Methods and Systems for Realizing High Resolution Three-Dimensional Optical Imaging

Methods and systems for realizing high resolution three-dimensional (3-D) optical imaging using diffraction limited low\u27 resolution optical signals. Using axial shift-based signal processing via computer based computation algorithm, three sets of high resolution optical data are detennined along the axial (or light beam propagation) direction using low resolution axial data. The three sets of low resolution data are generated by illuminating the 3~D object under observation along its three independent and orthogonal look directions (i.e., x. Y. and z) or by physically rotating the object by 90 degrees and also flipping the object by 90 degrees. The three sets of high resolution axial data is combined using a unique mathematical function to interpolate a 3-D image of the test object that is of much higher resolution than the diffiaction limited direct measurement 3-D resolution. Confocal microscopy or optical coherence tomography (OCT) are example methods to obtain the axial scan data sets

A High-Resolution Combined Scanning Laser- and Widefield Polarizing Microscope for Imaging at Temperatures from 4 K to 300 K

Polarized light microscopy, as a contrast-enhancing technique for optically anisotropic materials, is a method well suited for the investigation of a wide variety of effects in solid-state physics, as for example birefringence in crystals or the magneto-optical Kerr effect (MOKE). We present a microscopy setup that combines a widefield microscope and a confocal scanning laser microscope with polarization-sensitive detectors. By using a high numerical aperture objective, a spatial resolution of about 240 nm at a wavelength of 405 nm is achieved. The sample is mounted on a $^4$He continuous flow cryostat providing a temperature range between 4 K and 300 K, and electromagnets are used to apply magnetic fields of up to 800 mT with variable in-plane orientation and 20 mT with out-of-plane orientation. Typical applications of the polarizing microscope are the imaging of the in-plane and out-of-plane magnetization via the longitudinal and polar MOKE, imaging of magnetic flux structures in superconductors covered with a magneto-optical indicator film via Faraday effect or imaging of structural features, such as twin-walls in tetragonal SrTiO$_3$. The scanning laser microscope furthermore offers the possibility to gain local information on electric transport properties of a sample by detecting the beam-induced voltage change across a current-biased sample. This combination of magnetic, structural and electric imaging capabilities makes the microscope a viable tool for research in the fields of oxide electronics, spintronics, magnetism and superconductivity.Comment: 14 pages, 11 figures. The following article has been accepted by Review of Scientific Instruments. After it is published, it will be found at http://aip.scitation.org/journal/rs

Development of polarization-resolved optical scanning microscopy imaging techniques to study biomolecular organizations

Light, as electromagnetic radiation, conveys energy through space and time via fluctuations in electric and magnetic fields. This thesis explores the interaction of light and biological structures through polarization-resolved imaging techniques. Light microscopy, and polarization analysis enable the examination of biological entities. Biological function often centers on chromatin, the genetic material composed of DNA wrapped around histone proteins within cell nuclei. This structure's chiral nature gives rise to interactions with polarized light. This research encompasses three main aspects. Firstly, an existing multimodal Circular Intensity Differential Scattering (CIDS) and fluorescence microscopy are upgraded into an open configuration to be integrated with other modalities. Secondly, a novel cell classification method employing CIDS and a phasor representation is introduced. Thirdly, polarization analysis of fluorescence emission is employed for pathological investigations. Accordingly, the thesis is organized into three chapters. Chapter 1 lays the theoretical foundation for light propagation and polarization, outlining the Jones and Stokes-Mueller formalisms. The interaction between light and optical elements, transmission, and reflection processes are discussed. Polarized light's ability to reveal image contrast in polarizing microscopes, linear and nonlinear polarization-resolved microscopy, and Mueller matrix microscopy as a comprehensive technique for studying biological structures are detailed. Chapter 2 focuses on CIDS, a label-free light scattering method, including a single point angular spectroscopy mode and scanning microscopy imaging. A significant upgrade of the setup is achieved, incorporating automation, calibration, and statistical analysis routines. An intuitive phasor approach is proposed, enabling image segmentation, cell discrimination, and enhanced interpretation of polarimetric contrast. As a result, image processing programs have been developed to provide automated measurements using polarization-resolved laser scanning microscopy imaging integrated with confocal fluorescence microscopy of cells and chromatin inside cell nuclei, including the use of new types of samples such as progeria cells. Chapter 3 applies a polarization-resolved two-photon excitation fluorescence (2PEF) microscopy to study multicellular cancerous cells. A homemade 2PEF microscope is developed for colon cancer cell analysis. The integration of polarization and fluorescence techniques leads to a comprehensive understanding of the molecular orientation within samples, particularly useful for cancer diagnosis. Overall, this thesis presents an exploration of polarization-resolved imaging techniques for studying biological structures, encompassing theory, experimental enhancements, innovative methodologies, and practical applications

