MEMS Devices for Circumferential-scanned Optical Coherence Tomography Bio-imaging

Ph.DDOCTOR OF PHILOSOPH

Optical coherence microscopy and focal modulation microscopy for Real-time Deep Tissue imaging

Ph.DDOCTOR OF PHILOSOPH

Development of high-speed two-photon microscopy for biological and medical applications

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 2005.Includes bibliographical references (p. 135-144).Two-photon microscopy (TPM) is one of the most powerful microscopic technologies for in-vivo 3D tissue imaging up to a few hundred micrometers. It has been finding important applications in neuronal imaging, tumor physiology study, and optical biopsy. A practical limitation of TPM is its slow imaging speed (0.3 1 frames/s). We designed high-speed two-photon microscopes (HSTPMs) whose imaging speed is more than 10 times faster than traditional TPM, while their imaging depths, image contrast are comparable to those of TPM. The first high speed system is HSTPM based on polygonal mirror scanner. The scanning speed reaches 13 frames/s for typical tissues using a polygonal mirror scanner. This system is based on single-focus scanning and single-pixel signal collection. The usage of higher input power is required to compensate for the signal reduction due to higher scanning speed. However, since fluorescence signal is ultimately limited by the saturation of fluorophores due to their finite lifetimes, is the signal to noise ratio (SNR) of single focus scanning systems are also ultimately limited at high speed. This problem is circumvented in a second system based on parallelization by scanning specimens with multiple foci of excitation light and collecting signals with spatially resolved detectors. The imaging speed is increased proportional to the number of foci and similar excitation laser power per focus circumventing the problem of fluorophore saturation. However, it has been recognized that this method is severely limited for deep tissue imaging due to photon scattering.(cont.) We quantitatively measured the photon scattering effect and demonstrated that its image resolution is the same as conventional TPM but its image contrast is degraded to the faster signal decay with the increase of imaging depth. We designed a new MMM based on multi-anode photomultiplier tube (MAPMT) which utilizes the advantage of MMM in terms of parallelization but overcomes the emission photon scattering problem by optimizing the design detector geometry. This method achieved equivalent SNR as conventional TPM with imaging speed more than 10 times higher than TPM. We applied these HSTPMs to a number of novel biomedical applications focusing on studying biological problems that needs to resolve the high speed kinetics processes or or the imaging of large tissue sections with subcellular resolution to achieve the requisite statistical accuracy. In the study of transdermal drug delivery mechanisms with chemical enhancers,, large section imaging enables microscopic transport properties to be measured even in skin which is highly topographical heterogeneous. This methodology allowed us to identify the novel transport pathways through the stratum corneum of skin. In the study of tumor physiology, microvasculature in tumor tissue deep below the surface was characterized to be densely distributed and tortuous compared to that of normal tissue. The interaction of leukocyte and endothelium in tumor tissue was measured by imaging the kinetics of leukocyte interaction with blood vessel wall in tumor tissues using HSTPM. The capability of large section imaging was further applied to develop a 3D tissue cytometer with the advantages that cell-cell and cell- extracellular matrix interaction can be quantified in tissues.(cont.) The statistical accuracy of this instrument was verified by quantitatively measuring cell population ratios in engineered tissue constructs composed of a mixture of two cell subpopulations. Further, this 3D tissue cytometer was applied to screen and to identify rare recombination events in transgenic mice that carry novel fluorescent genetic reporters.by Ki Hean Kim.Ph.D

