Terahertz pulsed imaging and spectroscopy for biomedical and pharmaceutical applications

Terahertz (THz) radiation lies between the infrared and microwave regions of the electromagnetic spectrum.

Advances in THz technology have opened up many opportunities in this scientifically and technologically important spectroscopic region.

The THz frequency range excites large amplitude vibrational modes of molecules as well as probing the weak interactions between them.

Here we describe two techniques that utilize THz radiation, terahertz pulsed imaging (TPI) and terahertz pulsed spectroscopy (TPS).

Both have a variety of possible applications in biomedical imaging and pharmaceutical science.

TPI, a non-invasive imaging technique, has been used to image epithelial cancer ex vivo and recently in vivo.

The diseased tissue showed a change in absorption compared to normal tissue, which was confirmed by histology.

To understand the origins of the differences seen between diseased and normal tissue we have developed a TPS system.

TPS has also been used to study solids of interest in the pharmaceutical industry.

One particularly interesting example is ranitidine hydrochloride, which is used in treatment of stomach ulcers.

Crystalline ranitidine has two polymorphic forms known as form 1 and form 2.

These polymorphs have the same chemical formula but different crystalline structure that give rise to different physiochemical properties of the material.

Using TPS it is possible to rapidly distinguish between the two polymorphic forms.


Terahertz (THz) radiation is at the interface between infrared light and microwaves.

The THz gap, typically defined as the frequency range, 0.1 to 10 THz was a previously unexplored region of the electromagnetic spectrum, owing to a lack of suitable sources and detectors.1,2

Advances over the last ten years in laser and semiconductor technology have opened up many opportunities in this scientifically and technologically important spectroscopic region.

Radiation in this frequency range is of particular interest as it excites the intermolecular interactions, such as the librational and vibrational modes as well as probing the weak interactions between molecules.3

Here we describe two systems and example applications that utilize this frequency range.

Terahertz pulsed imaging (TPI) is a non-invasive, coherent, reflection imaging modality that explores this frequency region and has a current useable range of 0.1 to 3 THz, corresponding to a wavelength range of 3 mm to 100 µm.

These wavelengths are significantly larger than the scattering structures in tissue, and scattering using TPI should therefore be considerably reduced compared to techniques using near infrared or visible radiation.

The current lateral and axial resolutions attainable with our system at 3 THz are 200 µm and 20 µm respectively, making it a viable imaging modality.

As TPI is a coherent, time gated, low noise technique, both phase and amplitude information can be obtained, from which the absorption and refractive index of a medium can be determined.

TPI is a time-domain technique therefore depth information can readily be extracted.

Broadband detection allows for the acquisition of information over a wide spectral range at THz frequencies.

Terahertz pulsed spectroscopy (TPS) is similar to TPI but uses a transmission rather than reflection geometry, thus, is more suited to looking at the absorption spectra of liquid and solid samples.

Basal cell carcinoma and terahertz pulsed imaging

Epithelial cancers account for 85% of all cancers.4

One type, basal cell carcinoma (BCC) is the most common form of cancer worldwide in white populations.

In the UK the incidence rate has increased by 50% over the last 10 years,5 and has a reported annual incidence of around one million in the US.6

The diagnosis of BCC is based on visual assessment and possibly a biopsy.7

Well-defined, solid, cystic and superficial BCCs, and tumours of less than 20 mm in diameter are surgically excised.

A minimum margin of 4 mm is required to completely excise the tumour in over 95% of cases.8

Ill-defined tumours may extend 15 mm or more beyond the clinical edge.

In these cases Mohs' micrographic surgery (MMS) is the best technique as it allows review of all the margins and same-day closure of defects.9

A study was conducted to determine whether TPI could differentiate between BCC and normal tissue and to test whether it can help with outlining tumour margins prior to surgery.

Materials and methods – BCC and TPI

TPI system

A portable THz imager, TPI Scan (TeraView Ltd, UK), was used for ex vivo and in vivo measurements performed on samples and patients at Addenbrooke's Hospital, Cambridge, UK.

