Photoacoustic imaging for non‐invasive examination of the healthy temporal artery – systematic evaluation of visual function in healthy subjects

Photoacoustic (PA) imaging has the potential to become a non‐invasive diagnostic tool for giant cell arteritis, as shown in pilot experiments on seven patients undergoing surgery. Here, we present a detailed evaluation of the safety regarding visual function and patient tolerability in healthy subjects, and define the spectral signature in the healthy temporal artery.


Introduction
Surgical biopsy and histopathological analysis of the temporal artery are considered the gold standard in the diagnosis of giant cell arteritis (GCA). However, although this technique has high specificity, it has low sensitivity (Luqmani, Lee et al. 2016), and is associated with complications such as injury to the facial and trigeminal nerve, and peri-and postoperative haemorrhage (Guffey Johnson, Grossniklaus et al. 2009;Borchers & Gershwin 2012;Gunawardene & Chant 2014). Attempts have been made to implement non-invasive imaging techniques, in particular ultrasonography, for the diagnosis of GCA. However, ultrasound is highly operator-dependent (Hauenstein, Reinhard et al. 2012), and the sensitivity and specificity in the diagnosis of GCA have proved inadequate (Arida, Kyprianou et al. 2010;Borchers & Gershwin 2012;Buttgereit, Dejaco et al. 2016;Luqmani, Lee et al. 2016).
Photoacoustic (PA) imaging is currently one of the most rapidly developing biomedical imaging techniques, providing non-invasive, high-resolution images (Valluru & Willmann 2016). It is unique in that it uses pulsed laser light and optical absorption detected by ultrasound to provide high-resolution images with high spatial resolution. PA imaging provides an absorption spectrum of the tissue that depends on the molecular composition, and thus has a greater ability to discriminate between small differences in tissue composition than previously tested techniques. Another advantage of PA technology is that it is userindependent, since the spectral signature obtained is an objective measure. Encouraging results have been obtained in animal studies, showing detailed images of small blood vessels with high resolution (Jeon, Song et al. 2017). However, no clinical studies have been performed to evaluate the suitability of PA imaging for diagnosing GCA. We have recently adapted the PA technique for use in humans and resolved problems associated with motion artefacts and disturbances from other endogenous chromophores, in a previous study including seven patients undergoing surgery for suspected GCA (Sheikh et al., 2019). However, detailed investigation of safety regarding visual function or patient tolerability was not performed in that pilot study.
The main aim of the present study was to examine the temporal artery in 12 healthy subjects with PA imaging, and to evaluate the effects on visual function in detail, in terms of the visual acuity, colour vision and visual field, in order to confirm the safety of the technique, and to assess the patient tolerability of PA examination. The other aim was to characterize the spectral signature obtained the healthy temporal artery.

