3.1.1.

Palm

A small section of the lower palm at the base of the thumb of a volunteer was imaged at a laser wavelength of 584 nm. An area of 8×8 mm² was scanned. The resulting images, depicted in Fig. 2, are typical of the images obtained from the palms of other volunteers in other imaging experiments. Figure 2b is a cross-sectional or a B-scan image. The bright pixels (bright red, for color images) in this image indicate strong PA signals generated from the surface of the skin and blood vessels below it. From this image, the boundaries of the epidermis, the stratum corneum, and the epidermal-dermal junction are easily distinguishable. Approximately 200–300 μm below the surface of the palm is the epidermal–dermal junction [Fig. 2b]. Large concentrations of smaller blood vessels, namely capillaries in the dermal papillae, are found in this region. Below this depth are larger vessels, presumably of the subpapillary plexus. It is well known that acral skin, like that on the palm or sole of the foot, has a much thicker epidermal layer than skin elsewhere on the body. From the PA B-scan image, the epidermal thickness is approximated as 300 μm; this measurement is within the range of reported values from studies that used different measurement techniques. ^{15, 16} A maximum amplitude projection (MAP) of the 3-D image toward the skin surface is shown in Fig. 2c, and the cross-sectional slice shown in Fig. 2b is indicated. To make the vessels more clearly visible, the PA signal from and above the epidermal–dermal junction and the unresolved smaller papillary capillary vessels, were removed during construction of the MAP image. The image acquisition time for this image was relatively fast (~10 min); however, faster imaging times will reduce complications such as mechanical movement of the volunteer and enhance the spectroscopic capabilities necessary for functional measurements.

Fig. 2

(a) Photo of the palm of the volunteer. The red box indicates the imaged area 8 mm × 8 mm. (b) B-scan PA image taken along the dashed line in (c). Notable features including the stratum corneum, epidermal–dermal junction, and subpapillary blood vessels, are all labeled. Selected vessels have been labeled with color arrows for reference with the image in (c). (c) Maximum amplitude projection PA image taken from the palm using a 584 nm laser excitation wavelength. The green dashed line indicates the cross-section shown in (b). (Color online only.)

3.3.1.

Nevus

A common blue nevus in the forearm of a volunteer is shown in Fig. 4a. It was photoacoustically imaged and subsequently removed with a 3 mm punch biopsy, then histologically analyzed. Prior to excision, a 6 mm × 4 mm area encompassing the nevus was imaged with PAM at two different laser excitation wavelengths, 570 and 700 nm. The first wavelength, 570 nm, was chosen because it is an isosbestic wavelength for HbO₂ and HbR, and the signal at that wavelength can be used as a measure of total hemoglobin. At the longer excitation wavelength, 700 nm, the absorption of melanin, HbR, and HbO₂ is significantly weaker than at 570 nm, which enables deeper penetration of the laser light into the nevus. In the PA image acquired at 570 nm laser excitation wavelength, the surrounding microvasculature is clearly displayed, along with the nevus, in the MAP image [Fig. 4c]. In contrast, only the nevus and unresolved signal from some capillary vessels in the epidermal–dermal junction are visible in the PA images acquired at 700 nm [Fig. 4d]. The B-scan in Fig. 4b is a representative cross-section (along the scanning direction) of the nevus imaged with 570 nm light and shows the depth of the nevus and surrounding vessels.

Fig. 4

(a) Photo of a nevus located on the forearm of the volunteer. (b) B-scan PA image taken along the blue dashed line in (c). Notable features including the nevus, epidermal–dermal junction, and subpapillary blood vessels, are all labeled. (c) MAP image of a nevus acquired using 570 nm laser illumination. The nevus is clearly shown in “green scale,” and blood vessels are shown in red. The blue dashed line indicates the cross-section shown in (b) and the yellow dashed line indicates the cross-section depicted in Fig. 5c. (d) MAP image of a nevus acquired with a 700 nm laser excitation wavelength. The yellow line indicates the cross-section shown in Fig. 5d. (Color online only.)

