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

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

Optical imaging techniques for point-of-care diagnostics.

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

Lens-free computational imaging of capillary morphogenesis within three-dimensional substrates.

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.

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

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

Giga-pixel lensfree holographic microscopy and tomography using color image sensors.

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.

Lensfree on-chip tomographic microscopy employing multi-angle illumination and pixel super-resolution.

Tomographic imaging has been a widely used tool in medicine as it can provide three-dimensional (3D) structural information regarding objects of different size scales. In micrometer and millimeter scales, optical microscopy modalities find increasing use owing to the non-ionizing nature of visible light, and the availability of a rich set of illumination sources (such as lasers and light-emitting-diodes) and detection elements (such as large format CCD and CMOS detector-arrays). Among the recently developed optical tomographic microscopy modalities, one can include optical coherence tomography, optical diffraction tomography, optical projection tomography and light-sheet microscopy. These platforms provide sectional imaging of cells, microorganisms and model animals such as C. elegans, zebrafish and mouse embryos. Existing 3D optical imagers generally have relatively bulky and complex architectures, limiting the availability of these equipments to advanced laboratories, and impeding their integration with lab-on-a-chip platforms and microfluidic chips. To provide an alternative tomographic microscope, we recently developed lensfree optical tomography (LOT) as a high-throughput, compact and cost-effective optical tomography modality. LOT discards the use of lenses and bulky optical components, and instead relies on multi-angle illumination and digital computation to achieve depth-resolved imaging of micro-objects over a large imaging volume. LOT can image biological specimen at a spatial resolution of <1 µm x <1 µm x <3 µm in the x, y and z dimensions, respectively, over a large imaging volume of 15-100 mm(3), and can be particularly useful for lab-on-a-chip platforms.

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

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

Lensfree optofluidic microscopy and tomography.

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.

Partially coherent lensfree tomographic microscopy [Invited].

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.

Combined reflection and transmission microscope for telemedicine applications in field settings.

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.

Field-portable lensfree tomographic microscope.

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.

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.

Lens-free optical tomographic microscope with a large imaging volume on a chip.

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.

Optofluidic on-chip tomography.

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.

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

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

Multi-angle lensless digital holography for depth resolved imaging on a chip.

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.

Lensfree microscopy on a cellphone.

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

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

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

On-chip differential interference contrast microscopy using lensless digital holography.

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.

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.