Three-dimensional imaging in reflection phase microscopy with minimal axial scanning.

Reflection phase microscopy is a valuable tool for acquiring three-dimensional (3D) images of objects due to its capability of optical sectioning. The conventional method of constructing a 3D map is capturing 2D images at each depth with a mechanical scanning finer than the optical sectioning. This not only compromises sample stability but also slows down the acquisition process, imposing limitations on its practical applications. In this study, we utilized a reflection phase microscope to acquire 2D images at depth locations significantly spaced apart, far beyond the range of optical sectioning. By employing a numerical propagation, we successfully filled the information gap between the acquisition layers, and then constructed complete 3D maps of objects with substantially reduced number of axial scans. Our experimental results also demonstrated the effectiveness of this approach in enhancing imaging speed while maintaining the accuracy of the reconstructed 3D structures. This technique has the potential to improve the applicability of reflection phase microscopy in diverse fields such as bioimaging and material science.

Single-shot and phase-shifting digital holographic microscopy using a 2-D grating.

We demonstrate digital holographic microscopy that, while being based on phase-shifting interferometry, is capable of single-shot measurements. A two-dimensional (2-D) diffraction grating placed in a Fourier plane of a standard in-line holographic phase microscope generates multiple copies of a sample image on a camera sensor. The identical image copies are spatially separated with different overall phase shifts according to the diffraction orders. The overall phase shifts are adjusted by controlling the lateral position of the grating. These phase shifts are then set to be multiples of π/2. Interferograms composed of four image copies combined with a parallel reference beam are acquired in a single shot. The interferograms are processed through a phase-shifting algorithm to produce a single complex image. By taking advantage of the higher sampling capacity of the in-line holography, we can increase the imaging information density by a factor of 3 without compromising the imaging acquisition speed.

Single-shot digital holographic microscopy for quantifying a spatially-resolved Jones matrix of biological specimens.

Field-based polarization measurements are essential for the completeness of information when exploiting the complex nature of optical responses of target objects. Here, we demonstrate digital holographic microscopy for quantifying a polarization-sensitive map of an object with a single-shot measurement. Using the image-splitting device generating four different copies of an object image and a separate reference beam of an off-axis configuration enables single-shot and multi-imaging capability. With the use of two polarization filters, four complex field images containing an object's polarization response are obtained simultaneously. With this method, we can construct a complete set of 2-by-2 Jones matrix at every single point of the object's images, and thus clearly visualize the anisotropic structures of biological tissues with low level of birefringence. This method will facilitate the high-precision measurements for fast dynamics of the polarization properties of biological specimens.

Toward a miniature endomicroscope: pixelation-free and diffraction-limited imaging through a fiber bundle.

A fiber bundle is widely used for endoscopic imaging due to its direct image delivery capability. However, there exists an inevitable pixelation artifact, which limits spatial resolution to the diameter of individual fibers. In this Letter, we present a method that can eliminate this artifact and achieve diffraction-limited spatial resolution. We exploited the binary control of a digital micromirror device to measure a transmission matrix of a fiber bundle and to subsequently control mode mixing among individual fibers. In doing so, we achieved a 22 kHz scanning rate of a diffraction-limited focused spot and obtained fluorescence endoscope imaging (58 µm × 58 µm) with near video-rate (10.3 Hz) acquisition. Our study lays a foundation for developing an ultrathin and high-resolution microendoscope.

Investigating the correlation between early vascular alterations and cognitive impairment in Alzheimer's disease in mice with SD-OCT.

Vascular alterations have recently gained some attention with their strong association with Alzheimer's disease (AD). We conducted a label-free in vivo optical coherence tomography (OCT) longitudinal imaging using an AD mouse model. We achieved the tracking of the same individual vessels over time and conducted an in-depth analysis of temporal dynamics in vasculature and vasodynamics using OCT angiography and Doppler-OCT. The AD group showed an exponential decay in both vessel diameter and blood flow change with the critical timepoint before 20 weeks of age, which precedes cognitive decline observed at 40 weeks of age. Interestingly, for the AD group, the diameter change showed the dominance in arterioles over venules, but no such influence was found in blood flow change. Conversely, three mice groups with early vasodilatory intervention did not show any significant change in both vascular integrity and cognitive function compared to the wild-type group. We found early vascular alterations and confirmed their correlation with cognitive impairment in AD.

Scanner-free and wide-field endoscopic imaging by using a single multimode optical fiber.

