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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#160;</td>
			<td>Thorlabs</td>
			<td>LA1484-B</td>
			<td>For laser beam collimation</td>
		</tr>
		<tr>
			<td>Plano-convex lens&#160;</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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1.3K 조회수.  Washington University. 우리 모두 알고 있듯이 난소 암은 모든 부인과 악성 종양 중에서 가장 치명적이며, 난소 암이 초기 및 국소 단계에서 발견되면 수술과 화학 요법으로 환자의 약 70-90 %를 치료할 수 있지만 후기 단계에서 진단 될 때 20 % 이하에 불과합니다. 그리고 현재의 표준 치료 방법은 조기 난소암 발견에 대한 민감도가 낮은 것으로 알려져 있습니다. 또한 표준 치료 방법은 난소 부속기 병...

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

Spatial Separation of Molecular Conformers and Clusters

Gas-phase molecular physics and physical chemistry experiments commonly use supersonic expansions through pulsed valves for the production of cold molecular beams. However, these beams often contain multiple conformers and clusters, even at low rotational temperatures. We present an experimental methodology that allows the spatial separation of these constituent parts of a molecular beam expansion. Using an electric deflector the beam is separated by its mass-to-dipole moment ratio, analogous to a bender or an electric sector mass spectrometer spatially dispersing charged molecules on the basis of their mass-to-charge ratio. This deflector exploits the Stark effect in an inhomogeneous electric field and allows the separation of individual species of polar neutral molecules and clusters. It furthermore allows the selection of the coldest part of a molecular beam, as low-energy rotational quantum states generally experience the largest deflection. Different structural isomers (conformers) of a species can be separated due to the different arrangement of functional groups, which leads to distinct dipole moments. These are exploited by the electrostatic deflector for the production of a conformationally pure sample from a molecular beam. Similarly, specific cluster stoichiometries can be selected, as the mass and dipole moment of a given cluster depends on the degree of solvation around the parent molecule. This allows experiments on specific cluster sizes and structures, enabling the systematic study of solvation of neutral molecules.

We present a technique that allows the spatial separation of different conformers or clusters present in a molecular beam. An electrostatic deflector is used to separate species by their mass-to-dipole moment ratio, leading to the production of gas-phase ensembles of a single conformer or cluster stoichiometry.

Gas-phase molecular physics and physical chemistry experiments commonly use supersonic expansions through pulsed valves for the production of cold ...

Fast Imaging Technique to Study Drop Impact Dynamics of Non-Newtonian Fluids

In the field of fluid mechanics, many dynamical processes not only occur over a very short time interval but also require high spatial resolution for detailed observation, scenarios that make it challenging to observe with conventional imaging systems. One of these is the drop impact of liquids, which usually happens within one tenth of millisecond. To tackle this challenge, a fast imaging technique is introduced that combines a high-speed camera (capable of up to one million frames per second) with a macro lens with long working distance to bring the spatial resolution of the image down to 10 &#181;m/pixel. The imaging technique enables precise measurement of relevant fluid dynamic quantities, such as the flow field, the spreading distance and the splashing speed, from analysis of the recorded video. To demonstrate the capabilities of this visualization system, the impact dynamics when droplets of non-Newtonian fluids impinge on a flat hard surface are characterized. Two situations are considered: for oxidized liquid metal droplets we focus on the spreading behavior, and for densely packed suspensions we determine the onset of splashing. More generally, the combination of high temporal and spatial imaging resolution introduced here offers advantages for studying fast dynamics across a wide range of microscale phenomena.

Drop impact of non-Newtonian fluids is a complex process since different physical parameters influence the dynamics over a very short time (less than one tenth of a millisecond). A fast imaging technique is introduced in order to characterize the impact behaviors of different non-Newtonian fluids.

In the field of fluid mechanics, many dynamical processes not only occur over a very short time interval but also require high spatial resolution for ...

