*Review* **A Versatile THz Source from High-Brightness Electron Beams: Generation and Characterization**

**Enrica Chiadroni 1,\*, Alessandro Cianchi 2, Massimo Ferrario 1, Andrea Mostacci 3, Riccardo Pompili <sup>1</sup> and Vladimir Shpakov <sup>1</sup>**


Received: 24 April 2020; Accepted: 4 June 2020; Published: 8 June 2020

**Abstract:** Ultra-short electron bunches, such as those delivered by a high-brightness photo-injector, are suitable to produce high peak power THz radiation, both broad and narrow band, with sub-picosecond down to femtosecond pulse shaping. The features of this kind of source in the THz range of the electromagnetic spectrum are extremely appealing for frequency- and time-domain experiments in a wide variety of fields. The present manuscript will overview the method of generation and characterization of THz radiation produced by high-brightness electron beams, as those available at the SPARC\_LAB test facility.

**Keywords:** THz source; high-brightness electron beams; laser comb technique

#### **1. Introduction**

Terahertz (THz) science [1,2] is recently arousing interest worldwide for its impact in scientific research and technological progress because of the unique characteristics of this radiation. Indeed, THz radiation is non-ionizing, it penetrates in most insulating materials, e.g., plastic, ceramics, paper, and it is reflected by metals; moreover, it is absorbed by polar liquids, showing absorption spectra peculiar of the physical process. As a matter of fact, powerful THz sources are highly demanded, since they can provide efficient tools to investigate matter in the non-linear regime. Moreover, both biomedical and security applications can profit from the development of innovative THz sources, detection devices and characterization techniques.

THz radiation lies between the photonics and electronics bands of the electromagnetic spectrum. Effectively, this frequency region extends from 300 GHz up to 30 THz, corresponding to energies, in the meV range, of great interest for many physical, chemical and biological processes. Furthermore, molecular spectroscopy and imaging [3] can take advantage of a high peak power, tunable and narrow-band THz radiation.

Unlike conventional THz sources, such as those based on diodes, a new generation of sources, based on particle accelerators, allows a raising of the power, both average and peak, by many orders of magnitude. In particular, linac-based coherent THz radiation from relativistic high-brightness electron beams is a cutting-edge source [4–6] providing an efficient tool to investigate matter in the non-linear regime. Indeed, if the electron bunch longitudinal structure does not change during the emission process, the THz radiation is characterized by pulses whose temporal shape resembles the electron bunch one and with associated peak electric fields greater than 1–10 MV/cm [7].

The great advantage of a linac-based THz source lies in its versatility; indeed, the electron beam longitudinal distribution can be properly shaped to generate tunable, few-cycle and multi-cycle, coherent THz radiation with both broad and narrow spectral bandwidth. In particular, to enable the generation of narrow-band THz radiation, different approaches that foresee the longitudinal manipulation of the electron beam are currently used: temporal shaping of the photocathode drive laser pulse [8–11], mask insertion in a high dispersion, low beta function region of a beamline dogleg [12], transverse-to-longitudinal phase space exchange technique [13] to turn transverse modulation from a multi-slit into a longitudinal one, and magnetic undulator radiation [14–19]. Here, we report on the first approach, being the one developed and fully studied at the SPARC\_LAB test facility [20].

#### **2. Materials and Methods**

The key for high efficiency in a beam-based radiation source is to exploit the coherence enhancement effect boosted by tailoring the electron beam profile. For instance, coherent radiation in the THz range can be generated through ultra-short, i.e., sub-ps, relativistic electron bunches hitting a metal target as Transition Radiation (TR) [21]. Indeed, when a charged particle crosses the interface between two different refraction index media, TR is emitted both in forward and backward direction. The radiation results from the prompt change of the boundary conditions for the electromagnetic (EM) field carried by the particle in the first and second media. When a charged particle goes through a slit, opened in a metal target, only part of the particle field interacts with the screen, and the emitted radiation is called Diffraction Radiation (DR). Since the particle goes through the aperture without interacting with the screen, the emission process is not intercepting and non-invasive for the particle itself. Coherent Diffraction Radiation (CDR) is particularly appealing as non-disruptive longitudinal diagnostics [22].

When a bunch of *N* charged particles is considered, TR (DR) emission comes from each particle with the same intensity, but the position of each particle in the bunch has an impact on the emission, contributing with a phase term. Therefore, two regimes can be identified depending on the bunch length, *σz*, with respect to the emission wavelength, *λ*: at *λ σz*, each particle behaves independently from each other, resulting in incoherent emission, proportional to the number of electrons in the bunch, *N*; at *λ σz*, all electrons emit in phase and the amplitudes add constructively, resulting in coherent emission, proportional to *N*2. Therefore, the total emitted energy can be written as

$$\frac{d\mathcal{U}}{d\lambda} = \frac{d\mathcal{U}\_{sp}}{d\lambda} \left[ N + N(N-1)|F(\lambda)|^2 \right]. \tag{1}$$

*F*(*λ*) is the longitudinal bunch form factor, defined as *F*(*λ*) = <sup>∞</sup> <sup>−</sup><sup>∞</sup> *<sup>S</sup>*(*z*)*e*−2*πiz*/*λdz*, with *<sup>S</sup>*(*z*) = *S*3*D*(*r*) *dxdy* the longitudinal charge distribution, i.e., the projection of the three-dimensional normalized particle density distribution onto the z axis. The bunch longitudinal form factor sets the emission regime: at wavelengths longer than the bunch length, *F*(*λ*) → 1 and the spectrum is dominated by coherent emission, which extends to THz frequencies for sub-ps electron beams, with a huge intensity gain with respect to most existing THz sources thanks to the *N*<sup>2</sup> dependency.

#### *2.1. Broad Band Radiation*

Short, i.e., sub-ps, electron bunches drive the generation of high peak power radiation in the THz range. In linac-based facilities short electron bunches are typically produced by either magnetic or radio-frequency (RF) compression techniques. Magnetic compression, performed at high energy to overcome space charge effects, foresees a bunch with a time-energy correlation (or chirp) moving along an energy-dependent path length, represented by a dispersive, non-isochronous beam transport section. As alternative at lower energies, in the few MeV range, the RF compression scheme, known as velocity bunching [23] (VB), exploits the interaction with the electromagnetic fields of an accelerating cavity. A time-velocity correlation (or chirp) is provided to the electron bunch, inducing a longitudinal phase space rotation in the traveling RF wave potential due to the fact that electrons in the bunch tail are

faster than those in the bunch head. Since the beam is slightly relativistic, being slower than the phase velocity of the RF wave, when injected at the zero crossing field phase, it slips back to phases where the field is accelerating and, simultaneously, it is chirped and compressed. Therefore, compression and acceleration take place at the same time within the same accelerating section. Beyond the longitudinal dimension, the electron beam longitudinal shape also plays a significant role in the spectral content of the emission. In particular, asymmetric current profiles, caused by both longitudinal and transverse distortions of the phase space, and typical of compression techniques, promote emission at higher THz frequencies. Indeed, Figure 1 shows the longitudinal form factor as function of the frequency for four different beam longitudinal distributions, with the same RMS pulse duration (inset): Gaussian (black line), uniform (red line), triangular (blue line), and real measured one (green asterisks). The triangular-like longitudinal distribution results as the most efficient one to generate THz radiation, thanks to the steep rising edge, which is also shown in the longitudinally compressed beams (see green asterisks curve in the inset of Figure 1).

**Figure 1.** Longitudinal bunch profile (inset) and corresponding form factors for three different particle distribution with the same RMS pulse duration, i.e., 260 fs.

#### *2.2. Narrow-Band and Tunable Radiation*

One of the methods used to produce narrow-band and tunable THz radiation is based on the generation of Coherent Transition Radiation (CTR) from a train of sub-ps electron bunches hitting a metal target [10,24].

The generation of a train of sub-picosecond electron bunches with picosecond spacing has been conceived and demonstrated at the SPARC\_LAB test facility [25], applying the VB compression technique to a longitudinally modulated electron bunch as generated by a train of UV laser pulses hitting the photocathode, the so-called comb laser beam [26].

