The authors would like to acknowledge Madeleine Howell for her help with synthesis, Matthew Chest for his help designing the flow system, and Ethan Marschall for assistance with SolidWorks.

1. Nanoparticle Synthesis and Functionalization
NOTE: Synthesis of the FA-CuS NPs is achieved using a one pot synthesis method adapted from a previously published protocol21. 
CAUTION: All synthesis should occur in a ventilated chemical fume hood.
<ol>
	<li>Prior to synthesis, filter approximately 300 mL of deionized (DI) water though a 0.2 &#181;m sterile filter.</li>
	<li>Clean a 250 mL glass round bottom flask with a detergent solution and rinse with DI water. Add 0.0134 g of CuCl2 into 100 mL of DI water to create a 1 mM solution.</li>
	<li>Add 0.015 g of folic acid (FA) to the CuCl2 solution and stir for ~5 min using a magnetic stir bar.</li>
	<li>Add Na2S&#183;9H2O (0.024 g in 100 &#181;L DI water) over approximately 10 s to the reaction mixture utilizing a 200 &#181;L pipette. 
	NOTE: Upon addition of the Na2S&#183;9H2O, the solution will change color from a light yellow to a dark brown.</li>
	<li>Cap the reaction and place in an oil bath, set to 90 &#176;C, and continue stirring with a magnetic stir bar. After approximately 15 min, or when the oil bath has reached 85&#8722;90 &#176;C, allow the reaction to proceed for an additional hour. Your mixture should gradually turn to a dark green color. 
	NOTE: Make sure to vent the system while heating the reaction mixture to avoid pressure buildup.</li>
	<li>Remove the reaction vessel from the oil bath and briefly cool at room temperature for approximately 10 to 15 min before transferring to an ice bath.</li>
	<li>Once the reaction mixture has cooled below 20 &#176;C, adjust the pH to 10 utilizing 1M NaOH to dissolve the remaining folic acid into solution.</li>
	<li>Purify the FA-CuS reaction mixture using a 30 kDa centrifugation column. Add solution in 15 mL batches to the column and centrifuge at 3,082 x g for 15 min.</li>
	<li>Once all of the reaction mixture has been concentrated, recombine the concentrated fractions and wash 4x with 15 mL of pH 10 NaOH in the 30 kDa centrifugation column.</li>
	<li>For mass measurements, take 1/3 of the solution (~66 &#181;L) and split into three glass vials. Dry in a vacuum oven overnight at 40 &#176;C under a vacuum of ~27 mmHg.</li>
	<li>Dissolve the other 2/3 of concentrated solution into 250 &#181;L of PBS and store at 4 &#176;C until further use.</li>
	<li>Prior to utilizing the FA-CuS NPs, sonicate them for 30 min in a bath sonicator on a high setting.</li>
</ol>
2. NP Characterization
<ol>
	<li>Perform dynamic light scattering (DLS) Add 10 &#181;L of concentrated FA-CuS NPS in PBS solution from step 1.1.14 to 2 mL of DI water. Prior to characterization by DLS, sonicate the particles for 30 min in a bath sonicator on a high setting and filter through a 0.2 &#181;m sterile filter to remove residual dust.</li>
	<li>Perform transmission electron microscopy (TEM) : Add 10 &#181;L of concentrated FA-CuS NPS in PBS solution to a piece of wax paper. Invert a formvar coated copper grid on the top of the droplet and let sit for 2 min. Touch the edge of the formvar-coated grid to a piece of filter paper to remove excess liquid. Let air dry. Image the copper grid utilizing an electron microscope at an accelerating voltage of 80 kV. 
	NOTE: Typical results from FA-CuS NPS characterization are presented in Figure 1.</li>
</ol>
3. Cell Culture
<ol>
	<li>This protocol utilizes SKOV-3 cells. Unless otherwise noted, culture SKOV-3 cells in McCoy&#39;s 5A medium supplemented with 10% FBS, 100 U/mL penicillin, and 100 &#181;g/mL streptomycin, and maintain at 37 &#176;C in a humidified 5% CO2 incubator.</li>
</ol>
4. Fluorescent Tagging of FA-CuS NPS for Microscopy
<ol>
	<li>Add Texas-Red-X succinimidyl ester (0.2 mg dissolved in DMSO at a concentration of 10 mg/mL) to a solution containing 2 mg of FA-CuS NPs in 1 mL of 0.1 M NaHCO3 (pH ~9) buffer.</li>
	<li>Stir the reaction mixture using a magnetic stir bar for 1 h, away from light at room temperature.</li>
	<li>Concentrate the reaction mixture in a 4 mL 30 kDa MWCO centrifugation column by spinning at 4,000 x g for 10 min.</li>
	<li>Wash the concentrated solution 3x with 4 mL of 0.1 M NaHCO3 buffer (pH ~9) in a centrifugation column. Subsequently, wash the concentrated solution with 4 mL of DI water 3x or until only a trace amount of fluorescence remains visible in the flowthrough by UV-VIS.</li>
</ol>
5. FA-CuS NPS Uptake by Ovarian Cancer Cells
<ol>
