Minimum-phase-function-based Processing in Frequency-domain Optical Coherence Tomography Systems

Journal of the Optical Society of America. A, Optics, Image Science, and Vision. Jul, 2006  |  Pubmed ID: 16783430

We present a simple processing technique that uses the concept of minimum-phase functions to improve frequency-domain optical coherence tomography systems. Our approach removes the autocorrelation noise and therefore increases both the accessible depth range and the recovery accuracy. To our knowledge, this is the first time that the concept of minimum-phase functions has been applied to improve optical coherence tomography.

Differential Near-field Scanning Optical Microscopy

Nano Letters. Nov, 2006  |  Pubmed ID: 17090100

We theoretically and experimentally illustrate a new apertured near-field scanning optical microscopy (NSOM) technique, termed differential NSOM (DNSOM). It involves scanning a relatively large (e.g., 0.3-2 mum wide) rectangular aperture (or a detector) in the near-field of an object and recording detected power as a function of the scanning position. The image reconstruction is achieved by taking a two-dimensional derivative of the recorded power map. Unlike conventional apertured NSOM, the size of the rectangular aperture/detector does not determine the resolution in DNSOM; instead, the resolution is practically determined by the sharpness of the corners of the rectangular aperture/detector. Principles of DNSOM can also be extended to other aperture/detector geometries such as triangles and parallelograms.

Speckle Reduction in Optical Coherence Tomography Images Using Digital Filtering

Journal of the Optical Society of America. A, Optics, Image Science, and Vision. Jul, 2007  |  Pubmed ID: 17728812

Speckle noise is a ubiquitous artifact that limits the interpretation of optical coherence tomography images. Here we apply various speckle-reduction digital filters to optical coherence tomography images and compare their performance. Our results indicate that shift-invariant, nonorthogonal wavelet-transform-based filters together with enhanced Lee and adaptive Wiener filters can significantly reduce speckle and increase the signal-to-noise ratio, while preserving strong edges. The speckle reduction capabilities of these filters are also compared with speckle reduction from incoherent angular compounding. Our results suggest that by using these digital filters, the number of individual angles required to attain a certain level of speckle reduction can be decreased.

Ultra Wide-field Lens-free Monitoring of Cells On-chip

Lab on a Chip. Jan, 2008  |  Pubmed ID: 18094767

We experimentally and theoretically demonstrate the proof-of-principle of a new lens-free cell monitoring platform that involves using an opto-electronic sensor array to record the shadow image of cells onto the sensor plane. This technology can monitor/count cells over a field-of-view that is more than two orders of magnitude larger than that of a conventional light microscope. Furthermore, it does not require any mechanical scanning or optical elements, such as microscope objectives or lenses. We also show that this optical approach can conveniently be combined with microfluidic channels, enabling parallel on-chip monitoring of various different cell types, e.g., blood cells, NIH-3T3 fibroblasts, murine embryonic stem cells, AML-12 hepatocytes. An important application of this approach could be a miniaturized point-of-care technology to obtain CD4 T lymphocyte counts of HIV infected patients in resource limited settings.

Fluorescence Interferometry: Principles and Applications in Biology

Annals of the New York Academy of Sciences. 2008  |  Pubmed ID: 18596334

The use of fluorescence radiation is of fundamental importance for tackling measurement problems in the life sciences, with recent demonstrations of probing biological systems at the nanoscale. Usually, fluorescent light-based tools and techniques use the intensity of light waves, which is easily measured by detectors. However, the phase of a fluorescence wave contains subtle, but no less important, information about the wave; yet, it has been largely unexplored. Here, we introduce the concept of fluorescence interferometry to allow the measurement of phase information of fluorescent light waves. In principle, fluorescence interferometry can be considered a unique form of optical low-coherence interferometry that uses fluorophores as a light source of low temporal coherence. Fluorescence interferometry opens up new avenues for developing new fluorescent light-based imaging, sensing, ranging, and profiling methods that to some extent resemble interferometric techniques based on white light sources. We propose two experimental realizations of fluorescence interferometry that detect the interference pattern cast by the fluorescence fields. This article discusses their measurement capabilities and limitations and compares them with those offered by optical low-coherence interferometric schemes. We also describe applications of fluorescence interferometry to imaging, ranging, and profiling tasks and present experimental evidences of wide-field cross-sectional imaging with high resolution and large range of depth, as well as quantitative profiling with nanometer-level precision. Finally, we point out future research directions in fluorescence interferometry, such as fluorescence tomography of whole organisms and the extension to molecular interferometry by means of quantum dots and bioluminescence.

Multi-angle LUCAS for High-throughput On-chip Cytometry

Conference Proceedings : ... Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineering in Medicine and Biology Society. Conference. 2008  |  Pubmed ID: 19163046

We illustrate that by recording under-sampled diffraction patterns of cells at different illumination angles, we can achieve high-throughput on-chip characterization of a heterogeneous cell solution over an ultra large volume of approximately 5 ml. This platform, termed multi-angle LUCAS, is especially promising for cost-effective point-of-care cell counting applications.

Lensfree Holographic Imaging for On-chip Cytometry and Diagnostics

Lab on a Chip. Mar, 2009  |  Pubmed ID: 19255659

We experimentally illustrate a lensfree holographic imaging platform to perform on-chip cytometry. By controlling the spatial coherence of the illumination source, we record a 2D holographic diffraction pattern of each cell or micro-particle on a chip using a high resolution sensor array that has approximately 2 microm pixel size. The recorded holographic image is then processed by using a custom developed decision algorithm for matching the detected hologram texture to existing library images for on-chip characterization and counting of a heterogeneous solution of interest. The holographic diffraction signature of any microscopic object is significantly different from the classical diffraction pattern of the same object. It improves the signal to noise ratio and the signature uniformity of the cell patterns; and also exhibits much better sensitivity for on-chip imaging of weakly scattering phase objects such as small bacteria or cells. We verify significantly improved performance of this holographic on-chip cytometry approach by automatically characterizing heterogeneous solutions of red blood cells, yeast cells, E. coli and various sized micro-particles without the use of any lenses or microscope objectives. This lensless on-chip holography platform will especially be useful for point-of-care cytometry and diagnostics applications involving e.g., infectious diseases such as HIV or malaria.

