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
], scanning near-field microscopy [3
], photoacoustic microscopy [5
], ultrasonic imaging [6
], optical coherence tomography [7
], digital holography microscopy [9
], Raman scattering microscopy [12
], Coherent Raman Scattering (CRS) spectroscopic imaging [13
], two-photon fluorescence (TPF) [19
] and second-harmonic generation (SHG) imaging [25
], and super-resolved imaging techniques [28
Many efforts were done in THz technology, improving THz sources and detectors’ responses [32
], 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
], gas sensing [45
], chemical analysis [46
], new materials characterization in low-frequency range and non-destructive evaluation of composite materials and constructions [48
], astronomy [50
], microelectronics and security [51
], 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
]. (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
]. 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
]. 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−1
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
]. (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
]. 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.
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
]. 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
], alcohol perfused [36
], formalin fixed [148
], gelatin embedding [151
], lyophilized [153
], freezing [154
] 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
]—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
], 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
]. 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
]; the water content evaluation allows label-free differentiation for various types of tissues as an endogenous marker, distinguishing between healthy and pathological tissues [152
], see Figure 4
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
]. 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
TPI has been successfully applied to liver [164
], colon [165
], intestine [161
], brain [39
], skin [178
], ovarian [167
], oral [154
] and breast cancers [132
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
]. 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
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
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
], 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
] 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
]. 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
] 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
], 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
] 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.
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.
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
a 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 8
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.
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
]. 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
] 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
]. 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, Kamburoğlu 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
]. They demonstrated that TPI shows good performances for caries identification, compared to the most used techniques, see Figure 9