This work was supported by the NCI (R01CA151570, R01CA237664). The authors thank the entire GYN oncology group led by Dr. Mathew Powell for helping with recruiting patients, radiologists Drs. Cary Siegel, William Middleton, and Malak Itnai for helping with the US studies, and the pathologist Dr. Ian Hagemann for helping with the pathology interpretation of the data. The authors gratefully acknowledge the efforts of Megan Luther and the GYN study coordinators in coordinating the study schedules, identifying patients for the study, and obtaining informed consent.

All the research performed was approved by the Washington University Institutional Review Board.
1. System configuration: Optical illumination (Figure 1)
<ol>
	<li>Use an Nd:YAG laser pumping a pulsed, tunable (690-890 nm) Ti-sapphire laser at 10 Hz.</li>
	<li>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).</li>
	<li>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.</li>
	<li>Mount four multi-mode optical fibers with fiber chucks.</li>
	<li>Use four plano-convex lenses to focus the four laser beams into the four fibers.</li>
	<li>Due to laser safety considerations, cover all the optical components under a metal box to ensure that the optical path is not exposed.</li>
	<li>Attach the other ends of the four fibers to the transvaginal ultrasound probe, and enclose the probe and the fibers in a protective sheath. 
	&#8203;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.</li>
</ol>
2. System configuration: Ultrasonic detection and scanning scheme
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>Connect the internal trigger of the laser to the external trigger of the US system.</li>
	<li>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.</li>
</ol>
3. System calibration
<ol>
	<li>Set the laser pump energy to a fixed level.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Repeat steps 3.2-3.4 until the energy is satisfactory.</li>
	<li>Record the four fibers&#39; energy output at each wavelength, and enter the values in the USPAT display software. 
	&#8203;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.</li>
</ol>
4. A sample experimental procedure: Transvaginal USPAT imaging of the human ovary
<ol>
	<li>Preparation of the USPAT imaging system
	<ol>
		<li>Disinfect the endocavity US probe and the cover sheath with the standard ultrasound probe cleaning protocol at the institution.</li>
		<li>Turn on the clinical US system, start the US system software, and select the correct US transducer.</li>
		<li>Calibrate the laser system as in step 3.</li>
		<li>Enter the total pulse energy for each wavelength into the USPAT display software.</li>
		<li>Assemble the USPAT probe by enclosing the fibers and the probe inside the probe sheath.</li>
	</ol>
	</li>
	<li>Preparation of the patient
	<ol>
		<li>Follow the institution-specific protocol to obtain informed consent and prepare the patient.</li>
	</ol>
	</li>
	<li>Imaging
	<ol>
		<li>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.</li>
		<li>Select the desired depth in the USPAT control software.</li>
		<li>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.</li>
		<li>Repeat steps 4.3.1-4.3.3 to acquire more images and (if necessary) image the second lesion.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Nandy, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+ovarian+cancer:+Initial+application+of+coregistered+photoacoustic+tomography+and+US.">Evaluation of ovarian cancer: Initial application of coregistered photoacoustic tomography and US.</a> Radiology. 289 (3), 740-747 (2018).</li><li>Amidi, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+blood+oxygenation+saturation+in+ovarian+cancer+diagnosis+using+multi-spectral+photoacoustic+tomography.">Role of blood oxygenation saturation in ovarian cancer diagnosis using multi-spectral photoacoustic tomography.</a> Journal of Biophotonics. 14 (4), 202000368 (2021).</li><li>Dogan, B. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optoacoustic+imaging+and+gray-scale+US+features+of+breast+cancers:+Correlation+with+molecular+subtypes.">Optoacoustic imaging and gray-scale US features of breast cancers: Correlation with molecular subtypes.</a> Radiology. 292 (3), 564-572 (2019).</li><li>Menezes, G. L. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Downgrading+of+breast+masses+suspicious+for+cancer+by+using+optoacoustic+breast+imaging.">Downgrading of breast masses suspicious for cancer by using optoacoustic breast imaging.</a> Radiology. 288 (2), 355-365 (2018).</li><li>Neuschler, E. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+pivotal+study+of+optoacoustic+imaging+to+diagnose+benign+and+malignant+breast+masses:+A+new+evaluation+tool+for+radiologists.">A pivotal study of optoacoustic imaging to diagnose benign and malignant breast masses: A new evaluation tool for radiologists.</a> Radiology. 