Functional and molecular 3D mapping of angiosarcoma tumor using non-invasive laser speckle, hyperspectral, and photoacoustic imaging.

PURPOSE: The gold standard for skin cancer diagnosis is surgical excisional biopsy and histopathological examination. Several non-invasive diagnostic techniques exist, although they have not yet translated into clinical use. This is a proof-of-concept study to assess the possibility of imaging an angiosarcoma in the periocular area. METHODS: We use laser speckle, hyperspectral, and photoacoustic imaging to monitor blood perfusion and oxygen saturation, as well as the molecular composition of the tissue. The information obtained from each imaging modality was combined in order to yield a more comprehensive picture of the function, as well as molecular composition of a rapidly growing cutaneous angiosarcoma in the periocular area. RESULTS: We found an increase in perfusion coupled with a reduction in oxygen saturation in the angiosarcoma. We could also extract the molecular composition of the angiosarcoma at a depth, depicting both the oxygen saturation and highlighting the presence of connective tissue via collagen. CONCLUSIONS: We demonstrate the different physiological parameters that can be obtained with the different techniques and how these can be combined to provide detailed 3D maps of the functional and molecular properties of tumors useful in preoperative assessment.

Hyperspectral and Laser Speckle Contrast Imaging for Monitoring the Effect of Epinephrine in Local Anesthetics in Oculoplastic Surgery.

PURPOSE: Epinephrine is used in local anesthetics to induce vasoconstriction and thus reduce bleeding and prolong the anesthetic effect. Finding the optimal delay between the administration of the anesthetic and skin incision to ensure vasoconstriction and minimize bleeding is important and has recently become the subject of debate. This is the first study to assess blood perfusion and oxygen saturation (sO 2 ) simultaneously in response to a local anesthetic containing epinephrine in human oculoplastic surgery. METHODS: A local anesthetic consisting of lidocaine and epinephrine (20 mg/ml + 12.5 µg/ml) was injected in the eyelids of 9 subjects undergoing blepharoplasty. The perfusion and sO 2 of the eyelids were monitored using laser speckle contrast imaging and hyperspectral imaging, respectively. RESULTS: Laser speckle contrast imaging monitoring showed a decrease in perfusion over time centrally at the site of injection. Half-maximum effect was reached after 34 seconds, and full effect after 115 seconds, determined by exponential fitting. The drop in perfusion decreased gradually further away from the injection site and hypoperfusion was less prominent 4 mm from the injection site, with a spatially dependent half-maximum effect of 231 seconds. Hyperspectral imaging showed only a slight decrease in sO 2 of 11 % at the injection site. CONCLUSIONS: The optimal time delay for skin incision in oculoplastic surgery is approximately 2 minutes after the injection of lidocaine with epinephrine. Longer delay does not lead to a further decrease in perfusion. As sO 2 was only slightly reduced after injection, the results indicate that the use of epinephrine is safe in the periocular region.

Reperfusion of Free Full-Thickness Skin Grafts in Periocular Reconstructive Surgery Monitored Using Laser Speckle Contrast Imaging.

PURPOSE: Free skin grafts are frequently used in reconstructive surgery. However, little is known about the course of reperfusion due to the previous lack of reliable perfusion monitoring techniques. The aim of this study was to use state-of-the-art laser speckle contrast imaging to monitor free skin grafts in the periocular area. METHODS: Seven patients needing surgery due to tumor removal or cicatricial ectropion in the periocular region underwent reconstructive surgery using free skin grafts from either the contralateral upper eyelid or the upper inner arm. The free skin grafts measured 10-30 mm horizontally and 9-30 mm vertically. Blood perfusion was monitored using laser speckle contrast imaging immediately postoperatively (0 weeks) and at follow-up after 1, 3, and 7 weeks. RESULTS: All grafts were reperfused gradually during healing, the median value being 46% in the central part of the graft after 1 week and 79% after 3 weeks. The grafts were completely reperfused after 7 weeks. No difference was observed in the rate of reperfusion between the center and periphery of the grafts (p = not significant). The cosmetic and functional outcome was excellent in all but 1 patient, who developed ectropion that had to be surgically corrected. CONCLUSIONS: Skin grafts in the periorbital area are fully reperfused after 7 weeks. The periocular area is known to be well-vascularized and thus forgiving to reconstructive surgery. Future investigations of the reperfusion of free skin grafts in other parts of the body or in higher-risk populations should be carried out.