High Resolution Scanning Probes for Ferroelectric Thin Films

Advances in materials growth techniques enable precise control over the growth of novel functional materials such as ferroelectric thin films, which are interesting from both a physics and applications perspective. Physical properties of ferroelectric thin films differ a lot from their bulk counterparts, mainly due to the lattice mismatch at the film-substrate interface and differential thermal contraction experienced during growth. Those property anomalies are confined to a narrow range usually thinner than 1000 nm. High-resolution probes are important for understanding the spatial and temporal properties of these systems. We have developed mechanical and optical scanning probe techniques and used them to investigate various strain-engineered ferroelectric thin films. These optical and scanning probe techniques are designed to detect ferroelectric domain dynamics. Our experimental results either give direct evidence to verify material functionality, or reveal the relation between nano-scale dynamics to their macroscopic properties

Development of laser sources and interferometric approaches for polarization-based label-free microscopy

The project developed in this thesis describes the design and the experimental realization of optical methods which can probe the anisotropy of semitransparent media. The ability to manipulate polarized light enables a label-free imaging approach that can retrieve fundamental information about the sample structure without introducing any alteration within it. Such a potential is of great importance and methods like the ones based on polarization analysis are gaining more and more popularity in the biomedical and biophysical fields. Moreover, when they are coupled with fluorescence microscopy and nanoscopy, they may provide an invaluable tool for researchers. The optical method I developed mainly exploits the laser radiation emitted from tailored optical oscillators to dynamically generate polarization states. The realization of such states does not comprise any external active device. The resulting time-evolving polarization state once properly coupled to an optical system enables probing a sample to retrieve its anisotropies at a fast rate. The development of two different laser sources is presented together with the characterizations of their optical properties. One of them consists of a Helium-Neon laser modified by applying an external magnetic field to trigger the Zeeman effect in its active medium. The other one is a Dual-Comb source, that is a mode-locked (ML) laser generating a pair of mutually coherent twin beams. Moreover, the thesis delivers the theoretical model and the experimental realization of the optical method to probe the optical anisotropies of specimens. Finally, the technical realization of a custom laser scanning optical microscope and its imaging results obtained with such methods are reported

Agile optical confocal microscopy instrument architectures for high flexibility imaging

Ideally, a no-moving parts fast and agile scanning confocal microscope system is required that can produce true real-time 3-D scans with precision and repeatability. In this paper, such agile optical confocal microsopy designs are proposed that enable high speed precise non-invasive 3-D imaging. These compact confocal microscopes can provide real-time pin-point focussed imaging to enable confocal slices in-vivo, thus greatly reducing motion artifacts. These microscopes can be modified into interferometric microscopes for phase contrast imaging. The proposed microscopes can also greatly improve confocal fluorescence imaging as needed for cancer detection. An ultracompact confocal probe tip connected to a single ultra-thin fiber is another design option allowing flexibility for usage in tight cavities

Confocal Laser Scanning Microscopy As A Tool For The Investigation Of Tetracycline Fluorescence In Archaeologicalhuman Bone

Fluorochromes such as tetracycline have been used to label bone for histomorphometric analysis, measuring bone formation, growth, maintenance, and pathology. More recently, similar fluorescence has been observed in ancient human bone. Attributed to tetracycline (TC) exposure, this phenomenon could affect various aspects of health during life and/or preservation of remains postmortem. Standard epifluorescence microscopy is the most common tool employed in the analysis of these labels. Though valuable, this technique is limited by its inability to penetrate bone three-dimensionally and its inclusion of out-of-focus light, possibly disrupting accurate analysis. Confocal Laser Scanning Microscopy (CLSM) has been demonstrated as a valuable tool for three-dimensional histology. Its application to the study of compact bone fluorescence has been lacking, especially in archaeological and forensic sciences. In the following two papers, modern TC-controlled bone is compared to well preserved archaeological bone recovered from the Dakhleh Oasis, Egypt, using both standard wide-field and more modern confocal techniques for imaging and analysis. Spectral analysis via CLSM shows that both modern and ancient fluorescent labels in bone share the exact same fluorescence emission peak at 525 nm. Differences in the shape of the spectral curve and photobleaching characteristics are discussed. In addition, CLSM\u27s high-resolution two- and three-dimensional imaging capabilities (in polarized light, scattered light, and fluorescence light) are found to increase the flexibility and creativity of investigations into the occurrence of tetracycline labels in archaeological bone and could have added benefits for modern medical and anatomical experimentation

