A Coregistered Ultrasound and Photoacoustic Imaging Protocol for the Transvaginal Imaging of Ovarian Lesions

Summary

We report a coregistered ultrasound and photoacoustic imaging protocol for the transvaginal imaging of ovarian/adnexal lesions. The protocol may be valuable to other translational photoacoustic imaging studies, especially those using commercial ultrasound arrays for the detection of photoacoustic signals and standard delay-and-sum beamforming algorithms for imaging.

Abstract

Ovarian cancer remains the deadliest of all the gynecological malignancies due to the lack of reliable screening tools for early detection and diagnosis. Photoacoustic imaging or tomography (PAT) is an emerging imaging modality that can provide the total hemoglobin concentration (relative scale, rHbT) and blood oxygen saturation (%sO2) of ovarian/adnexal lesions, which are important parameters for cancer diagnosis. Combined with coregistered ultrasound (US), PAT has demonstrated great potential for detecting ovarian cancers and for accurately diagnosing ovarian lesions for effective risk assessment and the reduction of unnecessary surgeries of benign lesions. However, PAT imaging protocols in clinical applications, to our knowledge, largely vary among different studies. Here, we report a transvaginal ovarian cancer imaging protocol that can be beneficial to other clinical studies, especially those using commercial ultrasound arrays for the detection of photoacoustic signals and standard delay-and-sum beamforming algorithms for imaging.

Introduction

Photoacoustic imaging or tomography (PAT) is a hybrid imaging modality that measures the optical absorption distribution at US resolution and depths far beyond the tissue optical diffusion limit (~1 mm). In PAT, a nanosecond laser pulse is used to excite biological tissue, causing a transient temperature rise due to optical absorption. This leads to an initial pressure rise, and the resultant photoacoustic waves are measured by US transducers. Multispectral PAT involves the use of either a tunable laser or multiple lasers operating at different wavelengths to illuminate the tissue, thereby enabling the reconstruction of optical absorption maps at multiple wavelengths. Based on the differential absorption of oxygenated and deoxygenated hemoglobin in the near-infrared (NIR) window, multispectral PAT can compute the distributions of oxygenated and deoxygenated hemoglobin concentrations, the total hemoglobin concentration, and the blood oxygen saturation, which are all functional biomarkers related to tumor angiogenesis and blood oxygenation consumption or tumor metabolism. PAT has demonstrated success in many oncology applications, such as ovarian cancer^1,2, breast cancer^3,4,5, skin cancer⁶, thyroid cancer^7,8, cervical cancer⁹, prostate cancer^10,11, and colorectal cancer¹².

Ovarian cancer is the deadliest of all gynecological malignancies. Only 38% of ovarian cancers are diagnosed at an early (localized or regional) stage, where the 5 year survival rate is 74.2% to 93.1%. Most are diagnosed at a late stage, for which the 5 year survival rate is 30.8% or less¹³. Current clinical diagnosis methods, including transvaginal ultrasonography (TUS), Doppler US, serum cancer antigen 125 (CA 125), and human epididymis protein 4 (HE4), are shown to lack sensitivity and specificity for early ovarian cancer diagnosis^14,15,16. Additionally, a large portion of benign ovarian lesions may be difficult to diagnose accurately with current imaging technologies, which leads to unnecessary surgeries with increased healthcare costs and surgical complications. Thus, additional accurate non-invasive methods for the risk stratification of adnexal masses are needed to optimize the management and outcomes. Clearly, a technique that is sensitive and specific to early-stage ovarian cancer and more accurate in identifying malignant from benign lesions is needed.

Our group has developed a coregistered transvaginal US and PAT system (USPAT) for ovarian cancer diagnosis by combining a clinical US system, a custom-made probe sheath to house the optical fibers for light delivery, and a tunable laser¹. The total hemoglobin concentration (relative scale, rHbT) and the blood oxygen saturation (%sO2) derived from the USPAT system have demonstrated great potential for the detection of early-stage ovarian cancers and for accurately diagnosing ovarian lesions for effective risk assessment and the reduction of unnecessary benign lesion surgeries^1,2. The current system schematic is shown in Figure 1, and the control block diagram is shown in Figure 2. This strategy has the potential to be integrated into existing TUS protocols for ovarian cancer diagnosis while providing functional parameters (rHbT, %sO2) to improve the sensitivity and specificity of TUS.

Protocol

All the research performed was approved by the Washington University Institutional Review Board.

1. System configuration: Optical illumination (Figure 1)

1. Use an Nd:YAG laser pumping a pulsed, tunable (690-890 nm) Ti-sapphire laser at 10 Hz.
2. Expand the laser beam by first diverging the beam with a plano-concave lens and then collimating the beam with a plano-convex lens. Use two mirrors to direct the beam onto a beam splitter (described below).
3. Split the expanded laser beam into four beams with equal energy by splitting the original beam into two with a polarizing beam splitter and then splitting the two beams with two more second-stage beam splitters.
4. Mount four multi-mode optical fibers with fiber chucks.
5. Use four plano-convex lenses to focus the four laser beams into the four fibers.
6. Due to laser safety considerations, cover all the optical components under a metal box to ensure that the optical path is not exposed.
7. Attach the other ends of the four fibers to the transvaginal ultrasound probe, and enclose the probe and the fibers in a protective sheath.
   ​NOTE: The sheath and the acoustic window of the transducer are coated with highly reflective white paint to improve the illumination uniformity. This setup, including the use of four fibers for light delivery, was previously shown to be optimal for transvaginal applications¹⁷. See the discussion for more information.

