Infrared Spectroscopy and

Microscopy

Lisa Vaccari
SISSI beamline manager
 Outlines

• Infrared Spectroscopy: Basic Concepts

• FTIR Spectromicroscopy: Instrumentation

• A brief history of IR spectroscopy at SR facilities

• IRSR: Generation and properties

• SR FTIR Microscopy: Selected application for life Sciences

• New trends for IRSR

• FTIR Imaging and tomography

• IR nanoscopy: beyond diffraction limit with near-field IR
• Plasmonic for ultrasensitive SR FTIR microscopy
Infrared Spectroscopy: Basic Concepts
 Electromagnetic Spectrum: a closer
view into the IR spectral range

NIR MIR FIR

λ (μm) 0.74 3 30 300
ν (THz) 400 100 10 1
ν (cm-1) ~13000 ~3333 ~333 ~33
E (eV) 1.65 0.413 0.041 0.004
E (Kcal/mol) 37 10 1 0.1
 Infrared Spectroscopy: Basic
concepts
IR spectroscopy is an absorption spectroscopy that probes molecular
vibrations

The classical description of vibrational motion

The Hooke’s law - + - +
F(restoring force) = -k.x, k = Force constant [Nm-1] m1 m2
k
r0
Stronger the bond is,
higher the value of k is
1 k

2  x1 x2
m1m2

m1  m2
Any vibrational energy value is allowed by the
1 2 classical solution as well as null vibrational
ETotal Energy  kx
2 energy
 Infrared Spectroscopy: Basic
concepts
The anharmonic oscillator in quantum mechanics

Evib  hve [(n  1 2 )  xo(n  1 2) 2  higher terms]

Energy

ve  harmonicfre que ncy x0  anharmonicconstant

Dissociation

Selection rule Δn   integer

The intensity of an absorption band

is proportional to the population of
n=3 Second overtone the initial state from which a
n=2 First overtone transition takes place.
Fundamental frequency N1  (E  E0 )   ΔE 
n=1
 exp 1   exp  
n=0 E0 = Zero point Energy N0  k BT   k BT 

Displacement
Re
 Infrared Spectroscopy: Basic
concepts
Allowed and Forbidden vibrational Transitions
m2 A vibrating molecule is a dipole oscillator that
+   e  d produces a stationary alternating electric field.
The electric dipole moment oscillates at its
- m fundamental vibrational frequency, and hence EMR of
1 this frequency can be absorbed and induces vibrational
d
transitions
Vibrations that do not induce variation of the dipole moment of the molecule
are forbidden

How many vibrational transitions?

By exploiting molecular symmetries, it can be demonstrated that any vibration of a
molecule constituted by N atoms can be described by the suitable combinations of
3N-6 (3N-5 for linear molecules) simple harmonic vibrations, the so called
NORMAL MODES of vibration.
Normal modes of vibration are characterized by all the atoms moving in phase (they
pass thorough their equilibrium position at the same time), at the same frequency
but with different amplitudes.
 Infrared Spectroscopy: Basic
concepts
Stretching modes (ν)

Symmetric Stretching Antisymmetric Stretching

Deformation modes

Scissoring (δ) Rocking (r or ρ) Wagging (ω) Twisting (τ)

In plane deformations Out plane deformations

 Infrared Spectroscopy: Basic
concepts
An example:
Vibrational Spectrum
of liquid water

Overtones
and
Intermolecular bend = 50 cm-1
combination bands
Intermolecular stretch = 183 cm-1
L1 librations = 395 cm-1
L2 librations = 687 cm-1

ν1 = 3280 cm-1
Sym Stretching
ν2 = 1645 cm-1 Water librations, L
Bending

ν3 = 3490 cm-1
Asym Stretching

ν2 + L
Animation by Jens Dreyer, MBI
 Infrared Spectroscopy: Basic
concepts
FROM PEAK POSITION, INTENSITY AND WIDTH
NATURE OF ATOMS INVOLVED IN THE SPECIFIC VIBRATION
PARAMETERS OF THE ATOMIC BOND : BOND STRENGTH AND LENGHT
BOND CONFORMATION: DOUBLE BOND CIS/TRANS, PROTEINS’ CONFORMATION,….
CHEMICAL ENVIRONMENT (THROUGH MODULATION OF THE DIPOLE MOMENT)
ROTATIONAL MODES IN THE FIR REGION

FROM WHOLE SPECTRUM

NATURE OF THE MOLECULE: SPECTRAL FINGERPRINT=> MOLECULAR IDENTIFICATION
SAMPLE INTERACTIONS: FREE/BOUND WATER …
SAMPLE EVOLUTION: REACTION KINETIC, AGING, PHYSICO CHEMICAL TREATMENT,
CONSTRAINTS (PRESSURE, TEMPERATURE, pH) …
ATOMIC BOND ORIENTATION: POLARIZATION MEASURMENT

QUANTITATIVE or SEMI-QUANTITATIVE ANALYSIS

SIMPLE MIXTURES: BEER LAMBERT BOUGUER LAW
COMPLEX MIXTURE : PLS, CLS, ALS, MCR, PCR …
FTIR Spectromicroscopy: Instrumentation
 FTIR Spectromicroscopy:
Instrumentation
When dealing with molecular species (normal modes of vibration 3N-6), the
absorption profile at a single frequency (or limited spectral range) is scarcely useful.
Only a multi-frequency profile can account for the system complexity and its
interaction with the environment

An FTIR spectrum needs to be energy resolved over a large spectral range

The past instrumentation: Dispersive Interferometers

[Link]

