Ptychographic lensless coherent endomicroscopy through a flexible fiber bundle.

Conventional fiber-bundle-based endoscopes allow minimally invasive imaging through flexible multi-core fiber (MCF) bundles by placing a miniature lens at the distal tip and using each core as an imaging pixel. In recent years, lensless imaging through MCFs was made possible by correcting the core-to-core phase distortions pre-measured in a calibration procedure. However, temporally varying wavefront distortions, for instance, due to dynamic fiber bending, pose a challenge for such approaches. Here, we demonstrate a coherent lensless imaging technique based on intensity-only measurements insensitive to core-to-core phase distortions. We leverage a ptychographic reconstruction algorithm to retrieve the phase and amplitude profiles of reflective objects placed at a distance from the fiber tip, using as input a set of diffracted intensity patterns reflected from the object when the illumination is scanned over the MCF cores. Our approach thus utilizes an acquisition process equivalent to confocal microendoscopy, only replacing the single detector with a camera.

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.

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.

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.

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.

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.

Time-gated iterative phase conjugation for efficient light energy delivery in scattering media.

Light waves propagating through complex biological tissues are spatially spread by multiple light scattering, and this spread limits the working depth in optical bioimaging, phototherapy, and optogenetics. Here, we propose the iterative phase conjugation of time-gated backscattered waves for enhancing the light energy delivered to a target object embedded in a scattering medium. We demonstrate the enhancement of light energy delivered to a target object hidden behind a 200-µm-thick mouse skull by more than ten times in comparison with the initial random input. The maximum enhancement was reached in only 10 iterations, more than a hundred times smaller than existing methods based on either a time-gated reflection matrix or iterative feedback optimization of the time-gated reflection intensity. Consequently, the proposed method is less sensitive to sample perturbations. Furthermore, the number of images required for optimization remained almost unchanged with an increase in the illumination area, unlike existing methods, where the convergence time scales with the illumination area. The proposed method provides high operation speed over a wide illumination area, which can facilitate the use of wavefront shaping in practical applications.

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.

NFIB promotes cell survival by directly suppressing p21 transcription in TP53-mutated triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited treatment options and poor prognosis. There is an urgent need to identify and understand the key factors and signalling pathways driving TNBC tumour progression, relapse, and treatment resistance. In this study, we report that gene copy numbers and expression levels of nuclear factor IB (NFIB), a recently identified oncogene in small cell lung cancer, are preferentially increased in TNBC compared to other breast cancer subtypes. Furthermore, increased levels of NFIB are significantly associated with high tumour grade, poor prognosis, and reduced chemotherapy response. Concurrent TP53 mutations and NFIB overexpression (z-scores > 0) were observed in 77.9% of TNBCs, in contrast to 28.5% in non-TNBCs. Depletion of NFIB in TP53-mutated TNBC cell lines promotes cell death, cell cycle arrest, and enhances sensitivity to docetaxel, a first-line chemotherapeutic drug in breast cancer treatment. Importantly, these alterations in growth properties were accompanied by induction of CDKN1A, the gene encoding p21, a downstream effector of p53. We show that NFIB directly interacts with the CDKN1A promoter in TNBC cells. Furthermore, knockdown of combined p21 and NFIB reverses the docetaxel-induced cell growth inhibition observed upon NFIB knockdown, indicating that NFIB's effect on chemotherapeutic drug response is mediated through p21. Our results indicate that NFIB is an important TNBC factor that drives tumour cell growth and drug resistance, leading to poor clinical outcomes. Thus, targeting NFIB in TP53-mutated TNBC may reverse oncogenic properties associated with mutant p53 by restoring p21 activity. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Diversity in the structure of action potential-mediated neural connectivity within rat supra chiasmatic nucleus.

Action potential (AP)-mediated cell-to-cell communication is essential for the frequency-locking and phase-synchronization of the clock cells within the biological master clock, suprachiasmatic nucleus (SCN). Nevertheless, the morphology of its network connectivity is largely unexplored. Here, with an optimized optogenetic light-stimulation and scanning protocol, we report some key characteristics of the inhibitory receptive field (IRF), the area which brings inhibitory synaptic currents to a given target cell, and basic statistics of the inhibitory network connections of rat SCN clock cells. ChR2 transfected, slice cultures of rat SCN were stimulated by a blue power LED light in a repetitive box-scanning modes, while a target cell was whole-cell patched. The registered inhibitory postsynaptic currents, which were brought by light-induced APs of presynaptic neurons, were mostly GABAergic. The sizes and shapes of IRFs of SCN cells were very diverse, and the number of presynaptic cells making up the IRF of a given target cell followed an exponential distribution with an average value of 8.9 approximately, according to our clustering analysis which is based on a hybrid measure D, combining the physical distance r and the difference in the current amplitudes of two different sites. Although this estimate inevitably depends on the construct of the measure D, it is found not so sensitive on the parameter w, which weighs the relative significance of the current amplitude different with respect to the physical distance r: The average number of presynaptic neurons varies < 26% over a significant range of 0 < w < 30. On average, the presynaptic connection number density around a target cell falls off as an exponentially decreasing function of r. But, its space constant (~210.7 µm) is quite large that long-range (>210.7 µm) neural connections are abundant (>66.9%) within the SCN.