The TPI system used reflection geometry, as shown in Fig. 1.

Optical excitation was achieved using an ultrafast fiber laser (Femtolite, IMRA America Inc.) emitting 180 fs pulses centered at a wavelength of 780 nm, with a 48 MHz repetition rate and an average power of 25 mW.

A beamsplitter separated the laser light into two beams, an excitation beam and a detection beam.

Generation of the THz pulses was achieved by optical excitation of a gallium arsenide emitter.10

The THz pulses were collimated and focused onto the top surface of a z-cut quartz window by a pair of off-axis parabolic (OAP) mirrors.

The sample under investigation is placed onto the quartz window, as indicated in Fig. 1.

The angle of reflection was 30 degrees to the normal.

The THz pulses reflected from the tissue were re-collimated using another pair of OAP mirrors and focused onto a bow-tie photoconductive receiver on which the detection beam is also focused.

The system gave a usable frequency range of 0.1 to 3 THz with an average power of approximately 100 nW.

By sweeping the optical delay through the entire THz pulse at a rate of 15 Hz, the time-domain THz waveforms were obtained.

The entire THz optics indicated by the dashed box (Fig. 1), and hence the THz beam, were raster-scanned in the xy plane over a defined area.

A THz waveform is acquired at each xy point.

To remove any system response, the raw THz waveform of the tissue at each pixel was divided by a reference waveform in the frequency-domain and a numerical bandpass filter applied to remove high and low frequency noise.

The time resolution was approximately 200 fs which is limited by the laser pulse; assuming a refractive index of two in skin tissue in the THz frequency range, an axial resolution of approximately 20 µm close to the skin surface can be achieved.

The signal-to-noise ratio was approximately 5000∶1.

The time-domain waveform provided information to a depth on the order of 1 mm into the skin samples.2

The incident energy is over 1000 times lower than the maximum permissible exposure at similar frequencies (EN 60825-1, 1994).11

Cultured keratinocytes irradiated for longer times have shown no adverse change;12 therefore we assume the skin samples are unaffected by the THz radiation.

Tissue preparation

Excised skin tissue was obtained from the Department of Dermatology at Addenbrooke's Hospital, Cambridge, UK.

Appropriate consent was obtained for the study; all patient material was anonymised.

A sample typically consisted of a piece of suspected BCC with a margin of normal tissue surrounding it.

Prior to imaging, excess fluid was removed from the skin samples using surgical gauze.

The quartz window was covered with a transparent sterile dressing (Tegaderm™, 3M Healthcare, Germany).

This prevented contamination of the quartz window.

The skin sample was placed in direct contact with the covered quartz window (diameter 38 mm, thickness 2 mm), with the top surface of the skin facing the incident THz radiation.

No immersion medium was used between the quartz window and the tissue, and air gaps were minimized.

After imaging the tissue was photographed and placed in formalin and submitted for routine histology.

Data analysis

The TPI images were generated using an analysis technique called time post pulse (TPP), where TPP = E(t)/E(min).

TPP normalizes the THz pulse, E(t), at a time t, by the minimum peak value, E(min).

This analysis technique allows for the direct comparison of the relative change of THz waveforms and can differentiate between diseased and normal tissue.13

An example is given in Fig. 2.

As the THz pulse propagates through an absorptive medium, such as skin tissue, it broadens in time.2

The TPI images in Fig. 3 were produced by plotting the TPP value at a particular time, t, at each pixel over the area scanned.

Typically, t was chosen to provide the best contrast between the diseased and normal tissue.

A suture placed on the edge of the BCC allowed orientation of the histology sections and acted as a reference point for both the visible and THz images.

The histology cross-section was taken from the suture location through the centre of the tumour.

Results – BCC and TPI

Fig. 3 shows a comparison between the visible images, THz images and histology.

