Computational Image Analysis of Guided Acoustic Waves Enables Rheological Assessment of Sub-nanoliter Volumes.

We present a method for the computational image analysis of high frequency guided sound waves based upon the measurement of optical interference fringes, produced at the air interface of a thin film of liquid. These acoustic actuations induce an affine deformation of the liquid, creating a lensing effect that can be readily observed using a simple imaging system. We exploit this effect to measure and analyze the spatiotemporal behavior of the thin liquid film as the acoustic wave interacts with it. We also show that, by investigating the dynamics of the relaxation processes of these deformations when actuation ceases, we are able to determine the liquid's viscosity using just a lens-free imaging system and a simple disposable biochip. Contrary to all other acoustic-based techniques in rheology, our measurements do not require monitoring of the wave parameters to obtain quantitative values for fluid viscosities, for sample volumes as low as 200 pL. We envisage that the proposed methods could enable high throughput, chip-based, reagent-free rheological studies within very small samples.

Ferrodrop Dose-Optimized Digital Quantification of Biomolecules in Low-Volume Samples.

We present an approach to estimate the concentration of a biomolecule in a solution by sampling several nanoliter-scale volumes and determining if the volumes contain any biomolecules. In this method, varying volume fractions (nanoliter-scale) of a sample of nucleic acids are introduced to an array of uniform volume reaction wells (100 µL), which are then fluorescently imaged to determine if signal is above a threshold after nucleic acid amplification, all without complex instrumentation. The nanoliter volumes are generated and introduced using the simple positioning of a permanent magnet, and imaging is performed with a cellphone-based fluorescence detection scheme, both methods suitable for limited-resource settings. We use the length of time a magnetic field is applied to generate a calibrated number of nanoliter ferrodrops of sample mixed with ferrofluid at a step emulsification microfluidic junction. Each dose of ferrodrops is then transferred into larger microliter scale reaction wells on chip through a simple shift of the external magnet. Nucleic acid amplification is achieved using loop-mediated isothermal amplification (LAMP). By repeating each nanoliter dosage a number of times to calculate the probability of a positive signal at each dosage, we can use a binomial probability distribution to estimate the sample nucleic acid concentration. Using this approach we demonstrate detection of lambda DNA molecules down to 25 copies per microliter. The ability to dose separate nanoliter-scale volumes of a low-volume sample across wells in this platform is suited for multiplexed assays. This platform has the potential to be applied to a range of diseases by mixing a sample with magnetic nanoparticles.

Evaluation of a Mobile Phone-Based Microscope for Screening of Schistosoma haematobium Infection in Rural Ghana.

AbstractSchistosomiasis affects over 170 million people in Africa. Here we compare a novel, low-cost mobile phone microscope to a conventional light microscope for the label-free diagnosis of Schistosoma haematobium infections in a rural Ghanaian school setting. We tested the performance of our handheld microscope using 60 slides that were randomly chosen from an ongoing epidemiologic study in school-aged children. The mobile phone microscope had a sensitivity of 72.1% (95% confidence interval [CI]: 56.1-84.2), specificity of 100% (95% CI: 75.9-100), positive predictive value of 100% (95% CI: 86.3-100), and a negative predictive value of 57.1% (95% CI: 37.4-75.0). With its modest sensitivity and high specificity, this handheld and cost-effective mobile phone-based microscope is a stepping-stone toward developing a powerful tool in clinical and public health settings where there is limited access to conventional laboratory diagnostic support.

Highly Stable and Sensitive Nucleic Acid Amplification and Cell-Phone-Based Readout.

Key challenges with point-of-care (POC) nucleic acid tests include achieving a low-cost, portable form factor, and stable readout, while also retaining the same robust standards of benchtop lab-based tests. We addressed two crucial aspects of this problem, identifying a chemical additive, hydroxynaphthol blue, that both stabilizes and significantly enhances intercalator-based fluorescence readout of nucleic acid concentration, and developing a cost-effective fiber-optic bundle-based fluorescence microplate reader integrated onto a mobile phone. Using loop-mediated isothermal amplification on lambda DNA we achieve a 69-fold increase in signal above background, 20-fold higher than the gold standard, yielding an overall limit of detection of 25 copies/µL within an hour using our mobile-phone-based platform. Critical for a point-of-care system, we achieve a >60% increase in fluorescence stability as a function of temperature and time, obviating the need for manual baseline correction or secondary calibration dyes. This field-portable and cost-effective mobile-phone-based nucleic acid amplification and readout platform is broadly applicable to other real-time nucleic acid amplification tests by similarly modulating intercalating dye performance and is compatible with any fluorescence-based assay that can be run in a 96-well microplate format, making it especially valuable for POC and resource-limited settings.

High-throughput and automated diagnosis of antimicrobial resistance using a cost-effective cellphone-based micro-plate reader.