287 (2), 398-412 (2018).</li><li>von Knorring, T., Mogensen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoacoustic+tomography+for+assessment+and+quantification+of+cutaneous+and+metastatic+malignant+melanoma+-+A+systematic+review.">Photoacoustic tomography for assessment and quantification of cutaneous and metastatic malignant melanoma - A systematic review.</a> Photodiagnosis and Photodynamic Therapy. 33, 102095 (2021).</li><li>Han, S., Lee, H., Kim, C., Kim, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review+on+multispectral+photoacoustic+analysis+of+cancer:+Thyroid+and+breast.">Review on multispectral photoacoustic analysis of cancer: Thyroid and breast.</a> Metabolites. 12 (5), 382 (2022).</li><li>Kim, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiparametric+photoacoustic+analysis+of+human+thyroid+cancers+in+vivo.">Multiparametric photoacoustic analysis of human thyroid cancers in vivo.</a> Cancer Research. 81 (18), 4849-4860 (2021).</li><li>Basij, M., Karpiouk, A., Winer, I., Emelianov, S., Mehrmohammadi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual-illumination+ultrasound/photoacoustic+system+for+cervical+cancer+imaging.">Dual-illumination ultrasound/photoacoustic system for cervical cancer imaging.</a> IEEE Photonics Journal. 13 (1), 6900310 (2021).</li><li>Agrawal, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=development,+and+multi-characterization+of+an+integrated+clinical+transrectal+ultrasound+and+photoacoustic+device+for+human+prostate+imaging.">development, and multi-characterization of an integrated clinical transrectal ultrasound and photoacoustic device for human prostate imaging.</a> Diagnostics. 10 (8), 566 (2020).</li><li>Kothapalli, S. -. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+transrectal+ultrasound+and+photoacoustic+human+prostate+imaging.">Simultaneous transrectal ultrasound and photoacoustic human prostate imaging.</a> Science Translational Medicine. 11 (507), 2169 (2019).</li><li>Leng, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+rectal+cancer+treatment+response+using+coregistered+endorectal+photoacoustic+and+US+imaging+paired+with+deep+learning.">Assessing rectal cancer treatment response using coregistered endorectal photoacoustic and US imaging paired with deep learning.</a> Radiology. 299 (2), 349-358 (2021).</li><li>Surveillance, Epidemiology, and End Results Program. Cancer of the Ovary - Cancer Stat Facts. National Cancer Institute Available from: <a target="_blank" href="https://seer.cancer.gov/statfacts/html/ovary.html">https://seer.cancer.gov/statfacts/html/ovary.html</a> (2022)</li><li>Temkin, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Outcomes+from+ovarian+cancer+screening+in+the+PLCO+trial:+Histologic+heterogeneity+impacts+detection,+overdiagnosis+and+survival.">Outcomes from ovarian cancer screening in the PLCO trial: Histologic heterogeneity impacts detection, overdiagnosis and survival.</a> European Journal of Cancer. 87, 182-188 (2017).</li><li>Kobayashi, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+randomized+study+of+screening+for+ovarian+cancer:+A+multicenter+study+in+Japan.">A randomized study of screening for ovarian cancer: A multicenter study in Japan.</a> International Journal of Gynecological Cancer. 18 (3), 414-420 (2008).</li><li>Andreotti, R. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=O-RADS+US+risk+stratification+and+management+system:+A+consensus+guideline+from+the+ACR+ovarian-adnexal+reporting+and+data+system+committee.">O-RADS US risk stratification and management system: A consensus guideline from the ACR ovarian-adnexal reporting and data system committee.</a> Radiology. 294 (1), 168-185 (2020).</li><li>Salehi, H. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+of+optimal+light+delivery+system+for+coregistered+transvaginal+ultrasound+and+photoacoustic+imaging+of+ovarian+tissue.">Design of optimal light delivery system for coregistered transvaginal ultrasound and photoacoustic imaging of ovarian tissue.</a> Photoacoustics. 3 (3), 114-122 (2015).</li><li>Oppenheim, A. V., Schafer, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Digital Signal Processing. , (1975).</li><li>Zou, Y., Amidi, E., Luo, H., Zhu, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasound-enhanced+Unet+model+for+quantitative+photoacoustic+tomography+of+ovarian+lesions.">Ultrasound-enhanced Unet model for quantitative photoacoustic tomography of ovarian lesions.</a> Photoacoustics. 28, 100420 (2022).</li><li>Prince, J. L., Links, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Medical Imaging Signals and Systems. , (2006).</li><li>Kim, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Programmable+Real-time+Clinical+Photoacoustic+and+Ultrasound+Imaging+System.">Programmable Real-time Clinical Photoacoustic and Ultrasound Imaging System.</a> Scientific Reports. 6, 35137 (2016).</li></ol>