	<li>Prior to incubation with FA-CuS NPs, incubate SKOV-3 cells in a T75 flask with 8&#8722;15 mL of folic-acid-free RPMI-1640 media with 10% FBS and 1% penicillin/streptomycin for at least 24 h.</li>
	<li>Seed cells in 0.5 mL of folic-acid-free RPMI-1640 complete growth media at a density of 0.1 x 106 cells/mL into a 24 well plate.</li>
	<li>The following day, incubate cells with 400 &#181;g/mL FA-CuS NPS in 0.5 mL of folic-acid-free RPMI-1640 complete growth media for 2 h.</li>
	<li>Following this incubation, trypsinize the cells with 0.5 mL of 0.25% trypsin with EDTA. Add at least 1 mL of folic-acid-free RPMI-1640 complete growth media to neutralize the trypsin, and centrifuge the cells at 123 x g for 6 min.</li>
	<li>Remove the supernatant, resuspend the cells in 2 mL of PBS, and centrifuge at 123 x g for 6 min. Perform this wash step 2x to remove any unbound NPs.</li>
	<li>Resuspend the cells in 1&#8722;2 mL of PBS with 2% Tween solution.</li>
	<li>Count the cells using a hemocytometer and trypan blue. Further dilute cells if cell counts are too high. Dilute cells in PBS with 2% Tween to the chosen concentration for detection.</li>
	<li>The cells are now ready to be analyzed by the PAFC system.</li>
</ol>
6. Fluorescence Microscopy of FA-CuS NPS Uptake
<ol>
	<li>Repeat steps listed in step 5.1 and proceed with protocol below for microscopy.</li>
	<li>Seed cells at a density of 0.1 x 106 cells/mL in 0.5 mL of folic-acid-free RPMI-1640 complete growth media on glass coverslips in a 24 well plate.</li>
	<li>The following day, incubate the cells with fluorescently tagged FA-CuS NPs in triplicate, at concentrations of 100 &#181;g/mL, 200 &#181;g/mL, 300 &#181;g/mL, and 400 &#181;g/mL in 0.5 mL of folic-acid-free RPMI-1640 complete growth media.</li>
	<li>Incubate the cells with the NPs for 2 h in the 37 &#176;C incubator</li>
	<li>Following this incubation period, wash the cells 3x with PBS. 
	NOTE: For all wash steps, carefully add the solution on the side of the well plate to not disturb the cells. After addition, carefully tilt the plate and withdraw the solution from the side of the well.</li>
	<li>Incubate the cells with 0.5 mL of 3.7% paraformaldehyde (PFA) in PBS for 15 min and transfer the glass coverslips to a new 24 well plate. 
	CAUTION: PFA is a known carcinogen. Do all fixation in a ventilated chemical fume hood and wear appropriate personal protective equipment.</li>
	<li>Incubate the cells in a solution of 3.7% PFA with 0.1% Triton-X in PBS for 5 min.</li>
	<li>Wash the cells with 0.5 mL of PBS 3x for 5 min each and transfer the coverslips to a new plate.</li>
	<li>Incubate the cells with 0.5 mL of a PBS solution containing DAPI (20 &#181;L/mL of a 0.5 mg/mL stock solution is used for staining) for 5 min, away from light.</li>
	<li>Wash the cells with PBS 3x.</li>
	<li>Following the final PBS wash, mount the coverslips on slides with mounting medium.</li>
	<li>The cells are now ready to be imaged by fluorescence microscopy. Figure 2 shows an example of typical cell characterization by fluorescence microscopy.</li>
</ol>
7. Flow System Architecture
<ol>
	<li>Flow chamber construction 
	NOTE:A SolidWorks file of a 3D printed flow tank can be found in the Supplementary Materials.
	<ol>
		<li>Using the provided SolidWorks file, 3D print the flow tank using ABS thermoplastic or PLA plastic. Dimensions are provided below if SolidWorks is unavailable. Figure 3A shows a representation of this model. The body of the flow tank is 2.5 cm x 1.5 cm x 7.5 cm. The far ends of the flow tank include holes approximately 5 mm in diameter to allow for the entry of tubing containing the capillary tube. 
		NOTE: The flow tank has a 1 cm hole, perpendicular to the orientation of the capillary tube, for placement of the ultrasound transducer. A cylindrical extrusion with the same inner diameter as the hole extends 6 mm into the tank. For real-time imaging, the flow tank has a 1 mm x 3 mm slot directly below the capillary tube.</li>
		<li>After printing the 3D flow tank, clean and assemble the system for use.</li>
		<li>Place glass coverslips over the 1 mm x 3 mm slot and the 1 cm hole in the flow system.</li>
		<li>Carefully seal with silicone to prevent leakage.</li>
		<li>Fit the capillary tube into the silicone cured tubes. Insert the tubes into the flow chamber though the side of the flow tank such that the glass capillary tube is directly above and in front of the 3 mm slot and the 1 cm hole.</li>
		<li>Seal the tubing using silicone to prevent leakage.</li>
	</ol>
	</li>
	<li>Photoacoustic flow system setup 
