Imaging without lenses: achievements and remaining challenges of wide-field on-chip microscopy.

We discuss unique features of lens-free computational imaging tools and report some of their emerging results for wide-field on-chip microscopy, such as the achievement of a numerical aperture (NA) of ∼0.8-0.9 across a field of view (FOV) of more than 20 mm(2) or an NA of ∼0.1 across a FOV of ∼18 cm(2), which corresponds to an image with more than 1.5 gigapixels. We also discuss the current challenges that these computational on-chip microscopes face, shedding light on their future directions and applications.

Cellphone-based devices for bioanalytical sciences.

During the last decade, there has been a rapidly growing trend toward the use of cellphone-based devices (CBDs) in bioanalytical sciences. For example, they have been used for digital microscopy, cytometry, read-out of immunoassays and lateral flow tests, electrochemical and surface plasmon resonance based bio-sensing, colorimetric detection and healthcare monitoring, among others. Cellphone can be considered as one of the most prospective devices for the development of next-generation point-of-care (POC) diagnostics platforms, enabling mobile healthcare delivery and personalized medicine. With more than 6.5 billion cellphone subscribers worldwide and approximately 1.6 billion new devices being sold each year, cellphone technology is also creating new business and research opportunities. Many cellphone-based devices, such as those targeted for diabetic management, weight management, monitoring of blood pressure and pulse rate, have already become commercially-available in recent years. In addition to such monitoring platforms, several other CBDs are also being introduced, targeting e.g., microscopic imaging and sensing applications for medical diagnostics using novel computational algorithms and components already embedded on cellphones. This report aims to review these recent developments in CBDs for bioanalytical sciences along with some of the challenges involved and the future opportunities.

Lensfree super-resolution holographic microscopy using wetting films on a chip.

We investigate the use of wetting films to significantly improve the imaging performance of lensfree pixel super-resolution on-chip microscopy, achieving < 1 µm spatial resolution over a large imaging area of ~24 mm(2). Formation of an ultra-thin wetting film over the specimen effectively creates a micro-lens effect over each object, which significantly improves the signal-to-noise-ratio and therefore the resolution of our lensfree images. We validate the performance of this approach through lensfree on-chip imaging of various objects having fine morphological features (with dimensions of e.g., ≤0.5 µm) such as Escherichia coli (E. coli), human sperm, Giardia lamblia trophozoites, polystyrene micro beads as well as red blood cells. These results are especially important for the development of highly sensitive field-portable microscopic analysis tools for resource limited settings.

Lensfree On-Chip Microscopy and Tomography for Bio-Medical Applications.

Lensfree on-chip holographic microscopy is an emerging technique that offers imaging of biological specimens over a large field-of-view without using any lenses or bulky optical components. Lending itself to a compact, cost-effective and mechanically robust architecture, lensfree on-chip holographic microscopy can offer an alternative toolset addressing some of the emerging needs of microscopic analysis and diagnostics in low-resource settings, especially for telemedicine applications. In this review, we summarize the latest achievements in lensfree optical microscopy based on partially coherent on-chip holography, including portable telemedicine microscopy, cell-phone based microscopy and field-portable optical tomographic microscopy. We also discuss some of the future directions for telemedicine microscopy and its prospects to help combat various global health challenges.

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.

Color and monochrome lensless on-chip imaging of Caenorhabditis elegans over a wide field-of-view.

We demonstrate color and monochrome on-chip imaging of Caenorhabditis elegans samples over a wide field-of-view using incoherent lensless in-line holography. Digital reconstruction of the recorded lensless holograms rapidly creates the C. elegans images within <1 s over a field-of-view of >24 mm2. By digitally combining the reconstructed images at three different wavelengths (red, green and blue), color images of dyed samples are also acquired. This wide field-of-view and compact on-chip imaging modality also permits straightforward integration with microfluidic systems.

Lensfree microscopy on a cellphone.

We demonstrate lensfree digital microscopy on a cellphone. This compact and light-weight holographic microscope installed on a cellphone does not utilize any lenses, lasers or other bulky optical components and it may offer a cost-effective tool for telemedicine applications to address various global health challenges. Weighing approximately 38 grams (<1.4 ounces), this lensfree imaging platform can be mechanically attached to the camera unit of a cellphone where the samples are loaded from the side, and are vertically illuminated by a simple light-emitting diode (LED). This incoherent LED light is then scattered from each micro-object to coherently interfere with the background light, creating the lensfree hologram of each object on the detector array of the cellphone. These holographic signatures captured by the cellphone permit reconstruction of microscopic images of the objects through rapid digital processing. We report the performance of this lensfree cellphone microscope by imaging various sized micro-particles, as well as red blood cells, white blood cells, platelets and a waterborne parasite (Giardia lamblia).