Several measurements can be derived from the PA images that cannot be obtained with dermoscopy or photography, including the lesion's depth, thickness, and volume. To validate these measures, sections of the nevus were histologically analyzed. For comparison, it was necessary to select the correct “slice” of the 3-D data that corresponded to the same approximate section of the nevus in the histology section. To match histology with the PA data, the nevus was manually marked in photos from serial histology sections. The spacing between histology sections varied between 50 and 80 μm. A montage was made of the labeled photos and a MAP image of the nevus was formed from the histology data as shown in Fig. 5a. Based on the shape of the nevus and other key features, namely a prominent hair follicle disrupting the nevus at the edge, the histology and PA MAP images were co-registered, and “slices” of the PA data that most closely matched with the corresponding histology sample were selected [Figs. 5b, 5c, 5d]. Depth and thickness measurements were taken from the same position in the PA and histology data. PA data collected using 570 nm laser excitation light yielded the following measurements: 270 μm nevus thickness [Fig. 5e], 140 μm depth from the surface to the top of the nevus (410 μm total depth from the surface) [Fig. 5e], and a 2.66 mm width. The PA data collected with 700 nm laser light produced similar results: 262 μm nevus thickness [Fig. 5f], 135 μm depth from the surface to the top of the nevus (397 μm total depth from the surface) [Fig. 5f], and a 2.64 mm width. The edges of the nevus were defined as the points where the signal first exceeded two standard deviations from the average noise value. In both depth and lateral dimensions, differences in spatial measurements from both wavelengths were within the resolution of the system (15 μm axial and 45 μm lateral resolutions). These results are consistent, at least qualitatively, with the histology, which shows the nevus to be approximately 450 μm thick and located 150 μm deep below the surface (600 μm total depth from the surface), with a width of 2.18 mm. The measurements from the PA data are justifiably different from histology because of tissue distortion during excision, formalin fixation, and dehydration/paraffin embedding that result in tissue shrinkage artifact. We thus anticipated that the histological measurement of lesion width would be less. Taking into account that the histology section was generated from a 3 mm punch biopsy, the width of the nevus could be scaled to the entire tissue slice, yielding a more accurate measurement. From this calibration procedure, the width of the nevus was determined to be 2.7 mm, which agrees well with PA measurements. The smaller PAM values for depth (thickness and depth) compared to histology measurements are also to be expected from distortion of the tissue along the depth direction from excision, which relaxes the lateral tension of the removed tissue and causes the tissue to collapse in the lateral directions and elongate in the depth direction.

Fig. 5

(a) Maximum amplitude projection of a nevus generated from serial histology sections (top view, i.e., en face view, of the nevus) used to match histology with PA data. The nevus is shown in red, and a large hair follicle near the top was used to orient the image with the PA MAP image. (b) Histology section along the dashed line in (a), in which the location of the nevus thickness is indicated by the dashed line and two hash marks. A blood vessel used for orientation is circled in green. [(c) and (d)] Cross-sectional PA images of the nevus in the approximate region as (b) and are marked in Figs. 4b and 4c. (c) Image acquired with 570 nm laser illumination. The blood vessel circled in green matches with the vessels outlined in (b). (d) Image acquired using 700 nm laser illumination. The red dashed lines in both images (c) and (d) indicate the depth profiles shown in (e) and (f). Depth profile plots of the nevus are indicated by the dashed line in the cross-sectional images in (c) and (d). (e) Profile plot from the image acquired with a 570 nm laser excitation wavelength shows the nevus thickness to be 270 μm. (f) Profile plot from the image acquired with a 700 nm laser excitation wavelength shows the nevus thickness to be 262 μm. (Color online only.)

