Diapositiva 1

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The THz-BRIDGE Project Tera-Hertz radiation in
Biological Research, Investigation on Diagnostics
and study on potential Genotoxic Effects
An RD project funded by the European Commission
- 5th Framework Programme Quality of Life and
Management of Living Resources - Key Action
4-Environment and HealthContract
QLK4-CT2000-00129
The aim of this research project is to
investigate the interaction of Terahertz (THz)
radiation with biological systems. Present and
rapidly increasing future applications of
radiation in this spectral range, necessitate
preventive rather than reactive research. The
project follows a streamline of increasing
complexity from bio-molecules to cells, e.g.
membranes, chromosomal and DNA integrity. The
objectives of the project are to analyze the
physical mechanisms of interaction, to assess
risk of potential damage to biological activity,
both functional and morphological, and to guide
and accompany the development of microscopic
imaging at THz frequencies for biological and
biomedical applications.
The project will bridge the existing gap of
knowledge regarding the effects of Terahertz
(THz) radiation on biological systems. THz
radiation covers the frequency range between 100
GHz and 20 THz (i.e. a wavelength between 3 mm
and 15 µm), which spans the spectral interval
between the microwave- and the infrared regions
of the electromagnetic spectrum.

• RESEARCH OBJECTIVES OF THz-BRIDGE PROJECT
• To provide a spectroscopic database for selected
  enzymes, proteins, biological membranes and cells
  in the frequency range from 100 GHz to 20 THz
  under irradiation conditions that preserve the
  integrity and functionality of the biological
  samples. (Workpackage 1)
• To identify critical frequencies, which might
  induce damages on biological systems, and to
  determine the spectral regions for optimal
  contrast in imaging applications. (Workpackage 2)
• To assess potential risks due to the exposure of
  membranes, cells, and DNA to pulsed and CW THz
  radiation and to define exposure standards for
  THz biomedical imaging applications to monitor
  and recommend THz exposure conditions at specific
  occupational sites. (Workpackage 3)

THz-BRIDGE PROJECT KEY FACTS
Project start date February 1, 2001 Duration 36
months Total cost 2 MEuro - EU Contribution
1.4 MEuro
Interdisciplinary consortium 9 groups, 5
countries

• ENEA-Frascati (coordinator)
  (I)
• Forschungszentrum Rossendorf-Dresden
  (D)
• Tel-Aviv University - Dept. Physiology and
  Pharmacology (IL)
• Stuttgart University - I. Physics Institute
  (D)
• J.W. Goethe University - Biophysics Institute
  Frankfurt (D)
• IREA -Naples, IMS-Rome, DIE-University of Rome -
  (ICEmB) (I)
• National Hellenic Research Foundation Athens
  (EL)
• Teraview Limited Cambridge
  (UK)
• University of Nottingham - Biomedical School
  (UK)

Expected achievements and preliminary results
Role of T-rays in the biomedical field
Biological applications are based on the specific
spectroscopic fingerprints of biological matter
in the THz spectral regions.
Spectroscopic measurements are used to identify
the frequency intervals where THz radiation is
absorbed by a specific system.
The different values of the absorption
coefficient and index of refraction between water
and tissue carbonated proteins at such
frequencies, provide a unique contrast mechanism
for biomedical imaging applications.
First spectroscopic investigations have been
carried out on human blood, human hemoglobin,
melanin and uric acid in the THz region.
THE THz SOURCES

• Coherent radiation from Free Electron Lasers, Gas
  Lasers and Solid State Sources is available in a
  wide spectral range at the partner sites and at
  collaborating European FEL facilities.
• Features Wide tunability and power ranges,
  amplitude modulation
• Soft X-ray microscopy, SEM, Bio-labs available at
  ENEA, TAU and FZR

An exposure set-up for the irradiation of human
lymphocytes has been designed and constructed.
First irradiation experiments on whole blood have
been performed at a frequency of 120 GHz.
Following irradiation, the Micronucleus assay was
performed by ICEmB on human lymphocytes cultures.
Auston Switch
Optical Rectification
Free Electron Laser
PE0(z,t)2
The preliminary results obtained indicate that,
under the adopted experimental conditions, THz
radiation does not affect micronucleus frequency
and cell cycle kinetics in peripheral blood human
lymphocytes, as shown by comparing sham exposed
cultures with exposed ones. Preliminary exposure
of human skin cells to THz radiation has also
been undertaken.
The non-linear properties of selected
semiconductors are utilized to produce optical
rectification of ultrashort laser pulses. The non
linear polarization so generated travels in the
medium with a lower phase velocity, due to the
lattice vibration contribution. The result is an
emission in a typical Cerenkov cone
configuration.The rapid oscillations of the laser
E field are rectified and only the envelop
remains.
An ultrafast laser pulse is used to switch on a
circuit (a coplanar transmission line antenna). A
current transient is generated, producing a
time-dependent dipole moment that emits
radiation. The short duration of the pulse allows
THz emission.
In the above picture, the THz image of basal cell
carcinoma (BCC) obtained by Terahertz Pulse
Imaging (TPITM) is shown on the left. The red
areas indicate areas of cancer. On the right is
the visible image showing no obvious signs of
cancer growth.
In a FREE ELECTRON LASER radiation is produced by
a relativistic electron beam traveling through a
magnetic structure (undulator). FELs can produce
very short high power THz laser pulse.
Workplan WP 3
Workplan WP 2
Workplan WP1
Evaluation of Biological Effects In Vitro DNA
Bases and Human Lymphocytes ICEmB, TAU, NHRF,
ENEA, FZR-Dresden Evaluation of Biological
Effects In Vitro Membranes and epithelial-cell
cultures ICEmB, ENEA, UNOTT, FZR-Dresden, TVL
Safety issues at specific occupational sites
TVL, FZR-Dresden, UNOTT, TAU
Spectroscopy of Proteins, Enzymes, Biological
Membranes and Cells (Human Lymphocytes) UFRANK,
FZR-Dresden, USTUTT, ENEA, TVL