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.

High-throughput lens-free blood analysis on a chip.

We present a detailed investigation of the performance of lens-free holographic microscopy toward high-throughput on-chip blood analysis. Using a spatially incoherent source that is emanating from a large aperture, automated counting of red blood cells with minimal sample preparation steps at densities reaching up to approximately 0.4 x 10(6) cells/muL is presented. Using the same lens-free holographic microscopy platform, we also characterize the volume of the red blood cells at the single-cell level through recovery of the optical phase information of each cell. We further demonstrate the measurement of the hemoglobin concentration of whole blood samples as well as automated counting of white blood cells, also yielding spatial resolution at the subcellular level sufficient to differentiate granulocytes, monocytes, and lymphocytes from each other. These results uncover the prospects of lens-free holographic on-chip imaging to provide a useful tool for global health problems, especially by facilitating whole blood analysis in resource-poor environments.

Lensfree holographic imaging of antibody microarrays for high-throughput detection of leukocyte numbers and function.

Characterization of leukocytes is an integral part of blood analysis and blood-based diagnostics. In the present paper, we combine lensless holographic imaging with antibody microarrays for rapid and multiparametric analysis of leukocytes from human blood. Monoclonal antibodies (Abs) specific for leukocyte surface antigens (CD4 and CD8) and cytokines (TNF-alpha, IFN-gamma, IL-2) were printed in an array so as to juxtapose cell capture and cytokine detection antibody (Ab) spots. Integration of Ab microarrays into a microfluidic flow chamber (4 muL volume) followed by incubation with human blood resulted in capture of CD4 and CD8 T-cells on specific Ab spots. On-chip mitogenic activation of these cells induced release of cytokine molecules that were subsequently captured on neighboring anticytokine Ab spots. The binding of IL-2, TNF-alpha, and IFN-gamma molecules on their respective Ab spots was detected using horseradish peroxidase (HRP)-labeled anticytokine Abs and a visible color reagent. Lensfree holographic imaging was then used to rapidly ( approximately 4 s) enumerate CD4 and CD8 T-lymphocytes captured on Ab spots and to quantify the cytokine signal emanating from IL-2, TNF-alpha, and IFN-gamma spots on the same chip. To demonstrate the utility of our approach for infectious disease monitoring, blood samples of healthy volunteers and human immunodeficiency virus (HIV)-infected patients were analyzed to determine the CD4/CD8 ratio, an important HIV/AIDS diagnostic marker. The ratio obtained by lensfree on-chip imaging of CD4 and CD8 T-cells captured on Ab spots was in close agreement with conventional microscopy-based cell counting. The present paper, describing tandem use of Ab microarrays and lensfree holographic imaging, paves the way for future development of miniature cytometry devices for multiparametric blood analysis at the point of care or in a resource-limited setting.

A Smartphone-Based Rapid Telemonitoring System for Ebola and Marburg Disease Surveillance.

We have developed a digital and multiplexed platform for the rapid detection and telemonitoring of infections caused by Ebola and Marburg filoviruses. The system includes a flow cell assay cartridge that captures specific antibodies with microarrayed recombinant antigens from all six species of filovirus, and a smartphone fluorescent reader for high-performance interpretation of test results. Multiplexed viral proteins, which are expandable to include greater numbers of probes, were incorporated to obtain highest confidence results by cross-correlation, and a custom smartphone application was developed for data analysis, interpretation, and communication. The smartphone reader utilizes an opto-electro-mechanical hardware attachment that snaps at the back of a Motorola smartphone and provides a user interface to manage the operation, acquire test results, and communicate with cloud service. The application controls the hardware attachment to turn on LEDs and digitally record the optically enhanced images. Assay processing time is approximately 20 min for microliter amounts of blood, and test results are digitally processed and displayed within 15 s. Furthermore, a secure cloud service was developed for the telemonitoring of test results generated by the smartphone readers in the field. Assay system results were tested with sera from nonhuman primates that received a live attenuated EBOV vaccine. This integrated system will provide a rapid, reliable, and digital solution to prevent the rapid overwhelming of medical systems and resources during EVD or MVD outbreaks. Further, this disease-monitoring system will be useful in resource-limited countries where there is a need for dispersed laboratory analysis of recent or active infections.

Automated single-cell motility analysis on a chip using lensfree microscopy.

