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.

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.

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.

Cellular normoxic biophysical markers of hydroxyurea treatment in sickle cell disease.

Hydroxyurea (HU) has been used clinically to reduce the frequency of painful crisis and the need for blood transfusion in sickle cell disease (SCD) patients. However, the mechanisms underlying such beneficial effects of HU treatment are still not fully understood. Studies have indicated a weak correlation between clinical outcome and molecular markers, and the scientific quest to develop companion biophysical markers have mostly targeted studies of blood properties under hypoxia. Using a common-path interferometric technique, we measure biomechanical and morphological properties of individual red blood cells in SCD patients as a function of cell density, and investigate the correlation of these biophysical properties with drug intake as well as other clinically measured parameters. Our results show that patient-specific HU effects on the cellular biophysical properties are detectable at normoxia, and that these properties are strongly correlated with the clinically measured mean cellular volume rather than fetal hemoglobin level.

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.

High-resolution, dual-depth spectral-domain optical coherence tomography with interlaced detection for whole-eye imaging.

Dual-depth spectral-domain optical coherence tomography (SD-OCT) enables high-resolution in vivo whole-eye imaging. Two orthogonally polarized beams from a source are focused simultaneously on two axial positions of the anterior segment and the retina. For the detector arm, a 1×2 ultrafast optical switch sequentially delivers two spectral interference signals to a single spectrometer, which extends the in-air axial depth range up to 9.44 mm. An off-pivot complex conjugate removal technique doubles the depth range for all anterior segment imaging. The graphics-processing-unit-based parallel signal processing algorithm supports fast two- and three-dimensional image displays. The obtained high-resolution anterior and retinal images are measured biometrically. The dual-depth SD-OCT system has an axial resolution of ∼6.4 µm in air, and the sensitivity is 91.79 dB at 150 µm from the zero-delay line.

Transmission matrix of a scattering medium and its applications in biophotonics.

A conventional lens has well-defined transfer function with which we can form an image of a target object. On the contrary, scattering media such as biological tissues, multimode optical fibers and layers of disordered nanoparticles have highly complex transfer function, which makes them impractical for the general imaging purpose. In recent studies, we presented a method of experimentally recording the transmission matrix of such media, which is a measure of the transfer function. In this review paper, we introduce two major applications of the transmission matrix: enhancing light energy delivery and imaging through scattering media. For the former, we identified the eigenchannels of the transmission matrix with large eigenvalues and then coupled light to those channels in order to enhance light energy delivery through the media. For the latter, we solved matrix inversion problem to reconstruct an object image from the distorted image by the scattering media. We showed the enlargement of the numerical aperture of imaging systems with the use of scattering media and demonstrated endoscopic imaging through a single multimode optical fiber working in both reflectance and fluorescence modes. Our approach will pave the way of using scattering media as unique optical elements for various biophotonics applications.

Scanning color optical tomography (SCOT).

We have developed an interferometric optical microscope that provides three-dimensional refractive index map of a specimen by scanning the color of three illumination beams. Our design of the interferometer allows for simultaneous measurement of the scattered fields (both amplitude and phase) of such a complex input beam. By obviating the need for mechanical scanning of the illumination beam or detection objective lens; the proposed method can increase the speed of the optical tomography by orders of magnitude. We demonstrate our method using polystyrene beads of known refractive index value and live cells.

Dynamic speckle illumination wide-field reflection phase microscopy.

We demonstrate a quantitative reflection-phase microscope based on time-varying speckle-field illumination. Due to the short spatial coherence length of the speckle field, the proposed imaging system features superior lateral resolution, 520 nm, as well as high-depth selectivity, 1.03 µm. Off-axis interferometric detection enables wide-field and single-shot imaging appropriate for high-speed measurements. In addition, the measured phase sensitivity of this method, which is the smallest measurable axial motion, is more than 40 times higher than that available using a transmission system. We demonstrate the utility of our method by successfully distinguishing the motion of the top surface from that of the bottom in red blood cells. The proposed method will be useful for studying membrane dynamics in complex eukaryotic cells.