The authors have no relevant financial interests in the manuscript and no other potential conflicts of interest to disclose.

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 ...

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 cancer1,2, breast cancer3,4,5, skin cancer6, thyroid cancer7,8, cervical cancer9, prostate cancer10,11, 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 diagnosis14,15,16. 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 surgeries1,2. 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.

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

a coregistered ultrasound and photoacoustic imaging protocol for the transvaginal imaging of ovarian lesions

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.

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...

Watch this Scientific Journal Video about A Coregistered Ultrasound and Photoacoustic Imaging Protocol for the Transvaginal Imaging of Ovarian Lesions at JoVE.com

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

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.

Ovarian cancer remains the deadliest of all the gynecological malignancies due to the lack of reliable screening tools for early detection and diagnosis. ...

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Root-Locus Method

SCIENTISTS IN ACTION

1.4K Views.  Washington University. Zoals we allemaal weten dat eierstokkanker de dodelijkste van alle gynaecologische maligniteiten is, wanneer eierstokkanker in een eerder en gelokaliseerd stadium wordt ontdekt, kunnen chirurgie en chemotherapie ongeveer 70 tot 90% van de patiënten genezen, vergeleken met slechts 20% of minder wanneer het in de latere stadia wordt gediagnosticeerd. En van de huidige standaardzorgmethoden is bekend dat ze een lagere gevoeligheid hebben voor vroege detectie van eierstokkanker. Bovendien hebben...

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

Integrated Photoacoustic Ophthalmoscopy and Spectral-domain Optical Coherence Tomography

Both the clinical diagnosis and fundamental investigation of major ocular diseases greatly benefit from various non-invasive ophthalmic imaging technologies. Existing retinal imaging modalities, such as fundus photography1, confocal scanning laser ophthalmoscopy (cSLO)2, and optical coherence tomography (OCT)3, have significant contributions in monitoring disease onsets and progressions, and developing new therapeutic strategies. However, they predominantly rely on the back-reflected photons from the retina. As a consequence, the optical absorption properties of the retina, which are usually strongly associated with retinal pathophysiology status, are inaccessible by the traditional imaging technologies.
Photoacoustic ophthalmoscopy (PAOM) is an emerging retinal imaging modality that permits the detection of the optical absorption contrasts in the eye with a high sensitivity4-7 . In PAOM nanosecond laser pulses are delivered through the pupil and scanned across the posterior eye to induce photoacoustic (PA) signals, which are detected by an unfocused ultrasonic transducer attached to the eyelid. Because of the strong optical absorption of hemoglobin and melanin, PAOM is capable of non-invasively imaging the retinal and choroidal vasculatures, and the retinal pigment epithelium (RPE) melanin at high contrasts 6,7. More importantly, based on the well-developed spectroscopic photoacoustic imaging5,8 , PAOM has the potential to map the hemoglobin oxygen saturation in retinal vessels, which can be critical in studying the physiology and pathology of several blinding diseases 9 such as diabetic retinopathy and neovascular age-related macular degeneration.
Moreover, being the only existing optical-absorption-based ophthalmic imaging modality, PAOM can be integrated with well-established clinical ophthalmic imaging techniques to achieve more comprehensive anatomic and functional evaluations of the eye based on multiple optical contrasts6,10 . In this work, we integrate PAOM and spectral-domain OCT (SD-OCT) for simultaneously in vivo retinal imaging of rat, where both optical absorption and scattering properties of the retina are revealed. The system configuration, system alignment and imaging acquisition are presented.