Study of two-photon Line Excitation Array Detection microscopy

The functional meaning associated with neuronal activity in the mammalian brain is an active area of research limited by the available microscope instrumentation. Exploring this domain of neuroscience necessitates high-speed 3D imaging operating over 1 kHz volumetric scan rates with sub-cellular resolution, as action potentials propagate on sub-millisecond time scales. Monitoring these signals requires in vivo experimentation, so additional care must be taken to avoid invasive methods that may damage sample tissue to live animal subjects. Multi-photon imaging provides an opportunity for non-invasive microscopy with optical sectioning while simultaneously deeply penetrating brain tissue. However, current multi-photon microscopy methods are limited to 10-100 Hz volumetric imaging rates. This thesis explores and expands upon a potential high-speed 2-photon imaging technology, 2-photon Line Excitation Array Detection (2p-LEAD) microscopy. 2p-LEAD combines line scanning with detection via a multi-channel photomultiplier tube (PMT) array, with the potential to operate at 125 kHz frame rates. In the experimental prototype outlined in this thesis, a 1035 nm excitation line of 2.4 µm x 259 µm (1/e2 beam intensity diameter) is scanned at the focal plane. The resulting fluorescence is collected by a 16-channel linear PMT array. With a fast-scanning galvanometric mirror, we scan the excitation line at 3,000 FPS, generating a 170 µm x 75 µm fluorescence FOV imaged to a 16 x 320 pixel frame. Temporal focusing was implemented to improve optical sectioning and signal-to-noise ratio (SNR), by reducing the out of focus fluorescent signal. This reduction was achieved by dispersing the pulse-width from 300 fs at the focus to multiple picoseconds. 0.5-2 µm fluorescent polystyrene beads were imaged to characterize the system resolution of 1-5.3 µm laterally. Thus this research lays the groundwork for 2p-LEAD imaging at 125 kHz, with an acousto-optic deflector replacing the galvo-mirror as the primary scanning element, for high-speed neuronal imaging.Mechanical Engineerin

Laser-driven ion acceleration from carbon nano-targets with Ti:Sa laser systems

Over the past few decades, the generation of high energetic ion beams by relativistic intense laser pulses has attracted great attentions. Starting from the pioneering endeavors around 2000, several groups have demonstrated muliti-MeV (up to 58 MeV for proton by then) ion beams along with low transverse emittance and ps-scale pulse duration emitted from solid targets. Owing to those superior characteristics, laser driven ion beam is ideally suitable for many applications. However, the laser driven ion beam typically exhibits a large angular spread as well as a broad energy spectrum which for many applications is disadvantageous. The utilization of nano-targets as ion source provides a number of advantages over micrometer thick foils. The presented PhD work was intended to investigate laser driven ion acceleration from carbon nano-targets and demonstrate the potential feasibility for biological studies. Two novel nano-targets are employed: nm thin diamond-like-carbon (DLC) foil and carbon nanotubes foam (CNF). Both are self-produced in the technological laboratory at Ludwig-Maximilians-Universität München. Well-collimated proton beams with extremely small divergence (half angle) of 2 degrees are observed from DLC foils, one order of magnitude lower as compared to micrometer thick targets. Two-dimensional particle-in-cellsimulations indicate a strong influence from the electron density distribution on the divergence of protons. This interpretation is supported by an analytical model. In the same studies, the highest maximum proton energy was observed with a moderate laser intensity as low as 5*10^18W/cm^2. Parallel measurements of laser transmission and reflection are used to determine laser absorption in the nano-plasma, showing a strong correlation to the maximum proton energy. This observation indicates significance of absorbed laser energy rather than incident laser intensity and is supported by an analytical model. The ion energy also depends on pulse duration, a reduced optimum pulse duration is found as compared to micrometer thick targets. This behavior is attributed to a reduction of transverse electron spread due to the reduction of thickness from micrometer to nanometer. These remarkable proton bunch characteristics enabled irradiating living cells with a single shot dose of up to 7 Gray in one nanosecond, utilizing the Advanced Titanium: sapphire LASer (ATLAS)system at Max-Planck-Institut of Quantum Optics (MPQ). The experiments represent the first feasibility demonstration of a very compact laser driven nanosecond proton source for radiobiological studies by using a table-top laser system and advanced nano-targets. For the purpose of providing better ion sources for practical application, particularly in terms of energy increase, subsequent experiments were performed with the Astra Gemini laser system in the UK. The experiments demonstrate for the first time that ion acceleration can be enhanced by exploiting relativistic nonlinearities enabled by micrometer-thick CNF targets. When the CNF is attached to a nm-thick DLC foil, a significant increase of maximum carbon energy (up to threefold) is observed with circularly polarized laser pulses. A preferable enhancement of the carbon energy is observed with non-exponential spectral shape, indicating a strong contribution of the radiation pressure to the overall acceleration. In contrast, the linear polarization give rise to a more prominent proton acceleration. Proton energies could be increased by a factor of 2.4, inline with a stronger accelerating potential due to higher electron temperatures. Three-dimensional (3D) particle-in-cell (PIC) simulations reveal that the improved performance of the double-layer targets (CNF+DLC) can be attributed to relativistic self-focusing in near-critical density plasma. Interestingly, the nature of relativistic non-linearities, that plays a major role in laserwakefield-acceleration of electrons, can also apply to the benefit of laser driven ion acceleration