	NOTE: Figure 3B and Figure 3C show an example of the flow system architecture.
	<ol>
		<li>Connect the transducer to an ultrasound pulser/receiver. Amplify the signal with a 59 dB gain.</li>
		<li>Connect the output of the filter to a multipurpose reconfigurable oscilloscope equipped with a built-in field programmable gate array.</li>
		<li>Connect one of the tubes coming from the flow chamber to a T-junction, connected to two syringe pumps at each branch.</li>
		<li>Fill one of the syringe pumps with air and the other pump with the sample to be analyzed. Set the pump containing air to a flow rate of 40 &#181;L/min and the pump containing the sample to a flow rate of 20 &#181;L/min. The resulting two-phase flow will produce sample volumes of 1 &#181;L. At this flow rate, the system will test approximately 6.4 samples per minute. 
		NOTE: To maintain a consistent distribution of cells, lightly vortex each sample immediately before being tested. In addition, rotate the syringe every few minutes in order to prevent the cells from settling in the solution.</li>
		<li>Connect the remaining tube exiting the flow system to a container with 10% bleach, to dispose of cells after they exit the flow system. 
		NOTE: Before utilizing the flow system, check for leaks, as these can affect the flow. Cells must be contained within a closed system to maintain biological safety during the procedure.</li>
		<li>The design of the 3D printed tank allows for consistent and repeatable alignment between the transducer and laser light with minimal calibration. When placed correctly within the custom tank, the quartz capillary tube ensures that the transducer and laser are directly aligned.</li>
		<li>Place the section of the quartz capillary tube in direct alignment with the transducer, in the field of view of the microscope, allowing for careful placement of the optical fiber above the sample such that it illuminates the entire width of the tube.</li>
		<li>Irradiate the sample using an optical fiber channeling a diode-pumped solid state laser operating at a wavelength of 1,053 nm. The laser light incident on the sample and the transducer used to measure the photoacoustic effect are both unfocused.</li>
		<li>The energy of the laser incident on the sample is approximately 8 mJ and the 10 Hz laser rate is sufficient to illuminate each sample multiple times as it passes through the system.</li>
		<li>Place the flow system on top of an inverted microscope and ensure both the laser pulse and the path of the sample are visible as the sample passes though the flow system. Record flow using a microscope-mounted camera.</li>
		<li>Record the ultrasound acquisitions utilizing data acquisition software (see Table of Materials). Trigger ultrasound and pulsed laser using the FPGA. Utilize PBS with 2% Tween, and FA-CuS NPs at a concentration of 100 &#181;g/mL in PBS 2% Tween, as negative and positive controls, respectively.</li>
		<li>Utilizing a microscope-mounted camera, record both the firing of the laser and the passage of samples though the flow system. These recordings will be utilized to correlate the acoustic signal recorded by the transducer with the firing of the laser. As the samples pass in front of the firing of the laser, the signal can then be correlated to the resulting photoacoustic signal for analysis. At a sampling rate of 10 Hz, the laser will illuminate each plug several times.</li>
	</ol>
	</li>
</ol>
8. Post Processing
<ol>
	<li>For each signal acquisition, s(t), calculate the Hilbert transform, H[s(t)], in order to create an analytic signal.</li>
	<li>Create a complex envelope, se(t), by calculating the magnitude of the analytic signal, such that <img src="/files/ftp_upload/60279/60279eq1.jpg" /> and integrate the envelope to measure the total signal resulting from each acquisition. Compare the signals from each test group (i.e., PBS, tagged cells, FA-CuS NPs, cells alone) utilizing a t-test in R statistical software. Raw photoacoustic signals and their Hilbert transforms are presented in Figure 4.</li>
	<li>For image reconstruction, normalize the complex envelope based on the maximum peak across the whole run. If comparing a series of runs, normalize the complex envelope using the maximum peak across the entire series. Following normalization, convert each acquisition into a series of pixel values. Represent each series of pixel values as a column in the image reconstruction. Representative reconstructions of PBS and the FA-CuS NPs signals are shown in Figure 5, where both images were normalized using the maximum peak across both runs.</li>
</ol>