One way to generate comb laser pulses relies on birefringent crystals: an input laser pulse is decomposed into two orthogonally polarized pulses with a time separation proportional to the crystal length. A multi-peak laser profile is produced by stacking more birefringent crystals in the laser beam path. The so-generated comb laser pulse, illuminating a metallic photocathode in a RF gun, produces a train of short electron bunches thanks to the prompt temporal response of the metal, which allows for transverse and longitudinal shaping of the electron beam through laser manipulation. Downstream the photo-injector, the beam acquires an energy modulation because of the space charge forces and, if injected in a RF-compressor operating in the over-compression regime, the energy modulation turns back into density modulation. The train parameters, i.e., bunch charge, length, and time separation, can be fully controlled by properly adjust both laser and linac settings [27]. A train of sub-ps relativistic electron bunches intercepting an aluminum screen, emits CTR. Since the CTR emission is a surface phenomenon, it happens with femtosecond timescale and the electron bunch structure

does not change during the emission process; in addition, no interaction occurs between particles and radiation, resulting in a radiation pulse structure that resembles the multi-bunch longitudinal one with a spike-like spectrum at the frequency of the bunch distance and higher harmonics, as depicted in Figure 2.

**Figure 2.** Left: 4-bunches comb beam with different time separation (blue solid line, 1 ps; red dashed line, 0.7 ps). Right: Corresponding form factors highlighting the tunability of the narrow-band THz source.

Decreasing the sub-pulse length, the single pulse spectrum becomes larger and more harmonics of the sub-pulse repetition frequency appears in the comb spectrum, with the advantage that the energy at single frequency is the same as all the charge would be condensed in a single micro-pulse. In addition, the central THz frequency, and its harmonics, can be tuned by changing the micro-bunches time separation.

#### *2.3. Experimental Layout*

CTR is generated, at the SPARC\_LAB test facility, by highly relativistic electron beams hitting a 40 × 40 mm, aluminum coated silicon screen (380 μm thick with 40 nm coating). A proper optical system is needed to extract the CTR and transport it down to the detection apparatus and, eventually, to the sample. A sketch of the experimental layout, with light optics showing the radiation path, is depicted in Figure 3. Radiation is extracted from the vacuum pipe through a crystalline diamond window (700 μm thick, 0.3 deg wedge, 40 mm clear aperture), and collected by aluminum coated parabolic mirrors.

**Figure 3.** Experimental layout.

The coherent THz source is currently characterized by measuring the radiation spectrum in air (or dry air environment, i.e., 12% of relative humidity at 297 K) both through a set of discrete band-pass THz filters and an interferometer. The dry air system, a Kaeser Membrane Module dryer (FE-6D D-pack) working with compressed air, contributes to reduce water absorption in the Perspex box embedding the experimental apparatus (overall volume of about 200 litres), allowing measurement of radiation from very low charge bunches, of the order of 10 pC, needed for the production of ultra-short, fs-scale, electron bunches.

A two-beam interferometer is usually used for frequency domain measurements. For ps and sub-ps electron bunches a Martin–Puplett interferometer (MPI) [28] is preferred because of the beam splitter properties. Indeed, despite a Michelson interferometer, the beam splitter is a wire grid whose reflectivity coefficients depend on wires diameter and spacing. Furthermore, it splits polarizations, thus measuring the intensity of both polarizations: the difference interferogram can be normalized to the sum. Correlated fluctuations due to electron beam current instabilities are then canceled. The main limitation is however due to the high-frequency suppression introduced by the spacing between wires, which prevents the measurement of ultra-short bunches. Therefore, for bunches whose duration is from a few hundreds of fs down to a few tens of fs, the most suitable choice is a Michelson interferometer, which allows the covering of a wide spectral range, from 0.5 to 30 THz, by properly changing the thickness of the beam splitter from 24 μm down to 3.5 μm.

The autocorrelation measurements reported here have been performed using a Michelson interferometer with an additional detector (reference detector in Figure 3), which collects the radiation after a silicon beam splitter, placed before the Michelson interferometer box. The intensity measured by the measurement detector is normalized to the one measured by the reference detector, contributing to an increase of the signal to noise ratio; as a matter of fact this upgrade to the conventional Michelson interferometer allows the cancelation of correlated fluctuations due to electron beam current instabilities as in the two-beam MPI.

#### **3. Results and Discussion**

The versatility of linac-based THz sources stands in the possibility to tune the THz spectral content by properly setting the photo-injector regime and the UV photocathode laser longitudinal distribution for either narrow or broad band THz emission. In case of a longitudinally modulated electron beam, whose current profile and longitudinal phase space are shown in Figure 4 (left and right, respectively), the measured interferogram (Figure 5, left) presents the peculiar multi-peaks pattern, in particular (2*n* − 1) peaks, with *n* = 5 the number of bunches in the train. At zero optical path difference each sub-pulse within the two comb-like beamlet completely overlaps, resulting in the central maximum in the interferogram. As the path difference increases, the interferogram intensity decreases until four of the five pulses overlap, corresponding to the first side maxima. Further increasing the distance, the interferogram intensity keeps on decreasing resulting in the farther side maxima.

The spectrum corresponding to the autocorrelation function of the radiation produced by the 5 bunches beam is shown in Figure 5 (right). The main peak is at 0.65 THz corresponding to a 1.54 ps time separation between two of the five bunches, with a bandwidth (BW) of around 30%, whereas commercial THz filters have around 15–20% BW. Since the single bunch length in the train is of the order of 100 fs and less, a second and third harmonics arouse at around 1.25 THz and 1.95 THz. The peak at around 1 THz corresponds to the 0.8–0.9 ps distance of two of the four bunches, as shown in the electron beam current profile and longitudinal phase space.

Therefore, paying careful attention to a regular stacking of similar electron bunches, produces the suppression of all the frequencies apart the one corresponding to the succession itself, reaching in intensity at most the value of the single sub-pulse case. It is worth clarifying that to obtain the same radiation intensity at this frequency, in the case of a single bunch all the electrons *N* must be concentrated in the single bunch itself, with the drawback that space charge usually prevents to collect high charge in <100 fs. The width of this line depends on the number of pulses that constitutes the comb. Therefore the radiation produced by a longitudinally modulated beam (comb beam) is emitted at the frequency of comb and higher harmonics depending on the sub-bunch length, and the intensity at those frequencies is as if all electrons *N* would be concentrated in the single bunch whose duration is of the order of 100 fs or less. The transform limit is not violated, since the overall bandwidth is wide due to the fact that not only the fundamental frequency is populated, but also the higher harmonics. In addition, the shorter the pulse, the wider is the THz spectrum, because more harmonics show up. As a matter of fact, the photons of the total bandwidth are distributed differently, based on the temporal structure of the bunch, as its Fourier transform shows (Figure 5, right). Therefore the total peak power must be evaluated assuming that all the charge is collected in the micro-bunch duration, since it is the duration of the single micro-bunch in the train that sets the extension of the spectrum, thus the number of harmonics. Therefore, the term *narrow* for the frequency bandwidth of radiation from a train of micro-bunches refers to the bandwidth of each line of the frequency spectrum, which is naturally filtered from both the number and duration of bunches in the train.

**Figure 4.** Electron beam longitudinal profile (left) and longitudinal phase space of a 5-bunches ramp train (right).

**Figure 5.** Autocorrelation function (left) and relative spectrum (right) of a 5-pulses generated radiation.

The spectral properties of the THz radiation, as generated by a single sub-ps electron bunch can be also investigated sampling the signal by means of commercial band-pass THz filters [29]. Highly intense and broadband CTR has been produced, for instance, by a 110 MeV, 500 pC, 180 fs RMS beam impinging on an aluminum coated silicon screen, and its spectrum measured up to 5 THz, as shown in Figure 6.

**Figure 6.** Measured CTR spectrum sampled with THz band-pass filters center at 0.3–0.5–1–3–5 THz [11].

The drop below 0.3 THz in Figure 6 is due to the effect of the screen finite size with respect to the source size [30], being of the order of *γλ*/2*π* (*γ* the Lorentz factor and *λ* the emitted wavelength), combined with the fall of responsivity of the pyroelectric detector. On the other hand, the longitudinal extension of the bunch limits the generation above 5 THz. The integrated energy per pulse reaches up to 35 uJ as measured by a pyroelectric detector, whose responsivity and absorption curves have been provided by manufacturers. The pyrodetector is a 5 mm diameter THz-I-BNC GENTEC-EO, operating in the spectral range 0.1–30 THz, with responsivity ≈140 kV/W and a Noise Equivalent Power at 20 Hz ≈ <sup>4</sup> × <sup>10</sup>−<sup>10</sup> W/Hz0.5.

The main figures of merit of the SPARC\_LAB THz source for a single bunch to generate broadband THz radiation and five-ramp comb train to generate a narrow-band THz radiation are reported in Table 1 [11].