Quantitative cell motility studies are necessary for understanding biophysical processes, developing models for cell locomotion and for drug discovery. Such studies are typically performed by controlling environmental conditions around a lens-based microscope, requiring costly instruments while still remaining limited in field-of-view. Here we present a compact cell monitoring platform utilizing a wide-field (24 mm(2)) lensless holographic microscope that enables automated single-cell tracking of large populations that is compatible with a standard laboratory incubator. We used this platform to track NIH 3T3 cells on polyacrylamide gels over 20 hrs. We report that, over an order of magnitude of stiffness values, collagen IV surfaces lead to enhanced motility compared to fibronectin, in agreement with biological uses of these structural proteins. The increased throughput associated with lensfree on-chip imaging enables higher statistical significance in observed cell behavior and may facilitate rapid screening of drugs and genes that affect cell motility.

Toward giga-pixel nanoscopy on a chip: a computational wide-field look at the nano-scale without the use of lenses.

The development of lensfree on-chip microscopy in the past decade has opened up various new possibilities for biomedical imaging across ultra-large fields of view using compact, portable, and cost-effective devices. However, until recently, its ability to resolve fine features and detect ultra-small particles has not rivalled the capabilities of the more expensive and bulky laboratory-grade optical microscopes. In this Frontier Review, we highlight the developments over the last two years that have enabled computational lensfree holographic on-chip microscopy to compete with and, in some cases, surpass conventional bright-field microscopy in its ability to image nano-scale objects across large fields of view, yielding giga-pixel phase and amplitude images. Lensfree microscopy has now achieved a numerical aperture as high as 0.92, with a spatial resolution as small as 225 nm across a large field of view e.g., >20 mm(2). Furthermore, the combination of lensfree microscopy with self-assembled nanolenses, forming nano-catenoid minimal surfaces around individual nanoparticles has boosted the image contrast to levels high enough to permit bright-field imaging of individual particles smaller than 100 nm. These capabilities support a number of new applications, including, for example, the detection and sizing of individual virus particles using field-portable computational on-chip microscopes.

Optical imaging techniques for point-of-care diagnostics.

Improving access to effective and affordable healthcare has long been a global endeavor. In this quest, the development of cost-effective and easy-to-use medical testing equipment that enables rapid and accurate diagnosis is essential to reduce the time and costs associated with healthcare services. To this end, point-of-care (POC) diagnostics plays a crucial role in healthcare delivery in both developed and developing countries by bringing medical testing to patients, or to sites near patients. As the diagnosis of a wide range of diseases, including various types of cancers and many endemics, relies on optical techniques, numerous compact and cost-effective optical imaging platforms have been developed in recent years for use at the POC. Here, we review the state-of-the-art optical imaging techniques that can have a significant impact on global health by facilitating effective and affordable POC diagnostics.

Lensfree computational microscopy tools for cell and tissue imaging at the point-of-care and in low-resource settings.

The recent revolution in digital technologies and information processing methods present important opportunities to transform the way optical imaging is performed, particularly toward improving the throughput of microscopes while at the same time reducing their relative cost and complexity. Lensfree computational microscopy is rapidly emerging toward this end, and by discarding lenses and other bulky optical components of conventional imaging systems, and relying on digital computation instead, it can achieve both reflection and transmission mode microscopy over a large field-of-view within compact, cost-effective and mechanically robust architectures. Such high throughput and miniaturized imaging devices can provide a complementary toolset for telemedicine applications and point-of-care diagnostics by facilitating complex and critical tasks such as cytometry and microscopic analysis of e.g., blood smears, Papanicolaou (Pap) tests and tissue samples. In this article, the basics of these lensfree microscopy modalities will be reviewed, and their clinically relevant applications will be discussed.

Optical detection and sizing of single nanoparticles using continuous wetting films.

The physical interaction between nanoscale objects and liquid interfaces can create unique optical properties, enhancing the signatures of the objects with subwavelength features. Here we show that the evaporation on a wetting substrate of a polymer solution containing submicrometer or nanoscale particles creates liquid microlenses that arise from the local deformations of the continuous wetting film. These microlenses have properties similar to axicon lenses that are known to create beams with a long depth of focus. This enhanced depth of focus allows detection of single nanoparticles using a low-magnification microscope objective lens, achieving a relatively wide field-of-view, while also lifting the constraints on precise focusing onto the object plane. Hence, by creating these liquid axicon lenses through spatial deformations of a continuous thin wetting film, we transfer the challenge of imaging individual nanoparticles to detecting the light focused by these lenses. As a proof of concept, we demonstrate the detection and sizing of single nanoparticles (100 and 200 nm), CpGV granuloviruses, as well as Staphylococcus epidermidis bacteria over a wide field-of-view of 5.10 × 3.75 mm(2) using a 5× objective lens with a numerical aperture of 0.15. In addition to conventional lens-based microscopy, this continuous wetting-film-based approach is also applicable to lens-free computational on-chip imaging, which can be used to detect single nanoparticles over a large field-of-view of >20-30 mm(2). These results could be especially useful for high-throughput field analysis of nanoscale objects using compact and cost-effective microscope designs.

