Photoacoustic Microscopy Unlocks Secrets of Optical Absorption

In vivo, label-free subwavelength-resolution photoacoustic microscopy enables more precise measurement of optical absorption – and, therefore, offers more information.

Chi Zhang, Konstantin Maslov and Lihong V. Wang, Washington University, St. Louis

Mainstream optical microscopy technologies cannot sensitively measure the optical absorption that provides essential physiological information such as the concentration and oxygen saturation of hemoglobin.

In our research, we have implemented subwavelength-resolution photoacoustic microscopy (SW-PAM) to provide high-optical-absorption contrast by using a water-immersion optical objective with a 1.23 NA. SW-PAM can detect nonfluorescent endogenous pigments in vivo with 100 percent sensitivity to optical absorption.

We found that, by imaging gold nanospheres, SW-PAM approached the ultimate diffraction-limited optical resolution: 220 nm at a 532-nm wavelength. We imaged ex vivo cells, including melanoma, red blood and onion epidermal, using SW-PAM with subcellular organelles resolved. We also imaged in vivo vasculature and early-stage melanoma with 12:1 and 17:1 contrasts, respectively, without labeling. For all of these applications, SW-PAM showed contrasts orders of magnitude higher than those achieved with wide-field optical microscopy, ensuring the technique as a welcome complement to existing microscopy technologies.

Photoacoustic tomography (PAT)¹ is an emerging biomedical imaging technology that ultrasonically detects optical absorption in biological tissues. Because of the photoacoustic effect,² absorbed light is converted to heat and then induces acoustic waves. PAT can work in both the optical ballistic regime (<1 mm deep) and in the diffusive regime (up to several centimeters deep), with the same optical absorption contrast yet scalable resolution and depth.³ So far, PAT has been successfully applied to in vivo structural, functional and molecular imaging of biological tissues.^4-9

When light is focused into biological tissue and the resulting excited photo-acoustic signals are received by a focused ultrasonic transducer, the localized optical absorption within the optical or acoustic focal spot – whichever is smaller – is acquired. A high-resolution image of the tissue then can be obtained after point-by-point scanning of the tissue. This approach is called photoacoustic microscopy (PAM).

Although mainstream optical microscopy detects scattering (including confocal microscopy and optical coherence tomography) or fluorescence (including confocal and two-photon microscopy), PAM is sensitive to endogenous optical absorption with a relative sensitivity of 100 percent,^10,11 providing essential biological functional information. Because nonfluorescent hemoglobin and melanin are major sources of biological endogenous absorption in the visible and near-infrared spectral ranges, PAM finds broad application in imaging tumor angiogenesis, blood oxygen saturation and early-stage melanoma. Recently, by exciting DNA and RNA with ultraviolet light, PAM also imaged in vivo cell nuclei without labeling.¹²

In optical-resolution PAM, the image resolution is limited by the numerical aperture (NA) of the optical objective at the given wavelength. The first optical-resolution PAM system reached a resolution of 5 μm.¹³ In the past few years, the resolution has been improved for imaging of subcellular structures. Here we describe our subwavelength-resolution PAM, which has reached the finest diffraction-limited optical resolution.

Figure 1. Schematic diagram of subwavelength-resolution photoacoustic microscopy (SW-PAM). Reproduced with permission from Reference 15.

A system diagram of SW-PAM is shown in Figure 1.^14,15 We used a 532-nm Nd:YVO₄ laser as the irradiation source. A 1.5-ns laser pulse, focused by the 1.23-NA objective, irradiated the sample. An ultrasonic transducer with a central frequency of 40 MHz and an NA of 0.5 received the photoacoustic waves in transmission mode. The objective and the transducer mechanically scanned in raster mode in the X-Y plane to obtain a 3-D image, which will be shown as a two-dimensional maximum-amplitude projection image along the Z-axis.

We demonstrated the subwavelength resolution of SW-PAM by imaging the gold nanospheres (Figure 2a). The nanospheres had a diameter of 15 nm and, therefore, were ideal point absorbers to measure the point spread function of SW-PAM. The gray values of the typical spheres in the image were fitted by the theoretical Bessel-form function (Figure 2b). We measured the system transversal resolution as 220 ±20 nm, given by the full width half maximum of the Bessel-form point spread function. The theoretical resolution was calculated as 0.51 λ/NA ≈ 221 nm, in good agreement with the measured value.

Figure 2. Measuring the transverse spatial resolution of SW-PAM. (a) PAM image of gold nanospheres. (b) System point spread function calculated from the gold nanospheres. Circle: the averaged pixel value. Line: the theoretical Bessel-form function. Reproduced with permission from Reference 15.

In Figure 3, the subcellular structures are shown and the contrast of SW-PAM is compared with that of wide-field optical microscopy. In general, the PAM images have a dark background, while the optical microscopy images have a bright background. Melanosomes – organelles containing melanin – appear as bright dots (Figure 3a, left) in the PAM image, but as dark dots in the optical microscopy image (Figure 3a, middle).

