Fourier holographic endoscopy for imaging continuously moving objects.

Coherent fiber bundles are widely used for endoscopy, but conventional approaches require distal optics to form an object image and acquire pixelated information owing to the geometry of the fiber cores. Recently, holographic recording of a reflection matrix enables a bare fiber bundle to perform pixelation-free microscopic imaging as well as allows a flexible mode operation, because the random core-to-core phase retardations due to any fiber bending and twisting could be removed in situ from the recorded matrix. Despite its flexibility, the method is not suitable for a moving object because the fiber probe should remain stationary during the matrix recording to avoid the alteration of the phase retardations. Here, we acquire a reflection matrix of a Fourier holographic endoscope equipped with a fiber bundle and explore the effect of fiber bending on the recorded matrix. By removing the motion effect, we develop a method that can resolve the perturbation of the reflection matrix caused by a continuously moving fiber bundle. Thus, we demonstrate high-resolution endoscopic imaging through a fiber bundle, even when the fiber probe changes its shape along with the moving objects. The proposed method can be used for minimally invasive monitoring of behaving animals.

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.

Flexible-type ultrathin holographic endoscope for microscopic imaging of unstained biological tissues.

Ultrathin lensless fibre endoscopes offer minimally invasive investigation, but they mostly operate as a rigid type due to the need for prior calibration of a fibre probe. Furthermore, most implementations work in fluorescence mode rather than label-free imaging mode, making them unsuitable for general medical diagnosis. Herein, we report a fully flexible ultrathin fibre endoscope taking 3D holographic images of unstained tissues with 0.85-µm spatial resolution. Using a bare fibre bundle as thin as 200-µm diameter, we design a lensless Fourier holographic imaging configuration to selectively detect weak reflections from biological tissues, a critical step for label-free endoscopic reflectance imaging. A unique algorithm is developed for calibration-free holographic image reconstruction, allowing us to image through a narrow and curved passage regardless of fibre bending. We demonstrate endoscopic reflectance imaging of unstained rat intestine tissues that are completely invisible to conventional endoscopes. The proposed endoscope will expedite a more accurate and earlier diagnosis than before with minimal complications.

Single-shot multiple-depth macroscopic imaging by spatial frequency multiplexing.

We present a low-coherence interferometric imaging system designed for 3-dimensional (3-D) imaging of a macroscopic object through a narrow passage. Our system is equipped with a probe-type port composed of a bundle fiber for imaging and a separate multimode optical fiber for illumination. To eliminate the need for mechanical depth scanning, we employ a spatial frequency multiplexing method by installing a 2-D diffraction grating and an echelon in the reference arm. This configuration generates multiple reference beams, all having different path lengths and propagation directions, which facilitates the encoding of different depth information in a single interferogram. We demonstrate the acquisition of 9 depth images at the interval of 250 µm for a custom-made cone and a plaster teeth model. The proposed system minimizes the need for mechanical scanning and achieves a wide range of depth coverage, significantly increasing the speed of 3-D imaging for macroscopic objects.

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.

Locking Multi-Laser Frequencies to a Precision Wavelength Meter: Application to Cold Atoms.

We herein report a simultaneous frequency stabilization of two 780-nm external cavity diode lasers using a precision wavelength meter (WLM). The laser lock performance is characterized by the Allan deviation measurement in which we find σy=10-12 at an averaging time of 1000 s. We also obtain spectral profiles through a heterodyne spectroscopy, identifying the contribution of white and flicker noises to the laser linewidth. The frequency drift of the WLM is measured to be about 2.0(4) MHz over 36 h. Utilizing the two lasers as a cooling and repumping field, we demonstrate a magneto-optical trap of 87Rb atoms near a high-finesse optical cavity. Our laser stabilization technique operates at broad wavelength range without a radio frequency element.

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.

Multichannel near-infrared spectroscopy brain imaging system for small animals in mobile conditions.