• Survey of exposure conditions of technical
  personnel at specific occupational sites, where
  THz sources are employed or developed.
• Distribution of a questionnaire to collect
  information
• on the main radiation parameters of THz sources
  currently in use (e.g. frequency range, power
  level, modulation)
• on biological and potential biomedical
  applications of such sources
• on the exposure conditions (if any) of
  technical personnel
• on the safety measurements or precautions
  currently adopted
• Recommendations on safe exposure conditions.

• Development of biological samples, basic
  spectroscopic investigations in
• the spectral range 100 GHz - 20 THz
• Measurements of Reaction Induced Differential
  Spectra (RIDS)
• Time-resolved Spectroscopy at FEL Facilities
• Time Domain Spectroscopy (TDS) by solid state
  THz sources
• Assignment of absorption spectra to molecular
  bonds and functions

• Study of the exposition of human blood to THz
  radiation as a function of frequency, average
  and peak power, modulation conditions
• Genotoxicity Test on human lymphocytes
  Micronucleus, Comet, FISH
• Irradiation of DNA bases and measurement of the
  presence of fragments by mass spectroscopy
  techniques
• Study of the exposure of liposomes to THz
  radiation on-line evaluation of the carbonic
  anhydrase (CA) activity under THz exposure as a
  function of frequency, average and peak power,
  and of modulation conditions
• Investigations on epithelial cells keratinocytes
  cell lines (NHK) and corneal cells (HCE-T)
  (Alamar-Blue cell cell viability assay) effects
  on the adhesion molecules of epithelial tissues
  (Fluorescein Leakage assay on tight junctions)
• Modelling of the interaction mechanism

EXAMPLES OF SPECTROSCOPY MEASUREMENTS
Transmission of 120 GHz ENEA-FEL radiation was
measured in saline solution, culture medium (not
shown in the graph), serum and whole blood.
Results were compared to water absorption
Culture medium a 83 cm-1 Saline Solution a
79 cm-1 Whole blood a 75 cm-1 Serum a 71
cm-1 Water (non reported on the graph) 80 cm-1
EXAMPLE OF GENOTOXICITY TESTTHE MICRONUCLEI ASSAI
Whole blood samples are exposed to THz radiation
under controlled conditions
After preparation of cultures, lymphocytes are
induced to divide
After 44 h of growth Cytocalasin-B is added to
stop cell division at nuclear level
Survey on the use of THz radiation on the
THz-BRIDGE web site
The THz absorption spectra of uric acid and human
hemoglobin (Teraview Ltd.)
Significant progress has been made on the design
of spectroscopic cells and on the choice of
suitable materials transparent in the frequency
range of interest.
FURTHER INFORMATION

• THz-BRIDGE Web
  site
• http//www.frascati.enea.it/THz-B
  RIDGE
• FREE ACCESS AREA
• Project Description
• Progress reports
• Official document
• Contacts

Statistical analysis of experimental results
Slide preparation and observation of Micronuclei
frequency
EFFECTS ON MEMBRANE MODEL SYSTEM
A good model for the membrane system is composed
by carbonic-anydrase loaded liposomes vesicles.
With such a model it is possible to evaluate the
effects of THz radiation on the membrane
permeability
Polystyrene exhibits excellent optical properties
in the wavelength range between 200 mm and 3 mm
(frequency range 1.5 THz to 100 GHz). Other
materials like ZnTe can be used at shorter
wavelengths.
EU Scientific Officer Minna Wilkki - EUROPEAN
COMMISSION E-mail minna.wilkki_at_cem.eu.int Tel
32-2-299 5573 fax [phone]
Project Coordinator Dr. G.P. Gallerano ENEA
Frascati E-mail gallerano_at_frascati.enea.it Tel
39-06-94005223 fax [phone]

• The substrate has a very low self-diffusion rate
  across intact liposome bilayer.
• The substrate can be hydrolyzed by CA inside the
  liposomes only if it permeates across the
  bilayer.
• Any radiation induced increase of hydrolysis rate
  of substrate catalyzed by CA accounts for the
  effect on membrane permeability.
• The kinetic measurement is made following for 2-3
  minutes the appearance of reaction product
  p-nitrophenolate anion at its peak absorbance
  (?400 nm) on an Cary 50 Scan spectrophotometer

This work has been carried out with financial
support from the Commission of the European
Communities, specific RTD programme Quality of
Life and Management of Living Resources, Key
Action 4 Environment and Health - contract
QLK4-2000-00129.
Typical Time Domain Spectroscopy layout