Name: magnetic levitation coupled with portable imaging and analysis for disease diagnostics
Uploaded: 2017-02-19T15:00:00.000+00:00
Duration: 7 min 42 s
Description: Watch this Scientific Journal Video about Magnetic Levitation Coupled with Portable Imaging and Analysis for Disease Diagnostics at JoVE.com

The authors would like to acknowledge Dr. Matthew Heeney of Boston Children's Hospital/Dana-Farber Cancer Institute and Dr. Farzana Pashankar of Yale-New Haven Hospital for providing sickle cell patient samples. The authors would like to thank Chu H. Yu and Ashwini Joshi for their assistance in testing these samples and compiling the data.
S.T. acknowledges the American Heart Association Scientist Development Grant (15SDG25080056) and the University of Connecticut Research Excellence Program award for financial support of this research. S.K. acknowledges that this material is based upon work supported by the National Science Foundation Graduate Research Fellowship (DGE-1247393).

Ethical Statement: All procedures involving human blood samples were carried out according to the institutional regulations. All protocols were reviewed and approved by the Institutional Review Board. An informed consent was given by all participants.
1. Sample Preparation for Sickle Cell Disease Diagnosis5,8
<ol>
	<li>Prepare a 50 mM solution of gadobutrol in Hank&#39;s Balanced Salt Solution (HBSS).</li>
	<li>Dissolve 10 mM sodium metabisulfite in the gadolinium solution. 
	NOTE: Sodium metabisulfite is toxic by inhalation and strongly irritates skin and tissue. It is a corrosive acid when mixed with water. It may decompose to emit toxic oxide fumes of sulfur and sodium when heated to high temperature.</li>
	<li>Obtain blood sample via either fingerstick or venipuncture.
	<ol>
		<li>Draw blood using a lancing device with a clean, disposable lancet. Apply pressure near the pierced site and wipe the blood droplet formed three to four times before collecting blood using a pipette; take care not to &#34;milk&#34; the finger by squeezing the tissue, as this will result in contamination of the sample with tissue fluid.</li>
		<li>Alternatively, draw blood using standard venipuncture procedures14. 
		NOTE: If samples will be stored for more than a few hours, collect the blood into a vacutainer with an anticoagulant such as EDTA.</li>
	</ol>
	</li>
	<li>Add less than 1 &#181;L of blood to 100 &#181;l of the gadolinium-sodium metabisulfite solution.</li>
</ol>
2. Sample Preparation for WBC Cytometry7
<ol>
	<li>Obtain blood sample via either fingerstick or venipuncture, as in section 1.</li>
	<li>Optional: Lyse RBCs with an RBC lysis buffer. Pipette 5 &#181;L of blood into 500 &#181;L of RBC lysis buffer and incubate at room temperature for 3 - 5 min. 
	NOTE: this may be done to confirm the levitation range of an isolated population of WBCs. However, it is not necessary to perform this step, as the results presented here demonstrate that RBCs levitate at a distinctly lower position than WBCs.</li>
	<li>Dilute whole blood 1:1,000 in 25 mM Gd in HBSS 
	NOTE: if cell lysis was performed, dilute the lysed sample 9:1 lysed sample:250 mM Gd to obtain a sample containing 25 mM Gd.</li>
</ol>
3. Analysis of Samples Using the Magnetic Levitation Platform4,5,6,7,8
<ol>
	<li>Start the magnetic levitation device:
	<ol>
		<li>For the self-contained device, plug in the device, allow it to power up, and place on a flat, level surface.</li>
		<li>For the smartphone-compatible version, launch the smartphone application and swipe left to enter image capture mode, and place on a flat, level surface.</li>
	</ol>
	</li>
	<li>For static magnetic levitation:
	<ol>
		<li>Load the prepared sample into a square glass microcapillary tube by dipping the end into the solution and allowing the sample to fill the capillary via capillary action.</li>
		<li>Seal the end with a tube sealant by slowly pushing one end the capillary into the material.</li>
		<li>Insert the capillary between the magnets of the magnetic levitation device so that only 1 cm of the tube remains visible (please see Figure 3 for an illustration the sample preparation) . 
		NOTE: This step remains the same for both the smartphone-compatible and self-contained device.</li>
		<li>Wait 10 min without disturbing the device or the capillary.</li>
	</ol>
	</li>
	<li>For flow-assisted magnetic levitation:
	<ol>
		<li>Load the sample into the sample tube.</li>
		<li>Connect the inlet tubing between the sample container and microcapillary and connect the outlet tubing between the microcapillary and waste container.</li>
		<li>Set the LED intensity and flow parameters.</li>
	</ol>
	</li>
	<li>Ensure that cells are visible in the field of view, then press the capture button to capture an image (button 3 on the self-contained device and the camera button on the bottom of the screen in the smartphone application). 
	NOTE: If a USB is used to store the images, create a folder called &#34;images&#34; and insert the USB prior to turning on the device. If no drive is present, the device will store the images in its internal memory and transferred later. 
	NOTE: Several images may be captured and analyzed to reduce the risk of anomalies by moving the capillary in or out about &#189; cm between image capturing. 
	NOTE: If the number of cells within the field of view is too high or too low (as detected and reported on the user interface), shift the capillary in or out of the device or prepare another sample.</li>
	<li>Remove the sample and discard the sample according to the institutional or local regulations. Capillaries should be disposed as a sharp.</li>
</ol>
<img alt="Figure 3" src="/files/ftp_upload/55012/55012fig3.jpg" /> 
Figure 3: Sample Preparation and User Interface. (a) The sample preparation procedure involves lancing the subject&#39;s finger, forming a droplet of blood, transferring the blood drop to the sample testing solution, agitating the sample and loading into a capillary tube via capillary action, and inserting the sample into the magnetic levitation device. (b) These sample preparation steps are also displayed on the screen of the device to guide sample preparation. (c) The device includes four buttons: a button to zoom into the sample image in order to properly adjust the focus using the adjustment knob; a button to obtain a single measurement (a 5 s delay is implemented to allow time for the user to insert the sample); a time-lapse measurement (6 images are taken at 5 s intervals); and a button to power off the device after use. Reproduced, with permission, from Yenilmez, et al. 8<a href="http://cloudfront.jove.com/files/ftp_upload/55012/55012fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
4. Image Analysis4,5,6,7,8
<ol>
	<li>Run the image analysis software included on the device for the appropriate sample (cell distribution or cell type separation). 
	NOTE: For the self-contained device, the analysis is performed automatically and displayed on the graphical user interface. For the smartphone-compatible device, to perform the analysis, go to the gallery and select the desired video file to be analyzed.</li>
	<li>Observe and record the output of the analysis displayed on-screen. 
	NOTE: For cell distribution analysis (such as sickle cell disease diagnosis), the output will be the width of confinement of the cell population. 
	NOTE: For cell type separation (such as WBC identification), the output will be an image with the WBC population identified. In order to calculate the number of cells per microliter, multiply the average number of WBCs per image by a factor of 2,000. For example, if 5 WBCs were observed, this would indicate 10,000 WBCs/&#181;L. The normal range is generally considered to be 3,500 - 11,000 WBCs/&#181;L.</li>
	<li>Repeat the analysis by moving the sample tube in or out of the device about &#189; cm and capturing another image to be analyzed as described above. 
	NOTE: It is recommended to repeat the analysis 5 - 6 times per sample to avoid errors.</li>
</ol>