**Table 1.** SPARC\_LAB THz sources: radiation and electron beam parameters for a single bunch and a ramped five-bunch train (*σt*(*D*1) = 88 fs, *σt*(*D*2) = 127 fs, *σt*(*D*3) = 151 fs, *σt*(*D*4) = 165 fs, *σt*(*W*) = 100 fs).


† Systematic uncertainty due to missing detector calibration below 0.61 THz; † † the RMS duration, *σt*, for each bunch in the train is reported in the table caption; \* Defined as the FWHM; \*\* From measured results the time-bandwidth product is Δ*ν* t*<sup>p</sup>* = 0.72.

Linac-based THz sources are characterized by pulse energies reaching the multi-hundreds μJ regime [31] by increasing the charge, thanks to the *N*<sup>2</sup> dependence, and/or properly shaping the bunch longitudinal profile. In addition, taking profit of the beam longitudinal manipulation, the spectral bandwidth can be either tunable narrow or broad, making it extremely versatile. Finally, the repetition rate can span from a few Hz up to GHz, depending on the accelerator repetition rate, paving the way to an average power of the order of 10 W [16]. On the other hand, laser-driven THz sources, in particular those based on Optical Rectification [32] with organic crystals, have demonstrated to achieve hundred μJ pulses in tight focus resulting in electric and magnetic field larger than 10 MV/cm and 3 Tesla, over a broad 0.1–8 THz frequency range [33].

Compared to other linac-based sources [16], and based on the measured parameters, listed in Table 1, the SPARC\_LAB THz radiation is strongly competitive and extremely suitable for investigating non-linear THz spectroscopy and for testing novel detectors and materials. Indeed, thanks to MV/cm electric fields, which can excite fundamental quantum statuses of a given system, it allows for non-linear optic experiments [34].

#### **4. Conclusions**

In photo-injector-based facilities, THz radiation can be produced under different regimes, by a proper control of the bunch shaping and charge distribution. Indeed, taking profit of the prompt photoemission of electrons at the metal cathode, different electron longitudinal distribution can be tailored and manipulated by properly setting the UV photocathode laser and the photo-injector parameters.

In particular, the present manuscript reports the most valuable results obtained at SPARC\_LAB with both ultra-short single bunch and multi-bunches electron comb beams to provide high energy per pulse and broad and narrow spectral bandwidth THz radiation, respectively for non-linear and pump-probe experiments in solid-state physics and material science.

**Author Contributions:** E.C. planned and managed the THz activities at SPARC\_LAB, and wrote the manuscript; V.S. was involved in the project and setup of the experimental apparatus; M.F. coordinated the SPARC\_LAB test facility; A.C. took part to the preparation of the experiments; A.M. and E.C. carried out the data analysis; R.P. provided numerical simulations for photo-injector settings. All authors were involved in the operation of the experiments, extensively discussed the results and reviewed the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was partially funded by CSN5 of INFN.

**Acknowledgments:** Authors would like to acknowledge all the colleagues at the SPARC\_LAB test facility of the INFN Frascati and colleagues within the collaboration, in particular, S. Lupi (Sapienza University of Rome) and F. Giorgianni (PSI).

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References and Note**


© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

### *Review* **THz Pulsed Imaging in Biomedical Applications**

**Annalisa D'Arco 1,\*, Marta Di Fabrizio 2, Valerio Dolci 1,2,3, Massimo Petrarca 1,3 and Stefano Lupi 2,4**


Received: 13 December 2019; Accepted: 24 March 2020; Published: 1 April 2020

**Abstract:** Recent advances in technology have allowed the production and the coherent detection of sub-ps pulses of terahertz (THz) radiation. Therefore, the potentialities of this technique have been readily recognized for THz spectroscopy and imaging in biomedicine. In particular, THz pulsed imaging (TPI) has rapidly increased its applications in the last decade. In this paper, we present a short review of TPI, discussing its basic principles and performances, and its state-of-the-art applications on biomedical systems.

**Keywords:** terahertz spectroscopy; terahertz time-domain spectroscopy; terahertz pulsed imaging; near-field imaging; biomedical imaging; cancer diagnosis; endoscopy

#### **1. Introduction**

The increasing request for diagnosis techniques in the biomedical area, able to provide morphological, chemical and/or functional information for early, noninvasive and label-free detection, has led to hard cooperation in different scientific fields. Driven by technological advances, a plethora of new modern diagnostics systems is investigated and validated, as complementary biomedical techniques to the conventional ones, covering various scales, from macromolecules to tissues/organs. Among them, worthy of special mention are: infrared (IR) imaging [1,2], scanning near-field microscopy [3,4], photoacoustic microscopy [5], ultrasonic imaging [6], optical coherence tomography [7,8], digital holography microscopy [9–11], Raman scattering microscopy [12], Coherent Raman Scattering (CRS) spectroscopic imaging [13–23], two-photon fluorescence (TPF) [19,24] and second-harmonic generation (SHG) imaging [25–27], and super-resolved imaging techniques [28–31].

Many efforts were done in THz technology, improving THz sources and detectors' responses [32–38], and ensuring devices' flexibility and portability, in recent decades. This has stimulated a wide diffusion of THz systems for spectroscopic and imaging purposes, applicable in various science fields like biology and medicine [39–44], gas sensing [45], chemical analysis [46,47], new materials characterization in low-frequency range and non-destructive evaluation of composite materials and constructions [48,49], astronomy [50], microelectronics and security [51–53], agri-food industry [54], art conservation [55], etc. Thus, alongside these techniques, TPI has been developed and applied in the field of biomedicine.

THz radiation has a variety of properties that make this spectral region a viable spectroscopic imaging technique. First of all, (i) THz radiation has low photon energy (4 meV at 1 THz), coinciding with the energy levels related to rotational and vibrational molecule modes and intermolecular vibrations, such as hydrogen bonds. These low-frequency motions allow the identification of biomolecules, characterizing their spectral features in THz region [56,57]. (ii) In addition, the low photon energy is insufficient to cause atoms ionization. (iii) For this reason, it is suitable and attractive for noninvasive biomedical imaging, has a great potentiality and could be applied in vivo real-time studies without causing biological ionizing damage, unlike X- and γ-rays [42,43]. Whereas extremely low frequency electromagnetic fields have been long studied in relation to their possible human health effects, the biological effects of radiofrequency and THz signals have come under scrutiny. In particular, the effects of THz radiation are of interest because of the expanding THz technologies in biomedical applications. The prevailing view is that THz radiation, if thermal effects are not considered, does not damage directly the DNA, but it could act as a co-inductor for non-thermal effects [58]. Recent studies show evidence of potential thermal and non-thermal effects induced by exposure to THz radiation [59,60]. The biological response to this stimulus is due to different parameters: the physical settings of the THz radiation (frequency, mean and peak power, radiation intensity, continuous or pulsed radiation, the degree of coherence, etc.,); the physical and biological properties of the irradiated biological object (refractive index, absorption features, type of cells, tissues, organs) and the design of the exposure [60]. (iv) In addition, THz radiation is very sensitive to polar molecules, like water. (v) That is why the strong absorption, due to water molecules (220 cm−<sup>1</sup> for pure water at 1 THz and room temperature) [43], limits THz penetrability into the fresh tissues from tens to hundreds microns [42] and the capability of diagnostic only for superficial layers in vivo. (vi) The high sensitivity of water content can be used like an endogenous marker for the differentiation between fresh healthy and pathological tissues [42,43]. (vii) In particular, the time-domain spectroscopy (TDS) and imaging are insensitive to the thermal background and show high signal-to-noise ratio (SNR) [61]. (viii) TPI coherent detection ensures the record of temporal profile of the THz electric field; so, both the THz amplitude and phase can be estimated. From these, the broadband absorption coefficient and refractive index of a medium are determined, without using Kramers–Kronig relations [57,61]. The absorption is generally due to the chemical constituents of the material; thus, its spectral signatures are measured, providing useful information. As consequence, TPI has the potential to realize images with both morphological and functional information, in this frequency range. For all these reasons, THz radiation is considered very important and deserves special attention.

In this review, we introduce TPI, discussing its basic principles and performances, and the state-of-the-art applications on biomedical systems.

The review is organized as follows: in the first section, we outline the main features of pulsed THz systems and their key role in THz imaging setups, showing the equipment needed and some application ideas. Later, in the following section, after summarizing the main properties of the THz contrast image, we also focus on TPI challenges and relevant results achieved in the biomedical field.