This slow acquisition time limited the wide spreading of infrared spectroscopy until 1960s’,
when Fourier Transform Interferometer have been first proposed.
 FTIR Spectromicroscopy:
Instrumentation
The present instrumentation: Fourier Transform InfraRed Interferometers
Conventional sources
NIR: Tungsten lamp
MIR: Glow bar (SiC)
FIR: Hg-Arc

Beamsplitters
NIR: CaF2
MIR: KBr
FIR: Mylar, Silicon

Detectors
NIR – InGaAs, InSb, Ge, Si room
temperature detectors
MIR: Room temperature DLaTGS
Nitrogen cooled MCT
FIR – He Cooled Silicon Bolometer
Room temperature DLaTGS
Optical Path Difference _ OPD
2Δx=2vt v = mirror velocity
 FTIR Spectromicroscopy:
Instrumentation

For a single wavelength For a polychromatic source

I (x )  I (~)[1 cos( 2x~)]  
I ( x)  I (~)d~  I (~) cos(2x~)d~


I (ZPD)  2 I (~)d~  I 0


1
I ( x)  I 0  I (~) cos(2x~)d~
2


1
I ( x)  I 0  I ' ( x)  I (~) cos(2x~)d~
2


Fourier Transform (FT)  I (~)  


I ' ( x) cos(2x~)d x
 FTIR Spectromicroscopy:
Instrumentation
Detector Signal Detector Signal

OPD OPD

v0 V0 speed of change of OPD

i  v 0ti 
Detector Signal
VM mirror velocity
fi fi frequency
ti period

FT 
v0  vM
VM has to be KNOWN and CONSTANT OPD
He-Ne laser (632,80 nm) Spectrum

Spectrum

Frequency
fi  v 0 i
Frequency
 FTIR Spectromicroscopy:
Instrumentation
Continuum
Interferogram
source x
Fourier
Spectrum
Transform
ν
Apodization function  L max 
I (~)   I '(x ) cos( 2x~)dx  D (x )I '(x ) cos( 2x~)dx

L
 max 

Box-car Triangular
waveform waveform
 FTIR Spectromicroscopy:
Instrumentation
9 chambers = 5.13 m R= 0.00096cm-1

The spectral resolution or resolving

power ( ~ ) of a spectrograph is a
measure of its ability to resolve
features in the electromagnetic
spectrum.

1
~max 
(OPD) max
 FTIR Spectromicroscopy:
Instrumentation
0.020 1.0
0.018 Sample Single channel SSC
0.9
T(%) 
0.016
0.8
RSC
0.014
0.7

Abs (a.u.)
Abs (a.u.)

0.012
0.010 0.6
0.008
0.5
0.006
0.4
0.004
0.002 0.3

0.000 0.2
4000 3600 3200 2800 2400 2000 1600 1200 800 4000 3600 3200 2800 2400 2000 1600 1200 800
-1
 (cm )
-1
 (cm )

1.0
0.024 Reference Single channel 0.9  SSC 
0.022 Abs  log  
0.020
0.8
 RSC 
0.018 0.7
0.016
Abs (a.u.)
0.6
Abs (a.u.)

0.014
0.5
0.012
0.4
0.010
0.008 0.3
0.006 0.2
0.004
0.1
0.002
0.0
0.000 4000 3600 3200 2800 2400 2000 1600 1200 800
4000 3600 3200 2800 2400 2000 1600 1200 800
-1
-1  (cm )
 (cm )
 FTIR Spectromicroscopy:
Instrumentation
Spatially resolved chemical information on heterogeneous samples are obtained by
coupling FTIR spectrometers with specially designed Vis-IR microscopes

Schwarzschild objective

NA  n  sin 2
2θ = angular aperture
Working Distance
 FTIR Spectromicroscopy:
Instrumentation
In far-filed microscopies, and FTIR microscopy as well, lateral resolution,
δ, is diffraction limited


  0.66
nNA

Objective NA Wavelength δ

10 μm (1000cm-1) 15 μm
0.4
2.5 μm (4000cm-1)

10 μm (1000cm-1)
 4 μm

 9.5 μm
 
0.65
2.5 μm (4000cm-1)  2.5 μm
A brief history of IR spectroscopy at SR facilities
Once upon a time…..
 IR beamlines
The Cinderella Story

1976 Meyer and Lagarde (LURE, Orsay) published the first paper on IRSR
1981 Duncan and Yarwood observed at Daresbury the first IRSR emission
1985 The first IRSR spectrum (on N2O) is collected at Bessy (Berlin)
1986 The first beamline was opened to users at UVSOR (Japan)
1987 Started the brilliant story of IR-beamlines at NSLS Brookhaven (USA)
1992 In Europe: Orsay (France), Lund (Sweden), Daresbury (GB)
1995 First international workshop on IRSR, Rome (Italy)
2001 First IR beamline in Italy (SINBAD@DAΦNE)
2006 Second beamline in Italy (SISSI@Elettra)

Opening of the IR1 beamline at LNLS (Brazil)

New IR beamlines are under construction
 SR-IR beamlines
More than 40 IRSR beamlines worldwide
IRSR: Generation and properties
 IRSR Generation
Bending Magnet IRSR
Extrapolation of the Schwinger
equations (1949) by WD Ducan and
GP William (1980s)

Infrared synchrotron radiation from electron

storage rings; Appl Opt. 1983 22(18):2914.