Study subjects
Twelve study subjects, aged 60 years or older, and generally healthy, were included in the study. The median age was 67 years (range 60-73 years), four were 4 male and 6 female. Diabetes mellitus (n = 1), systemic hypertension (n = 3) or previous cardiovascular events were not reasons for exclusion. The ankle-brachial pressure index was within the normal range, that is 1.0-1.2, indicating the absence of peripheral vascular disease. All patients were of similar skin type, that is Fitzpatrick type I and II. The exclusion criterion was any medical condition caused by inflammation. The study was approved by the Ethics Committee at Lund University, Sweden.
Ultrasound was first performed using the ultrasound part of a Vevo LAZR-X system (FUJIFILM VisualSonics Inc., Toronto, ON, Canada). A 25-MHz handheld ultrasound transducer (MX250) was used to locate the superficial temporal artery and its parietal and frontal branches. The 40-MHz transducer (MX400) was then used to obtain a more detailed image. There were no halo signs or compression signs in the examined temporal arteries, confirming the absence of GCA (Luqmani, Lee et al. 2016) (Aschwanden, Daikeler et al. 2013). The 40-MHz transducer was then attached to an adjustable arm (Mounting Accessory, GCX Corporation, Petaluma, CA, USA) to allow scanning with a linear stepper motor (VisualSonics Inc., Toronto, ON, Canada). Colour Doppler ultrasound scanning was performed to provide accurate location of the artery in preparation for PA scanning.
PA imaging was performed using the Vevo LAZR-X system. Two planar laser beams, located on either side of the ultrasound linear array, illuminate the skin surface, and the PA signal is detected using an MX400 ultrasound linear array transducer. PA imaging was first performed using the Spectro mode, in which hyperspectral PA images were collected from 680 to 970 nm in 5-nm increments, to obtain spectral signatures of the tissues. Thereafter, a 3D PA scan was performed using the stepper motor (step size 500 lm), starting with a single wavelength scan at 820 nm to check the PA signal. Finally, a multiwavelength 3D scan was performed at 12 wavelengths (700, 720, 740, 760, 780, 800, 820, 840, 860, 880, 900 and 940 nm). The laser pulse energy was 55 mJ at 680 nm and decreased slightly with increasing wavelength, showing a variation in energy per pulse of approximately 2-5 mJ. The spectral signatures of the vessel wall (not including the lumen), subcutaneous tissue and skin were analysed using the VisualSonics Vevo LAB 3.1.0 software and MATLAB R2017b (MathWorks Inc., Natick, MA, USA). Further details of the method can be found in Sheikh et al. (Sheikh et al., 2019).
Visual function was tested before and after PA imaging to evaluate the safety of the method. Best corrected visual acuity was measured with the Snellen letter chart (Ortho-KM, Lund, Sweden). Colour vision was tested with both Sahlgren's Saturation Test (SST, VISUMETRICS AB, G€ oteborg, Sweden) and the Ishihara colour vision test (Luxvision, US Ophthalmic, Doral, USA), since combining these tests allows discrimination between congenital and acquired defects (Frisen & Kalm 1981). Neither visual acuity nor colour vision was affected by PA examination. The visual field was measured with a Humphrey visual field analyzer, using the 24-2 test protocol (Carl Zeiss Meditec AG, Jena, Germany). A slight improvement was seen in the results after PA imaging, which could be due to a learning effect. Detailed results are given in Table 1.
We anticipated that the risk of PA imaging affecting visual function would be minimal since the PA probe is  subjects. However, it was of very short duration and was not perceived as worrisome by the subjects. We therefore concluded that PA imaging was safe with regard to visual function. The subjects' experience of the PA examination was assessed using a visual analog scale (VAS) ranging from 0 to 100. The results show that the level of discomfort was low (median 8, range 1 to 17). Only little heat was felt from the probe (5, range 1 to 37), and only little light sensation was reported (22, range 5 to 80) on the VAS 0-100 scale. None of the examined subjects reported any negative experiences of the PA examination.
Photoacoustic imaging showed that the artery could be clearly delineated in the 3D scans. The multiwavelength 3D images were analysed using the spectral unmixing function in the Vevo LAB 3.1.0 software, providing clear visualization of the overall artery architecture and its extension. The spectral signature of the artery wall was clearly differentiated from those of the subcutaneous tissue (p < 0.05 in the wavelength range 830-895 nm) and skin (p < 0.05 in the wavelength range 795-940 nm) (Fig. 1). This is one of the first studies on human vasculature using PA imaging. A few studies have previously been reported on PA imaging of blood vessels in humans, for example, vessels of the skin (Zafar, Breathnach et al. 2015;Xu, Yang et al. 2016), coronary arteries (Daeichin, Wu et al. 2016), the radial artery (Bok, Hysi et al. 2017;Karlas, Reber et al. 2017), the tibialis posterior and dorsalis pedis arteries (Taruttis et al., 2016), the carotid artery (Kruizinga et al., 2014), the digital arteries (Hai, Zhou et al. 2015) and the palmar digital arteries (Matsumoto, Asao et al. 2018).
Limitations of the present study were that all the participants in this study had similar skin types (Fitzpatrick type I and II), and it was not possible to determine the effect of spectral colouring due to superficial tissue chromophores such as melanin. Melanin is an endogenous chromophore that may affect light propagation through the skin. Other factors that may affect the spectrum are the arterial depth, that is, the amount of tissue the light must propagate through, and the amount of haemoglobin and its oxygenation status.
There is some noise in the data, as can be seen from the irregularity of the plots in Fig. 1. The standard deviation in the results was high, particularly when the arteries were imaged with laser wavelengths shorter than 800 nm. This could be due to small variations in the energy between the laser pulses, motion artefacts, or the scattering of light by blood and other chromophores. Post-processing of the measured data, using spectral unmixing together with Monte Carlo simulations, may be necessary/useful to compensate for the variations in chromophores and measurement depth between patients. The problem of motion artefacts could be solved by the application of software to correct for motion artefacts in the image, or the use of electrocardiography to trigger image capture to compensate for arterial pulsation.
In conclusion, the present study shows that PA imaging of the temporal artery is well tolerated and safe with regard to visual function. The artery wall was clearly delineated, and unique spectral signatures were obtained for the artery, compared to the surrounding tissues. Further studies will be required to determine whether PA imaging can be used to identify anomalies in the temporal artery for the diagnosis of GCA. The next step in the development of PA imagining into a clinical diagnostic tool will require a larger clinical trial in which patients with suspected GCA are examined before undergoing surgical biopsy.