#### **2. THz Pulsed System**/**Imaging**

Over the past two decades, THz generation and detection technology has considerably advanced, and several devices are commercially available now [33–37,62,63]. Their development has led to various spectroscopic and imaging techniques in the THz spectral window. According to THz sources used, the systems are divided into two general categories: the continuous wave (CW) systems, which produce a single or several discrete frequencies, and pulsed systems, characterized by a broadband frequency output, introduced for the first time as potential imaging tool in 1995 by Hu and Nuss [64]. All of them feature a large variety of properties, as well as, frequencies, power and sensitivity. In this paragraph, we briefly discuss the most common TPI systems that are the aim of our review, with a strong emphasis on THz pulsed sources and detectors.

#### *2.1. Generation and Detection of THz Pulsed Radiation*

In TPI systems, a broadband frequency emission, from tens or hundreds of GHz up to several THz, is obtained. Currently, various modalities can be listed for generating and detecting pulsed THz radiation [65,66]: biased photoconductive antennas (PCAs) [67–69], optical rectification (OR) in nonlinear optical (NLO) crystals [70–72], THz production by plasma in air [73–75] and carrier tunneling in coupled double quantum well structures [76–78]. They exhibit different physical properties concerning powers, used materials, covered spectral range, SNR and dynamic range (DR). Among them, the most established approaches, based on PCAs and on OR, require pumping made by expensive infrared femtosecond lasers, often emitting in the near-infrared (NIR) region. For the first mentioned, a pulsed laser beam illuminates PCA gap, made of high resistive semiconductor thin film with two electrical contact pads. Thus, in the presence of the applied bias voltage and laser beam, photocurrent is generated and the static bias field accelerates the free carriers producing broadband frequency THz waves into the free space.

OR ensures the THz broadband generation through NLO centrosymmetric crystals [70–72], extended from 0.1 THz and beyond 40 THz [57,79,80], such as organic NLO crystals. 4-N,N-dimethylamino -4'-N'-methyl -stilbazolium 2,4,6-trimethylbenzenesulfonate (DSTMS) [81–84] and 4-N,N-dimethylamino-4'-N'-methyl-stilbazolium tosylate (DAST) show efficient generation from 0.3 to >16 THz in the phase-matching condition between 720 nm and 1650 nm. The NLO materials need to have high second-order NLO susceptibility, high transparency at both pump and THz frequency, and high optical damage threshold, in order to satisfy the phase-matching condition between the fundamental optical pump and generated THz waves [85,86]. When intense NIR laser beams propagate through some crystals, second order nonlinear effects occur, developing a DC or low frequency polarization [38]. This leads to the radiation of an electromagnetic single-cycle pulse with a wide frequency spectrum, ranging roughly from zero frequency to some maximum value [38].

Another way to produce THz radiation is by charges acceleration. As consequence, the charges radiate electromagnetic waves, that under particular conditions, lie in the THz region of the electromagnetic spectrum. The processes can be done either within semiconductors, in vacuum or in air [87–90]: via electrons acceleration by the intense laser pulse [87,88], by an applied bias voltage across an air gap, similarly to PCA, or by nonlinear four-wave mixing of the fundamental and the second harmonic frequencies of the laser beam, in air or in various gases [89,90].

Besides OR, photoconduction or photocurrent, surge is another primary mechanism for THz generation from semiconductor materials [57,59]. When semiconductor quantum wells (QWs) are under a bias, the mechanism for THz generation is attributed to the creation of polarized electron–hole pairs [91–93].

In its turn, two schemes are ideal to THz pulse detection, based on incoherent and coherent techniques. In terms of incoherent techniques, power-to-signal transducers, suitable for incoming energy measurements, are used. For coherent ones instead, the detectors give information concerning the power, frequency spectral range and phase of the incoming electrical signals [57]. Among THz incoherent detectors commercially available, we can mention bolometers [94]; Golay-cells, providing stable performances and high sensitivity; and pyroelectric detectors [95] and thermopiles with low sensitivity. Commercial bolometers, resistive temperature sensors, widely used in THz spectral region, are cryogenic ones. The cooling of the devices ensures high sensitivity, with noise equivalent powers (NEPs) ranging from 10−<sup>12</sup> W/Hz1/<sup>2</sup> to 10−<sup>14</sup> W/Hz1/2. Golay-cell detectors [96] measure the thermal energy of THz propagating through a window [61]. Their spectral performances are strongly dependent on the material used for the window. In the case of diamond and crystal quartz windows, the spectral range covers from millimeters-wave to visible frequencies [61], while for HDPE (High-Density Polyethylene) window the spectral range is upper bounded at ~20 THz. Power DR can vary from hundreds nW up to 1 mW, with power DR from hundreds nW up to 1 mW. These devices can reach excellent NEPs at room temperature—around ~10−<sup>10</sup> W/Hz1/<sup>2</sup> [61]. Instead, pyroelectric devices show lower sensitivity (NEPs ~10<sup>−</sup>9–10−<sup>10</sup> W/Hz1/2) compared to bolometers and Golay performances. Table 1 summarizes some performances of the most used incoherent THz detectors.


**Table 1.** Main incoherent detectors suitable for THz spectral range with their performances:noise equivalent powers NEPs, THz coverage and detector bandwidth, linked to the detector response time (the higher the bandwidth, the faster the response time). Bolometer, Golay cell and Pyroelectric are pure

Schottky diode 10<sup>−</sup>14–10−<sup>11</sup> 0.1–2 102–106 <sup>1</sup> High-Density Polyethylene (HDPE) window; <sup>2</sup> Diamond window.

Pyroelectric 10<sup>−</sup>10–10−<sup>9</sup> 0.1–10 10–10<sup>8</sup>

THz cameras were the object of intense research activities in recent years. Actually, various THz cameras, based either on cooled and uncooled bolometers or semiconductor devices, are commercially available. Among the THz thermal cameras, the Golay cells are slow detectors and are difficult to integrate into array detectors. Consequently, the imaging scheme for them is a single-pixel detector [97]. The pyroelectric cameras, like the Pyrocam series sold by Ophir Optics, can operate with pulsed and CW sources, as well as thermopile cameras [98,99]. Generally, they are widely commercially available, and their low sensitivity makes them less attractive for THz imaging. The microbolometric cameras are arrays of bolometers; because of the relatively higher sensitivity they are considered the prime candidates for THz imaging. When cooled at cryogenic temperatures, they can achieve NEPs around 10−<sup>16</sup> W/Hz1/2. With proper design modifications, microbolometric cameras at room temperature also achieve high enough sensitivity.

Many such imaging techniques cannot be realized with existing THz cameras, but require a coherent detection scheme. Among commercial coherent or heterodyne detectors, largely used, we mention: Schottkly diodes [100,101], PCAs and NLO crystals or via electro-optical (EO) effect in nonlinear crystals [70–72]. Schottkly diodes [100,101] show limited spectral bandwidth around 1–2 THz and NEPs up to 10−<sup>14</sup> W/Hz1/2, and actually they are the object of research. PCAs (frequency range limited <10 THz and NEPs around 10–12 W/Hz1/2) can detect a THz electric field profile with a coherent detection scheme. The probe beam, derived from the same laser used to generate the THz radiation, irradiates receiver PCA, which has no external bias voltage. The laser pulses gate the detector acting as a photocurrent switch by generating charge carriers. The incoming THz radiation, on the receiver, induces the bias voltage, thus the photocarriers are accelerated [67,69]. The transient current is measured by a low-current amplifier and a lock-in technique, that are referenced against a modulation placed on the PCA emitter bias voltage.

In EO rectification NLO crystals, instead, the THz field induces birefringence in them; at a given instant, the polarization state of the probe laser pulse changes proportionally to the THz field amplitude [25,102]. Then, a balanced photodiode is used for measuring these changes in birefringence, reconstructing the THz pulse. In recent years, great interest in THz sensors and cameras is focused on the development of devices with high sensitivity, which operate at room temperature and fast operation speed [103], such as optoelectronic sensors and detectors [104–107].

#### *2.2. TPI Equipment*

The switch from the spectroscopy system to the imaging one is based on the possibility to collect spectroscopic information on large sample areas. For this purpose, the point-by-point signal collection requires a mechanism for scanning: moving the beam or the sample holder. The first case is realized with a beam motion or oscillating objective; using for example, galvo-mirrors and/or piezoelectric rotator or stages. In the second case, the lateral translation of the specimen stage is combined with a stationary illumination; for this purpose, a 3D linear translation stage is used, allowing sample holder motion along the THz beam (1D motion) and perpendicularly to it (2D motion).