<ol>
	<li>Tasoglu, S., Khoory, J., Tekin, H., Thomas, C., Karnoub, A., Ghiran, I., Demirci, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Levitational+Image+Cytometry+with+Temporal+Resolution.">Levitational Image Cytometry with Temporal Resolution.</a> Advanced Materials. 27 (26), 3901-3908 (2015).</li><li>Tasoglu, S., Yu, C. H., Liadudanskaya, V., Guven, S., Migliaresi, C., Demirci, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+Levitational+Assembly+for+Living+Material+Fabrication.">Magnetic Levitational Assembly for Living Material Fabrication.</a> Advanced Healthcare Materials. 4 (10), 1469-1476 (2015).</li><li>Tasoglu, S., Yu, C. H., Gungordu, H. I., Guven, S., Vural, T., Demirci, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guided+and+magnetic+self-assembly+of+magnetoceptive+gels.">Guided and magnetic self-assembly of magnetoceptive gels.</a> Nature Communications. 5, 4702 (2014).</li><li>Amin, R., Knowlton, S., Yenilmez, B., Hart, A., Joshi, A., Tasoglu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Smart-phone+Attachable,+Flow-Assisted+Magnetic+Focusing+Device.">Smart-phone Attachable, Flow-Assisted Magnetic Focusing Device.</a> RSC Advances. 6, 93922-93931 (2016).</li><li>Knowlton, S. M., Sencan, I., Aytar, Y., Khoory, J., Heeney, M. M., Ghiran, I. C., Tasoglu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sickle+Cell+Detection+Using+a+Smartphone.">Sickle Cell Detection Using a Smartphone.</a> Sci Rep. 5, 15022 (2015).</li><li>Knowlton, S., Yu, C. H., Jain, N., Ghiran, I. C., Tasoglu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Smart-Phone+Based+Magnetic+Levitation+for+Measuring+Densities.">Smart-Phone Based Magnetic Levitation for Measuring Densities.</a> PLoS One. 10 (8), e0134400 (2015).</li><li>Yenilmez, B., Knowlton, S., Tasoglu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-Contained+Handheld+Magnetic+Platform+for+Point+of+Care+Cytometry+in+Biological+Samples+.">Self-Contained Handheld Magnetic Platform for Point of Care Cytometry in Biological Samples .</a> Advanced Materials Technologies. 1, 1600144 (2016).</li><li>Yenilmez, B., Knowlton, S., Yu, C. H., Heeney, M., Tasoglu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-Free+Sickle+Cell+Disease+Diagnosis+Using+a+Low-Cost,+Handheld+Platform.">Label-Free Sickle Cell Disease Diagnosis Using a Low-Cost, Handheld Platform.</a> Adv Mat Tech. 1 (5), 1600100 (2016).</li><li>Bender, M. A., Douthitt Seibel, G., Pagon, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> GeneReviews. , (1993).</li><li>Kaul, D. K., Fabry, M. E., Windisch, P., Baez, S., Nagel, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Erythrocytes+in+sickle+cell+anemia+are+heterogeneous+in+their+rheological+and+hemodynamic+characteristics.">Erythrocytes in sickle cell anemia are heterogeneous in their rheological and hemodynamic characteristics.</a> J Clin Invest. 72 (1), 22-31 (1983).</li><li>Joiner, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gardos+pathway+to+sickle+cell+therapies?.">Gardos pathway to sickle cell therapies?.</a> Blood. 111 (8), 3918-3919 (2008).</li><li>Finch, J. T., Perutz, M. F., Bertles, J. F., D&#246;bler, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+Sickled+Erythrocytes+and+of+Sickle-Cell+Hemoglobin+Fibers.">Structure of Sickled Erythrocytes and of Sickle-Cell Hemoglobin Fibers.</a> Proc Natl Acad Sci. 70 (3), 718-722 (1973).</li><li>Lew, V. L., Etzion, Z., Bookchin, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dehydration+response+of+sickle+cells+to+sickling-induced+Ca(++)+permeabilization.">Dehydration response of sickle cells to sickling-induced Ca(++) permeabilization.</a> Blood. 99 (7), 2578-2585 (2002).</li><li>Ernst, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> NCCLS Procedures for the Collection of Diagnostic Blood Specimens by Venipuncture: Approved Standard-Sixth Addition. 27 (26), (2007).</li></ol>

