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Beyond the lateral resolution limit by phase imaging

Beyond the lateral resolution limit by phase imaging

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Conference on three-dimensional and multidimensional microscopy - image acquisition and processing XVIII

作者： Cotte, Y. Toy, M. Fatih Depeursinge, C. Ecole Polytech Fed Lausanne Microvis & Microdiagnost Grp CH-1015 Lausanne Switzerland

ISBN: (纸本)9780819484413

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).

关键词： Point spread function microscopy super-resolution digital holography phase imaging phase vortices

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Refractive index reconstruction of biological samples from multimodal phase microscopy

Refractive index reconstruction of biological samples from m...

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Conference on three-dimensional and multidimensional microscopy - image acquisition and processing XVIII

作者： Sierra, Heidy DiMarzio, Charles A. Brooks, Dana H. Rutgers State Univ Dept Biomed Engn 599 Taylor Rd Piscataway NJ 08854 USA Northeastern Univ Dept Elect & Comp Engn Boston MA 02115 USA

ISBN: (纸本)9780819484413

The extraction of quantitative information is important to better understand cellular activity in biological processes. In particular the optical refractive index can be used to analyze the results of cellular processes such as the average dry mass of biological samples. Phase microscopy modalities are widely used to image unstained biological samples because of their ability to obtain high-contrast images without introducing exogenous agents. The most common phase modalities are predominantly qualitative. However quantitative phase microscopy can provide more specific information about optical thickness and refractive index. In biological samples with several internal inhomogeneities and thickness variations, refractive index calculation becomes challenging to achieve by direct analysis of the images. Here we present a multimodal iterative method to reconstruct the spatial distribution of refractive index, combining information from two phase microscopy techniques. We use a constrained boundary iterative method under the assumption that the index of refraction inside the object can be approximated as piecewise constant. The boundary locations of all inhomogeneities are obtained by leveraging measurements from DIC and quantitative phase imaging modalities, and then the index of refraction is estimated based on those boundaries and a quantitative forward model for one modality. Simulations have confirmed the reliability of the proposed method. Experiments with measurement from mouse embryos at several development stages show that the proposed approach can reconstruct the distribution of the refractive index of these samples.

关键词： Differential Interference Contrast (DIC) Multimodal Phase Imaging Inversion Techniques three-dimensional Reconstruction Index of Refraction Reconstruction Optical Quadrature microscopy (OQM)

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Whole cell imaging based on wide-field interferometric phase microscopy and its application to cardiomyocytes

Whole cell imaging based on wide-field interferometric phase...

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Conference on three-dimensional and multidimensional microscopy - image acquisition and processing XVIII

作者： Shaked, Natan T. Satterwhite, Lisa L. Bursac, Nenad Wax, Adam Duke Univ Dept Biomed Engn Fitzpatrick Inst Photon Durham NC 27708 USA

ISBN: (纸本)9780819484413

Whole cell imaging is a novel technique using which the time-dependent quantitative phase profiles of live unstained biological cells are analyzed numerically to learn on the cell functionally. Dynamic phase profiles of the sample are first acquired by wide-field digital interferometry (WFDI), a quantitative holographic approach, without the need for scanning or using exogenous contrast agents. The resulting phase profiles are proportional to the multiplication between the cell thickness profile and its integral refractive index profile. However, many morphological parameters, including cell volume and cell force distribution, are based on the cell thickness profile, rather than on its WFDI phase profile. For cells with heterogeneous refractive index structure, more than a single exposure is typically needed to decouple thickness from integral refractive index using the phase profile, with the risk of losing transient acquisition. The presented whole-cell-imaging approach show that the WFDI phase profiles are useful for numerically analyzing cells even in cases where decoupling of thickness and integral refractive index is not possible or desired. We thus define new numerical parameters that directly utilize the WFDI phase profile and demonstrate their usefulness for characterizing contracting cardiomyocytes, cells with complex and highly-dynamic refractive-index structure.

关键词： Cell analysis interference microscopy phase holography cardiomyocytes

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Dynamic phase imaging utilizing a 4-dimensional microscope system

Dynamic phase imaging utilizing a 4-dimensional microscope s...