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The authors have nothing to disclose.

This protocol is a straightforward method for the detection of ovarian CTCs utilizing PAFC and a targeted CuS contrast agent. Many methods have been explored for the detection of ovarian CTCs, including microfluidic devices, RT-PCR, and fluorescence flow cytometry23,24,25. These range in complexity, cost, and accuracy, limiting their effectiveness in clinical settings. PAFC introduces several advantages over these traditional me...

Ovarian cancer is one of the deadliest gynecological malignancies and resulted in an estimated 184,800 deaths worldwide in 20181. Multiple studies have shown the correlation between ovarian cancer progression (i.e., metastasis) and the presence of CTCs2,3,4. The most common method for detection and isolation of CTCs utilizes the Cellsearch system, which targets the EpCam receptor5. EpCam expression, however, is downregulated in epithelial to mesenchymal transition, which has been implicated in cancer metastasis6. Despite advances, current clinical technologies still suffer from low accuracy, high cost, and complexity. Due to these drawbacks, new technologies for the discovery and enumeration of ovarian CTCs has become an important area for research.
Recently, PAFC emerged as an effective method for the noninvasive detection of cancer cells, analysis of nanomaterials, and identification of bacteria7,8,9. PAFC differs from traditional fluorescence flow cytometry by detecting analytes in flow by utilizing photoacoustics. The photoacoustic effect is generated when laser light is absorbed by a material that causes thermoelastic expansion, producing an acoustic wave that can be detected by an ultrasound transducer10,11. Advantages of PAFC over traditional flow cytometry methods include simplicity, ease of translation to clinical settings, and the detection of CTCs at unprecedented depths in patient samples12,13. Recent studies have utilized PAFC systems for the detection of cells using endogenous and exogenous contrast14,15. Near infrared (NIR) light-absorbing contrast agents such as indocyanine green dye, and metal NPs (e.g., gold and CuS) have been used for the selective labeling of cells and tissues in combination with photoacoustic imaging16,17,18. Due to the improved penetration depth of NIR light within biological tissues, photoacoustic detection of absorbers can be performed at greater depths for clinical applications. Because of its great potential for use in the clinic, the combination of targeted NIR contrast agents with PAFC has generated considerable interest for the detection of CTCs.
PAFC in combination with targeted contrast agents provides an improved approach for high-throughput analysis of patient samples with enhanced accuracy and targeted detection of CTCs. One of the principal detection strategies for CTCs is the specific targeting of membrane proteins present on the cell of interest. One notable characteristic of ovarian CTCs is the overexpression of folate receptors located on their outer membrane19. Folate receptor targeting is an ideal strategy for the identification of ovarian CTCs in blood because endogenous cells, which have higher expression of folic acid receptors, are generally luminal and have limited exposure to the bloodstream20. Copper sulfide NPs (CuS NPs) have recently been recognized for their ability to target folate receptors expressed on cancerous cells21. Combined with their biocompatibility, ease of synthesis, and absorption deep in the NIR, these NP contrast agents make an ideal targeting strategy for the detection of ovarian CTCs utilizing PAFC.
This work describes the preparation of FA-CuS NPs and their use for the detection of ovarian cancer cells in a photoacoustic flow system. CuS NPs are modified with folic acid to specifically target ovarian CTCs and emit a photoacoustic signal when stimulated with a 1,053 nm laser. The results indicate the successful detection of ovarian cancer cells incubated with these photoacoustic contrast agents within the PAFC system. These results show detection of ovarian cancer cells down to concentrations of 1 cell/&#181;L, and fluorescence microscopy confirms successful uptake of these particles by SKOV-3 ovarian cancer cells22. This work provides a detailed description of the FA-CuS NPs synthesis, preparation of samples for fluorescence microscopy, construction of the photoacoustic flow system, and the photoacoustic detection of ovarian cancer cells. The presented method shows successful identification of ovarian CTCs in flow utilizing FA-CuS NPs. Future work will focus on the clinical application of this technology towards the early detection of ovarian cancer metastasis.