Successful Free Bilamellar Eyelid Grafts for the Repair of Upper and Lower Eyelid Defects in Patients and Laser Speckle Contrast Imaging of Revascularization.

PURPOSE: It is generally believed that large eyelid defects must be repaired using a vascularized flap for 1 lamella, while the other can be a free graft. Recent studies indicate that the pedicle of a tarsoconjunctival flap does not contribute to blood perfusion. The purpose of this study was to explore whether large eyelid defects can be repaired using a free bilamellar eyelid autograft alone. METHODS: Ten large upper and lower eyelid defects resulting from tumor excision were reconstructed using bilamellar grafts harvested from the contralateral or opposing eyelid. Revascularization of the flap was monitored during healing using laser speckle contrast imaging, and the surgical outcome was assessed. RESULTS: The functional and cosmetic results were excellent. All grafts survived and there was no tissue necrosis. Only 1 patient underwent revision after 4 days as the sutures came loose. Two patients developed minimal ectropion but needed no reoperation. All patients were satisfied with the surgical results. Perfusion monitoring showed that the grafts were gradually revascularized, exhibiting 50% perfusion after 4 weeks and 90% perfusion after 8 weeks. CONCLUSIONS: A free bilamellar eyelid graft appears to be an excellent alternative to the tarsoconjunctival flap procedure in the reconstruction of both upper and lower eyelid defects, especially in patients who cannot tolerate visual axis occlusion or the 2-stage procedure of the conventional staged flap procedure.

Novel Evidence Concerning Lacrimal Sac Movement Using Ultra-High-Frequency Ultrasound Examinations of Lacrimal Drainage Systems.

PURPOSE: Current hypothesis regarding the mechanism of active tear drainage is based on studies performed ex vivo or under nonphysiological conditions. Novel ultra-high-frequency ultrasound has the advantage of generating images with superior resolution, enabling measurements of low flow in small vessels, and the tracking of tissue motion in real time. The purpose of this study was to investigate the lacrimal drainage system and active drainage using this modality. METHODS: The upper lacrimal drainage system was investigated with 40-70 MHz ultrasound in 22 eyes in 13 patients. Irrigation confirmed a lacrimal obstruction in 10 eyes. Motion tracking was used to map movement of the lateral lacrimal sac wall and to measure flow when possible. RESULTS: The anatomy of the upper lacrimal drainage system was mapped in vivo, including the proximal canaliculi, which have not previously been imaged. The lacrimal sac lumen is slit shaped in its resting state but is distended when irrigated or if a nasolacrimal duct obstruction is present. Thus, the healthy lacrimal sac is not a cavity, and the medial retinaculum does not act against a stretched structure. Motion tracking visualized the "lacrimal pump," showing that the direction of motion of the lateral lacrimal sac wall is mainly in the sagittal plane during blinking. CONCLUSIONS: Ultra-high-frequency ultrasound allows detailed physiological monitoring of the upper lacrimal drainage system in vivo. Our findings suggest that current theories of active tear drainage need to be reappraised.

Spectral Signatures in the Different Layers of the Human Eyelid by Photoacoustic Imaging.

