Computational Imaging Methods for Improving Resolution in Biological Microscopy

Optical microscopy is an essential tool for biological research, as it allows for non-invasive imaging of small animals. However, optical microscopy has its limits. Due to the low light level, fluorescence microscopy prohibits high speed imaging, making it difficult to study fast dynamic biological processes. In addition, optical blur due to the diffraction of light results in limited spatial resolution, particularly when using objective lenses with low numerical apertures. In this thesis, we propose computational imaging methods to overcome these limitations using a combination of novel image acquisition procedures and reconstruction algorithms.The first part of this thesis deals with improving temporal resolution in fluorescence microscopy to image rapid, repeating processes. We take advantage of multiple acquisitions, each taken with different time delays or temporally modulated illumination patterns, to recover high frequency information that is lost with traditional imaging. We demonstrate our method to image the beating heart in live embryonic zebrafish with reduced motion blur and high resolution in time.The second part of this thesis deals with reducing spatial blur in optical projection tomography, a form of optical microscopy that uses multiple 2D projections to reconstruct a 3D image of an object. We propose a method to reduce the optical distortion (as characterized by the system's optical point spread function) that can be implemented with a scanning acquisition approach combined with a modified filtered backprojection algorithm for reconstruction. We demonstrate our method to image blood vessels in larval zebrafish with high spatial resolution and reduced out-of-focus blur.The final part of this thesis deals with the dimensional limitation of 2D sensors for measuring 3D motion in microscopy. We propose a method to combine two-dimensional motion estimates from multiple views to recover out-of-plane velocity and reconstruct a divergence-free, three-dimensional velocity field. We demonstrate our method to measure, for the first time, dynamic blood flow in 3D inside the beating heart of a live zebrafish using optical microscopy.This thesis provides new tools that integrate custom image acquisition procedures and image reconstruction algorithms to overcome the resolution limitations -- temporal, spatial, and out-of-plane velocity resolution -- in optical microscopy. The methods presented in this thesis, in particular the single camera, active illumination method for temporal superresolution in fluorescence microscopy, will be directly applicable to a broad range of biological studies and will open up new perspectives for imaging small organisms in 3D (and time) with high spatio-temporal resolution

Characterization of a Hyperspectral Chromotomographic Imaging Ground System

A field-deployable hyperspectral chromotomographic imager has been developed and tested as a risk-reduction prototype to assist design of a space-based system. The instrument uses a high-speed video camera looking through a rotating direct-vision prism to simultaneously observe the full field of view in all visible wavelength channels. The chromotomographic process multiplexes the spectral and spatial so an advanced reconstruction algorithm is required to separate the spectral channels. A physics-based model of the instrument was developed to assist in future trade-space choices for design of the spaced-based system. Additionally, the model is used for the development and assessment of the filtered backprojection reconstruction algorithm. Laboratory experiments from the field-deployable instrument were collected, and the results are compared to physics-based model predictions. Results from the simulated and experimental data show that the instrument and algorithm are capable of detecting spectral and spatial information of complex scene

Development and Characterization of a Chromotomosynthetic Hyperspectral Imaging System

A chromotomosynthetic imaging (CTI) methodology based upon mathematical reconstruction of a set of 2-D spectral projections to collect high-speed (100 Hz) 3-D hyperspectral data cube has been proposed. The CTI system can simultaneously provide usable 3-D spatial and spectral information, provide high-frame rate slitless 1-D spectra, and generate 2-D imagery equivalent to that collected with no prism in the optical system. The wavelength region where prism dispersion is highest (500 nm) is most sensitive to loss of spectral resolution in the presence of systematic error, while wavelengths 600 nm suffer mostly from a shift of the spectral peaks. The quality of the spectral resolution in the reconstructed hyperspectral imagery was degraded by as much as a factor of two in the blue spectral region with less than 1° total angular error in mount alignment in the two axes of freedom. Even with no systematic error, spatial artifacts from the reconstruction limit the ability to provide adequate spectral imagery without specialized image reconstruction techniques as targets become more spatially and spectrally uniform

An Experimental Evaluation of Image Quality for Various Scenarios In a Chromotomographic System With a Spinning Prism

A lab and eld based hyperspectral chromotomographic imager has been developed at the Air Force Institute of Technology. It is a prototype used to study the requirements for a space-based system. The imager uses a high speed visible band camera behind a direct-vision prism to image both spatial dimensions and the spectral dimension at the same time. Capturing all 3 simultaneously allows for the hyperspectral imaging of transient events. The prism multiplexes the spectral and spatial information, so tomographic reconstruction algorithms must be used to separate hyperspectral channels. Experiments were conducted to compare reconstructed image quality as a function of spatial, spectral, and temporal complexity. To test spatial complexity, a simulated point source, a standard bar chart, and an amorphous object were imaged. The more simple objects were reconstructed well. When objects dispersed by the prism overlapped in the raw data, reconstructions diminished. To test spectral complexity, atomic emission lines and white light with different types of optical filters were imaged. Because of the way the prism disperses light, shorter wavelengths were reconstructed better than longer ones. The temporal complexity experiments determined 3/4 of a prism revolution is sufficient (with approximately 60 projections) to reconstruct a scene as long as there were a sufficient number of frames in the recording. As few as 12 frames are sufficient in a complete revolution. Finally, a fireball was imaged in a eld experiment. The chromotomographic imager constructed the spectrum of the fireball with very wide spectral lines. Hindrances to reconstruction, such as a cone of missing hyperspectral information, are discussed. Additionally, a comparison of and improvements to the reconstruction algorithms are suggested