Fig. 3a shows a clinical photograph of a male patient with a featureless, primary, clinically ill-defined BCC on the forehead.

The black outline approximately represents the tissue removed and imaged.

The resulting THz image of the excised tissue is shown in Fig. 3b.

The false colour THz image shows a strong contrast between the diseased tissue shown as red ‘hot spots’ and the surrounding normal tissue which shows a more uniform green/yellow colour throughout.

The X marks the location of the suture and the dotted black line indicates the axis of the vertical histology section.

The histology (Fig. 3c) shows a nodular BCC, and the suture is indicated on the left with an ‘X’.

Nodules of tumour are indicated with an ‘*’.

These nodules of tumour in the histology correlate well with location of areas of contrast in the THz image.

Fig. 3d shows a clinical photograph of a bland looking lesion on the right temple of a female patient.

The black outline approximately represents the tissue removed and imaged.

The resulting THz image of the excised tissue is shown in Fig. 3e and the histology in Fig. 3f.

This histology in Fig. 3f shows a sclerotic basal cell carcinoma which extends over the region between the two asterisks which corresponds to the area of contrast seen on the THz image.

Discussion – BCC and TPI

The study describes the application of TPI for imaging BCC ex vivo over a region of the electromagnetic spectrum not previously investigated.

The BCCs showed an increase in absorption of THz compared to normal tissue.

The level of contrast observed in the THz images was sufficient to identify tumour margins when compared to histology.

However, the source of this contrast is still under investigation.

Simple diatomic polar molecules, for example water, readily absorb THz radiation.14

Water has a strong broadband absorption around 5.4 THz, arising from the stretching of the hydrogen bond between molecules, the tail of which extends to the lower frequencies, with significant absorption in the operating range of our TPI system.3,15

Increased water content in malignant tissues has been observed in many different tumour types, and is often used as a marker for malignancy.16–18

The change in absorption in diseased regions that gives rise to contrast in TPI images might be attributed to a change in the intermolecular vibrational modes of water molecules with other functional groups.

Introduction to terahertz pulsed spectroscopy (TPS)

To understand the nature of the contrast mechanism observed in TPI images (for example in Figs. 3b and 3e), it is necessary to understand the spectroscopy of materials in the THz region.

To this end we have constructed a THz spectrometer.

Terahertz pulsed spectroscopy (TPS) is a quick and easy-to-use technique and has recently been applied to a range of materials.

Walther et al19. have used the technique to study a range of saccharides.

They observe a series of distinct absorption lines which originate from the lowest intermolecular vibrational modes.

However, when the sugars are melted to form an amorphous state, the sharp features observed in the crystalline form disappear to give a broad, featureless absorption spectrum.

This is because the long-scale intermolecular interactions (or phonon modes) have been broken down leading to random orientation of the molecules.

The same group also report on some anomalous behaviour in the peak positions of the phonon vibrations when the sample is cooled to 5 K. Because there is considerable lack of theoretical understanding of weak non-covalent forces assigning and understanding the source of these vibrational features is not possible.

However, several groups are beginning to use density functional theory (DFT) to predict and try to understand THz spectra.

For example, Shen et al20. have used DFT to try to explain the THz absorption spectra of purine and adenine.

The use of the theory is still at an early stage but with more groups using THz technology a deeper understanding of the meaning of the spectra will be obtained.

A molecule that has been well studied and is believed to be of great importance to TPI is water.

Water is a good example of a molecule that undergoes intermolecular interaction, more commonly known, as hydrogen bonding.

Water molecules can form up to four hydrogen bonds forming a tetrahedral structure.21

In low frequency Raman spectroscopy some investigators have reported observation of an absorption band at 1.8 THz.

The origin of this band is believed to be due to a hydrogen bond bending motion, because of the selection rules this band is not observed in the THz absorption spectrum.

There is another strong hydrogen bonding transitions at 5.4 THz due to a hydrogen-bonding stretching mode.