Authors Stephanie Knowlton and Savas Tasoglu are founders of and have an equity interest in mBiotics, a startup working to commercialize the magnetic levitation platform described herein for point of care diagnostic solutions. A provisional patent entitled "Magnetic Levitation with On-Board Optical Imaging and Image Analysis for Density-Based Separation, Identification, And Measurement" has been filed with UCONN Case No. 16-027 on the technology described here.

Critical Steps within the Protocol 
Critical factors in this process include the proper alignment of the magnets. If the magnets become dislodged or separated more than normal within the device, this can affect the results. To control for this fault or others in the process, a density-controlled particle, such as polystyrene microspheres, can be used periodically to control for changes over time. Further, levitation time is important to allow the cells to reach equilibrium. For red blood cells, 10 min is sufficient ...

Here, we present a technology platform and a technique which uses magnetic levitation coupled with automated imaging and analysis to analyze the density distribution of a patient&#39;s cells as an indicator for disease. This versatile approach for density-based cytometric analysis can ultimately be applied to a range of disease diagnostics. However, in order to be compatible with point-of-care testing and use in developing countries, the technique must satisfy requirements for low cost, portability, and usability. The device and consumables must be easily obtained at a low cost. The sample preparation must be simple, analysis should be automated with minimal requirements for user input or interpretation, and results should be returned quickly. Further, the device must be compact and portable to be useful in clinical settings as well as developing countries. Thus, we have developed a device and method to use magnetic levitation in point-of-care-compatible technology by coupling automated imaging and image analysis to return results regarding the density distribution of a population of a patient&#39;s cells.
Point-of-care technologies offer a notable advantage over current clinical laboratory testing procedures. The technology currently available is too expensive to be owned by a clinician or too complex to be carried out by medical staff. Many of these procedures require labor-intensive protocols which must be carried out by a trained technician. For these reasons, patient samples such as blood or urine are generally collected in the physician&#39;s office then transferred to a remote, centralized testing laboratory for clinical testing, which may take several days for the physician to receive the results of the test. This can cause delays or complications in the course of treatment in some cases, makes this testing very costly and inefficient (causing a financial burden on insurance payers), and further makes many diagnostics inaccessible in low-resource settings and developing countries.
Here, we present a magnetic levitation technique coupled with automated imaging and analysis in both a device with embedded imaging and processing (Figure 1) and a smartphone-compatible device (Figure 2). These magnetic levitation-based devices represent a broadly applicable platform technology which has the potential to be applied to a range of different medical diagnostic applications. The magnetic levitation approach functions based on an equilibrium between two forces: a magnetic force and a buoyancy force1,2,3. When a particle is suspended in a paramagnetic medium and inserted into a magnetic field generated by two magnets with like poles facing each other, a magnetic force acts on the particle in the direction toward the centerline between the two magnets. The buoyancy force is caused by the relative density of the particle compared to the suspending medium and is upward in the case of particles less dense than the medium and downward in the case of particles denser than the surrounding medium. Based on these two forces, particles will reach an equilibrium levitation position in the field which balances these two forces; this position is directly related to the density of the particle, with denser particles levitating lower in the field than less dense particles. An imaging module, either a built-in smartphone camera4,5,6 or independent optical components equipped with a magnifying lens7,8, are used to visualize the positions of the particles. Image processing, either through a smartphone application4,5,6 or an embedded processing unit7,8, then processes the captured images to quantify the spatial distribution and, therefore, the density distribution of the population. In order to analyze larger samples (such as those with only a few particles of interest per milliliter, flow can be integrated directly into the device such the particles are levitated and analyzed as they pass through the imaging region (Figure 2).
<img alt="Figure 1" src="/files/ftp_upload/55012/55012fig1.jpg" /> 
Figure 1: Self-contained Magnetic Levitation Platform. (a) Compact magnetic levitation device including a magnetic focusing module, imaging components (a light-emitting diode (LED), an optical lens, and a camera detector), and a processing unit with a display screen. (b) Magnetic field strength in the cross-section of the area between the magnets where the sample is inserted. The field strength is greatest at the surface of the magnets and approaches zero at the centerline between them. (c) Particles, such as cells, within the magnetic field experience several forces: a magnetic force (Fm) toward the centerline between the magnetics, with magnitude varying based on the position of the particle; a gravitational force (Fg&#39;) which depends on the particle density relative to that of the suspending medium, and a drag force (Fd) resisting the particle motion. Reproduced, with permission, from Yenilmez, et al.8 <a href="http://cloudflare.jove.com/files/ftp_upload/55012/55012fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55012/55012fig2.jpg" /> 