Flame front propagation velocity measurement and in-cylinder combustion reconstruction using POET

The objective of this thesis is to develop an intelligent diagnostic technique POET (Passive Optical Emission Tomography) for the investigation of in cylinder combustion chemiluminescence. As a non-intrusive optical system, the POET system employs 40 fibre optic cables connected to 40 PMTs (Photo Multiplier Tube) to monitor the combustion process and flame front propagation in a modified commercial OHV (Over Head Valve) Pro 206 IC engine. The POET approach overcomes several limitations of present combustion research methods using a combination of fibre optic detection probes, photomultipliers and a tomographic diagnostics. The fibre optic probes are placed on a specially designed cylinder head gasket for non-invasively inserting cylinder. Each independent probe can measure the turbulent chemiluminescence of combustion flame front at up to 20 kHz. The resultant intensities can then be gathered tomographically using MART (Multiplicative Algebraic Reconstruction Technique) software to reconstruct an image of the complete flame-front. The approach is essentially a lensless imaging technique, which has the advantage of not requiring a specialized engine construction with conventional viewing ports to visualize the combustion image. The fibre optic system, through the use of 40, 2m long thermally isolated fibre optic cables can withstand combustion temperatures and is immune from electronic noise, typically generated by the spark plug. The POET system uses a MART tomographic methodology to reconstruct the turbulent combustion process. The data collected has been reconstructed to produce a temporal and spatial image of the combustion flame front. The variations of lame turbulence are monitored by sequences of reconstructed images. Therefore, the POET diagnostic technique reduces the complications of classic flame front propagation measurement systems and successfully demonstrates the in-cylinder combustion process. In this thesis, a series of calibration exercises have been performed to ensure that the photomultipliers of the POET system have sufficient temporal and spatial resolution to quantitatively map the flow velocity turbulence and chemiluminescence of the flame front. In the results, the flame has been analyzed using UV filters and blue filters to monitor the modified natural gas fuel engine. The flame front propagation speed has been evaluated and it is, on average, 12 m/s at 2280 rpm. Sequences of images have been used to illustrate the combustion explosion process at different rpm

Characterization of multiphase flows integrating X-ray imaging and virtual reality

Multiphase flows are used in a wide variety of industries, from energy production to pharmaceutical manufacturing. However, because of the complexity of the flows and difficulty measuring them, it is challenging to characterize the phenomena inside a multiphase flow. To help overcome this challenge, researchers have used numerous types of noninvasive measurement techniques to record the phenomena that occur inside the flow. One technique that has shown much success is X-ray imaging. While capable of high spatial resolutions, X-ray imaging generally has poor temporal resolution. This research improves the characterization of multiphase flows in three ways. First, an X-ray image intensifier is modified to use a high-speed camera to push the temporal limits of what is possible with current tube source X-ray imaging technology. Using this system, sample flows were imaged at 1000 frames per second without a reduction in spatial resolution. Next, the sensitivity of X-ray computed tomography (CT) measurements to changes in acquisition parameters is analyzed. While in theory CT measurements should be stable over a range of acquisition parameters, previous research has indicated otherwise. The analysis of this sensitivity shows that, while raw CT values are strongly affected by changes to acquisition parameters, if proper calibration techniques are used, acquisition parameters do not significantly influence the results for multiphase flow imaging. Finally, two algorithms are analyzed for their suitability to reconstruct an approximate tomographic slice from only two X-ray projections. These algorithms increase the spatial error in the measurement, as compared to traditional CT; however, they allow for very high temporal resolutions for 3D imaging. The only limit on the speed of this measurement technique is the image intensifier-camera setup, which was shown to be capable of imaging at a rate of at least 1000 FPS. While advances in measurement techniques for multiphase flows are one part of improving multiphase flow characterization, the challenge extends beyond measurement techniques. For improved measurement techniques to be useful, the data must be accessible to scientists in a way that maximizes the comprehension of the phenomena. To this end, this work also presents a system for using the Microsoft Kinect sensor to provide natural, non-contact interaction with multiphase flow data. Furthermore, this system is constructed so that it is trivial to add natural, non-contact interaction to immersive visualization applications. Therefore, multiple visualization applications can be built that are optimized to specific types of data, but all leverage the same natural interaction. Finally, the research is concluded by proposing a system that integrates the improved X-ray measurements, with the Kinect interaction system, and a CAVE automatic virtual environment (CAVE) to present scientists with the multiphase flow measurements in an intuitive and inherently three-dimensional manner