BACKGROUND AND OBJECTIVES: The eyelids are susceptible to a number of skin cancers, which are challenging to excise radically without sacrificing excessive healthy tissue. Photoacoustic (PA) imaging is an emerging non-invasive biomedical imaging modality that could potentially be used for intraoperative micrographic control of the surgical margins of eyelid tumors. In this study, non-cancerous human eyelid tissue was characterized using PA as a first step in the development of this technique. STUDY DESIGN/MATERIALS AND METHODS: Twelve full-thickness samples from nine patients were analyzed ex vivo using PA imaging. Two-dimensional PA images were acquired using 59 wavelengths in the range of 680-970 nm to obtain the spectral signatures of the skin, orbicularis oculi muscle, and the tarsal plate. Three-dimensional images were obtained by scanning the tissues using a linear stepping motor. Spectral unmixing was performed to visualize the chromophore distribution. RESULTS: The resulting PA spectra could be used to differentiate between the orbicularis oculi muscle and the other two structures (P < 0.05). The signals from the skin and the tarsal plate were more similar in appearance, probably due to similarities in their molecular composition. Spectral unmixing provided a clear visualization of the overall architecture of the eyelids. CONCLUSIONS: PA imaging can be used to differentiate between the orbicularis oculi muscle and the eyelid skin and tarsal plate. The main structures of human eyelids could be visualized in three dimensions using PA imaging. This technique could potentially be used to examine eyelid tumors intraoperatively in the future. However, further studies on tumors in vivo are needed before considering such clinical use. Lasers Surg Med. © 2019 Wiley Periodicals, Inc.

Perfusion in Upper Eyelid Flaps: Effects of Rotation and Stretching Measured With Laser Speckle Contrast Imaging in Patients.

PURPOSE: The aim of this study was to investigate how the blood perfusion in human upper eyelid skin flaps is affected by the length of the flap and the degree of stretching and rotation of the flap. METHODS: Twenty-nine upper eyelids were dissected as part of a blepharoplastic procedure in patients. The 1-cm wide proximal end of the flap remains attached, to mimic a random pattern skin flap (hereafter called a "skin flap"). Blood perfusion was measured with laser speckle contrast imaging before and after the flap was stretched with forces of 0.5, 1, and 2 N. The flap was then rotated 90°, and the same tensions were applied. RESULTS: Blood perfusion decreased gradually from the base to the tip of the flap. The flap was only well perfused in the proximal 1 cm (60% at 0.5 cm and 37% at 1.0 cm) and was minimally perfused beyond 2 cm (22% at 2.0 cm). Stretching the nonrotated flaps affected perfusion slightly (decreased to 43% at 0.5 cm). Simply rotating the flaps by 90° had no significant effect on the perfusion. The combination of rotation (90°) and stretching reduced the perfusion to 22% at 2 N, when measured 0.5 cm from the base. CONCLUSIONS: Blood perfusion in upper eyelid skin flaps decreases rapidly with distance from the base of the flap. Rotating and stretching the skin flap reduces blood perfusion even further, leading to minimal perfusion in this kind of flap at distances greater than 1.5 cm from the base.

Blood Perfusion in Rotational Full-Thickness Lower Eyelid Flaps Measured by Laser Speckle Contrast Imaging.

PURPOSE: Large upper eyelid defects can be repaired by rotational full-thickness lower eyelid flaps. The aim was to measure the blood perfusion in such flaps, and how it is affected by the length of the flaps, and the degree of rotation and stretching. METHODS: Nine patients underwent the Quickert procedure for entropion repair in which a full-thickness eyelid flap of approximate width 0.5 cm and length 2 cm was dissected in the lower eyelid. This generates a full-thickness eyelid flap similar to that used to repair large upper eyelid defects. Perfusion was measured using laser speckle contrast imaging, before and after the flap was rotated 90° and 120°, and stretched using forces of 0.5, 1, and 2 N. RESULTS: Blood perfusion decreased gradually from the base to the tip of the flap; being 75% of the reference value 0.5 cm from the base, 63% at 1.0 cm, 55% at 1.5, 23% at 1.75 cm, and 4% at 2.0 cm. Rotating the flaps by 90° or 120° had little effect on the perfusion. Stretching reduced the perfusion from 63% to 32% at 2 N, when measured at 1 cm. The combination of stretching and rotation did not lead to any further decrease. CONCLUSIONS: Blood perfusion in lower eyelid rotational flaps seems to be more sensitive to stretching than to rotation. Provided the flap is no longer than 1.5 cm, the perfusion will be greater than 20%, even when rotated, which should be sufficient for adequate survival and healing.

