BIOLOGICAL (Human Body) EFFECTS OF TERAHERTZ (THZ) RADIATION http://docs.wixstatic.com/ugd/a5089d_721ea3c553c94f40852820d6e355598c.pdf Biological Effects of Terahertz Radiation Abstract Terahertz (THz) imaging and sensing technologies are increasingly being used in a host of medical, military, and security applications. For example, THz systems are now being tested at international airports for security screening purposes, at major medical centers for cancer and burn diagnosis, and at border patrol checkpoints for identification of concealed explosives, drugs, and weapons. Recent advances in THz have regarding the with this . Biological effects studies are a valuable type of basic science research because they serve to enhance our fundamental understanding of the mechanisms that govern THz interactions with . Such studies are also important because they often times lay the foundation for the development of future applications. In addition, from a practical standpoint, THz is also for the and for the safe use of THz . Given the importance and timeliness of THz bioeffects data, the purpose of this review is twofold. First, to provide readers with a common reference, which contains the necessary background concepts in biophysics and THz technology, that are required to both conduct and evaluate THz biological research. Second, to provide a critical review of the scientific literature. Keywords Terahertz . THz . Thermal effects . Microarray . Cellular effects . Gene expression . Invited review . Biological effects . Review article . Radiation CONTENTS 1.Introduction …………………………………….. 2. Background: composition and function of biological structures ……………………………. 3. Terahertz interactions with biological materials 3.1. Fundamental principles 3.2. Biological origin of tissue absorption properties 3.3. Thermal response of tissue. G. J. Wilmink (*) : J. E. Grundt 711th Human Performance Wing, Radio Frequency Radiation Branch, Air Force Research 4. Thermal effects in biological materials 4.1. Organisms and tissues 4.2. Mammalian cells 4.3. Cellular organelles 4.4. Biological macromolecules 4.5. Microthermal effects 5. Terahertz biological effects research 5.1. Sources 5.2. Detectors 5.3. Equipment used for controlled exposures and dosimetry 5.4. General challenges and considerations 6. Methodology and study-by-study analysis of the THz bioeffects literature 6.1. Organism level studies 6.1.1. Vertebrates 6.1.2. Insects 6.1.3. Plants 6.2. Excised tissues. applications 1 stimulated renewed interest biological effects associated frequency range biological systems biological effects research necessary accurate health hazard evaluation, development of empirically-based safety standards, systems Comment [i]: carbon nanotubes (CNTs) and graphene have emerged as extraordinary low-dimensional systems with a variety of outstanding electronic and photonic properties, 1 − 7 including those ideally suited for terahertz (THz) devices 6.3. Mammalian cells 6.4. Cellular organelles: lipid membranes 6.5. Biological macromolecules 7. Summary and future prospects Introduction energy of THz is level type of because of below the from eVs). Thus, are . This fundamental distinction is important vastly free to other free in that are is that only to cause direct These direct effects are they result in the can cause In contrast, , . terahertz portion electromagnetic does not but it can cause . For many years, data has been scarce at THz frequencies because suitable sources were not widely available. However, a recent surge in research activity has resulted in the development of many new types of sources and components. These new THz technologies have bridged the proverbial “THz Gap,” and are increasingly being integrated into a host of practical medical, military, and security applications. For instance, THz imaging and Electromagnetic Spectrum Frequency (Hz) Spectral bands 10 5 10 6 10 7 10 8 10 10 9 10 10 11 10 12 10 13 10 14 10 15 10 16 10 17 10 18 10 19 10 20 10 21 Radio waves -wave THz IR VIS UV X- rays – rays Frequency (THz): Wavelength ( m): Wavenumber (cm-1): Period (picoseconds): Photon energy (meV): Temperature (K): h c/ k = 1/ 0.1 3000 3.3 10.0 0.4 1.0 300 33.4 1.0 4.1 47.8 10.0 30 334.0 0.1 41.0 478.0 from from bulk frequency spectrum region occupies located . The THz region is between 2 1 The (THz) a large of the (EM) that is the (IR) and (MW) typically defined to include the frequencies ranging from 0.1 to 10 THz, where 1 THz equals 1012 Hz. In terms of other frequently used units, this range corresponds to the following: wavelength 1 (303000 m); wavenumber k (3.3334 cm-1); period t (0.110 picoseconds), temperature T (4.8478 K), and photon energy E (0.441 milli-electron volts) (Fig. 1). It is important to note that the infrared photons several orders magnitude energy required to ionize, or remove, valence electrons biological molecules (typically, several “T-rays” classified non-ionizing radiation nonionizing a nd ionizing radiation generate different effects in biological s tructures. Perhaps, the most noteworthy difference ionizing radiation particles carry o water and to enough energy ionization effects t biomolecules. particularly harmful biological structures formation of highly r eactive radicals nonionizing secondary or indirect damage biomolecules radiation generate radicals biological structures, thermal effects indistinguishable effects observed heating because microwave regions as a which T = h /kB 4.8 Fig. 1 The Terahertz (THz) band of the electromagnetic spectrum. sensing techniques are presently used at major airports for security screening purposes [1, 2], at major medical centers for cancer and burn diagnosis [38], and at border patrol checkpoints for identification of concealed explosives, drugs, and weapons [911]. Widespread deployment of new THz applications has prompted increased scientific interest regarding the biological effects associated with this frequency range. In recent years, many timely investigations have been performed to investigate the possible biological effects associated with THz radiation [1222]. Unfortunately, however, a comprehensive review has not yet appeared in the literature which both discusses the fundamental interaction mechanisms, and also critically reviews the bioeffects studies that have been conducted to date. Thus, the purpose of this review is twofold. First, to provide readers with a common reference, which contains the necessary background concepts in biophysics and THz technology that are required to both conduct and evaluate THz biological research. Second, to provide a review and analysis of the studies reported in the literature on the topic of THz bioeffects. This review is divided into seven sections. The first section provides a general introduction to the THz spectral band. The the of and an of the irradiation of materials. The fourth section summarizes the primary thermal effects that are observed in biological materials at an organism, tissue, cellular, organelle, and molecular level. The concepts described in this section are valuable because they provide the foundation to understand THz-induced effects at all levels of biological organization. In addition, they give the reader the tools to determine whether the effects observed in THz reports can be fully attributable to the temperature rise generated during exposure. The fifth section surveys the major types of THz sources, detectors, and equipment that are used in biological research. This section also addresses the common challenges and considerations that investigators face in this field. Following the description of THz technologies, the sixth section then describes our methodology to survey the literature. This section provides a comprehensive review and “study-by-study” analysis of the THz bioeffects reports that appear in the literature. The review concludes with a summary section that addresses challenges and future opportunities in this field. 