Figure 2: Smartphone-compatible Flow-assisted Magnetic Levitation Platform. (a-c) Front (a), side (b), and back (c) views of magnetic levitation device (d) The components of the device include: 1) Magnetic levitation module, including permanent magnets, a magnifying lens, and an LED and light diffuser, 2) smartphone case, 3) electronics, including a microcontroller, pump driver, and Bluetooth receiver, 4) micro-pump holder, 5) adjustable orifice, 6) waste tube holder, 7) battery holder, 8) sample holder, 9) dual-purpose stand and cover. (e) Flow schematic, showing pumping of the sample through the magnetic field. (f) Cross-section of the magnetic levitation module, showing how particles of different densities will align as they are pumped through the field; less dense particles, such as Particle 1, will equilibrate at a higher levitation height than denser particles, such as Particle 2. Reproduced, with permission, from Amin, et al.1 <a href="http://cloudflare.jove.com/files/ftp_upload/55012/55012fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The minimum requirements for use of any sample for density distribution analysis in this system include the ability to obtain a suspension of cells or particles greater than approximately 5 &#181;m and less than approximately 250 &#181;m in size (for imaging and image processing) and its compatibility with mixing in a solution of a paramagnetic solution such as the gadobutrol used here. For disease diagnostics, compatible applications include those in which (i) cells of interest inherently have an altered density when they carry a disease compared to healthy controls, (ii) a density change can be induced in the cell by addition of a reagent or some alternative treatment for a short incubation time, or (iii) different cell types are being identified in a single sample and inherently (or via some treatment) have unique characteristic densities.
Sickle cell disease is a genetic disorder causing a mutated form of hemoglobin, HbS, to be produced in a person&#39;s red blood cells (RBCs), which can result in intermittent vaso-occlusive events and chronic hemolytic anemia9. It is diagnosed using either hemoglobin isoelectric focusing, high-performance liquid chromatography (HPLC) fractionation, or hemoglobin electrophoresis which are highly accurate but must be performed in a clinical testing laboratory because they are incompatible with point-of-care settings. Solubility and paper-based tests for sickle cell disease have been proposed, but generally require subjective user interpretation and confirmatory testing. Here, we use a density-based approach to identify sickle RBCs, which attain a higher density than RBCs from people without sickle cell disease. The mechanism involves polymerization of the mutated form of hemoglobin, HbS, which causes RBC dehydration in sickle cell disease RBCs under deoxygenated conditions10,11,12,13.
This density-based approach can also be applied to separate cells of different types on the basis of density: white blood cells (WBCs) and RBCs7. WBCs are generally responsible for fighting infections in the body. WBC cytometry can be used to quantify the number of these cells in the blood and serves as a useful diagnostic tool. WBC counts higher than normal (generally considered greater than 11,000 cells per &#181;L) may indicate infection, immune system disorders, or leukemia. WBC counts below the normal range (around 3,500 cells per &#181;L) may be caused by autoimmune disorders or conditions which damage bone marrow. Unlike alternative technologies, the process presented here does not rely on lysis of the RBCs or stains in order to identify WBCs. This cell-based test takes advantage of the unique inherent densities of the two cell types to perform separation, as the WBC population density has been reported to be lower than that of the RBC population as calculated previously using density gradient centrifugation1,5,8.
Compared to alterative testing at remote locations, this test is rapid, with simple sample preparation (Figure 3), separation of cells in the device within 10 - 15 min, and automated imaging and analysis which requires less than 1 min. In this way, the device can return results quickly to better inform medical decisions, allow treatment to be administered immediately to alleviate physical and psychological pain, and reduce the risk of complications associated with a delay in medical care. This technique can be performed on-site either in clinical settings due to simple sample preparation and automated imaging and analysis which returns a result with minimal user input or interpretation. Because of the use of a simple approach using permanent magnets for sample analysis and the use of either a smartphone or simple electrical components for imaging and image processing, the device as well as the per-test costs are minimal compared to some sophisticated testing procedures.

<table><tbody><tr><td>Gadavist (Bayer)</td><td>Jefferson Medical and Imaging</td><td>2068062</td><td>Gadavist contains 1M gadobutrol, a chelate of gadolinium. We purchased 2 ml vials with 15/ca.</td></tr><tr><td>Square glass microcapillary tubes</td><td>Vitrocom</td><td>8270</td><td>50 mm length is sufficient</td></tr><tr><td>Sodium metabisulfite</td><td>Sigma-Aldrich</td><td>S9000</td><td>Chemical formula: Na&lt;sub&gt;2&lt;/sub&gt;S&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;5&lt;/sub&gt;</td></tr><tr><td>Leica Microsystems Critoseal tube sealant</td><td>Fisher Scientific</td><td>02-676-20</td><td></td></tr><tr><td>Hank&#039;s Balanced Salt Solution</td><td>Sigma-Aldrich</td><td>H9269&amp;nbsp;SIGMA</td><td></td></tr><tr><td>Trypsin-EDTA</td><td>Sigma-Aldrich</td><td>T4049</td><td>Or other reagent as recommended for the cell type used</td></tr><tr><td>MICROLET 2 Adjustable Lancing Device</td><td>Walgreens</td><td>246567</td><td>Any lancing device is acceptable when used according to biosafety protocols</td></tr><tr><td>Microlet Lancets</td><td>Walgreens</td><td>667474</td><td>Must be dispoable and not reused</td></tr><tr><td>Hausser Bright-Line Phase Hemacytometer</td><td>Fisher Scientific</td><td>02-671-6</td><td>Or any preferred method for cell counting</td></tr><tr><td>ACK Lysing Buffer</td><td>ThermoFisher</td><td>A1049201&amp;nbsp;</td><td></td></tr></tbody></table>