The acquisition speed is one of the most critical challenges when using THz radiation for biomedical imaging, and it requires more attention for the implementation of clinical devices. The imaging speed depends on the specific image scheme chosen: using a point source and detector, the acquisition is slow, due to the point-by-point sample scanning. Although, the two-dimensional galvo-scanner, combined with a fast detector or sophisticated signal processing techniques [108–110], improves the scanning speed and the acquisition on a large field of view.

The object surface is illuminated by the THz beam, and it is sampled on a discrete grid, scanned in continuous or pixel-by-pixel raster modes. The information collected in each pixel is digitalized, transferred to PC from a data acquisition card (DAQ) and quantized in a finite number of bits for preand post-image processing.

#### 2.2.1. TPI Far-Field Systems

A typical pulsed THz wave generation and detection system is a pump-probe setup. It can be considered as an extended THz time-domain spectroscopy (THz-TDS) method. The basic idea of THz-TDS and TPI systems is illustrated in Figure 1a.

**Figure 1.** This is an example of TPI system switched by PCAs. (**a**) The schematic layout of common TPI system based on photoconductive switches. Here, Fs laser stands for femtosecond laser, BS for beamsplitter, DL is the delay line, LS are lenses, LIA, DAQ and PC stand for lock-in amplifier, data acquisition card and desktop computer, respectively. (**b**) The schematic layout of two potential sample orientation: transmission and reflection modes. The standard transmission imaging configuration involves the specimen placed between THz low-absorption material plates or standing in free space. The generated THz signal travels at a normal incidence to the sample surface and the transmitted one is received on the other side of the sample. Instead, for reflection mode, the THz signal illuminates one sample surface and it is reflected by the same surface.

TPI system, shown in Figure 1a, is powered by a femtosecond laser. A beamsplitter splits the initial laser beam into two parts: the pump and the probe beams. The pump beam is modulated via an optical or mechanical chopper or by modulating the THz emitter bias voltage, and then is focused on the THz emitter. The generated THz radiation is then collimated and focused onto a target sample. When a THz pulse illuminates a target, a train of THz pulses will be reflected from the various interfaces or transmitted. The transmitted (Figure 1b) and/or the reflected (Figure 1b) electric fields are, then, recollimated and refocused onto THz detector using THz transparent lenses or parabolic mirrors. The electrical signal is filtered and detected by lock-in amplifier detection. The analog output of the lock-in amplifier is collected and digitalized by a dedicated DAQ. A delay line is used in order to temporally sample the THz electric field. Thus, for each individual pulse in the detected signal, the amplitude and phase are different and are measured too.

If the pump and probe optical paths are equal, the THz electric field is measured at one fixed instant in time. In order to sample the whole THz pulse, a delay line is introduced in the optical scheme, to delay pump and probe beams. Generally, this can be performed using a mechanical translation stage, that moves at fixed steps; but rapid-scanning delay lines can also be used to speed the acquisition [111,112].

For example, we reported our home-made TPI system based on THz-TDS (see Figure 2a). It was implemented in transmission configuration.

It generates broadband THz radiation (0.1–3.5 THz) via photoconductive switches, used both for THz emission and detection. We use a mode-locked ultrafast laser (FemtoFiber NIRpro, Toptica), at 780 nm with a 100 fs temporal pulse width and a repetition rate of 80 MHz to illuminate twin G10620-11 Hamamatsu PCAs. The femtosecond laser is split into two beams by a 50:50 beamsplitter—each beam has 15 mW average power. Dielectric mirrors deflect the beams towards emitter and detector PCAs. TPX lenses are used to collimate and focalize THz radiation. The object is aligned in the focus of the THz beam, perpendicularly to the propagating THz radiation. The point-by-point spectral signal collection is obtained by scanning a large area of the object with 3D axes stage (Thorlabs). After transmission through the sample, THz pulses are collimated and refocused on the THz-detector PCA by TPX lenses. Simultaneously, the probe beam is used to gate the THz-detector PCA. The THz electric field as a function of time is measured with a delay line [112]. The output signal from the THz-detector, extracted by a lock-in amplifier (Stanford, SR830), is transferred to a National Instrument acquisition card (NI 6361-BNC connector) allowing the data collection. Our system exhibits performances comparable with the commercial systems, and being a home-made spectrometer, it can switch on the other two spectroscopic configurations using metallic flip mirrors, inserted in the THz collimated region. We can select between THz–TDS refection spectroscopy and attenuated total reflection (ATR) spectroscopy, see Figure 2a.

In Figure 2b, the temporal electric field profile, in free-space, as a function of time, is reported with relative power spectrum, for the scheme in transmission.

**Figure 2.** (**a**) Schematic layout of our TPI system. In the scheme, Fs laser stands for femtosecond laser, BS for beamsplitter, DL for delay line, LS is referred to lenses, THz det. and THz emit. are THz detector and THz emitter respectively. With two metal flip mirrors, introduced in the collimated THz beam region, one can select the scheme for THz spectroscopic imaging in transmission (B) or reflection (C) or ATR (A) spectroscopy. (**b**) The temporal electric field profile, in free-space, as a function of time, is reported with relative power spectrum.

#### 2.2.2. TPI Near-Field Systems

The major limitation of THz far-field imaging systems is due to the problem of the diffraction-limited spatial resolution central to THz wavelengths, compared, for example, to visible radiation [113,114]. The optical resolution R in the object plane is generally defined by the Rayleigh criterion [115–118], described as:

$$R = \frac{0.61 \cdot \lambda}{NA} \approx \frac{\lambda}{2NA}$$

at the wavelength λ and numerical aperture NA. The minimal resolvable feature corresponds to the minimum size, in the object plane, of the smallest object that the optical system can resolve: as the resolution is directly proportional to the wavelength and inversely proportional to the numerical aperture of the optical components, at 1 THz the resolution turns out to be around 0.5 mm [113]. In fact, 1 THz corresponds to 0.3 mm and one must consider the loss of resolution caused mostly by lenses on the beam path. For this reason, effective and wide spreading of THz images in biomedical research and diagnostics is difficult. The wavelength-limited resolution is caused by diffraction of propagating waves, so this problem can be overcome by collecting THz pulses in near-field (NF), namely at a distance with the sample comparable to the wavelength. In this way, evanescent waves, that propagate only very close to the sample and then rapidly decay, can be detected. That is the reason why THz detector must be placed in proximity of the sample. Several techniques have been explored to enable sub-wavelength THz imaging [37,114,119,120]. With this purpose, detectors and sources of different shapes can be used; in particular, Hunsche et al. [121] proposed and demonstrated, for the first time, a method deriving from the confocal microscopy. It uses pinholes that act as spatial filter, reducing the THz beam size to sub-wavelength dimensions and blocking outside the cone of light. This results in increased lateral and depth resolutions [122,123]. However, the aperture introduces an immense loss of THz light intensity at deep sub-wavelength resolutions (<λ/10) [124], caused by the low frequency cut-off [125] and optical coupling loss. Following, it is possible to place a small detector or emitter directly behind the aperture [126–128]. The approaches without apertures, instead, are better set to achieve deep sub-wavelength resolutions, because the low-frequency cut-off is avoided. The sharp metallic tips for local field enhancement, tip apex, small detectors and/or sources elements with sub-wavelength dimensions are placed in proximity of the sample surface or in direct-contact. In this way, high resolutions are achieved (even λ/100), with the only limitations imposed by the tip diameter [129]. This means that NF resolution is not limited by diffraction so that, in this way it becomes independent of the wavelength.

The direct-contact approach, instead, is generally employed with EO sampling using THz-TDS. The sample could be then in contact with the detection crystal, leading to a direct measurement of the THz electric field in NF region. In [130], the deposition of various metal structures onto a gallium phosphite (GaP) crystal can improve the resolution by imaging at 20 μm.

#### **3. Basics of THz Imaging**

TPI can be viewed as an extension of the THz-TDS method. The classic way to obtain THz imaging from THz-TDS is illustrated above. In order to image the objects, a 2D point-by-point raster scanning of them, combined with a coherent detection, is performed; and the individual temporal profiles are recorded for each point-pixel. The raw one-dimensional time or extracted frequency data are converted into physical values [57,61,64,131,132]. The normalization is chosen to enhance the image contrast, and it can be summarized in the diagram (Figure 3).

