Name: detection and isolation of circulating melanoma cells using photoacoustic flowmetry
Uploaded: 2011-11-25T13:00:00
Duration: 9 min 52 s
Description: Watch this Scientific Journal Video about Detection and Isolation of Circulating Melanoma Cells using Photoacoustic Flowmetry at JoVE.com

We acknowledge the support of the Department of Biological Engineering and the Christopher S. Bond Life Sciences Center at the University of Missouri. We acknowledge grant support from Missouri Life Sciences Research Board 09-1034 and NIH R21CA139186-0. We also thank the Life Sciences Undergraduate Research Opportunity Program at the University of Missouri, the University of Missouri Molecular Cytology Core, and the University of Missouri College of Engineering for financial support.

1. Blood sample preparation
<ol>
	<li>Draw approximately 10 mL of whole blood from a Stage IV Cancer patient into two 5 mL vacutainers.</li>
	<li>Centrifuge the tubes for ten minutes at 3,000 revolutions per minute (rpm).</li>
	<li>The blood sample will separate into three different layers: the bottom layer comprises the erythrocytes, the middle layer, also called the buffy coat, contains leukocytes and melanoma cells, and the top layer contains plasma and platelets. Remove as much plasma as possible without disturbing the buffy coat.</li>
	<li>Place 250 &mu;L of Histopaque 1077 in two 1 mL Wintrobe tubes. </li>
	<li>Using a 9&Prime; Pasteur Pipette, extract the remaining plasma, the entire buffy coat, and even the top layer of erythrocytes, making sure to collect &lt; 500 &mu;L.</li>
	<li>Place the solution atop the Histopaque layer and centrifuge the Wintrobe tubes in 15 mL conical tubes for 10 minutes at 3000 rpm. </li>
	<li>The blood will again separate, but this time the erythrocytes will be separated by the Histopaque, allowing for easy extraction of the buffy coat. Use a 9&Prime; Pasteur Pipette to obtain the entire buffy coat and place it in a 2 mL eppendorf tube. </li>
	<li>Suspend the buffy coat in 1.5 mL of PBS with 2% v/v Tween 20. </li>
</ol>
2. Flow Chamber Construction
<ol>
	<li>Obtain an acrylic cylinder, 20 mm outer diameter, 17 mm inner diameter, and 8 mm tall. Drill three 5 mm diameter holes through the side of the cylinder at 90 degree angles from each other. Cut three pieces of polymer tubing (1.6 mm inner diameter, 5 mm outer diameter), and place them into the drilled holes.</li>
	<li>Stretch Parafilm across the bottom of the acrylic ring to make a water tight seal. The parafilm forms a shallow bowl with the acrylic ring and will hold the acrylamide solution that will form a flow chamber.</li>
	<li>Suspend a 1.6 mm diameter wire through the pieces of tubing that are across from each other, making sure that the wire alone is visible through the middle of the ring. Fill the third piece of tubing with a 1.6 mm diameter wire and position the tube 1 mm away from the suspended wire. These wires prevent acrylamide from entering the tubing. After the acrylamide solidifies, the wires will be extracted, leaving a cylindrical flow path and a place to position an optical fiber focuses onto the flow path.</li>
	<li>Add 5 mL of prepared acrylamide [20 g acrylamide (Sigma Aldrich), 0.7 g bisacrylamide (Sigma Aldrich), 100 mL of distilled water] to a small beaker. Measure out 0.02 g of ammonium persulfate (Sigma) and add it to the beaker. Mix with a stir bar for 3 minutes. Add 20 &mu;L tetramethylethylenediamene (Fisher Scientific) to the beaker. Quickly pour the mixture into the acrylic ring, filling to the top. After a few seconds, the solution will gel, making a flow chamber mold. Carefully pull the wires out, making sure not to disturb the acrylamide gel. The flow chamber is now ready for use.</li>
</ol>
3. Photoacoustic flowmetry set-up
<ol>
	<li>Acoustically couple a polyvinylidene fluoride piezoelectric acoustic transducer (Panametrics) to a flow chamber using ultrasound gel directly above the flow path.</li>
	<li>Using a Bayonet Neill-Concelman (BNC) cable, connect the transducer to the input of a RITEC Broadband Receiver-640A (lowpass filter: out, highpass filter: 1 MHz, imput impedance: 50 Ohms, amplification: 35 dB). Connect the output of the filter to the oscilloscope (Tektronix TDS 2024B). To trigger the oscilloscope, a photodiode (Thorlabs) is set up near the transducer and is plugged into the oscilloscope using a BNC cable.</li>
