ABSORPTION AND
DIFFUSION MEASUREMENT
OF BIOLOGICAL SAMPLES
USING A
FREE ELECTRON LASER
M. D’Arienzo,A. Doria, G.P. Gallerano, E.
Giovenale, A. Lai, G. Messina, D. Piccinelli
ENEA C.R.Frascati, Via Enrico Fermi 45,
00044 Frascati (Italy)
1
Why Terahertz?

The region of the spectrum of electromagnetic radiation laying
between 100 GHz and 20 THz is usually referred to as the "THz
gap", since it is considered a rather poorly explored region.
Terahertz radiation corresponds to:
FREQUENCY
WAVELENGTH
WAVENUMBER
ENERGY
(0.1 - 20) THz
(3000 - 15) mm
(3 - 700) cm-1
( 0.4 - 80 ) meV
2
The ENEA Free Electron
Laser


Bunker
Microtron
In a Free Electron Laser (FEL) a
beam of high energy electrons
interacts with a suitable magnetic
structure ( in the specific case, an 8
periods undulator) to generate
coherent electromagnetic radiation.
The Compact FEL produces a "train"
of micropulses of about 50 ps
duration, with 330 ps spacing
between adjacent pulses. The
overall duration of the train
(macropulse) is several
microseconds. Macropulses can be
generated up to a maximum
repetition frequency of 20 Hz.
3
The ENEA Free Electron
Laser
FEL PROPERTIES

Electron beam energy: 2.3 - 5 Mev
 Spectral Range: 90-150 GHz
 Bandwidth : 7 %
 Maximum peak power : 10kW
COMPACT MM-WAVE FEL DESIGN PARAMETERS
Electron Energy (MeV)
2.3
Energy spread
1%
I-peak (A)
4
Und. period (cm)
2.5
I-av (A)
0.2
N (num. of periods)
8
Micropulse duration (ps) 15
Undulator par. K
1
Waveguide hor. gap
10.67
Macropulse duration (ms) 4
(mm)
Waveguide vert. gap
Norm. emittance (cm rad) 0.02
4.32
(mm)
4
Experimental and theoretical
activities
Within the THz-BRIDGE project the ENEA team carried out different experimental
activities among which the irradiation of:
HUMAN BLOOD
 CULTURE MEDIUM
 SERUM
 SALINE SOLUTION
 LIPOSOMES
TRIS-SALINE SOLUTION
 LYMPHOCITES


Some theoretical calculations have been carried out to modelize the interaction of
THz radiation with scattering elements within the various samples. In particular
scattering of 120 GHz radiation with lymphocytes and lyposomes has been studied.
5
Irradiation Set-Up

a
b
d
A
c
The radiation is carried in the
control room by a light pipe (a);
here it is delivered into the sample
by a metallic cone (TDS, THz
Delivery System) studied and
designed to optimize coupling
between the light and the sample
(b). The transmitted radiation
impinges on a beam splitter (c) and
is finally collected by a pyroelettric
detector (d).
The THz beam coming
from the light pipe into
the TDS is first focused
down to a 17.5 mm
diameter aperture by
means of a conical
section and is then let
B
expand by diffraction to
about 52 mm diameter to
match the required 6
irradiation area.
Sample Preparation

The biological samples have been
irradiated inside polystyrene Petri
dishes given the excellent
transmission properties of this
material in the THz range.
Transmission through polystyrene dishes
and optical properties of polystyrene in the THz
P o ly s ty r e n e (d = 1 .1 6 5 m m )
region.
3 .0
1.0
0 .0 1 0
'
T=300K
Transmission
0.8
0 .0 0 8
2 .5
"
0.6
'
0.4
2 .0
0 .0 0 6
"
0 .0 0 4
1 .5
0.2
0.0
0
10
Polystyrene
d = 1.18 m m
0 .0 0 2
1 .0
10
1
10
2
10
-1
Frequency (cm )
3
10
4
0
2
4
6
8
10
12
F re q u e n c y (c m
-1
14
)
16
18
7
Experimental results
1,0000