A broad featureless absorption spectrum of water has been reported by Ronne and Keiding.21

The broad spectrum is believed to be due to the superposition of a continuum of water active rotational and translational modes.

The sensitivity of TPS leads us to investigate the possibility of distinguishing between polymorphs.

Walther et al19. has reported that it is possible to distinguish between different isomers of hydroxybenzoic acid.

This is a result of isomers having different crystalline structures arising from the different packing density due to functional groups being in different positions within the parent molecule.

However, it is not obvious that the same would be true for polymorphs.

Taday et al22. have reported for the first time that it is possible to discriminate between the polymorphs of ranitidine HCl.

We report here the temperature dependent spectrum of the two polymorphs which should aid in the assignment of the features observed.


Many materials of pharmaceutical interest can exist in more than one solid form.23

These different forms have different crystalline structures which can lead to different physiochemical properties of a material.

These different crystalline structures are known as polymorphs.

The formation of different polymorphs can be controlled during crystallization by solvent used, cooling rate and the degree of super saturation of the solution.

Once in the crystalline form the polymorphic state can change by incorrect storage or during tablet preparation.

Ranitidine hydrochloride (HCl) is used in treatment of duodenal-gastric ulceration and Zollinger–Ellison syndrome.

Ranitidine HCl exists in two polymorphic forms.24

Form 1 is obtained by crystallizing from an ethanolic solution after the addition of ethyl acetate,25 while form 2 is obtained from a solution of isopropanol–HCl.26

The two forms have equal solubilities and there is no difference in bioavailability.27,28

The molecular structure of ranitidine HCl is shown in Fig. 4.

Materials and methods – Polymorph detection and TPS

Ranitidine HCl forms 1 and 2 were obtained from Neuland Laboratories Limited and were used without any further purification.

The drugs were lightly crushed and mixed with polyethylene (PE) powder at a percentage weight of 25%.

The mixture was compressed into discs of thickness about 1 mm with a compression of about 2 tons.

It is possible to convert one polymorphic into the other by compressing a tablet.

To check that this did not occur with ranitidine HCl, a series of polyethylene disc mixtures were prepared at different compressions between 1 to 2 tons.

The THz spectra of these disc showed that there was no interconversion between the two polymorphic states.

The sample was placed in a helium-cooled cryostat and held at temperatures as low as 4 K. It should be noted, however, that at this stage of the development the temperature at the sample position has not been accurately determined.

All temperatures stated in this report are recorded at the heat exchanger of the cryostat.

TPS system

The apparatus employed for TPS is similar to that used for TPI but uses a transmission rather than reflection geometry and is described in .ref. 22

Briefly the laser used in these experiments was a Ti:Sapphire laser (Vitesse, Coherent Inc), that produces 90 fs near-bandwidth limited pulses.

A schematic of the experimental setup is shown in Fig. 5(a).

The laser beam is split into two.

One beam is used to generate the THz-radiation; the other beam is used as a probe to detect the THz-radiation using electro-optic sampling (EOS),29 a typical waveform is shown in Fig. 5(b).

By changing the delay and monitoring the change to the probe beam on a pair of balanced photodiodes, a THz waveform is obtained.

The waveform is obtained in the time-domain and a fast-Fourier transform (FFT) can be performed to convert the refractive index, n, to obtain spectral information shown in Fig. 5(c).

The useful bandwidth of the waveform to the frequency domain to obtain spectral information shown in Fig. 5(c).

The useful bandwidth of detection is from about 300 GHz to about 3 THz (10–100 cm–1).

The range of the temporal scan determines the spectral resolution of a system.

A resolution of the order 2.5 GHz (0.08 cm–1) can be achieved with our system.

The THz-field transmitted through a sample is attenuated by dispersion and absorption.

The ratio of the electric field strength before, Es(ω), and after transmission, Er(ω), is given by where d is the thickness of the sample, ω the frequency of the radiation, c the speed of light in vacuo and T[n(ω)] are the reflection losses at the sample surface.