2 Background: composition and function of biological structures THz-induced biological effects are influenced by two general factors: the THz exposure (i.e., etc.) and the of the . This section provides background on the chemical composition and function of skin, the . It also discusses energy deposition processes and temperature transients that result from THz of THz with and 3 second section describes composition function biological structures: skin tissue, mammalian cells, organelles, biological macromolecules. The third section provides fundamental mechanisms governing interaction radiation biological materials overview the parameters frequency, power, exposure duration, composition and/or properties biological target largest and primary biological target for THz radiation. Please note, the is also an an to properties. 2.1 and Skin of two (Fig. 2a). The and cell for THz in this we have not . This section also serves to provide the necessary foundation that is required to understand the biological origin of tissue optical important for this cells are type of skin cell. The of a toasaquamousepithelial a biological target effort details make section more concise tissue . The main function of the epidermis is is by 95% of all to and to provide a physical barrier that not only protects against water loss, but also prevents harmful external agents from entering. This protective barrier epithelial tissue consists of five distinct layers or strata: (sc), (sl), (sg), (ss), and (sb) (Fig. 2a). as they the to the include: increases in keratin production, decreases in water content, decreases in cellular metabolism, loss of nuclei and organelles, and cellular flattening. a b Plasma membrane Nucleus Cytosol Inorganic ions RNA H2O Ribosome DNA Cytoskeleton Mitochondria Endoplasmic reticulum O2 ATP Golgi apparatus Lysosome H+ . In brief, these changes cornea 4 radiation; however, provided Human skin: structure chemical composition consists primary layers: an outer epidermis and an underlying dermis epidermis consists of water, keratin proteins, melanin granules, several types, including langerhans, melanocytes, keratinocytes keratinization achieved keratinocytes. in the Roughly keratinocytes, thus, they are the most common Keratinocytes are genetically programmed undergo cellular differentiation process known as keratinization. process results formation layered barrier referred stratified squamous tissue. S stratum corneum lucidum Keratinocytes granulosum spinosum basale undergo several phenotypic changes progress from inner outer stratum Protein Extracellular matrix Fig. 2 (ab). a. Skin anatomy. Histological cross section of porcine skin tissue (Hematoxylin and Eosin stain at 40X magnification). Epidermis (epi), basement membrane (bm), dermis (d). Legend for magnification: stratum corneum (sc), stratum lucidum (sl), stratum granulosum (sg), stratum spinosum (ss), and stratum basale (sb) b. Cellular chemistry and morphology. Image created with Ingenuity IPA software. Although keratinocytes in the sb layer are devoid of keratin, they do have high concentrations of melanin, a pigment responsible for skin (i.e., Fitzpatrick skin type). Melanin granules are produced by melanocytes, and they are transferred to keratinocytes via cytocrine secretion mechanisms. To date, studies have not been performed to characterize the optical properties of melanin at THz frequencies; however, comparable studies have been conducted at optical frequencies. These studies report that the absorption coefficient (a) of melanin decreases with wavelength, and can be approximated as: a (cm-1) = 1.70×1012 ×1-3.48 (nanometers, nm) [23]. Assuming this trend continues into the THz region, melanin absorption is probably weak at THz frequencies (i.e., a 10-4 cm-1). In addition to contributing to skin color, the sb layer also The BM is the and in of the the to few of pattern 5 color It and have c assists primarily formation basement membrane (BM). layer that of type IV .I separates epidermis dermis. consists collagen, laminin, entactin, sulfated proteoglycans nterestingly, date, studies haracterized the optical properties these biomolecules frequencies [24]. Such information would likely improve the accuracy of computational models that are currently used to predict THz-tissue interactions. Immediately below the epidermis lies the dermis. The dermis provides skin with shape and structural integrity, and it ranges in thickness across the human body between 0.3 and 4 millimeters (Fig. 2a). The dermis consists of dermal fibroblasts that are anchored in an extracellular matrix (ECM). The ECM consists of fibrillar collagen embedded in a ground substance material. It is interesting to note that healthy fibrillar collagen exhibits a characteristic with a of ~60 nm, this The significance of this feature will be described in greater detail in Section 4.1. is of and that their own [25]. Due to this property, large volumes of water typically reside in the at THz banding thermally damaged c gylcosaminoglycans ground substance of the dermis. This is the its THz is at THz is to note , thus, (see ollagen loses Ground substance primarily comprised Sections 3.1-3.3). 2.2 Structure and chemical composition of mammalian cells Cells in the human body come in a wide variety of sizes and shapes; however, virtually all cells (Fig. 2b). First, all cells are enclosed by an outer protective barrier known as the plasma membrane. The plasma membrane provides a selective barrier between intracellular contents and extracellular fluids. The is composed of a periodicity water, collagen, are whereas signature banding pattern. elastin, proteoglycans, (GAGs). GAGs hydrophilic molecules volume sequester water volumes roughly 1000 times property important because water primary chromophore frequencies presence strongly governs where energy deposited share certain characteristics plasma membrane phospholipid bilayer, which contains integral proteins Comment [i]: Carbon C 60 would then bind with these carbons and make a person even more sensitive to a terra hertz hit Comment [i]: are are Comment [i]: that for and small digesting Lysosomes compartments responsible damaged macromolecules, which are collected during phagocytosis autophagy, endocytosis, processes Mitochondria are a of that are all second `the the cell,’ the of in the of is to of class cytoplasmic organelles present in nearly cells. Nicknamed powerhouse mitochondria main function generate chemical energy adenosine form triphosphate (ATP). and . The phospholipid bilayer is comprised of two elements: polar are the outer exact of on the cell type, and ratio of cholesterol composition, membranes 6 hydrophilic surface, and in the tails are in the interior of . The degree of saturation of the carbon-carbon hydrocarbon which bonds in the hydrocarbon tails governs the structure and order of the bilayer, where saturated hydrocarbons chains are more restricted and unsaturated chains are more fluid [26]. Overall, the properties and present bilayer interior heads, which oriented towards thermal sensitivity plasma depend membrane saturated versus [27]. Two distinct regions exist inside the plasma membrane of all cells: and cytoplasm (Fig. 2b). The cytosol makes up the largest volume of cells, and it is primarily composed of and ions (i.e., unsaturated hydrocarbons roles in a thick and ), and cytoskeleton filaments. to cells, and they also play key . The is [27]. that that are cytosol water, organic inorganic sodium, potassium, magnesium, calcium, phosphate, chloride Cytoskeleton filaments provide structural