Name: magnetic levitation coupled with portable imaging and analysis for disease diagnostics
Uploaded: 2017-02-19T15:00:00
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 ...

<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. 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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 border="0" cellpadding="0" cellspacing="0" width="938">
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		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Gadavist (Bayer)</td>
			<td style="width:257px;">Jefferson Medical and Imaging</td>
			<td style="width:119px;">2068062</td>
			<td style="width:241px;">Gadavist contains 1M gadobutrol, a chelate of gadolinium. We purchased 2 mL vials with 15/ca.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Square glass microcapillary tubes</td>
			<td style="width:257px;">Vitrocom</td>
			<td style="width:119px;">8270</td>
			<td>50 mm length is sufficient</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">Sodium metabisulfite</td>
			<td style="width:257px;">Sigma-Aldrich</td>
			<td style="width:119px;">S9000</td>
			<td>Chemical formula: Na2S2O5</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Leica Microsystems Critoseal tube sealant</td>
			<td style="width:257px;">Fisher Scientific</td>
			<td style="width:119px;">02-676-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Hank&#39;s Balanced Salt Solution</td>
			<td style="width:257px;">Sigma-Aldrich</td>
			<td style="width:119px;">H9269&#160;SIGMA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Trypsin-EDTA</td>
			<td style="width:257px;">Sigma-Aldrich</td>
			<td style="width:119px;">T4049</td>
			<td>Or other reagent as recommended for the cell type used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">MICROLET 2 Adjustable Lancing Device</td>
			<td style="width:257px;">Walgreens</td>
			<td style="width:119px;">246567</td>
			<td>Any lancing device is acceptable when used according to biosafety protocols</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Microlet Lancets</td>
			<td style="width:257px;">Walgreens</td>
			<td style="width:119px;">667474</td>
			<td>Must be dispoable and not reused</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Hausser Bright-Line Phase Hemacytometer</td>
			<td style="width:257px;">Fisher Scientific</td>
			<td style="width:119px;">02-671-6</td>
			<td>Or any preferred method for cell counting</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">ACK Lysing Buffer</td>
			<td style="width:257px;">ThermoFisher</td>
			<td style="width:119px;">A1049201&#160;</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 ...