magnetic levitation coupled with portable imaging and analysis for disease diagnostics

Currently, many clinical diagnostic procedures are complex, costly, inefficient, and inaccessible to a large population in the world. The requirements for specialized equipment and trained personnel require that many diagnostic tests be performed at remote, centralized clinical laboratories. Magnetic levitation is a simple yet powerful technique and can be applied to levitate cells, which are suspended in a paramagnetic solution and placed in a magnetic field, at a position determined by equilibrium between a magnetic force and a buoyancy force. Here, we present a versatile platform technology designed for point-of-care diagnostics which uses magnetic levitation coupled to microscopic imaging and automated analysis to determine the density distribution of a patient's cells as a useful diagnostic indicator. We present two platforms operating on this principle: (i) a smartphone-compatible version of the technology, where the built-in smartphone camera is used to image cells in the magnetic field and a smartphone application processes the images and to measures the density distribution of the cells and (ii) a self-contained version where a camera board is used to capture images and an embedded processing unit with attached thin-film-transistor (TFT) screen measures and displays the results. Demonstrated applications include: (i) measuring the altered distribution of a cell population with a disease phenotype compared to a healthy phenotype, which is applied to sickle cell disease diagnosis, and (ii) separation of different cell types based on their characteristic densities, which is applied to separate white blood cells from red blood cells for white blood cell cytometry. These applications, as well as future extensions of the essential density-based measurements enabled by this portable, user-friendly platform technology, will significantly enhance disease diagnostic capabilities at the point of care.

For cell density distribution analysis, which is the technique used for sickle cell disease diagnosis, the aim is to identify the width of the distribution of the cell population. Blood cells from patients without sickle cell disease will be confined within a predictable width. Cells from patients with sickle cell disease will be distributed throughout a wider region, with a downward skew in the cell distribution (see Figure 4.) For any particular application, a threshold...

Watch this Scientific Journal Video about Magnetic Levitation Coupled with Portable Imaging and Analysis for Disease Diagnostics at JoVE.com

Magnetic Levitation Coupled with Portable Imaging and Analysis for Disease Diagnostics

We present a magnetic levitation technique coupled with automated imaging and analysis in both a smartphone-compatible device and a device with embedded imaging and processing. This is applied to measure the density distribution of cells with two demonstrated biomedical applications: sickle cell disease diagnosis and separating white and red blood cells.

Currently, many clinical diagnostic procedures are complex, costly, inefficient, and inaccessible to a large population in the world. The requirements for ...

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8.7K Views.  University of Connecticut. We present a magnetic levitation technique coupled with automated imaging and analysis in both a smartphone-compatible device and a device with embedded imaging and processing. This is applied to measure the density distribution of cells with two demonstrated biomedical applications: sickle cell disease diagnosis and separating white and red blood cells.

Video: Magnetic Levitation Coupled with Portable Imaging and Analysis for Disease Diagnostics

Registered Bioimaging of Nanomaterials for Diagnostic and Therapeutic Monitoring 

Nanomedications can be carried by blood borne monocyte-macrophages into the reticuloendothelial system (RES; spleen, liver, lymph nodes) and to end organs. The latter include the lung, RES, and brain and are operative during human immunodeficiency virus type one (HIV-1) infection. Macrophage entry into tissues is notable in areas of active HIV-1 replication and sites of inflammation. In order to assess the potential of macrophages as nanocarriers, superparamagnetic iron-oxide and/or drug laden particles coated with surfactants were parenterally injected into HIV-1 encephalitic mice. This was done to quantitatively assess particle and drug biodistribution. Magnetic resonance imaging (MRI) test results were validated by histological coregistration and enhanced image processing. End organ disease as typified by altered brain histology were assessed by MRI. The demonstration of robust migration of nanoformulations into areas of focal encephalitis provides '"proof of concept" for the use of advanced bioimaging techniques to monitor macrophage migration. Importantly, histopathological aberrations in brain correlate with bioimaging parameters making the general utility of MRI in studies of cell distribution in disease feasible. We posit that using such methods can provide a real time index of disease burden and therapeutic efficacy with translational potential to humans.

Registered Bioimaging of Nanomaterials for Diagnostic and Therapeutic Monitoring

Bioimaging methods used to assess cell biodistribution of nanoparticles are applicable for therapeutic and diagnostic monitoring of nanoformulated compounds. The methods described herein are sensitive and specific when assessed by histological coregistration. The methodologies provide a translational pathway from rodent to human applications.

Nanomedications can be carried by blood borne monocyte-macrophages into the reticuloendothelial system (RES; spleen, liver, lymph nodes) and to end ...