1/ 3
PBM ( )  4.38 10  I   H  bw   
14
photons s -1 [1]

I  Curre nt(A)
 H  Horizontal Colle ctionAngle (rads)
bw  Bandwidth(%)
 ,   Radius of the ring, Wave le ngth(same units)

P(λ) as obtained in [1], in the spectral range 1 to 104 μm (104 to 1 cm-1), THz/FIR MIR/NIR
for a current of 1 A, a horizontal angle θH = 100 mrads and ρ = 5 m.
Comparison with the emission for a BB source at 2000K.
 IRSR Generation
Bending Magnet IRSR
IBI

Constant Field
Emission
0

 1000    BM
 V  Nat  1.66     μm ;    m
  
Angular range into which 90% of the
emitted photons travel
λ [μm] υ [cm-1] THz θV-Nat Very large extraction apertures are
needed for IR beamlines for:
1 10000 300 9.2

10 1000 30 19.8 • Maximizing the flux (θH)

100 100 3 42.2 • Allowing efficient extraction of

lower energy components of IR
1000 10 0.3 90.3
synchrotron emission (θv)
Calculated for Elettra ρ = 5.5 m.
 IRSR Generation
Bending Magnet IRSR

Vertical opening angle depends on the electron energy (through bending

magnet radius)

INDUS DIAMOND
0.45 GeV 3.0 GeV
45 mrad H X 30 mrad V 45 mrad H X 30 mrad V
BM BM

255μμm
15
15 15

100 μm
15

10 m
15

10
10 10
10 2.5
30 mrad

30 mrad
55 55
Position

Vertical Position
25
Vertical Position
Position

00 2.5
5μμμm
10
100 mm 0
Vertical

Vertical
Vertical

-5
-5 -5

-10
-10 -10
-10

-15mm
-15mm
-15mm -15mm
-15mm
-15mm

-25mm -20mm
-20 -15 -10
-10
-10 -5 00
0 5 10
10 15 20mm
20
20mm
20 25 -20
-20mm
-20mm
-20 -10
-10 0 10
10
10 20mm
20
20
20mm

45 mrad
Horizontal
HorizontalPosition
Horizontal
Horizontal Position
Position Horizontal Position
45 mrad

By courtesy of Paul Dumas, SOLEIL

 IRSR Generation
Edge Radiation
Edge radiation is produced when electrons experience a changing magnetic field
(entering or exit a BM, where B is constant).
IBI

IBI
0
0

BM
Straight section
BM
 4
PEdge    I  I   A;    mrad
1   2 2 2
 
  Lore ntzFactor;  Fine Structure Constant
• Edge radiation has a ring structure characterized by interference pattern
• Being Θ max ~ 1/ γ ~10 mrads, it is spatially confined and intrisically bright
• It is radially polarized

By courtesy of Paul Dumas, SOLEIL

 IRSR Generation
Edge Radiation

SOLEIL SOLEIL
2.75 GeV 2.75 GeV
45 mrad H X 30 mrad V 30 mrad H X 30 mrad V
BM BM
Horizontal Position
HorizontalPosition
Horizontal Position
20mm
20
20mm
20 1010
10 0 00 -10
-10
-10 -20
-20mm
-20
-20mm

30mm 30
30mm
30
-15mm
-15mm

105 μ
100
25 μmm -10
-10
2020
20
20
100
10
25 μm
μ m
m
530μmrad
30 mrad

30 mrad
-5-5 1010
10
10
Vertical Position
Vertical Position

Vertical Position
Vertical Position
Position
00
0 000

Vertical
Vertical
55
-10-10
-10
-10
1010

-20-20
-20
-20
1515

-30mm
-30-30
-30mm
-30mm
-30 -20
-20 -10
-10 0000 10
10 2020 30
30mm
45 mrad -30mm
-30mm -20 -20 -10-10
30 mrad
Horizontal
10
Position
HorizontalPosition
Horizontal
Horizontal Position
10
Position
2020 3030

By courtesy of Paul Dumas, SOLEIL

 IRSR
The brightness advantage
BB temperature= 2000K
SOLEIL Synchrotron 500 mA
Photon Flux ( Watts/cm-1)

1E-4

Synchrotron IR is much
brighter !
1E-5

Synchrotron Flux @ SOLEIL

1E-6

100 1000
Wavenumbers ( cm-1)

3.8 10 20  bw  I
B      μm ; I   A; bw  %
 2

By courtesy of Paul Dumas, SOLEIL

 IRSR
The brightness advantage

It is the brightness, B, that determines the signal to noise ratio, S/N, in a infrared
experiment via the formula

1/ 2
Particularly useful for
100 A microscopy and
%N( Noise%) 
B t1/ 2D *
spectroscopy of small
sample/diluted systems
 Exploitation of IRSR advantages

Flux Adavantage • Higher S/N FIR and THz

in FIR and THz • Faster data collection spectroscopy

Broad band • Complete data collection Spectroscopy and

nature Microscopy

• BM Linear polarization Spectroscopy and

Polarization
• ER circular polarization Microscopy

Brigthness • Higher S/N ratio

Microscopy
advantage • Faster data collection
 IR beamlines: A special design

SISSI beamline layout

Synchrotron Infrared Source for Spectroscopy and Imaging

Radiation is collected M1 Plane mirror a=3.5 m d=1.5m

over a large solid M2 Ellipsoidal mirror b=1.0m e=1.0m
65 mrad (H) x 25 mrad (V) M3 Plane mirror c=11.5m f=2.5m
M4 Ellipsoidal mirror

c d

a b M1 Plane mirror
e f
M2 Ellipsoidal mirror
M3 Plane mirror
M4 Ellipsoidal mirror

First Chamber in the Ring Tunnel

 IR beamlines: A special design
IRSR intensity distribution at the
The air-cooled extraction mirror
extraction mirror (M1) FIR