A single multimode fiber is considered an ideal optical element for endoscopic imaging due to the possibility of direct image transmission via multiple spatial modes. However, the wave distortion induced by the mode dispersion has been a fundamental limitation. In this Letter, we propose a method for eliminating the effect of mode dispersion and therefore realize wide-field endoscopic imaging by using only a single multimode fiber with no scanner attached to the fiber. Our method will potentially revolutionize endoscopy in various fields encompassing medicine and industry.

Full-field and single-shot quantitative phase microscopy using dynamic speckle illumination.

We developed an off-axis quantitative phase microscopy that works for a light source with an extremely short spatial coherence length in order to reduce the diffraction noise and enhance the spatial resolution. A dynamic speckle wave whose coherence length is 440 nm was used as an illumination source. To implement an off-axis interferometry for a source of low spatial coherence, a diffraction grating was inserted in the reference beam path. In doing so, an oblique illumination was generated without rotation of the wavefront, which leads to a full-field and single-shot phase recording with improved phase sensitivity of more than a factor of 10 in comparison with coherent illumination. The spatial resolution, both laterally and axially, and the depth selectivity are significantly enhanced due to the wide angular spectrum of the speckle wave. We applied our method to image the dynamics of small intracellular particles in live biological cells. With enhanced phase sensitivity and speed, the proposed method will serve as a useful tool to study the dynamics of biological specimens.

Synthetic aperture microscopy for high resolution imaging through a turbid medium.

We report on synthetic aperture microscopy through a highly turbid medium. We first recorded a transmission matrix for the turbid medium with an angular basis of 20,000 complex images covering 0.6 NA. This effectively converts the medium into a lens of the same NA. Distorted images of a target object are then taken at 500 different angles of illumination covering 0.6 NA. For each of the distorted images, the original object image is reconstructed from the transmission matrix by the recently developed turbid lens imaging (TLI) technique. All 500 reconstructed images are synthesized to enhance the NA to 1.2 and thereby generate an object image with twice the enhanced spatial resolution of the individual images. Our method of applying aperture synthesis for TLI makes it possible to enhance the resolving power without increasing the number of transmission matrix elements. This relieves the demand for data acquisition and processing that has impeded the practicality of TLI.

Overcoming the diffraction limit using multiple light scattering in a highly disordered medium.

We report that disordered media made of randomly distributed nanoparticles can be used to overcome the diffraction limit of a conventional imaging system. By developing a method to extract the original image information from the multiple scattering induced by the turbid media, we dramatically increase a numerical aperture of the imaging system. As a result, the resolution is enhanced by more than 5 times over the diffraction limit, and the field of view is extended over the physical area of the camera. Our technique lays the foundation to use a turbid medium as a far-field superlens.

The effect of pupil transmittance on axial resolution of reflection phase microscopy.

A reflection phase microscope (RPM) can be equipped with the capability of depth selection by employing a gating mechanism. However, it is difficult to achieve an axial resolution close to the diffraction limit in real implementation. Here, we systematically investigated the uneven interference contrast produced by pupil transmittance of the objective lens and found that it was the main cause of the practical limit that prevents the axial resolution from reaching its diffraction limit. Then we modulated the power of illumination light to obtain a uniform interference contrast over the entire pupil. Consequently, we could achieve an axial resolution fairly close to the diffraction limit set by the experimental conditions.

Application of M1 macrophage as a live vector in delivering nanoparticles for in vivo photothermal treatment.

Introduction: To enhance photothermal treatment (PTT) efficiency, a delivery method that uses cell vector for nanoparticles (NPs) delivery has drawn attention and studied widely in recent years. Objectives: In this study, we demonstrated the feasibility of M1 activated macrophage as a live vector for delivering NPs and investigated the effect of NPs loaded M1 stimulated by Lipopolysaccharide on PTT efficiency in vivo. Methods: M1 was used as a live vector for delivering NPs and further to investigate the effect of NPs loaded M1 on PTT efficiency. Non-activated macrophage (MФ) was stimulated by lipopolysaccharide (LPS) into M1 and assessed for tumor cell phagocytic capacity towards NPs. Results: We found M1 exhibited a 20-fold higher uptake capacity of NPs per cell volume and 2.9-fold more active infiltration into the tumor site, compared with non-activated macrophage MФ. We injected M1 cells peritumorally and observed that these cells penetrated into the tumor mass within 12 h. Then, we conducted PTT using irradiation of a near-infrared laser for 1 min at 1 W/cm2. As a result, we confirmed that using M1 as an active live vector led to a more rapid reduction in tumor size within 1 day indicating that the efficacy of PTT with NPs-loaded M1 is higher than that with NPs-loaded MФ. Conclusion: Our study demonstrated the potential role of M1 as a live vector for enhancing the feasibility of PTT in cancer treatment.