Integrating Microfluidics and Lensless Imaging for Point-of-care Testing

Biosensors & Bioelectronics. Jul, 2009  |  Pubmed ID: 19467854

We demonstrate an integrated platform that merges a microfluidic chip with lensless imaging to target CD4(+) T-lymphocyte counts for HIV point-of-care testing at resource-limited settings. The chips were designed and fabricated simply with a laser cutter without using expensive cleanroom equipment. To capture CD4(+) T-lymphocytes from blood, anti-CD4 antibody was immobilized on only one side of the microfluidic chip. These captured cells were detected through an optically clear chip using a charge coupled device (CCD) sensor by lensless shadow imaging techniques. Gray scale image of the captured cells in a 24 mm x 4 mm x 50 microm microfluidic chip was obtained by the lensless imaging platform. The automatic cell counting software enumerated the captured cells in 3s. Captured cells were also imaged with a fluorescence microscope and manually counted to characterize functionality of the integrated platform. The integrated platform achieved 70.2+/-6.5% capture efficiency, 88.8+/-5.4% capture specificity for CD4(+) T-lymphocytes, 96+/-1.6% CCD efficiency, and 83.5+/-2.4% overall platform performance (n=9 devices) compared to the gold standard, i.e. flow cytometry count. The integrated system gives a CD4 count from blood within 10 min. The integrated platform points a promising direction for point-of-care testing (POCT) to rapidly capture, image and count subpopulations of cells from blood samples in an automated matter.

High-throughput Lensfree Imaging and Characterization of a Heterogeneous Cell Solution on a Chip

Biotechnology and Bioengineering. Feb, 2009  |  Pubmed ID: 18853435

A high-throughput on-chip imaging platform that can rapidly monitor and characterize various cell types within a heterogeneous solution over a depth-of-field of approximately 4 mm and a field-of-view of approximately 10 cm(2) is introduced. This powerful system can rapidly image/monitor multiple layers of cells, within a volume of approximately 4 mL all in parallel without the need for any lenses, microscope-objectives or any mechanical scanning. In this high-throughput lensless imaging scheme, the classical diffraction pattern (i.e., the shadow) of each micro-particle within the entire sample volume is detected in less than a second using an opto-electronic sensor chip. The acquired shadow image is then digitally processed using a custom developed "decision algorithm" to enable both the identification of the particle location in 3D and the characterization of each micro-particle type within the sample volume. Through experimental results, we show that different cell types (e.g., red blood cells, fibroblasts, etc.) or other micro-particles all exhibit uniquely different shadow patterns and therefore can be rapidly identified without any ambiguity using the developed decision algorithm, enabling high-throughput characterization of a heterogeneous solution. This lensfree on chip cell imaging platform shows a significant promise especially for medical diagnostic applications relevant to global health problems, where compact and cost-effective diagnostic tools are urgently needed in resource limited settings.

Lensfree Color Imaging on a Nanostructured Chip Using Compressive Decoding

Applied Physics Letters. Nov, 2010  |  Pubmed ID: 21173866

We demonstrate subpixel level color imaging capability on a lensfree incoherent on-chip microscopy platform. By using a nanostructured substrate, the incoherent emission from the object plane is modulated to create a unique far-field diffraction pattern corresponding to each point at the object plane. These lensfree diffraction patterns are then sampled in the far-field using a color sensor-array, where the pixels have three different types of color filters at red, green, and blue (RGB) wavelengths. The recorded RGB diffraction patterns (for each point on the structured substrate) form a basis that can be used to rapidly reconstruct any arbitrary multicolor incoherent object distribution at subpixel resolution, using a compressive sampling algorithm. This lensfree computational imaging platform could be quite useful to create a compact fluorescent on-chip microscope that has color imaging capability.

Holographic Opto-fluidic Microscopy

Optics Express. Dec, 2010  |  Pubmed ID: 21197025

Over the last decade microfluidics has created a versatile platform that has significantly advanced the ways in which micro-scale organisms and objects are controlled, processed and investigated, by improving the cost, compactness and throughput aspects of analysis. Microfluidics has also expanded into optics to create reconfigurable and flexible optical devices such as reconfigurable lenses, lasers, waveguides, switches, and on-chip microscopes. Here we present a new opto-fluidic microscopy modality, i.e., Holographic Opto-fluidic Microscopy (HOM), based on lensless holographic imaging. This imaging modality complements the miniaturization provided by microfluidics and would allow the integration of microscopy into existing on-chip microfluidic devices with various functionalities. Our imaging modality utilizes partially coherent in-line holography and pixel super-resolution to create high-resolution amplitude and phase images of the objects flowing within micro-fluidic channels, which we demonstrate by imaging C. elegans, Giardia lamblia, and Mulberry pollen. HOM does not involve complicated fabrication processes or precise alignment, nor does it require a highly uniform flow of objects within microfluidic channels.

Lensfree Sensing on a Microfluidic Chip Using Plasmonic Nanoapertures

Applied Physics Letters. Nov, 2010  |  Pubmed ID: 21203381

We demonstrate lensfree on-chip sensing within a microfluidic channel using plasmonic nanoapertures that are illuminated by a partially coherent quasimonochromatic source. In this approach, lensfree diffraction patterns of metallic nanoapertures located at the bottom of a microfluidic channel are recorded using an optoelectronic sensor-array. These lensfree diffraction patterns can then be rapidly processed, using phase recovery techniques, to back propagate the optical fields to an arbitrary depth, creating digitally focused complex transmission patterns. Cross correlation of these patterns enables lensfree on-chip sensing of the local refractive index surrounding the near-field of the plasmonic nanoapertures. Based on this principle, we experimentally demonstrate lensfree sensing of refractive index changes as small as ∼2×10(-3). This on-chip sensing approach could be quite useful for development of label-free microarray technologies by multiplexing thousands of plasmonic structures on the same microfluidic chip, which can significantly increase the throughput of sensing.

Lensfree Fluorescent On-Chip Imaging Using Compressive Sampling

Optics and Photonics News. Dec, 2010  |  Pubmed ID: 21546979

Lensfree Holographic Imaging of Antibody Microarrays for High-throughput Detection of Leukocyte Numbers and Function

Analytical Chemistry. May, 2010  |  Pubmed ID: 20359168

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.

Wide Field-of-view Lens-free Fluorescent Imaging on a Chip

Lab on a Chip. Apr, 2010  |  Pubmed ID: 20379564

We demonstrate an on-chip fluorescent detection platform that can simultaneously image fluorescent micro-objects or labeled cells over an ultra-large field-of-view of 2.5 cm x 3.5 cm without the use of any lenses, thin-film filters and mechanical scanners. Such a wide field-of-view lensless fluorescent imaging modality, despite its limited resolution, might be very important for high-throughput screening applications as well as for detection and counting of rare cells within large-area microfluidic devices.

On-chip Differential Interference Contrast Microscopy Using Lensless Digital Holography

Optics Express. Mar, 2010  |  Pubmed ID: 20389485

We introduce the use of a birefringent crystal with lensless digital holography to create an on-chip differential interference contrast (DIC) microscope. Using an incoherent source with a large aperture, in-line holograms of micro-objects are created, which interact with a uniaxial crystal and an absorbing polarizer, encoding differential interference contrast information of the objects on the chip. Despite the fact that a unit fringe magnification and an incoherent source with a large aperture have been used, holographic digital processing of such holograms rapidly recovers the differential phase contrast image of the specimen over a large field-of-view of approximately 24 mm(2).