2. System configuration: Ultrasonic detection and scanning scheme

1. Use a programmable clinical US system.
   NOTE: A programmable system means the raw ultrasound data is accessible, and custom data acquisition protocols and processing algorithms can be programmed.
2. Connect an additional monitor to the US system to run the USPAT display software for the real-time visualization of the rHbT, %sO2 maps, and other functional parameters.
3. Connect the internal trigger of the laser to the external trigger of the US system.
4. Use a time-division multiplexing approach during coregistered mode; specifically, for each wavelength, sequentially acquire five consecutive PAT frames and one coregistered US frame. Average the PAT frames to improve the signal-to-noise ratio. The total data acquisition time for four wavelengths is around 15 s.

3. System calibration

1. Set the laser pump energy to a fixed level.
2. For each wavelength (750 nm, 780 nm, 800 nm, and 830 nm), check the per pulse energy output at each fiber tip to make sure the calculated energy density at each selected wavelength is at the expected value given in Table 1.
3. If energy output is lower than expected, fine-tune the optical alignment by adjusting the mirror and beam splitter angles. This step is not always needed.
4. Repeat steps 3.2-3.4 until the energy is satisfactory.
5. Record the four fibers' energy output at each wavelength, and enter the values in the USPAT display software.
   ​NOTE: These values are used to calibrate the calculation of the rHbT. The laser energy fluctuates over time, and calibration ensures that the quantitative parameters computed from the multispectral PAT data are as accurate as possible.

4. A sample experimental procedure: Transvaginal USPAT imaging of the human ovary

1. Preparation of the USPAT imaging system
   1. Disinfect the endocavity US probe and the cover sheath with the standard ultrasound probe cleaning protocol at the institution.
   2. Turn on the clinical US system, start the US system software, and select the correct US transducer.
   3. Calibrate the laser system as in step 3.
   4. Enter the total pulse energy for each wavelength into the USPAT display software.
   5. Assemble the USPAT probe by enclosing the fibers and the probe inside the probe sheath.
2. Preparation of the patient
   1. Follow the institution-specific protocol to obtain informed consent and prepare the patient.
3. Imaging
   1. Locate the target ovary using pulse-echo US.
      NOTE: This step is done by the study physician, who is free to adjust the imaging parameters on the clinical US machine, such as the depth, the dynamic range, and the TGC.
   2. Select the desired depth in the USPAT control software.
   3. Click Scan in the control software to start the coregistered USPAT B-mode data acquisition. Watch the USPAT image display software to review the coregistered US and PAT B-mode images and reconstructed functional maps in real time.
   4. Repeat steps 4.3.1-4.3.3 to acquire more images and (if necessary) image the second lesion.

Results

Here, we show examples of malignant and normal ovarian lesions imaged by USPAT. Figure 3 shows a 50 year old premenopausal woman with bilateral multicystic adnexal masses revealed by contrast-enhanced CT. Figure 3A shows the US image of the left adnexa with the ROI marking the suspicious solid nodule inside the cystic lesion. Figure 3B shows the PAT rHbT map superimposed onto the US and shown in red. The rHbT showed extensive diffus...

Discussion

Optical illumination
The number of fibers used is based on two factors: light illumination uniformity and system complexity. It is critical to have a uniform light illumination pattern at the skin surface to avoid hot spots. It is also important to keep the system simple and robust with a minimal number of fibers. The use of four separate fibers has previously been shown to be optimal for creating uniform illumination at depths of several millimeters and beyond. Additionally, the light coupling to ...

Materials

Name	Company	Catalog Number	Comments
Clinical US imaging system	Alpinion Medical Systems	EC-12R	Fully programmable clinical US system
Dielectric mirror	Thorlabs	BB1-E03	Used to reflect light along the optical path
Endocavity US transducer	Alpinion Medical Systems	EC3-10	Transvaginal ultrasound probe
Laser power meter	Coherent	LabMax TOP	Used to measure laser energy
Multi-mode optical fiber	Thorlabs	FP1000ERT	Couple laser light to the endocavity ultrasound probe
Non-polarizing beam splitter plate	Thorlabs	BSW11	For splitting laser beam into sensors to measure energy
Plano-concave lens	Thorlabs	LC1715	For laser beam expansion
Plano-convex lens	Thorlabs	LA1484-B	For laser beam collimation
Plano-convex lens	Thorlabs	LA1433-B	Used to focus light into four optical fibers
Polarizing beam splitter cube	Thorlabs	PBS252	For splitting laser beam into four beams
Protective probe shealth	Custom 3D printed		Hold and protect the four optical fibers at the tip of the ultrasound probe
Right angle prism mirror	Thorlabs	MRA25-E03	Used to reflect light along the optical path
Tunable laser system	Symphotic TII	LS-2145-LT50PC	Light source for multispectral PAT
USPAT control software	Custom developed in C++		Controls acquisition parameters of the ultrasound machine and the laser wavelength
USPAT image display software	Custom developed in C++		Displays the US/PAT B-scans and sO2/rHbT maps in real time