Photoacoustic ophthalmology (PAOM), an optical-absorption-based imaging modality, provides the complementary evaluation of the retina to the currently available ophthalmic imaging technologies. We report the using of PAOM integrated with spectral-domain optical coherence tomography (SD-OCT) for simultaneous multimodal retinal imaging in rats.

Both the clinical diagnosis and fundamental investigation of major ocular diseases greatly benefit from various non-invasive ophthalmic imaging ...

Bioengineering

Photoacoustic Cystography

Conventional pediatric cystography, which is based on diagnostic X-ray using a radio-opaque dye, suffers from the use of harmful ionizing radiation. The risk of bladder cancers in children due to radiation exposure is more significant than many other cancers. Here we demonstrate the feasibility of nonionizing and noninvasive photoacoustic (PA) imaging of urinary bladders, referred to as photoacoustic cystography (PAC), using near-infrared (NIR) optical absorbents (i.e. methylene blue, plasmonic gold nanostructures, or single walled carbon nanotubes) as an optical-turbid tracer. We have successfully imaged a rat bladder filled with the optical absorbing agents using a dark-field confocal PAC system. After transurethral injection of the contrast agents, the rat's bladders were photoacoustically visualized by achieving significant PA signal enhancement. The accumulation was validated by spectroscopic PA imaging. Further, by using only a laser pulse energy of less than 1 mJ/cm2 (1/20 of the safety limit), our current imaging system could map the methylene-blue-filled-rat-bladder at the depth of beyond 1 cm in biological tissues in vivo. Both in vivo and ex vivo PA imaging results validate that the contrast agents were naturally excreted via urination. Thus, there is no concern regarding long-term toxic agent accumulation, which will facilitate clinical translation.

Photoacoustic cystography (PAC) has a great potential to map urinary bladders, a radiation sensitive internal organ in pediatric patients, without using any ionizing radiation or toxic contrast agent. Here we demonstrate the use of PAC for mapping urinary bladders with an injection of optical-opaque tracers in rats in vivo.

Conventional pediatric cystography, which is based on diagnostic X-ray using a radio-opaque dye, suffers from the use of harmful ionizing radiation. The ...

Universal Hand-held Three-dimensional Optoacoustic Imaging Probe for Deep Tissue Human Angiography and Functional Preclinical Studies in Real Time

The exclusive combination of high optical contrast and excellent spatial resolution makes optoacoustics (photoacoustics) ideal for simultaneously attaining anatomical, functional and molecular contrast in deep optically opaque tissues. While enormous potential has been recently demonstrated in the application of optoacoustics for small animal research, vast efforts have also been undertaken in translating this imaging technology into clinical practice. We present here a newly developed optoacoustic tomography approach capable of delivering high resolution and spectrally enriched volumetric images of tissue morphology and function in real time. A detailed description of the experimental protocol for operating with the imaging system in both hand-held and stationary modes is provided and showcased for different potential scenarios involving functional and molecular studies in murine models and humans. The possibility for real time visualization in three dimensions along with the versatile handheld design of the imaging probe make the newly developed approach unique among the pantheon of imaging modalities used in today&#8217;s preclinical research and clinical practice.