<table><tbody><tr><td>0.025% Trypsin With EDTA</td><td>Corning</td><td>25-053-Cl</td><td></td></tr><tr><td>0.2 &amp;micro;m 1,000 mL Vacuum Filtration Unit</td><td>VWR</td><td>10040-440</td><td>For filtering larger volumes of DI water.</td></tr><tr><td>0.2 &amp;micro;m sterile syringe filter</td><td>VWR</td><td>28145-477</td><td></td></tr><tr><td>3D Printed Tank</td><td></td><td>Custom-made</td><td></td></tr><tr><td>Acquisition Card</td><td>National Instruments</td><td>PXIe-5170R</td><td>250 MS/s, 8-Channel, 14-bit</td></tr><tr><td>Alconox</td><td>Sigma-Aldrich</td><td>242985-1.8KG</td><td>Detergent used for cleaning glassware.</td></tr><tr><td>Amicon Ultra-15 Centrifugal Filters</td><td>Millipore</td><td>UFC903024</td><td></td></tr><tr><td>Amicon Ultra-4 Centrifugal Filters</td><td>Millipore</td><td>UFC803024</td><td></td></tr><tr><td>Bright-Line Hematocytometer</td><td>Hausser Scientific</td><td>1492</td><td></td></tr><tr><td>Copper(II) Chloride</td><td>ACROS ORGANICS</td><td>206532500</td><td></td></tr><tr><td>Coupling Objective</td><td>Thorlabs</td><td>LMH-10x-532</td><td>To couple pulsed light to optical fiber.</td></tr><tr><td>Coupling Stage</td><td>Newport</td><td>F-91-C1-T</td><td>Stage for coupling pulsed light to objective. Holds FP-1A and LMH-10x-532</td></tr><tr><td>CPX Series Digital Ultrasonic Cleaning Bath</td><td>Fisherbrand</td><td>Model CPX3800</td><td></td></tr><tr><td>Data Acquisition software</td><td>National Instruments</td><td>NI LabVIEW 2017 (32-bit)</td><td>LabVIEW used to synchronize laser pulses with data acquisition.</td></tr><tr><td>Data Processing Software</td><td>Mathworks</td><td>Matlab R2016a</td><td>Reconstructions and graphs produced using Matlab software.</td></tr><tr><td>FBS</td><td>Sigma-Aldrich</td><td>F2442-500ML</td><td></td></tr><tr><td>Fiber Chuck</td><td>Newport</td><td>FPH-DJ</td><td>Used to hold the bare fiber.</td></tr><tr><td>Fiber Coupler</td><td>Newport</td><td>FP-1A</td><td>3-Axis stage for positioning fiber chuck and optical fiber at the focus of the objective.</td></tr><tr><td>Folic Acid</td><td>Sigma-Aldrich</td><td>F7876-10G</td><td></td></tr><tr><td>Formvar Coated TEM Grids</td><td>Electron Microscopy Sciences</td><td>FCF300-CU-SB</td><td></td></tr><tr><td>Masterflex Tubing</td><td>Cole Parmer</td><td>EW-96420-14</td><td></td></tr><tr><td>McCoy&#039;s 5A Medium</td><td>ATCC</td><td>30-2007</td><td></td></tr><tr><td>Norm-Ject 10 mL Syringes</td><td>HENKE SASS WOLF</td><td>4100-X00V0</td><td></td></tr><tr><td>Optical Fiber</td><td>Thorlabs</td><td>FG550LEC</td><td>Used to expose sample to pulsed light.</td></tr><tr><td>PBS</td><td>Alfa Aesar</td><td>J62036</td><td></td></tr><tr><td>Penicillin Streptomycin</td><td>GIBCO</td><td>15140-122</td><td></td></tr><tr><td>Pulsed Laser</td><td>RPMC Lasers Inc</td><td>Quantus-Q1D-1053</td><td>Pulsed laser source with specifications 1053 nm, 8 ns pulse, 10 Hz maximum.</td></tr><tr><td>Pulser/Receiver</td><td>Olympus</td><td>5077PR</td><td>Receives, filters, and amplifies photoacoustic signals. Operated with 59 dB Gain.</td></tr><tr><td>Quartz Capillary Tube</td><td>Sutter Instrument</td><td>QF150-75-10</td><td></td></tr><tr><td>RPMI Midum 1640 (1x) Folic Acid Free</td><td>Gibco</td><td>27016-021</td><td></td></tr><tr><td>Silicone</td><td>Momentive Performance Materials, Inc.</td><td>GE284</td><td></td></tr><tr><td>SKOV-3 Cells</td><td>ATCC</td><td>HTB-77</td><td></td></tr><tr><td>Sodium Bicarbonate</td><td>Sigma-Aldrich</td><td>S5761</td><td></td></tr><tr><td>Sodium Carbonate</td><td>Sigma-Aldrich</td><td>S7795-500G</td><td></td></tr><tr><td>Sodium Hydroxide Beads</td><td>BDH</td><td>BDH9292-500G</td><td></td></tr><tr><td>Sodium Sulfide Nonahydrate</td><td>Sigma-Aldrich</td><td>431648-50G</td><td></td></tr><tr><td>Syringe Pumps</td><td>New Era Pump Systems Inc</td><td>DUAL-1000</td><td></td></tr><tr><td>Texas Red-X-Succinimydl ester</td><td>Invitrogen</td><td>1949071</td><td></td></tr><tr><td>Transducer</td><td>Olynmpus</td><td>V214-BB-RM</td><td>Ultrasound detector with central frequency of 50 MHz and -6 dB fractional bandwidth of 82%.</td></tr><tr><td>Trypan Blue Solution 0.4%</td><td>Amresco</td><td>K940-100ML</td><td></td></tr><tr><td>Tween 20</td><td>Sigma-Aldrich</td><td>P7949-100ML</td><td></td></tr><tr><td>Ultrasound Gel</td><td>Parker Laboratories Inc.</td><td>Aquasonic 100</td><td>Ultrasound gel for transducer coupling</td></tr></tbody></table>

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.