Magnetically-Assisted Remote Controlled Microcatheter Tip Deflection under Magnetic Resonance Imaging

X-ray fluoroscopy-guided endovascular procedures have several significant limitations, including difficult catheter navigation and use of ionizing radiation, which can potentially be overcome using a magnetically steerable catheter under MR guidance.
	The main goal of this work is to develop a microcatheter whose tip can be remotely controlled using the magnetic field of the MR scanner. This protocol aims to describe the procedures for applying current to the microcoil-tipped microcatheter to produce consistent and controllable deflections.
	A microcoil was fabricated using laser lathe lithography onto a polyimide-tipped endovascular catheter. In vitro testing was performed in a waterbath and vessel phantom under the guidance of a 1.5-T MR system using steady-state free precession (SSFP) sequencing. Various amounts of current were applied to the coils of the microcatheter to produce measureable tip deflections and navigate in vascular phantoms.
	The development of this device provides a platform for future testing and opportunity to revolutionize the endovascular interventional MRI environment.

Current applied to an endovascular microcatheter with microcoil tip made by laser lathe lithography can achieve controllable deflections under magnetic resonance (MR) guidance, which may improve speed and efficacy of navigation of vasculature during various endovascular procedures.

X-ray fluoroscopy-guided  endovascular procedures have several significant limitations, including  difficult catheter navigation and use of ionizing ...

Frequency Mixing Magnetic Detection Scanner for Imaging Magnetic Particles in Planar Samples

The setup of a planar Frequency Mixing Magnetic Detection (p-FMMD) scanner for performing Magnetic Particles Imaging (MPI) of flat samples is presented. It consists of two magnetic measurement heads on both sides of the sample mounted on the legs of a u-shaped support. The sample is locally exposed to a magnetic excitation field consisting of two distinct frequencies, a stronger component at about 77 kHz and a weaker field at 61 Hz. The nonlinear magnetization characteristics of superparamagnetic particles give rise to the generation of intermodulation products. A selected sum-frequency component of the high and low frequency magnetic field incident on the magnetically nonlinear particles is recorded by a demodulation electronics. In contrast to a conventional MPI scanner, p-FMMD does not require the application of a strong magnetic field to the whole sample because mixing of the two frequencies occurs locally. Thus, the lateral dimensions of the sample are just limited by the scanning range and the supports. However, the sample height determines the spatial resolution. In the current setup it is limited to 2 mm. As examples, we present two 20 mm &#215; 25 mm p-FMMD images acquired from samples with 1 &#181;m diameter maghemite particles in silanol matrix and with 50 nm magnetite particles in aminosilane matrix. The results show that the novel MPI scanner can be applied for analysis of thin biological samples and for medical diagnostic purposes.

A scanner for imaging magnetic particles in planar samples was developed using the planar frequency mixing magnetic detection technique. The magnetic intermodulation product response from the nonlinear nonhysteretic magnetization of the particles is recorded upon a two-frequency excitation. It can be used to take 2D images of thin biological samples.

The setup of a planar Frequency Mixing Magnetic Detection (p-FMMD) scanner for performing Magnetic Particles Imaging (MPI) of flat samples is presented. ...

Engineering

Remote Magnetic Actuation of Micrometric Probes for in situ 3D Mapping of Bacterial Biofilm Physical Properties

Bacterial adhesion and growth on interfaces lead to the formation of three-dimensional heterogeneous structures so-called biofilms. The cells dwelling in these structures are held together by physical interactions mediated by a network of extracellular polymeric substances. Bacterial biofilms impact many human activities and the understanding of their properties is crucial for a better control of their development &#8212; maintenance or eradication &#8212; depending on their adverse or beneficial outcome. This paper describes a novel methodology aiming to measure in situ the local physical properties of the biofilm that had been, until now, examined only from a macroscopic and homogeneous material perspective. The experiment described here&#160;involves introducing magnetic particles into a growing biofilm to seed local probes that can be remotely actuated without disturbing the structural properties of the biofilm. Dedicated magnetic tweezers were developed to exert a defined force on each particle embedded in the biofilm. The setup is mounted on the stage of a microscope to enable the&#160;recording of time-lapse images of the particle-pulling period. The particle trajectories are then extracted from the pulling sequence and the local viscoelastic parameters are derived from each particle displacement curve, thereby providing the 3D-spatial distribution of the parameters. Gaining insights into the biofilm mechanical profile is essential from an engineer&#39;s point of view for biofilm control purposes but also from a fundamental perspective to clarify the relationship between the architectural properties and the specific biology of these structures.

Remote Magnetic Actuation of Micrometric Probes for in situ 3D Mapping of Bacterial Biofilm Physical Properties

This paper shows an original methodology based on the remote actuation of magnetic particles seeded in a bacterial biofilm and the development of dedicated magnetic tweezers to measure in situ the local mechanical properties of the complex living material built by micro-organisms at interfaces.

Bacterial adhesion and growth on interfaces lead to the formation of three-dimensional heterogeneous structures so-called biofilms. The cells dwelling in ...

