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.

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.

Recent advances in optical imaging through deep tissue: imaging probes and techniques.

Optical imaging has been essential for scientific observations to date, however its biomedical applications has been restricted due to its poor penetration through tissues. In living tissue, signal attenuation and limited imaging depth caused by the wave distortion occur because of scattering and absorption of light by various molecules including hemoglobin, pigments, and water. To overcome this, methodologies have been proposed in the various fields, which can be mainly categorized into two stategies: developing new imaging probes and optical techniques. For example, imaging probes with long wavelength like NIR-II region are advantageous in tissue penetration. Bioluminescence and chemiluminescence can generate light without excitation, minimizing background signals. Afterglow imaging also has high a signal-to-background ratio because excitation light is off during imaging. Methodologies of adaptive optics (AO) and studies of complex media have been established and have produced various techniques such as direct wavefront sensing to rapidly measure and correct the wave distortion and indirect wavefront sensing involving modal and zonal methods to correct complex aberrations. Matrix-based approaches have been used to correct the high-order optical modes by numerical post-processing without any hardware feedback. These newly developed imaging probes and optical techniques enable successful optical imaging through deep tissue. In this review, we discuss recent advances for multi-scale optical imaging within deep tissue, which can provide reseachers multi-disciplinary understanding and broad perspectives in diverse fields including biophotonics for the purpose of translational medicine and convergence science. Methodologies for multi-scale optical imaging within deep tissues are discussed in diverse fields including biophotonics for the purpose of translational medicine and convergence science. Recent imaging probes have tried deep tissue imaging by NIR-II imaging, bioluminescence, chemiluminescence, and afterglow imaging. Optical techniques including direct/indirect and coherence-gated wavefront sensing also can increase imaging depth.

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.

Single-shot multi-depth full-field optical coherence tomography using spatial frequency division multiplexing.

Fast 3D volumetric imaging has been essential for biology, medicine and industrial inspections, and various optical coherence tomography (OCT) methods have been developed to meet such needs. Point-scanning based approaches, such as swept-source OCT and spectral domain OCT, can obtain a depth information at once, but they require lateral scan for full 3D imaging. On the contrary, full-field OCT needs the scanning of imaging depth while it records a full lateral information at once. Here, we present a full-field OCT system that can obtain multi-depth information at once by a single-shot recording. We combine a 2D diffraction grating and a custom-made echelon to prepare multiple reference beams having different pathlengths and propagating angles. By recording a single interference image between the reflected wave from a sample and these multiple reference beams, we reconstruct full-field images at multiple depths associated with the pathlengths of the individual reference beams. We demonstrated the single-shot recording of 7 different depth images at 10 µm for biological tissues. Our method can potentially be useful for applications where high-speed recording of multiple en-face images is crucial.

Laser scanning reflection-matrix microscopy for aberration-free imaging through intact mouse skull.

A mouse skull is a barrier for high-resolution optical imaging because its thick and inhomogeneous internal structures induce complex aberrations varying drastically from position to position. Invasive procedures creating either thinned-skull or open-skull windows are often required for the microscopic imaging of brain tissues underneath. Here, we propose a label-free imaging modality termed laser scanning reflection-matrix microscopy for recording the amplitude and phase maps of reflected waves at non-confocal points as well as confocal points. The proposed method enables us to find and computationally correct up to 10,000 angular modes of aberrations varying at every 10 × 10 µm2 patch in the sample plane. We realized reflectance imaging of myelinated axons in vivo underneath an intact mouse skull, with an ideal diffraction-limited spatial resolution of 450 nm. Furthermore, we demonstrated through-skull two-photon fluorescence imaging of neuronal dendrites and their spines by physically correcting the aberrations identified from the reflection matrix.

Single-shot imaging of microscopic dynamic scenes at 5 THz frame rates by time and spatial frequency multiplexing.

Femtosecond-scale ultrafast imaging is an essential tool for visualizing ultrafast dynamics in many scientific fields. We present a single-shot ultrafast microscopy that can capture more than a dozen frames at a time with the frame rate of 5 THz. We combine a spatial light modulator and a custom-made echelon for efficiently generating a large number of reference pulses with designed time delays and propagation angles. The single-shot recording of the interference image between these reference pulses with a sample pulse allows us to retrieve the stroboscopic images of the dynamic scene at the timing of the reference pulses. We demonstrated the recording of 14 temporal snapshots at a time, which is the largest to date, with the optimal temporal resolution set by the laser output pulse. This will have profound impacts on uncovering the interesting spatio-temporal dynamics yet to be explored.

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.

Displacement measurement using an optoelectronic oscillator with an intra-loop Michelson interferometer.

We report on measurement of small displacements with sub-nanometer precision using an optoelectronic oscillator (OEO) with an intra-loop Michelson interferometer. In comparison with conventional homodyne and heterodyne detection methods, where displacement appears as a power change or a phase shift, respectively, in the OEO detection, the displacement produces a shift in the oscillation frequency. In comparison with typical OEO sensors, where the frequency shift is proportional to the OEO oscillation frequency in radio-frequency domain, the frequency shift in our method with an intra-loop interferometer is proportional to an optical frequency. We constructed a hybrid apparatus and compared characteristics of the OEO and heterodyne detection methods.

Cavity-modified collective Rayleigh scattering of two atoms.

We report on the observation of cooperative radiation of exactly two neutral atoms strongly coupled to the single mode field of an optical cavity, which is close to the lossless-cavity limit. Monitoring the cavity output power, we observe constructive and destructive interference of collective Rayleigh scattering for certain relative distances between the two atoms. Because of cavity backaction onto the atoms, the cavity output power for the constructive two-atom case (N=2) is almost equal to the single-emitter case (N=1), which is in contrast to free-space where one would expect an N^{2} scaling of the power. These effects are quantitatively explained by a classical model as well as by a quantum mechanical model based on Dicke states. We extract information on the relative phases of the light fields at the atom positions and employ advanced cooling to reduce the jump rate between the constructive and destructive atom configurations. Thereby we improve the control over the system to a level where the implementation of two-atom entanglement schemes involving optical cavities becomes realistic.

Bayesian feedback control of a two-atom spin-state in an atom-cavity system.

We experimentally demonstrate real-time feedback control of the joint spin-state of two neutral cesium atoms inside a high finesse optical cavity. The quantum states are discriminated by their different cavity transmission levels. A Bayesian update formalism is used to estimate state occupation probabilities as well as transition rates. We stabilize the balanced two-atom mixed state, which is deterministically inaccessible, via feedback control and find very good agreement with Monte Carlo simulations. On average, the feedback loop achieves near optimal conditions by steering the system to the target state marginally exceeding the time to retrieve information about its state.

Dependence of transverse and longitudinal resolutions on incident Gaussian beam widths in the illumination part of optical scanning microscopy.

We studied both theoretically and experimentally the intensity distribution of a Gaussian laser beam when it was focused by an objective lens with its numerical aperture up to 0.95. Approximate formulas for full width at half-maximum (FWHM) of the intensity distribution at focus were derived for very large and very small initial beam waists with respect to the entrance pupil radius of the objective lens. In experiments, the energy flux through a 0.5 microm pinhole was measured for various pinhole positions. It was found in theoretical analysis and confirmed in experiments that the FWHMs at focus in the transverse and longitudinal directions do not increase much from the ultimate FWHMs until the input beam waist is reduced below half of the entrance pupil radius.