Origin of Enhancement of Orbital Magnetic Moment in SiO2-Coated Fe3O4 Nanocomposites Studied by X-ray Magnetic Circular Dichroism.

In this study, magnetic Fe3O4 nanoparticles (NPs) were dispersed uniformly by varying the thickness of the SiO2 coating, and their electronic and magnetic properties were investigated. X-ray diffraction confirmed the structural configuration of monophase inverse-spinel Fe3O4 NPs in nanometer size. Scanning electron microscopy revealed the formation of proper nonporous crystallite particles with a clear core-shell structure with silica on the surface of Fe3O4 NPs. The absorption mechanism studied through the zeta potential indicates that SiO2-coated Fe3O4 nanocomposites (SiO2@Fe3O4 NCs) possess electrostatic interactions to control their agglomeration in stabilizing suspensions by providing a protective shield of amorphous SiO2 on the oxide surface. High-resolution transmission electron microscopy images demonstrate a spherical morphology having an average grain diameter of ∼11-17 nm with increasing thickness of SiO2 coating with the addition of a quantitative presence and proportion of elements determined through elemental mapping and electron energy loss spectroscopy studies. Synchrotron-based element-specific soft X-ray absorption spectroscopy and X-ray magnetic circular dichroism (XMCD) techniques have been involved in the bulk-sensitive total fluorescence yield mode to understand the origin of magnetization in SiO2@Fe3O4 NCs. The magnetization hysteresis of Fe3O4 was determined by XMCD. At room temperature, the magnetic coercivity (Hc) is as high as 1 T, which is about 2 times more than the value of the thin film and about 5 times more pronounced than that of NPs. For noninteracting single-domain NPs with the Hc spread from 1 to 3 T, the Stoner-Wohlfarth model provided an intriguing explanation for the hysteresis curve. These curves determine the different components of Fe oxides present in the samples that derive the remnant magnetization involved in each oxidation state of Fe and clarify which Fe component is responsible for the resultant magnetism and magnetocrystalline anisotropy based on noninteracting single-domain particles.

Modeling the depth-sectioning effect in reflection-mode dynamic speckle-field interferometric microscopy.

Unlike most optical coherence microscopy (OCM) systems, dynamic speckle-field interferometric microscopy (DSIM) achieves depth sectioning through the spatial-coherence gating effect. Under high numerical aperture (NA) speckle-field illumination, our previous experiments have demonstrated less than 1 µm depth resolution in reflection-mode DSIM, while doubling the diffraction limited resolution as under structured illumination. However, there has not been a physical model to rigorously describe the speckle imaging process, in particular explaining the sectioning effect under high illumination and imaging NA settings in DSIM. In this paper, we develop such a model based on the diffraction tomography theory and the speckle statistics. Using this model, we calculate the system response function, which is used to further obtain the depth resolution limit in reflection-mode DSIM. Theoretically calculated depth resolution limit is in an excellent agreement with experiment results. We envision that our physical model will not only help in understanding the imaging process in DSIM, but also enable better designing such systems for depth-resolved measurements in biological cells and tissues.

Non-axial-scanning multifocal confocal microscopy with multiplexed volume holographic gratings.

Confocal imaging techniques offer an optical sectioning capability to acquire three-dimensional information from various volumetric samples by discriminating the desired in-focus signals from the out-of-focus background. However, confocal, in general, requires a point-by-point scan in both the lateral and axial directions to reconstruct three-dimensional images. In addition, axial scanning in confocal is slower than scanning in lateral directions. In this Letter, a non-axial-scanning multifocal confocal microscope incorporating multiplexed holographic gratings in illumination and dual detection for depth discrimination is presented. Further, we demonstrate the ability of the proposed confocal microscopy to image ex vivo tissue structures simultaneously at different focal depths without mechanical or electro-optic axial scanning.

Talbot multi-focal holographic fluorescence endoscopy for optically sectioned imaging.

A wide-field multi-plane endoscopic system incorporating multiplexed volume holographic gratings and Talbot illumination to simultaneously acquire optically sectioned fluorescence images of tissue structures from different depths is presented. The proposed endoscopic system is configured such that multiple Talbot-illumination planes occur inside a volumetric sample and serve as the input focal planes for the subsequent multiplexed volume holographic imaging gratings. We describe the design, implementation, and experimental data demonstrating this endoscopic system's ability to obtain optically sectioned multi-plane fluorescent images of tissue samples in wide-field fashion without scanning in lateral and axial directions.