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Conference on three-dimensional and multidimensional microscopy - image acquisition and processing XVIII

作者： Creath, Katherine 4D Technol Corp Tucson AZ 85706 USA

ISBN: (纸本)9780819484413

This paper describes a new, novel interference Linnik microscope system and presents images and data of live biological samples. The specially designed optical system enables instantaneous 4-dimensional video measurements of dynamic motions within and among live cells without the need for contrast agents. This "label-free", vibration insensitive imaging system enables measurement of biological objects in reflection using harmless light levels with a variety of magnifications and wavelengths with fields of view from several hundred microns up to a millimeter. At the core of the instrument is a phase measurement camera (PMC) enabling simultaneous measurement of multiple interference patterns utilizing a pixelated phase mask taking advantage of the polarization properties of light. Utilizing this technology enables the creation of phase image movies in real time at video rates so that dynamic motions and volumetric changes can be tracked. Objects are placed on a reflective surface in liquid under a coverslip. Phase values are converted to optical thickness data enabling volumetric, motion and morphological studies. Data from a number of different organisms such as flagellates and rotifers will be presented, as will measurements of human breast cancer cells with the addition of various agents that break down the cells. These data highlight examples of monitoring different biological processes and motions.

关键词： phase imaging interference microscopy polarization interferometry cellular imaging cell dynamics optical thickness measurement

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Two dimensional scanning probe using off-axis magnetic force of single solenoid for 3D OCT imaging

Two dimensional scanning probe using off-axis magnetic force...

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Conference on three-dimensional and multidimensional microscopy - image acquisition and processing XVIII

作者： Min, Eun Jung Shin, Jun Geun Kim, Yuri Lee, Byeong Ha GIST Sch Informat & Commun 261 Cheomdan Gwagiro Kwangju 500712 South Korea GIST Grad Program Med Syst Engn Kwangju 500712 South Korea

ISBN: (纸本)9780819484413

We propose a single piece optical fiber-based two-dimensional scanning hand-held probe suitable for three-dimensional optical coherence tomography. The probe consists of only a single piece of optical fiber loaded with a bead of ferromagnetic material, which acts as a vibrating cantilever. The fiber cantilever is two dimensionally actuated with a single miniaturized solenoid. For effective beam focusing, a fiber lens is formed at the end of the fiber. The inductance and input current of the solenoid were 100 mu H and 216 mA, respectively. The iron-bead on the fiber is located at the off-axis of solenoid for two-dimensional scanning. Then, by modulating the input current to the solenoid, it was possible to mechanically oscillate the fiber cantilever in an elliptically spiral pattern. With the proposed probe, 2-dimensional scanning could be experimentally achieved in a rate of 4 s/vortex across a scanning area of approximately 30 mm(2), which could be controlled with the length of the fiber or/and the weight of the iron-bead. three-dimensional tomographic image of a coin was successfully obtained with the spectral domain optical coherence tomography equipped with the proposed scanner. It is expected that the scheme of 2-dimentional scanning with a single actuator might be useful in various real-time imaging applications including OCT owing to the advantages of low cost, low power consumption, simple fabrication process and versatile design.

关键词： fiber-based hand-held probe ferromagnetic material spectral domain OCT real-time in vivo imaging

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Development of a digital holographic microscopy system integrated with atomic force microscope

Development of a digital holographic microscopy system integ...

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Conference on three-dimensional and multidimensional microscopy - image acquisition and processing XVIII

作者： Cardenas, Nelson Ingle, Ninad Yu, Lingfeng Mohanty, Samarendra Univ Texas Arlington Dept Phys Biophys & Physiol Grp POB 19059 Arlington TX 76019 USA Univ Texas Arlington Dept Mat Sci Engn Arlington TX 76019 USA Nanoscope Technol LLC Arlington TX 76012 USA

ISBN: (纸本)9780819484413

Atomic Force Microscope (AFM) imaging, due to the scanning method of recording, requires significant recording time for examination of wide sample area. In contrast, digital holographic microscopy (DHM), owing to the wide-field method, allows recording of the hologram in very fast rate which could be numerically analyzed to reveal surface of the sample with axial resolution at the nanometer scale. However, DHM yields quantitative phase properties of the sample, and therefore sensitive to changes in refractive index along with physical thickness. Therefore, to accurately determine the refractive index map, it is imperative to estimate the physical thickness map of the sample. This was achieved by AFM imaging. Further, since the transverse resolution of DHM is limited by diffraction limit, co-registration of AFM image provided higher transverse resolution at nanometer scale. The interference of the AFM probe was observed to be minimal during simultaneous AFM and DHM recording due to the transparent nature and bent configuration of the optical fiber based AFM cantilever. Integration of DHM and AFM led to realization of a powerful platform for nanoscale imaging. The integrated AFM-DHM system was built on an inverted fluorescence microscope to enable fluorescence imaging of the sample. The integrated system was employed to analyze fluorescent polystyrene microspheres, two-photon polymerized microstructures and red blood cells.

