The study of interfacial structures is of utmost importance not only for various research fields such as cell biology and display systems but also their sub-disciplines. One of the traditional means of imaging buried structures rely on the use optical sectioning with superresolution microscopy. Although it exceeds diffraction limit in resolution, there are various shortcomings to utilize this methodology such as its reliance on fluorescent markers, long exposure times to high cost of the imaging system. Ultimately, these limitations position the existing technologies unideal for live cell imaging, including the imaging of surface proteins of a living cell. A label free quantitative phase imaging method is realized in this project to enable imaging of an interface between different media. This system is based on an off-axis holographic microscope and uses a high numerical aperture (NA) microscope objective to achieve total internal reflection (TIR). Existing literature on total internal reflection holographic microscopy utilizes prism to achieve TIR which limits the working distance of objective hence magnification. Our system relies on a 100x objective with 1.49 NA to improve resolution and magnification. Complex field which is reflected from the sample can be recovered by using digital holography principles. The resolution of the system can further be enhanced by combining several illumination angles and utilizing synthetic aperture reconstruction.
Photolithography has become a powerful tool in the fabrication of micro-optical elements following the advancements in grayscale approaches. However, hitting tight design tolerance goals require precise control of all parameters such as temperature, resist nonlinearity or preventing vignetting. In this work we took an alternative route to these problems by combining maskless lithography with digital holography. Addition of digital holography enables the use of feedback by measuring the quantitative phase of specimen near real time and in situ nondestructively. After each near UV exposure, phase retardation map of exposed photopolymer is measured with digital holography part of the system. Any deviation from target phase is corrected by changing the pattern displayed on the mask. We showed that the proposed method reduces the standard deviation of resulting phase compared to traditional one-shot grayscale lithography. It also does not require any precalibration of photoresist and relaxes the constraints for uniform UV illumination in sample plane.
Refractive index tomography as an emerging technique enables the 3D morphological investigation of cells with no marker. Here, refractive index tomographic imaging of myelinating glial cells is presented. Myelin as a signal insulation layer around an axon is formed by the wrapping of Schwann cells or oligodendrocytes. Microscopic investigation of myelination traditionally requires fluorescent markers. Glial cells generally wrap the axon for more than ten layers. This multilayer formation has alternating and uniform layers of protein and lipid. Earlier studies on the structure of the myelin sheath have shown that the thickness period is lower than 20nm including the thickness of the extracellular medium after each layer. Direct observation of an individual layer is not possible (using classical microscopy techniques) due to dimensions being very small compared to the wavelength of the illumination light. However, periodic nature of the layers enables the differentiation of a myelinated axon from an unmyelinated one. Rapid change of the integrated refractive index and the Bragg fiber like structure alters the transmission behavior as a function of wavelength and incidence angle. With the 3D sectioning capability of refractive index tomography, these features can be easily identified.
Digital holographic microscopy (DHM) has been gaining interest from cell biology community because of its label free
nature and quantitative phase signal output. Besides, fast shutter time, image reconstruction by numerical propagation of
the wave fields, and numerical compensation of the aberrations are other intrinsic advantages of this technique that can
be explored for harsh imaging conditions. In the frame of this work, a transmission type DHM is developed with a
decoupled epifluorescence microscopy mode for cytomorphological monitoring under zero gravity and hyper gravity.
With the implemented automatic post processing routines, real time observation of the cell morphology is proven to be
feasible under the influence of mechanical disturbances of zero gravity platforms. Post processing of holograms is
composed from dynamic numerical compensation of holograms, robust autofocusing and phase image registration.
Experiments on live myoblast cells are carried out on two different platforms; random positioning machine (RPM), a
ground base microgravity simulation platform, and parabolic flight campaign (PFC), a fixed wing plane flight providing
short durations of alternating gravity conditions. Results show clear perinuclear phase increase. During seconds scale
microgravity exposure, measurable scale morphological modifications are observed with the accumulated effect of
repetitive exposures and short breaks.