	<li>A t-connector (Small Parts) is attached to one arm of the flow chamber. Two syringe pumps (Braintree Scientific) are connected to the other branches of the connector. One syringe contains air, while the other will contain the cell suspension prepared in part 1. Set the flow rates to 100 &mu;L/minute.</li>
	<li>Extract the small tubing of the flow chamber until a small pocket of air forms, and insert a 1 mm diameter core optical fiber with a 0.37 numerical aperture in that slot. Fill the pocket with water to reduce interface signals and increase the amount of laser light applied to the flow path. Due to the numerical aperture of the optical fiber and short distance to the flow path, the laser will only irradiate one slug of sample at any given time, ensuring that the sample directly in front of the laser is the same sample creating photoacoustic signals. The laser energy should be approximately 28 mJ/cm2 with a spot size of 7 mm2.</li>
	<li>Turn both syringe pumps on. When the two immiscible fluids reach the t-junction they will be partitioned into homogeneous slugs and flow past the transducer.</li>
	<li>Irradiate the flowing sample with a nanosecond pulsed laser to generate photoacoustic waves in the melanoma cells. As melanin is a broadband optical absorber, any visible laser wavelength can be used. </li>
	<li>If a signal appears on the oscilloscope when a slug is atop the transducer, the slug contains melanoma, and should be tracked through the system until dropping into a collection vial.</li>
</ol>
4. Immunocytochemistry
<ol>
	<li>Take the captured slugs to a Cytospin 4 Centrifuge (Thermo Scientific) in order to fix them to glass slides. Place glass slides into Cytofunnels (Shandon EZ Single Cytofunnel with Brown Filter Card Thermo Scientific) and load them into the Cytospin 4 Centrifuge. Put 100 &mu;L of 1% Bovine Serum Albumin (Sigma) in PBS into each funnel and then centrifuge at 600 rpm for 3 minutes to help the captured cells stick to the glass slides.</li>
	<li>After centrifugation, add the cell samples to the Cytofunnels and then centrifuge at 600 rpm for 3 minutes. Then spray the slides with an ethanol based fixative.</li>
	<li>Wash the slides in PBS for 10 minutes twice.</li>
	<li>To block nonspecific binding, incubate each slide with a mixture of 10 &mu;L of Goat Serum (Sigma) and 90 &mu;L of PBS for 1 hour at room temperature.</li>
	<li>After the incubation, decant the slides and incubate with the primary antibody solution for 1 hour in a humidified chamber at room temperature. Each slide should contain 1% MART-1 antibody raised in rabbit, 1% CD-45 antibody raised in mouse, 10% Goat serum, and 89% PBS.</li>
	<li>After incubation, wash the slides in PBS for 10 minutes three times.</li>
	<li>The slides are then incubated in a secondary antibody solution, which has fluorophore bound to the antibodies. The mixture contains 0.05% goat anti-rabbit antibody, 0.05% goat anti-mouse antibody, 0.1% goat serum, and 99% PBS. The slides are incubated in the dark for 1 hour at room temperature.</li>
	<li>After incubation, decant the slides wash in PBS for 5 minutes three times.</li>
	<li>To stain for a nucleus, incubate the cells with 0.1 &mu;g/mL of DAPI (Invitrogen) for 1 minute. Then decant the slides and wash in PBS.</li>
	<li>Mount a coverslip over the cell sample using 20 &mu;L of a resin-based mounting media per slide. Seal the coverslip with clear nail polish to preserve the slide. Now the slides are ready to be imaged with a fluorescent microscope.</li>
</ol>
5. Representative Results: 
<img src="/files/ftp_upload/3559/3559fig1.jpg" alt="Figure 1"/> Figure 1.&nbsp;
The photoacoustic waveforms from white blood cells and melanoma cells are shown here. The white blood cells, having no inherent pigmentation, produce no photoacoustic waves and manifest as a flat line of electronic noise (left). The pigmented melanoma cells produces a robust photoacoustic wave (right).
<img src="/files/ftp_upload/3559/3559fig2.jpg" alt="Figure 2"/> Figure 2.&nbsp;
After immunocytochemical staining, cultured melanoma cells show DAPI signals in blue while MART1 is highlighted in green.
<img src="/files/ftp_upload/3559/3559fig3.jpg" alt="Figure 3"/> Figure 3.&nbsp;
Overlaying images for DAPI and MART1, as in Figure 2, with CD45 as an indicator of leukocytes, this figure shows melanoma cells among several white blood cells.