9
10

10 meV
10
8
~ 80 cm-1

30X15 cm
10
10

9
10
100 meV
8
10 ~ 805 cm-1

11
10

10
10

9
10
500 meV
8
10
~ 4000 cm-1

The focusing 11
10
Ellipsoidal mirror (M2) 10
10
NIR
35X18 cm 10
9
1 eV
10
8
~ 8000 cm-1
 IR beamlines: A special design

@ Australian Synchrotron @ Soleil

Edge radiation to
“high
« ER »
resolution”
spectrometer

BM

Bending magnet Microscope 2

radiation to “microscope” Branche ER

Microscope 1
Branche BM
 Performance of SISSI beamline
Conventional
Source Diffraction Limited FTIR Microscopy is IRSR
practically achievable only with IRSR
Min Max

~ 20 μm
~ 30 μm
~ 50 μm
 SISSI branchlines

Hyperion
Vertex 70
3000

2nd Branch
Life Sciences
Optimized for FTIRM in the MIR
Vertex 70v

Open to users since 2007

1st Branch
Solid State
Hyperion
2000

Optimized for spectroscopy from Thz to Vis

Open to users since January 2006
 SR-FTIR Mapping
The spatial resolution is achieved
by beam confinement

Vis Camera

Chemical map of the Sample

Motorized
apertures

Motorized XYZ stage

Integration of a Specific Band

Motorized
apertures
To Single point
Detector

Acquired Spectra
SR FTIR Microscopy: Selected application for life
Sciences
 Biology and life sciences:
The spectroscopic point of view

FTIR Spectroscopy

Cell

Samples conventionally studied

Tissue by FTIR Microspectroscopy
Organ

FTIR Microscopy allows to investigate several aspects of

the biochemistry of cells and tissues
 Biology and life sciences:
The spectroscopic point of view
Lipids Carbohydrates
2
2.5
1.5 2

1.5
1
1
0.5
0.5
0 0
3900 3400 2900 2400 1900 1400 900 400 3400 2400 1400 400
-0.5 -0.5

Nucleic Acids
2.5
SINGLE CELL
2
SPECTRUM
1.5

1
Chemical AmideI
Proteins

0.5

0
Imaging 0.12

0.10
Lipid
s
CH2/CH3
AmideII Nucleic Acids-
Carbohydrates
3900 3400 2900 2400 1900 1400 900 400 0.08 asPO2- sPO2-

Abs (a.u.)
0.06

Lipid ester C-C/C-O

0.04

Proteins 0.02
3
0.00
2.5
2 -0.02
3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000
1.5 -1
Wavenumber (cm )
1
0.5
0 Band intensity, position, width and shape (band components)
3900 3400 2900 2400 1900 1400 900 400 -0.5
are sensitive to subtle biochemical changes of bio-specimens
Compositional and structural information at tissue, cellular and sub-cellular level
can be achieved by exploiting SR brightness advantage
 Soft X-Ray Radiation Damage On Fixed Cells
Investigated with SR FTIRM, AFM and XRM
TwinMic Beamline at
Elettra • Radiation damage induced by X-rays on biological
Functionality and toxicity of Zn in wheat samples is one of the remaining bottlenecks for their
ultrastructural characterization by X-ray microscopy
techniques
• X-ray nanofocusing is a today reality but the extent
to which the lateral resolution can be pushed without
unacceptable bio-sample degradation is still an open
question

C/O Zn Mg Fe 8 mm

M. Regvar, D. Eichert, B. Kaulich, A. Gianoncelli, P. Pongrac, K. Vogel-Mikus, I. Kreft, New insights into globoids of protein storage vacuoles in wheat
aleurone using synchrotron soft X-ray microscopy, Journal of Experimental Botany, Vol. 62, No. 11, 3929–3939, 2011.
 Soft X-Ray Radiation Damage On Fixed Cells
Investigated with SR FTIRM, AFM and XRM
The very same radiation
Literaturethat induces damage is
Survey
exploited for probing it
Radiation
damage is
dose Radiolytic effects
dependent undergone by a
specific molecule
Hydrated strongly depend
samples on the sample
undergo more
Radiation relevant
architecture
damage changes with Genetic
depends on respect to fixed material is
sample ones
extremely
preparation sensitive to
Cryo-XRM
better preserves
ionizing
the structural radiation
integrity of bio-
samples
 Soft X-Ray Radiation Damage On Fixed Cells
Investigated with SR FTIRM, AFM and XRM

Hek293T cells (human

embryonic kidney)

Cell growth on 100 nm Si3N4 membranes

Step Cell fixation with PFA 3.7% and overnight air drying
0
Cell drying in vacuum @ TwinMic
Step (p < 10-5 mbar) for 1:30 hour
1
Step Low Dose STXM mapping @ TwinMic 1 keV
Estimated dose: 2*106 Gy
2
Step High Dose STXM mapping @ TwinMic 1 keV Estimated
dose: 2*107 Gy
3 Cumulative estimated dose: 2.2*107 Gy

Step Very high dose STXM mapping @ TwinMic 1 keV

Estimated dose: 6*108 Gy
4 Cumulative estimated dose: 6.2*108 Gy
A. Gianoncelli, L. Vaccari, G. Kourousias, D. Cassese, DE Bedolla, S. Kenig, P. Storici,. M. Lazzarino and M. Kiskinova. Soft X-Ray
Microscopy Radiation Damage On Fixed Cells Investigated With Synchrotron Radiation FTIR Microscopy. Scientific Reports 2015 5,
article number 10250
 Outcomes of AFM

• Minimal cell shrinkage

• Evident degradation/thinning of pseudopodia terminations
• Appreciable thickness variations, especially on the nuclear region at Step 4
• Outstanding topographical changes: nanometric pits and bulges increase in
number and size when increasing dose
 Outcomes of XRM

