Tracing multiple scattering trajectories for deep optical imaging in scattering media.

Multiple light scattering hampers imaging objects in complex scattering media. Approaches used in real practices mainly aim to filter out multiple scattering obscuring the ballistic waves that travel straight through the scattering medium. Here, we propose a method that makes the deterministic use of multiple scattering for microscopic imaging of an object embedded deep within scattering media. The proposed method finds a stack of multiple complex phase plates that generate similar light trajectories as the original scattering medium. By implementing the inverse scattering using the identified phase plates, our method rectifies multiple scattering and amplifies ballistic waves by almost 600 times. This leads to a significant increase in imaging depth-more than three times the scattering mean free path-as well as the correction of image distortions. Our study marks an important milestone in solving the long-standing high-order inverse scattering problems.

Mapping nanoscale topographic features in thick tissues with speckle diffraction tomography.

Resolving three-dimensional morphological features in thick specimens remains a significant challenge for label-free imaging. We report a new speckle diffraction tomography (SDT) approach that can image thick biological specimens with ~500 nm lateral resolution and ~1 µm axial resolution in a reflection geometry. In SDT, multiple-scattering background is rejected through spatiotemporal gating provided by dynamic speckle-field interferometry, while depth-resolved refractive index maps are reconstructed by developing a comprehensive inverse-scattering model that also considers specimen-induced aberrations. Benefiting from the high-resolution and full-field quantitative imaging capabilities of SDT, we successfully imaged red blood cells and quantified their membrane fluctuations behind a turbid medium with a thickness of 2.8 scattering mean-free paths. Most importantly, we performed volumetric imaging of cornea inside an ex vivo rat eye and quantified its optical properties, including the mapping of nanoscale topographic features of Dua's and Descemet's membranes that had not been previously visualized.

Computational conjugate adaptive optics microscopy for longitudinal through-skull imaging of cortical myelin.

Myelination processes are closely related to higher brain functions such as learning and memory. While their longitudinal observation has been crucial to understanding myelin-related physiology and various brain disorders, skull opening or thinning has been required to secure clear optical access. Here we present a high-speed reflection matrix microscope using a light source with a wavelength of 1.3 µm to reduce tissue scattering and aberration. Furthermore, we develop a computational conjugate adaptive optics algorithm designed for the recorded reflection matrix to optimally compensate for the skull aberrations. These developments allow us to realize label-free longitudinal imaging of cortical myelin through an intact mouse skull. The myelination processes of the same mice were observed from 3 to 10 postnatal weeks to the depth of cortical layer 4 with a spatial resolution of 0.79 µm. Our system will expedite the investigations on the role of myelination in learning, memory, and brain disorders.

High-throughput volumetric adaptive optical imaging using compressed time-reversal matrix.

Deep-tissue optical imaging suffers from the reduction of resolving power due to tissue-induced optical aberrations and multiple scattering noise. Reflection matrix approaches recording the maps of backscattered waves for all the possible orthogonal input channels have provided formidable solutions for removing severe aberrations and recovering the ideal diffraction-limited spatial resolution without relying on fluorescence labeling and guide stars. However, measuring the full input-output response of the tissue specimen is time-consuming, making the real-time image acquisition difficult. Here, we present the use of a time-reversal matrix, instead of the reflection matrix, for fast high-resolution volumetric imaging of a mouse brain. The time-reversal matrix reduces two-way problem to one-way problem, which effectively relieves the requirement for the coverage of input channels. Using a newly developed aberration correction algorithm designed for the time-reversal matrix, we demonstrated the correction of complex aberrations using as small as 2% of the complete basis while maintaining the image reconstruction fidelity comparable to the fully sampled reflection matrix. Due to nearly 100-fold reduction in the matrix recording time, we could achieve real-time aberration-correction imaging for a field of view of 40 × 40 µm2 (176 × 176 pixels) at a frame rate of 80 Hz. Furthermore, we demonstrated high-throughput volumetric adaptive optical imaging of a mouse brain by recording a volume of 128 × 128 × 125 µm3 (568 × 568 × 125 voxels) in 3.58 s, correcting tissue aberrations at each and every 1 µm depth section, and visualizing myelinated axons with a lateral resolution of 0.45 µm and an axial resolution of 2 µm.

In vivo visualization of butterfly scale cell morphogenesis in Vanessa cardui.