Figure 1A shows a typical TEM image of the synthesized nanoparticles. The average size of the typical nanoparticle is approximately 8.6 nm &#177; 2.5 nm. Nanoparticle measuring was performed in ImageJ. Threshold and watershed functions were applied to separate the particles for measurement. The horizontal and vertical diameters of each particle were measured perpendicular to each other and further averaged. For DLS, a representative measureme...

Watch this Scientific Journal Video about Ovarian Cancer Detection Using Photoacoustic Flow Cytometry at JoVE.com

Ovarian Cancer Detection Using Photoacoustic Flow Cytometry

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 ...

ovarian-cancer-detection-using-photoacoustic-flow-cytometry

Research

JoVE Journal

Bioengineering

6.0K Views.  Arizona State University. Huidige klinische eierstokkanker detectie maatregelen zijn niet-specifieke en gebrek aan zorg detectie van circulerende tumorcellen. Ons fotoakoetische stroomsysteem maakt het mogelijk om patiëntmonsters te testen op specifieke eierstokkankermarkers in de bloedstroom. Deze procedure biedt een unieke platformtechnologie met verbeteringen ten opzichte van de huidige technieken op het gebied van eenvoud, lage kosten, veelbelovende detectielimieten en het vermogen om te worden toegepast op brede...

Video: Ovarian Cancer Detection Using Photoacoustic Flow Cytometry

Detection and Isolation of Circulating Melanoma Cells using Photoacoustic Flowmetry

Circulating tumor cells (CTCs) are those cells that have separated from a macroscopic tumor and spread through the blood and lymph systems to seed secondary tumors1,2,3. CTCs are indicators of metastatic disease and their detection in blood samples may be used to diagnose cancer and monitor a patient&prime;s response to therapy. Since CTCs are rare, comprising about one tumor cell among billions of normal blood cells in advanced cancer patients, their detection and enumeration is a difficult task. We exploit the presence of pigment in most melanoma cells to generate photoacoustic, or laser induced ultrasonic waves in a custom flow cytometer for detection of circulating melanoma cells (CMCs)4,5. This process entails separating a whole blood sample using centrifugation and obtaining the white blood cell layer. If present in whole blood, CMCs will separate with the white blood cells due to similar density. These cells are resuspended in phosphate buffered saline (PBS) and introduced into the flowmeter. Rather than a continuous flow of the blood cell suspension, we induced two phase flow in order to capture these cells for further study. In two phase flow, two immiscible liquids in a microfluidic system meet at a junction and form alternating slugs of liquid6,7. PBS suspended white blood cells and air form microliter slugs that are sequentially irradiated with laser light. The addition of a surfactant to the liquid phase allows uniform slug formation and the user can create different sized slugs by altering the flow rates of the two phases. Slugs of air and slugs of PBS with white blood cells contain no light absorbers and hence, do not produce photoacoustic waves. However, slugs of white blood cells that contain even single CMCs absorb laser light and produce high frequency acoustic waves. These slugs that generate photoacoustic waves are sequestered and collected for cytochemical staining for verification of CMCs.

We have developed a flow cytometer using laser induced ultrasound to detect circulating melanoma cells as an early indicator of metastatic disease.

Circulating tumor cells (CTCs) are those cells that have separated from a macroscopic tumor and spread through the blood and lymph systems to seed ...

Surface-enhanced Resonance Raman Scattering Nanoprobe Ratiometry for Detecting Microscopic Ovarian Cancer via Folate Receptor Targeting