Label-free microendoscopy using a micro-needle imaging probe for in vivo deep tissue imaging.

We report a label-free imaging method for microendoscopy that uses a needle-type imaging probe. We inserted a thin GRIN lens that had been attached to a fiber bundle into a medical-grade needle that was used as an imaging probe. The introduction of the needle probe into biological tissue allows for direct access to deep regions that we otherwise could not achieve because of the multiple light scattering. To minimize invasiveness, we introduced the illuminating probe on the tissue surface, using an oblique back-illumination configuration. We achieved three-dimensional depth imaging by changing the depth of penetration. Since only the imaging probe goes deep into the tissue while leaving the illumination channels outside, the achievable signal depends on the location of the illumination channels. We explored this point and investigated the optimal condition for the illumination distance in a systematic way. We also applied this method to ex vivo, as well as in vivo, imaging of a mouse brain, and confirmed that we had visualized the microvasculature embedded deep within the brain.

Enhancement of aberrantly modified integrin­mediated cell motility in multicellular tumor spheroids.

Multicellular tumor spheroids (MTSs) of malignant cells can display cell­cell and cell­matrix interactions, different from monolayer cultures. The objective of the present study was to examine difference in intercellular and cell­matrix interaction between monolayered cultures and spheroid cultures. Expression levels of cell adhesion molecules (CAMs) and epithelial­mesenchymal transition (EMT) signaling molecules in monolayered cells and MTS cells were compared. The motility of single cells dispersed from each culture was evaluated using a live­cell imaging device. The effect of an E­cadherin neutralizing antibody, DECMA, was also compared between the two cultures. Among various CAMs, only E­cadherin was increased in MTSs. The motility of single cells dispersed from MTSs was higher than that from monolayered cells. Compared with monolayered cells, the molecular weight (MW) of ß1 integrin was decreased during MTS formation, particularly during the early stage. This notable reduction was maintained when DECMA was used to treat MTSs. Additionally, the expression levels of the EMT signaling molecules Snail and ILK increased more in MTSs than in monolayered cells. The blocking of E­cadherin elicited increased expression levels of EMT molecules and cellular motility only in MTSs. In conclusion, the alteration of E­cadherin expression and presence of low­MW ß1 integrin in MTS may enhance cell motility via the upregulation of EMT signaling molecules that may be intensified by blocking E­cadherin.

Correction: Correction: Two distinct actin waves correlated with turns-and-runs of crawling microglia.

[This corrects the article DOI: 10.1371/journal.pone.0222692.].

Correction: Two distinct actin waves correlated with turns-and-runs of crawling microglia.

[This corrects the article DOI: 10.1371/journal.pone.0220810.].

Inversion-free image recovery from strong aberration using a minimally sampled transmission matrix.

A transmission matrix (TM), a characteristic response for an input-output relation of an optical system, has been used for achieving diffraction-limited and aberration-free images through highly-aberrant imaging systems. However, its requirement of acquiring a huge-size TM along with its heavy computational load limit its widespread applications. Here we propose a method for TM-based image reconstruction, which is more efficient in terms of data manipulation and computational time. Only 10% of the TM elements for a fish-eye (FE) lens with strong aberration were sampled compared to that required for the image reconstruction by the conventional inversion method. The missing information was filled in by an iterative interpolation algorithm working in k-space. In addition, as a replacement of the time-consuming matrix inversion process, a phase pattern was created from the minimally sampled TM in order to compensate for the angle-dependent phase retardation caused by the FE lens. The focal distortion could be corrected by applying the phase correction pattern to the angular spectrums of the measured object images. The remaining spatial distortion could also be determined through the geometrical transformation also determined by the minimally sampled TM elements. Through the use of these procedures, the object image can be reconstructed 55 times faster than through the use of the usual inversion method using the full-sized TM, without compromising the reconstruction performances.

Two distinct actin waves correlated with turns-and-runs of crawling microglia.