Wide-field optical detection of nanoparticles using on-chip microscopy and self-assembled nanolenses.

The direct observation of nanoscale objects is a challenging task for optical microscopy because the scattering from an individual nanoparticle is typically weak at optical wavelengths. Electron microscopy therefore remains one of the gold standard visualization methods for nanoparticles, despite its high cost, limited throughput and restricted field-of-view. Here, we describe a high-throughput, on-chip detection scheme that uses biocompatible wetting films to self-assemble aspheric liquid nanolenses around individual nanoparticles to enhance the contrast between the scattered and background light. We model the effect of the nanolens as a spatial phase mask centred on the particle and show that the holographic diffraction pattern of this effective phase mask allows detection of sub-100 nm particles across a large field-of-view of >20 mm2. As a proof-of-concept demonstration, we report on-chip detection of individual polystyrene nanoparticles, adenoviruses and influenza A (H1N1) viral particles.

Integrated rapid-diagnostic-test reader platform on a cellphone.

We demonstrate a cellphone-based rapid-diagnostic-test (RDT) reader platform that can work with various lateral flow immuno-chromatographic assays and similar tests to sense the presence of a target analyte in a sample. This compact and cost-effective digital RDT reader, weighing only ~65 g, mechanically attaches to the existing camera unit of a cellphone, where various types of RDTs can be inserted to be imaged in reflection or transmission modes under light-emitting diode (LED)-based illumination. Captured raw images of these tests are then digitally processed (within less than 0.2 s per image) through a smart application running on the cellphone for validation of the RDT, as well as for automated reading of its diagnostic result. The same smart application then transmits the resulting data, together with the RDT images and other related information (e.g., demographic data), to a central server, which presents the diagnostic results on a world map through geo-tagging. This dynamic spatio-temporal map of various RDT results can then be viewed and shared using internet browsers or through the same cellphone application. We tested this platform using malaria, tuberculosis (TB) and HIV RDTs by installing it on both Android-based smartphones and an iPhone. Providing real-time spatio-temporal statistics for the prevalence of various infectious diseases, this smart RDT reader platform running on cellphones might assist healthcare professionals and policymakers to track emerging epidemics worldwide and help epidemic preparedness.

Lensfree computational microscopy tools for cell and tissue imaging at the point-of-care and in low-resource settings.

The recent revolution in digital technologies and information processing methods present important opportunities to transform the way optical imaging is performed, particularly toward improving the throughput of microscopes while at the same time reducing their relative cost and complexity. Lensfree computational microscopy is rapidly emerging toward this end, and by discarding lenses and other bulky optical components of conventional imaging systems, and relying on digital computation instead, it can achieve both reflection and transmission mode microscopy over a large field-of-view within compact, cost-effective and mechanically robust architectures. Such high throughput and miniaturized imaging devices can provide a complementary toolset for telemedicine applications and point-of-care diagnostics by facilitating complex and critical tasks such as cytometry and microscopic analysis of e.g., blood smears, Pap tests and tissue samples. In this article, the basics of these lensfree microscopy modalities will be reviewed, and their clinically relevant applications will be discussed.

Holographic pixel super-resolution in portable lensless on-chip microscopy using a fiber-optic array.

We report a portable lensless on-chip microscope that can achieve <1 µm resolution over a wide field-of-view of ∼ 24 mm(2) without the use of any mechanical scanning. This compact on-chip microscope weighs ∼ 95 g and is based on partially coherent digital in-line holography. Multiple fiber-optic waveguides are butt-coupled to light emitting diodes, which are controlled by a low-cost micro-controller to sequentially illuminate the sample. The resulting lensfree holograms are then captured by a digital sensor-array and are rapidly processed using a pixel super-resolution algorithm to generate much higher resolution holographic images (both phase and amplitude) of the objects. This wide-field and high-resolution on-chip microscope, being compact and light-weight, would be important for global health problems such as diagnosis of infectious diseases in remote locations. Toward this end, we validate the performance of this field-portable microscope by imaging human malaria parasites (Plasmodium falciparum) in thin blood smears. Our results constitute the first-time that a lensfree on-chip microscope has successfully imaged malaria parasites.