During metamorphosis, the wings of a butterfly sprout hundreds of thousands of scales with intricate microstructures and nano-structures that determine the wings' optical appearance, wetting characteristics, thermodynamic properties, and aerodynamic behavior. Although the functional characteristics of scales are well known and prove desirable in various applications, the dynamic processes and temporal coordination required to sculpt the scales' many structural features remain poorly understood. Current knowledge of scale growth is primarily gained from ex vivo studies of fixed scale cells at discrete time points; to fully understand scale formation, it is critical to characterize the time-dependent morphological changes throughout their development. Here, we report the continuous, in vivo, label-free imaging of growing scale cells of Vanessa cardui using speckle-correlation reflection phase microscopy. By capturing time-resolved volumetric tissue data together with nanoscale surface height information, we establish a morphological timeline of wing scale formation and gain quantitative insights into the underlying processes involved in scale cell patterning and growth. We identify early differences in the patterning of cover and ground scales on the young wing and quantify geometrical parameters of growing scale features, which suggest that surface growth is critical to structure formation. Our quantitative, time-resolved in vivo imaging of butterfly scale development provides the foundation for decoding the processes and biomechanical principles involved in the formation of functional structures in biological materials.

Time-resolved detection of early-arriving ballistic waves in a quasi-diffusive regime.

Ballistic waves directly carry image information in imaging through a scattering medium, but they are often obscured by much intense multiple-scattered waves. Detecting early arriving photons has been an effective method to extract ballistic waves in the transmission-mode imaging. However, it has been difficult to identify the temporal distribution of ballistic waves relative to the multiple scattering waves in the quasi-diffusive regime. Here, we present a method to separately quantify ballistic and multiple-scattered waves at their corresponding flight times even when multiple scattering is much stronger than the ballistic waves. This is realized by measuring the transmission matrix of an object embedded within scattering medium and comparing the coherent accumulation of ballistic waves with their incoherent addition. To further elucidate the temporal behavior of ballistic waves in quasi-diffusive regime, we analyze the flight time difference between ballistic and multiple-scattered waves and the effect of coherence gating on their relative intensities for the scattering medium of different thicknesses. The presented method to distinctively detect the temporal behavior of ballistic and multiple-scattered waves will lay a foundation to exploit multiple-scattered waves for deep-tissue imaging.

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.

Optical transfer function of time-gated coherent imaging in the presence of a scattering medium.

Optical imaging of objects embedded within scattering media such as biological tissues suffers from the loss of resolving power. In our previous work, we proposed an approach called collective accumulation of single scattering (CASS) microscopy that attenuates this detrimental effect of multiple light scattering by combining the time-gated detection and spatial input-output correlation. In the present work, we perform a rigorous theoretical analysis on the effect of multiple light scattering to the optical transfer function of CASS microscopy. In particular, the spatial frequency-dependent signal to noise ratio (SNR) is derived depending on the intensity ratio of the single- and multiple-scattered waves. This allows us to determine the depth-dependent resolving power. We conducted experiments using a Siemens star-like target having various spatial frequency components and supported the theoretical derived SNR spectra. Our study provides a theoretical framework for understanding the effect of multiple light scattering in high-resolution and deep-tissue optical imaging.

Label-free neuroimaging in vivo using synchronous angular scanning microscopy with single-scattering accumulation algorithm.

Label-free in vivo imaging is crucial for elucidating the underlying mechanisms of many important biological systems in their most native states. However, the applicability of existing modalities has been limited to either superficial layers or early developmental stages due to tissue turbidity. Here, we report a synchronous angular scanning microscope for the rapid interferometric recording of the time-gated reflection matrix, which is a unique matrix characterizing full light-specimen interaction. By applying single scattering accumulation algorithm to the recorded matrix, we removed both high-order sample-induced aberrations and multiple scattering noise with the effective aberration correction speed of 10,000 modes/s. We demonstrated in vivo imaging of whole neural network throughout the hindbrain of the larval zebrafish at a matured stage where physical dissection used to be required for conventional imaging. Our method will expand the scope of applications for optical imaging, where fully non-invasive interrogation of living specimens is critical.

Optical imaging featuring both long working distance and high spatial resolution by correcting the aberration of a large aperture lens.

High-resolution optical imaging within thick objects has been a challenging task due to the short working distance of conventional high numerical aperture (NA) objective lenses. Lenses with a large physical diameter and thus a large aperture, such as microscope condenser lenses, can feature both a large NA and a long working distance. However, such lenses suffer from strong aberrations. To overcome this problem, we present a method to correct the aberrations of a transmission-mode imaging system that is composed of two condensers. The proposed method separately identifies and corrects aberrations of illumination and collection lenses of up to 1.2 NA by iteratively optimizing the total intensity of the synthetic aperture images in the forward and phase-conjugation processes. At a source wavelength of 785 nm, we demonstrated a spatial resolution of 372 nm at extremely long working distances of up to 1.6 mm, an order of magnitude improvement in comparison to conventional objective lenses. Our method of converting microscope condensers to high-quality objectives may facilitate increases in the imaging depths of super-resolution and expansion microscopes.