Color and Monochrome Lensless On-chip Imaging of Caenorhabditis Elegans over a Wide Field-of-view

Lab on a Chip. May, 2010  |  Pubmed ID: 20390127

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.

Compact, Light-weight and Cost-effective Microscope Based on Lensless Incoherent Holography for Telemedicine Applications

Lab on a Chip. Jun, 2010  |  Pubmed ID: 20401422

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.

Lensfree Microscopy on a Cellphone

Lab on a Chip. Jul, 2010  |  Pubmed ID: 20445943

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).

High-throughput Lens-free Blood Analysis on a Chip

Analytical Chemistry. Jun, 2010  |  Pubmed ID: 20450181

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 On-chip Imaging Using Nanostructured Surfaces

Applied Physics Letters. Apr, 2010  |  Pubmed ID: 20502644

We introduce the use of nanostructured surfaces for lensfree on-chip microscopy. In this incoherent on-chip imaging modality, the object of interest is directly positioned onto a nanostructured thin metallic film, where the emitted light from the object plane, after being modulated by the nanostructures, diffracts over a short distance to be sampled by a detector-array without the use of any lenses. The detected far-field diffraction pattern then permits rapid reconstruction of the object distribution on the chip at the subpixel level using a compressive sampling algorithm. This imaging modality based on nanostructured substrates could especially be useful to create lensfree fluorescent microscopes on a compact chip.

Smart Technology for Global Access to Healthcare

SPIE Newsroom. Mar, 2010  |  Pubmed ID: 20582244

Multi-angle Lensless Digital Holography for Depth Resolved Imaging on a Chip

Optics Express. Apr, 2010  |  Pubmed ID: 20588819

A multi-angle lensfree holographic imaging platform that can accurately characterize both the axial and lateral positions of cells located within multi-layered micro-channels is introduced. In this platform, lensfree digital holograms of the micro-objects on the chip are recorded at different illumination angles using partially coherent illumination. These digital holograms start to shift laterally on the sensor plane as the illumination angle of the source is tilted. Since the exact amount of this lateral shift of each object hologram can be calculated with an accuracy that beats the diffraction limit of light, the height of each cell from the substrate can be determined over a large field of view without the use of any lenses. We demonstrate the proof of concept of this multi-angle lensless imaging platform by using light emitting diodes to characterize various sized microparticles located on a chip with sub-micron axial and lateral localization over approximately 60 mm(2) field of view. Furthermore, we successfully apply this lensless imaging approach to simultaneously characterize blood samples located at multi-layered micro-channels in terms of the counts, individual thicknesses and the volumes of the cells at each layer. Because this platform does not require any lenses, lasers or other bulky optical/mechanical components, it provides a compact and high-throughput alternative to conventional approaches for cytometry and diagnostics applications involving lab on a chip systems.

Lensless Wide-field Fluorescent Imaging on a Chip Using Compressive Decoding of Sparse Objects

Optics Express. May, 2010  |  Pubmed ID: 20588904

We demonstrate the use of a compressive sampling algorithm for on-chip fluorescent imaging of sparse objects over an ultra-large field-of-view (>8 cm(2)) without the need for any lenses or mechanical scanning. In this lensfree imaging technique, fluorescent samples placed on a chip are excited through a prism interface, where the pump light is filtered out by total internal reflection after exciting the entire sample volume. The emitted fluorescent light from the specimen is collected through an on-chip fiber-optic faceplate and is delivered to a wide field-of-view opto-electronic sensor array for lensless recording of fluorescent spots corresponding to the samples. A compressive sampling based optimization algorithm is then used to rapidly reconstruct the sparse distribution of fluorescent sources to achieve approximately 10 microm spatial resolution over the entire active region of the sensor-array, i.e., over an imaging field-of-view of >8 cm(2). Such a wide-field lensless fluorescent imaging platform could especially be significant for high-throughput imaging cytometry, rare cell analysis, as well as for micro-array research.

Lensfree On-chip Microscopy over a Wide Field-of-view Using Pixel Super-resolution

Optics Express. May, 2010  |  Pubmed ID: 20588977

We demonstrate lensfree holographic microscopy on a chip to achieve approximately 0.6 microm spatial resolution corresponding to a numerical aperture of approximately 0.5 over a large field-of-view of approximately 24 mm2. By using partially coherent illumination from a large aperture (approximately 50 microm), we acquire lower resolution lensfree in-line holograms of the objects with unit fringe magnification. For each lensfree hologram, the pixel size at the sensor chip limits the spatial resolution of the reconstructed image. To circumvent this limitation, we implement a sub-pixel shifting based super-resolution algorithm to effectively recover much higher resolution digital holograms of the objects, permitting sub-micron spatial resolution to be achieved across the entire sensor chip active area, which is also equivalent to the imaging field-of-view (24 mm2) due to unit magnification. We demonstrate the success of this pixel super-resolution approach by imaging patterned transparent substrates, blood smear samples, as well as Caenoharbditis Elegans.

Detection of Waterborne Parasites Using Field-portable and Cost-effective Lensfree Microscopy

Lab on a Chip. Sep, 2010  |  Pubmed ID: 20694255

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.

Contributors to the Emerging Investigators Issue

Lab on a Chip. Sep, 2010  |  Pubmed ID: 20717627

Compact and Light-weight Automated Semen Analysis Platform Using Lensfree On-chip Microscopy

Analytical Chemistry. Oct, 2010  |  Pubmed ID: 20836503

We demonstrate a compact and lightweight platform to conduct automated semen analysis using a lensfree on-chip microscope. This holographic on-chip imaging platform weighs ∼46 g, measures ∼4.2 × 4.2 × 5.8 cm, and does not require any lenses, lasers or other bulky optical components to achieve phase and amplitude imaging of sperms over ∼24 mm(2) field-of-view with an effective numerical aperture of ∼0.2. Using this wide-field lensfree on-chip microscope, semen samples are imaged for ∼10 s, capturing a total of ∼20 holographic frames. Digital subtraction of these consecutive lensfree frames, followed by appropriate processing of the reconstructed images, enables automated quantification of the count, the speed and the dynamic trajectories of motile sperms, while summation of the same frames permits counting of immotile sperms. Such a compact and lightweight automated semen analysis platform running on a wide-field lensfree on-chip microscope could be especially important for fertility clinics, personal male fertility tests, as well as for field use in veterinary medicine such as in stud farming and animal breeding applications.