Routine antimicrobial susceptibility testing (AST) can prevent deaths due to bacteria and reduce the spread of multi-drug-resistance, but cannot be regularly performed in resource-limited-settings due to technological challenges, high-costs, and lack of trained professionals. We demonstrate an automated and cost-effective cellphone-based 96-well microtiter-plate (MTP) reader, capable of performing AST without the need for trained diagnosticians. Our system includes a 3D-printed smartphone attachment that holds and illuminates the MTP using a light-emitting-diode array. An inexpensive optical fiber-array enables the capture of the transmitted light of each well through the smartphone camera. A custom-designed application sends the captured image to a server to automatically determine well-turbidity, with results returned to the smartphone in ~1 minute. We tested this mobile-reader using MTPs prepared with 17 antibiotics targeting Gram-negative bacteria on clinical isolates of Klebsiella pneumoniae, containing highly-resistant antimicrobial profiles. Using 78 patient isolate test-plates, we demonstrated that our mobile-reader meets the FDA-defined AST criteria, with a well-turbidity detection accuracy of 98.21%, minimum-inhibitory-concentration accuracy of 95.12%, and a drug-susceptibility interpretation accuracy of 99.23%, with no very major errors. This mobile-reader could eliminate the need for trained diagnosticians to perform AST, reduce the cost-barrier for routine testing, and assist in spatio-temporal tracking of bacterial resistance.

Cellphone-Based Hand-Held Microplate Reader for Point-of-Care Testing of Enzyme-Linked Immunosorbent Assays.

Standard microplate based enzyme-linked immunosorbent assays (ELISA) are widely utilized for various nanomedicine, molecular sensing, and disease screening applications, and this multiwell plate batched analysis dramatically reduces diagnosis costs per patient compared to nonbatched or nonstandard tests. However, their use in resource-limited and field-settings is inhibited by the necessity for relatively large and expensive readout instruments. To mitigate this problem, we created a hand-held and cost-effective cellphone-based colorimetric microplate reader, which uses a 3D-printed opto-mechanical attachment to hold and illuminate a 96-well plate using a light-emitting-diode (LED) array. This LED light is transmitted through each well, and is then collected via 96 individual optical fibers. Captured images of this fiber-bundle are transmitted to our servers through a custom-designed app for processing using a machine learning algorithm, yielding diagnostic results, which are delivered to the user within ∼1 min per 96-well plate, and are visualized using the same app. We successfully tested this mobile platform in a clinical microbiology laboratory using FDA-approved mumps IgG, measles IgG, and herpes simplex virus IgG (HSV-1 and HSV-2) ELISA tests using a total of 567 and 571 patient samples for training and blind testing, respectively, and achieved an accuracy of 99.6%, 98.6%, 99.4%, and 99.4% for mumps, measles, HSV-1, and HSV-2 tests, respectively. This cost-effective and hand-held platform could assist health-care professionals to perform high-throughput disease screening or tracking of vaccination campaigns at the point-of-care, even in resource-poor and field-settings. Also, its intrinsic wireless connectivity can serve epidemiological studies, generating spatiotemporal maps of disease prevalence and immunity.

Imaging and sizing of single DNA molecules on a mobile phone.

DNA imaging techniques using optical microscopy have found numerous applications in biology, chemistry and physics and are based on relatively expensive, bulky and complicated set-ups that limit their use to advanced laboratory settings. Here we demonstrate imaging and length quantification of single molecule DNA strands using a compact, lightweight and cost-effective fluorescence microscope installed on a mobile phone. In addition to an optomechanical attachment that creates a high contrast dark-field imaging setup using an external lens, thin-film interference filters, a miniature dovetail stage and a laser-diode for oblique-angle excitation, we also created a computational framework and a mobile phone application connected to a server back-end for measurement of the lengths of individual DNA molecules that are labeled and stretched using disposable chips. Using this mobile phone platform, we imaged single DNA molecules of various lengths to demonstrate a sizing accuracy of <1 kilobase-pairs (kbp) for 10 kbp and longer DNA samples imaged over a field-of-view of ∼2 mm2.

Cost-effective and rapid blood analysis on a cell-phone.

We demonstrate a compact and cost-effective imaging cytometry platform installed on a cell-phone for the measurement of the density of red and white blood cells as well as hemoglobin concentration in human blood samples. Fluorescent and bright-field images of blood samples are captured using separate optical attachments to the cell-phone and are rapidly processed through a custom-developed smart application running on the phone for counting of blood cells and determining hemoglobin density. We evaluated the performance of this cell-phone based blood analysis platform using anonymous human blood samples and achieved comparable results to a standard bench-top hematology analyser. Test results can either be stored on the cell-phone memory or be transmitted to a central server, providing remote diagnosis opportunities even in field settings.

Cost-effective and compact wide-field fluorescent imaging on a cell-phone.