Speckle-based volume holographic microscopy for optically sectioned multi-plane fluorescent imaging.

Structured illumination microscopy has been widely used to reconstruct optically sectioned fluorescence images in wide-field fashion; however, it still requires axial scanning to obtain multiple depth information of a volumetric sample. In this paper, a new imaging scheme, called speckle-based volume holographic microscopy system, is presented. The proposed system incorporates volumetric speckle illumination and multiplexed volume holographic gratings to acquire multi-plane images with optical sectioning capability, without any axial scanning. We present the design, implementation, and experimental image data demonstrating the proposed system's ability to simultaneously obtain wide-field, optically sectioned, and multi-depth resolved images of fluorescently labeled microspheres and tissue structures.

In-line digital holographic imaging in volume holographic microscopy.

A dual-plane in-line digital holographic imaging method incorporating volume holographic microscopy (VHM) is presented to reconstruct objects in a single shot while eliminating zero-order and twin-image diffracted waves. The proposed imaging method is configured such that information from different axial planes is acquired simultaneously using multiplexed volume holographic imaging gratings, as used in VHM, and recorded as in-line holograms where the corresponding reference beams are generated in the fashion of Gabor's in-line holography. Unlike conventional VHM, which can take axial intensity information only at focal depths, the proposed method digitally reconstructs objects at any axial position. Further, we demonstrate the proposed imaging technique's ability to effectively eliminate zero-order and twin images for single-shot three-dimensional object reconstruction.

Three dimensional HiLo-based structured illumination for a digital scanned laser sheet microscopy (DSLM) in thick tissue imaging.

Laser sheet based microscopy has become widely accepted as an effective active illumination method for real time three-dimensional (3D) imaging of biological tissue samples. The light sheet geometry, where the camera is oriented perpendicular to the sheet itself, provides an effective method of eliminating some of the scattered light and minimizing the sample exposure to radiation. However, residual background noise still remains, limiting the contrast and visibility of potentially interesting features in the samples. In this article, we investigate additional structuring of the illumination for improved background rejection, and propose a new technique, "3D HiLo" where we combine two HiLo images processed from orthogonal directions to improve the condition of the 3D reconstruction. We present a comparative study of conventional structured illumination based demodulation methods, namely 3Phase and HiLo with a newly implemented 3D HiLo approach and demonstrate that the latter yields superior signal-to-background ratio in both lateral and axial dimensions, while simultaneously suppressing image processing artifacts.

Full-field phase modulation characterization of liquid-crystal spatial light modulator using digital holography.

A direct quantitative phase measurement method to characterize intrinsic phase modulation from an entire active area of transmissive twisted-nematic liquid-crystal spatial light modulator (TN-LCSLM) is presented using digital holography (DH). The change in birefringence of liquid crystal material with respect to addressed gray scale produces phase modulation of wavefront transmitted through TN-LCSLM. Existing methods for phase modulation characterization of LCSLM mainly provides point measurement on its total active region. In this paper, the DH method is evolved to extract quantitative phase information of an entire active area from a single digital hologram formed using the complex wavefront transmitted through TN-LCSLM.

Studying nucleic envelope and plasma membrane mechanics of eukaryotic cells using confocal reflectance interferometric microscopy.

Mechanical stress on eukaryotic nucleus has been implicated in a diverse range of diseases including muscular dystrophy and cancer metastasis. Today, there are very few non-perturbative methods to quantify nuclear mechanical properties. Interferometric microscopy, also known as quantitative phase microscopy (QPM), is a powerful tool for studying red blood cell biomechanics. The existing QPM tools, however, have not been utilized to study biomechanics of complex eukaryotic cells either due to lack of depth sectioning, limited phase measurement sensitivity, or both. Here, we present depth-resolved confocal reflectance interferometric microscopy as the next generation QPM to study nuclear and plasma membrane biomechanics. The proposed system features multiple confocal scanning foci, affording 1.5 micron depth-resolution and millisecond frame rate. Furthermore, a near common-path interferometer enables quantifying nanometer-scale membrane fluctuations with better than 200 picometers sensitivity. Our results present accurate quantification of nucleic envelope and plasma membrane fluctuations in embryonic stem cells.

Speckle illumination holographic non-scanning fluorescence endoscopy.