Ovarian cancer represents the deadliest gynecologic malignancy. Most patients present at an advanced stage (FIGO stage III or IV), when local metastatic spread has already occurred. However, ovarian cancer has a unique pattern of metastatic spread, in that tumor implants are initially contained within the peritoneal cavity. This feature could enable, in principle, the complete resection of tumor implants with curative intent. Many of these metastatic lesions are microscopic, making them hard to identify and treat. Neutralizing such micrometastases is believed to be a major goal towards eliminating tumor recurrence and achieving long-term survival. Raman imaging with surface enhanced resonance Raman scattering nanoprobes can be used to delineate microscopic tumors with high sensitivity, due to their bright and bioorthogonal spectral signatures. Here, we describe the synthesis of two &#39;flavors&#39; of such nanoprobes: an antibody-functionalized one that targets the folate receptor &#8212; overexpressed in many ovarian cancers &#8212; and a non-targeted control nanoprobe, with&#160;distinct spectra. The nanoprobes are co-administered intraperitoneally to mouse models of metastatic human ovarian adenocarcinoma. All animal studies were approved by the Institutional Animal Care and Use Committee of Memorial Sloan Kettering Cancer Center. The peritoneal cavity of the animals is surgically exposed, washed, and scanned with a Raman microphotospectrometer. Subsequently, the Raman signatures of the two nanoprobes are decoupled using a Classical Least Squares fitting algorithm, and their respective scores divided to provide a ratiometric signal of folate-targeted over untargeted probes. In this way, microscopic metastases are visualized with high specificity. The main benefit of this approach is that the local application into the peritoneal cavity &#8212; which can be done conveniently during the surgical procedure &#8212; can tag tumors without subjecting the patient to systemic nanoparticle exposure. False positive signals stemming from non-specific binding of the nanoprobes onto visceral surfaces can be eliminated by following a ratiometric approach where targeted and non-targeted nanoprobes with distinct Raman signatures are applied as a mixture. The procedure is currently still limited by the lack of a commercial wide-field Raman imaging camera system, which once available will allow for the application of this technique in the operating theater.

Ovarian cancer forms metastases throughout the peritoneal cavity. Here, we present a protocol to make and use folate-receptor targeted surface-enhanced resonance Raman scattering nanoprobes that reveal these lesions with high specificity via ratiometric imaging. The nanoprobes are administered intraperitoneally to living mice, and the derived images correlate well with histology.

Ovarian cancer represents the deadliest gynecologic malignancy. Most patients present at an advanced stage (FIGO stage III or IV), when local metastatic ...

Cancer Research

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 ...

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 percent of women with ovarian cancer develop new metastases, and the recurrence is often fatal2. It is, therefore, necessary to understand how secondary metastases arise in order to develop better treatments for intermediate and late stage ovarian cancer. Ovarian cancer metastasis occurs when malignant cells detach from the primary tumor site and disseminate throughout the peritoneal cavity. The disseminated cells can form multicellular clusters, or spheroids, that will either remain unattached, or implant onto organs within the peritoneal cavity3 (Figure 1, Movie 1). 
All of the organs within the peritoneal cavity are lined with a single, continuous, layer of mesothelial cells4-6 (Figure 2). However, mesothelial cells are absent from underneath peritoneal tumor masses, as revealed by electron micrograph studies of excised human tumor tissue sections3,5-7 (Figure 2). This suggests that mesothelial cells are excluded from underneath the tumor mass by an unknown process. 
Previous in vitro experiments demonstrated that primary ovarian cancer cells attach more efficiently to extracellular matrix than to mesothelial cells8, and more recent studies showed that primary peritoneal mesothelial cells actually provide a barrier to ovarian cancer cell adhesion and invasion (as compared to adhesion and invasion on substrates that were not covered with mesothelial cells)9,10. This would suggest that mesothelial cells act as a barrier against ovarian cancer metastasis. The cellular and molecular mechanisms by which ovarian cancer cells breach this barrier, and exclude the mesothelium have, until recently, remained unknown. 
Here we describe the methodology for an in vitro assay that models the interaction between ovarian cancer cell spheroids and mesothelial cells in vivo (Figure 3, Movie 2). Our protocol was adapted from previously described methods for analyzing ovarian tumor cell interactions with mesothelial monolayers8-16, and was first described in a report showing that ovarian tumor cells utilize an integrin &#x2013;dependent activation of myosin and traction force to promote the exclusion of the mesothelial cells from under a tumor spheroid17. This model takes advantage of time-lapse fluorescence microscopy to monitor the two cell populations in real time, providing spatial and temporal information on the interaction. The ovarian cancer cells express red fluorescent protein (RFP) while the mesothelial cells express green fluorescent protein (GFP). RFP-expressing ovarian cancer cell spheroids attach to the GFP-expressing mesothelial monolayer. The spheroids spread, invade, and force the mesothelial cells aside creating a hole in the monolayer. This hole is visualized as the negative space (black) in the GFP image. The area of the hole can then be measured to quantitatively analyze differences in clearance activity between control and experimental populations of ovarian cancer and/ or mesothelial cells. This assay requires only a small number of ovarian cancer cells (100 cells per spheroid X 20-30 spheroids per condition), so it is feasible to perform this assay using precious primary tumor cell samples. Furthermore, this assay can be easily adapted for high throughput screening.