Magnetic Field Mapping using Off-Axis Electron Holography in the Transmission Electron Microscope

Off-axis electron holography is a powerful technique that involves the formation of an interference pattern in a transmission electron microscope (TEM) by overlapping two parts of an electron wave, one of which has passed through a region of interest on a specimen and the other is a reference wave. The resulting off-axis electron hologram can be analyzed digitally to recover the phase difference between the two parts of the electron wave, which can then be interpreted to provide quantitative information about local variations in electrostatic potential and magnetic induction within and around the specimen. Off-axis electron holograms can be recorded while a specimen is subjected to external stimuli such as elevated or reduced temperature, voltage, or light. The protocol that is presented here describes the practical steps that are required to record, analyze, and interpret off-axis electron holograms, with a primary focus on the measurement of magnetic fields within and around nanoscale materials and devices. Presented here are the steps involved in the recording, analysis, and processing of off-axis electron holograms, as well as the reconstruction and interpretation of phase images and visualization of the results. Also discussed are the need for optimization of the specimen geometry, the electron optical configuration of the microscope, and the electron hologram acquisition parameters, as well as the need for the use of information from multiple holograms to extract the desired magnetic contributions from the recorded signal. The steps are illustrated through a study of specimens of B20-type FeGe, which contain magnetic skyrmions and were prepared with focused ion beams (FIBs). Prospects for the future development of the technique are discussed.

Guidelines are presented for recording and interpreting off-axis electron holograms to provide quantitative images of magnetic fields in nanoscale materials and devices in the transmission electron microscope.

Off-axis electron holography is a powerful technique that involves the formation of an interference pattern in a transmission electron microscope (TEM) by ...

Rapid Homogeneous Detection of Biological Assays Using Magnetic Modulation Biosensing System

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or separation step. When the IL-8 target was present, a 'sandwich'-based assay attached magnetic beads with IL-8 capture antibody to streptavidin coupled fluorescent protein via the IL-8 target and a biotinylated IL-8 antibody. The magnetic beads are maneuvered into oscillatory motion by applying an alternating magnetic field gradient through two electromagnetic poles. The fluorescent proteins, which are attached to the magnetic beads are condensed into the detection area and their movement in and out of an orthogonal laser beam produces a periodic fluorescent signal that is demodulated using synchronous detection. The magnetic modulation biosensing system was previously used to detect the coding sequences of the non-structural Ibaraki virus protein 3 (NS3) complementary DNA (cDNA) [2]. The techniques that are demonstrated in this work for external manipulation and condensation of particles may be used for other applications, e.g. delivery of magnetically-coupled drugs in-vivo or enhancing the contrast for in-vivo imaging applications.

Magnetic modulation biosensing system is utilized to rapidly, sensitively and simply detect biological assays, such as DNA molecules and proteins.

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or ...

Biology

Guided Tumor Inoculation and Multimodal Imaging in Mice

A Dorsal Skinfold Window Chamber Tumor Mouse Model for Combined Intravital Microscopy and Magnetic Resonance Imaging in Translational Cancer Research

Preclinical intravital imaging such as microscopy and optical coherence tomography have proven to be valuable tools in cancer research for visualizing the tumor microenvironment and its response to therapy. These imaging modalities have micron-scale resolution but have limited use in the clinic due to their shallow penetration depth into tissue. More clinically applicable imaging modalities such as CT, MRI, and PET have much greater penetration depth but have comparatively lower spatial resolution (mm scale). 
To translate preclinical intravital imaging findings into the clinic, new methods must be developed to bridge this micro-to-macro resolution gap. Here we describe a dorsal skinfold window chamber tumor mouse model designed to enable preclinical intravital and clinically applicable (CT and MR) imaging in the same animal, and the image analysis platform that links these two disparate visualization methods. Importantly, the described window chamber approach enables the different imaging modalities to be co-registered in 3D using fiducial markers on the window chamber for direct spatial concordance. This model can be used for validation of existing clinical imaging methods, as well as for the development of new ones through direct correlation with "ground truth" high-resolution intravital findings. 
Finally, the tumor response to various treatments-chemotherapy, radiotherapy, photodynamic therapy-can be monitored longitudinally with this methodology using preclinical and clinically applicable imaging modalities. The dorsal skinfold window chamber tumor mouse model and imaging platforms described here can thus be used in a variety of cancer research studies, for example, in translating preclinical intravital microscopy findings to more clinically applicable imaging modalities such as CT or MRI.

Author Spotlight: Integrating High-Resolution Intravital Imaging and MRI to Enhance Stereotactic Body Radiation Therapy Planning

Translation of Intravital microscopy findings is challenged by its shallow depth penetration into tissue. Here we describe a dorsal window chamber mouse model that enables co-registration of intravital microscopy and clinically applicable imaging modalities (e.g., CT, MRI) for direct spatial correlation, potentially streamlining clinical translation of intravital microscopy findings.

Preclinical intravital imaging such as microscopy and optical coherence tomography have proven to be valuable tools in cancer research for visualizing the ...

Cancer Research

Quantification of Cellular Densities and Antigenic Properties using Magnetic Levitation

The described method was developed based on the principles of magnetic levitation, which separates cells and particles based on their density and magnetic properties. Density is a cell type identifying property, directly related to its metabolic rate, differentiation, and activation status. Magnetic levitation allows a one-step approach to successfully separate, image and characterize circulating blood cells, and to detect anemia, sickle cell disease, and circulating tumor cells based on density and magnetic properties. This approach is also amenable to detecting soluble antigens present in a solution by using sets of low- and high-density beads coated with capture and detection antibodies, respectively. If the antigen is present in solution, it will bridge the two sets&#160;of beads, generating a new bead-bead complex, which will levitate in between the rows of antibody-coated beads. Increased concentration of the target antigen in solution will generate a larger number of bead-bead complexes when compared to lower concentrations of antigen, thus allowing for quantitative measurements of the target antigen. Magnetic levitation is advantageous to other methods due to its decreased sample preparation time and lack of dependance on classical readout methods. The image generated is easily captured and analyzed using a standard microscope or mobile device, such as a smartphone or a tablet.