We demonstrate wide-field fluorescent and darkfield imaging on a cell-phone with compact, light-weight and cost-effective optical components that are mechanically attached to the existing camera unit of the cell-phone. For this purpose, we used battery powered light-emitting diodes (LEDs) to pump the sample of interest from the side using butt-coupling, where the pump light was guided within the sample cuvette to uniformly excite the specimen. The fluorescent emission from the sample was then imaged using an additional lens that was positioned right in front of the existing lens of the cell-phone camera. Because the excitation occurs through guided waves that propagate perpendicular to our detection path, an inexpensive plastic colour filter was sufficient to create the dark-field background required for fluorescent imaging, without the need for a thin-film interference filter. We validate the performance of this platform by imaging various fluorescent micro-objects in 2 colours (i.e., red and green) over a large field-of-view (FOV) of ∼81 mm(2) with a raw spatial resolution of ∼20 µm. With additional digital processing of the captured cell-phone images, through the use of compressive sampling theory, we demonstrate ∼2 fold improvement in our resolving power, achieving ∼10 µm resolution without a trade-off in our FOV. Further, we also demonstrate darkfield imaging of non-fluorescent specimen using the same interface, where this time the scattered light from the objects is detected without the use of any filters. The capability of imaging a wide FOV would be exceedingly important to probe large sample volumes (e.g., >0.1 mL) of e.g., blood, urine, sputum or water, and for this end we also demonstrate fluorescent imaging of labeled white-blood cells from whole blood samples, as well as water-borne pathogenic protozoan parasites such as Giardia Lamblia cysts. Weighing only ∼28 g (∼1 ounce), this compact and cost-effective fluorescent imaging platform attached to a cell-phone could be quite useful especially for resource-limited settings, and might provide an important tool for wide-field imaging and quantification of various lab-on-a-chip assays developed for global health applications, such as monitoring of HIV+ patients for CD4 counts or viral load measurements.

Wide-field fluorescent microscopy on a cell-phone.

We demonstrate wide-field fluorescent imaging on a cell-phone, using compact and cost-effective optical components that are mechanically attached to the existing camera unit of the cell-phone. Battery powered light-emitting diodes (LEDs) are used to side-pump the sample of interest using butt-coupling. The pump light is guided within the sample cuvette to excite the specimen uniformly. The fluorescent emission from the sample is then imaged with an additional lens that is put in front of the existing lens of the cell-phone camera. Because the excitation occurs through guided waves that propagate perpendicular to the detection path, an inexpensive plastic color filter is sufficient to create the dark-field background needed for fluorescent imaging. The imaging performance of this light-weight platform (~28 grams) is characterized with red and green fluorescent microbeads, achieving an imaging field-of-view of ~81 mm(2) and a spatial resolution of ~10 µm, which is enhanced through digital processing of the captured cell-phone images using compressive sampling based sparse signal recovery. We demonstrate the performance of this cell-phone fluorescent microscope by imaging labeled white-blood cells separated from whole blood samples as well as water-borne pathogenic protozoan parasites such as Giardia Lamblia cysts.

Detection of waterborne parasites using field-portable and cost-effective lensfree microscopy.

Protection of human health and well-being through water quality management is an important goal for both the developed and the developing parts of the world. In the meantime, insufficient disinfection techniques still fail to eliminate pathogenic contaminants in freshwater as well as recreational water resources. Therefore, there is a significant need for screening of water quality to prevent waterborne outbreaks and incidents of water-related diseases. Toward this end, here we investigate the use of a field-portable and cost-effective lensfree holographic microscope to image and detect pathogenic protozoan parasites such as Giardia Lamblia and Cryptosporidium Parvum at low concentration levels. This compact lensless microscope (O. Mudanyali et al., Lab Chip, 2010, 10, 1417-1428), weighing approximately 46 grams, achieves a numerical aperture of approximately 0.1-0.2 over an imaging field of view that is more than an order of magnitude larger than a typical 10X objective lens, and therefore may provide an important high-throughput analysis tool for combating waterborne diseases especially in resource limited settings.

Compact, light-weight and cost-effective microscope based on lensless incoherent holography for telemedicine applications.

Despite the rapid progress in optical imaging, most of the advanced microscopy modalities still require complex and costly set-ups that unfortunately limit their use beyond well equipped laboratories. In the meantime, microscopy in resource-limited settings has requirements significantly different from those encountered in advanced laboratories, and such imaging devices should be cost-effective, compact, light-weight and appropriately accurate and simple to be usable by minimally trained personnel. Furthermore, these portable microscopes should ideally be digitally integrated as part of a telemedicine network that connects various mobile health-care providers to a central laboratory or hospital. Toward this end, here we demonstrate a lensless on-chip microscope weighing approximately 46 grams with dimensions smaller than 4.2 cm x 4.2 cm x 5.8 cm that achieves sub-cellular resolution over a large field of view of approximately 24 mm(2). This compact and light-weight microscope is based on digital in-line holography and does not need any lenses, bulky optical/mechanical components or coherent sources such as lasers. Instead, it utilizes a simple light-emitting-diode (LED) and a compact opto-electronic sensor-array to record lensless holograms of the objects, which then permits rapid digital reconstruction of regular transmission or differential interference contrast (DIC) images of the objects. Because this lensless incoherent holographic microscope has orders-of-magnitude improved light collection efficiency and is very robust to mechanical misalignments it may offer a cost-effective tool especially for telemedicine applications involving various global health problems in resource limited settings.