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.

High-resolution adaptive optical imaging within thick scattering media using closed-loop accumulation of single scattering.

Thick biological tissues give rise to not only the multiple scattering of incoming light waves, but also the aberrations of remaining signal waves. The challenge for existing optical microscopy methods to overcome both problems simultaneously has limited sub-micron spatial resolution imaging to shallow depths. Here we present an optical coherence imaging method that can identify aberrations of waves incident to and reflected from the samples separately, and eliminate such aberrations even in the presence of multiple light scattering. The proposed method records the time-gated complex-field maps of backscattered waves over various illumination channels, and performs a closed-loop optimization of signal waves for both forward and phase-conjugation processes. We demonstrated the enhancement of the Strehl ratio by more than 500 times, an order of magnitude or more improvement over conventional adaptive optics, and achieved a spatial resolution of 600 nm up to an imaging depth of seven scattering mean free paths.

Label-free characterization of ultra violet-radiation-induced changes in skin fibroblasts with Raman spectroscopy and quantitative phase microscopy.

Minimizing morbidities and mortalities associated with skin cancers requires sustained research with the goal of obtaining fresh insights into disease onset and progression under specific stimuli, particularly the influence of ultraviolet rays. In the present study, label-free profiling of skin fibroblasts exposed to time-bound ultra-violet radiation has been performed using quantitative phase imaging and Raman spectroscopy. Statistically significant differences in quantifiable biophysical parameters, such as matter density and cell dry mass, were observed with phase imaging. Accurate estimation of changes in the biochemical constituents, notably nucleic acids and proteins, was demonstrated through a combination of Raman spectroscopy and multivariate analysis of spectral patterns. Overall, the findings of this study demonstrate the promise of these non-perturbative optical modalities in accurately identifying cellular phenotypes and responses to external stimuli by combining molecular and biophysical information.

Controlled generation of single photons in a coupled atom-cavity system at a fast repetition-rate.

We have demonstrated high-speed controlled generation of single photons in a coupled atom-cavity system. A single 85Rb atom, pumped with a nanosecond-pulse laser, generates a single photon into the cavity mode, and the photon is then emitted out the cavity rapidly. By employing cavity parameters for a moderate coupling regime, the single-photon emission process was optimized for both high efficiency and fast bit rates up to 10 MHz. The temporal single-photon wave packet was studied by means of the photon-arrival-time distribution relative to the pump pulse and the efficiency of the single-photon generation was investigated as the pump power. The single-photon nature of the emission was confirmed by the second-order correlation of emitted photons.

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.

Continuous control of the coupling constant in an atom-cavity system by using elliptic polarization and magnetic sublevels.

Atom-cavity coupling constant is a key parameter in cavity quantum electrodynamics for describing the interaction between an atom and a quantized electromagnetic field in a cavity. This paper reports a novel way to tune the coupling constant continuously by inducing an averaging of the atomic dipole moment over degenerate magnetic sublevels with elliptic polarization of the cavity field. We present an analytic solution of the stationary-state density matrix for this system with consideration of F -> F +1 hyperfine transition under a weak excitation condition. We rigorously show that the stationary-state emission spectra of this system can be approximated by that of a non-degenerate two-level atom with an effective coupling constant as a function of the elliptic angle of the cavity field only. A precise condition for this approximation is derived and its physical meaning is interpreted in terms of a population-averaged transition strength and its variance. Our results can be used to control the coupling constant in cavity quantum electrodynamics experiments with a degenerate two-level atom with magnetic sublevels. Possible applications of our results are discussed.

Quasieigenstate coalescence in an atom-cavity quantum composite.

We report the first direct observation of an exceptional point (EP) in an open quantum composite of a single atom and a high-Q cavity mode. The atom-cavity coupling constant was made a continuous variable by utilizing the multisublevel nature of a single rubidium atom when it is optimally coupled to the cavity mode. The spectroscopic properties of quasieigenstates of the atom-cavity composite were experimentally investigated near the EP. Branch-point singularity of quasieigenenergies was observed and its 4pi symmetry was demonstrated. Consequently, the cavity transmission at the quasieigenstate was observed to exhibit a critical behavior at the EP.