Xin Heng

Lensless high-resolution on-chip optofluidic microscopes for Caenorhabditis elegans and cell imaging

Low-cost and high-resolution on-chip microscopes are vital for reducing cost and improving efficiency for modern biomedicine and bioscience. Despite the needs, the conventional microscope design has proven difficult to miniaturize. Here, we report the implementation and application of two high-resolution (≈0.9 μm for the first and ≈0.8 μm for the second), lensless, and fully on-chip microscopes based on the optofluidic microscopy (OFM) method. These systems abandon the conventional microscope design, which requires expensive lenses and large space to magnify images, and instead utilizes microfluidic flow to deliver specimens across array(s) of micrometer-size apertures defined on a metal-coated CMOS sensor to generate direct projection images. The first system utilizes a gravity-driven microfluidic flow for sample scanning and is suited for imaging elongate objects, such as Caenorhabditis elegans; and the second system employs an electrokinetic drive for flow control and is suited for imaging cells and other spherical/ellipsoidal objects. As a demonstration of the OFM for bioscience research, we show that the prototypes can be used to perform automated phenotype characterization of different Caenorhabditis elegans mutant strains, and to image spores and single cellular entities. The optofluidic microscope design, readily fabricable with existing semiconductor and microfluidic technologies, offers low-cost and highly compact imaging solutions. More functionalities, such as on-chip phase and fluorescence imaging, can also be readily adapted into OFM systems. We anticipate that the OFM can significantly address a range of biomedical and bioscience needs, and engender new microscope applications.

Methods and application areas of endoscopic optical coherence tomography

We review the current state of research in endoscopic optical coherence tomography (OCT). We first survey the range of available endoscopic optical imaging techniques. We then discuss the various OCT-based endoscopic methods that have thus far been developed. We compare the different endoscopic OCT methods in terms of their scan performance. Next, we examine the application range of endoscopic OCT methods. In particular, we look at the reported utility of the methods in digestive, intravascular, respiratory, urinary and reproductive systems. We highlight two additional applications--biopsy procedures and neurosurgery--where sufficiently compact OCT-based endoscopes can have significant clinical impacts.

Characterization of light collection through a subwavelength aperture from a point source

We experimentally measure and theoretically model the light transmission characteristics of subwavelength apertures. The characterization consists of translating a point source at varying vertical height and lateral displacement from the aperture and measuring the resulting transmission. We define the variation of the transmission with lateral source displacement as the collection mode point spread function (CPSF). This transmission geometry is particularly relevant to subwavelength aperture based imaging devices and enables determination of their resolution. This study shows that the achieved resolutions degrade as a function of sample height and that the behavior of sensor devices based on the use of apertures for detection is different from those devices where the apertures are used as light sources. In addition, we find that the measured CPSF is dependent on the collection numerical aperture (NA). Finally, we establish that resolution beyond the diffraction limit for a nominal optical wavelength of 650 nm and nominal medium refractive index of 1.5 is achievable with subwavelength aperture based devices when the aperture size is smaller than 225 nm.

Slanted Hole Array Beam Profiler (SHArP) - a High-Resolution Portable Beam Profiler Based on a Linear Aperture Array

We demonstrate a novel high-resolution portable beam profiler based on a slanted linear array of small apertures, termed a slanted hole array beam profiler (SHArP). The apertures are directly fabricated on a metal-coated CMOS imaging sensor. With a single linear scan, the aperture array can establish a virtual grid of sampling points for beam profiling. With our prototype, we demonstrate beam profiling of Gaussian beams over an area of 66.5 microm x 66.5 microm with a resolution of 0.8 microm (compare with the CMOS pixel size of 10 microm). The resolution can be improved into the range of submicrometers by fabricating smaller apertures. The good correspondence between the measured and calculated beam profiles proves the fidelity of our new beam profiling scheme.

An optical tweezer actuated, nanoaperture-grid based Optofluidic Microscope implementation method

We report a novel grid based Optofluidic Microscope (OFM) method where a closely spaced 2D grid of nanoapertures (diameter = 100 nm, separation = 2.5 mum) provided patterned illumination. We achieved a one-to-one mapping of the light transmissions through the nanoapertures onto a high-speed CCD camera. By optically tweezing a targeted sample across the grid in a controlled fashion and recording the time varying light reception from the nanoapertures, we were able to generate high-resolution images of the sample. The achievable resolution limit of the prototype was ~ 110 nm (Sparrows criterion) under optimal conditions. We demonstrated the technique by imaging polystyrene beads and pollen spores.