关键词： Digital Holographic microscopy Atomic Force Microscope Surface topography nanoscopic imaging

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In-vivo Third-Harmonic Generation microscopy at 1550nm three-dimensional long-term time-lapse studies in living C. elegans embryos

In-vivo Third-Harmonic Generation Microscopy at 1550nm three...

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Conference on three-dimensional and multidimensional microscopy - image acquisition and processing XVIII

作者： Aviles-Espinosa, Rodrigo Santos, Susana I. C. O. Brodschelm, Andreas Kaenders, Wilhelm G. Alonso-Ortega, Cesar Artigas, David Loza-Alvarez, Pablo ICFO Inst Photon Sci Mediterranean Techn PkAv Canal Olimp S-N Castelldefels 08860 Barcelona Spain TOPTICA Photon AG D-82166 Graefelfing Germany Univ Politecn Cataluna Dept Signal Theory & Commun E-08034 Barcelona Spain

ISBN: (纸本)9780819484413

In-vivo microscopic long term time-lapse studies require controlled imaging conditions to preserve sample viability. Therefore it is crucial to meet specific exposure conditions as these may limit the applicability of established techniques. In this work we demonstrate the use of third harmonic generation (THG) microscopy for long term time-lapse three-dimensional studies (4D) in living Caenorhabditis elegans embryos employing a 1550 nm femtosecond fiber laser. We take advantage of the fact that THG only requires the existence of interfaces to generate signal or a change in the refractive index or in the chi(3) nonlinear coefficient, therefore no markers are required. In addition, by using this wavelength the emitted THG signal is generated at visible wavelengths (516 nm) enabling the use of standard collection optics and detectors operating near their maximum efficiency. This enables the reduction of the incident light intensity at the sample plane allowing to image the sample for several hours. THG signal is obtained through all embryo development stages, providing different tissue/structure information. By means of control samples, we demonstrate that the expected water absorption at this wavelength does not severely compromise sample viability. Certainly, this technique reduces the complexity of sample preparation (i.e. genetic modification) required by established linear and nonlinear fluorescence based techniques. We demonstrate the non-invasiveness, reduced specimen interference, and strong potential of this particular wavelength to be used to perform long-term 4D recordings.

关键词： Multiphoton processes Nonlinear microscopy Fiber Laser Biological in-vivo imaging three dimensional Time-lapse imaging

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Computational Model of Optical Scattering by Elastin in Lung

Computational Model of Optical Scattering by Elastin in Lung

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Conference on three-dimensional and multidimensional microscopy - image acquisition and processing XVIII

作者： Swedish, Tristan B. Robinson, Joseph P. Silva, Maricris R. Gouldstone, Andrew Kaeli, David DiMarzio, Charles A. Northeastern Univ Dept Elect & Comp Engn 360 Huntington Ave Boston MA 02115 USA Northeastern Univ CenSSIS Boston MA 02115 USA Northeastern Univ Dept Mech & Ind Engn Boston MA 02115 USA

ISBN: (纸本)9780819484413

Little is understood about the detailed micromechanical properties of lung in vivo. Attempts to improve imaging are hampered by heterogeneity of the tissue. One common ex vivo technique is optical coherence tomography (OCT). Simulated OCT with a Finite-Difference Time-Domain (FDTD) computer model elucidates the relationship between captured images and the physical geometry of the lung. Parallel computation and improved processing power make accurate coherent imaging models feasible. A previous FDTD model of pulsed laser wave propagation in the lung produced images that displayed many of the properties of experimental images. The model was improved with the addition of elastin and increased computational volume. Elastin plays an important role in the simulation because the combination of its fibrous structure and high index of refraction acts as an excellent scatterer of light. This strong scattering increases the signal reported by the simulated OCT scan in areas where elastin is most abundant, improving visualization of the structure as more light is reflected back from the heterogeneous elastin network. However, scattering by elastin decreases the depth of penetration and leads to images that are more difficult to interpret. Gaining a better understanding of how lung structures affect light propagation will lead to improved signal processing, instrumentation, and the development of new probing techniques. This image modeling technique can also be applied to other imaging modalities such as confocal and other laser scanning methods.