Optical sectioning endoscopy such as confocal endoscopy offers capabilities to obtain three-dimensional (3D) information from various biological samples by discriminating between the desired in-focus signals and out-of-focus background. However, in general confocal images are formed through point-by-point scanning and the scanning time is proportional to the 3D space-bandwidth product. Recently, structured illumination endoscopy has been utilized for optically sectioned wide-field imaging, but it still needs axial scanning to acquire images from different depths of focal plane. Here, we report wide-field, multiplane, optical sectioning endoscopic imaging, incorporating 3D active speckle-based illumination and multiplexed volume holographic gratings, to simultaneously obtain images of fluorescently labeled tissue structures from different depths, without the need of scanning. We present the design, and implementation, as well as experimental data, demonstrating this endoscopic system's ability to obtain optically sectioned multiplane fluorescent images of tissue samples, with cellular level resolution in wide-field fashion, and no need for mechanical or optical axial scanning.(A) Schematic drawing of the SIHN endoscopy to simultaneously acquire multiplane images from different depths. (B) Uniform, and (C) SIHN illuminated images of standard fluorescence beads (25 µm in diameter) for the two axial planes. (D) Intensity profile on fluorescently labeled signal (ie, in-focus) and background (ie, out-of-focus) of microspheres.

Automated fluorescence intensity and gradient analysis enables detection of rare fluorescent mutant cells deep within the tissue of RaDR mice.

Homologous recombination (HR) events are key drivers of cancer-promoting mutations, and the ability to visualize these events in situ provides important information regarding mutant cell type, location, and clonal expansion. We have previously created the Rosa26 Direct Repeat (RaDR) mouse model wherein HR at an integrated substrate gives rise to a fluorescent cell. To fully leverage this in situ approach, we need better ways to quantify rare fluorescent cells deep within tissues. Here, we present a robust, automated event quantification algorithm that uses image intensity and gradient features to detect fluorescent cells in deep tissue specimens. To analyze the performance of our algorithm, we simulate fluorescence behavior in tissue using Monte Carlo methods. Importantly, this approach reduces the potential for bias in manual counting and enables quantification of samples with highly dense HR events. Using this approach, we measured the relative frequency of HR within a chromosome and between chromosomes and found that HR within a chromosome is more frequent, which is consistent with the close proximity of sister chromatids. Our approach is both objective and highly rapid, providing a powerful tool, not only to researchers interested in HR, but also to many other researchers who are similarly using fluorescence as a marker for understanding mammalian biology in tissues.

Quantitative phase microscopy of red blood cells during planar trapping and propulsion.

Red blood cells (RBCs) have the ability to undergo morphological deformations during microcirculation, such as changes in surface area, volume and sphericity. Optical waveguide trapping is suitable for trapping, propelling and deforming large cell populations along the length of the waveguide. Bright field microscopy employed with waveguide trapping does not provide quantitative information about structural changes. Here, we have combined quantitative phase microscopy and waveguide trapping techniques to study changes in RBC morphology during planar trapping and transportation. By using interference microscopy, time-lapsed interferometric images of trapped RBCs were recorded in real-time and subsequently utilized to reconstruct optical phase maps. Quantification of the phase differences before and after trapping enabled study of the mechanical effects during planar trapping. During planar trapping, a decrease in the maximum phase values, an increase in the surface area and a decrease in the volume and sphericity of RBCs were observed. QPM was used to analyze the phase values for two specific regions within RBCs: the annular rim and the central donut. The phase value of the annular rim decreases whereas it increases for the central donut during planar trapping. These changes correspond to a redistribution of cytosol inside the RBC during planar trapping and transportation.

Near-common-path interferometer for imaging Fourier-transform spectroscopy in wide-field microscopy.

Imaging Fourier-transform spectroscopy (IFTS) is a powerful method for biological hyperspectral analysis based on various imaging modalities, such as fluorescence or Raman. Since the measurements are taken in the Fourier space of the spectrum, it can also take advantage of compressed sensing strategies. IFTS has been readily implemented in high-throughput, high-content microscope systems based on wide-field imaging modalities. However, there are limitations in existing wide-field IFTS designs. Non-common-path approaches are less phase-stable. Alternatively, designs based on the common-path Sagnac interferometer are stable, but incompatible with high-throughput imaging. They require exhaustive sequential scanning over large interferometric path delays, making compressive strategic data acquisition impossible. In this paper, we present a novel phase-stable, near-common-path interferometer enabling high-throughput hyperspectral imaging based on strategic data acquisition. Our results suggest that this approach can improve throughput over those of many other wide-field spectral techniques by more than an order of magnitude without compromising phase stability.