**Figure 3.** Schematic summary concerning the image contrast in THz-TDS imaging.

In time domain, when there is interaction with the sample, the THz pulse can be attenuated, delayed or broadened, compared to the reference without the sample, generally measured in air. The electric field amplitude, at a fixed time, represents a contrast modality to normalize the image. In addition, the normalized amplitude of the main peak, as the ratio between maxima of the sample and reference electric fields, provides image information regarding the absorption, reflection or scattering losses in the object. Finally, the knowledge of time delay of the main peak with respect to the reference allows mapping the optical path changes, providing material/thickness contrast [133]. For example, the time-of-flight (TOF) technique permits to estimate the depth information about target internal dielectric profiles. A THz pulse is launched to the sample and the reflected echo is measured in amplitude or/and phase. The TOF information of the echo pulse indicates the presence of the boundaries, or inner structures along the THz propagation path, which extracts the one-dimensional depth profile. Thus, by performing a 2D scan, a 3D image of the object can be visualized, viewing into a layered structure or inside the optically opaque materials. 3D imaging potentiality is suitable for the implementation of various imaging techniques, largely used in biomedical contexts. Applying the Fourier transform on the temporal electric pulse, one gets the amplitude and phase of the spectrum. The first gives a general indication of the losses; the second one is related to the refractive index. Fixing the frequency, one can visualize the object in the frequency domain, the amplitude and the phase images [134]. This offers more contrast, because of the sufficiently different refractive indexes of the materials than the losses, which provide a meaningful contrast.

Summarizing, the THz-TDS measurements allow the extraction of both amplitude and phase of THz waveform. The complex refractive index and absorption coefficient can be obtained using the Fresnel coefficients [135,136], and from them the complex permittivity [137,138]. The knowledge of sample spectral behavior ensures to extract information concerning its morphological structure and the optical properties: the variation of refractive indexes could differentiate tissues, and could be related to the pathological status. Moreover, many materials are relatively transparent to THz radiation, and are used in the development of 3D THz imaging modalities that constitute an active research field [107,139]. Tomographic slices are employed for object/tissue 2D or 3D reconstructions, in order to obtain their internal properties and features [111]. The method tried a natural extension for the object rotation towards THz computer tomography (CT) [140,141]. In addition to THz CT, there are several other tomographic techniques based on THz radiation, including THz interaction tomography, THz tomosynthesis, time-of-flight pulsed imaging and 3D THz holography [139].

#### **4. THz Pulsed Imaging: Uses, Advantages and Challenges for Biomedical Applications**

As briefly discussed in the introduction and in the previous paragraph, the unique spectral features of THz radiation make this technology particularly interesting in the biomedical field—for ex vivo and in vivo experiments and/or analyses. During the past decades, systematic spectroscopic investigations carried out in the THz frequency region on biological targets, as biomacromolecule, cells and tissues, demonstrated differences in their optical properties. In the last five years only, the rate of imaging experiments in the THz domain is increasing.

An unavoidable issue, when talking about THz imaging, is water: THz is particularly sensitive to water content, exhibiting a strong absorption [142] and consequently limiting THz penetration depth.

In fact, the strong absorption of water limits the penetration depth in fresh tissues between tens and hundreds of microns. For example, into the human skin THz waves can penetrate only a few hundred microns [143]. In vivo clinical measurements, it limits the probing on superficial layers of the target and the reflection mode becomes the suitable choice [144,145]. The limitation could be overcome in the case of ex vivo examinations, where histopathological evaluations are required. Researchers overcome these limitations using thin and/or fixed samples and appropriate geometry. The transmitted TPI is performed on thin prepared tissues, suitable for in vitro, and reflected TPI allows the investigation of surface features, reliable in vivo and ex vivo imaging.

However, difficulties in extrapolating measurements on tissue to ex vivo include, for example, saline uptake from the sample storage environment, changes in hydration level during the measurement, temperature- and humidity-dependent and scattering effects. Considering that, the human body contains a large amount of water (~60%) and THz is heavily absorbed by water [142], fresh tissue can vastly alter THz spectroscopy measurements. As fresh tissue dehydrates when left exposed to the air, many contrast features are reduced during the data acquisition. Therefore, for biomedical studies, sample conservation is crucial.

Several research groups have investigated excised and fixed tissues considering numerous approaches; among them are dehydration [146,147], alcohol perfused [36], formalin fixed [148–150], gelatin embedding [151,152], lyophilized [153], freezing [154,155] and fixation in paraffin emulsion and embedding [156].

Formalin fixing, in order to preserve and fix excised tissues, is a common histopathological routine for diagnosis. The formalin reduces the sample variation due to the dehydration process [149,154]—the tissues' water content is replaced by formalin.

In addition, lyophilization could represent an interesting alternative to fresh tissue for THz spectroscopic measurements [153]; its pros are fast and effective water removal, and structural preservation. The problems with handling fresh tissue, such as time variability in the THz bandwidth, dynamic range and sample thickness, are therefore overcome [153]. Several studies proposed the freezing of biological sample, as an alternative technique to increase the penetration depth of THz waves in the tissues, as the absorption coefficient of ice is one order of magnitude lower than the absorption coefficient of water [143]. The discussed technique has a limitation in clinical use; the process of freezing might cause necrosis. Some suggestions are proposed by using a penetration-enhancing agent (PEA) to increase the THz radiation delivery depth. The treatment with PEAs, that are cost-effective and easy-to-find, biocompatible and nontoxic to the human body, could also ensure long preservation time for ex vivo tissues and the application in vivo. Oh et al. [157] used glycerol as PEA, easily absorbed by human skin and tissue and with an absorption coefficient much lower than that of water in the THz spectral region. They treated abdominal mouse skin with glycerol and demonstrated the enhancement of penetration depth with the reconstruction of a metal blade hidden under the tissue by the second pulse reflection of it [157].

Various alternative approaches are suggested. Oleic acid is proposed by Wang et al. [158] for trapping moisture inside thin tissue slices, in transmission acquisition mode. They realized a sandwich configuration, by placing a frozen layer of oleic acid above and below the frozen tissue sample, between tsurpica windows and waited for complete thawing, before starting the measurements at room temperature. Oleic acid is highly transparent to THz radiation and its refractive index matched tsurpica. The oleic acid layers were used to keep up the tissue hydration, showing good performances for 70 min. Fan et al. [151] used gelatin embedding for porcine skin slices reflection measurements. By looking at the THz image data and computing the optical parameters of the gelatin-embedded

sample, they found a successful method to preserve the sample for at least 35 h, both for imaging and spectroscopy purposes.

Since the first demonstration of THz imaging [64], TPI and spectroscopy show their ability to differentiate human tissues [141,159], with clear differences between the tissue properties, in particular regarding absorption coefficients [160]. Fatty tissue consists of mainly hydrocarbon chains, largely nonpolar molecules, thus its absorption coefficient and refractive index are much smaller than the ones of muscle, kidney and/or liver tissues [141].

Concerning the optical characterization at the low frequency region, many spectroscopic works are present in the literature [161–167]. One significant work about the ability in distinguishing and classifying the THz response of different types of tissues is Ref. [131], which is mentioned for the chirped probe pulse technique. It offers a significant improvement concerning the slow acquisition time, and it could have great potential in imaging acquisition.

In addition, the high sensitivity of THz waves to water content in the tissues becomes the key contrast factor in many biomedical applications [168,169]; the water content evaluation allows label-free differentiation for various types of tissues as an endogenous marker, distinguishing between healthy and pathological tissues [152,168], see Figure 4.

**Figure 4.** Refractive index n and absorption coefficient α in THz spectral region, observed by Gavdush et al. [152], and H&E-stained histology of gelatin-embedded ex vivo human tissues: intact tissue, gliomas of grade I to IV, and edema. (**a**)–(**c**) grade I; (**d**)–(**f**) grade II; (**g**)–(**i**) grade III; and (**j**)–(**l**) grade IV. The THz optical properties of gliomas are compared with equal data for the intact and edematous tissues, averages within the entire set of brain tissues specimens [152].

In the case of cancer, the contrast between healthy and malign tissue is originated from the structural variation and different water content [170]. In fact, the presence of cancer induces, in many cases, increased blood supply to the affected tissues and a local increase in tissue water content, which suggests water contrast for THz cancer imaging [171]. As a result, it involves a higher refractive index and absorption coefficient [39,169,172]. As proof, Figure 5 shows refractive indexes and absorption coefficients of normal and cancer regions of a fresh rat brain tissue sample, as a function of frequency [39].

