In vivo photoacoustic lifetime imaging of tumor hypoxia in small animals

Abstract

Tumor hypoxia is an important factor in assessment of both cancer progression and cancer treatment efficacy. This has driven a substantial effort toward development of imaging modalities that can directly measure oxygen distribution and therefore hypoxia in tissue. Although several approaches to measure hypoxia exist, direct measurement of tissue oxygen through an imaging approach is still an unmet need. To address this, we present a new approach based on in vivo application of photoacoustic lifetime imaging (PALI) to map the distribution of oxygen partial pressure (pO2) in tissue. This method utilizes methylene blue, a dye widely used in clinical applications, as an oxygen-sensitive imaging agent. PALI measurement of oxygen relies upon pO2-dependent excitation lifetime of the dye. A multimodal imaging system was designed and built to achieve ultrasound (US), photoacoustic, and PALI imaging within the same system. Nude mice bearing LNCaP xenograft hindlimb tumors were used as the target tissue. Hypoxic regions were identified within the tumor in a combined US/PALI image. Finally, the statistical distributions of pO2 in tumor, normal, and control tissues were compared with measurements by a needle-mounted oxygen probe. A statistically significant drop in mean pO2 was consistently detected by both methods in tumors.

Fig. 1

Schematic of in vivo multimodal imaging system. The system was capable of generating ultrasound (US), photoacoustic imaging (PAI), and photoacoustic lifetime imaging (PALI) images using the same hardware. The animal was illuminated by two laser systems triggered by an FPGA module. Both pulse-echo US and PA signals were acquired by a conventional phased-array US transducer and then amplified, digitized, and stored in the US system. Data were transferred to a PC for image processing and display.

Fig. 2

Experimental setup and multimodal imaging results of a two-tube phantom experiment. (a) The phantom consists of two plastic tubes containing methylene blue (MB) aqueous solution at high ${\mathrm{pO}}_2$ (150 mmHg, right) and low ${\mathrm{pO}}_2$ (50 mmHg, left). Both tubes were illuminated by the excitation beam (650-nm laser) and probe beam (810-nm laser). (b) Photo of the phantom and the US transducer. UPAT, ultrasound phased-array transducer. (c) Transient PA image of the two tubes by the 810-nm laser at a pump–probe delay of 0.5 μs displayed in a linear scale. The two clusters of PA signals represent the location of the wall–dye interface in the two tubes. The phantom was illuminated by two lasers from the left, resulting in higher PA amplitude in the left tube. (d) PALI image (color) superposed on US (gray scale) image. Inner and outer walls of both tubes are indicated by dashed lines.

Fig. 3

In vivo imaging platform for small animals. The nude mouse was fixed within a water tank with its head above the water level. Two laser beams overlapped on the tumor-bearing hindlimb, while the US transducer was positioned next to the illuminated spot and aligned for multimodal imaging. TBM, tumor-bearing mouse; UPAT, ultrasound phased-array transducer. Red dashed line, the 650-nm laser beam; green dotted line, the 810-nm laser beam.

Fig. 4

Multimodal imaging of the tumor-bearing mice. (a) US image of the left tumor-bearing hindlimb of a mouse. The area of the tumor is enclosed by a red dashed line. (b) PAI representing the amplitude of background absorption at 810 nm. Amplitude is displayed in linear scale. (c) PAI of the transient absorption of MB with an 810-nm laser at a pump–probe delay of 0.25 μs. (d) PALI of ${\mathrm{pO}}_2$ in color scale superimposed on US image. (e) Transient PA amplitudes of two representative pixels within the tumor and in normal control tissue, respectively. The triangles and circles are the averaged transient PA amplitudes of tumor and normal control tissue, respectively. The error bars represent the standard deviation of 100 recordings. Both the sets of data were fitted with an exponential curve as shown by the dashed line. (f) Animal after imaging procedure. An arrow indicates the MB stained imaging region. (g) Open-skin view of the tumor site. Note that the tissue was still stained with MB after the imaging process, thereby confirming stable MB staining of the tissue for a period of $>1\mathrm{h}$. The red arrow indicates the site of the tumor.

Fig. 5

Histogram of ${\mathrm{pO}}_2$ in tumor tissue, normal tissue, and control mice. (a) Top row: histogram of ${\mathrm{pO}}_2$ values extracted from PALI. Bottom row: histogram of ${\mathrm{pO}}_2$ values measured by the oxygen probe. Data from tumor tissue, normal tissue (adjacent to tumor site), and control tissue (no tumor present) are displayed in left, middle, and right columns, respectively. (b) PALI showing the regions of tumor (red dashed line) and normal (yellow dashed line) sites in a tumor-bearing hindlimb of a mouse. (c) PALI showing the control tissue of a tumor-free hindlimb. The image does not show a substantial area of low ${\mathrm{pO}}_2$ as compared to Fig. 4(d).