Lensfree On-chip Holography Facilitates Novel Microscopy Applications

SPIE Newsroom. May, 2010  |  Pubmed ID: 21643449

Combined Reflection and Transmission Microscope for Telemedicine Applications in Field Settings

Lab on a Chip. Aug, 2011  |  Pubmed ID: 21709875

We demonstrate a field-portable upright and inverted microscope that can image specimens in both reflection and transmission modes. This compact and cost-effective dual-mode microscope weighs only ∼135 grams (<4.8 ounces) and utilizes a simple light emitting diode (LED) to illuminate the sample of interest using a beam-splitter cube that is positioned above the object plane. This LED illumination is then partially reflected from the sample to be collected by two lenses, creating a reflection image of the specimen onto an opto-electronic sensor-array that is positioned above the beam-splitter cube. In addition to this, the illumination beam is also partially transmitted through the same specimen, which then casts lensfree in-line holograms of the same objects onto a second opto-electronic sensor-array that is positioned underneath the beam-splitter cube. By rapid digital reconstruction of the acquired lensfree holograms, transmission images (both phase and amplitude) of the same specimen are also created. We tested the performance of this field-portable microscope by imaging various micro-particles, blood smears as well as a histopathology slide corresponding to skin tissue. Being compact, light-weight and cost-effective, this combined reflection and transmission microscope might especially be useful for telemedicine applications in resource limited settings.

Optofluidic Fluorescent Imaging Cytometry on a Cell Phone

Analytical Chemistry. Sep, 2011  |  Pubmed ID: 21774454

Fluorescent microscopy and flow cytometry are widely used tools in biomedical sciences. Cost-effective translation of these technologies to remote and resource-limited environments could create new opportunities especially for telemedicine applications. Toward this direction, here we demonstrate the integration of imaging cytometry and fluorescent microscopy on a cell phone using a compact, lightweight, and cost-effective optofluidic attachment. In this cell-phone-based optofluidic imaging cytometry platform, fluorescently labeled particles or cells of interest are continuously delivered to our imaging volume through a disposable microfluidic channel that is positioned above the existing camera unit of the cell phone. The same microfluidic device also acts as a multilayered optofluidic waveguide and efficiently guides our excitation light, which is butt-coupled from the side facets of our microfluidic channel using inexpensive light-emitting diodes. Since the excitation of the sample volume occurs through guided waves that propagate perpendicular to the detection path, our cell-phone camera can record fluorescent movies of the specimens as they are flowing through the microchannel. The digital frames of these fluorescent movies are then rapidly processed to quantify the count and the density of the labeled particles/cells within the target solution of interest. We tested the performance of our cell-phone-based imaging cytometer by measuring the density of white blood cells in human blood samples, which provided a decent match to a commercially available hematology analyzer. We further characterized the imaging quality of the same platform to demonstrate a spatial resolution of ~2 μm. This cell-phone-enabled optofluidic imaging flow cytometer could especially be useful for rapid and sensitive imaging of bodily fluids for conducting various cell counts (e.g., toward monitoring of HIV+ patients) or rare cell analysis as well as for screening of water quality in remote and resource-poor settings.

Lensfree Optofluidic Microscopy and Tomography

Annals of Biomedical Engineering. Sep, 2011  |  Pubmed ID: 21887590

Microfluidic devices aim at miniaturizing, automating, and lowering the cost of chemical and biological sample manipulation and detection, hence creating new opportunities for lab-on-a-chip platforms. Recently, optofluidic devices have also emerged where optics is used to enhance the functionality and the performance of microfluidic components in general. Lensfree imaging within microfluidic channels is one such optofluidic platform, and in this article, we focus on the holographic implementation of lensfree optofluidic microscopy and tomography, which might provide a simpler and more powerful solution for three-dimensional (3D) on-chip imaging. This lensfree optofluidic imaging platform utilizes partially coherent digital in-line holography to allow phase and amplitude imaging of specimens flowing through micro-channels, and takes advantage of the fluidic flow to achieve higher spatial resolution imaging compared to a stationary specimen on the same chip. In addition to this, 3D tomographic images of the same samples can also be reconstructed by capturing lensfree projection images of the samples at various illumination angles as a function of the fluidic flow. Based on lensfree digital holographic imaging, this optofluidic microscopy and tomography concept could be valuable especially for providing a compact, yet powerful toolset for lab-on-a-chip devices.

Lensfree Super-resolution Holographic Microscopy Using Wetting Films on a Chip

Optics Express. Aug, 2011  |  Pubmed ID: 21935102

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.

Field-portable Reflection and Transmission Microscopy Based on Lensless Holography

Biomedical Optics Express. Sep, 2011  |  Pubmed ID: 21991559

We demonstrate a lensfree dual-mode holographic microscope that can image specimens in both transmission and reflection geometries using in-line transmission and off-axis reflection holography, respectively. This field-portable dual-mode holographic microscope has a weight of ~200 g with dimensions of 15 x 5.5 x 5cm, where a laser source is powered by two batteries. Based on digital in-line holography, our transmission microscope achieves a sub-pixel lateral resolution of ≤2 µm over a wide field-of-view (FOV) of ~24 mm(2) due to its unit fringe magnification geometry. Despite its simplicity and ease of operation, in-line transmission geometry is not suitable to image dense or connected objects such as tissue slides since the reference beam gets distorted causing severe aberrations in reconstruction of such objects. To mitigate this challenge, on the same cost-effective and field-portable assembly we built a lensless reflection mode microscope based on digital off-axis holography where a beam-splitter is used to interfere a tilted reference wave with the reflected light from the object surface, creating an off-axis hologram of the specimens on a CMOS sensor-chip. As a result of the reduced space-bandwidth product of the off-axis geometry compared to its in-line counterpart, the imaging FOV of our reflection mode is reduced to ~9 mm(2), while still achieving a similar sub-pixel resolution of ≤2 µm. We tested the performance of this compact dual-mode microscopy unit by imaging a US-air force resolution test target, various micro-particles as well as a histopathology slide corresponding to skin tissue. Due to its compact, cost-effective, and lightweight design, this dual-mode lensless holographic microscope might especially be useful for field-use or for conducting microscopic analysis in resource-poor settings.

Partially Coherent Lensfree Tomographic Microscopy [Invited]

Applied Optics. Dec, 2011  |  Pubmed ID: 22193016

Optical sectioning of biological specimens provides detailed volumetric information regarding their internal structure. To provide a complementary approach to existing three-dimensional (3D) microscopy modalities, we have recently demonstrated lensfree optical tomography that offers high-throughput imaging within a compact and simple platform. In this approach, in-line holograms of objects at different angles of partially coherent illumination are recorded using a digital sensor-array, which enables computing pixel super-resolved tomographic images of the specimen. This imaging modality, which forms the focus of this review, offers micrometer-scale 3D resolution over large imaging volumes of, for example, 10-15 mm(3), and can be assembled in light weight and compact architectures. Therefore, lensfree optical tomography might be particularly useful for lab-on-a-chip applications as well as for microscopy needs in resource-limited settings.