We provide herein a detailed description of the experimental protocol for imaging with a newly developed hand-held optoacoustic (photoacoustic) system for three-dimensional functional and molecular imaging in real time. The demonstrated powerful performance and versatility may define new application areas of the optoacoustic technology in preclinical research and clinical practice.

The exclusive combination of high optical contrast and excellent spatial resolution makes optoacoustics (photoacoustics) ideal for simultaneously ...

Non-Invasive Ultrasound Assessment of Endometrial Cancer Progression in Pax8-Directed Deletion of the Tumor Suppressors Arid1a and Pten in Mice

Uterine cancers can be studied in mice due to the ease of handling and genetic manipulation in these models. However, these studies are often limited to assessing pathology post-mortem in animals euthanized at multiple time points in different cohorts, which increases the number of mice needed for a study. Imaging mice in longitudinal studies can track the progression of disease in individual animals, reducing the number of mice needed. Advances in ultrasound technology have allowed for the detection of micrometer-level changes in tissues. Ultrasound has been used to study follicle maturation in ovaries and xenograft growth but has not been applied to morphological changes in the mouse uterus. This protocol examines the juxtaposition of pathology with in vivo imaging comparisons in an induced endometrial cancer mouse model. The features observed by ultrasound were consistent with the degree of change seen by gross pathology and histology. Ultrasound was found to be highly predictive of the observed pathology, supporting the incorporation of ultrasonography into longitudinal studies of uterine diseases such as cancer in mice.

Non-Invasive Ultrasound Assessment of Endometrial Cancer Progression in Pax8-Directed Deletion of the Tumor Suppressors Arid1a and Pten in Mice

This protocol describes a method for monitoring the progression of morphological changes over time in the uterus in an inducible mouse model of endometrial cancer using ultrasound imaging with correlation to gross and histological changes.

Uterine cancers can be studied in mice due to the ease of handling and genetic manipulation in these models. However, these studies are often limited to ...

Cancer Research

Murine Model for Non-invasive Imaging to Detect and Monitor Ovarian Cancer Recurrence

Epithelial ovarian cancer is the most lethal gynecologic malignancy in the United States. Although patients initially respond to the current standard of care consisting of surgical debulking and combination chemotherapy consisting of platinum and taxane compounds, almost 90% of patients recur within a few years. In these patients the development of chemoresistant disease limits the efficacy of currently available chemotherapy agents and therefore contributes to the high mortality. To discover novel therapy options that can target recurrent disease, appropriate animal models that closely mimic the clinical profile of patients with recurrent ovarian cancer are required. The challenge in monitoring intra-peritoneal (i.p.) disease limits the use of i.p. models and thus most xenografts are established subcutaneously. We have developed a sensitive optical imaging platform that allows the detection and anatomical location of i.p. tumor mass. The platform includes the use of optical reporters that extend from the visible light range to near infrared, which in combination with 2-dimensional X-ray co-registration can provide anatomical location of molecular signals. Detection is significantly improved by the use of a rotation system that drives the animal to multiple angular positions for 360 degree imaging, allowing the identification of tumors that are not visible in single orientation. This platform provides a unique model to non-invasively monitor tumor growth and evaluate the efficacy of new therapies for the prevention or treatment of recurrent ovarian cancer.

We describe a non-invasive animal imaging platform that allows the detection, quantification, and monitoring of ovarian cancer growth and recurrence. This intra-peritoneal xenograft model mimics the clinical profile of patients with ovarian cancer.

Epithelial ovarian cancer is the most lethal gynecologic malignancy in the United States. Although patients initially respond to the current standard of ...