Electron Channeling Contrast Imaging for Rapid III-V Heteroepitaxial Characterization

Misfit dislocations in heteroepitaxial layers of GaP grown on Si(001) substrates are characterized through use of electron channeling contrast imaging (ECCI) in a scanning electron microscope (SEM). ECCI allows for imaging of defects and crystallographic features under specific diffraction conditions, similar to that possible via plan-view transmission electron microscopy (PV-TEM). A particular advantage of the ECCI technique is that it requires little to no sample preparation, and indeed can use large area, as-produced samples, making it a considerably higher throughput characterization method than TEM. Similar to TEM, different diffraction conditions can be obtained with ECCI by tilting and rotating the sample in the SEM. This capability enables the selective imaging of specific defects, such as misfit dislocations at the GaP/Si interface, with high contrast levels, which are determined by the standard invisibility criteria. An example application of this technique is described wherein ECCI imaging is used to determine the critical thickness for dislocation nucleation for GaP-on-Si by imaging a range of samples with various GaP epilayer thicknesses. Examples of ECCI micrographs of additional defect types, including threading dislocations and a stacking fault, are provided as demonstration of its broad, TEM-like applicability. Ultimately, the combination of TEM-like capabilities &#8211; high spatial resolution and richness of microstructural data &#8211; with the convenience and speed of SEM, position ECCI as a powerful tool for the rapid characterization of crystalline materials.

The use of electron channeling contrast imaging in a scanning electron microscope to characterize defects in III-V/Si heteroexpitaxial thin films is described. This method yields similar results to plan-view transmission electron microscopy, but in significantly less time due to lack of required sample preparation.

Misfit dislocations in heteroepitaxial layers of GaP grown on Si(001) substrates are characterized through use of electron channeling contrast imaging ...

Measurement of Scattering Nonlinearities from a Single Plasmonic Nanoparticle

Plasmonics, which are based on the collective oscillation of electrons due to light excitation, involve strongly enhanced local electric fields and thus have potential applications in nonlinear optics, which requires extraordinary optical intensity. One of the most studied nonlinearities in plasmonics is nonlinear absorption, including saturation and reverse saturation behaviors. Although scattering and absorption in nanoparticles are closely correlated by the Mie theory, there has been no report of nonlinearities in plasmonic scattering until very recently. 
Last year, not only saturation, but also reverse saturation of scattering in an isolated plasmonic particle was demonstrated for the first time. The results showed that saturable scattering exhibits clear wavelength dependence, which seems to be directly linked to the localized surface plasmon resonance (LSPR). Combined with the intensity-dependent measurements, the results suggest the possibility of a common mechanism underlying the nonlinear behaviors of scattering and absorption. These nonlinearities of scattering from a single gold nanosphere (GNS) are widely applicable, including in super-resolution microscopy and optical switches. 
In this paper, it is described in detail how to measure nonlinearity of scattering in a single GNP and how to employ the super-resolution technique to enhance the optical imaging resolution based on saturable scattering. This discovery features the first super-resolution microscopy based on nonlinear scattering, which is a novel non-bleaching contrast method that can achieve a resolution as low as l/8 and will potentially be useful in biomedicine and material studies.

Saturable and reverse saturable scattering were discovered in isolated plasmonic particles and adopted as a novel non-bleaching contrast method in super-resolution microscopy. Here the experimental procedures of detecting and extracting nonlinear scattering are explained in detail, as well as how to enhance resolution with the aid of saturated excitation microscopy.

Plasmonics, which are based on the collective oscillation of electrons due to light excitation, involve strongly enhanced local electric fields and thus ...