The overall goal of this process is to perform density-based cellular analysis for various medical diagnostic applications by measuring the altered distribution of a cell population or by separating cell types based on their characteristic densities. This method can help solve a key challenge in biomedical engineering to perform important cell ways medical diagnostics in clinical settings or developing countries. The main advantage of this technique is that it does not require a lot of equipment or preparation and it can be done by almost anyone.The implications of this technique extend toward sickle cell diagnosis because this device can quantify the increased density and decreased levitation rate caused by low oxygen conditions in sickle cells. Though this method is useful for medical diagnostic tests as demonstrated here, it can also be applied to other density based cytometric tests by altering the sample and modifying the software. First, prepare 10 milliliters of a 50 millimolar solution of gadobutrol and Hanks balanced salt solution.Then add sodium metabisulfite to achieve a 10 millimolar concentration in the gadolinium solution. Place 100 microliters of the gadolinium sodium metabisulfite stock solution in a microcentrifuge tube. Obtain a blood sample by standard venipuncture techniques or by finger stick.To collect the fingerstick sample, apply pressure near the pierced site without squeezing the tissue. Wipe the pierced site at least three times, and then collect the blood sample with a micropipettor. Mix up to one microliter of the blood sample with the gadolinium sodium metabisulfite solution in the tube to obtain the sample for sickle cell disease diagnosis.First, prepare both 25 and 250 millimolar solutions of gadobutrol and Hanks balanced salt solution. Then, acquire a blood sample using the previously described fingerstick or venipuncture techniques. Dilute about one microliter of the blood sample by a factor of 1000 with a 25 millimolar gadobutrol solution to obtain the sample for white blood cell cytometry.To prepare an additional sample for determining the levitation range of white blood cells alone add 5 microliters of the blood sample to 500 microliters of red blood cell lysis buffer. Incubate the mixture at room temperature for three to five minutes. Then, prepare a solution of one part 250 millimolar gadolinium solution and nine parts lysate to obtain the lysate sample solution with a gadolinium concentration of 25 millimolar.To begin sample analysis, turn on the device and place it on a level surface. Draw the sample into a square glass capillary tube. Once the tube is half full, seal the end with capillary tube sealant.Insert the capillary into the magnetic levitation module of the device, so that only one centimeter of the tube remains visible. Allow the sample to sit undisturbed in the device for 10 minutes to equilibrate. Then, check that the cells of interest are visible on the screen.Press the single measurement button to save the image. Record the analysis results automatically displayed on screen. If the device indicates that too many or too few cells are onscreen, move the capillary slightly in or out of the device and equilibrate the sample for another 10 minutes.After saving the first image, move the capillary in or out by about 0.5 centimeters. Allow the sample to equilibrate, and then acquire another image for analysis. Acquire data from at least five images from different locations on the sample to avoid errors.Save the images to the device or to a USB drive. Once finished, remove the sample from the device and dispose of it appropriately. First, fit a smartphone into the magnetic levitation device.Ensure that the smartphone camera is in line with the magnetic levitation module. Secure the smartphone upright on a stand on a level surface. Insert the capillary into the levitation module and attach the micropump tubing to each end.Place the sample vial in the sample holder. Then, launch the smartphone app. Turn on the micropump and set the parameters for flow rate and LED brightness on the first screen.Once the sample is flowing smoothly, swipe left to enter image capture mode. Acquire at least five different videos. Then, in the analysis section of the app, select flow assisted analysis and open each file to be analyzed.When subjected to magnetic levitation, blood cells from patients without sickle cell disease are confined to a predictable width. Cells from patients with sickle cell diseases are distributed over a wider region that trends downwards. This is reflected in the analysis of the confinement widths.The healthy control cells appear in a narrow band, whereas the sickle cells appear in a broader band with far more variation in overall width. Individual cell types with discrete density ranges can also be differentiated by this method with the best separation achieved at low gadolinium concentrations. Here, the levitation height of white blood cells was determined from a sample of which red blood cells had been lysed before analysis.This known levitation height was used for white blood cell counting. To calculate the number of white blood cells per microliter of a sample diluted by one to 1000, the number of cells observed in the image is multiplied by 2000. Once mastered, this technique can be done in only 10 minutes if it's performed properly.Following this procedure, other researchers can use this approach to develop medical diagnostic tests for a range of medical conditions.

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Hybrid &#181;CT-FMT imaging and image analysis

Fluorescence-mediated tomography (FMT) enables longitudinal and quantitative determination of the fluorescence distribution in vivo and can be used to assess the biodistribution of novel probes and to assess disease progression using established molecular probes or reporter genes. The combination with an anatomical modality, e.g., micro computed tomography (&#181;CT), is beneficial for image analysis and for fluorescence reconstruction. We describe a protocol for multimodal &#181;CT-FMT imaging including the image processing steps necessary to extract quantitative measurements. After preparing the mice and performing the imaging, the multimodal data sets are registered. Subsequently, an improved fluorescence reconstruction is performed, which takes into account the shape of the mouse. For quantitative analysis, organ segmentations are generated based on the anatomical data using our interactive segmentation tool. Finally, the biodistribution curves are generated using a batch-processing feature. We show the applicability of the method by assessing the biodistribution of a well-known probe that binds to bones and joints.

Hybrid  &amp;#181;CT-FMT imaging and image analysis

We describe a protocol for hybrid imaging, combining fluorescence-mediated tomography (FMT) with micro computed tomography (&#181;CT). After fusion and reconstruction, we perform interactive organ segmentation to extract quantitative measurements of the fluorescence distribution.

Fluorescence-mediated tomography (FMT) enables longitudinal and quantitative determination of the fluorescence distribution in vivo and can be used to ...