support intracellular transport and (pH ~7.17.2) liquid that cellular division cytoplasm alkaline contains all organelles Organelles are vital in specialized membrane-bound compartments provide cellular functions. The primary organelles present virtually all cells are and the Golgi lysosomes, mitochondria, endoplasmic reticulum, complex (Fig. 2b). Lysosomes are small compartments that are responsible for digesting damaged macromolecules, which are collected during autophagy, endocytosis, and phagocytosis processes. Digestion is performed within lysosomes by lipases, proteases, and other pH-sensitive hydrolase enzymes [28]. use to maintain their acidic (pH<4.8) [26]. In addition to their Lysosomes membrane waste disposal functions, lysosomes also assist in the [29] (Fig. 2b). and of are a in all cells. hydrogen proton pumps highly internal environment repair `the is to sealing damaged plasma membranes Mitochondria cytoplasmic organelles present nearly Nicknamed powerhouse function mitochondria generate c hemical energy adenosine triphosphate (ATP). mitochondria, aerobic respiration mechanisms, Kreb's cycle and oxidation phosphorylation, mediate conversion biochemical energy from nutrients oxygen addition to producing mitochondria function transient storage (Ca+2), a cation required cellular functions, transduction, apoptosis, cellular proliferation class of . Calcium entry into the mitochondrial matrix is driven by a steep electrochemical proton gradient provided by the mitochondrial membrane potential (m) equal to -100 to 220 mV [30]. Interestingly, maintenance of a low m is directly linked to the formation and generation of free radicals and reactive oxygen species (ROS) [31]. In the such as the that are of the cell,' the main of the into the form of ATP. In ATP, of and in the form of second also as a site for calcium for many such as signal Furthermore, data at m`s that ROS than 140 mV [30]. recent and suggest play roles in ATP and redox (Fig. 2b). The (ER) and production i ncreases exponentially greater Overall, mitochondria critical production, cellular metabolism, signal transduction, endoplasmic reticulum Comment [i]: The (ER) and the are the final two that are cells The of the ER is to and and o the The of the ER is to and and o the endoplasmic reticulum Golgi complex major cytoplasmic organelles present in most primary function synthesize deliver proteins, nascent lipids, steroids t Golgi complex primary function synthesize deliver nascent proteins, lipids, steroids t Golgi complex the Golgi Golgi . Once delivered to the golgi complex, these macromolecules are then packaged and delivered to their final destination the cell (Fig. 2b). complex nucleus organelle. from THz are the final two largest contains perhaps nucleus of the . Therefore, knowledge of the fundamental principles governing these processes is necessary to understand the biological effects associated with THz irradiation. This section provides the following: an overview of the principles governing the interaction and propagation of THz radiation in biological materials (Section 3.1): an examination of the biological origins of absorption and scattering phenomenon (Section 3.2); and a discussion on energy deposition, temperature transients, and thermal responses of biological materials (Section 3.3).1080 3.1 of When THz photons interact with a material, a fraction of the are at the , and the remaining photons are transmitted into the material. Figure 3a is a graphical representation of an incident THz interactions with biological materials. a. Image illustrating THz beam attenuation in biological materials. major majority cytoplasmic organelles genetic is and beam and can that material 7 are present of the ER is to and and steroids to the in most cells . The primary function synthesize The is the and The (i.e., DNA and gene the most of a cell's to as the cell's to 3 The (i.e., the DNA [26] (Fig. 2b). deliver RNA), and it is .' The nascent with proteins, sometimes lipids, complex referred within important `control center primary of the a site for and functions nucleus are to cellular regulate cellular expression, provide mRNA transcription, facilitate replication Terahertz interactions biological materials interaction of THz by two spot size, e ); and (ii) the (i.e., index of . As a radiation biological with : (i) THz e materials influenced primary elements xposure parameters frequency, xposure duration, irradiance, profile properties) composition and properties both of these of and of biological materials refraction, absorption properties, scattering result, elements impact propagation, spatial distribution energy, thermal effects resulting irradiation biological materials Fundamental principles THz-material interactions photons reflected material boundary Comment [i]: Factors that impact the terrahertz beams– Two directly the of the to (i) the of 1 THz and (ii) the of n contribute amount specular reflectance loses at a air (n1) and the (n2 of factors material interface: angle incidence beam; incoming index refraction mismatch between sample material 8 THz wave being reflected and transmitted into skin. Assuming a unit incident irradiance o (Wm-2), the light transmitted into the tissue can be defined as: T=1 – Rs, where T and Rs represent the ratio of transmitted and specular reflected photons, respectively. (n2). The relationship between these entities can be computed using Snell's Law, n1 sin 1 = n2 sin 2, where 1 is the angle of incidence in air (n1 1), and 2 is the angle of refraction in the material (n2). a Incident THz wave ( o) b 1r Angle of refraction ( ) 30 25 20 15 10 5 0 0.4 0.2 0.0 0 10 20 30 40 50 60 70 80 90 Angle of incidence ( ) 0.4 0.5 0.6 0.7 0.8 0.9 1.0 c 1.0 0.8 0.6 Rs 0.25 0.20 0.15 0.10 0.05 0.00 1 1.5 2 2.5 3 Tissue index of refraction (n2) 1.3 43.4 231 1.4 46.7 214 1.5 1.6 Specular reflection (Rs) 2 Transmitted (T) ( z) 0.1 3.3 0.2 6.7 0.3 10.0 Frequency (THz) Frequency (cm-1) Wavelength (μm) 1.1 Two factors directly c ontribute to the amount of specular reflectance loses at a material interface: (i) the angle of incidence 1 of the incoming THz beam; and (ii) the index of refraction n mismatch between air (n1) and the sample material 1.2 40.0 250 13.3 16.7 750 600 20.0 23.3 26.7 30.0 500 428 375 333 33.4 36.7 300 273 50.0 53.4 200 187 3000 1500 1000 d Index of 4.0 refraction 3.0 (n) 2.0 Water and Skin Water: Wilmink et al. (2011) Water: Jepsen et al. (2007) Water: Nazarov et al. (2010) Skin: Wilmink et al. (2011) 4.0 3.0 2.0 40 30 20 10 0 400 300 200 100 0 300 200 100 0 e 40 30 Specular reflection 20 losses (%) 10 0 f 400 Absorption 300 coefficient 200 μa 100 0 300 Optical Penetration 200 Depth 100 0 g Fig. 3 (ag) THz interactions with biological materials. a. Image illustrating THz beam attenuation in biological materials. bc. Graphical representations illustrating the relationship between angle of incidence, angle of refraction, tissue index of refraction, and magnitude of specular reflection. dg. Real index of refraction (n), specular reflection loses (Rs), absorption coefficient (a). and optical penetration depth (). Water spectra: Wilmink et al. [182], blue line; Jepsen et al. [41], red line; Nazarov et al. [183]; green line. Skin spectra: Wilmink et al. [182], black line. J Infrared Milli Terahz Waves (2011) 32:1074 9 1122 Neglecting polarization, approximated using the the of can be 1 tan2 ðq1 À q2 Þ sin2 ðq1 À q 2 Þ ; ð1Þ þ 2 Rs 1⁄4 2 tan2 ðq1 þ q2 Þ sin ðq1 þ q 2 Þ Figure 3b is plot of 1 versus the Fresnel relation: of Rs the to: n1Àn22;ð2ÞRs1⁄4n1þn2Figure3cisagraphical representation that illustrates the relationship between a material’s index of refraction and the magnitude of Rs. The data that in a of in in Rs. Figure 3d contains published data for the index