Molecular contrast optical coherence tomography: a pump-probe scheme using indocyanine green as a contrast agent

The use of indocyanine green (ICG), a U.S. Food and Drug Administration approved dye, in a pump-probe scheme for molecular contrast optical coherence tomography (MCOCT) is proposed and demonstrated for the first time. In the proposed pump-probe scheme, an optical coherence tomography (OCT) scan of the sample containing ICG is first acquired. High fluence illumination (approximately 190 kJ/cm2) is then used to permanently photobleach the ICG molecules--resulting in a permanent alteration of the overall absorption of the ICG. A second OCT scan is next acquired. The difference of the two OCT scans is used to determine the depth resolved distribution of ICG within a sample. To characterize the extent of photobleaching in different ICG solutions, we determine the cumulative probability of photobleaching, phi(B,cum), defined as the ratio of the total photobleached ICG molecules to the total photons absorbed by the ground state molecules. An empirical study of ICG photobleaching dynamics shows that phi(B,cum) decreases with fluence as well as with increasing dye concentration. The quantity phi(B,cum) is useful for estimating the extent of photobleaching in an ICG sample (MCOCT contrast) for a given fluence of the pump illumination. The paper also demonstrates ICG-based MCOCT imaging in tissue phantoms as well as within stage 54 Xenopus laevis.

Harmonically-related diffraction gratings-based interferometer for quadrature phase measurements

We demonstrate the use of shallow diffraction gratings for quadrature phase interferometry. A single shallow diffraction grating-based Michelson interferometer yields only trivial (0(o) or 180(o) ) phase shift between different output ports. In comparison, a combination of two parallel shallow diffraction gratings can be useful to achieve desired phase shifts (e.g., 90 (o) for quadrature phase interferometry). We show that the phase at different output ports of a grating-pair based interferometer can be adjusted by shearing the two gratings with respect to each other. Two harmonically-related diffraction gratings are used to demonstrate phase shift control at the output ports of a modified Michelson interferometer. Our experimental data is in good agreement with theory.

Full field phase imaging using a harmonically matched diffraction grating pair based homodyne quadrature interferometer

In this letter, the authors present a novel quadrature interferometry method based on the use of a harmonically matched shallow grating pair. Unlike a simple beam splitter or single shallow grating, the grating pair can confer a nontrivial interference phase shift (other than 0° or 180°) between the output ports of the interferometer. Using the grating pair as the beam splitter/combiner, the authors implement a homodyne quadrature full field phase interferometer and demonstrate the system’s capability to acquire phase and amplitude images.

Homodyne en face optical coherence tomography

We demonstrate, for what we believe to be the first time, the use of a 3 x 3 fiber-optic coupler to realize a homodyne optical coherence tomography (OCT) system for en face imaging of highly scattering tissues and turbid media. The homodyne OCT setup exploits the inherent phase shifts between different output ports of a 3 x 3 fiber-optic coupler to extract amplitude information of a sample. Our homodyne en face OCT system features a measured resolution of 14 microm axially and 9.4 microm laterally with a 90 dB signal-to-noise ratio at 10 micros integration time. En face OCT imaging of a stage 52 Xenopus laevis was successfully demonstrated at a depth of 600 microm within the sample.

InstantScope: a low-cost whole slide imaging system with instant focal plane detection.

We report the development of a high-throughput whole slide imaging (WSI) system by adapting a cost-effective optomechanical add-on kit to existing microscopes. Inspired by the phase detection concept in professional photography, we attached two pinhole-modulated cameras at the eyepiece ports for instant focal plane detection. By adjusting the positions of the pinholes, we can effectively change the view angle for the sample, and as such, we can use the translation shift of the two pinhole-modulated images to identify the optimal focal position. By using a small pinhole size, the focal-plane-detection range is on the order of millimeter, orders of magnitude longer than the objectives depth of field. We also show that, by analyzing the phase correlation of the pinhole-modulated images, we can determine whether the sample contains one thin section, folded sections, or multiple layers separated by certain distances - an important piece of information prior to a detailed z scan. In order to achieve system automation, we deployed a low-cost programmable robotic arm to perform sample loading and