A major concern with PA data pertains to the accuracy of the measured thickness of the lesion. From a single PA measurement, it is not necessarily guaranteed that the entire thickness of the lesion has been recorded because the PA data will not inherently indicate if light has fully penetrated the lesion. However, by imaging with two different wavelengths, it is possible to confirm that the entire thickness of the lesion was accurately recorded, if the measurements at both wavelengths are consistent. The primary absorber in a pigmented lesion is melanin and the optical absorption of melanin decays exponentially with increasing optical wavelength. Thus if one wavelength is sufficiently longer, the absorption coefficient of melanin can be an order of magnitude (or more) lower at that wavelength, which results in much deeper light penetration. The PA signal is proportional to the product of the absorption coefficient, μ_a, and the local laser fluence, which can be assumed to decay exponentially with μ_a inside a specific target (i.e., nevus, blood vessel, etc.). For example, using two wavelengths, λ₁ and λ₂, if μ_a ^λ1 = 5 μ_a ^λ2, at a depth of d = 1/μ_a ^λ1 into the target, the PA signal from λ₁ will be approximately 11 times greater than the PA signal from λ₂, or PA(d)^λ1 ~ 11PA(d)^λ2. So, if the absorption coefficient is significantly different and the initial laser fluences are roughly equivalent at the two wavelengths, similar thickness measurements will confirm that light has fully penetrated the lesion at both wavelengths and both measurements are accurate. If the thickness measurements are different, it is guaranteed that the shorter measure is inaccurate and it remains unknown if the larger value is correct or not. However, this scenario does not represent a fundamental limitation of photoacoustic imaging because utilizing longer excitation wavelengths and/or increasing the illumination intensity will enable an accurate lesion thickness measurement. The data presented here show similar thickness values using both 570 and 700 nm laser light, which validates the accuracy of the measurement. Furthermore, a blood vessel is clearly evident directly below the nevus in the PA image collected with a 570 nm laser wavelength (1). The presence and detection of this blood vessel confirms that a sufficient number of photons penetrated the nevus, enough to generate the PA signal from the blood vessel. Consequently, if the nevus extended to the depth of the blood vessel seen beneath it, then a detectable PA signal should have been generated from the nevus itself at this depth. The absence of a PA signal from the nevus at the depth of this vessel suggests that the lower boundary of the nevus was detected.

Video 1

In this video, there are two figures. The upper figure is the MAP image of the nevus and surrounding vasculature taken from the forearm of the volunteer. The lower figure shows the consecutively acquired B-scan images. The position of each B-scan image or cross-section in relation to the MAP image is indicated by the red bar on the MAP image. (QuickTime, 1 MB)1

With better characterization and analysis of pigmented lesions, there is great potential to improve the diagnosis and assessment of clinically atypical nevi and melanoma. Using clinical criteria that include asymmetry, border irregularity, color variation, diameter, and evolution, early melanomas that do not exhibit these criteria are missed. Conversely, many benign lesions (atypical nevi) may be needlessly biopsied. The use of dermoscopy has aided clinical assessment; however, this tool is useful only in the evaluation of surface characteristics and is not effectively employed by all clinicians. With PAM, additional characteristics of pigmented lesions may be noninvasively evaluated, including vasculature, lesion architecture, and 3-D symmetry. In suspected melanoma, measurements including lesion thickness and tumor volume may also be assessed in vivo. Once multiple pigmented lesions have been imaged, analyzed, and compared with histologic diagnoses, we anticipate that algorithms will be devised using PAM parameters to aid in the diagnosis of melanoma. This work is currently ongoing. Such information could be helpful not only in diagnosis of melanoma, but potentially in planning definitive surgery.

In conclusion, PAM has been used to collect in vivo images of the microvasculature and a pigmented lesion. Images of the microvasculature clearly show the structure of the vessels and the differences between vascular networks in acral and nonacral skin, and they demonstrate potential for numerous functional measurements. A benign nevus was photoacoustically imaged, biopsied, and histologically analyzed. Thickness and depth measurements of the nevus were consistent in both PA images, acquired using different laser wavelengths, and agreed well with the histology measurements, including the effect of tissue distortion during sample preparation. These results exhibit the capabilities and potential of PAM in dermatological applications.

Patients: All of the experiments were conducted in accordance with the human studies protocols approved by the Institutional Review Board at Washington University in St. Louis. The Declaration of Helsinki protocols were followed and all volunteers freely submitted their written informed consent.