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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.025% Trypsin With EDTA</td>
			<td>Corning</td>
			<td>25-053-Cl</td>
			<td></td>
		</tr>
		<tr>
			<td>0.2 &#181;m 1000 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 &#181;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&#39;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 .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

5.9K Views.  Arizona State University. 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.

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 ...

Analyzing Cellular Internalization of Nanoparticles and Bacteria by Multi-spectral Imaging Flow Cytometry

Nanoparticulate systems have emerged as valuable tools in vaccine delivery through their ability to efficiently deliver cargo, including proteins, to antigen presenting cells1-5. Internalization of nanoparticles (NP) by antigen presenting cells is a critical step in generating an effective immune response to the encapsulated antigen. To determine how changes in nanoparticle formulation impact function, we sought to develop a high throughput, quantitative experimental protocol that was compatible with detecting internalized nanoparticles as well as bacteria. To date, two independent techniques, microscopy and flow cytometry, have been the methods used to study the phagocytosis of nanoparticles. The high throughput nature of flow cytometry generates robust statistical data. However, due to low resolution, it fails to accurately quantify internalized versus cell bound nanoparticles. Microscopy generates images with high spatial resolution; however, it is time consuming and involves small sample sizes6-8. Multi-spectral imaging flow cytometry (MIFC) is a new technology that incorporates aspects of both microscopy and flow cytometry that performs multi-color spectral fluorescence and bright field imaging simultaneously through a laminar core. This capability provides an accurate analysis of fluorescent signal intensities and spatial relationships between different structures and cellular features at high speed.
	Herein, we describe a method utilizing MIFC to characterize the cell populations that have internalized polyanhydride nanoparticles or Salmonella enterica serovar Typhimurium. We also describe the preparation of nanoparticle suspensions, cell labeling, acquisition on an ImageStreamX system and analysis of the data using the IDEAS application. We also demonstrate the application of a technique that can be used to differentiate the internalization pathways for nanoparticles and bacteria by using cytochalasin-D as an inhibitor of actin-mediated phagocytosis.

In this article, we describe a method utilizing multi-spectral imaging flow cytometry to quantify the internalization of polyanhydride nanoparticles or bacteria by RAW 264.7 cells.

Nanoparticulate  systems have emerged as valuable tools in vaccine delivery through their  ability to efficiently deliver cargo, including proteins, to ...

Evaluation of Polymeric Gene Delivery Nanoparticles by Nanoparticle Tracking Analysis and High-throughput Flow Cytometry

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a technology for regenerative medicine2. Unlike viruses, which have significant safety issues, polymeric nanoparticles can be designed to be non-toxic, non-immunogenic, non-mutagenic, easier to synthesize, chemically versatile, capable of carrying larger nucleic acid cargo and biodegradable and/or environmentally responsive. Cationic polymers self-assemble with negatively charged DNA via electrostatic interaction to form complexes on the order of 100 nm that are commonly termed polymeric nanoparticles. Examples of biomaterials used to form nanoscale polycationic gene delivery nanoparticles include polylysine, polyphosphoesters, poly(amidoamines)s and polyethylenimine (PEI), which is a non-degradable off-the-shelf cationic polymer commonly used for nucleic acid delivery1,3 . Poly(beta-amino ester)s (PBAEs) are a newer class of cationic polymers4 that are hydrolytically degradable5,6 and have been shown to be effective at gene delivery to hard-to-transfect cell types such as human retinal endothelial cells (HRECs)7, mouse mammary epithelial cells8, human brain cancer cells9 and macrovascular (human umbilical vein, HUVECs) endothelial cells10.
A new protocol to characterize polymeric nanoparticles utilizing nanoparticle tracking analysis (NTA) is described. In this approach, both the particle size distribution and the distribution of the number of plasmids per particle are obtained11. In addition, a high-throughput 96-well plate transfection assay for rapid screening of the transfection efficacy of polymeric nanoparticles is presented. In this protocol, poly(beta-amino ester)s (PBAEs) are used as model polymers and human retinal endothelial cells (HRECs) are used as model human cells. This protocol can be easily adapted to evaluate any polymeric nanoparticle and any cell type of interest in a multi-well plate format.

A protocol for nanoparticle tracking analysis (NTA) and high-throughput flow cytometry to evaluate polymeric gene delivery nanoparticles is described. NTA is utilized to characterize the nanoparticle particle size distribution and the plasmid per particle distribution. High-throughput flow cytometry enables quantitative transfection efficacy evaluation for a library of gene delivery biomaterials.