Lensless Fluorescent On-chip Microscopy Using a Fiber-optic Taper

Conference Proceedings : ... Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineering in Medicine and Biology Society. Conference. Aug, 2011  |  Pubmed ID: 22255702

We demonstrate a lensfree on-chip fluorescent microscopy platform that can image fluorescently labeled cells over -60 mm(2) field-of-view with <4 urn spatial resolution. In this lensfree imaging system, micro-objects of interest are directly located on a tapered fiber-optic faceplate which has > 5-fold higher density of fiber-optic waveguides in its top facet compared to the bottom facet. For excitation, an incoherent light source (e.g., a simple light emitting diode - LED) is used to pump fluorescent objects through a glass hemi-sphere interface. Upon interacting with the entire sample volume, the excitation light is rejected by total internal reflection process occurring at the bottom of the sample substrate. Fluorescent emission from the objects is then collected by the smaller facet of the tapered faceplate and is delivered to a detector-array with an image magnification of ∼2.4X. A compressive sampling based decoding algorithm is used for sparse signal recovery, which further increases the space-bandwidth-product of our lensfree on-chip fluorescent imager. We validated the performance of this lensfree imaging platform using fluorescent micro-particles as well as labeled water-borne parasites (e.g., Giardia Muris cysts). Such a compact and wide-field fluorescent microscopy platform could be valuable for cytometry and rare cell imaging applications as well as for micro array research.

Wide-field Fluorescent Microscopy on a Cell-phone

Conference Proceedings : ... Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineering in Medicine and Biology Society. Conference. Aug, 2011  |  Pubmed ID: 22255900

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.

Portable and Cost-effective Pixel Super-resolution On-chip Microscope for Telemedicine Applications

Conference Proceedings : ... Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineering in Medicine and Biology Society. Conference. Aug, 2011  |  Pubmed ID: 22256247

We report a field-portable lensless on-chip microscope with a lateral resolution of <1μm and a large field-of-view of ∼24mm(2). This microscope is based on digital in-line holography and a pixel super-resolution algorithm to process multiple lensfree holograms and obtain a single high-resolution hologram. In its compact and cost-effective design, we utilize 23 light emitting diodes butt-coupled to 23 multi-mode optical fibers, and a simple optical filter, with no moving parts. Weighing only ∼95 grams, we demonstrate the performance of this field-portable microscope by imaging various objects including human malaria parasites in thin blood smears.

Optofluidic On-chip Tomography

Conference Proceedings : ... Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineering in Medicine and Biology Society. Conference. Aug, 2011  |  Pubmed ID: 22256312

The first demonstration of optofluidic tomography is presented. Using partially coherent illumination, holograms of objects are recorded at multiple viewing angles, as they flow through a microfluidic channel placed directly on the top of an opto-electronic sensor array. These lensfree holograms are then digitally processed to compute pixel super-resolved tomograms of micro-objects to achieve sectional opto-fluidic imaging on a chip.

Cost-effective and Compact Wide-field Fluorescent Imaging on a Cell-phone

Lab on a Chip. Jan, 2011  |  Pubmed ID: 21063582

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.

Field-portable Lensfree Tomographic Microscope

Lab on a Chip. Jul, 2011  |  Pubmed ID: 21573311

We present a field-portable lensfree tomographic microscope, which can achieve sectional imaging of a large volume (∼20 mm(3)) on a chip with an axial resolution of <7 μm. In this compact tomographic imaging platform (weighing only ∼110 grams), 24 light-emitting diodes (LEDs) that are each butt-coupled to a fibre-optic waveguide are controlled through a cost-effective micro-processor to sequentially illuminate the sample from different angles to record lensfree holograms of the sample that is placed on the top of a digital sensor array. In order to generate pixel super-resolved (SR) lensfree holograms and hence digitally improve the achievable lateral resolution, multiple sub-pixel shifted holograms are recorded at each illumination angle by electromagnetically actuating the fibre-optic waveguides using compact coils and magnets. These SR projection holograms obtained over an angular range of ±50° are rapidly reconstructed to yield projection images of the sample, which can then be back-projected to compute tomograms of the objects on the sensor-chip. The performance of this compact and light-weight lensfree tomographic microscope is validated by imaging micro-beads of different dimensions as well as a Hymenolepis nana egg, which is an infectious parasitic flatworm. Achieving a decent three-dimensional spatial resolution, this field-portable on-chip optical tomographic microscope might provide a useful toolset for telemedicine and high-throughput imaging applications in resource-poor settings.

Optofluidic Tomography on a Chip

Applied Physics Letters. Apr, 2011  |  Pubmed ID: 21580801

Using lensfree holography we demonstrate optofluidic tomography on a chip. A partially coherent light source is utilized to illuminate the objects flowing within a microfluidic channel placed directly on a digital sensor array. The light source is rotated to record lensfree holograms of the objects at different viewing directions. By capturing multiple frames at each illumination angle, pixel super-resolution techniques are utilized to reconstruct high-resolution transmission images at each angle. Tomograms of flowing objects are then computed through filtered back-projection of these reconstructed lensfree images, thereby enabling optical sectioning on-a-chip. The proof-of-concept is demonstrated by lensfree tomographic imaging of C. elegans.

Lensfree Fluorescent On-chip Imaging of Transgenic Caenorhabditis Elegans over an Ultra-wide Field-of-view

PloS One. 2011  |  Pubmed ID: 21253611

We demonstrate lensfree on-chip fluorescent imaging of transgenic Caenorhabditis elegans (C. elegans) over an ultra-wide field-of-view (FOV) of e.g., >2-8 cm(2) with a spatial resolution of ∼10 µm. This is the first time that a lensfree on-chip platform has successfully imaged fluorescent C. elegans samples. In our wide-field lensfree imaging platform, the transgenic samples are excited using a prism interface from the side, where the pump light is rejected through total internal reflection occurring at the bottom facet of the substrate. The emitted fluorescent signal from C. elegans samples is then recorded on a large area opto-electronic sensor-array over an FOV of e.g., >2-8 cm(2), without the use of any lenses, thin-film interference filters or mechanical scanners. Because fluorescent emission rapidly diverges, such lensfree fluorescent images recorded on a chip look blurred due to broad point-spread-function of our platform. To combat this resolution challenge, we use a compressive sampling algorithm to uniquely decode the recorded lensfree fluorescent patterns into higher resolution images, demonstrating ∼10 µm resolution. We tested the efficacy of this compressive decoding approach with different types of opto-electronic sensors to achieve a similar resolution level, independent of the imaging chip. We further demonstrate that this wide FOV lensfree fluorescent imaging platform can also perform sequential bright-field imaging of the same samples using partially-coherent lensfree digital in-line holography that is coupled from the top facet of the same prism used in fluorescent excitation. This unique combination permits ultra-wide field dual-mode imaging of C. elegans on a chip which could especially provide a useful tool for high-throughput screening applications in biomedical research.