The melanosomes in the PAM image have a contrast-to-background ratio of 54.5 ±0.4:1 and a contrast-to-noise ratio (CNR) of 49 dB, while the optical microscopy image has a much lower contrast of 0.79 ±0.04:1 and a lower CNR of 25 dB. This difference occurs because PAM is sensitive to absorption only, but optical microscopy shows both absorption and scattering (the latter is relatively close between melanosomes and other areas).

Figure 3. Ex vivo images of cells. (a) Melanoma cells imaged by PAM, optical microscopy and PAM combined with an optical microscopy image of the blue-stained nuclei. CN: cell nucleus. (b) PAM and optical microscopy images of red blood cells. (c) PAM and optical microscopy images of onion epidermal cells. Reproduced with permission from Reference 15.

In the PAM image, the dark holes inside the cells with few melanosomes are nuclei, confirmed by staining them with fluorescent dye (Figure 3a, right). In comparison, the nuclei in the optical microscopy image are less visible because of the low contrast. The hemoglobin contrast disparity also can be observed by imaging red blood cells, as shown in Figure 3b (the two images show different areas of the cell sample). The characteristic doughnut shape of a red blood cell is clearly shown. Moreover, rich contrasts are observed by PAM in the purple onion epidermal cells (Figure 3c).

We also imaged the melanin distribution in a black mouse ear (Harlan Laboratories Inc., Indianapolis, C57BL/6NHsd) in vivo with SW-PAM. We acquired two images, focusing at ~10 and ~30 μm deep, respectively, as shown in Figure 4. Most melanocytes reside in the basal layer of the epidermis, whose thickness is ~10 μm in this case.¹⁶ Therefore, single melanosomes can be clearly identified in the shallower layer (Figure 4a). In the deeper layer (Figure 4b), more skin structures, such as the sebaceous glands, are shown, but melanosomes are now out of focus.

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Figure 4. Melanin distribution in a black mouse ear at different depths. (a) ~10 μm deep (the close-up image indicates melanosomes). (b) ~30 μm deep. SG: sebaceous gland. Reproduced with permission from Reference 14.

It is worth noting that the signals from the deeper layer are weaker, but Figure 4b appears to have higher contrast because it is scaled to the full color range. The optical microscopy image of the ear, although not shown here, has extremely low contrast. These results suggest potential applications of PAM in quantifying melanin distribution in vivo, which is important for detecting melanoma as well as determining individualized radiant exposure in dermatological laser therapies.¹⁷

The ear of a nude mouse (Harlan Laboratories, Hsd:Athymic Nude-Foxn1^nu) also was imaged in vivo. Here, we used an objective with a 0.60 NA (providing 400-nm resolution) to extend the focal zone for the thick layer of vessels. As shown in Figure 5a, we imaged all the blood vessels, including capillaries, with high contrast and with few background signals. In some areas, motionless red blood cells can be identified with the characteristic doughnut shape (Figure 5b). Next, we injected 10 μl of suspension containing 1 million B16 melanoma cells into the ear to induce a melanoma tumor.

Figure 5. Monitoring melanoma growing on a nude mouse ear. (a) PAM image of the ear before injecting melanoma cells. (b) PAM image, where in vivo red blood cells (RBCs) can be identified (indicated in the close-up image). (c) PAM image of the ear four days after injecting melanoma cells. MT: melanoma tumor. (d) Optical Microscopy image, in which the melanoma is obscure. Reproduced with permission from Reference 14.

We imaged the same ear again, as shown in Figure 5c, four days later. The melanoma generated stronger photo-acoustic signals than the vessels and was easily identified. The vasculature and melanoma had contrasts of 12 ±1:1 (33-dB CNR) and 17 ±1:1 (36-dB CNR), respectively. The contrast difference between the vasculature and the melanoma can be further increased if the laser wavelength is changed to, for example, 650 nm. Under wide-field optical microscopy (Figure 5d), the melanoma is obscure, with a contrast of 0.27 ±0.02:1 (21-dB CNR).

As shown by Figures 2 to 5, SW-PAM can resolve structures as small as sub-cellular organelles, both ex vivo and in vivo. Melanoma and vasculature can be visualized with high contrast. Therefore, SW-PAM has superior potential to detect melanoma in the early stage, to count red blood cells as an in vivo flow cytometer and to measure blood flow velocity in capillaries.

Moreover, although the current transmission-mode configuration of SW-PAM limits the thickness of tissues that can be imaged, we are extending SW-PAM to reflection mode for applications in more anatomical sites. As a result, combining it with scaled-up macroscopy – deep-penetrating photoacoustic tomography^4-18 – may bridge microscopic research and clinical practice.

Meet the authors

Chi Zhang is a graduate student, Konstantin Maslov a research associate professor and Lihong V. Wang the Gene K. Beare Distinguished Professor at the Optical Imaging Laboratory in the Department of Biomedical Engineering at Washington University in St. Louis; e-mail: [email protected]. This work was sponsored in part by National Institutes of Health grants R01 EB000712, R01 EB008085, R01 CA113453901, U54 CA136398 and 5P60 DK02057933. Lihong Wang has a financial interest in Microphotoacoustics Inc. and Endra Inc. Neither supported this work.

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