This paper describes a magnetic levitation-based method that can specifically detect the presence of antigens, either soluble or membrane-bound, by quantifying changes in the levitation height of capture beads with fixed densities.

The described method was developed based on the principles of magnetic levitation, which separates cells and particles based on their density and magnetic ...

Cardiac Magnetic Resonance Imaging

Source: Frederick W. Damen and <a href="https://jove.unicartagenaproxy.elogim.com/author/Craig+J._Goergen">Craig J. Goergen</a>, Weldon School of Biomedical Engineering, Purdue University, West Lafayette, Indiana
In this video, high field, small-bore magnetic resonance imaging (MRI) with physiological monitoring is demonstrated to acquire gated cine loops of the murine cardiovascular system. This procedure provides a basis for assessing left-ventricular function, visualizing vascular networks, and quantifying motion of organs due to respiration. Comparable small animal cardiovascular imaging modalities include high-frequency ultrasound and micro-computed tomography (CT); however, each modality is associated with trade-offs that should be considered. While ultrasound does provide high spatial and temporal resolution, imaging artifacts are common. For example, dense tissue (i.e., the sternum and ribs) can limit imaging penetration depth, and hyperechoic signal at the interface between gas and liquid (i.e., pleura surrounding the lungs) can blur contrast in nearby tissue. Micro-CT in contrast does not suffer from as many in-plane artifacts, but does have lower temporal resolution and limited soft-tissue contrast. Furthermore, micro-CT uses X-ray radiation and often requires the use of contrast agents to visualize vasculature, both of which are known to cause side effects at high doses including radiation damage&#160; and renal injury. Cardiovascular MRI provides a nice compromise between these techniques by negating the need for ionizing radiation and providing the user with the ability to image without contrast agents (although contrast agents are often used for MRI).
This data was acquired with a triggering Fast Low Angle SHot (FLASH) MRI sequence that was gated off of the R-peaks in the cardiac cycle and expiratory plateaus in respiration. These physiological events were monitored through subcutaneous electrodes and a pressure-sensitive pillow that was secured against the abdomen. To ensure the mouse was properly warmed, a rectal temperature probe was inserted and used to control the output of a MRI-safe heating fan. Once the animal was inserted into the bore of the MRI scanner and navigation sequences were run to confirm positioning, the gated FLASH imaging planes were prescribed and data acquired. Overall, high field MRI is a powerful research tool that can provide soft tissue contrast for the study of small animal disease models.

Cardiac MRI: Principle and Imaging

Source: Frederick W. Damen and Craig J. Goergen, Weldon School of Biomedical Engineering, Purdue University, West Lafayette, Indiana
In this video, high ...

JoVE Science Education

Biomedical Engineering

A Magnetic Nanoparticle-Based Immunoassay Using a Microfluidic Device

Source: Hern&#225;ndez-Ortiz, J. A., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/63899">Computer Numerical Control Micromilling of a Microfluidic Acrylic Device with a Staggered Restriction for Magnetic Nanoparticle-Based Immunoassays.</a> J. Vis. Exp. (2022).
This video demonstrates a microfluidic system for a magnetic nanoparticle-based immunoassay. The detection system utilizes a microfluidic device incorporating a magnetic trap within its channels. Pre-formed immunocomplexes, comprised of antigen-coated magnetic nanoparticles bound to primary and peroxidase-conjugated secondary antibodies, are introduced into the channels. The immunocomplexes become captured in the porous magnetic trap upon applying an external magnetic field. Using a fluorogenic substrate emitting fluorescence upon catalysis by the peroxidases, the captured immunocomplexes are detected under a microscope.

1. Device preparation

	Fill the channels with distilled water using a syringe. Make sure there are no leaks or resistance to flow. Immerse the device in ...

Magnetic Levitation Coupled with Portable Imaging and Analysis for Disease Diagnostics

Summary

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Chapters in this video

Registered Bioimaging of Nanomaterials for Diagnostic and Therapeutic Monitoring

Magnetically-Assisted Remote Controlled Microcatheter Tip Deflection under Magnetic Resonance Imaging

Frequency Mixing Magnetic Detection Scanner for Imaging Magnetic Particles in Planar Samples

Remote Magnetic Actuation of Micrometric Probes for in situ 3D Mapping of Bacterial Biofilm Physical Properties

Magnetic Field Mapping using Off-Axis Electron Holography in the Transmission Electron Microscope

Rapid Homogeneous Detection of Biological Assays Using Magnetic Modulation Biosensing System

A Dorsal Skinfold Window Chamber Tumor Mouse Model for Combined Intravital Microscopy and Magnetic Resonance Imaging in Translational Cancer Research

Quantification of Cellular Densities and Antigenic Properties using Magnetic Levitation

Cardiac Magnetic Resonance Imaging

A Magnetic Nanoparticle-Based Immunoassay Using a Microfluidic Device