关键词： Optical Coherence Tomography (OCT) Confocal Reflectance microscopy (CRM) Computational microscopy Lung Alveolar Structure Elastin Finite Difference Time Domain (FDTD)

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Design and analysis of confocal-spectral microscopy using wavelength scanning scheme

Design and analysis of confocal-spectral microscopy using wa...

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Conference on three-dimensional and multidimensional microscopy - image acquisition and processing XVIII

作者： Do, Dukho Chun, Wanhee Jeong, Hyeongjun Gweon, Dae-Gab Korea Adv Inst Sci & Technol Dept Mech Engn Nano Opto Mechatron Lab Taejon 305701 South Korea

ISBN: (纸本)9780819484413

The multi-color, or spectral fluorescence microscopy has ability to detect fluorescence spectral signals which are useful in case of studying interactions and phenomena between biological samples. Recently, commercial devices are combining with confocal microscope so to enhance lateral resolution and to have axial direction discernment. Also Acousto-Optic Tunable Filter(AOTF) is used instead of dichroic mirror to divide excitation and emission signals with mininum light efficiency. In addition, AOTF is used in spectral fluorescence microscopy have many advantages, these are very fast switching speed and high resolution in wavelength selection. However it uses acousto-optic interactions in birefringence material, Tellurium Dioxide(TeO(2)), the excitation light interacts with appropriate acoustic signal so that it is diffracted to 1 or -1 order path. But the fluorescence signals from a sample propagate in 0 order path with small different angle according to the polarization state. In this paper, a confocal-spectral microscopy is proposed with the new kind of spectral detector design having wavelength scanning galvano mirror. It makes possible to detect broad wavelength fluorescence signal by single PMT with simply rotating the galvano mirror. Also a new birefringent material, calcite(CaCO(3)) is used to compensate polarization effect. The proposed spectral confocal microscopy with unique spectrometer body has many advantages in comparison with commercial devices. In terms of detection method, it can be easily applied to other imaging modalities. Hence this system will be adapted in many applications.

关键词： wavelength scanning confocal-spectral birefringence compensation spectral detector

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Pupil Engineering for a Confocal Reflectance Line-Scanning Microscope

Pupil Engineering for a Confocal Reflectance Line-Scanning M...

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Conference on three-dimensional and multidimensional microscopy - image acquisition and processing XVIII

作者： Patel, Yogesh G. Rajadhyaksha, Milind DiMarzio, Charles A. Northeastern Univ Dept Elect & Comp Engn 440 Dana Bldg360 Huntington Ave Boston MA 02115 USA Northeastern Univ Dept Mech & Ind Engn Boston MA 02115 USA Serv Dermatol Dept Med Memorial Sloan Kettering Canc Ctr New York NY 10022 USA

ISBN: (纸本)9780819484413

Confocal reflectance microscopy may enable screening and diagnosis of skin cancers noninvasively and in real-time, as an adjunct to biopsy and pathology. Current confocal point-scanning systems are large, complex, and expensive. A confocal line-scanning microscope, utilizing a of linear array detector can be simpler, smaller, less expensive, and may accelerate the translation of confocal microscopy in clinical and surgical dermatology. A line scanner may be implemented with a divided-pupil, half used for transmission and half for detection, or with a full-pupil using a beamsplitter. The premise is that a confocal line-scanner with either a divided-pupil or a full-pupil will provide high resolution and optical sectioning that would be competitive to that of the standard confocal point-scanner. We have developed a confocal line-scanner that combines both divided-pupil and full-pupil configurations. This combined-pupil prototype is being evaluated to determine the advantages and limitations of each configuration for imaging skin, and comparison of performance to that of commercially available standard confocal point-scanning microscopes. With the combined configuration, experimental evaluation of line spread functions (LSFs), contrast, signal-to-noise ratio, and imaging performance is in progress under identical optical and skin conditions. Experimental comparisons between divided-pupil and full-pupil LSFs will be used to determine imaging performance. Both results will be compared to theoretical calculations using our previously reported Fourier analysis model and to the confocal point spread function (PSF). These results may lead to a simpler class of confocal reflectance scanning microscopes for clinical and surgical dermatology.

关键词： confocal microscopy Fourier optics point-scanning line-scanning full-pupil divided-pupil

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