Wide-field Lensless Fluorescent Microscopy Using a Tapered Fiber-optic Faceplate on a Chip

The Analyst. Sep, 2011  |  Pubmed ID: 21283900

We demonstrate lensless fluorescent microscopy over a large field-of-view of ~60 mm(2) with a spatial resolution of <4 µm. In this on-chip fluorescent imaging modality, the samples are placed on a fiber-optic faceplate that is tapered such that the density of the fiber-optic waveguides on the top facet is >5 fold larger than the bottom one. Placed on this tapered faceplate, the fluorescent samples are pumped from the side through a glass hemisphere interface. After excitation of the samples, the pump light is rejected through total internal reflection that occurs at the bottom facet of the sample substrate. The fluorescent emission from the sample is then collected by the smaller end of the tapered faceplate and is delivered to an opto-electronic sensor-array to be digitally sampled. Using a compressive sampling algorithm, we decode these raw lensfree images to validate the resolution (<4 µm) of this on-chip fluorescent imaging platform using microparticles as well as labeled Giardia muris cysts. This wide-field lensfree fluorescent microscopy platform, being compact and high-throughput, might provide a valuable tool especially for cytometry, rare cell analysis (involving large area microfluidic systems) as well as for microarray imaging applications.

Holographic Pixel Super-resolution in Portable Lensless On-chip Microscopy Using a Fiber-optic Array

Lab on a Chip. Apr, 2011  |  Pubmed ID: 21365087

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.

Lens-free Optical Tomographic Microscope with a Large Imaging Volume on a Chip

Proceedings of the National Academy of Sciences of the United States of America. May, 2011  |  Pubmed ID: 21504943

We present a lens-free optical tomographic microscope, which enables imaging a large volume of approximately 15 mm(3) on a chip, with a spatial resolution of < 1 μm × < 1 μm × < 3 μm in x, y and z dimensions, respectively. In this lens-free tomography modality, the sample is placed directly on a digital sensor array with, e.g., ≤ 4 mm distance to its active area. A partially coherent light source placed approximately 70 mm away from the sensor is employed to record lens-free in-line holograms of the sample from different viewing angles. At each illumination angle, multiple subpixel shifted holograms are also recorded, which are digitally processed using a pixel superresolution technique to create a single high-resolution hologram of each angular projection of the object. These superresolved holograms are digitally reconstructed for an angular range of ± 50°, which are then back-projected to compute tomograms of the sample. In order to minimize the artifacts due to limited angular range of tilted illumination, a dual-axis tomography scheme is adopted, where the light source is rotated along two orthogonal axes. Tomographic imaging performance is quantified using microbeads of different dimensions, as well as by imaging wild-type Caenorhabditis elegans. Probing a large volume with a decent 3D spatial resolution, this lens-free optical tomography platform on a chip could provide a powerful tool for high-throughput imaging applications in, e.g., cell and developmental biology.

High-throughput Screening of Large Volumes of Whole Blood Using Structured Illumination and Fluorescent On-chip Imaging

Lab on a Chip. Dec, 2012  |  Pubmed ID: 23047492

Undiluted blood samples are difficult to image in large volumes since blood constitutes a highly absorbing and scattering medium. As a result of this limitation, optical imaging of rare cells (e.g., circulating tumour cells) within unprocessed whole blood remains a challenge, demanding the use of special microfluidic technologies. Here we demonstrate a new fluorescent on-chip imaging modality that can rapidly screen large volumes of absorbing and scattering media, such as undiluted whole blood samples, for detection of fluorescent micro-objects at low concentrations (for example ≤50-100 particles/mL). In this high-throughput imaging modality, a large area microfluidic device (e.g., 7-18 cm(2)), which contains for example ~0.3-0.7 mL of undiluted whole blood sample, is directly positioned onto a wide-field opto-electronic sensor-array such that the fluorescent emission within the microchannel can be detected without the use of any imaging lenses. This microfluidic device is then illuminated and laterally scanned with an array of Gaussian excitation spots, which is generated through a spatial light modulator. For each scanning position of this excitation array, a lensfree fluorescent image of the blood sample is captured using the opto-electronic sensor-array, resulting in a sequence of images (e.g., 144 lensfree frames captured in ~36 s) for the same sample chip. Digitally merging these lensfree fluorescent images based on a maximum intensity projection (MIP) algorithm enabled us to significantly boost the signal-to-noise ratio (SNR) and contrast of the fluorescent micro-objects within whole blood, which normally remain undetected (i.e., hidden) using conventional uniform excitation schemes, involving plane wave illumination. This high-throughput on-chip imaging platform based on structured excitation could be useful for rare cell research by enabling rapid screening of large volume microfluidic devices that process whole blood and other optically dense media.

A Mathematical Framework for Combining Decisions of Multiple Experts Toward Accurate and Remote Diagnosis of Malaria Using Tele-microscopy

PloS One. 2012  |  Pubmed ID: 23071544

We propose a methodology for digitally fusing diagnostic decisions made by multiple medical experts in order to improve accuracy of diagnosis. Toward this goal, we report an experimental study involving nine experts, where each one was given more than 8,000 digital microscopic images of individual human red blood cells and asked to identify malaria infected cells. The results of this experiment reveal that even highly trained medical experts are not always self-consistent in their diagnostic decisions and that there exists a fair level of disagreement among experts, even for binary decisions (i.e., infected vs. uninfected). To tackle this general medical diagnosis problem, we propose a probabilistic algorithm to fuse the decisions made by trained medical experts to robustly achieve higher levels of accuracy when compared to individual experts making such decisions. By modelling the decisions of experts as a three component mixture model and solving for the underlying parameters using the Expectation Maximisation algorithm, we demonstrate the efficacy of our approach which significantly improves the overall diagnostic accuracy of malaria infected cells. Additionally, we present a mathematical framework for performing 'slide-level' diagnosis by using individual 'cell-level' diagnosis data, shedding more light on the statistical rules that should govern the routine practice in examination of e.g., thin blood smear samples. This framework could be generalized for various other tele-pathology needs, and can be used by trained experts within an efficient tele-medicine platform.

Lens-free Computational Imaging of Capillary Morphogenesis Within Three-dimensional Substrates

Journal of Biomedical Optics. Dec, 2012  |  Pubmed ID: 23235893

Endothelial cells cultured in three-dimensional (3-D) extracellular matrices spontaneously form microvessels in response to soluble and matrix-bound factors. Such cultures are common for the study of angiogenesis and may find widespread use in drug discovery. Vascular networks are imaged over weeks to measure the distribution of vessel morphogenic parameters. Measurements require micron-scale spatial resolution, which for light microscopy comes at the cost of limited field-of-view (FOV) and shallow depth-of-focus (DOF). Small FOVs and DOFs necessitate lateral and axial mechanical scanning, thus limiting imaging throughput. We present a lens-free holographic on-chip microscopy technique to rapidly image microvessels within a Petri dish over a large volume without any mechanical scanning. This on-chip method uses partially coherent illumination and a CMOS sensor to record in-line holographic images of the sample. For digital reconstruction of the measured holograms, we implement a multiheight phase recovery method to obtain phase images of capillary morphogenesis over a large FOV (24 mm2) with ≈ 1.5 μm spatial resolution. On average, measured capillary length in our method was within approximately 2% of lengths measured using a 10 × microscope objective. These results suggest lens-free on-chip imaging is a useful toolset for high-throughput monitoring and quantitative analysis of microvascular 3-D networks.