Diffraction optical tomography using a quantitative phase imaging unit.

A simple and practical method to measure three-dimensional (3-D) refractive index (RI) distributions of biological cells is presented. A common-path self-reference interferometry consisting of a compact set of polarizers is attached to a conventional inverted microscope equipped with a beam scanning unit, which can precisely measure multiple 2-D holograms of a sample with high phase stability for various illumination angles, from which accurate 3-D optical diffraction tomograms of the sample can be reconstructed. 3-D RI tomograms of nonbiological samples such as polystyrene microspheres, as well as biological samples including human red blood cells and breast cancer cells, are presented.

Depth-selective microscopic observation of a photomobile liquid crystal polymer under UV illumination.

By using the depth selective imaging method, we studied the UV induced change in a photomobile liquid crystalline polymer film. With 1 µm depth resolution, each slice inside the film was selectively observed. A network-like structure mixed with the ordered and disordered regions of molecules in the middle of the film, and a rubbed polymer layer at the bottom of the film were observed. In each slice of the film, the phase change induced by UV light was observed strongly dependent on the director direction, which indicates the ordering change of the liquid crystalline molecules in the director direction. It took several tens of seconds for the ordering change caused by the collaborative interaction between the molecules. Furthermore, it was suggested that the UV induced change travelled from the bottom layer to the middle layer on the micron order.

Disorder-mediated enhancement of fiber numerical aperture.

The numerical aperture (NA) of a multimode optical fiber sets the limit of the information transport capacity along the spatial degree of freedom. In this Letter, we report that the application of a highly disordered medium can overcome the capacity limit set by the fiber NA. Specifically, we coated the input surface of a multimode fiber with a disordered medium made of ZnO nanoparticles and transported a wide-field image through the fiber with a spatial resolution beyond the diffraction limit given by the fiber NA. This was made possible because multiple scatterings induced by the disordered medium physically increased the NA of the entire system. Our study will lead to enhancing the spatial resolution of fiber-based endoscopic imaging and also improving the information transport capacity in optical communications.

Measurement of the time-resolved reflection matrix for enhancing light energy delivery into a scattering medium.

Multiple scatterings occurring in a turbid medium attenuate the intensity of propagating waves. Here, we propose a method to efficiently deliver light energy to the desired target depth in a scattering medium. We measure the time-resolved reflection matrix of a scattering medium using coherent time-gated detection. From this matrix, we derive and experimentally implement an incident wave pattern that optimizes the detected signal corresponding to a specific arrival time. This leads to enhanced light delivery at the target depth. The proposed method will lay a foundation for efficient phototherapy and deep-tissue in vivo imaging in the near future.

Experimental measurement of the number of modes for a multimode optical fiber.

For a multimode optical fiber, the number of modes (N(m)) can be calculated by analytic theory when the fiber is straight, twist-free, and strain-free. In practice, however, the fiber is subject to distortions that modify its mode characteristics. In this Letter, we present an experimental method to interrogate the mode properties of a multimode optical fiber. We experimentally measured the transmission matrix of a multimode optical fiber and performed singular value decomposition. We proved, both theoretically and experimentally, that the rank of the transmission matrix is equal to N(m). We expect that the suggested method will contribute to the fields of the biomedical optics and optical communications where optical fiber is widely used.

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.

Tunneling-induced spectral broadening of a single atom in a three-dimensional optical lattice.

We have investigated the spectral broadening in the near-resonance fluorescence spectrum of a single rubidium atom trapped in a three-dimensional (3D) optical lattice in a strong Lamb-Dicke regime. Besides the strong Rayleigh peak, the spectrum exhibited weak Stokes and anti-Stokes Raman sidebands. The line width of the Rayleigh peak for low potential depths was well explained by matter-wave tunneling between the first-two lowest vibrational states of 3D anisotropic harmonic potentials of adjacent local minima of the optical lattice.

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.

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.