Multi-angle illumination with pixel super-resolution enables lensfree on-chip tomography
Three-dimensional microscopy techniques have found increasing use in the life sciences because they provide high-resolution structural information of biological specimens.1 Numerous approaches to achieve 3D optical imaging have been demonstrated, for example, optical coherence tomography, confocal microscopy, diffraction tomography,2 optical projection tomography,3 and light-sheet microscopy.4 Many of these imaging platforms have complex and relatively bulky architectures, which restrict their use to advanced settings and impede their integration with lab-on-a-chip platforms. To address these issues, we recently introduced a compact and cost-effective tomographic microscopy platform for use in resource-limited settings and lab-on-a-chip devices.
Lensfree optical tomography (LOT) is an on-chip 3D microscopy technique for high-throughput imaging of biological specimens at a spatial resolution of <1μm×<1μm×<3μm in the x, y, and z dimensions, respectively.5 LOT relies on digital holographic microscopy to achieve sectional imaging of micro-sized objects. The technique uses simple light sources, such as LEDs, and an optoelectronic sensor array, such as a CCD or CMOS chip. The 3D structure of the target object is digitally reconstructed by measuring in-line transmission holograms of the sample as a function of the illumination angle. Additionally, by removing the lenses, we can reduce the size and complexity of the imaging system while increasing the field-of-view and depth-of-field that can be probed. Overall, LOT can perform sectional imaging of a large sample volume—approximately 15–100mm3—in a single data acquisition step.5
In LOT, the sample of interest is placed directly on a sensor array and is illuminated using partially-coherent light, such as an LED that is spatially filtered by a pinhole with a diameter of ∼0.05–0.1mm: see Figure 1(a). This partially-coherent source emanating through a large aperture simultaneously enables hologram recording of micro-objects on the sensor-chip while significantly reducing the coherent noise terms from speckle and multiple-reflections. To achieve tomographic imaging, lensfree holograms are recorded at different illumination angles within a range of ±50° with 2° increments. We limit the angular range to ±50° because the degraded pixel response at higher incident angles added significant artifacts to the holograms. To compensate for the resulting missing spatial frequency information, a dual-axis tomography scheme is used, where illumination is varied along two orthogonal arcs: see Figure 1(a).
At each angle of illumination, we acquire multiple sub-pixel shifted holograms by shifting the light source to different positions with ∼50–100μm increments, which do not need to be precisely known. Using pixel super-resolution (PSR) algorithms, we can digitally synthesize a single high-resolution hologram from multiple lower resolution holograms.6, 7 Next, we reconstruct these high-resolution holograms using iterative phase recovery techniques to obtain 2D projection images of the objects for each angle of illumination. Finally, we back-project these projection images to compute tomographic images of the specimens, such as cells or microorganisms like Caenorhabditis elegans: see Figure 1(b).5
Owing to its architectural simplicity, LOT also lends itself to a compact, light-weight, and cost-effective architecture (see Figure 2). To this end, we also demonstrated a field-portable tomographic microscope based on LOT.8 In this portable device, which weighs ∼110g, separate LEDs coupled to multimode optical fibers (∼0.1mm core diameter) are devoted to each illumination angle. The tips of these fibers are electromagnetically actuated to achieve source-shifting using small magnets and coils to record shifted holograms and implement PSR at each viewing angle. Angular increments of 4° along a single-arc were used in this device, as opposed to 2° in the dual-axis bench-top demonstration. We measured the axial resolution (along the z-axis) to be <7μm and obtained a lateral resolution of ∼1μm.8
In summary, we developed a device capable of achieving tomographic microscopy of micro-objects over large imaging volumes. LOT can provide a useful tool for lab-on-a-chip platforms and high-throughput imaging applications in low-resource settings.5–8 We hope to extend the angular range of illumination to, for example, ±80° using emerging sensor-array architectures. These may offer improved pixel-response at even higher incident angles and should allow us to achieve near-isotropic 3D resolution at the sub-micrometer scale.
Aydogan Ozcan received his PhD from Stanford University's electrical engineering department. After a postdoctoral fellowship at Stanford, he was appointed as a faculty member at Harvard Medical School, Wellman Center for Photomedicine. He joined UCLA in 2007, where he is currently an associate professor.