**Figure 5.** Optical characterization of a fresh rat brain tissue sample [39]. (**a**) Refractive index and (**b**) Absorption coefficient (cm<sup>−</sup>1) of normal and tumor regions, from 0.8 to 1.5 THz. Both refractive index and absorption coefficient in the tumor region are higher than in normal tissue due to the higher water content in cancer tissue.

TPI has been successfully applied to liver [164,173,174], colon [165,175,176], intestine [161], brain [39,156,177], skin [178], ovarian [167], oral [154] and breast cancers [132,163,179].

For instance, a particular attention deserves the oral-, gastric- and intestinal- neoplasia, because an early/rapid diagnosis is required. However, it may be quite difficult due to oral-gastric-intestinal apparatus anatomy. Sim et al. [154] used a TPI reflection system in the frequency range of 0.2–1.2 THz to assess oral carcinoma. Seven oral tissues from four patients were analyzed, and then the authors compared the optical, frozen and room temperature THz, histopathological images. They found that THz images at −20 ◦C showed better contrast between healthy and cancer regions compared to the room temperature ones (20 ◦C). Additionally, frozen temperature images demonstrated better area correlation than the histopathological images [154]. The recent and detailed review of Danciu et al. [180] takes an overview of diagnostics methods and current available data on THz-based detection for digestive cancers. Some current in vitro and ex vivo progress is highlighted for identifying specific digestive neoplasia [180].

Instead, TPI has revealed the contrast between normal and neoplastic tissue regions [159,181]. One of the first applications on human ex vivo wet tissue involved imaging in excised basal cell carcinoma [182]; the regions had recognition of healthy skin and basal cell carcinoma (BBC), both in vitro [96] or in vivo and ex vivo [183,184].

According to Wallace et al. [184], TPI could differentiate between basal cell carcinoma (BCC) and normal tissue both in vivo and ex vivo, and it is under test whether TPI can facilitate the tumor margins delineation, prior to surgery. The BCC's areas have different THz properties compared to healthy tissue. The general clinical image does not identify the skin cancer distribution with accuracy in depth, under the skin surface. However, THz images, as shown in [184], clearly display the cancer distribution on the skin and also the extent of the neoplasm invasion into the skin, with a depth of 250 μm.

Comparing ex vivo and in vivo images of the same tumors, similar contrast levels can be found, however tissue deformation and shrinkage after excision leads to an exact match in the contrast pattern. Nevertheless, the contrast level, present in the THz images, was enough to map diseased tissue margins, when compared to histology. Reese et al. [185], using THz pulsed imaging for studying freshly excised colon cancer, have differentiated normal and diseased colon tissues, with a good contrast for their recognition. Moreover, Wahaia et al. [186] measured colon tissue samples both fixed in formalin and embedded in paraffin. In the first case, water is still present, and in the second one, it is eliminated. They still obtained good image contrast between healthy and cancerous regions. As a result, water is not the only factor contributing to the contrast between the two different tissue

areas. Thus, the tumor boundaries in THz images can be recognized, and these are in accordance with the visible images, indicating that the THz imaging technique could be useful for diagnosing cancers. Potentially, this technique could be employed as a complementary label-free technique, allowing surgeons to determine tumor margins in real time.

While different kinds of cancer can be differentiated from healthy tissue according to the high sensitivity of THz radiation to water content, the strong potential of TPI in cancer assessment is not limited to fresh tissues, as it has been efficiently demonstrated for discrimination of pathological and healthy dehydrated tissue as well [132,174,177].

Afterwards, TPI has been used to identify tumor borders on excised breast carcinoma [187]. While THz imaging has proven potential contrast between breast cancer and healthy tissue, both in fresh and formalin-fixed, paraffin-embedded (FFPE) tumors [154,179,188,189], all published works were performed on flat sections of the tumor. The application of THz imaging to a three-dimensional sample can be exploited to produce cross section images by in-depth scanning, without slicing the tumor. Bowman et al. [190], in their work, illustrated the powerful THz pulsed imaging applications to two different carcinoma samples: infiltrating ductal carcinoma and lobular carcinoma, both embedded in paraffin blocks. THz pulsed images showed clear definition of the cancer boundaries in the block. The results were correlated in 3D with histopathology sections sliced throughout the blocks. THz images highlighted the cancer regions only when there were interfaces inside the tissue, thus near to the sample surface. In all the histopathology images, instead, the infiltrating ductal carcinoma and lobular carcinoma were clearly visible, at any section. Furthermore, the THz 3D block images could be sectioned into planar (x-y, x-z, and y-z) images in order to produce in-plane and in-depth tissue images, thus successfully identifying cancer regions, without slicing the tissue. These results demonstrate the effectiveness of THz pulsed imaging for tumor edge identification, when diseased tissue is near the surgical excision.

Reid et al. [176] conducted a study on normal, tumor and dysplastic freshly excised colon samples from 30 patients, using a conventional THz-TDS system, in reflection mode. The study showed a sensitivity of 82% and a specificity of 77% in discriminating between normal and pathological tissues, while a sensitivity of 89% and a specificity of 71% in differentiating normal and dysplastic samples [176]. Another study [191], using diverse intelligent analysis methods (neural networks, decision trees, and support vector machines) re-evaluated the data provided by [191]. This method increased the sensitivity to 90%–100% and the specificity to 86%–90%, improving the overall diagnosis precision.

With the aim of reducing patient re-operation rates and improving the ability of resection margin in breast conserving surgery, Santaolalla et al. [192] applied a multivariate Bayesian classifier to the samples waveform produced by TPI probe system. They can discriminate tumor from benign breast tissue, obtaining a sensitivity of 96% and a specificity of 95%.

Nowadays, in vivo THz detection investigates mostly superficial normal and diseased tissues, close to the epithelial layer (i.e., breast, skin). Observing the significant contrast in THz dielectric permittivity responses of healthy skin, dysplastic and non-dysplastic skin nevi, Zaytsev et al. [193,194] distinguished the precursor of melanoma, with a non-invasive in vivo analysis, proving the efficiency of THz pulsed radiation in the early diagnosis of the melanomas.

To facilitate the use of TPI to scan tumor resection margins intraoperatively, Teraview Ltd. (Cambridge, UK) has proposed and developed a handheld probe system [195]. TPI handheld probe measures tissue sample positioned in histology cassette, in reflection mode. The TPI handheld probe ensures to discriminate benign from malignant breast tissue in an ex vivo setting. The purpose of the THz device is to give valid support for rapid histopathological evaluation of excised tissues in surgery [195].

Despite this, in the last ten years, researchers began to extend THz applications to inner tissues, organs or hollow cavities of the human body. The request is for endoscopic access, achievable using highly bendable waveguides with low transmission loss [196–198]. In 2009, Ji et al. [199] fabricated and developed a miniaturized fiber-coupled THz endoscope apparatus. It emits and collects THz

radiation using an optical fiber linked to a femtosecond laser. The measurement is performed close to the reflective surface of a profound tissue or an organ [199]. The researchers tested the device by looking at THz reflections from mouth, tongue and palm skin tissues and demonstrated that the moisture of internal organs is a significant confounding issue. Some years later, an innovative THz prototype, with single-channel detection based on flexible metal-coated THz waveguides [197,198] and a specific polarization exposure method, was integrated into a commercial optical endoscope and demonstrated its potentiality, successfully differentiating between normal and cancer colonic tissues [175]. This also makes it a suitable tool for investigating the water content and hydration profile of the skin. By using some basic image processing techniques like intensity windowing, histogram manipulation, edge detection and region growing [200–202], the discrimination of healthy and cancer regions by THz images can be considerably improved.

The same group also developed THz otoscope that enables the detection of otitis media [203], a disease that causes fluid and purulence in the posterior part of the tympanic membrane within the middle ear. The THz otoscope is able to measure the change in water content on the tympanic membrane; therefore, it is applicable as a medical device for otitis media diagnosis [203].

In [204] and in Figure 6, an imaging device was proposed as a potential tool for the detection of deterioration in the feet of diabetic patients. Assuming that dehydration of the feet skin of diabetics, owing to peripheral vascular disease, is a central element of their deterioration process, they take advantage of THz pulsed imaging in order to obtain a promising diagnostic tool. The results are encouraging and provide key elements that will allow the design of a clinical trial in the future.

**Figure 6.** Platform for early screening of diabetic foot syndrome [204]. (**a**) THz setup design. It consists of an elevated surface where two high-density polyethylene windows are used to place the patient's feet. A chair is also provided in order to maintain the patient's position and avoid motion of the feet during the diagnostic image acquisition. The space under the patient sitting area is used to place the THz-TDS spectrometer and a raster scanning system [204]. (**b**) Photograph of the assembled setup during the testing. (**c**) Schematic layout of the raster-scanning imaging system, placed on the platform under the windows as indicated in (**a**).