Use of a Multi-compartment Dynamic Single Enzyme Phantom for Studies of Hyperpolarized Magnetic Resonance Agents

Imaging of hyperpolarized substrates by magnetic resonance shows great clinical promise for assessment of critical biochemical processes in real time. Due to fundamental constraints imposed by the hyperpolarized state, exotic imaging and reconstruction techniques are commonly used. A practical system for characterization of dynamic, multi-spectral imaging methods is critically needed. Such a system must reproducibly recapitulate the relevant chemical dynamics of normal and pathological tissues. The most widely utilized substrate to date is hyperpolarized [1-13C]-pyruvate for assessment of cancer metabolism. We describe an enzyme-based phantom system that mediates the conversion of pyruvate to lactate. The reaction is initiated by injection of the hyperpolarized agent into multiple chambers within the phantom, each of which contains varying concentrations of reagents that control the reaction rate. Multiple compartments are necessary to ensure that imaging sequences faithfully capture the spatial and metabolic heterogeneity of tissue. This system will aid the development and validation of advanced imaging strategies by providing chemical dynamics that are not available from conventional phantoms, as well as control and reproducibility that is not possible in vivo.

A multi-compartment dynamic phantom is used to simulate some biology of interest for metabolic studies using hyperpolarized magnet resonance agents.

Imaging of hyperpolarized substrates by magnetic resonance shows great clinical promise for assessment of critical biochemical processes in real time. Due ...

Nanostructured Ag-zeolite Composites as Luminescence-based Humidity Sensors

Small silver clusters confined inside zeolite matrices have recently emerged as a novel type of highly luminescent materials. Their emission has high external quantum efficiencies (EQE) and spans the whole visible spectrum. It has been recently reported that the UV excited luminescence of partially Li-exchanged sodium Linde type A zeolites [LTA(Na)] containing luminescent silver clusters can be controlled by adjusting the water content of the zeolite. These samples showed a dynamic change in their emission color from blue to green and yellow upon an increase of the hydration level of the zeolite, showing the great potential that these materials can have as luminescence-based humidity sensors at the macro and micro scale. Here, we describe the detailed procedure to fabricate a humidity sensor prototype using silver-exchanged zeolite composites. The sensor is produced by suspending the luminescent Ag-zeolites in an aqueous solution of polyethylenimine (PEI) to subsequently deposit a film of the material onto a quartz plate. The coated plate is subjected to several hydration/dehydration cycles to show the functionality of the sensing film.

A protocol for the synthesis of moisture-responsive luminescent Ag-zeolite composites is described in this report.

Small silver clusters confined inside zeolite matrices have recently emerged as a novel type of highly luminescent materials. Their emission has high ...

Demonstration of a Hyperlens-integrated Microscope and Super-resolution Imaging

The use of super-resolution imaging to overcome the diffraction limit of conventional microscopy has attracted the interest of researchers in biology and nanotechnology. Although near-field scanning microscopy and superlenses have improved the resolution in the near-field region, far-field imaging in real-time remains a significant challenge. Recently, the hyperlens, which magnifies and converts evanescent waves into propagating waves, has emerged as a novel approach to far-field imaging. Here, we report the fabrication of a spherical hyperlens composed of alternating silver (Ag) and titanium oxide (TiO2) thin layers. Unlike a conventional cylindrical hyperlens, the spherical hyperlens allows for two-dimensional magnification. Thus, incorporation into conventional microscopy is straightforward. A new optical system integrated with the hyperlens is proposed, allowing for a sub-wavelength image to be obtained in the far-field region in real time. In this study, the fabrication and imaging setup methods are explained in detail. This work also describes the accessibility and possibility of the hyperlens, as well as practical applications of real-time imaging in living cells, which can lead to a revolution in biology and nanotechnology.

The use of a hyperlens has been regarded as a novel super-resolution imaging technique due to its advantages in real-time imaging and its simple implementation with conventional optics. Here, we present a protocol describing the fabrication and imaging applications of a spherical hyperlens.

The use of super-resolution imaging to overcome the diffraction limit of conventional microscopy has attracted the interest of researchers in biology and ...