Mass Thickness ρt   ln
I I0 
μ*
Step 2 (~ 106 Gy)

Mass absorption
coefficient
Step 3 (~ 107 Gy)
Element Mass fraction

C 0.5
N 0.16
H 0.07
O 0.25
Step 4 (~ 108 Gy) P+S 0.02
g/cm2

• Mass Thickness decreases with increasing dose

Combining XRM and AFM

XRM cell images

normalized over
AFM cell thickness

ρ   ln
I I0 
g/cm3
μ* t

• Progressive reduction of
the cell density with
increasing X-ray dose
 FTIRM Outcomes

Step 0

Step 1
Lipids
2988-2830 cm-1

Step 2
Proteins
1702-1480 cm-1
0 1 Step 3
Nucleic acids
Min Max Step 4 1270-1190 cm-1
FTIRM Outcomes

Lipids Proteins Nucleic acids

 FTIRM Outcomes

Dehydration Low-Dose Medium-Dose High-Dose

General Conclusions
 FTIR Microscopy and Prion Research
Aberrant metabolism of the Prion Protein (PrP)
As revealed by FTIRM

PrPC PrPSC – PrPCJD

Alpha-helical beta- plated sheet

PNAS 1998 vol. 95 no. 23 13363-13383

Protease sensitive Protease resistant

PNAS 1999 vol. 96 no. 26 15137-15142

As revealed by FTIR spectroscopy
PrPC 42% α-helix; 3% β-sheet
PrPSc < α-helix; > β-sheet Phenotype dependent

Amide I band – 1700-1600 cm-1

1695-1675 Antiparallel β-sheet/ Aggregated strands
1670-1660 310 - Helix
1660-1648 α-helix
1648-1640 Random coil
1640-1625 β-sheet
1628-1610 Aggregated strands
Prion rods PrP amyloid plaques
Ariane Kretlow, Qi Wang, Janina Kneipp, Peter Lasch, Michael Beekes, Lisa Miller, Dieter Naumann. FTIR-microspectroscopy of prion-infected
nervous tissue. Biochimica et Biophysica Acta (BBA) - Biomembranes, 2006, 1758 (7), 948-959
 A multi-technique approach
for cellular analysis

PrPC Rocky
PrPSc
Mountain
prion strain
GT1 Sc-GT1

AFM outcomes

GT1 and Sc-GT1 have

comparable pyramid like
shape and effective cell
height

Prion Laboratory , Neurobiology Sector, SISSA Prof. G. Legname, A. Didonna

Alessandro Didonna, Lisa Vaccari, Alpan Bek, and Giuseppe Legname. Infrared Microspectroscopy: A
Multiple-Screening Platform for Investigating Single-Cell Biochemical Perturbations upon Prion
Infection. ACS Chemical Neuroscience 2011 2 (3), 160-174
 Outcomes of FTIRM on
individual single cells_1
Proteinase K Western blot assay FTIR Microscopy on whole cells
Chronic infection of the neural cell line Healthy and Infected cells can be discerned

PrPSC is resistant to resistant to

proteolytic degradation

Execution time: few minutes

Execution time: many hours
Multiple cell sensitivity

Single cell sensitivity

HCA on SVN 1st derivatives of IR cell microspectra
[1800-1150 cm-1, Euclidean distances, Ward’s algorithm]
 Multivariate statistical
analysis: Cluster Analysis
D k ((ak )  (bk )) 2
1. Spectral distance calculation
Distance between spectra a and b can be calculated with
many algorithms. Euclidean spectral distance between a
and b spectra is calculated over the all sampled k points
(or within specific intervals).

2. Spectral distance matrix 3. Spectra clustering

a b c d e
a 0

b 44 0
N Clusters
c 11 54 0

d 100 68 97 0 1 a-b-c-d-e

e 120 92 115 21 0 2 ac-b-d-e

3 ac-b-de
There are many methods available to
calculate spectral distances between a 4 abc-de Dendrogram
newly-created cluster and all the other
spectra or clusters. 5 abcde
 Multivariate statistical
analysis: PCA
Principal component Analysis
p d
p

Scores
Spectral Data = Factors
Matrix d
n n
n spectra with p data points; d scores for each spectrum (d<n); d factors with p data points (d<n)

5 spectra Scores Factors

Factor 4
Factor3

Factor 2
 Outcomes of FTIRM on
individual single cells_2

Aspartate Aspartic acid

Glutamate Glutamic acid

~ -27% ~ -27%
FTIRM

~ -31%
~ -26%

Proteins Nucleic Lipids

ac.
 Outcomes of FTIRM at
Proteins subcellular
Lipids level: GT1
Phospholipids
1700-1480 cm-1v 3000-2800 cm-1 1750-1720 cm-1

5μm Proteins Lipids Phospholipids

5μm Proteins Lipids Phospholipids

 Outcomes of FTIRM at
Proteins
subcellular
Lipids
level: ScGT1
Phospholipids
1700-1480 cm-1v 3000-2800 cm-1 1750-1720 cm-1

5μm Proteins Lipids Phospholipids

5μm Proteins Lipids Phospholipids

 Conclusions

• Down-regulation of Lipid and Protein metabolism

• The lower concentration of cellular proteins and lipids is not related to variations
in shape and volume of the infected cells
One of the distinctive histophatological hallmarks of prion disease is spongiosis
 GT1 cell line seems to undergo in vitro the same alterations experienced in vivo
• Lipid signal almost colocalizes with the protein one in GT1 cells, while it extends
into the cytoplasm for Sc-GT1
• The phospholipid signal is not almost exclusively detectable at nuclear level for
Sc-GT1 cells, while it extends into the cytosolic compartment
• Conversion from carboxylate to carboxylic forms of aspartic and glutamic
aminoacids has been elicited. This conversion is incompatible with cytosolic pH
of live cells
Extended cytosolic vesicles and discrete vesicular foci characterize prion infection
 Direct Involvement of the lysosomal compartment in prion propagation