of of and skin at THz frequencies [32 43]. The data shows that water’s index ranges 3.5 at 0.1 THz to 2.0 at 1.6 THz, magnitude of Rs. The data that the for than 60 to the illustrate exponentially of incidence Assuming angle of is Eq. 1 incidence magnitude normal magnitude material, increases 10 specular reflectance angles greater degrees. reduces clearly show material’s index increases appreciable increases refraction water porcine while skin’s from refraction result Comment [i]: the hijacking of people with hydrogen through terrahertz frequencies ~ the hydrogen has a network of a “collective manner” which means it will amplify the TZ freq hit 11 accounting for Rs, the transmission T of THz photons can then be determined using Beer-Lambert’s law: 6z 1⁄4 eðÀma zÞ ; ð3Þ T1⁄4 6o where o is the incidence irradiance (Wm-2), z is the irradiance (Wm-2) after a path index ranges from 2.2 at 0.1 THz to 2.0 at 1.6 THz. Corresponding Rs values are provided in Fig. 3e. The data show that only 70 to 90% of the THz is into . the airtissue leads to losses. After incidence irradiance transmitted materials Clearly, is of a is absorption a per to as the mean depth (m). This value is equal to 1/e, or to the traveling is through length material, coefficient material. absorption coefficient (cm-1) defined probability photon absorbed infinitesimal length reciprocal referred absorption length optical penetration irradiance corresponds where roughly incidence irradiance. addition absorption, scattering properties of materials impact spatial distribution deposited photons. defined refractive length mammalian z in the The that a . The or the to the depth coefficient s (cm-1) is unit path in is particularly molecules, large 51]. In occur at 1.5 THz [49- also interface the In and a is the of the as the unit path 37% of the the of can also Analogous to the absorption coefficient, the scattering as the of per . arises from index of the cells. Photons scatter most strongly by structures whose size matches the incident wavelength. Thus, in the and of than most wavelengths probability . Due to this to be an absorption-dominated case. 3.2 Biological origin and absorption properties of skin at THz frequencies feature, THz tissue interactions are Many and [4448]. (i.e., DNA, to tissue but most data at THz that water is the chief tissue many that to its . One such property is the ability with THz of water molecules to readily engage in both inter- and intra- molecular hydrogen bonding with neighboring molecules. As a result of these interactions, water molecules bond that in a of this of water are quite . Interestingly, the are the at a of 5.6 THz [4951]. In addition, due to the slow relaxation time of bulk water substantial because biologic photon at and THz scattering infinitesimal Scattering spatial variations tissue, extracellular constituents, scattering biological materials strong visible near-IR wavelengths, and weak at weak at THz longer wavelengths. Biological is are scattering waves several orders magnitude larger biological structures assumed biological macromolecules proteins, tryptophan, carbohydrates) contribute absorption, suggest chromophore Water exhibits unique radiation properties contribute frequencies strong i nteraction create extensive dynamic hydrogen networks behave collective manner intermolecular stretching vibrations network, which origin macroscopic dynamics, strong frequency intermolecular bending vibrations addition to water, many other at THz f biological macromolecules exhibit collective vibrational modes requencies [5256]. to the tissue at THz Overall, biological macromolecules contribute absorption, believed chromophore frequencies optical p roperties water, primary constituent biological tissues, well-characterized frequencies. Figure ) contains absorption values corresponding many but water is to be the main The of skin and the of . all are at THz 3(f-g the and 12 optical penetration frequencies. depths absorption coefficient water value between shows optical penetration of the this depth THz is is then hundred microns lower frequencies, and roughly microns at higher Thermal response tissue When energy transmitted into biological materials, optical energy absorbed target chromophores. Once absorbed, energy converted heat, process generates significant thermal transients, which driving force precursor photothermal processes () for water and ex vivo skin. The of and skin are and range in 100 cm-1 at 0.1 THz to 300 cm-1 at 1.6 THz [42, 57]. The data also that the at THz is a few fifty THz by into 3.3 is and in the are the and for all . Thus, in the absence of photochemical processes and phase comparable porcine transitions, all a by is converted into the THz and the energy absorbed THz-exposed temperature rise. for skin, the rate and of that is into the can be This rate is as the rate of heat S Sðr; zÞ 1⁄4 ma ðr; zÞ6o ðr; zÞ; ð4Þ where o (r,z) is the irradiance at a point located in the tissue at (r,z), and a is the local absorption coefficient at point (r,z). S in Eq. 4, the local rise in the can be the ð5Þ T is the local r,tisthe and c is the $T ðr; zÞ 1⁄4 S ðr; zÞ$t ; rc rise in ) at an oftheheat , isthe heat in Once the calculated location where energy absorption terms are determined, the Pennes’ bioheat equation, which is based on the heat diffusion equation, can be used to model heat transfer [58, 59]: rc @T 1⁄4 kr2 T þ S þ q @t ð6Þ where is the density of the water or tissue (kg m-3), c is the specific heat (J kg- 1K-1), T is the temperature (Kelvin), t is the exposure duration (seconds), is the (Wm-1K-1), S is the heat of generation , and q is the rate of perfusion. Equation 6 can then be converted into 3-D Cartesian domains: @T @ @T @ @T @ @T 1⁄4 k þ k þ k þsþq rc @t @x @x @y @y @z @z ð7Þ can then be used with such as and exposure tissue Using incident irradiance absorption coefficient amount deposited tissue defined (Kelvin energy calculated. typically generation (Wm-3): Then using computed tissue using (gm-3), tissue’s temperature following relationship: temperature duration specific capacity (Jg-1K-1). arbitrary tissue density thermal conductivity and and of THz in For further details These equations on M&S we refer the reader to several books [58, 60]. 4 in to THz can a . in the by to . Thus, knowledge computational modeling simulation (M&S) techniques, Monte Carlo Finite- difference-time-domain (FDTD) methods, to model propagation deposition photons biological tissues. biological materials Biological materials exposed Thermal energy effects undergo changes t various levels Common effects include tissue coagulation, structural protein damage, cell death, activation intracellular stress responses, and THz of THz disruption of cellular organelle functions. Furthermore, since energy is strongly absorbed biological tissues, higher levels power are likely generate pronounced thermal effects biological materials of Comment [i]: Meaning there is more to this in the way in cam alter and impact you but this will be a limited expose’ Comment [i]: How the pleotropic effects of hyperthermia. 