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	<li>Pantel, K., Brakenhoff, R., Brandt, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+clinical+relevance+and+specific+biological+properties+of+disseminating+tumour+cells.">Detection clinical relevance and specific biological properties of disseminating tumour cells.</a> Nature Reviews Cancer. 8, 329-340 (2008).</li><li>Pantel, K., Brakenhoff, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissecting+the+metastatic+cascade.">Dissecting the metastatic cascade.</a> Nature Reviews Cancer. 4, 448-456 (2004).</li><li>Yu, M., Stott, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+tumor+cells:+approaches+to+isolation+and+characterization.">Circulating tumor cells: approaches to isolation and characterization.</a> The Journal of Cell Biology. 192, 373-373 (2011).</li><li>Weight, R. 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John A. Viator is founder and president of Viator Technologies Inc, a company formed to commercialize photoacoustic technologies for the improvement of human health.

Detecting CTCs is still a research intensive field with very few clinical applications due to the difficult problem of isolating the rare tumor cells. Many other techniques are being evaluated for CTC detection, including RT-PCR, microfluidic cell capture, immunomagnetic capture, and other methods8-12. However, photoacoustic detection of CMCs shows promise as it is label free and provides a rapid and accurate means to capture small, light absorbing particles. 
The protocol described to isolate white blood cells is highly efficient, yet a low number of red blood cells exist in the sample. These rouge contaminating cells also absorb laser light, but at a greatly decreased level. To ensure the red blood cells do not interfere with our melanoma detection system, five healthy patient samples were introduced through the system and none gave a signal, indicating that red blood cells are present at low enough concentrations to be disregarded. 
A series of concentration studies have been conducted using cultured melanoma cells and the photoacoustic flow system has proven sensitive down to 1 cell/&mu;L (unpublished data). These studies are on going and will soon include trials using cancer patient samples.
This photoacoustic technique is also being evaluated for use in non-pigmented CTCs, such as breast and prostate cancer by attaching exogenous chromophores. We have performed preliminary work using gold nanoparticles on cancer cells13. These tests have shown that nanoparticles can provide the necessary optical absorption to generate photoacoustic waves. The remaining technical challenge is to attach such particles to cancer cells selectively.

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>DMEM</td>
 <td>Invitrogen</td>
 <td>ABCD1234</td>
 <td>&nbsp;</td>
 </tr>
</tbody>
</table>

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.

Watch this Scientific Journal Video about Detection and Isolation of Circulating Melanoma Cells using Photoacoustic Flowmetry at JoVE.com

Detection and Isolation of Circulating Melanoma Cells using Photoacoustic Flowmetry

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 ...