Subcellular compartment/s where the PrPC to PrPSC conversion takes place is

still the subject of intense debate
Our results may reflect the role of lysosomal compartment as conversion sites
 FTIR Microscopy and Cancer Research
Glioblastomas
Gliomas are an heterogeneous group of primary brain tumors
Glioblastoma is the most malignant one (only 5% 5-years survival)
Cancer Stem Cells Theory

Only a small subpopulation of cancer

stem cells is able to initiate tumor
growth and drive development

CSCs not removed by surgery are

responsible for tumor recurrence

CSCs are resistant to standard radio-

and chemo-therapies

Inducing differentiation of stem cells is

the most promising therapeutic
approach nowadays under investigation
Fast methods for monitor the presence/abundance of stem cells and the
efficiency of stem-differentiating agent are needed
 Stem cell differentiation as
revealed by FTIRM
CONTROL STEM CELLS
NCH421K Human Stem Cell Like Gliomblastoma cell line
IR light

Astrocytes differentiation marker Stemness markers

ATRA-DIFFERENTIATED CELLS
Saša Kenig, Diana Bedolla, Giovanni Birarda, Valentina Faoro, Elisa Mitri, Alessandro Vindigni, Paola Storici, Lisa Vaccari.
Fourier transform infrared microspectroscopy reveals biochemical changes associated with glioma stem cell
differentiation. Biophysical Chemitry 2015 207, 90-96
 Stem cell differentiation as
revealed by FTIRM
Same cell cycle distribution
ATRA-DIFFERENTIATED – STEM CELLS

Same, minimal level of apoptosis

Concentration (μg/mL)
4

0
Ctrl Atra

Different
Higher set of genes/proteins
glycogen
ATRA differentiatedlevelcells expressed byby
in ATRA-differentiated
are characterized differentiated
a lower lipidcells
cells concentration
Decreased
In
Thecancer leveland
cells,
decrement of phosphorilated
glioma
mostly as well,
affects proteins
glycogen
methylene inlevels
differentiated
moieties are often
(shorter cells  Down
increased
lipids chains)  regulation of
higher fluidity
kinase inhibitors
Glycogen
of plasma serves and
as
membranes high level ofenergy
supplemental proteinsubstrate
of ATRA-differentiatedkinase inwhen
cells glioma CSCs supply is insufficient
glucose
 Stem cell differentiation as
revealed by FTIRM
All the aforementioned differences are detected at single cell level by using a label-
free, non-damaging and fast method.
To determine abundancy of stem cells and efficiency of ATRA-induced
differentiation of glioma stem cell model NCH412K is possible.

What’s about primary cells? This is still an open question

 Water: two sides of the same coin

“Water is life’s matter and matrix, mother and

medium. There is no life without water”
Albert Szent-Gyorgyi, Hugarian biochemist, Nobel Prize for Medicine in 1937

With small exceptions water still remains a

‘nightmare’ for many spectroscopy and microscopy
techniques
 FTIRM of live cells:
The physiological Conditions
1- For fully exploiting the label-free capabilities of FTIRM
2- For collecting data of major biological relevance
3- For monitoring dynamic cellular processes

Cell Dry mass

~ 50% of proteins
~ 15% of carbohydrates
~ 15% of nucleic acids
~ 10% of lipids
~ 15% other molecules

Cellular water
~ 70% of the cell weight
 Microtechnologies for IR-Live

G. Birarda et al., IR-Live: Fabrication of a low-cost plastic microfluidic device for infrared spectromicroscopy of living cells , Lab Chip, 2016,
Accepted Manuscript (DOI: 10.1039/C5LC01460C )
Mitri E., Birarda G., Vaccari L., Kenig S., Tormen M., Grenci G. SU-8 bonding protocol for the fabrication of microfluidic devices dedicated to
FTIR microspectroscopy of live cells . Lab on a Chip 2014 14(1), 210-8
Mitri E., Pozzato A., Coceano G., Cojoc D., Vaccari L., Tormen M., Grenci G. Highly IR-transparent microfluidic chip with surface-modified BaF
2 optical windows for Infrared Microspectroscopy of living cells. Microelectronic Engineering 2013 107, 6-9
 Advantages of live cell analysis

Air dried Ethanol Formalin Live

U937 leukemic monocytes

FTIRM on live
cells

FTIRM on fixed
cells

• Ethanol dramatically alters both membrane order (increased disorder) and composition. It also
induces protein aggregation and precipitation, as well as a massif loss of cellular material
•Reliable spectroscopic
Formalin assessment
well preserves of nucleic
both cellular acid content
membranes and structure
and protein secondary canstructure,
be done similarly
only under
to
hydrated
unfixed air-dried cells. However, unfixed cells areconditions
not stable in time
• Nucleic acid structure is affected by both formalin and ethanol as well as by air-drying. Dehydration
induces the B-DNA to A-DNA transition (1120  1240 cm-1), limiting FTIRM diagnostic capabilities
L. Vaccari, G. Birarda, L. Businaro, S. Pacor and G. Grenci. Infrared Microspectroscopy of Living Cells in Microfluidic Devices (MD-IRMS):
Toward a Powerful Label-Free Cell-Based Assay. Analytical Chemistry 2012, 84 (11), 4768–4775
 Cell life: Cell cycle analysis by FTIRM