Freely crawling cells are often viewed as randomly moving Brownian particles but they generally exhibit some directional persistence. This property is often related to their zigzag motile behaviors that can be described as a noisy but temporally structured sequence of "runs" and "turns." However, its underlying biophysical mechanism is largely unexplored. Here, we carefully investigate the collective actin wave dynamics associated with the zigzag-crawling movements of microglia (as primary brain immune cells) employing a number of different quantitative imaging modalities including synthetic aperture microscopy and optical diffraction tomography, as well as conventional fluorescence imaging and scanning electron microscopy. Interestingly, we find that microglia exhibit two distinct types of actin waves working at two quite different time scales and locations, and they seem to serve different purposes. One type of actin waves is fast "peripheral ruffles" arising spontaneously with an oscillating period of about 6 seconds at some portion of the leading edge of crawling microglia, where the vigorously biased peripheral ruffles seem to set the direction of a new turn (in 2-D free space). When the cell turning events are inhibited with a physical confinement (in 1-D track), the peripheral ruffles still exist at the leading edge with no bias but showing phase coherence in the cell crawling direction. The other type is "dorsal actin waves" which also exhibits an oscillatory behavior but with a much longer period of around 2 minutes compared to the fast "peripheral ruffles". Dorsal actin waves (whether the cell turning events are inhibited or not) initiate in the lamellipodium just behind the leading edge, travelling down toward the core region of the cell and disappear. Such dorsal wave propagations seem to be correlated with migration of the cell. Thus, we may view the dorsal actin waves are connected with the "run" stage of cell body, whereas the fast ruffles at the leading front are involved in the "turn" stage.

Quantification of focal adhesion dynamics of cell movement based on cell-induced collagen matrix deformation using second-harmonic generation microscopy.

Mechanical interactions of living cells with the surrounding environment via focal adhesion (FA) in three dimensions (3-D) play a key role in dynamic biological events, such as tissue regeneration, wound healing, and cancer invasion. Recently, several methods for observing 3-D cell-extracellular matrix (ECM) interactions have been reported, lacking solid and quantitative analysis on the dynamics of the physical interaction between the cell and the ECM. We measured the submicron displacements of ECM deformation in 3-D due to protrusion-retraction dynamics during cell migration, using second-harmonic generation without labeling the matrix structures. We then quantitatively analyzed the mechanical deformation between the ECM and the cells based on spatiotemporal volumetric correlations. The greatest deformations within the collagen matrix were found to occur at sites of colocalization of the FA site-related proteins vinculin and actin, which confirms that FA sites play a critical role in living cells within the ECM as a point for adhesion, traction, and migration. We believe that this modality can be used in studies of cell-ECM interaction during angiogenesis, wound healing, and metastasis.

Reflection phase microscopy using spatio-temporal coherence of light.

Many disease states are associated with cellular biomechanical changes as markers. Label-free phase microscopes are used to quantify thermally driven interface fluctuations, which allow the deduction of important cellular rheological properties. Here, the spatio-temporal coherence of light was used to implement a high-speed reflection phase microscope with superior depth selectivity and higher phase sensitivity. Nanometric scale motion of cytoplasmic structures can be visualized with fine details and three-dimensional resolution. Specifically, the spontaneous fluctuation occurring on the nuclear membrane of a living cell was observed at video rate. By converting the reflection phase into displacement, the sensitivity in quantifying nuclear membrane fluctuation was found to be about one nanometer. A reflection phase microscope can potentially elucidate biomechanical mechanisms of pathological and physiological processes.

In vivo photothermal treatment with real-time monitoring by optical fiber-needle array.

Photothermal treatment (PTT) using gold nanoshells (gold-NSs) is accepted as a method for treating cancer. However, owing to restrictions in therapeutic depth and skin damage caused by excessive light exposure, its application has been limited to lesions close to the epidermis. Here, we demonstrate an in vivo PTT method that uses gold-NSs with a flexible optical fiber-needle array (OFNA), which is an array of multiple needles in which multimode optical fibers are inserted, one in each, for light delivery. The light for PTT was directly administrated to subcutaneous tissues through the OFNA, causing negligible thermal damage to the skin. Enhancement of light energy delivery assisted by the OFNA in a target area was confirmed by investigation using artificial tissues. The ability of OFNA to treat cancer without causing cutaneous thermal damage was also verified by hematoxylin and eosin (H&E) staining and optical coherence tomography in cancer models in mice. In addition, the OFNA allowed for observation of the target site through an imaging fiber bundle. By imaging the activation of the injected gold-NSs, we were able to obtain information on the PTT process in real-time.