Guidelines and Experience Using Imaging Biomarker Explorer (IBEX) for Radiomics

Imaging Biomarker Explorer (IBEX) is an open-source tool for medical imaging radiomics work. The purpose of this paper is to describe how to use IBEX's graphical user interface (GUI) and to demonstrate how IBEX calculated features have been used in clinical studies. IBEX allows for the import of DICOM images with DICOM radiation therapy structure files or Pinnacle files. Once the images are imported, IBEX has tools within the Data Selection GUI to manipulate the viewing of the images, measure voxel values and distances, and create and edit contours. IBEX comes with 27 preprocessing and 132 feature choices to design feature sets. Each preprocessing and feature category has parameters that can be altered. The output from IBEX is a spreadsheet that contains: 1) each feature from the feature set calculated for each contour in a data set, 2) image information about each contour in a data set, and 3) a summary of the preprocessing and features used with their selected parameters. Features calculated from IBEX have been used in studies to test the variability of features under different imaging conditions and in survival models to improve current clinical models.

Guidelines and Experience Using Imaging Biomarker Explorer IBEX for Radiomics

We describe IBEX, an open-source tool designed for medical imaging radiomics studies, and how to use this tool. In addition, some published works that have used IBEX for uncertainty analysis and model building are showcased.

Imaging Biomarker Explorer (IBEX) is an open-source tool for medical imaging radiomics work. The purpose of this paper is to describe how to use IBEX's ...

Novel Photoacoustic Microscopy and Optical Coherence Tomography Dual-modality Chorioretinal Imaging in Living Rabbit Eyes

Photoacoustic ocular imaging is an emerging ophthalmic imaging technology that can noninvasively visualize ocular tissue by converting light energy into sound waves and is currently under intensive investigation. However, most reported work to date is focused on the imaging of the posterior segment of the eyes of small animals, such as rats and mice, which poses challenges for clinical human translation due to small eyeball sizes. This manuscript describes a novel photoacoustic microscopy (PAM) and optical coherence tomography (OCT) dual-modality system for posterior segment imaging of the eyes of larger animals, such as rabbits. The system configuration, system alignment, animal preparation, and dual-modality experimental protocols for in vivo, noninvasive, label-free chorioretinal imaging in rabbits are detailed. The effectiveness of the method is demonstrated through representative experimental results, including retinal and choroidal vasculature obtained by the PAM and OCT. This manuscript provides a practical guide to reproducing the imaging results in rabbits and advancing photoacoustic ocular imaging in larger animals.

This manuscript describes the novel setup and operating procedure of a photoacoustic microscopy and optical coherence tomography dual-modality system for noninvasive, label-free chorioretinal imaging of larger animals, such as rabbits.

Photoacoustic ocular imaging is an emerging ophthalmic imaging technology that can noninvasively visualize ocular tissue by converting light energy into ...

Three-Dimensional Ultrasonic Needle Tip Tracking with a Fiber-Optic Ultrasound Receiver

Ultrasound is frequently used for guiding minimally invasive procedures, but visualizing medical devices is often challenging with this imaging modality. When visualization is lost, the medical device can cause trauma to critical tissue structures. Here, a method to track the needle tip during ultrasound image-guided procedures is presented. This method involves the use of a fiber-optic ultrasound receiver that is affixed within the cannula of a medical needle to communicate ultrasonically with the external ultrasound probe. This custom probe comprises a central transducer element array and side element arrays. In addition to conventional two-dimensional (2D) B-mode ultrasound imaging provided by the central array, three-dimensional (3D) needle tip tracking is provided by the side arrays. For B-mode ultrasound imaging, a standard transmit-receive sequence with electronic beamforming is performed. For ultrasonic tracking, Golay-coded ultrasound transmissions from the 4 side arrays are received by the hydrophone sensor, and subsequently the received signals are decoded to identify the needle tip&#39;s spatial location with respect to the ultrasound imaging probe. As a preliminary validation of this method, insertions of the needle/hydrophone pair were performed in clinically realistic contexts. This novel ultrasound imaging/tracking method is compatible with current clinical workflow, and it provides reliable device tracking during in-plane and out-of-plane needle insertions.

Accurate and efficient visualization of invasive medical devices is extremely important in many ultrasound-guided minimally invasive procedures. Here, a method for localizing the spatial position of a needle tip relative to the ultrasound imaging probe is presented.

Ultrasound is frequently used for guiding minimally invasive procedures, but visualizing medical devices is often challenging with this imaging modality. ...