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ABSORPTION AND ABSORPTION AND DIFFUSION MEASUREMENT DIFFUSION MEASUREMENT OF BIOLOGICAL SAMPLES OF BIOLOGICAL SAMPLES

USING A USING A FREE ELECTRON LASERFREE ELECTRON LASER

M. D’Arienzo,A. Doria, G.P. Gallerano, E. M. D’Arienzo,A. Doria, G.P. Gallerano, E. Giovenale, A. Lai, G. Messina, D. Giovenale, A. Lai, G. Messina, D.

PiccinelliPiccinelli

ENEA C.R.Frascati, Via Enrico Fermi 45, ENEA C.R.Frascati, Via Enrico Fermi 45, 00044 Frascati (Italy)00044 Frascati (Italy)

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Why Terahertz?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 (0.1 - 20) THz WAVELENGTH (3000 - 15) m WAVENUMBER (3 - 700) cm-1

ENERGY ( 0.4 - 80 ) meV

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The ENEA Free Electron The ENEA Free Electron Laser Laser

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. Microtron

Bunker

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The ENEA Free Electron The ENEA Free Electron Laser Laser

Electron beam energy: 2.3 - 5 Mev Spectral Range: 90-150 GHz

Bandwidth : 7 % Maximum peak power : 10kW

FEL PROPERTIES

COMPACT MM-WAVE FEL DESIGN PARAMETERSElectron 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

Macropulse duration (s) 4Waveguide hor. gap(mm)

10.67

Norm. emittance (cm rad) 0.02Waveguide vert. gap(mm)

4.32

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Experimental and Experimental and theoretical activitiestheoretical activities

HUMAN BLOODHUMAN BLOOD CULTURE MEDIUMCULTURE MEDIUM

SERUMSERUM SALINE SOLUTIONSALINE SOLUTION

LIPOSOMESLIPOSOMES TRIS-SALINE SOLUTIONTRIS-SALINE SOLUTION

LYMPHOCITES LYMPHOCITES

Within the THz-BRIDGE project the ENEA team carried out different experimentalactivities among which the irradiation of:

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.

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Irradiation Set-UpIrradiation Set-Up The radiation is carried in the The radiation is carried in the

control room by a light pipe (a); control room by a light pipe (a); here it is delivered into the here it is delivered into the sample by a metallic cone (TDS, sample by a metallic cone (TDS, THz Delivery System) studied THz Delivery System) studied and designed to optimize and designed to optimize coupling between the light and coupling between the light and the sample (b). The transmitted the sample (b). The transmitted radiation impinges on a beam radiation impinges on a beam splitter (c) and is finally collected splitter (c) and is finally collected by a pyroelettric detector (d).by a pyroelettric detector (d).

a

b

cd

A B

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 expand by diffraction to about 52 mm diameter to match the required irradiation area.

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Sample PreparationSample Preparation The biological samples have been The biological samples have been

irradiated inside polystyrene Petri irradiated inside polystyrene Petri dishes given the excellent dishes given the excellent

transmission properties of this transmission properties of this material in the THz range.material in the THz range.

100 101 102 103 1040.0

0.2

0.4

0.6

0.8

1.0

Polystyrened = 1.18 mm

Tra

nsm

issio

n

Frequency (cm-1)

0 2 4 6 8 10 12 14 16 181.0

1.5

2.0

2.5

3.0

'

'

0.002

0.004

0.006

0.008

0.010

28 June 2001file:biowall.opj

T=300K

Polystyrene (d=1.165mm)

"

"

Frequency (cm-1)

Transmission through polystyrene dishes and optical properties of polystyrene in the

THz region.

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Experimental results Experimental results

Culture medium (not shown in the graph): = 83 cm-1

Saline Solution: = 79 cm-1

Whole blood: = 75 cm-1

Serum: = 71 cm-1

0,0001

0,0010

0,0100

0,1000

1,0000

0,00 0,05 0,10 0,15

nominal thickness (cm)

I/I0

Phys. Sol.

Serum

Whole blood

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 the exponential attenuation law, it is responsible of the difference in transmission between whole blood

and physiologic solution.

100 101 102 103 10410-1

100

101

102

103

104

105

Absorption coefficient

Frequency (cm-1)

(cm

-1)

Water absorption of THz radiation

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Investigations on cell Investigations on cell membrane functionalitymembrane 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).

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Irradiation on LiposomesIrradiation 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 membranecontaining a water filled

vescicule.Mean size of a liposome: 50 nm

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Experimental results on Experimental results on liposomes and tris-saline liposomes and tris-saline

solutionsolution

Experimental absorbing coefficients

EMPTY LIPOSOMES: (503) cm-1 FILLED LIPOSOMES: (553) cm-1

TRIS-SALINE SOLUTION: (438) cm-1 TRIS-SALINE SOL. With ENZYME: (488) cm-

1

Systematic offset owed

to “meniscus effect”

at the edge of the Petri

dishes

0 100 200 300 400 500 600 700 800 900 1000 1100 12005

4

3

2

1

0

1

5

Thickness (m)

----------- Lyposomes with enzime----------- “Empty” Lyposome----------- Trisaline solution with enzie----------- Trisaline solution

lnItrans.