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

The mesothelial clearance assay described here takes advantage of fluorescently labeled cells and time-lapse video microscopy to visualize and quantitatively measure the interactions of ovarian cancer multicellular spheroids and mesothelial cell monolayers. This assay models the early steps of ovarian cancer metastasis.

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 ...

Medicine

Evanescent Field Based Photoacoustics: Optical Property Evaluation at Surfaces

Here, we present a protocol to estimate material and surface optical properties using the photoacoustic effect combined with total internal reflection. Optical property evaluation of thin films and the surfaces of bulk materials is an important step in understanding new optical material systems and their applications. The method presented can estimate thickness, refractive index, and use absorptive properties of materials for detection. This metrology system uses evanescent field-based photoacoustics (EFPA), a field of research based upon the interaction of an evanescent field with the photoacoustic effect. This interaction and its resulting family of techniques allow the technique to probe optical properties within a few hundred nanometers of the sample surface. This optical near field allows for the highly accurate estimation of material properties on the same scale as the field itself such as refractive index and film thickness. With the use of EFPA and its sub techniques such as total internal reflection photoacoustic spectroscopy (TIRPAS) and optical tunneling photoacoustic spectroscopy (OTPAS), it is possible to evaluate a material at the nanoscale in a consolidated instrument without the need for many instruments and experiments that may be cost prohibitive.

Here we present a protocol to estimate material and surface optical properties using the photoacoustic effect combined with total internal reflection. This technique evanescent field-based photoacoustics can be used to create a photoacoustic metrology system to estimate materials&#39; thicknesses, bulk and thin film refractive indices, and explore their optical properties.

Here, we present a protocol to estimate material and surface optical properties using the photoacoustic effect combined with total internal reflection. ...

Engineering

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 ...

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.

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. ...

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 ...

In Vitro Photoacoustic Flow Cytometry: A Non-invasive Flow Photoacoustic Technique to Detect Nanoparticle-bearing Circulating Ovarian Cancer Cells

Source: Joel F. Lusk et al. <a href="https://www.jove.com/t/60279">Ovarian Cancer Detection Using Photoacoustic Flow Cytometry.</a> J. Vis. Exp. (2020)
In this video, we present a photoacoustic flow cytometric detection of ovarian cancer cells bearing nanoparticles in a circulating in vitro flow chamber. This non-invasive technique successfully identifies circulating tumor cells in a flow system utilizing a contrast agent.

Source: Joel F. Lusk et al. Ovarian Cancer Detection Using Photoacoustic Flow Cytometry. J. Vis. Exp. (2020)
In this video, we present a photoacoustic ...

JoVE Encyclopedia of Experiments

Gynecologic Cancer

Assays & techniques

Modeling Ovarian Cancer Communication for Imaging Mass Spectrometry Analysis: A Method to Identify Small Molecule Chemical Communicators in Metastatic Ovarian Tumor Development

Place the 8-well divider on top of the ITO-treated slide and insert plastic dividers diagonally into wells. Prepare cell cultures as done previously and combine 100 microliters of cell suspension and 100 microliters of liquefied agarose in a 2-milliliter tube. Immediately, plate 150 microliters of cell agarose mixture on one side of the divider while keeping downward pressure on the divider.
Allow agarose to cool and solidify for approximately 1 minute and then remove the divider. Use forceps to place ovary explant in the center of the empty half of the well. Add 150 microliters of cell agarose mixture over the top of the ovary. Incubate the slide at 37 degrees Celsius and 5% carbon dioxide in a humidified incubator.
After four days, remove the chamber divider from the agarose plugs. With a flat spatula, detach the sides of the agarose from the chamber and gently pull the chamber upward. If any agarose plugs are moved, gently reposition them so that they are not touching one another.
Place the slide in a 37 degree Celsius oven for approximately four hours and rotate 90 degrees every hour to ensure even heat distribution throughout the sample. The slide must be completely dried; otherwise, it could lead to an explosion of the sample in the high vacuum environment of the MALDI-TOF mass spectrometer.
Drying the slide and monitoring the progress is the most critical step to achieve high spatial resolution and quality mass spectra. If flaking or excessive wrinkling occur, we do not recommend collecting the mass spectrometry data.
With the parameters set upon the matrix sprayer, apply matrix solution to the slide. To use phosphorus red as calibrant, add 1 microliter of phosphorus red to a clear spot on the slide. To use the peptide mixture as calibrant, mix it with matrix on parafilm in a 1 to 1 ratio to aid ionization and spot 0.5 to 1 microliter onto the slide. Wait for calibrant to dry before imaging mass spectrometry data acquisition.

Ovarian Cancer Detection Using Photoacoustic Flow Cytometry

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