detection-isolation-circulating-melanoma-cells-using-photoacoustic

Research

JoVE Journal

Bioengineering

Three-dimensional Optical-resolution Photoacoustic Microscopy

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As the mainstays of in vivo three-dimensional (3-D) optical microscopy, single-/multi-photon fluorescence microscopy and optical coherence tomography (OCT) have demonstrated their extraordinary sensitivities to fluorescence and optical scattering contrasts, respectively. However, the optical absorption contrast of biological tissues, which encodes essential physiological/pathological information, has not yet been assessable. 
The emergence of biomedical photoacoustics has led to a new branch of optical microscopy optical-resolution photoacoustic microscopy (OR-PAM)1, where the optical irradiation is focused to the diffraction limit to achieve cellular1 or even subcellular2 level lateral resolution. As a valuable complement to existing optical microscopy technologies, OR-PAM brings in at least two novelties. First and most importantly, OR-PAM detects optical absorption contrasts with extraordinary sensitivity (i.e., 100%). Combining OR-PAM with fluorescence microscopy3 or with optical-scattering-based OCT4 (or with both) provides comprehensive optical properties of biological tissues. Second, OR-PAM encodes optical absorption into acoustic waves, in contrast to the pure optical processes in fluorescence microscopy and OCT, and provides background-free detection. The acoustic detection in OR-PAM mitigates the impacts of optical scattering on signal degradation and naturally eliminates possible interferences (i.e., crosstalks) between excitation and detection, which is a common problem in fluorescence microscopy due to the overlap between the excitation and fluorescence spectra. 
Unique for optical absorption imaging, OR-PAM has demonstrated broad biomedical applications since its invention, including, but not limited to, neurology5, 6, ophthalmology7, 8, vascular biology9, and dermatology10. In this video, we teach the system configuration and alignment of OR-PAM as well as the experimental procedures for in vivo functional microvascular imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology capable of imaging optical absorption contrasts in vivo with cellular resolution and sensitivity. Here, we provide a visualized instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, typical in vivo experimental procedures, and functional imaging schemes.

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As ...

Rapid Isolation of Viable Circulating Tumor Cells from Patient Blood Samples

Circulating tumor cells (CTC) are cells that disseminate from a primary tumor throughout the circulatory system and that can ultimately form secondary tumors at distant sites. CTC count can be used to follow disease progression based on the correlation between CTC concentration in blood and disease severity1. As a treatment tool, CTC could be studied in the laboratory to develop personalized therapies. To this end, CTC isolation must cause no cellular damage, and contamination by other cell types, particularly leukocytes, must be avoided as much as possible2. Many of the current techniques, including the sole FDA-approved device for CTC enumeration, destroy CTC as part of the isolation process (for more information see Ref. 2). A microfluidic device to capture viable CTC is described, consisting of a surface functionalized with E-selectin glycoprotein in addition to antibodies against epithelial markers3. To enhance device performance a nanoparticle coating was applied consisting of halloysite nanotubes, an aluminosilicate nanoparticle harvested from clay4. The E-selectin molecules provide a means to capture fast moving CTC that are pumped through the device, lending an advantage over alternative microfluidic devices wherein longer processing times are necessary to provide target cells with sufficient time to interact with a surface. The antibodies to epithelial targets provide CTC-specificity to the device, as well as provide a readily adjustable parameter to tune isolation. Finally, the halloysite nanotube coating allows significantly enhanced isolation compared to other techniques by helping to capture fast moving cells, providing increased surface area for protein adsorption, and repelling contaminating leukocytes3,4. This device is produced by a straightforward technique using off-the-shelf materials, and has been successfully used to capture cancer cells from the blood of metastatic cancer patients. Captured cells are maintained for up to 15 days in culture following isolation, and these samples typically consist of &gt;50% viable primary cancer cells from each patient. This device has been used to capture viable CTC from both diluted whole blood and buffy coat samples. Ultimately, we present a technique with functionality in a clinical setting to develop personalized cancer therapies.

Circulating tumor cells are isolated from the blood of cancer patients without inflicting cellular damage. Isolation of tumor cells is accomplished using a bimolecular surface of E-selectin in addition to antibodies against epithelial markers. A nanotube coating specifically promotes cancer cell adhesion resulting in high capture purities.

Circulating tumor cells (CTC) are cells that disseminate from a primary tumor throughout the circulatory system and that can ultimately form secondary ...