Revascularization of Free Skin Grafts Overlying Modified Hughes Tarsoconjunctival Flaps Monitored Using Laser-Based Techniques.

PURPOSE: It has recently been shown that the flap pedicle does not supply blood to a tarsoconjunctival graft in the modified Hughes procedure in patients. This raises questions concerning the rate of revascularization of the free skin graft commonly used to reconstruct the anterior lamella. The aim of this study was, thus, to monitor the course of revascularization in free skin grafts overlying modified Hughes tarsoconjunctival flaps, using laser-based techniques. METHODS: Free skin grafts from the upper eyelid or upper arm in 9 patients were used to cover a tarsoconjunctival flap according to the modified Hughes procedure. Blood perfusion was monitored using laser speckle contrast imaging, and vascular reactivity was studied with laser Doppler velocimetry after heating the tissue to 44°C. Measurements were made at the time of surgery (baseline) and at 1, 3, 8, and 16 weeks postoperatively. RESULTS: The gradual increase in perfusion of the free skin grafts during the healing process indicates revascularization. A slight increase in perfusion was seen already after 1 week. Perfusion reached 50% of the baseline after 3 weeks, and complete restoration of perfusion was seen after 8 weeks. The vascular function monitored with heat-induced hyperemia increased in a similar fashion. CONCLUSIONS: Full-thickness skin grafts revascularize within 3 to 8 weeks, despite overlying a tarsoconjunctival flap, which has recently been reported to be avascular. This provides further evidence that it should be possible to repair large eyelid defects using free full-thickness eyelid grafts.

Perfusion Monitoring Shows Minimal Blood Flow From the Flap Pedicle to the Tarsoconjunctival Flap.

BACKGROUND: A previous study in pigs has shown that the pedicle of the tarsoconjunctival flap does not appear to have adequate blood perfusion. The aim of this study was to monitor perfusion in tarsoconjunctival flaps in patients with large lower eyelid defects resulting from tumor surgery. METHODS: The modified Hughes procedure was performed in 13 patients. Blood perfusion was monitored using laser Doppler velocimetry and laser speckle-contrast imaging. RESULTS: Blood flow decreased gradually from the pedicle base to the end of the flap and was 19% at the flap base, 11% in the middle of the flap, and 4% in the distal end of the flap. The flaps survived, and there was no tissue necrosis. CONCLUSIONS: Tarsoconjunctival tissue survival does not seem to be dependent on a conjunctival flap. Free tarsoconjunctival grafts or composite grafts might be considered as viable alternatives in reconstruction of major eyelid defects.

Optimal Epinephrine Concentration and Time Delay to Minimize Perfusion in Eyelid Surgery: Measured by Laser-Based Methods and a Novel Form of Extended-Wavelength Diffuse Reflectance Spectroscopy.