Fabrication of a Functionalized Magnetic Bacterial Nanocellulose with Iron Oxide Nanoparticles

In this study, bacterial nanocellulose (BNC) produced by the bacteria Gluconacetobacter xylinus is synthesized and impregnated in situ with iron oxide nanoparticles (IONP) (Fe3O4) to yield a magnetic bacterial nanocellulose (MBNC). The synthesis of MBNC is a precise and specifically designed multi-step process. Briefly, bacterial nanocellulose (BNC) pellicles are formed from preserved G. xylinus strain according to our experimental requirements of size and morphology. A solution of iron(III) chloride hexahydrate (FeCl3&#183;6H2O) and iron(II) chloride tetrahydrate (FeCl2&#183;4H2O) with a 2:1 molar ratio is prepared and diluted in deoxygenated high purity water. A BNC pellicle is then introduced in the vessel with the reactants. This mixture is stirred and heated at 80 &#176;C in a silicon oil bath and ammonium hydroxide (14%) is then added by dropping to precipitate the ferrous ions into the BNC mesh. This last step allows forming in situ magnetite nanoparticles (Fe3O4) inside the bacterial nanocellulose mesh to confer magnetic properties to BNC pellicle. A toxicological assay was used to evaluate the biocompatibility of the BNC-IONP pellicle. Polyethylene glycol (PEG) was used to cover the IONPs in order to improve their biocompatibility. Scanning electron microscopy (SEM) images showed that the IONP were located preferentially in the fibril interlacing spaces of the BNC matrix, but some of them were also found along the BNC ribbons. Magnetic force microscope measurements performed on the MBNC detected the presence magnetic domains with high and weak intensity magnetic field, confirming the magnetic nature of the MBNC pellicle. Young&#39;s modulus values obtained in this work are also in a reasonable agreement with those reported for several blood vessels in previous studies.

Here, we present a protocol to make a bacterial nanocellulose (BNC) magnetic for applications in damaged blood vessel reconstruction. The BNC was synthesized by G. xylinus strain. On the other hand, magnetization of the BNC was realized through in situ precipitation of Fe2+ and Fe3+ ferrous ions inside the BNC mesh.

In this study, bacterial nanocellulose (BNC) produced by the bacteria Gluconacetobacter xylinus is synthesized and impregnated in situ with iron oxide ...

Optimized Setup and Protocol for Magnetic Domain Imaging with In Situ Hysteresis Measurement

This paper elaborates the sample preparation protocols required to obtain optimal domain patterns using the Bitter method, focusing on the extra steps compared to standard metallographic sample preparation procedures. The paper proposes a novel bespoke rig for dynamic domain imaging with in situ BH (magnetic hysteresis) measurements and elaborates the protocols for the sensor preparation and the use of the rig to ensure accurate BH measurement. The protocols for static and ordinary dynamic domain imaging (without in situ BH measurements) are also presented. The reported method takes advantage of the convenience and high sensitivity of the traditional Bitter method and enables in situ BH measurement without interrupting or interfering with the domain wall movement processes. This facilitates establishing a direct and quantitative link between the domain wall movement processes&#8211;microstructural feature interactions in ferritic steels with their BH loops. This method is anticipated to become a useful tool for the fundamental study of microstructure&#8211;magnetic property relationships in steels and to help interpret the electromagnetic sensor signals for non-destructive evaluation of steel microstructures.

Optimized Setup and Protocol for Magnetic Domain Imaging with In Situ Hysteresis Measurement

This paper elaborates the sample and sensor preparation procedures and the protocols for using the test rig particularly for dynamic domain imaging with in situ BH measurements in order to achieve optimal domain pattern quality and accurate BH measurements.

This paper elaborates the sample preparation protocols required to obtain optimal domain patterns using the Bitter method, focusing on the extra steps ...

Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics

Proteases are multi-functional enzymes that specialize in the hydrolysis of peptide-bonds and control broad biological processes including homeostasis and allostasis. Moreover, dysregulated protease activity drives pathogenesis and is a functional biomarker of diseases such as cancer; therefore, the ability to detect protease activity in vivo may provide clinically relevant information for biomedical diagnostics. The goal of this protocol is to create nanosensors that probe for protease activity in vivo by producing a quantifiable signal in urine. These protease nanosensors consist of two components: a nanoparticle and substrate. The nanoparticle functions to increase circulation half-life and substrate delivery to target disease sites. The substrate is a short peptide sequence (6-8 AA), which is designed to be specific to a target protease or group of proteases. The substrate is conjugated to the surface of the nanoparticle and is terminated by a reporter, such as a fluorescent marker, for detection. As dysregulated proteases cleave the peptide substrate, the reporter is filtered into urine for quantification as a biomarker of protease activity. Herein we describe construction of a nanosensor for matrix metalloproteinase 9 (MMP9), which is associated with tumor progression and metastasis, for detection of colorectal cancer in a mouse model.

Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics

Proteases are tightly regulated enzymes involved in fundamental biological processes, and dysregulated protease activity drives progression of complex diseases such as cancer. This method&#39;s goal is to create nanosensors that measure protease activity in vivo by producing a cleavage signal that is detectable from host urine and discriminates disease.

Proteases are multi-functional enzymes that specialize in the hydrolysis of peptide-bonds and control broad biological processes including homeostasis and ...

Using Nanoplasmon-Enhanced Scattering and Low-Magnification Microscope Imaging to Quantify Tumor-Derived Exosomes

Infected or malignant cells frequently secrete more exosomes, leading to elevated levels of disease-associated exosomes in the circulation. These exosomes have the potential to serve as biomarkers for disease diagnosis and to monitor disease progression and treatment response. However, most exosome analysis procedures require exosome isolation and purification steps, which are usually time-consuming and labor-intensive, and thus of limited utility in clinical settings. This report describes a rapid procedure to analyze specific biomarkers on the outer membrane of exosomes without requiring separate isolation and purification steps. In this method, exosomes are captured on the surface of a slide by exosome-specific antibodies and then hybridized with nanoparticle-conjugated antibody probes specific to a disease. After hybridization, the abundance of the target exosome population is determined by analyzing low-magnification dark-field microscope (LMDFM) images of the bound nanoparticles. This approach can be easily adopted for research and clinical use to analyze membrane-associated exosome biomarkers linked to disease.

Clinical translation of exosome-derived biomarkers for diseased and malignant cells is hindered by the lack of rapid and accurate quantification methods. This report describes the use of low-magnification dark-field microscope images to quantify specific exosome subtypes in small volume serum or plasma samples.

Infected or malignant cells frequently secrete more exosomes, leading to elevated levels of disease-associated exosomes in the circulation. These exosomes ...

Measuring the Spin-Lattice Relaxation Magnetic Field Dependence of Hyperpolarized [1-13C]pyruvate

The fundamental limit to in vivo imaging applications of hyperpolarized 13C-enriched compounds is their finite spin-lattice relaxation times. Various factors affect the relaxation rates, such as buffer composition, solution pH, temperature, and magnetic field. In this last regard, the spin-lattice relaxation time can be measured at clinical field strengths, but at lower fields, where these compounds are dispensed from the polarizer and transported to the MRI, the relaxation is even faster and difficult to measure. To have a better understanding of the amount of magnetization lost during transport, we used fast field-cycling relaxometry, with magnetic resonance detection of 13C nuclei at ~0.75 T, to measure the nuclear magnetic resonance dispersion of the spin-lattice relaxation time of hyperpolarized [1-13C]pyruvate. Dissolution dynamic nuclear polarization was used to produce hyperpolarized samples of pyruvate at a concentration of 80 mmol/L and physiological pH (~7.8). These solutions were rapidly transferred to a fast field-cycling relaxometer so that relaxation of the sample magnetization could be measured as a function of time using a calibrated small flip angle (3&#176;-5&#176;). To map the T1 dispersion of the C-1 of pyruvate, we recorded data for different relaxation fields ranging between 0.237 mT and 0.705 T. With this information, we determined an empirical equation to estimate the spin-lattice relaxation of the hyperpolarized substrate within the mentioned range of magnetic fields. These results can be used to predict the amount of magnetization lost during transport and to improve experimental designs to minimize signal loss.