Serum
Whole blood
0,1000
0,0100
Water absorption of THz radiation
0,0010
5
10
Absorption coefficient
4
10
3
10
-1

Culture medium (not shown in the
graph): a = 83 cm-1
Saline Solution: a = 79 cm-1
Whole blood: a = 75 cm-1
Serum: a = 71 cm-1
a (cm )

I/I0
Phys. Sol.
0,0001
2
10
0,00
1
10
0,05
0,10
0,15
0
10
nominal thickness (cm)
-1
10
0
10
1
10
2
3
10
10
-1
Frequency (cm )
4
10
Such values are close to the absorption coefficient of water at room temperature. The value of the absorption
coefficient measured for whole blood shows that less than 1% of the incident radiation penetrates through
1 mm thickness. Although weak scattering by blood cells does not cause a significant displacement from
8
the exponential attenuation law, it is responsible of the difference in transmission between whole blood
and physiologic solution.
Investigations on cell membrane
functionality
The interaction of cellular systems with their environment occurs primarily
through the cell membrane. Studies on different kind of cells indicate that
millimeter-wave radiation may alter membrane structural and functional
properties that control cellular response. One of the membrane functions is
the transport of substances into and out of cell. This transport through the
membrane can involve the lipid bilayer as well as the membrane proteins.
A very simple membrane model, such as that provided by liposomes, will be
used to study the permeability of a simple bilayer in response to THz radiation
in absence of interfering reactions. This model consists of lipid vesicles enclosing
in their interior a soluble enzyme, such as the carbonic anhydrase (CA).
9
Irradiation on Liposomes
Liposomes are microscopic, fluid-filled pouches whose walls are made of layers of
phospholipids identical to the phospholipids that make up cell membranes. It was found
that phospholipids combined with water immediately formed a sphere because one end of
each molecule is water soluble, while the opposite end is water insoluble. During the
experiment two kinds of liposomes have been irradiated:

“Empty Liposomes”
“Liposomes filled with carbonic
anhydrase enzyme”
A liposome membrane
containing a water filled
vescicule.
Mean size of a liposome: 50 nm
10
Experimental results on
liposomes and tris-saline solution
1
0
Systematic offset owed
to “meniscus effect”
at the edge of the Petri
-----------------------------------------
1
 Itrans.

 Iinc. 
Lyposomes with enzime
“Empty” Lyposome
Trisaline solution with enzie
Trisaline solution
ln
2
dishes
3
4
-5
5
0
100
200
300
400
500
600
700
800
900
1000
1100
Thickness (mm)
Experimental absorbing coefficients
EMPTY LIPOSOMES: (503) cm-1
TRIS-SALINE SOLUTION:
FILLED LIPOSOMES: (553) cm-1
TRIS-SALINE SOL. With ENZYME: (488) cm-1
(438) cm-1
11
1200
Theoretical approach: Mie
simulations I

A model has been developed in which liposomes are considered a bilayer system
made of two stratified spheres:
An external shell (lipid membrane whose refractive index is the same of fat at
120 GHz
1.6-0.5i)
An inner core four times bigger (the water or ahnydrase vesicle whose refractive
index can be approximated to water at 120 GHz
3.4-1.9i)
Water vesicle
3.4-1.9i
Membrane
n=1.6-0.5i
Mie calculations show the reduced cross sections defined as:
versus Mie parameter defined as:

R
2
2  R
 medium
Mie parameter in this case is ~ 0.001
12
Theoretical approach: Mie
simulations II
2

0

4

0

4

0

4
T .ext 3.4-i1.9  1.6-i0.5 
T .sca  3.4-i1.9  1.6-i0.5 
T .abs 3.4-i1.9  1.6-i0.5 
Reduced cross section of the core:
The extinction is owed to
absorption of radiation up to a
value of the Mie parameter ~ 0.8
Extinction