Iinc.

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Theoretical approach: Mie Theoretical approach: Mie simulations Isimulations I

A model has been developed in which liposomes are considered a A model has been developed in which liposomes are considered a bilayer system made of two stratified spheres:bilayer system made of two stratified spheres:

Membranen=1.6-0.5i

Water vesicle3.4-1.9i

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)

Mie calculations show the reduced cross sections defined as:

versus Mie parameter defined as:

R2

2 Rmedium

Mie parameter in this case is ~ 0.001

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Theoretical approach: Mie Theoretical approach: Mie simulations IIsimulations II

T.ext 3.4 i 1.9 1.6 i 0.50

4 0

T.sca 3.4 i 1.9 1.6 i 0.50

4 0

T.abs 3.4 i 1.9 1.6 i 0.50

4 0

0

Extinction

Scattering

Absorption

0

2

0 0.5 1

T.ext 3.4 i 1.9 1.6 i 0.50

4 0

T.sca 3.4 i 1.9 1.6 i 0.50

4 0

T.abs 3.4 i 1.9 1.6 i 0.50

4 0

0

0

2

0 10.5

Extinction

Scattering

Absorption

Reduced cross section of the core:

The extinction is owed to absorption of radiation up to a value of the Mie parameter ~

0.8

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

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Irradiation on Irradiation on Lymphocytes ILymphocytes 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 regimeBallistic regime

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Scattering and AbsorptionScattering and Absorption

Lhymphocytes radius ~5 Lhymphocytes radius ~5 mm 2-Propanol refractive index at 120 GHz ~1.3-0.53i ( dielectric constant ~1.4-1.5i, ENEA measurement)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) Water refractive index at 120 GHz ~3.4-1.9i (dielectric constant ~9-1.4i)

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) :

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

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Irradiation on Irradiation on Lymphocytes IILymphocytes II

Lymphocytes in blood can be considered as scattering elements within an absorbing 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 liquid solution (plasma). The high absorption of the water content in the plasma

(95% in volume) “hides” the optical properties of the single particles.(95% in volume) “hides” the optical properties of the single particles.

2-Propanol (or Isopropylic alcohol) presented the lowest absorption coefficient

(4.50.3) 1/cm

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.

The survival time of lymphocytes in 2-propanol has been measured and it resulted in about 10 minutes.

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Experimental results onExperimental results on 2-propanol and 2-propanol and lymphocytes Ilymphocytes I

0 200 400 600 800 1000 12002

1.5

1

0.5

0

12000

Absorption coefficient:

2-Propanol = (4.5±0.5) cm-1

2-Propanol + lymphocytes = (5±0.5) cm-1

Density of lymphocytes in 2-propanol: mL

1106 5 Or, similarly 3

1600

mm

Within the experimental error it’ s impossible to distinguish the two curves (and thus the two absorption coefficients)

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Experimental results onExperimental results on 2-propanol and lymphocytes 2-propanol and lymphocytes

IIII

NO RESONANT ABSORPTION OF LYMPHOCYTES AT 120 NO RESONANT ABSORPTION OF LYMPHOCYTES AT 120 GHzGHz

Since the absorption coefficient is related to the absorption cross section by the equation:

absN We can use the upper limit of (0.5 cm-1 ) to estimate the experimental absorption

cross section of the process:

27105.8 cmabs

27108.7 cmabs

We can also calculate the geometric cross section (being the medium radius of a lymphocyte 5 m):

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Theoretical approach: Mie Theoretical approach: Mie simulationssimulations

From the experiment we can deduce that lymphocytes have small scattering and absorbing properties.Below are reported Mie simulation of 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 : lymphocytes in 2-propanol and blood for extinction, scattering and absorbing reduced cross section versus Mie parameter :

0 0.5 1 1.5 2 2.5 30

0.5

1

1.5

2

0 0.5 1 1.5 2 2.5 30

0.5

1

1.51.472

0

30

Thus, a guessed reasonable (yet not true) value of the refractive index has been considered for lymphocytes.

Lymphocytes refractive index: 1.5-0.5iMie parameter ~ 0.04

Lymphocytes in:

PropanolBlood

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CONCLUSIONSCONCLUSIONS 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”. 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).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).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). Lymphocytes present poor absorption of radiation (low imaginary part of the refractive index). In particular there are no resonances at 120 GHz.In particular there are no resonances at 120 GHz.