4.1 Thermal effects on organisms and biological tissues The type and severity of thermal effects depends on many factors: (i) exponentially dependent on terrhertz impacts Comment [i]: Results of temperature; (ii) hyper thermia — linearly dependent on excess heat damage- the duration of exposure; (iii) – Hyperthermia causes organism, tissue, and several effects at the cell type; (iv) tissue organism and tissue architecture and level. The most macroscopic common effects environment (i.e., include: (i) activation blood perfusion, of acute hydration levels); inflammatory and (v) metabolism, responses; (ii) tissue physiology, and dessication and microenvironment of necrosis; and (iii) cellular constituents irreversible structural protein denaturation, birefringence loss, and visible tissue whitening [6163] [Fig. 4(a-b)]. The sensory nerves that 13 of conventional thermal effects and their respective time-temperature histories is essential for proper analysis of THz bioeffects studies. In this section, we shall provide a summary of the principle thermal effects observed in biological structures. Clearly, this field is too broad for a section of modest length, so we have to only the signs of Here we the of cells, cellular . This section is organized with the intention of providing the reader with a framework to understand the (i.e. pH, O2, CO2, ATP, and ]. These factors vary widely in different biological materials, thus, each vastly . Clearly, given the large number of variables that contribute to thermosensitivity, it is t the exact of all that can be used to relate tissue level. The most . This response, which typically lasts several days, to not only but also to the The Ca+2 a 1 Tissue and ECM 32 4 Cytoskeleton 2A chosen highlight signature hyperthermic damage. describe thermal effects organisms, tissues, and extracellular proteins, mammalian organelles, biological macromolecules pleotropic effects of hyperthermia. 4.1 Thermal effects on organisms and biological on many tissues The type and (i) severity exponentially of thermal effects depends factors: dependent on temperature; (ii) l inearly dependent and cell type; (iv) tissue (i.e., blood on the duration of exposure; and (iii) organism, tissue, architecture macroscopic environment perfusion, hydration l evels); and (v) metabolism, physiology, and microenvironment of c ellular constituents glucose, metabolite levels)[27 material exhibits different thermal sensitivities difficult o determine thermosensitivity a particular biological material. However, biological materials exhibit similar response t rends dosimetry with observed effects . Hyperthermia causes several e ffects at the organism and common effects include: (i) activation of acute inflammatory responses; (ii) tissue dessication and necrosis; and (iii) irreversible tissue structural whitening protein [6163] [Fig. denaturation, birefringence The loss, and visible 4(a-b)]. sensory nerves that reside in skin perceive hyperthermia to be a harmful stimulus, and consequently response respond t o it by activating an acute inflammatory functions remove injurious stimuli, initiate wound healing cascade. blebbing, permeability, perforation, bursts protein damage Intracellular protein damage Protein aggregation reside in skin perceive hyperthermia to be a harmful stimulus, and consequently respond to it by Ca+2 Ca+2 5 destabilize & Disrupt DNA replication & repair Mitochondria 3B 2B HSF HSF 3A HSF HSF HSF 5A 5B Production of H2O2 Om HSF ATP Ribosome 14 disintegrate actin detach from plasma membrane Unfold “Sticky” Nuclear proteins destabilize reactive oxygen species (ROS) Cellular stress response (CSR) Fibrillar collagen Inhibit aerobic glycolysis H-bonds hydrophobic region Denaturation Stress granules HSE-Hsp70 promotor 2C HSP70 15 redox status inhibit burst swelling permeability + Birefringence loss HSF HSF HSF HSF 2D 6 Lysosome H+ ATP ROS Visible tissue whitening Protein Refolding Hydrolytic enzymes Destruction, membrane leakage b 100 90 80 70 60 50 40 30 20 10 0 0.01 0.1 100 90 80 70 60 50 40 30 20 Necrosis Cell Hyperthermia Hypothermia Tissue dessication Irreversible structural protein damage death: apoptosis Visible tissue 10 0 cold Temperature (°C) 1 + C): ASP “Heat damage shock” Cell Pain Mild stress death 0.001 Homeostasis Severe stress “Cold shock” Moderate stress Severe stress Visible tissue d amage (blister) (Pearce 2010) Tissue necrosis ( Moritz 1947) Cellular death (Wilmink 2007) Cell membrane bledding (Borelli 1986) Cellular survival + high CSR (Wilmink 2009) Cellular survival moderate CSR (Wilmink 2009) Cellular survival + mild CSR Pain (Wilmink 2009) Cellular survival + no CSR (Wilmink 2009) (Moritz 1948) Reduced cell growth + C): C prolonged G1 phase (Nishiyama 1997) stress (25-35 IRP + RBM3 (Nishiyama 1997) Moderate stress (15-25 C): Cold HSPs ( Kaneko 1997) Severe cold stress (0-15 (Grand 1995) induced-apoptosis (Fujita 1999, Gregory 1994) Cold-induced-necrosis (Fujita 1999, Gregory 1994) Mild cold 10 100 1000 c Temperature (°C) 56 54 52 50 48 46 44 42 40 16 Exposure duration (minutes) Stress Region Region 1 10 100 DNA + high CSR Cell Mild cell Fig. 4 (AC) (4), with to & 1: Cell & & 2: 3: Cell survival stimulation Region death organelle damage Rupture plasma nuclear membrane (Dressler 2005) Disrupt mitochondria membrane p otential (Dressler 2005) Lysosomal membranes leak (Hume 19 78) Mitochondrial damage (Cole 1988) death (Wilmink 2007) Heat-induced-apoptosis (Harmon 1990) Heat- induced-necrosis (Harmon 1990) Plasma membrane proteins (Lepock 1983) DNA Roti double s trand breaks (Takahashi 2008) Inhibit repair (Roti 2010) Cell Cell + mild CSR Roti Cell s & membrane deformation survival (Wilmink 2009) survival + moderate (Wilmink 2009) progression survival (Wilmink 2009) + no CSR Inhibit (Roti 1986) urvival (Wilmink 2009) flattening cytoskeleton destabilization (Dressler 2005) Stimulate cellular growth metabolism & of and CSR Cell cell cycle Cell Exposure duration (minutes) Thermal on effects tissues, extracellular cells, organelles, biological macromolecules. cartoon a. A and (2), p (5), and l graphic illustrating the effects radiation tissue collagen proteins intracellular proteins lasma membranes (3), actin cytoskeleton mitochondria ysosomes (6). b. Thermal effects tissues exposure of i are temperature of are: the plotted versus and exposure duration. Thermal effects cells, organelles, macromolecules and . cardinal signs signs nflammation pain, heat, redness, swelling. These primarily result of the increased blood facilitate the movement of white blood region. Biological tissues typically b ecome desiccated of THz (1), flow that is used cells into the and of c. injured tissue necrotic to 80 loss that the tissue and extracellular collagen proteins (Pathway 1, far left). The image provided in the figure is a histological cross section of heated skin for (see Section 2.1). Figure 4a contains a cartoon graphic illustrating the effect of hyperthermia on of is 17 when exposed elevated temperatures ranging between -100°C several seconds. In at for contrast, irreversible damage extracellular proteins can 50 such as occur considerably lower temperatures ranging between -70°C several minutes [61] [Fig. 4(b)]. In fact, are or they structural proteins, fibrillar collagen, often damaged when tissue temperatures reach 60°C for 1 minute longer [64]. When structural proteins are is a irreversibly damaged become visibly whiter . Tissue whiting consequent o f the biefringence occurs when regular arrangement collagen molecules disrupted stained with a Gomori Trichrome collagen stain. The the in the skin the in the Often times, tissue effects are more subtle, and are not clearly visible. For and . It is important to note that visible tissue damage is used as a biological endpoint for the determination of safety standards image that red, illustrates denatured collagen wounded region stains whereas healthy collagen untreated skin . region stains green these instances, tissue damage assessment requires microscopic techniques, such as transmission polarizing, transmission electron, multi-photon microscopy at optical and higher THz frequencies (i.e. ANSI standards). Thus, knowledge of the thermal effects of tissue is important for the determination of safety standards at THz is cells The level the [6669]. The all cell lines can be [65]. A in Fig. 4b. 4.2 of [6669], and (iv) cell via plot of on (ii) (iii) at a and 2, and of [70]. Consistent