Cell cycle stages Cell growth Cell cycle analysis by FC

G1/GO
S
G2/M

Experimental Design
FC FTIR
M
HCA Centroids HCA on FTIRM FC Analysis

PhI
PhII
From PI-FC to FTIRM
 FC Analysis
HCA on FTIRM From PI-FC to FTIRM

Discrimination between G1/G0, S and G2/M phases of the cell cycle is possible in-situ on live
PhI band
intensity

cells by FTIRM with a degree of accuracy comparable to PI-FC

Bedolla DE, Kenig S, Mitri E, Ferraris P, Marcello A, Grenci G, Vaccari L. Determination of cell cycle phases in live B16 melanoma cells using
IRMS. Analyst 2013, 138 (14), 4015–21
 From PI-FC to FTIRM

FTIRM can discriminateRNA/(RNA+DNA)

between progressive G1 and G0 quiescent cell cycle
≈ 0.5 G1, S, G2, M
RNAG0stages
≈ ½ RNAG1

G0 G1 G0 G1 G0 G1
G0 ≈ 90% G0 < 1% G0 ≈ 20%

Diana E. Bedolla, Saša Kenig, Elisa Mitri, Paola Storici, Lisa Vaccari. Further insights into the assessment of cell cycle phases by FTIR
microspectroscopy. Vibrational Spectroscopy 2014, 75, 127–35
 In-cell RNA spectral features
The spectral features of a molecule in a cell can
not be entirely described by the isolated molecule
0.010% Triton X100, PBS, RT, 5 min.
0.2mg/mL RNase, PBS, RT, 30 min.
RNase
10-30 pg/cell
80-85% m-RNA
Triton 0.01%

r-RNA
r-RNA
t-RNA
Ctrl
Minor RNA forms

• Permeabilization at very low Triton concentrations does not affect the

biochemical cellular composition

• Only three well defined spectral contributions are affected by RNase treatment,
28S centered at & “classically” assigned to:
o ~ 1240 cm-1: Asym. Stretch. of phosphodiester group of RNA + phospolipids +
18S
Amide III (in which proportion?)
o ~ 1123 cm-1: Sym. Stretch. of phosphodiester group of RNA & Asym C-O
Stretch. of Ribose
o ~ 996 cm-1: Sym. C-O Stretch. of Ribose
 From AO-FC to FTIRM

Results of FC with Acridine Orange

Acridine orange: Nucleic acid selective fluorescent dye
DNA probe: Exc: 502 nm Em: 525 (green)
RNA probe: Exc: 460nm; Em: 650 nm(red)

FTIRM Results
New method for baseline correction
New developed program for spectral band fitting
0.05
DNA (1220 cm-1 from PhI)
0.04
0.03
0.02
0.01
0

0 0.01 0.02 0.03 0.04 0.05

RNA (1240 cm-1 from PhI)

 Real-time cellular dynamics
Heat Shock Response
Regular temperature Temperature increase
HSP bonded to Native folding
HSP proteins Folded proteins Misfolded proteins Misfolded proteins restoring

Inactive Heat-shock
Active HSF New HSP proteins
transcription factors (HSF)

Experimental
Experimental
Design Design Mild 39.5±1°C
Severe 42.5±1°C
Deleterious 45.5±1°C
 Initial State

β-sheet
Initial state

α-helix
 Immediate Response: 5-10
minutes
Initial state

5 minutes
 Immediate Response: 5-10
minutes
Initial state 5 min

10 minutes
 Early Response: 15-35 minutes

Initial state 5 min 10 min

15-25
minutes
 Early Response: 15-35 minutes

Initial state 5 min 10 min 10-25 min

30
minutes
 Late Response: 40-120 minutes

Initial state 5 min 10 min 15-25 min 30 min

40
minutes
 The final State: 130 minutes

Initial state 5 min 10 min 15-25 min 30 min 40 min

Final
state

83
 Time-Resolved FTIRM in Live Cells

Fluidics for FTIR allows the safe monitoring of live

cells providing more reliable data and information
on the cellular dynamics

Elisa Mitri, Saša Kenig, Giovanna Coceano, Diana E. Bedolla, Massimo Tormen, Gianluca Grenci, and Lisa Vaccari. Time-Resolved FT-IR
Microspectroscopy of Protein Aggregation Induced by Heat-Shock in Live Cells. Analytical Chemistry, 2015, 87(7): 3670-77
 Where is the future of SR-FTIR?

Technology Science

Future is where FTIRM’s limitations are

• Slow data acquisition

• Limited lateral resolution
• Limited sensitivity (micromolar regime)
 From FTIR Microscopy to FTIR
Imaging
Speeding data acquisition by using 2D multiplex MIR detectors
Dichroic Mirror

Bidimensional
Detector
64x64 pixel =4016 spectra

50 µm

The spatial resolution is guaranteed by pixel size

of the detector
 From FTIR Microscopy to FTIR
Imaging

IRENE (SRC, Wisconsin): a dedicated beamline

Wide horizontal extraction: 320X27 mrad (HXV)

UHV
From M.J. Nasse et al., High-resolution Fourier-transform infrared chemical imaging with multiple synchrotron
beams, Nature Methods, 8:413 (2011)
 From FTIR Microscopy to FTIR
Imaging

FTIRI at U10 (NSLS, NewYork) with an external coupling box

85X40 mrad (HXV)

From E. Stavitski et al., Dynamic Full-Field Infrared Imaging with Multiple Synchrotron Beams, Anal. Chem., 2013,
85 (7), pp 3599–3605
 Potentialities of FTIR Imaging

 ~100 fold pixel area reduction (5μm  0.5 μm) maintaining almost
unaffected the spectral quality
 ~ 10 fold S/N gain
 ~ 102-103 data-acquisition time speeding
 Enhanced chemical contrast
 Improved sensitivity (in the femto-molar range)

