1. 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) A Free Electron Laser (FEL) has been selected as a source to perform the transmission measurements on biological samples. Advantages of FELs are the ability of producing radiation at the desired wavelength, wide range tunability and high power emission. It is then easy to realize a THz FEL, utilizing a low energy (few MeV) electron beam. Exploiting new peculiarities of coherent radiation emission it is possible to realize a broad band FEL THz radiator. Electron beam energy: 2.3 - 5 Mev Spectral Range: 90-150 GHz Bandwidth : 7 % Maximum peak power : 10kW Average peak power (4  s): 1.5 kW CW power: ~ 30 mW ENEA is involved in a large EU project, called THz-BRIDGE. Objective of the project is to set-up a spectroscopic database of biological materials in the THz region, test THz irradiation effects on biological samples in vitro and provide safety procedures for people exposed to THz radiation. So there is a great interest in investigating the optical properties of biological samples in the THz region, both to build a spectroscopic database and to obtain parameters that will allow us to correctly calculate the absorbed dose when irradiating biological samples with THz radiation. Absorption Measurements have been carried out on Whole Human Blood, Serum, Plasma, Water, Saline Solution, Culture medium, Lymphocytes, and other biological samples. Theoretical calculations have been carried out to model the interaction of THz radiation with scattering elements within the various samples. In particular scattering of 120 GHz radiation with lymphocytes and liposomes has been studied. ENEA is involved in a large EU project, called THz-BRIDGE. Objective of the project is to set-up a spectroscopic database of biological materials in the THz region, test THz irradiation effects on biological samples in vitro and provide safety procedures for people exposed to THz radiation. So there is a great interest in investigating the optical properties of biological samples in the THz region, both to build a spectroscopic database and to obtain parameters that will allow us to correctly calculate the absorbed dose when irradiating biological samples with THz radiation. Absorption Measurements have been carried out on Whole Human Blood, Serum, Plasma, Water, Saline Solution, Culture medium, Lymphocytes, and other biological samples. Theoretical calculations have been carried out to model the interaction of THz radiation with scattering elements within the various samples. In particular scattering of 120 GHz radiation with lymphocytes and liposomes has been studied. Motivations of the Experiment The Source Radiation generated from the FEL was transported into the control room by a light pipe and was delivered to the biological sample, contained into a polystyrene Petri dish. The transmitted radiation impinges on a beam splitter and is finally collected by a pyroelettric detector. Transmission through polystyrene dishes and optical properties of polystyrene in the THz region. Absorption Measurements Culture medium (not shown in the graph):  = 83 cm -1 Saline Solution:  = 79 cm -1 Whole blood:  = 75 cm -1 Serum:  = 71 cm -1 Absorption coefficient 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 can be considered responsible of the difference in transmission between whole blood and physiologic solution. Water absorption spectrum Optical properties of Lymphocytes 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 We can calculate the Rayleigh scattering and absorption cross sections of lymphocytes in 2-propanol and in blood, considering for the latter one the same optical properties of water (which is basically made of) : CALCULATION PARAMETERS Lymphocytes radius ~5  m 2-Propanol refractive index at 120 GHz ~2.3-0.53i (dielectric constant ~1.4-1.5i, ENEA measurement) Water refractive index at 120 GHz ~3.4-1.9i (dielectric constant ~9-1.4i) Thus we found that 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 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 Isopropyl alcohol) presented the lowest absorption coefficient: (4.5±0.3) cm -1 The survival time of lymphocytes in 2-propanol has been measured and it resulted to be about 15 minutes. 2-Propanol:  = (4.5±0.5) cm -1 2-Propanol + lymphocytes:  = (5±0.5) cm -1 Density of lymphocytes in 2-propanol: Within the experimental error it’ s impossible to distinguish the two curves (and thus it is only possible to define an upper limit to the value of Lymphocytes absorption cross section), so we can deduce that there is NO RESONANT ABSORPTION OF LYMPHOCYTES AT 120 GHz Since the absorption coefficient is related to the absorption cross section by the equation: We can use the upper limit of a (0.5 cm -1 ) to estimate the experimental absorption cross section of the process This value is very close to the geometric cross section, being the medium radius of a lymphocyte 5  m: Theoretical approach: Mie simulations Mie theory has been used to calculate scattering and absorption cross sections for lymphocytes in blood and in propanol. Lymphocytes exhibit small scattering and absorbing properties at 120 GHz, where the Mie parameter for lymphocytes is about 0.03. Higher scattering contributions at shorter wavelengths is expected. CONCLUSIONS Optical properties of blood are similar to water (high absorption at 120 GHz where rotational and vibrational states are excited). Scattering contribution to extinction coefficient at 120 GHz is negligible (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 around 120 GHz. These results can be applied to the dose calculation when irradiating human blood with THz radiation 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 laser pulses. Emission frequency is ruled by the electron beam energy, the undulator parameter K (proportional to the magnetic field) and the spatial period of the undulator: For our measurement the ENEA Compact FEL was utilized. ENEA FEL emits around 120 GHz with a peak power exceeding 10 kW. Emission parameters are reported below. The ENEA 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. FEL EMISSION PARAMETERS The Sample Absorption Measurements have been carried out on Whole Human Blood, Serum, Plasma, Water, Saline Solution, Culture medium, Lymphocytes, and other biological samples. Absorption measurements have been carried out also on materials to be used to hold the biological samples, like polystyrene and polyethylene. Polystyrene has been found to exhibit good optical properties in the spectral region around 120 GHz, and was thus selected as material for spectroscopic cells Absorption coefficient measurements: 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. REDUCED CROSS SECTIONS FOR LYMPHOCITES IN 2-PROPANOL 00.511.522.53 0 0.5 1 1.5 00.511.522.53 0 0.5 1 1.5 MIE PARAMETER REDUCED CROSS SECTION   r 2  2  r  Extinction Absorption Scattering Zone of interest for lymphocytes at 120 GHz Calculations for lymphocites in water