with this theory, Dressler et al. demonstrated that cells exposed to mild heat stress (40 and 42°C for 30 min) maintained their structural integrity, but their F-actin network appeared mildly destabilized, giving the cells a [70]. Clearly, mild hyperthermia can lead to subtle morphological effects; such also and mild and 72]. cells 46°C for 30-50 min (Fig. 4c, : (i) size, ECM and and (iv) and of cells is cell type a into 1, and mild 40-42°C are such such as cell to be a of 18 frequencies summary hyperthermic tissue effects provided Thermal effects mammalian most common thermal effects observed cellular include following: (i) stimulation of cell growth metabolism; morphological changes (i.e., swelling, blebbing, shrinking); activation cellular stress response (CSR) mechanisms death apoptotic necrotic pathways thermal sensitivity dependent; however, typically share similar response trend. This trend generalized and grouped three distinct time-temperature dosimetry regions, where region region region 3 comprise the (Fig. 4a, c). not to can lead to and effects observed for increasing levels hyperthermia Temperatures ranging between typically lethal most cells (Fig. 4c, Region 1). However, exposures subtle morphological alterations, flattening membrane ruffling. These effects are believed result of cytoskeleton reorganization destabilization actin-plasma membrane connections exposures intracellular (4042°C for 1030 min) has been shown to of a flattened signaling appearance such as [66, 69, 71] (Fig. 4c). In fact, pathways, cellular growth metabolic processes thermal stress increase the g rowth of cells by 20% [66, 69, 71, 42– (i.e., to of cell cycle however, activate Mammalian metabolic rates exposed to temperatures ranging between Region 2) typically exhibit s everal signature effects dramatically altered cellular (ii) (iii) i morphology shape, irregularities, and roughness); reduced adhesion intracellular actin cytoskeleton; nhibition progression; activation molecular the Specifically, Dressler et al. demonstrated that cells to 45°C for 30 min a a in cell size, and [70]. In addition to these 43– of cell cycle . For structural and morphological effects, 44°C for 15-50 min can also the of CSR 19 defense reaction called cellular spheroidal stress and i cell response (CSR). exposed exhibit disintegrated actin network, rough rregular plasma membrane, decrease shape temperatures ranging between result in the impairment triggering mechanisms observed undergo a progression transient intracellular and/or is well cause exposed block, while when exposed exhibited lasting G2 phase S-phase DNA block mechanism documented literature general consensus activate these mechanisms combat and survive hyperthermic stress . CSR mechanisms i nvolve many pathways, i ncluding redox, sensing repair, molecular c haperones, proteolysis, energy metabolism, apoptosis group evolutionary conserved proteins emerged mediators . These proteins, collectively referred to as by cells minimal stress proteins, significantly upregulated immediately exposure t stress most widely studied family minimal stress proteins shock proteins example, cells to 45°C for 15 min were G2 to 45°C for 30 min, they CSR and the to a long and a late [73]. The in the [6669, 7477], is that cells to and and [78]. Many proteins are associated with these pathways; however, a of 44 have as core [79] are aftero.The of are the heat (Hsps), which includes Hsp70, Hsp40, Hsp60, and Hsp105 [6669, 7477] (Fig. 4a, c). with cells than 46°C of of etc); or (Fig. 4b). and (iii) they [8183]. At to the effects of hypothermia- based effects will become clearer in subsequent sections. In THz cells cold to of such on can to temperatures Mammalian exposed severe thermal stress greater ablative regime) exhibit gross alterations to most cellular components (Fig. 4c, Region 3). These effects i nclude the collapse cellular membranes shape, complete destabilization and d isintegration actin cytoskeleton network, rupture of both via plasma and and nuclear death apoptotic necrotic pathways general, preferentially activate apoptotic pathways w hen resources available (ATP, oxygen, however, w hen resources unavailable, cells die via necrosis pathways [ 80]. Finally, addition in can also to hyperthermia, hypothermia shock temperatures cause severe cellular effects shock effects are t (i) Mild (25 mild cold ypically divided into (15 cells three time-temperature categories: 35°C); (ii) Moderate 25°C); Severe 15°C). Under shock conditions express cold- inducible proteins (CIRP), while apoptosis-specific at more m oderate conditions express HSPs proteins ( ASPs) point clear to reader e decided address hypothermia (i.e., (Fig. 4a, c). In cold Cold (0 both this it may not be [70], and cell cells they have the are and why we hav . The relevance and importance of the of . 4.3 is brief, many bioeffects studies expose under shock conditions, thus, knowledge hypothermia-induced cellular effects critical differentiate origin effects Thermal effects cellular organelles Hyperthermia cause direct effects cellular Comment [i]: One solution is to increase saturated fats in diet not omega 3s which woul complement the terrahertz tech by further causing the cells to break down and cause the hydrogen in the fluids to accentuate the frequency organelles. Damage organelles consequent damage to the outer we shall membranes major organelle. section, briefly describe thermal effects frequently observed on the and plasma membrane, cytoskeleton, mitochondria, lysosomes, nucleus . Heat can cause and its gross several changes to the These of plasma membrane interconnected cytoskeleton. effects include morphological changes, redistribution membrane proteins, actin-plasma membrane detachment, membrane perforation, c hanges in membrane permeability, spikes intracellular calcium The of of to with cell type, the and ensitivity to 20 is often times a direct of of each In this the that are actin in vital cellular protection, several groups have provided compelling evidence that the plasma membrane may be the most component [70, 87]. In fact, a recent study showed that morphology changes were observed in cells heated at 45°C for 30 min. These effects are to be a direct result of from the levels [70, 8486] (Fig. 4a, c). Although surprising, given the fact that the plasma membrane provides thermosensitive cellular membranes believed cytoskeleton detachment . Data from this study also showed that these over the 40 to 56°C are range [70]. on the These properties plasma membrane effects continuously enhanced temperature thermal s plasma depends composition and on the levels of and the ratio on properties of the local membrane. depend cholesterol, membrane fats. Thus, since of cells also cell type. The effect that thermosensitivity has been in These studies specifically showed that cells supplemented with polyunsaturated proteins, saturated unsaturated membrane has on composition varies thermal sensitivity depends membrane fatty acids had increased thermosensivity, whereas cells fatty acids had highlighted composition several studies with [88, 89]. Heat can (i) disrupt the redox status of cells; (v) ATP (Fig. 4a, c) [27]. mild heat stress (40 and are (Bax) and 2 [91]. [88, 89]. supplemented saturated decreased t hermosensivity several interrelated effects on 90]; (ii) of ROS; (iv) mitochondria: membrane potential (m)[84, change increased bursts stimulate mitochondrial enzyme activity (i.e. citrate synthase)[87]; inhibit production; destabilize intracellular proteins Membrane disruption typically under pronounced under severe Membrane disruptions mediated signaling of pro-apoptotic family members, particular caspase cause (iii) cause and (vi) does not occur 45°C for 30 min), but it becomes quite (50-56°C for 30 min) [90]. to be via the Bcl-2 in conditions believed antiapopotic In , are to the of by50%,andtheevelofATP[87, 91].Infact,athatcellsheirATP combination redox, almost by [87]. to a 20 min hea can also lead to the can lead to of CSR . Heat can on [9295]. Heat increase and DNA 41 and 43°C for 90 min [95]. In in and into the can such as 45°C for 90 min, can in the of . such as and . The nuclear membrane has long been considered the most thermoresilinient cellular organelle. This theory seemed reasonable given the fact that temperatures of 56°C for 30 min are required to puncture the nuclear membrane [70]. A that the of that are is the to date [96]. The nuclear matrix plays several vital functions, including DNA at 45°C of both 21 mechanisms, these effects believed directly affect cellular generation ROS, reduce citrate synthase activity ultimately reduce production l recent study showed decrease t content nearly 60% when exposed t shock These primary effects destabilization cytosolic and nuclear proteins, which activation damage cause several effects lysosomal membranes stress at can temperatures membranes ranging between durations trigger increases lysosomal enzyme activity addition, higher temperatures, make lysosomal leaky, resulting release hydrogen hydrolytic enzymes cytosol Thus, these primary effects cause drastic secondary effects, proteolysis deficiencies impaired cellular function more recent report, however, provided definitive evidence nucleolus is a repository stress stress responsive proteins released in that a o as the response to thermal [96]. This work also provided evidence critical underlying nuclear structure, referred t nuclear matrix, most temperature s ensitive subcellular component identified replication, RNA and DNA . heat shock has of the cell, and thus processing, the to may be the key cell the thermal effects of the nucleus, we refer the reader to the following articles [73, 84, 97, 98]. 4.4 on The repair Thus, potential disrupt many critical functions mechanism underlying death . For further details on Thermal effects biological macromolecules Comment [i]: How convenient to be able to utilize something and not have the capacity to validate if it has been used thermal effects result of to excitation groups, of the the CSR (50-60°C) cause (40-48°C) cause the and are a direct result that (Fig. 4a). In observed exceeds at a and 22 level are a direct . At a molecular level, when rises increase the When this cellular provided organelle damage intracellular biomolecules biological tissues are heated, the of the ensuing (i.e., DNA) undergo conformational changes and/or denature. Thermal effects to macromolecules are typically subdivided into two temperature kinetic energy tissue’s water and the biomolecules. energy energy by the i ntramolecular bonds, which holds the molecules together, biological molecules lipids, proteins, mRNA, where higher thermal ablation te mperatures irreversible damage, and hyperthermic t emperatures reversible damage of of .C ommon reversible effects include deactivation enzymes , protein unfolding or denaturation, acceleration cellular metabolism . These and and DNA. When cells the effects disruption of the of hydrogen disulfide bonds maintain tertiary structure proteins intracellular proteins are reversibly damaged, mammalian typically activate mechanisms (described above) to repair damage contrast, when proteins irreversibly damaged, cells activate proteolysis mechanisms degrade proteins lysosomes. are to in For more details on the thermal effects on macromolecules we refer the reader to Urano et al. [27] 4.5 are . Thus, in may Microthermal biological effects Hydrated biological tissues known strongly to THz absorb radiation at THz to cause frequencies high-power radiation is a ssumed thermal effects biological materials. In addition conventional thermal effects, however, several researchers proposed radiation induce low-level thermal nonthermal appropriately, microthermal effects. to have that THz also , , or more These theories were initially hypothesized by Frohlich et al. in 1971 [99, 100], and more recent studies propose that these are the direct coherent of [99] or linear/nonlinear resonance mechanisms [101, 102]. In recent years, several research groups have conducted extensive efforts to develop more established theoretical frameworks to support the concept of “nonthermal” effects [101, 103, 104]. These that microthermal effects mediated through excitation biomolecules studies suggest nonthermal radiation coupling mechanisms oscillates (picoseconds) natural phonon frequencies molecules radiation create localized are openings believed t “bubbles” between strands. openings o drive double- stranded (dsDNA) to “unzip” and tools are not it is interfere transcription processes Modern available detect microthermal effects; therefore, difficult, impossible, existence effects experimentally of THz to DNA may may be due to the fact that THz (i.e., THz = 1012 Hz) on the same time scale as the of biological [101, 102]. Interestingly, these models also contend that the coupling the DNA DNA [101, 102]. Such However, since many observed effects reported in the THz be by the rise (i.e., . As a result, the concept of these effects has remained a constant subject of debate [105, 106]. verify the of such with to if not to bioeffects literature cannot readily explained temperature generated during irradiation conventional thermal effects) we Comment [i]: Don’t you Just love it– biological research- means testing on mankind and life forms of the planet 23 that it is that the reader is aware of these potential and . 5 Terahertz biological research Although an extensive review on THz sources, detectors, and technologies is beyond the scope of this review, a brief overview of equipment commonly used in THz biological research is valuable for subsequent bioeffects discussions. For additional details about THz technologies we refer the reader to several excellent reviews and books [107116]. In this section, we provide an overview of the main sources, detectors, and equipment used in THz biological research. We then proceed by describing the systems and tools used to control exposure conditions and . We conclude with a section describing the common challenges faced in THz biological research. 5.1 Terahertz sources THz sources are typically categorized according to their principle operational scheme. The most used in THz believe mechanisms conduct dosimetry common (i) direct schemes biological research following: generation sources; solid-state electronic devices (frequency up-conversion accelerating electrons-based The three most sources . 