Bacterial Detection &amp; Identification Using Electrochemical Sensors

Electrochemical sensors are widely used for rapid and accurate measurement of blood glucose and can be adapted for detection of a wide variety of analytes. Electrochemical sensors operate by transducing a biological recognition event into a useful electrical signal. Signal transduction occurs by coupling the activity of a redox enzyme to an amperometric electrode. Sensor specificity is either an inherent characteristic of the enzyme, glucose oxidase in the case of a glucose sensor, or a product of linkage between the enzyme and an antibody or probe.
Here, we describe an electrochemical sensor assay method to directly detect and identify bacteria. In every case, the probes described here are DNA oligonucleotides. This method is based on sandwich hybridization of capture and detector probes with target ribosomal RNA (rRNA). The capture probe is anchored to the sensor surface, while the detector probe is linked to horseradish peroxidase (HRP). When a substrate such as 3,3',5,5'-tetramethylbenzidine (TMB) is added to an electrode with capture-target-detector complexes bound to its surface, the substrate is oxidized by HRP and reduced by the working electrode. This redox cycle results in shuttling of electrons by the substrate from the electrode to HRP, producing current flow in the electrode.

We describe an electrochemical sensor assay method for rapid bacterial detection and identification. The assay involves a sensor array functionalized with DNA oligonucleotide capture probes for ribosomal RNA (rRNA) species-specific sequences. Sandwich hybridization of target rRNA with the capture probe and a horseradish peroxidase-linked DNA oligonucleotide detector probe produces a measurable amperometric current.

Electrochemical  sensors are widely used for rapid and accurate measurement of blood glucose and  can be adapted for detection of a wide variety of ...

Integrated Photoacoustic Ophthalmoscopy and Spectral-domain Optical Coherence Tomography

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

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

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

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 ...

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells similar to pericyte-derived mesoangioblasts (iPSC-derived mesoangioblast-like stem/progenitor cells: IDEMs) can be established from iPSCs generated from patients affected by different forms of muscular dystrophy. Patient-specific IDEMs can be genetically corrected with different strategies (e.g. lentiviral vectors, human artificial chromosomes) and enhanced in their myogenic differentiation potential upon overexpression of the myogenesis regulator MyoD. This myogenic potential is then assessed in vitro with specific differentiation assays and analyzed by immunofluorescence. The regenerative potential of IDEMs is further evaluated in vivo, upon intramuscular and intra-arterial transplantation in two representative mouse models displaying acute and chronic muscle regeneration. The contribution of IDEMs to the host skeletal muscle is then confirmed by different functional tests in transplanted mice. In particular, the amelioration of the motor capacity of the animals is studied with treadmill tests. Cell engraftment and differentiation are then assessed by a number of histological and immunofluorescence assays on transplanted muscles. Overall, this paper describes the assays and tools currently utilized to evaluate the differentiation capacity of IDEMs, focusing on the transplantation methods and subsequent outcome measures to analyze the efficacy of cell transplantation.

Induced pluripotent stem cell (iPSC)-derived myogenic progenitors are promising candidates for cell therapy strategies to treat muscular dystrophies. This protocol describes transplantation and functional measurements required to evaluate the engraftment and differentiation of iPSC-derived mesoangioblasts (a type of muscle progenitors) in mouse models of acute and chronic muscle regeneration.

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells ...

Label-free Single Molecule Detection Using Microtoroid Optical Resonators

Detecting small concentrations of molecules down to the single molecule limit has impact on areas such as early detection of disease, and fundamental studies on the behavior of molecules. Single molecule detection techniques commonly utilize labels such as fluorescent tags or quantum dots, however, labels are not always available, increase cost and complexity, and can perturb the events being studied. Optical resonators have emerged as a promising means to detect single molecules without the use of labels. Currently the smallest particle detected by a non-plasmonically-enhanced bare optical resonator system in solution is a 25 nm polystyrene sphere1. We have developed a technique known as Frequency Locking Optical Whispering Evanescent Resonator (FLOWER) that can surpass this limit and achieve label-free single molecule detection in aqueous solution2. As signal strength scales with particle volume, our work represents a &#62; 100x improvement in the signal to noise ratio (SNR) over the current state of the art. Here the procedures behind FLOWER are presented in an effort to increase its usage in the field.

We have developed a label-free biosensing system based on optical resonator technology known as Frequency Locking Optical Whispering Evanescent Resonator (FLOWER) that is capable of detecting single molecules in solution. Here the procedures behind this work are described and presented.