Significance: We propose a customized animal-specific head cap and an near-infrared spectroscopy (NIRS) system to obtain NIRS signals in mobile small animals. NIRS studies in mobile small animals provide a feasible solution for comprehensive brain function studies. Aim: We aim to develop and validate a multichannel NIRS system capable of performing functional brain imaging along with a closed-box stimulation kit for small animals in mobile conditions. Approach: The customized NIRS system uses light-weight long optical fibers, along with a customized light-weight head cap to securely attach the optical fibers to the mouse. A customized stimulation box was designed to perform various stimuli in a controlled environment. The system performance was tested in a visual stimulation task on eight anesthetized mice and eight freely moving mice. Results: Following the visual stimulation task, we observed a significant stimulation-related oxyhemoglobin (HbO) increase in the visual cortex of freely moving mice during the task. In contrast, HbO concentration did not change significantly in the visual cortex of anesthetized mice. Conclusions: We demonstrate the feasibility of a wearable, multichannel NIRS system for small animals in a less confined experimental design.

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.

Cross-scanning optical coherence tomography angiography for eye motion correction.

We propose a cross-scanning optical coherence tomography (CS-OCT) system to correct eye motion artifacts in OCT angiography images. This system employs a dual-illumination configuration with two orthogonally polarized beams, each of which simultaneously perform raster scanning in perpendicular direction with each other over the same area. In the reference arm, a polarization delay unit is used to acquire the two orthogonally polarized interferograms with a single photo detector by introducing different optical delay lines. The two cross-scanned volume data are affected by the same eye motion but in two orthogonal directions. We developed a motion correction algorithm, which removes artifacts in the slow axis of each angiogram using the other and merges them through a nonrigid registration algorithm. In this manner, we obtained a motion-corrected angiogram within a single volume scanning time without additional eye-tracking devices.

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.].

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.

Hemodynamic correlation imaging of the mouse brain for application in unilateral neurodegenerative diseases.

We developed a single-camera two-channel hemodynamic imaging system that uses near-infrared light to monitor the mouse brain in vivo with an exposed, un-thinned, and intact skull to explore the effect of Parkinson's disease on the resting state functional connectivity of the brain. To demonstrate our system's ability to monitor cerebral hemodynamics, we first performed direct electrical stimulation of an anesthetized healthy mouse brain and detected hemodynamic changes localized to the stimulated area. Subsequently, we developed a unilaterally lesioned 6-hydroxydopamine (hemi-parkinsonian) mouse model and detected the differences in functional connectivity between the normal and hemi-parkinsonian mouse brains by comparing the hemispheric hemodynamic correlations during the resting state. Seed-based correlation for the oxy-hemoglobin channel from the left and right hemispheres of healthy mice was much higher and more symmetric than in hemi-parkinsonian mice. Through a k-means clustering of the hemodynamic signals, the healthy mouse brains were segmented according to brain region, but the hemi-parkinsonian mice did not show a similar segmentation. Overall, this study highlights the development of a spatial multiplexing hemodynamic imaging system that reveals the resting state hemodynamic connectivity in healthy and hemi-parkinsonian mice.

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.

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.

Full ocular biometry through dual-depth whole-eye optical coherence tomography.

We propose a new method of determining the optical axis (OA), pupillary axis (PA), and visual axis (VA) of the human eye by using dual-depth whole-eye optical coherence tomography (OCT). These axes, as well as the angles "α" between the OA and VA and "κ" between PA and VA, are important in many ophthalmologic applications, especially in refractive surgery. Whole-eye images are reconstructed based on simultaneously acquired images of the anterior segment and retina. The light from a light source is split into two orthogonal polarization components for imaging the anterior segment and retina, respectively. The OA and PA are identified based on their geometric definitions by using the anterior segment image only, while the VA is detected through accurate correlation between the two images. The feasibility of our approach was tested using a model eye and human subjects.

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.

Removal of back-reflection noise at ultrathin imaging probes by the single-core illumination and wide-field detection.

Thin waveguides such as graded-index lenses and fiber bundles are often used as imaging probes for high-resolution endomicroscopes. However, strong back-reflection from the end surfaces of the probes makes it difficult for them to resolve weak contrast objects, especially in the reflectance-mode imaging. Here we propose a method to spatially isolate illumination pathways from detection channels, and demonstrate wide-field reflectance imaging free from back-reflection noise. In the image fiber bundle, we send illumination light through individual core fibers and detect signals from target objects through the other fibers. The transmission matrix of the fiber bundle is measured and used to reconstruct a pixelation-free image. We demonstrated that the proposed imaging method improved 3.2 times on the signal to noise ratio produced by the conventional illumination-detection scheme.

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.