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a ...

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 ...

Wide-field Fluorescent Microscopy and Fluorescent Imaging Flow Cytometry on a Cell-phone

Fluorescent microscopy and flow cytometry are widely used tools in biomedical research and clinical diagnosis. However these devices are in general relatively bulky and costly, making them less effective in the resource limited settings. To potentially address these limitations, we have recently demonstrated the integration of wide-field fluorescent microscopy and imaging flow cytometry tools on cell-phones using compact, light-weight, and cost-effective opto-fluidic attachments. In our flow cytometry design, fluorescently labeled cells are flushed through a microfluidic channel that is positioned above the existing cell-phone camera unit. Battery powered light-emitting diodes (LEDs) are butt-coupled to the side of this microfluidic chip, which effectively acts as a multi-mode slab waveguide, where the excitation light is guided to uniformly excite the fluorescent targets. The cell-phone camera records a time lapse movie of the fluorescent cells flowing through the microfluidic channel, where the digital frames of this movie are processed to count the number of the labeled cells within the target solution of interest. Using a similar opto-fluidic design, we can also image these fluorescently labeled cells in static mode by e.g. sandwiching the fluorescent particles between two glass slides and capturing their fluorescent images using the cell-phone camera, which can achieve a spatial resolution of e.g. ~ 10 &mu;m over a very large field-of-view of ~ 81 mm2. This cell-phone based fluorescent imaging flow cytometry and microscopy platform might be useful especially in resource limited settings, for e.g. counting of CD4+ T cells toward monitoring of HIV+ patients or for detection of water-borne parasites in drinking water.

We review our recent results on the integration of fluorescent microscopy and imaging flow cytometry tools on a cell-phone using compact and cost-effective opto-fluidic attachments. These cell-phone based micro-analysis devices might be useful for cytometric analysis, such as performing various cell counting tasks as well as for high-throughput screening of e.g., water samples in resource limited settings.

Fluorescent microscopy and flow cytometry are widely used tools in biomedical research and clinical diagnosis. However these devices are in general ...

Localization and Relative Quantification of Carbon Nanotubes in Cells with Multispectral Imaging Flow Cytometry

Carbon-based nanomaterials, like carbon nanotubes (CNTs), belong to this type of nanoparticles which are very difficult to discriminate from carbon-rich cell structures and de facto there is still no quantitative method to assess their distribution at cell and tissue levels. What we propose here is an innovative method allowing the detection and quantification of CNTs in cells using a multispectral imaging flow cytometer (ImageStream, Amnis). This newly developed device integrates both a high-throughput of cells and high resolution imaging, providing thus images for each cell directly in flow and therefore statistically relevant image analysis. Each cell image is acquired on bright-field (BF), dark-field (DF), and fluorescent channels, giving access respectively to the level and the distribution of light absorption, light scattered and fluorescence for each cell. The analysis consists then in a pixel-by-pixel comparison of each image, of the 7,000-10,000 cells acquired for each condition of the experiment. Localization and quantification of CNTs is made possible thanks to some particular intrinsic properties of CNTs: strong light absorbance and scattering; indeed CNTs appear as strongly absorbed dark spots on BF and bright spots on DF with a precise colocalization.
This methodology could have a considerable impact on studies about interactions between nanomaterials and cells given that this protocol is applicable for a large range of nanomaterials, insofar as they are capable of absorbing (and/or scattering) strongly enough the light.

In this study, the main purpose was to monitor the cellular uptake and eventual exocytosis of carbon nanotubes (CNTs). From this perspective, we proposed here a unique method using multispectral imaging flow cytometry allowing a quantification and localization of CNTs in a statistically relevant number of cells.

Carbon-based nanomaterials, like carbon nanotubes (CNTs), belong to this type of nanoparticles which are very difficult to discriminate from carbon-rich ...