Field-portable Wide-field Microscopy of Dense Samples Using Multi-height Pixel Super-resolution Based Lensfree Imaging

Lab on a Chip. Feb, 2012  |  Pubmed ID: 22334329

We report a field-portable lensfree microscope that can image dense and connected specimens with sub-micron resolution over a large field-of-view of ∼30 mm(2) (i.e., ∼6.4 mm ×∼4.6 mm) using pixel super-resolution and iterative phase recovery techniques. Weighing ∼122 grams with dimensions of 4 cm × 4 cm × 15 cm, this microscope records lensfree in-line holograms of specimens onto an opto-electronic sensor-array using partially coherent illumination. To reconstruct the phase and amplitude images of dense samples (with >0.3 billion pixels in each image, i.e., >0.6 billion pixels total), we employ a multi-height imaging approach, where by using a mechanical interface the sensor-to-sample distance is dynamically changed by random discrete steps of e.g., ∼10 to 80 μm. By digitally propagating back and forth between these multi-height super-resolved holograms (corresponding to typically 2-5 planes), phase and amplitude images of dense samples can be recovered without the need for any spatial masks or filtering. We demonstrate the performance of this field-portable multi-height lensfree microscope by imaging Papanicolaou smears (also known as Pap tests). Our results reveal the promising potential of this multi-height lensfree computational microscopy platform for e.g., pathology needs in resource limited settings.

Maskless Imaging of Dense Samples Using Pixel Super-resolution Based Multi-height Lensfree On-chip Microscopy

Optics Express. Jan, 2012  |  Pubmed ID: 22330550

Lensfree in-line holographic microscopy offers sub-micron resolution over a large field-of-view (e.g., ~24 mm²) with a cost-effective and compact design suitable for field use. However, it is limited to relatively low-density samples. To mitigate this limitation, we demonstrate an on-chip imaging approach based on pixel super-resolution and phase recovery, which iterates among multiple lensfree intensity measurements, each having a slightly different sample-to-sensor distance. By digitally aligning and registering these lensfree intensity measurements, phase and amplitude images of dense and connected specimens can be iteratively reconstructed over a large field-of-view of ~24 mm² without the use of any spatial masks. We demonstrate the success of this multi-height in-line holographic approach by imaging dense Papanicolaou smears (i.e., Pap smears) and blood samples.

Lens-free Imaging for Biological Applications

Journal of Laboratory Automation. Feb, 2012  |  Pubmed ID: 22357607

Lens-free (or lensless) imaging is emerging as a cost-effective, compact, and lightweight detection method that can serve numerous biological applications. Lens-free imaging can generate high-resolution images within a field-portable platform, which is ideal for affordable point-of-care devices aiming at resource-limited settings. In this mini-review, we first describe different modes of operation for lens-free imaging and then highlight several recent biological applications of this emerging platform technology.

Quantum Dot Enabled Detection of Escherichia Coli Using a Cell-phone

The Analyst. Jun, 2012  |  Pubmed ID: 22396952

We report a cell-phone based Escherichia coli (E. coli) detection platform for screening of liquid samples. In this compact and cost-effective design attached to a cell-phone, we utilize anti-E. coli O157:H7 antibody functionalized glass capillaries as solid substrates to perform a quantum dot based sandwich assay for specific detection of E. coli O157:H7 in liquid samples. Using battery-powered inexpensive light-emitting-diodes (LEDs) we excite/pump these labelled E. coli particles captured on the capillary surface, where the emission from the quantum dots is then imaged using the cell-phone camera unit through an additional lens that is inserted between the capillary and the cell-phone. By quantifying the fluorescent light emission from each capillary tube, the concentration of E. coli in the sample is determined. We experimentally confirmed the detection limit of this cell-phone based fluorescent imaging and sensing platform as ∼5 to 10 cfu mL(-1) in buffer solution. We also tested the specificity of this E. coli detection platform by spiking samples with different species (e.g., Salmonella) to confirm that non-specific binding/detection is negligible. We further demonstrated the proof-of-concept of our approach in a complex food matrix, e.g., fat-free milk, where a similar detection limit of ∼5 to 10 cfu mL(-1) was achieved despite challenges associated with the density of proteins that exist in milk. Our results reveal the promising potential of this cell-phone enabled field-portable and cost-effective E. coli detection platform for e.g., screening of water and food samples even in resource limited environments. The presented platform can also be applicable to other pathogens of interest through the use of different antibodies.

Lensfree Computational Microscopy Tools for Cell and Tissue Imaging at the Point-of-care and in Low-resource Settings

Analytical Cellular Pathology (Amsterdam). 2012  |  Pubmed ID: 22433451

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.

Integrated Rapid-diagnostic-test Reader Platform on a Cellphone

Lab on a Chip. Aug, 2012  |  Pubmed ID: 22596243

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.

Distributed Medical Image Analysis and Diagnosis Through Crowd-sourced Games: a Malaria Case Study

PloS One. 2012  |  Pubmed ID: 22606353

In this work we investigate whether the innate visual recognition and learning capabilities of untrained humans can be used in conducting reliable microscopic analysis of biomedical samples toward diagnosis. For this purpose, we designed entertaining digital games that are interfaced with artificial learning and processing back-ends to demonstrate that in the case of binary medical diagnostics decisions (e.g., infected vs. uninfected), with the use of crowd-sourced games it is possible to approach the accuracy of medical experts in making such diagnoses. Specifically, using non-expert gamers we report diagnosis of malaria infected red blood cells with an accuracy that is within 1.25% of the diagnostics decisions made by a trained medical professional.

Nanofabrication Using Near-field Optical Probes

Journal of Laboratory Automation. Aug, 2012  |  Pubmed ID: 22713756

Nanofabrication using near-field optical probes is an established technique for rapid prototyping and automated maskless fabrication of nanostructured devices. In this review, we present the primary types of near-field probes and their physical processing mechanisms. Highlights of recent developments include improved resolution by optimizing the probe shape, incorporation of surface plasmonics in probe design, broader use in biological and magnetic storage applications, and increased throughput using probe arrays as well as high-speed writing and patterning.