Reassignment of scattered emission photons in multifocal multiphoton microscopy.

Multifocal multiphoton microscopy (MMM) achieves fast imaging by simultaneously scanning multiple foci across different regions of specimen. The use of imaging detectors in MMM, such as CCD or CMOS, results in degradation of image signal-to-noise-ratio (SNR) due to the scattering of emitted photons. SNR can be partly recovered using multianode photomultiplier tubes (MAPMT). In this design, however, emission photons scattered to neighbor anodes are encoded by the foci scan location resulting in ghost images. The crosstalk between different anodes is currently measured a priori, which is cumbersome as it depends specimen properties. Here, we present the photon reassignment method for MMM, established based on the maximum likelihood (ML) estimation, for quantification of crosstalk between the anodes of MAPMT without a priori measurement. The method provides the reassignment of the photons generated by the ghost images to the original spatial location thus increases the SNR of the final reconstructed image.

Talbot holographic illumination nonscanning (THIN) fluorescence microscopy.

Optical sectioning techniques offer the ability to acquire three-dimensional information from various organ tissues by discriminating between the desired in-focus and out-of-focus (background) signals. Alternative techniques to confocal, such as active structured illumination, exist for fast optically sectioned images, but they require individual axial planes to be imaged consecutively. In this article, an imaging technique (THIN), by utilizing active Talbot illumination in 3D and multiplexed holographic Bragg filters for depth discrimination, is demonstrated for imaging in vivo 3D biopsy without mechanical or optical axial scanning.

Improving signal-to-noise ratio of structured light microscopy based on photon reassignment.

In this paper, we report a method for 3D visualization of a biological specimen utilizing a structured light wide-field microscopic imaging system. This method improves on existing structured light imaging modalities by reassigning fluorescence photons generated from off-focal plane excitation, improving in-focus signal strength. Utilizing a maximum likelihood approach, we identify the most likely fluorophore distribution in 3D that will produce the observed image stacks under structured and uniform illumination using an iterative maximization algorithm. Our results show the optical sectioning capability of tissue specimens while mostly preserving image stack photon count, which is usually not achievable with other existing structured light imaging methods.

Quasi-physical phase compensation in digital holographic microscopy.

In digital holographic microscopy, if an optical setup is well aligned, the phase curvature introduced by the microscope objective (MO) together with the illuminating wave to the object wave is a spherical phase curvature. It can be physically compensated by introducing the same spherical phase curvature in the reference beam. Digital holographic microscopy setups based on the Michelson interferometric configuration with MO and an adjustable lens are presented, which can well perform the quasi-physical phase compensation during the hologram recording. In the reflection mode, the adjustable lens serves as both the condensing lens and the compensation lens. When the spatial frequency spectra of the hologram become a point spectrum, one can see that the phase curvature introduced by imaging is quasi-physically compensated. A simple plane numerical reference wavefront used for the reconstruction can give the correct quantitative phase map of the test object. A theoretical analysis and experimental demonstration are given. The simplicity of the presented setup makes it easy to align it well at lower cost.

Amplitude and phase analysis in digital dynamic holography.

Lensless in-line digital holographic interferometry has the potential for vibration analysis of objects smaller than 5 mm in diameter. This is particularly useful for dynamic characterization of microelectromechanical systems devices. To achieve this, there is a need to magnify the object wave, which is done using a diverging beam. It is observed that an increase in the object-to-CCD distance increases the sensitivity of the amplitude-modulated time-average fringes. At the same time the effect on phase information that represents the mean static deformation of a vibrating object is studied. It is also observed that a reduction in the object-to-CCD distance increases the phase sensitivity as evidenced by the double-exposure time-average fringes. The experimental observation and a theoretical explanation for this contradictory phenomenon are presented.

Time-averaged in-line digital holographic interferometry for vibration analysis.

Time-averaged in-line digital holography is applied for vibration analysis. In particular, by use of a double-exposure approach, simultaneous determination of vibration mode shape and mean static state deformation during a vibration cycle are obtained. The subtraction of two numerically reconstructed digital holograms recorded at the same resonant frequency but with a small difference in amplitude shows the mixing of Bessel-type time-averaged fringes owing to vibration and of the double-exposure fringes owing to differences in the mean deformation of the object. It is shown that separation of these fringe patterns can be readily accomplished numerically. An experimental demonstration of this effect by use of in-line digital holography for relatively small membranes is demonstrated.