FTIR spectro-microtomography

From M. Martin et al., 3D spectral imaging with synchrotron Fourier

transform infrared spectro-microtomography, Nature Methods, 10: 861
(2013)
 FTIR spectro-microtomography

From 2D To 3D
 Vibrational characterization at the
nanoscale

Understanding how natural systems are organized at the nanoscale

and how this organization contributes to their function will improve
human ability to interact with them and to build nano-organized
synthetic platforms with improved functional efficiency

There is nowadays a large variety of tools for characterizing materials’

morphology, structure and function at the nanoscale, while chemical
composition analysis through far-field infrared microscopy yields
information at a larger length scale
 Vibrational characterization at the
nanoscale
Scattering-type Scanning Near-field Infrared Microscopy: s-SNIM
Fritz Keilmann and Rainer Hillenbrand
Optical oscillation modes of plasmon particles observed in direct space by phase-
contrast near-field microscopy , Applied Physics B 73, 239 (2001)

Working principle
Lateral resolution
~
AFM tip radius

E sca   eff E0  se i
1
  tip  
 eff 
  m tip (1   )1  
3
 16 ( z  R) 
 sample  1   m

 sample  1   m
 Vibrational characterization at the
nanoscale
Scattering-type Scanning Near-field Infrared Microscopy: s-SNIM
A sample affects the s-SNOM signal only through its dielectric value εs taken at the wavelength
of illumination. This provides the basis to view s-SNOM as a nanospectroscopic tool, to
measure the local dielectric function for identifying nanosystems according to their known
(far-field) optical and infrared dielectric properties.

Working principle
Lateral resolution
~
AFM tip radius

E sca   eff E0  se i
1
  tip  
 eff 
  m tip (1   )1  
3
 16 ( z  R) 
 sample  1   m

 sample  1   m
 Vibrational characterization at the
nanoscale
Esca   eff E0
 eff  se i
Relative amplitude
Phase shift
Wavelength 633 nm
Material Si: εs = 15
Tip Au: R=10 nm; εs = -10+2i

z/R
 Vibrational characterization at the
nanoscale
Measured signal
Background scattering + Near field scattering

Background scattering >> Near field scattering

Demodulation of the dectector signal at

second of higher armonics of the
oscillation frequency of the tip
 Vibrational characterization at the
nanoscale
From laser s-SNIM
Structural analysis and mapping of individual protein complexes by infrared
nanospectroscopy resolution
Iban Amenabar et al., published in Nature Communications 4, Article number: 2890doi:10.1038/ncomms3890

To broadband s-SNIM
Infrared-spectroscopic nanoimaging with a thermal source
F. Huth, M. Schnell, J. Wittborn, N. Ocelic & R. Hillenbrand, published in Nature Materials 10, 352–356 (2011)

Broadband sources offer

better spectral accuracy
and more efficient data
collection, improving
recognition capabilities
 Vibrational characterization at the
nanoscale

From laser/conventional IR sources to IR-SR

Hans A. Bechtel et al.,

Ultrabroadband infrared
nanospectroscopic imaging
PNAS 111 (20) 7191-7196 (2014)

s-SNIM @ SR Facilities
• Advance Light Source
• ANKA
• Spring8
• Brazilian Synchrotron Light Laboratory
• Diamond
 Structural characterization of
functional proteins by ultrasensitive SR
Collective Enhanced IR Absorption
Microscopy (SR-CEIRA Microscopy)

Background
• FTIR spectroscopy is a very sensitive technique for the structural
characterization of proteins in solution (natural environment)
• It offers complementary information to CD, SAXS and NMR spectroscopy
for proteins difficult to crystallize
Amide I deconvolution

FTIR spectroscopy is sensitive

to micromolar concentration at
the best.
 Structural characterization of
functional proteins by ultrasensitive SR
Collective Enhanced IR Absorption
Microscopy (SR-CEIRA Microscopy)
Surface Enhanced IR Absorption spectroscopy (SEIRA) offers the possibility to
defeat the sensitivity limits of FTIR spectroscopy

• SEIRA with randomly oriented substrates

• Randomly oriented metallic substrate – E field enhancement up to 10-102
• SEIRA with single engineerized nanoantennas
• Single metallic nanoantennas/nanogap - E field enhancement up to 103
• CEIRA with ordered arrays of nanoantennas - E field enhancement up to 104-105
 Structural characterization of
functional proteins by ultrasensitive SR
Collective Enhanced IR Absorption
Microscopy (SR-CEIRA Microscopy)

The reasons for SR-CEIRA Microscopy

• Increasing the intensity of the incoming electric field, the surface enhancement effect
is maximized
SR Brilliance
• Not only Amide I band is interesting for structural characterization of proteins
(despite largely used)
SR broadband and Flexibility of fabrication approaches

The project

Four spectral regions Regions Of Interest (ROIs) optimized for SR-CEIRA:

•Amide I (1695-1610 cm-1) and Amide II (1590-1480 cm-1)
• Amide III band (~1350-1250 cm-1)
• Stretching modes of lateral aliphatic chains of aminoacids (3000-2800 cm-1)
•C-O-C and phosphate linkages, especially relevant for glyco- and phosphorilated-proteins),
and (below 1200 cm-1)
 Structural characterization of functional proteins
by ultrasensitive SR Collective Enhanced IR
Absorption Microscopy (SR-CEIRA Microscopy)
IRSR signal
enhancement by CEIRA

IRSR light polarized parallel to

antennas

IRSR light polarized

perpendicular to antennas
IRSR signal of a BSA monolayer
enhanced by CEIRA
Thank you for your attention