REAL-TIME RAMAN SPECTROSCOPY AND WIDE-ANGLE X-RAY DIFFRACTION DURING SINGLE-LAYER AND MULTI-LAYER BLOWN FILM EXTRUSION

Properties exhibited by blown films are controlled by the microstructure developed during their processing. Therefore, real-time measurement of microstructure during the blown film extrusion can help in better control and optimization of the process needed to obtain desired properties. The objectives of this research were (i) to conduct real-time microstructural measurements during single-layer and multi-layer blown film extrusion of polypropylene (PP) and low-density polyethylene (LDPE) using real-time Raman spectroscopy; (ii) to conduct real-time wide-angle X-ray diffraction (WAXD) during single-layer blown film extrusion of LDPE to obtain crystallinity and orientation values during the process; and (iii) to investigate the effect of blown film coextrusion on the microstructure of PP/LDPE bilayer films. The potential of real-time Raman spectroscopy as a rapid microstructure monitoring tool for better process control during blown film extrusion is demonstrated. Real-time polarized Raman spectroscopy was conducted to measure the orientation development during blown film extrusion of low-density polyethylene (LDPE). Polarized Raman spectra were obtained at different locations along the blown film line, starting from the molten state near the die and extending up to the solidified state near nip-rolls. The trans C-C symmetrical stretching vibration of PE at 1130 cm-1 was analyzed for films possessing uniaxial symmetry. For the given peak, the principal axis of the Raman tensor is coincident with the c-axis of the orthorhombic crystal, and was used to obtain second (2(cos θ)\u3e)( ) and fourth ( ) moments of the orientation distribution function. The orientation parameters (P2, P4) were found to increase along the axial distance in the film line even past the frost-line height (FLH). The P2 values also showed an increasing trend with crystalline evolution during extrusion consistent with past observations that molecular orientation takes place even after the blown film diameter is locked into place. It was also found that the integral ratio (I1132/I1064), obtained from a single, ZZ-backscattered mode, can provide a reasonable estimate of molecular orientation. Although Raman spectroscopy is a convenient technique, it is not a primary measurement technique to obtain crystallinity and orientation in fibers or films. So for the first time, a real-time wide-angle X-ray diffraction (WAXD) technique was attempted during blown film extrusion. WAXD patterns were obtained at different axial positions in the film line starting from a location near the die up to the nip-roller. The composite X-ray diffraction patterns from the bubble were analyzed and quantified for crystallinity values. From the evolution of (110) and (200) peaks in the WAXD pattern, it was inferred that the crystallization process started near the frost-line height (FLH) and showed a steep increase at lower axial distance near the freeze line and then a gradual increase at higher axial distance in the film line. The differences in the profiles for crystallinity were also evident with changing processing conditions. The crystallinity results obtained using WAXD were found to be consistent with those from simultaneous real-time Raman spectroscopic measurements. Thus, for the first time, real-time WAXD technique was successfully used for measurement of microstructure during the single-layer blown film extrusion of low density polyethylene (LDPE). Multi-layer blown films are of significant industrial importance to make packaging films with desirable properties through combination of two or more polymers. Therefore, real-time Raman measurements were extended from single-layer blown film extrusion to multi-layer blown film extrusion. Online spectroscopic measurements were carried out to estimate crystalline growth of the individual components of a bicomponent low-density polyethylene (LDPE) and polypropylene (PP) film (LDPE/PP). The 1296-1305 cm-1 band, observed predominantly for PE, was only slightly masked by the contribution from the PP layer. In contrast, the 809-841 cm-1 band was unique for PP and unaffected by the presence of the PE layer and 1418 cm-1 band was unique for PE. These distinct peaks enabled successful deconvolution of the superimposed spectra to enable crystallinity measurements during coextrusion of LDPE/PP films. Such real-time results have not been reported earlier in the literature for multi-layer films. Finally, real-time Raman spectroscopy results were used to develop an understanding of processing-microstructure relationship for the blown film process. For bilayer films (PP/LDPE), the onset crystallization-time difference for PP and LDPE components was found to be an important parameter, which controls the orientation and morphology of the coextruded films. Although overall molecular orientation within PP and LDPE multiple layers was not affected, single-layer LDPE films displayed some row-nucleation, but not the LDPE layer in coextruded films. Also, there was a slight decrease of crystalline a-axis orientation for coextruded LDPE layer as compared to that for single-layer LDPE films. Thus, as one component of experimental research being conducted at the Center for Advanced Engineering Fibers and Films (CAEFF), this study was successful in generating real-time experimental results during the film formation that are critical for validating modeling results being generated in companion studies