Corneal topography with high-speed swept source OCT in clinical examination

We present the applicability of high-speed swept source (SS) optical coherence tomography (OCT) for quantitative evaluation of the corneal topography. A high-speed OCT device of 108,000 lines/s permits dense 3D imaging of the anterior segment within a time period of less than one fourth of second, minimizing the influence of motion artifacts on final images and topographic analysis. The swept laser performance was specially adapted to meet imaging depth requirements. For the first time to our knowledge the results of a quantitative corneal analysis based on SS OCT for clinical pathologies such as keratoconus, a cornea with superficial postinfectious scar, and a cornea 5 months after penetrating keratoplasty are presented. Additionally, a comparison with widely used commercial systems, a Placido-based topographer and a Scheimpflug imaging-based topographer, is demonstrated

Defect and thickness inspection system for cast thin films using machine vision and full-field transmission densitometry

Quick mass production of homogeneous thin film material is required in paper, plastic, fabric, and thin film industries. Due to the high feed rates and small thicknesses, machine vision and other nondestructive evaluation techniques are used to ensure consistent, defect-free material by continuously assessing post-production quality. One of the fastest growing inspection areas is for 0.5-500 micrometer thick thin films, which are used for semiconductor wafers, amorphous photovoltaics, optical films, plastics, and organic and inorganic membranes. As a demonstration application, a prototype roll-feed imaging system has been designed to inspect high-temperature polymer electrolyte membrane (PEM), used for fuel cells, after being die cast onto a moving transparent substrate. The inspection system continuously detects thin film defects and classifies them with a neural network into categories of holes, bubbles, thinning, and gels, with a 1.2% false alarm rate, 7.1% escape rate, and classification accuracy of 96.1%. In slot die casting processes, defect types are indicative of a misbalance in the mass flow rate and web speed; so, based on the classified defects, the inspection system informs the operator of corrective adjustments to these manufacturing parameters. Thickness uniformity is also critical to membrane functionality, so a real-time, full-field transmission densitometer has been created to measure the bi-directional thickness profile of the semi-transparent PEM between 25-400 micrometers. The local thickness of the 75 mm x 100 mm imaged area is determined by converting the optical density of the sample to thickness with the Beer-Lambert law. The PEM extinction coefficient is determined to be 1.4 D/mm and the average thickness error is found to be 4.7%. Finally, the defect inspection and thickness profilometry systems are compiled into a specially-designed graphical user interface for intuitive real-time operation and visualization.M.S.Committee Chair: Tequila Harris; Committee Member: Levent Degertekin; Committee Member: Wayne Dale

Automated 3D model generation for urban environments [online]

Abstract In this thesis, we present a fast approach to automated generation of textured 3D city models with both high details at ground level and complete coverage for birds-eye view. A ground-based facade model is acquired by driving a vehicle equipped with two 2D laser scanners and a digital camera under normal traffic conditions on public roads. One scanner is mounted horizontally and is used to determine the approximate component of relative motion along the movement of the acquisition vehicle via scan matching; the obtained relative motion estimates are concatenated to form an initial path. Assuming that features such as buildings are visible from both ground-based and airborne view, this initial path is globally corrected by Monte-Carlo Localization techniques using an aerial photograph or a Digital Surface Model as a global map. The second scanner is mounted vertically and is used to capture the 3D shape of the building facades. Applying a series of automated processing steps, a texture-mapped 3D facade model is reconstructed from the vertical laser scans and the camera images. In order to obtain an airborne model containing the roof and terrain shape complementary to the facade model, a Digital Surface Model is created from airborne laser scans, then triangulated, and finally texturemapped with aerial imagery. Finally, the facade model and the airborne model are fused to one single model usable for both walk- and fly-thrus. The developed algorithms are evaluated on a large data set acquired in downtown Berkeley, and the results are shown and discussed