Sensitive Detection of Proteopathic Seeding Activity with FRET Flow Cytometry

Increasing evidence supports transcellular propagation of toxic protein aggregates, or proteopathic seeds, as a mechanism for the initiation and progression of pathology in several neurodegenerative diseases, including Alzheimer's disease and the related tauopathies. The potentially critical role of tau seeds in disease progression strongly supports the need for a sensitive assay that readily detects seeding activity in biological samples. 
By combining the specificity of fluorescence resonance energy transfer (FRET), the sensitivity of flow cytometry, and the stability of a monoclonal cell line, an ultra-sensitive seeding assay has been engineered and is compatible with seed detection from recombinant or biological samples, including human and mouse brain homogenates. The assay employs monoclonal HEK 293T cells that stably express the aggregation-prone repeat domain (RD) of tau harboring the disease-associated P301S mutation fused to either CFP or YFP, which produce a FRET signal upon protein aggregation. The uptake of proteopathic tau seeds (but not other proteins) into the biosensor cells stimulates aggregation of RD-CFP and RD-YFP, and flow cytometry sensitively and quantitatively monitors this aggregation-induced FRET. The assay detects femtomolar concentrations (monomer equivalent) of recombinant tau seeds, has a dynamic range spanning three orders of magnitude, and is compatible with brain homogenates from tauopathy transgenic mice and human tauopathy subjects. With slight modifications, the assay can also detect seeding activity of other proteopathic seeds, such as &#945;-synuclein, and is also compatible with primary neuronal cultures. The ease, sensitivity, and broad applicability of FRET flow cytometry makes it useful to study a wide range of protein aggregation disorders.

Cell-to-cell transfer of protein aggregates, or proteopathic seeds, may underlie the progression of pathology in neurodegenerative diseases. Here, a novel FRET flow cytometry assay is described that enables specific and sensitive detection of seeding activity from recombinant or biological samples.

Increasing evidence supports transcellular propagation of toxic protein aggregates, or proteopathic seeds, as a mechanism for the initiation and ...

Modeling Ovarian Cancer Multicellular Spheroid Behavior in a Dynamic 3D Peritoneal Microdevice

Ovarian cancer is characterized by extensive peritoneal metastasis, with tumor spheres commonly found in the malignant ascites. This is associated with poor clinical outcomes and currently lacks effective treatment. Both the three-dimensional (3D) environment and the dynamic mechanical forces are very important factors in this metastatic cascade. However, traditional cell cultures fail to recapitulate this natural tumor microenvironment. Thus, in vivo-like models that can emulate the intraperitoneal environment are of obvious importance. In this study, a new microfluidic platform of the peritoneum was set up to mimic the situation of ovarian cancer spheroids in the peritoneal cavity during metastasis. Ovarian cancer spheroids generated under a non-adherent condition were cultured in microfluidic channels coated with peritoneal mesothelial cells subjected to physiologically relevant shear stress. In summary, this dynamic 3D ovarian cancer-mesothelium microfluidic platform can provide new knowledge on basic cancer biology and serve as a platform for potential drug screening and development.

To study ovarian tumor progression in a physiologically relevant model, multicellular spheroids were cultured in a microdevice under simulated fluid flow. This dynamic 3D model emulates the intraperitoneal environment with the cellular and mechanical components where ovarian cancer metastasis occurs.

Ovarian cancer is characterized by extensive peritoneal metastasis, with tumor spheres commonly found in the malignant ascites. This is associated with ...

Isolation of Primary Murine Retinal Ganglion Cells (RGCs) by Flow Cytometry

Neurodegenerative diseases often have a devastating impact on those affected. Retinal ganglion cell (RGC) loss is implicated in an array of diseases, including diabetic retinopathy and glaucoma, in addition to normal aging. Despite their importance, RGCs have been extremely difficult to study until now due in part to the fact that they comprise only a small percentage of the wide variety of cells in the retina. In addition, current isolation methods use intracellular markers to identify RGCs, which produce non-viable cells. These techniques also involve lengthy isolation protocols, so there is a lack of practical, standardized, and dependable methods to obtain and isolate RGCs. This work describes an efficient, comprehensive, and reliable method to isolate primary RGCs from mice retinae using a protocol based on both positive and negative selection criteria. The presented methods allow for the future study of RGCs, with the goal of better understanding the major decline in visual acuity that results from the loss of functional RGCs in neurodegenerative diseases.

Isolation of Primary Murine Retinal Ganglion Cells RGCs by Flow Cytometry

Millions of people suffer from retinal degenerative diseases that result in irreversible blindness. A common element of many of these diseases is the loss of retinal ganglion cells (RGCs). This detailed protocol describes the isolation of primary murine RGCs by positive and negative selection with flow cytometry.

Neurodegenerative diseases often have a devastating impact on those affected. Retinal ganglion cell (RGC) loss is implicated in an array of diseases, ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.