Crowd-sourced BioGames: Managing the Big Data Problem for Next-generation Lab-on-a-chip Platforms

Lab on a Chip. Oct, 2012  |  Pubmed ID: 22918378

We describe a crowd-sourcing based solution for handling large quantities of data that are created by e.g., emerging digital imaging and sensing devices, including next generation lab-on-a-chip platforms. We show that in cases where the diagnosis is a binary decision (e.g., positive vs. negative, or infected vs. uninfected), it is possible to make accurate diagnosis by crowd-sourcing the raw data (e.g., microscopic images of specimens/cells) using entertaining digital games (i.e., ) that are played on PCs, tablets or mobile phones. We report the results and the analysis of a large-scale public experiment toward diagnosis of malaria infected human red blood cells (RBCs), where binary responses from approximately 1000 untrained individuals from more than 60 different countries are combined together (corresponding to more than 1 million cell diagnoses), resulting in an accuracy level that is comparable to those of expert medical professionals. This platform holds promise toward cost-effective and accurate tele-pathology, improved training of medical personnel, and can also be used to manage the "Big Data" problem that is emerging through next generation digital lab-on-a-chip devices.

Imaging Without Lenses: Achievements and Remaining Challenges of Wide-field On-chip Microscopy

Nature Methods. Sep, 2012  |  Pubmed ID: 22936170

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.

Giga-pixel Lensfree Holographic Microscopy and Tomography Using Color Image Sensors

PloS One. 2012  |  Pubmed ID: 22984606

We report Giga-pixel lensfree holographic microscopy and tomography using color sensor-arrays such as CMOS imagers that exhibit Bayer color filter patterns. Without physically removing these color filters coated on the sensor chip, we synthesize pixel super-resolved lensfree holograms, which are then reconstructed to achieve ~350 nm lateral resolution, corresponding to a numerical aperture of ~0.8, across a field-of-view of ~20.5 mm(2). This constitutes a digital image with ~0.7 Billion effective pixels in both amplitude and phase channels (i.e., ~1.4 Giga-pixels total). Furthermore, by changing the illumination angle (e.g., ± 50°) and scanning a partially-coherent light source across two orthogonal axes, super-resolved images of the same specimen from different viewing angles are created, which are then digitally combined to synthesize tomographic images of the object. Using this dual-axis lensfree tomographic imager running on a color sensor-chip, we achieve a 3D spatial resolution of ~0.35 µm × 0.35 µm × ~2 µm, in x, y and z, respectively, creating an effective voxel size of ~0.03 µm(3) across a sample volume of ~5 mm(3), which is equivalent to >150 Billion voxels. We demonstrate the proof-of-concept of this lensfree optical tomographic microscopy platform on a color CMOS image sensor by creating tomograms of micro-particles as well as a wild-type C. elegans nematode.

High-throughput Lensfree 3D Tracking of Human Sperms Reveals Rare Statistics of Helical Trajectories

Proceedings of the National Academy of Sciences of the United States of America. Oct, 2012  |  Pubmed ID: 22988076

Dynamic tracking of human sperms across a large volume is a challenging task. To provide a high-throughput solution to this important need, here we describe a lensfree on-chip imaging technique that can track the three-dimensional (3D) trajectories of > 1,500 individual human sperms within an observation volume of approximately 8-17 mm(3). This computational imaging platform relies on holographic lensfree shadows of sperms that are simultaneously acquired at two different wavelengths, emanating from two partially-coherent sources that are placed at 45° with respect to each other. This multiangle and multicolor illumination scheme permits us to dynamically track the 3D motion of human sperms across a field-of-view of > 17 mm(2) and depth-of-field of approximately 0.5-1 mm with submicron positioning accuracy. The large statistics provided by this lensfree imaging platform revealed that only approximately 4-5% of the motile human sperms swim along well-defined helices and that this percentage can be significantly suppressed under seminal plasma. Furthermore, among these observed helical human sperms, a significant majority (approximately 90%) preferred right-handed helices over left-handed ones, with a helix radius of approximately 0.5-3 μm, a helical rotation speed of approximately 3-20 rotations/s and a linear speed of approximately 20-100 μm/s. This high-throughput 3D imaging platform could in general be quite valuable for observing the statistical swimming patterns of various other microorganisms, leading to new insights in their 3D motion and the underlying biophysics.

Optical Imaging Techniques for Point-of-care Diagnostics

Lab on a Chip. Jan, 2013  |  Pubmed ID: 23044793

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.

Cost-effective and Rapid Blood Analysis on a Cell-phone

Lab on a Chip. Mar, 2013  |  Pubmed ID: 23392286

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.

High-throughput 3D Imaging of Sperm

Molecular Reproduction and Development. Feb, 2013  |  Pubmed ID: 23401146

Lensfree Computational Microscopy Tools for Cell and Tissue Imaging at the Point-of-Care and in Low-Resource Settings

Studies in Health Technology and Informatics. 2013  |  Pubmed ID: 23542940

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.

On-chip Biomedical Imaging

IEEE Reviews in Biomedical Engineering. 2013  |  Pubmed ID: 23558399

Lab-on-a-chip systems have been rapidly emerging to pave the way toward ultra-compact, efficient, mass producible and cost-effective biomedical research and diagnostic tools. Although such microfluidic and microelectromechanical systems have achieved high levels of integration, and are capable of performing various important tasks on the same chip, such as cell culturing, sorting and staining, they still rely on conventional microscopes for their imaging needs. Recently, several alternative on-chip optical imaging techniques have been introduced, which have the potential to substitute conventional microscopes for various lab-on-a-chip applications. Here we present a critical review of these recently emerging on-chip biomedical imaging modalities, including contact shadow imaging, lens-free holographic microscopy, fluorescent on-chip microscopy and lens-free optical tomography.

A Personalized Food Allergen Testing Platform on a Cellphone

Lab on a Chip. Feb, 2013  |  Pubmed ID: 23254910

We demonstrate a personalized food allergen testing platform, termed iTube, running on a cellphone that images and automatically analyses colorimetric assays performed in test tubes toward sensitive and specific detection of allergens in food samples. This cost-effective and compact iTube attachment, weighing approximately 40 grams, is mechanically installed on the existing camera unit of a cellphone, where the test and control tubes are inserted from the side and are vertically illuminated by two separate light-emitting-diodes. The illumination light is absorbed by the allergen assay, which is activated within the tubes, causing an intensity change in the acquired images by the cellphone camera. These transmission images of the sample and control tubes are digitally processed within 1 s using a smart application running on the same cellphone for detection and quantification of allergen contamination in food products. We evaluated the performance of this cellphone-based iTube platform using different types of commercially available cookies, where the existence of peanuts was accurately quantified after a sample preparation and incubation time of ~20 min per test. This automated and cost-effective personalized food allergen testing tool running on cellphones can also permit uploading of test results to secure servers to create personal and/or public spatio-temporal allergen maps, which can be useful for public health in various settings.