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.

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.

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.

Lensfree On-chip Holography Facilitates Novel Microscopy Applications

SPIE Newsroom. May, 2010  |  Pubmed ID: 21643449

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.

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.

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.