Both the refractive index n(ω) and the absorption coefficient α(ω) can be determined from the ratios of the measured THz-fields.

We report here only the absorption coefficient α(ω) of the material.

Results and discussion

Fig. 6 shows the THz absorption spectra for forms 1 and 2 of ranitidine HCl for three different temperatures, 5 K, 150 K and 300 K. The data are fitted to a series of Lorentzian lineshape functions.

From the room temperature spectra (300 K) one can see there are major differences between the two polymorphs.

The spectra recorded at different temperatures may allow us to understand which features are due to intermolecular interactions (or phonon modes) and which are due to intramolecular vibrations.

At this stage of the research there has been no normal mode or DFT calculation on ranitidine HCl, so assignment of the transitions observed in the spectra is not possible.

A detailed assignment of the vibrational modes in the THz region is a challenging task.

We limited ourselves to a description of the spectra observed.

In the temperature dependent spectra of form 1 of ranitidine HCl, the feature seen at 2.54 THz (84.7 cm–1) in the 300 K spectrum disappears in the 150 K and 5 K spectra.

This could be explained by the fact that the vibrational potential for this particular hydrogen bond stretch is anharmonic and as the temperature increases the bond softens causing a red shift in the peak position.

However, the feature at 2.75 THz (91.7 cm1) is not affected by temperature change and thus hides the 2.54 THz feature.

Again, the 2.75 THz feature could be a hydrogen bond with a harmonic potential or an intramolecular vibration which is not affected by temperature change.

Another interesting feature in the form 1 spectra is the considerable change in intensity in the peak at 1.78 THz (59.3 cm–1).

At the moment without good theoretical background it is difficult to understand.

But, we speculate that there are competing modes within this peak and as one mode dominates the other we observe a variation in intensity.

The spectrum of form 2 of ranitidine HCl is considerably different from that of form 1 which is in good agreement with the data obtained in .ref. 22

This remains true for all temperatures which were measured in these experiments, so over the range 5–300 K there is no phase change between the polymorphs.

It is noted that form 2 appears to be more temperature sensitive than form 1, with most of the major peaks red-shifting with increase of temperature.

It is clear from our work and others that a greater theoretical understanding is required.


Over the past year results emerging from new applications of terahertz technologies have grown considerably.

Here we have presented two examples.

It has been shown that TPS is a technique that can provide information about the physical state of a pharmaceutical active ingredient during the processing and storage of a tablet formulation.

Moreover, there is scope for further development of this technique for these and other applications.

Advanced data processing methods such as chemometrics will facilitate a greater understanding of solid dose formulations.

TPS has been used in the investigation of the crystalline structure of materials.

This is of considerable interest to the pharmaceutical industry where the lack of identification of polymorphs could lead to loss of patent protection (e.g. GSK Paxil™/Seroxal™) or if found during manufacture the company could be penalised by their national regulator.

The technique has a fast data acquisition rate; currently raw data waveforms can be obtained in less than 100 ms, and it is envisaged that further developments will reduce this to less than 100 µs.

This will open opportunities for incorporation of the technology into a high throughput polymorph screening system.

TPI has been used to image epithelial cancer ex vivo.

The diseased tissue showed a change in absorption compared to normal tissue, which correlated well with regions of tumour seen in histology.

This suggests that TPI may have potential as a tool to identify boundaries of tumours.

These measurements are now being conducted in vivo and will be published at a later date.

Also, TPS measurements are being performed on malignant and healthy tissue to determine sources of contrast and spectral features which may allow diagnosis of many conditions.

The origin of this useful and interesting spectral behaviour in polymorphs as well as that of water and biomolecules which contribute to the contrast in TPI images of tissue are now being explored and are an important area of scientific investigation.

TPI is one amongst several imaging techniques that are being evaluated as diagnostic tools for skin lesions and tumour margin assessment.

TPI may prove advantageous in distinguishing type, lateral spread and depth of tumours.