We present a theory to extend the classical Abbe resolution limit by introducing a spatially varying phase into the illumination beam of a phase imaging system. It allows measuring lateral and axial distance differences between point sources to a higher accuracy than intensity imaging alone. Various proposals for experimental realization are debated. Concretely, the phase of point scatterers' interference is experimentally visualized by high numerical aperture (NA = 0.93) digital holographic microscopy combined with angular scanning. Proof-of-principle measurements are presented by using sub-wavelength nanometric holes on an opaque metallic film. In this manner, Rayleighs classical two-point resolution condition can be rebuilt. With different illumination phases, enhanced bandpass information content is demonstrated, and its spatial resolution is theoretically shown to be potentially signal-to-noise ratio limited.
We present a theory stating how to overcome the classical Rayleigh-resolution limit. It is based upon a new
resolution criterion in phase of coherent imaging process and its spatial resolution is thought to be only SNR
limited. Recently, the experimental observation of systematically occurring phase singularities in coherent
imaging of sub-Rayleigh distanced objects has been reported.1 The phase resolution criterion relies on the
unique occurrence of phase singularities. A priori, coherent imaging system's resolution can be extended to
Abbe's limit.2 However, by introducing a known phase difference, the lateral as well as the longitudinal resolution
can be tremendously enlarged.
The experimental setup is based on Digital Holographic Microscopy (DHM), an interferometric method
providing access to the complex wave front. In off-axis transmission configuration, sub-wavelength nano-metric
holes on a metallic film acts as the customized high-resolution test target. The nano-metric apertures are drilled
with focused ion beam (FIB) and controlled by scanning electron microscopy (SEM). In this manner, Rayleighs
classical two-point resolution condition can be rebuilt by interfering complex fields emanated from multiple
single circular apertures on an opaque metallic film. By introducing different offset phases, enhanced resolution
is demonstrated. Furthermore, the measurements can be exploited analytically or within the post processing of
sampling a synthetic complex transfer function (CTF).
Previous investigations on mammalian cells have shown that microgravity, either that experienced in space, or simulated on earth, causes severe cellular modifications that compromise tissue determination and function. The aim of this study is to investigate, in real time, the morphological changes undergone by cells experiencing simulated microgravity by using digital holographic microscopy (DHM). DHM analysis of living mouse myoblasts (C2C12) is undertaken under simulated microgravity with a random positioning machine. The DHM analysis reveals cytoskeletal alterations similar to those previously reported with conventional methods, and in agreement with conventional brightfield fluorescence microscopy a posteriori investigation. Indeed, DHM is shown to be able to noninvasively and quantitatively detect changes in actin reticular formation, as well as actin distribution, in living unstained samples. Such results were previously only obtainable with the use of labeled probes in conjunction with conventional fluorescence microscopy, with all the classically described limitations in terms of bias, bleaching, and temporal resolution.
A dual mode microscope is developed to study morphological evolution of mouse myoblast cells under simulated
microgravity in real time. Microscope operates in Digital Holographic Microscopy (DHM) and widefield epifluorescence
microscopy modes in a time sequential basis. DHM provides information on real time cellular morphology.
EGFP transfected actin filaments in mouse myoblast cells function as the reporter for the fluorescence microscopy mode.
Experimental setup is fixed in the RPM to observe microgravity induced dynamic changes in live cells. Initial results
revealed two different modifications. Disorganized structures become visible in the formed lamellipodias, and proteins
accumulate in the perinuclear region.
Novel thermo-mechanical detector arrays with integrated diffraction grating for optical readout were designed and
fabricated. Parylene was used as the structural material due to its high thermal isolation and mismatch properties.
Calculations reveal that the NETD performance of a thermo-mechanical array using Parylene can be significantly better
than SiNx based designs and offer a theoretical NETD value <10mK assuming an optical readout with a high dynamic
range detector array. Finite Element simulations were performed with length of the bimaterial leg as the optimization
parameter. It was observed that only a few microns of isolation leg supported 30 fps applications, leaving rest of the leg
to be bimaterial and providing large thermo-mechanical deflections.
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