OBJECTIVE: This study investigates the hypoperfusion effects of epinephrine in local anesthesia in eyelid surgery. A novel form of extended-wavelength diffuse reflectance spectroscopy was evaluated. METHODS: Blood perfusion in porcine eyelid flaps was measured using laser Doppler velocimetry and laser speckle contrast imaging, whereas the tissue response was measured using diffuse reflectance spectroscopy with a broad spectrum (450-1550 nm). Epinephrine was either injected cumulatively, 0.1 (1:10,000,000), 1.0 (1:1,000,000), 10 (1:100 000), and 100 µg/ml (1:10 000), to determine the dose-response relation, or given as a single dose (10 µg/ml). Control experiments were performed with saline or lidocaine. RESULTS: Increasing concentrations of epinephrine resulted in a gradual decrease in tissue perfusion, measured by laser Doppler velocimetry and laser speckle contrast imaging, approaching a minimum after the injection of 10 µg/ml. Similar tissue response was observed with diffuse reflectance spectroscopy. The time from the injection of epinephrine (10 µg/ml) to the stabilization of hypoperfusion was 75 seconds. After administration of 10 µg/ml epinephrine, about 20% of the blood perfusion remained, supporting the use of epinephrine in eyelid flaps with a narrow pedicle. CONCLUSIONS: 10 µg/ml epinephrine appears to be adequate for vasoconstriction before oculoplastic surgery. Incisions need only be delayed for about 1 minute. Extended-wavelength diffuse reflectance spectroscopy appears to be a promising technique for monitoring the tissue response following changes in blood perfusion in plastic surgery reconstructions. However, more rigorous validation of the technique is required before it can be implemented in clinical practice.

Blood Perfusion in Human Eyelid Skin Flaps Examined by Laser Speckle Contrast Imaging-Importance of Flap Length and the Use of Diathermy.

PURPOSE: It is well known that blood perfusion is important for the survival of skin flaps. As no study has been conducted to investigate how the blood perfusion in human eyelid skin flaps is affected by the flap length and diathermy, the present study was carried out to investigate these in patients. METHODS: Fifteen upper eyelids were dissected as part of a blepharoplastic procedure, releasing a 30-mm long piece of skin, while allowing the 5 mm wide distal part of the skin to remain attached, to mimic a skin flap (hereafter called a "skin flap"). Blood perfusion was measured before and after repeated diathermy, using laser speckle contrast imaging. RESULTS: Blood perfusion decreased from the base to the tip of the flap: 5 mm from the base, the perfusion was 69%, at 10 mm it was 40%, at 15 mm it was 20%, and at 20 mm it was only 13% of baseline values. Diathermy further decreased blood perfusion (measured 15 mm from the base) to 13% after applying diathermy for the first time, to 6% after the second and to 4% after the third applications of diathermy. CONCLUSIONS: Blood perfusion falls rapidly with distance from the base of skin flaps on the human eyelid, and diathermy reduces blood perfusion even further. Clinically, it may be advised that flaps with a width of 5 mm be no longer than 15 mm (i.e., a width:length ratio of 1:3), and that the use of diathermy should be carefully considered.

Detailed measurements of the four extraocular rectus muscles' contribution to the perfusion of the anterior segment of the eye.

BACKGROUND/AIMS: Anterior segment ischaemia (ASI) is a rare but serious complication of strabismus surgery, which may be caused by damage to the anterior ciliary arteries that run along the rectus muscles. To avoid ASI, clinical praxis is to operate on a maximum of two rectus muscles at a time. The aim of this study was to perform a detailed study of the contribution of the four ocular rectus muscles to the perfusion of the anterior segment using laser speckle contrast imaging (LSCI). METHODS: The four rectus muscles were successively detached during enucleation in nine patients with suspected uveal melanoma. Perfusion of the anterior segment was monitored with LSCI during the detachment of each of the rectus muscles, and after the ophthalmic artery had been severed. RESULTS: Perfusion of the anterior segment, measured in the paralimbal tissue, decreased gradually as the four rectus muscles were detached, showing a decrease to 98% when the first (lateral rectus) muscle was detached, to 88% when the second (medial rectus), and 69% when the third (inferior rectus) muscles were detached. The decrease was more pronounced and statistically significant when the fourth (superior rectus) muscle was detached (to 28%, p=0.0102). CONCLUSION: LSCI is a valuable perfusion monitoring tool, as it has the capability to visualise the gradual decrease in anterior segment perfusion as the rectus muscles are successively detached. Further studies are needed to determine how many rectus muscles can be safely detached during strabismus surgery without risking ASI.

