8.or.jp/dlmonitor.html)

Thank you for your attention!
Ferenc Borondics
Mid-IR beamline
Canadian Light Source
ferenc.borondics@lightsource.ca
http://midir.lightsource.ca

34