ABSORPTION AND DIFFUSION MEASUREMENT OF BIOLOGICAL SAMPLES USING A FREE ELECTRON LASER

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)

2. 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) mm WAVENUMBER (3 - 700) cm-1 ENERGY ( 0.4 - 80 ) meV

3. The ENEA Free Electron 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. Bunker Microtron

4. 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

5. 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.

6. a b c d B A Irradiation Set-Up • 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 expand by diffraction to about 52 mm diameter to match the required irradiation area.

7. 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 region.

8. 1,0000 Phys. Sol. I/I0 Serum Whole blood 0,1000 0,0100 0,0010 0,0001 0,00 0,05 0,10 0,15 nominal thickness (cm) Experimental results • 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 Water absorption of THz radiation 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.

9. 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).

10. 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: “Liposomesfilled with carbonic anhydrase enzyme” “Empty Liposomes” A liposome membrane containing a water filled vescicule. Mean size of a liposome: 50 nm

11. ----------- Lyposomes with enzime ----------- “Empty” Lyposome ----------- Trisaline solution with enzie ----------- Trisaline solution 1 - 5 Thickness (m) 0 1 2 3 4 5 0 100 200 300 400 500 600 700 800 900 1000 1100 1200 Experimental results on liposomes and tris-saline solution Systematic offset owed to “meniscus effect” at the edge of the Petri dishes Experimental absorbing coefficients EMPTY LIPOSOMES: (503) cm-1 FILLED LIPOSOMES: (553) cm-1 TRIS-SALINE SOLUTION: (438) cm-1 TRIS-SALINE SOL. With ENZYME: (488) cm-1

12. An external shell (lipid membrane whose refractive index is the same of fat at 120 GHz 1.6-0.5i) Water vesicle 3.4-1.9i Membrane n=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) Theoretical approach: Mie simulations I • A model has been developed in which liposomes are considered a bilayer system made of two stratified spheres: Mie calculations show the reduced cross sections defined as: versus Mie parameter defined as: Mie parameter in this case is ~ 0.001

13. Reduced cross section of the core: The extinction is owed to absorption of radiation up to a value of the Mie parameter ~ 0.8 2 2 Extinction Extinction 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 Absorption Absorption Scattering Scattering 1 0 0 0 0.5 0.5 0 1 Theoretical approach: Mie simulations II

14. ..which are the absorbing properties of lymphocytes. 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 scattering properties of lymphocytes (diffusive regime or ballistic?) Diffusive regime Ballistic regime

15. 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

16. 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.

17. 0 0.5 1 1.5 2 0 200 400 600 800 1000 1200 0 1200 Experimental results on 2-propanol and lymphocytes I Absorption coefficient: 2-Propanol = (4.5±0.5) cm-1 2-Propanol + lymphocytes  = (5±0.5) cm-1 Or, similarly Density of lymphocytes in 2-propanol: Within the experimental error it’ s impossible to distinguish the two curves (and thus the two absorption coefficients)

18. 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: We can use the upper limit of a (0.5 cm-1) to estimate the experimental absorption cross section of the process: We can also calculate the geometric cross section (being the medium radius of a lymphocyte 5 m):

19. 1.5 2 1.472 1.5 1 1 0.5 0.5 0 0 0 0 0.5 1 1.5 2 2.5 3 0 0.5 1 1.5 2 2.5 3 0 3 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 Lymphocytes in: Propanol Blood

20. 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.