Medicine

In Vivo, Percutaneous, Needle Based, Optical Coherence Tomography of Renal Masses

Optical coherence tomography (OCT) is the optical equivalent of ultrasound imaging, based on the backscattering of near infrared light. OCT provides real time images with a 15 &#181;m axial resolution at an effective tissue penetration of 2-3 mm. Within the OCT images the loss of signal intensity per millimeter of tissue penetration, the attenuation coefficient, is calculated. The attenuation coefficient is a tissue specific property, providing a quantitative parameter for tissue differentiation. 
Until now, renal mass treatment decisions have been made primarily on the basis of MRI and CT imaging characteristics, age and comorbidity. However these parameters and diagnostic methods lack the finesse to truly detect the malignant potential of a renal mass. A successful core biopsy or fine needle aspiration provides objective tumor differentiation with both sensitivity and specificity in the range of 95-100%. However, a non-diagnostic rate of 10-20% overall, and even up to 30% in SRMs, is to be expected, delaying the diagnostic process due to the frequent necessity for additional biopsy procedures. 
We aim to develop OCT into an optical biopsy, providing real-time imaging combined with on-the-spot tumor differentiation. This publication provides a detailed step-by-step approach for percutaneous, needle based, OCT of renal masses.

In Vivo, Percutaneous, Needle Based, Optical Coherence Tomography of Renal Masses

Optical coherence tomography (OCT) is a high resolution imaging technique that allows analysis of tissue specific optical properties providing the means for tissue differentiation. We developed needle based OCT, providing real-time imaging combined with on-the-spot tumor differentiation. This publication describes a method for percutaneous, needle based OCT of renal masses.

Optical coherence tomography (OCT) is the optical equivalent of ultrasound imaging, based on the backscattering of near infrared light. OCT provides real ...

Quantitation of Intra-peritoneal Ovarian Cancer Metastasis

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancy in the United States. Mortality is due to diagnosis of 75% of women with late stage disease, when metastasis is already present. EOC is characterized by diffuse and widely disseminated intra-peritoneal metastasis. Cells shed from the primary tumor anchor in the mesothelium that lines the peritoneal cavity as well as in the omentum, resulting in multi-focal metastasis, often in the presence of peritoneal ascites. Efforts in our laboratory are directed at a more detailed understanding of factors that regulate EOC metastatic success. However, quantifying metastatic tumor burden represents a significant technical challenge due to the large number, small size and broad distribution of lesions throughout the peritoneum. Herein we describe a method for analysis of EOC metastasis using cells labeled with red fluorescent protein (RFP) coupled with in vivo multispectral imaging. Following intra-peritoneal injection of RFP-labelled tumor cells, mice are imaged weekly until time of sacrifice. At this time, the peritoneal cavity is surgically exposed and organs are imaged in situ. Dissected organs are then placed on a labeled transparent template and imaged ex vivo. Removal of tissue auto-fluorescence during image processing using multispectral unmixing enables accurate quantitation of relative tumor burden. This method has utility in a variety of applications including therapeutic studies to evaluate compounds that may inhibit metastasis and thereby improve overall survival.

Ovarian cancer metastasis is characterized by numerous diffuse intra-peritoneal lesions, such that accurate visual quantitation of tumor burden is challenging. Herein we describe a method for in situ and ex vivo quantitation of metastatic tumor burden using red fluorescent protein (RFP)-labeled tumor cells and optical imaging.

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancy in the United States.  Mortality is due to diagnosis of 75% of ...

Ovarian Cancer Detection Using Photoacoustic Flow Cytometry

Many studies suggest that the enumeration of circulating tumor cells (CTCs) may show promise as a prognostic tool for ovarian cancer. Current strategies for the detection of CTCs include flow cytometry, microfluidic devices, and real-time polymerase chain reaction (RT-PCR). Despite recent advances, methods for the detection of early ovarian cancer metastasis still lack the sensitivity and specificity required for clinical translation. Here, a novel method is presented for the detection of ovarian circulating tumor cells by photoacoustic flow cytometry (PAFC) utilizing a custom three dimensional (3D) printed system, including a flow chamber and syringe pump. This method utilizes folic acid-capped copper sulfide nanoparticles (FA-CuS NPs) to target SKOV-3 ovarian cancer cells by PAFC. This work demonstrates the affinity of these contrast agents for ovarian cancer cells. The results show NP characterization, PAFC detection, and NP uptake by fluorescence microscopy, thus demonstrating the potential of this novel system to detect ovarian CTCs at physiologically relevant concentrations.