Measuring the Spin-Lattice Relaxation Magnetic Field Dependence of Hyperpolarized [1-13C]pyruvate

We present a protocol to measure the magnetic field dependence of the spin-lattice relaxation time of 13C-enriched compounds, hyperpolarized by means of dynamic nuclear polarization, using fast field-cycled relaxometry. Specifically, we have demonstrated this with [1-13C]pyruvate, but the protocol could be extended to other hyperpolarized substrates.

The fundamental limit to in vivo imaging applications of hyperpolarized 13C-enriched compounds is their finite spin-lattice relaxation times. Various ...

High-Dimensionality Flow Cytometry for Immune Function Analysis of Dissected Implant Tissues

The success of implanting laboratory-grown tissue or a medical device in an individual is subject to the immune response of the recipient host. Considering an implant as a foreign body, a hostile and dysregulated immune response may result in the rejection of the implant, while a regulated response and regaining of homeostasis can lead to its acceptance. Analyzing the microenvironments of implants dissected out under in vivo or ex vivo&#160;settings can help in understanding the pattern of immune response, which can ultimately help in developing new generations of biomaterials. Flow cytometry is a well-known technique for characterizing immune cells and their subsets based on their cell surface markers. This review describes a protocol based on manual dicing, enzymatic digestion, and filtration through a cell strainer for the isolation of uniform cell suspensions from dissected implant tissue. Further, a multicolor flow cytometry staining protocol has been explained, along with steps for initial cytometer settings to characterize and quantify these isolated cells by flow cytometry.

Isolation of cells from dissected implants and their characterization by flow cytometry can significantly contribute to understanding the pattern of immune response against implants. This paper describes a precise method for the isolation of cells from dissected implants and their staining for flow cytometric analysis.

The success of implanting laboratory-grown tissue or a medical device in an individual is subject to the immune response of the recipient host. ...

Quantifying Fibrillar Collagen Organization with Curvelet Transform-Based Tools

Fibrillar collagens are prominent extracellular matrix (ECM) components, and their topology changes have been shown to be associated with the progression of a wide range of diseases including breast, ovarian, kidney, and pancreatic cancers. Freely available fiber quantification software tools are mainly focused on the calculation of fiber alignment or orientation, and they are subject to limitations such as the requirement of manual steps, inaccuracy in detection of the fiber edge in noisy background, or lack of localized feature characterization. The collagen fiber quantitation tool described in this protocol is characterized by using an optimal multiscale image representation enabled by curvelet transform (CT). This algorithmic approach allows for the removal of noise from fibrillar collagen images and the enhancement of fiber edges to provide location and orientation information directly from a fiber, rather than using the indirect pixel-wise or window-wise information obtained from other tools. This CT-based framework contains two separate, but linked, packages named &#8220;CT-FIRE&#8221; and &#8220;CurveAlign&#8221; that can quantify fiber organization on a global, region of interest (ROI), or individual fiber basis. This quantification framework has been developed for more than ten years and has now evolved into a comprehensive and user-driven collagen quantification platform. Using this platform, one can measure up to about thirty fiber features including individual fiber properties such as length, angle, width, and straightness, as well as bulk measurements such as density and alignment. Additionally, the user can measure fiber angle relative to manually or automatically segmented boundaries. This platform also provides several additional modules including ones for ROI analysis, automatic boundary creation, and post-processing. Using this platform does not require prior experience of programming or image processing, and it can handle large datasets including hundreds or thousands of images, enabling efficient quantification of collagen fiber organization for biological or biomedical applications.

Here, we present a protocol to use a curvelet transform-based, open-source MATLAB software tool for quantifying fibrillar collagen organization in the extracellular matrix of both normal and diseased tissues. This tool can be applied to images with collagen fibers or other types of line-like structures.

Fibrillar collagens are prominent extracellular matrix (ECM) components, and their topology changes have been shown to be associated with the progression ...