Laser speckle contrast imaging enables perfusion monitoring of the anterior segment during strabismus surgery: a study on the horizontal rectus muscles.

BACKGROUND: A dreaded complication of strabismus surgery is anterior segment ischaemia (ASI), caused by damage to the anterior ciliary arteries. To avoid ASI, a maximum of two rectus muscles are operated on at a time. However, these surgical protocols are based on empirical observations of clinical outcome, rather than objective perfusion measurements. There is no method available for perioperative, real-time perfusion measurements during ocular muscle surgery. The aims of this study were to investigate whether laser speckle contrast imaging (LSCI) could be used for such measurements, and to monitor perfusion during strabismus surgery on one or two horizontal rectus muscles. METHODS: Forty-four eyes in 44 patients with horizontal strabismus underwent corrective surgery involving medial and/or lateral rectus muscle detachment. Perfusion in the adjacent paralimbal and iris tissue was monitored with LSCI. RESULTS: When the first horizontal rectus muscle was detached perfusion in the adjacent paralimbal tissue decreased by 23% (p<0.0001), and by 12% (p<0.0001) when the second muscle was detached. The iris perfusion decreased by 5% (p<0.05) when the first muscle was detached but showed no significant decrease as the second muscle was cut. CONCLUSION: This is the first study showing that perfusion of the anterior segment can be monitored non-invasively with LSCI during strabismus surgery. In this cohort, two horizontal rectus muscles were detached with only a small decrease in the anterior segment circulation. Future studies are required for complete mapping of the effect of surgery on multiple ocular muscles on the anterior segment circulation.

Laser speckle contrast imaging enables perfusion monitoring of the anterior segment during eye muscle surgery.

We demonstrate that laser speckle contrast imaging can be used to monitor blood perfusion noninvasively during the detachment of ocular muscles, which may be a valuable tool for reducing the risk of anterior segment ischemia as a complication of strabismus surgery.

Mapping the architecture of the temporal artery with photoacoustic imaging for diagnosing giant cell arteritis.

Photoacoustic (PA) imaging is rapidly emerging as a promising clinical diagnostic tool. One of the main applications of PA imaging is to image vascular networks in humans. This relies on the signal obtained from oxygenated and deoxygenated hemoglobin, which limits imaging of the vessel wall itself. Giant cell arteritis (GCA) is a treatable, but potentially sight- and life-threatening disease, in which the artery wall is infiltrated by leukocytes. Early intervention can prevent complications making prompt diagnosis of importance. Temporal artery biopsy is the gold standard for diagnosing GCA. We present an approach to imaging the temporal artery using multispectral PA imaging. Employing minimally supervised spectral analysis, we produce histology-like images where the artery wall is clearly discernible from the lumen and further differentiate between PA spectra from biopsies diagnosed as GCA- and GCA+ in 77 patients.

Photoacoustic imaging of periorbital skin cancer ex vivo: unique spectral signatures of malignant melanoma, basal, and squamous cell carcinoma.

Radical excision of periorbital skin tumors is difficult without sacrificing excessive healthy tissue. Photoacoustic (PA) imaging is an emerging non-invasive biomedical imagi--ng modality that has potential for intraoperative micrographic control of surgical margins. This is the first study to assess the feasibility of PA imaging for the detection of periocular skin cancer. Eleven patients underwent surgical excision of periocular skin cancer, one of which was a malignant melanoma (MM), eight were basal cell carcinomas (BCCs), and two squamous cell carcinomas (SCCs). Six tumors were located in the eyelid, and five in periocular skin. The excised samples, as well as healthy eyelid samples, were scanned with PA imaging postoperatively, using 59 wavelengths in the range 680-970 nm, to generate 3D multispectral images. Spectral unmixing was performed using endmember spectra for oxygenated and deoxygenated Hb, melanin, and collagen, to iden--tify the chromophore composition of tumors and healthy eyelid tissue. After PA scanning, the tumor samples were examined histopathologically using standard hematoxylin and eosin staining. The PA spectra of healthy eyelid tissue were dominated by melanin in the skin, oxygenated and deoxygenated hemoglobin in the orbicularis oculi muscle, and collagen in the tarsal plate. Multiwavelength 3D scanning provided spectral information on the three tumor types. The spectrum from the MM was primarily reconstructed by the endmember melanin, while the SCCs showed contributions primarily from melanin, but also HbR and collagen. BCCs showed contributions from all four endmembers with a predominance of HbO2 and HbR. PA imaging may be used to distinguish different kinds of periocular skin tumors, paving the way for future intraoperative micrographic control.