A protocol is presented to detect circulating ovarian tumor cells utilizing a custom-made photoacoustic flow system and targeted folic acid-capped copper sulfide nanoparticles.

Many studies suggest that the enumeration of circulating tumor cells (CTCs) may show promise as a prognostic tool for ovarian cancer. Current strategies ...

Multispectral Optoacoustic Tomography for Functional Imaging in Vascular Research

Microcirculatory impairment has been recognized in various disease processes, underlying this growing theme within vascular research. In recent years, the development of live imaging systems has set the (analytical) pace in both basic and clinical research, with the objective of creating new instruments capable of providing real-time, quantifiable endpoints with clinical interest and application. Near-infrared spectroscopy (NIRS), positron emission tomography (PET), computed tomography (CT), and magnetic resonance imaging (MRI) are available, among other techniques, but cost, image resolution, and reduced contrast are recognized as common challenges. Optoacoustic tomography (OT) offers a new perspective on vascular functional imaging, combining state-of-the-art optical absorption and spatial resolution capacities (from micrometer optical to millimeter acoustic resolution) with tissue depth. In this study, we tested the applicability of multispectral optoacoustic tomography (MSOT) for functional imaging. The system uses a tunable optical parametric oscillator (OPO) pumped by an Nd: YAG laser, providing excitation pulses sensed by a 3D probe at wavelengths from 680 nm to 980 nm. Images obtained from the human forearm were reconstructed through a specific algorithm (supplied within the manufacturer&#39;s software) based on the response of specific chromophores. Maximal Oxygenated Hemoglobin (Max HbO2) and Deoxygenated Hemoglobin (Max Hb), Total Hemoglobin (HbT), and mean Oxygen Saturation (mSO2) to vascular density (&#181;Vu), inter-unit average distances (&#950;Ad), and capillary blood volume (mm3) may be measured using this system. The applicability potential found with this OT system is relevant. Ongoing software developments will surely improve the utility of this imaging system.

The present protocol describes the acquisition of multispectral optoacoustic images of in vivo human skin vasculature. These include the quantification of hemoglobin and melanin, regarded as chromophores of interest for functional analysis.

Microcirculatory impairment has been recognized in various disease processes, underlying this growing theme within vascular research. In recent years, the ...

Imaging the Abdominal Aorta with Point-of-Care Ultrasound

An Approach to Point-Of-Care Ultrasound Evaluation of the Abdominal Aorta

Disorders of the abdominal aorta, including aneurysms and dissection, have potentially high rates of morbidity and mortality. While computed tomography (CT) is the current gold standard to image the abdominal aorta, the process of obtaining a CT may be time-consuming, requires the use of intravenous contrast dye, and involves exposure to ionizing radiation. Point-of-care Ultrasound (POCUS) can be performed at the bedside and has excellent sensitivity and specificity for the diagnosis of abdominal aortic aneurysm and excellent specificity for the diagnosis of abdominal aortic dissection. Additionally, POCUS is non-invasive, cost-effective, lacks ionizing radiation, requires no intravenous contrast dye, and can be performed without taking the patient from a critical care area. Screening for abdominal aortic aneurysm (AAA) can be done in primary care settings as well. 
This article will review the approach to POCUS of the abdominal aorta to evaluate such critical pathology. In this paper, we will review the sonographic anatomy of the abdominal aorta as well as the choice of the ultrasound probe, description of POCUS image acquisition, and some pearls and pitfalls of using POCUS to aid in the diagnosis of potentially life-threatening abdominal aortic pathology.

Author Spotlight: Using Point-of-Care Ultrasound for Comprehensive Evaluation of the Abdominal Aorta

This protocol reviews the steps to image the abdominal aorta with point-of-care ultrasound. We discuss image acquisition, troubleshooting imaging pitfalls and artifacts, and the recognition of life-threatening abdominal aortic pathology.

Disorders of the abdominal aorta, including aneurysms and dissection, have potentially high rates of morbidity and mortality. While computed tomography ...

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

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