Concerning brain tumors, Ji et al. in Figure 7 and in [205] demonstrated the effectiveness of THz reflectometry imaging (TRI) in distinguishing cancer and normal regions within a brain tissue section. Relatively high intensity regions (depicted in red) are spatially well correlated to the tumor areas found with green fluorescent protein (GFP) and Hematoxylin and eosin (H&E) techniques.

**Figure 7.** Brain tumor discrimination with different imaging techniques [205]. (**a**) MRI (magnetic resonance imaging). (**b**) White light imaging. (**c**) GFP (green fluorescent protein) imaging. (**d**) H&E (Hematoxylin and eosin) stained imaging. (**e**) OCT (optical coherence tomography) imaging. (**f**) THz reflectometry imaging (TRI). (**g**) 5-ALA-induced ppIX fluorescence imaging. TRI images show red regions (relatively high intensity) that are in agreement with tumor regions images obtained with GFP and H&E modalities, while in white light images, cancer regions are not identified.

Yamaguchi et al. [39], instead, take advantage of different optical properties (like refractive indexes and absorption coefficients) in normal and tumor regions to produce a THz image of a fresh rat brain tissue sample, see Figure 8.

**Figure 8.** Brain tumor images of rat fresh tissue using different techniques: (**a**) THz spectroscopy and (**b**) HE-stained (hematoxylin and eosin) image of the same tissue section [39]. The THz image has been realized by computing a Tumor probability, where zero value corresponds to healthy tissue, based on the different refractive indexes of tumor and normal tissue regions. The red area in (**a**) shows the tumor region and is well in agreement with the dark purple area in HE-stained image (**b**).

Figure 8a shows the THz image based on the refractive index information (expressed as tumor probability), that results higher in the tumor than in the normal region (see also Figure 5). The red area locates the diseased tissue with great accordance to the HE-stained (hematoxylin and eosin) image of the same tissue section, where a darker purple region is visible (Figure 8b) [39].

Table 2 summarizes the main kind of tumor studied with TPI, with their relative references. Most works are ex vivo, while some papers for skin cancer are available in vivo and in vitro too.


**Table 2.** Different kind of tumors investigated with TPI and their references.

Additionally, THz radiation is [211] a non-ionizing one, such that it is biologically safe for in vivo applications [212]. Moreover, the limited penetration depth of THz radiation has focused medical imaging research into the dentistry area [213]. TPI has been found to be an interesting technique for dental tissue (enamel, dentine and pulp) discrimination because refractive index differences enable the three tissue regions to be identified [66,148,214–217]. TPI is not the only possible technique for dental disease monitoring [218]: (i) visual caries examination, for example, loss of enamel translucency in the region between the contacting proximal surfaces of two adjacent teeth, (ii) X-ray imaging, (iii) electrical impedance measurements, (iv) ultrasound and (v) fluorescence-based methods [218,219] can also be adopted. However, many disadvantages can be assessed to some of these techniques: (i) is not possible at early detection stages, (i) and (ii) are difficult for posterior teeth, (ii) is potentially harmful for patients' health because it uses ionizing radiation. For these reasons, additional strategies for dental health monitoring, with a particular emphasis on diagnosis at an earlier stage of formation and the use of non-ionizing radiations, are widely requested [218].

Arnone et al. used TPI in order to distinguish different animal and human tissues, in particular enamel, dentine and pulp, in human teeth. They realized two different kind of images with the same data set: TOF and absorption images. Being enamel ~99% mineral and dentine ~70% mineral, they show different refractive indexes, that result in different THz TOFs. The produced TOF image shows enamel only and enamel and dentine regions. In the absorption image, instead, the inner pulp region of tooth is visible, because it shows a strong absorption, thanks to the additional material in that tooth area [214]. Zinov'eva et al. [215] did some transmission images of human permanent molar tooth slices, at different THz frequencies, ranging from 0.2 to 1.5 THz. The teeth used in the experiments were processed to produce artificial lesions by chemical demineralization. The images clearly resolved enamel and dentine areas. In addition, demineralized tooth regions have shown an increase of THz transmission signal in comparison with healthy tooth tissue. This difference can be used to trace demineralization development in dental tissues [215]. Artificial demineralization detection has been the first step towards dental caries detection because caries regions are the result of mineral loss from enamel, causing a change in enamel refractive indexes and absorption coefficients. These changes can be exploited to identify lesions not visible to the naked eye. Early detection is important because initial stages of demineralization are reversible [66,148]. Crawley et al. [148] calculated enamel, dentine and caries absorption coefficients and refractive indexes in the range between 0.3 and 2.0 THz, in THz transmission geometry. Like [214], they produced a THz absorption map in a 210 μm thick tooth slice and a TOF image, but with more accurate spatial resolution. Caries are correctly detected by the absorption image because average absorption coefficient of carious enamel is typically 35% larger than the one associated with healthy enamel, in that frequency range, using TPI. Enamel and dentine are precisely identified using TOF data because they are related to different refractive indexes. In reflection geometry, instead, some images have been created by plotting the change in THz pulse height at a specific time delay after reflection from the sample surface. In these images, both caries are detected, and dentine and enamel are differentiated. Moreover, they investigated a hypomineralization region of a 200 μm thick tooth slice and demonstrated that TPI images can distinguish hypomineralization from enamel caries (demineralization) [216]. Finally, they demonstrated that even a TPI image of a tooth hemisection (much thicker with respect to previous slices) can discriminate caries inside it.

A 3D study of dental tissue has been reported in [217]. The enamel-dentine junction in 12 human incisor teeth has been detected in 91% of the cases. A series of ~100 μm deep steps were chemically produced in order to alter enamel thickness. They were imaged and the authors demonstrated that they accurately and reliably make direct measurements of enamel thickness; this is necessary for monitoring enamel erosion, a common dental disorder.

Finally, in order to verify TPI validity and effectiveness in dental caries detection, Kamburo ˘glu et al. [218] compared TPI (static images and movie video) with common radiological techniques: intraoral photostimulable phosphor late (PSP) and cone beam CT (CBCT) for the detection of dental caries ex vivo [218–220]. They demonstrated that TPI shows good performances for caries identification, compared to the most used techniques, see Figure 9.

**Figure 9.** Tooth caries identification with different imaging techniques [218]. (**a**) White light image. (**b**) THz static image at 0.35 THz. (**c**) THz image from video movie. (**d**) CBCT (cone beam CT) image. (**e**) PSP (photostimulable phosphor late) image. Red arrows indicate dental caries in tooth tissue. The five images are in agreement in identifying caries regions.

#### **5. Conclusions**

In summary, the rapid THz technological advances have led THz radiation to emerge as a promising and useful tool in medicine. We have outlined the detection techniques of THz pulsed radiation and biomedical applications, based on the use of TPI systems.

In this perspective, we have summarized the prominent advantages in the use of THz radiation, with particular attention to THz pulsed radiation. Briefly, we have illustrated THz pulsed sources, and subsequently incoherent and coherent detectors, that are commercially available and/or of interest for technological research. The common schematic layouts for reflection and transmission imaging modes are discussed too.

Concerning the THz radiation properties, we have discussed the major application of TPI in the field of biomedicine, reporting many examples of ex vivo and in vivo studies, including cases of histopathological imaging of cancers, which are suitable targets. The enhancement of the sensing capabilities and penetration depth within tissues, in medical applications, were addressed. This represents a delicate point to question, and in particular the use of PEAs should require more attention, and many extended trials should be carried out to allow the application of TPI systems in clinical fields and hospitals.

Although THz technology is rapidly increasing as a medical imaging modality, and THz imaging applications in biomedical fields have drawn extensive interest, the technologies are maturing and the principles have been demonstrated in order to direct efforts towards the realization that THz clinical applications are viable in the real world.

**Author Contributions:** Writing-Original Draft Preparation, A.D. and M.D.F.; Writing-Review & Editing, all authors; Formal Analysis, M.D.F. and V.D.; Supervision, S.L. and M.P.; Project Administration, S.L.; Funding Acquisition, S.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** We acknowledge the financial support of the Bilateral Cooperation Agreement between Italy and Japan of the Italian Ministry of Foreign Affairs and of the International Cooperation (MAECI), in the framework of the project of major relevance N. PGR00728.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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