Detecting small concentrations of molecules down to the single molecule limit has impact on areas such as early detection of disease, and fundamental ...

Preparation and Photoacoustic Analysis of Cellular Vehicles Containing Gold Nanorods

Gold nanorods are attractive for a range of biomedical applications, such as the photothermal ablation and the photoacoustic imaging of cancer, thanks to their intense optical absorbance in the near-infrared window, low cytotoxicity and potential to home into tumors. However, their delivery to tumors still remains an issue. An innovative approach consists of the exploitation of the tropism of tumor-associated macrophages that may be loaded with gold nanorods in vitro. Here, we describe the preparation and the photoacoustic inspection of cellular vehicles containing gold nanorods. PEGylated gold nanorods are modified with quaternary ammonium compounds, in order to achieve a cationic profile. On contact with murine macrophages in ordinary Petri dishes, these particles are found to undergo massive uptake into endocytic vesicles. Then these cells are embedded in biopolymeric hydrogels, which are used to verify that the stability of photoacoustic conversion of the particles is retained in their inclusion into cellular vehicles. We are confident that these results may provide new inspiration for the development of novel strategies to deliver plasmonic particles to tumors.

We show the preparation and address the feasibility of cellular vehicles containing gold nanorods for the photoacoustic imaging of cancer.

Gold nanorods are attractive for a range of biomedical applications, such as the photothermal ablation and the photoacoustic imaging of cancer, thanks to ...

Circulating MicroRNA Quantification Using DNA-binding Dye Chemistry and Droplet Digital PCR

Circulating (of cell-free) microRNAs (miRNAs) are released from cells into the blood stream. The amount of specific microRNAs in the circulation has been linked to a disease state and has the potential to be used as disease biomarker. A sensitive and accurate method for circulating microRNA quantification using a dye-based chemistry and droplet digital PCR technology has been recently developed. Specifically, using Locked Nucleic Acid (LNA)-based miRNA-specific primers with a green fluorescent DNA-binding dye in a compatible droplet digital PCR system it is possible to obtain the absolute quantification of specific miRNAs. Here, we describe how performing this technique to assess miRNA amount in biological fluids, such as plasma and serum, is both feasible and effective.

A sensitive and accurate method for cell-free microRNAs quantification using a dye-based chemistry and droplet digital PCR technology is described.

Circulating (of cell-free) microRNAs (miRNAs) are released from cells into the blood stream. The amount of specific microRNAs in the circulation has been ...

Switchable Acoustic and Optical Resolution Photoacoustic Microscopy for In Vivo Small-animal Blood Vasculature Imaging

Photoacoustic microscopy (PAM) is a fast-growing invivo imaging modality that combines both optics and ultrasound, providing penetration beyond the optical mean free path (~1 mm in skin) with high resolution. By combining optical absorption contrast with the high spatial resolution of ultrasound in a single modality, this technique can penetrate deep tissues. Photoacoustic microscopy systems can have either a low acoustic resolution and probe deeply or a high optical resolution and probe shallowly. It is challenging to achieve high spatial resolution and large depth penetration with a single system. This work presents an AR-OR-PAM system capable of both high-resolution imaging at shallow depths and low-resolution deep-tissue imaging of the same sample in vivo. A lateral resolution of 4 &#181;m with 1.4 mm imaging depth using optical focusing and a lateral resolution of 45 &#181;m with 7.8 mm imaging depth using acoustic focusing were successfully demonstrated using the combined system. Here,&#160;in vivo&#160;small-animal blood vasculature imaging is performed to demonstrate its biological imaging capability.

Switchable Acoustic and Optical Resolution Photoacoustic Microscopy for In Vivo Small-animal Blood Vasculature Imaging

Here a switchable acoustic resolution (AR) and optical resolution (OR) photoacoustic microscopy (AR-OR-PAM) system capable of both high resolution imaging at shallow depth and low resolution deep tissue imaging on the same sample in vivo is demonstrated.

Photoacoustic microscopy (PAM) is a fast-growing invivo imaging modality that combines both optics and ultrasound, providing penetration beyond the ...