 0 

Absorption

Scattering
 0 


 0 

0
0.5
0
1
0
2

0

4

0

4

0

4
T .ext 3.4-i1.9  1.6-i0.5 
T .sca  3.4-i1.9  1.6-i0.5 
T .abs 3.4-i1.9  1.6-i0.5 
Extinction

 0 

Absorption

 0 
Scattering


 0 

0
0
0.5
0
1
Reduced cross section of the
external membrane: in the range
0-1 for the Mie parameter,
extinction of radiation is again
owed to absorption rather than
scattering
13
Irradiation on Lymphocytes I
What kind of information do we expect to find out from the
experiment?
..which are the optical properties of lymphocytes.
..if there is any resonant absorption at 120 GHz

?
..which are the absorbing properties of lymphocytes.
..which are the scattering properties of lymphocytes
(diffusive regime or ballistic?)
Diffusive regime
Ballistic regime
14
Scattering and Absorption
Approximately we can calculate the Rayleigh scattering and absorbing cross sections of
lhympocytes in 2-propanol and in blood, considering for the latter one the same optical
properties of water (which is basically made of) :



Lhymphocytes radius ~5 mm
2-Propanol refractive index at 120 GHz ~1.3-0.53i ( dielectric constant ~1.4-1.5i,
ENEA measurement)
Water refractive index at 120 GHz ~3.4-1.9i (dielectric constant ~9-1.4i)
Thus scattering is almost absent in the process. All the electromagnetic energy is
absorbed by the particle. The absorbing properties are described by the complex
dielectric constant of the particle
BALLISTIC REGIME
15
Irradiation on Lymphocytes II

Lymphocytes in blood can be considered as scattering elements within an absorbing
liquid solution (plasma). The high absorption of the water content in the plasma
(95% in volume) “hides” the optical properties of the single particles.
Thus, there is a need to find a THz transparent liquid solution as surrounding medium.
Alcoholic solutions showed weakly absorbing properties in the THz range of frequency.
2-Propanol (or Isopropylic alcohol) presented the lowest absorption coefficient
(4.50.3) 1/cm
The survival time of lymphocytes in 2-propanol has been measured and it
resulted in about 10 minutes.
16
Experimental results on
2-propanol and lymphocytes I
0
Absorption coefficient:
0.5
2-Propanol
a= (4.5±0.5) cm-1
1
1.5
2-Propanol + lymphocytes
a = (5±0.5) cm-1
2
0
200
400
600
800
1000
1200
0
5
Density of lymphocytes in 2-propanol: 6  10
1200
1
mL
Or, similarly
600
1
mm 3
Within the experimental error it’ s impossible to distinguish the two
curves (and thus the two absorption coefficients)
17
Experimental results on
2-propanol and lymphocytes II

NO RESONANT ABSORPTION OF LYMPHOCYTES AT 120 GHz
Since the absorption coefficient is related to the absorption cross section by the equation:
a  N   abs
We can use the upper limit of a (0.5 cm-1 ) to estimate the experimental absorption cross section
of the process:
 abs  8.5 10-7 cm2
We can also calculate the geometric cross section (being the medium radius of a lymphocyte 5 mm):
 abs  7.8 10-7 cm2
18
Theoretical approach: Mie
simulations

From the experiment we can deduce that lymphocytes have small scattering
and absorbing properties.Below are reported Mie simulation of lymphocytes
in 2-propanol and blood for extinction, scattering and absorbing reduced
cross section versus Mie parameter :
Thus, a guessed reasonable (yet not true) value of the refractive index has been
considered for lymphocytes.
Lymphocytes refractive index: 1.5-0.5i
Mie parameter ~ 0.04
2
1.4721.5
1.5
Lymphocytes
in:
Propanol
Blood
1
0.5
00
0.5
1
1.5
2
2.5
3
1
0.5
0 0
0
0
0.5
1
1.5
2
2.5
3
3
19
CONCLUSIONS

Liposomes membrane do not scatter radiation at all; the extinction of
radiation is completely owed to absorption. Even if bigger than the
scattering cross section, the absorption cross section is still extremely
small. The same is true for the “core vesicles”.

The optical properties of blood are similar to water (high absorption at
120 GHz where rotational and vibrational states are excited).

Its components seem not to scatter 120 GHz radiation (evident
exponential decay of transmitted signal).

Lymphocytes present poor absorption of radiation (low imaginary part of
the refractive index). In particular there are no resonances at 120 GHz.
20