Electric and Magnetic Field Devices for Stimulation of Biological Tissues

Electric fields (EFs) and magnetic fields (MFs) have been widely used by tissue engineering to improve cell dynamics such as proliferation, migration, differentiation, morphology, and molecular synthesis. However, variables such stimuli strength and stimulation times need to be considered when stimulating either cells, tissues or scaffolds. Given that EFs and MFs vary according to cellular response, it remains unclear how to build devices that generate adequate biophysical stimuli to stimulate biological samples. In fact, there is a lack of evidence regarding the calculation and distribution when biophysical stimuli are applied. This protocol is focused on the design and manufacture of devices to generate EFs and MFs and implementation of a computational methodology to predict biophysical stimuli distribution inside and outside of biological samples. The EF device was composed of two parallel stainless-steel electrodes located at the top and bottom of biological cultures. Electrodes were connected to an oscillator to generate voltages (50, 100, 150 and 200 Vp-p) at 60 kHz. The MF device was composed of a coil, which was energized with a transformer to generate a current (1 A) and voltage (6 V) at 60 Hz. A polymethyl methacrylate support was built to locate the biological cultures in the middle of the coil. The computational simulation elucidated the homogeneous distribution of EFs and MFs inside and outside of biological tissues. This computational model is a promising tool that can modify parameters such as voltages, frequencies, tissue morphologies, well plate types, electrodes and coil size to estimate the EFs and MFs to achieve a cellular response.

This protocol describes the step-by-step process to build both electrical and magnetic stimulators used to stimulate biological tissues. The protocol includes a guideline to simulate computationally electric and magnetic fields and manufacture of stimulator devices.

Electric fields (EFs) and magnetic fields (MFs) have been widely used by tissue engineering to improve cell dynamics such as proliferation, migration, ...

Quantification of Mouse Heart Left Ventricular Function, Myocardial Strain, and Hemodynamic Forces by Cardiovascular Magnetic Resonance Imaging

Mouse models have contributed significantly to understanding genetic and physiological factors involved in healthy cardiac function, how perturbations result in pathology, and how myocardial diseases may be treated. Cardiovascular magnetic resonance imaging (CMR) has become an indispensable tool for a comprehensive in vivo assessment of cardiac anatomy and function. This protocol shows detailed measurements of mouse heart left ventricular function, myocardial strain, and hemodynamic forces using 7-Tesla CMR. First, animal preparation and positioning in the scanner are demonstrated. Survey scans are performed for planning imaging slices in various short- and long-axis views. A series of prospective ECG-triggered short-axis (SA) movies (or CINE images) are acquired covering the heart from apex to base, capturing end-systolic and end-diastolic phases. Subsequently, single-slice, retrospectively gated CINE images are acquired in a midventricular SA view, and in 2-, 3-, and 4-chamber views, to be reconstructed into high-temporal resolution CINE images using custom-built and open-source software. CINE images are subsequently analyzed using dedicated CMR image analysis software.
Delineating endomyocardial and epicardial borders in SA end-systolic and end-diastolic CINE images allows for the calculation of end-systolic and end-diastolic volumes, ejection fraction, and cardiac output. The midventricular SA CINE images are delineated for all cardiac time frames to extract a detailed volume-time curve. Its time derivative allows for the calculation of the diastolic function as the ratio of the early filling and atrial contraction waves. Finally, left ventricular endocardial walls in the 2-, 3-, and 4-chamber views are delineated using feature-tracking, from which longitudinal myocardial strain parameters and left ventricular hemodynamic forces are calculated. In conclusion, this protocol provides detailed in vivo quantification of the mouse cardiac parameters, which can be used to study temporal alterations in cardiac function in various mouse models of heart disease.

This study describes a comprehensive cardiovascular magnetic resonance imaging (CMR) protocol to quantify the left ventricular functional parameters of the mouse heart. The protocol describes the acquisition, post-processing, and analysis of the CMR images as well as assessment of different cardiac functional parameters.

Mouse models have contributed significantly to understanding genetic and physiological factors involved in healthy cardiac function, how perturbations ...