Regional motion correction for in vivo photoacoustic imaging in humans using interleaved ultrasound images.

In translation from preclinical to clinical studies using photoacoustic imaging, motion artifacts represent a major issue. In this study the feasibility of an in-house algorithm, referred to as intensity phase tracking (IPT), for regional motion correction of in vivo human photoacoustic (PA) images was demonstrated. The algorithm converts intensity to phase-information and performs 2D phase-tracking on interleaved ultrasound images. The radial artery in eight healthy volunteers was imaged using an ultra-high frequency photoacoustic system. PA images were motion corrected and evaluated based on PA image similarities. Both controlled measurements using a computerized stepping motor and free-hand measurements were evaluated. The results of the controlled measurements show that the tracking corresponded to 97 ± 6% of the actual movement. Overall, the mean square error between PA images decreased by 52 ± 15% and by 43 ± 19% when correcting for controlled- and free-hand induced motions, respectively. The results show that the proposed algorithm could be used for motion correction in photoacoustic imaging in humans.

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

PURPOSE: 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. METHODS: Photoacoustic scanning of the temporal artery was performed in 12 healthy subjects using 59 wavelengths (from 680 nm to 970 nm). Visual function was tested before and after the examination. The subjects' experience of the examination was rated on a 0-100 VAS scale. Two- and three-dimensional PA images were generated from the spectra obtained from the artery. RESULTS: Photoacoustic imaging did not affect the best corrected visual acuity, colour vision (tested with Sahlgren's Saturation Test or the Ishihara colour vision test) or the visual field. The level of discomfort was low, and only little heat and light sensation were reported. The spectral signature of the artery wall could be clearly differentiated from those of the subcutaneous tissue and skin. Spectral unmixing provided visualization of the chromophore distribution and overall architecture of the artery. CONCLUSIONS: Photoacoustic imaging of the temporal artery is well tolerated and can be performed without any risk to visual function, including the function of the retina and the optic nerve. The spectral signature of the temporal artery is specific, which is promising for future method development.

Comparison of photoacoustic imaging and histopathological examination in determining the dimensions of 52 human melanomas and nevi ex vivo.

Surgical excision followed by histopathological examination is the gold standard for the diagnosis and staging of melanoma. Reoperations and unnecessary removal of healthy tissue could be reduced if non-invasive imaging techniques were available for presurgical tumor delineation. However, no technique has gained widespread clinical use to date due to shallow imaging depth or the absence of functional imaging capability. Photoacoustic (PA) imaging is a novel technology that combines the strengths of optical and ultrasound imaging to reveal the molecular composition of tissue at high resolution. Encouraging results have been obtained from previous animal and human studies on melanoma, but there is still a lack of clinical data. This is the largest study of its kind to date, including 52 melanomas and nevi. 3D multiwavelength PA scanning was performed ex vivo, using 59 excitation wavelengths from 680 nm to 970 nm. Spectral unmixing over this broad wavelength range, accounting for the absorption of several tissue chromophores, provided excellent contrast between healthy tissue and tumor. Combining the results of spectral analysis with spatially resolved information provided a map of the tumor borders in greater detail than previously reported. The tumor dimensions determined with PA imaging were strongly correlated with those determined by histopathological examination for both melanomas and nevi.