Bright-field and fluorescence chip-scale microscopy for biological imaging

Optical microscopy is an essential tool in biological science and one of the gold standards for medical examinations. Miniaturization of microscopes can be a crucial stepping stone towards realizing compact, cost-effective and portable platforms for biomedical research and healthcare. This thesis reports on implementations of bright-field and fluorescence chip-scale microscopes for a variety of biological imaging applications. The term “chip-scale microscopy” refers to lensless imaging techniques realized in the form of mass-producible semiconductor devices, which transforms the fundamental design of optical microscopes.

Our strategy for chip-scale microscopy involves utilization of low-cost Complementary metal Oxide Semiconductor (CMOS) image sensors, computational image processing and micro-fabricated structural components. First, the sub-pixel resolving optofluidic microscope (SROFM), will be presented, which combines microfluidics and pixel super-resolution image reconstruction to perform high-throughput imaging of fluidic samples, such as blood cells. We discuss design parameters and construction of the device, as well as the resulting images and the resolution of the device, which was 0.66 µm at the highest acuity. The potential applications of SROFM for clinical diagnosis of malaria in the resource-limited settings is discussed.

Next, the implementations of ePetri, a self-imaging Petri dish platform with microscopy resolution, are presented. Here, we simply place the sample of interest on the surface of the image sensor and capture the direct shadow images under the illumination. By taking advantage of the inherent motion of the microorganisms, we achieve high resolution (~1 µm) imaging and long term culture of motile microorganisms over ultra large field-of-view (5.7 mm × 4.4 mm) in a specialized ePetri platform. We apply the pixel super-resolution reconstruction to a set of low-resolution shadow images of the microorganisms as they move across the sensing area of an image sensor chip and render an improved resolution image. We perform longitudinal study of Euglena gracilis cultured in an ePetri platform and image based analysis on the motion and morphology of the cells. The ePetri device for imaging non-motile cells are also demonstrated, by using the sweeping illumination of a light emitting diode (LED) matrix for pixel super-resolution reconstruction of sub-pixel shifted shadow images. Using this prototype device, we demonstrate the detection of waterborne parasites for the effective diagnosis of enteric parasite infection in resource-limited settings.

Then, we demonstrate the adaptation of a smartphone’s camera to function as a compact lensless microscope, which uses ambient illumination as its light source and does not require the incorporation of a dedicated light source. The method is also based on the image reconstruction with sweeping illumination technique, where the sequence of images are captured while the user is manually tilting the device around any ambient light source, such as the sun or a lamp. Image acquisition and reconstruction is performed on the device using a custom-built android application, constructing a stand-alone imaging device for field applications. We discuss the construction of the device using a commercial smartphone and demonstrate the imaging capabilities of our system.

Finally, we report on the implementation of fluorescence chip-scale microscope, based on a silo-filter structure fabricated on the pixel array of a CMOS image sensor. The extruded pixel design with metal walls between neighboring pixels successfully guides fluorescence emission through the thick absorptive filter to the photodiode layer of a pixel. Our silo-filter CMOS image sensor prototype achieves 13-µm resolution for fluorescence imaging over a wide field-of-view (4.8 mm × 4.4 mm). Here, we demonstrate bright-field and fluorescence longitudinal imaging of living cells in a compact, low-cost configuration.

An investigation of a method of underwater propulsion by direct gas injection

This report presents the results of an investigation of a method of underwater propulsion. The propelling system utilizes the energy of a small mass of expanding gas to accelerate the flow of a large mass of water through an open ended duct of proper shape and dimensions to obtain a resultant thrust. The investigation was limited to making a large number of runs on a hydroduct of arbitrary design, varying between wide limits the water flow and gas flow through the device, and measuring the net thrust caused by the introduction and expansion of the gas.

In comparison with the effective exhaust velocity of about 6,000 feet per second observed in rocket motors, this hydroduct model attained a maximum effective exhaust velocity of more than 27,000 feet per second, using nitrogen gas. Using hydrogen gas, effective exhaust velocities of 146,000 feet per second were obtained. Further investigation should prove this method of propulsion not only to be practical but very efficient.

This investigation was conducted at Project No. 1, Guggenheim Aeronautical Laboratory, California Institute of Technology, Pasadena, California.

I. Anisotropy and crystal structure of ferromagnetic thin films. II. Investigations into magnetic microstructure by Lorentz microscopy

The induced magnetic uniaxial anisotropy of Ni-Fe alloy films has been shown to be related to the crystal structure of the film. By use of electron diffraction, the crystal structure or vacuum-deposited films was determined over the composition range 5% to 85% Ni, with substrate temperature during deposition at various temperatures in the range 25° to 500° C. The phase diagram determined in this way has boundaries which are in fair agreement with the equilibrium boundaries for bulk material above 400°C. The (α+ ɤ) mixture phase disappears below 100°C.

The measurement of uniaxial anisotropy field for 25% Ni-Fe alloy films deposited at temperatures in the range -80°C to 375°C has been carried out. Comparison of the crystal structure phase diagram with the present data and those published by Wilts indicates that the anisotropy is strongly sensitive to crystal structure. Others have proposed pair ordering as an important source of anisotropy because of an apparent peak in the anisotropy energy at about 50% Ni composition. The present work shows no such peak, and leads to the conclusion that pair ordering cannot be a dominant contributor.

Width of the 180° domain wall in 76% Ni-Fe alloy films as a function of film thickness up to 1800 Å was measured using the defocused mode of Lorentz microscopy. For the thinner films, the measured wall widths are in good agreement with earlier data obtained by Fuchs. For films thicker than 800 Å, the wall width increases with film thickness to about 9000 Å at 1800 Å film thickness. Similar measurements for polycrystalline Co films with thickness from 200 to 1500 Å have been made. The wall width increases from 3000 Å at 400 Å film thickness to about 6000 Å at 1500 Å film thickness. The wall widths for Ni-Fe and Co films are much greater than predicted by present theories. The validity of the classical determination of wall width is discussed, and the comparison of the present data with theoretical results is given.

Finally, an experimental study of ripple by Lorentz microscopy in Ni-Fe alloy films has been carried out. The following should be noted: (1) the only practical way to determine experimentally a meaningful wavelength is to find a well-defined ripple periodicity by visual inspection of a photomicrograph. (2) The average wavelength is of the order of 1µ. This value is in reasonable agreement with the main wavelength predicted by the theories developed by others. The dependence of wavelength on substrate deposition temperature, alloy composition and the external magnetic field has been also studied and the results are compared with theoretical predictions. (3) The experimental fact that the ripple structure could not be observed in completely epitaxial films gives confirmation that the ripple results from the randomness of crystallite orientation. Furthermore, the experimental observation that the ripple disappeared in the range 71 and 75% Ni supports the theory that the ripple amplitude is directly dependent on the crystalline anisotropy. An attempt to experimentally determine the order of magnitude of the ripple angle was carried out. The measured angle was about 0.02 rad. The discrepancy between the experimental data and the theoretical prediction is serious. The accurate experimental determination of ripple angle is an unsolved problem.