5.1.1 direct Direct generation sources common generation sources far-infrared (FIR), electrically-pumped quantum cascade lasers. commonly biological research. source dating 1960’s, oldest sources typically tunable consist high following components power carbon vacuum envelope container gases; medium: low and (iv) pressure molecular methanol (CH3OH) intracavity waveguides propagate radiation transverse direction action achieved molecules, are solid state, and Of these, the FIR laser is the most With initial uses are the laser for THz used back to the FIR laser is one of the of the . In brief, lasing using the pump laser to excite the levels of gas have in the THz FIR lasers that make them an ideal for THz . First, they provide high levels of average output power, with values on the order of 100 mW at many frequency lines. Notably, these power values are the highest levels of all commercially available bench-top THz sources [43, 119122] (Fig. 5a). Second, FIRs are widely tunable to hundreds of discrete frequency lines across the THz spectral band. Fortunately for FIR users, tuning or “hopping” to each discrete frequency line is straightforward and is achieved by simply adjusting the pump laser wavelength, gas type and pressure. FIRs THz radiation that is high wave (CW), and ~50 kHz). Finally, and perhaps most importantly, FIR sources are easy to operate and maintain. The main drawbacks of FIR lasers are their large footprint, weight, and expense. Systems can cost in excess of several hundred thousand dollars. In summary, FIR laser sources provide high output power, a wide range of operational frequencies, and high laser beam quality; they are a very for THz . Such sources are particularly useful to researchers who are interested in investigating the the early THz [117, (i) diode (CO2) laser; (ii) to house molecular gases, such as to THz 118]. FIR lasers pump source: a laser cavity with a (iii) gain in is important effects (ii) laser THz laser ); (iii) vibrational which transition frequencies spectrum. exhibit several characteristics source bioeffects studies Third, generate quality, coherent, monochromatic, continuous narrow linewidth (typically therefore, attractive source biological research 24 induced biological effects frequency dependence of THz . 5.1.2 Electronic THz sources using frequency up-conversion schemes In recent years, numerous electronic devices have been developed to generate modest power levels of THz radiation at frequencies less than 1 THz. Electronic devices typically consist of a microwave synthesizer or oscillator, and a frequency multiplier element, which consists of an array of schottky barrier diodes (SBDs). In the to and the SBD to of the to THz (i.e., frequency up-conversion)[123]. Electronic sources exhibit several design and performance features that make them useful devices for THz biological research studies. First, they are capable of providing high levels of Power (mW) 10 10 10 10 10 10 10 10 54 10 54 FEL IMPATT FIR gas laser 10 10 10 10 10 10 10 103 210 103 2 1 0 -1 -2 -3 -1 -2 -3 BWOs Electronic sources 1 0.1 10 Frequency (THz) b c IR Camera Electric heater brief, oscillator functions functions radiation generate “seed” microwave radiation, array multiply the frequency incoming microwave frequencies CO2 Laser Spectrometer Transparent windows THz FIR Laser Mirrors N2 Input d IR Camera THz FIR Laser Well plate e Single well f THz Gold plated mirror Fig. 5 (af) THz biological research: Sources and Equipment. a. Frequency-power spectrum for several THz sources. b. Image of a exposure chamber created using a cell culture incubator (Reprinted with permission from [145]). c. Macroscopic image of in vitro THz exposure setup: FIR THz laser source, CO2 laser spectrometer, temperature-controlled exposure chamber, and electric heater. (Modified and reprinted with permission from [120]). d. Magnification of THz transmission and delivery optics: electric shutter, flat gold plated mirror, parabolic silver plated mirror, well plate holder (adjustable in XYZ), and IR camera. e. Magnification of THz-culture plate interaction. f. Sample representative image of THz beam profile at airwell interface measured with Pyrocam III detector array. 25 beam Electric shutter Comment [i]: Type of devices to increase power out put 26 100 mW) at lower THz frequencies. they CW THz radiation. Finally, they are rugged, compact, and operate at room temperature. Due to the above properties, solid state THz sources are frequently used in both basic and . However, despite their incredible efficiency at lower THz frequencies, such approaches are limited because they are only capable of generating a milliwatt of power at higher frequencies [111]. In fact, recent data indicates that the output power of electronic sources drops off between 1/f2 and 1/f3 with increases in frequency [111]. In recent years, several groups have addressed the above challenges, resulting in the generation of higher power Gunn diodes [124, 125], [131]. For example, InP Gunn diodes can now generate greater than 100 mW of power at 0.1 THz and 0.1 mW at 0.48 THz [124, 125] (Fig. 5a). Several groups are also developing more advanced frequency- multiplier systems, such as varactors and varistors. Overall, electronic solid- state devices are a reliable source of low frequency THz radiation, and they are frequently used in THz bioeffects studies. With future advances in fabrication techniques, such sources may generate higher levels of output power at higher THz frequencies, and may increased use in THz biological research. 5.1.3 Accelerating electron-based THz sources Accelerating electron-based THz sources, such as backward wave oscillators (BWOs) and free electron lasers average output power (approximately Second, generate narrow line-width (10-6), electronic applied research frequency multiplier transit-time units based on SBDs, impact ionization avalanche devices (IMPATTs) [126, 127], tunneling tunneling transit time diodes (TUNNETT) [128130], and resonant diodes (RTDs) (FELs), are frequently used for THz bioeffects investigations. Interestingly, despite their striking differences both in appearance and size, BWOs and FELs both function using the same general operation principle. both use a of and an to and an sources system magnets . The primary difference between these systems is that the external structure in a BWO control, accelerate, collimate, is a comb grating, while in a FEL it is a wiggler system. In both systems, the external structure functions to create a periodic acceleration of the electrons in the beam, which in turn results in the generation of THz radiation. In the subsequent section, we shall explain several important features of BWOs and FELs. BWOs are table-top devices that use electron-vacuum tubes to generate THz radiation. These devices are referred to as BWOs because they use an electron beam that travels in the of a travelling EM wave. For years after their first demonstration in 1951 [133135], BWOs were primarily developed and used in Russia. However, in recent years, several companies have increased their efforts to commercialize BWOs for use in the US and Europe. Conventional BWOs consist of a magnetic housing system (~1 Tesla), high voltage power supply (typically, 26.5 kV), comb grating, cooling system, waveguide, and electron gun (cathode and anode). The frequency of the wave generated by a BWO is controlled by the velocity of the electron beam. Therefore, the output THz frequency can be directly adjusted by altering the bias voltage. Conventional BWO sources are tunable over a wide range of frequencies (0.035- 1.42 THz), provide modest power levels (0.2100 mW), and offer narrow linewidths (110 MHz). BWOs were utilized in several of the initial THz bioeffects studies [136]. However, these devices have several performance and commercialization drawbacks, which have greatly limited their use. First, BWOs are quite expensive because they require sophisticated engineering and development approaches. Second, they have limited portability due to their cumbersome magnetic housing system (i.e., 27 L, 100 lbs, 1 Tesla). Finally, they have short working lifetimes (approximately 500 h). This is primarily because electron vacuum tubes wear down quickly due to their consistent exposure to extreme temperatures (1200°C), voltages (6.5 kV), and pressures (10-8 Torr). Over the past few decades, several FELs have been developed to create high power THz radiation. Four THz FELs are located in the United States (Jefferson Laboratory, University of California Santa Barbara (UCSB), University of Hawaii, and Stanford University), and several are located in Japan, Korea, Netherlands, Germany, Australia, France, Russia, and Italy. THz FELs consist of two primary components: a large electron accelerator (linac or

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