Iterative optimization of time-gated reflectance for the efficient light energy delivery within scattering media.

In complex media, light waves are diffused both in space and time due to multiple light scattering, and its intensity is attenuated with the increase of propagation depth. In this paper, we propose an iterative wavefront shaping method for enhancing time-gated reflection intensity, which leads to efficient light energy delivery to a target object embedded in a highly scattering medium. We achieved an over 10 times enhancement of reflectance at the specific flight time and demonstrated the focusing of light energy to the target object. Since the proposed method does not require reflection matrix measurement, it will be highly suited to samples in mechanically dynamic conditions.

Size homeostasis in adherent cells studied by synthetic phase microscopy.

The coupling of the rate of cell growth to the rate of cell division determines cell size, a defining characteristic that is central to cell function and, ultimately, to tissue architecture. The physiology of size homeostasis has fascinated generations of biologists, but the mechanism, challenged by experimental limitations, remains largely unknown. In this paper, we propose a unique optical method that can measure the dry mass of thick live cells as accurately as that for thin cells with high computational efficiency. With this technique, we quantify, with unprecedented accuracy, the asymmetry of division in lymphoblasts and epithelial cells. We can then use the Collins-Richmond model of conservation to compute the relationship between growth rate and cell mass. In attached epithelial cells, we find that due to the asymmetry in cell division and size-dependent growth rate, there is active regulation of cell size. Thus, like nonadherent cells, size homeostasis requires feedback control.

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.

Holistic random encoding for imaging through multimode fibers.

The input numerical aperture (NA) of multimode fiber (MMF) can be effectively increased by placing turbid media at the input end of the MMF. This provides the potential for high-resolution imaging through the MMF. While the input NA is increased, the number of propagation modes in the MMF and hence the output NA remains the same. This makes the image reconstruction process underdetermined and may limit the quality of the image reconstruction. In this paper, we aim to improve the signal to noise ratio (SNR) of the image reconstruction in imaging through MMF. We notice that turbid media placed in the input of the MMF transforms the incoming waves into a better format for information transmission and information extraction. We call this transformation as holistic random (HR) encoding of turbid media. By exploiting the HR encoding, we make a considerable improvement on the SNR of the image reconstruction. For efficient utilization of the HR encoding, we employ sparse representation (SR), a relatively new signal reconstruction framework when it is provided with a HR encoded signal. This study shows for the first time to our knowledge the benefit of utilizing the HR encoding of turbid media for recovery in the optically underdetermined systems where the output NA of it is smaller than the input NA for imaging through MMF.

Exploring anti-reflection modes in disordered media.

Sensing and manipulating targets hidden under scattering media are universal problems that take place in applications ranging from deep-tissue optical imaging to laser surgery. A major issue in these applications is the shallow light penetration caused by multiple scattering that reflects most of incident light. Although advances have been made to eliminate image distortion by a scattering medium, dealing with the light reflection has remained unchallenged. Here we present a method to minimize reflected intensity by finding and coupling light into the anti-reflection modes of a scattering medium. In doing so, we achieved more than a factor of 3 increase in light penetration. Our method of controlling reflected waves makes it readily applicable to in vivo applications in which detector sensors can only be positioned at the same side of illumination and will therefore lay the foundation of advancing the working depth of many existing optical imaging and treatment technologies.

Speckle suppression via sparse representation for wide-field imaging through turbid media.

Speckle suppression is one of the most important tasks in the image transmission through turbid media. Insufficient speckle suppression requires an additional procedure such as temporal ensemble averaging over multiple exposures. In this paper, we consider the image recovery process based on the so-called transmission matrix (TM) of turbid media for the image transmission through the media. We show that the speckle left unremoved in the TM-based image recovery can be suppressed effectively via sparse representation (SR). SR is a relatively new signal reconstruction framework which works well even for ill-conditioned problems. This is the first study to show the benefit of using the SR as compared to the phase conjugation (PC) a de facto standard method to date for TM-based imaging through turbid media including a live cell through tissue slice.

Far-field control of focusing plasmonic waves through disordered nanoholes.

Control of near-field waves is the key to going beyond the diffraction limit in imaging and manipulating target objects. Here we present the focusing of plasmonic waves, a type of near-field waves, by the wavefront shaping of far-field waves. We coupled far-field waves to a random array of holes on a thin gold film to generate speckled plasmonic waves. By controlling the phase pattern of the incident waves with the wavelength of 637 nm, we demonstrated the focusing of plasmonic waves down to 170 nm at arbitrary positions. Our study shows the possibility of using disordered nanoholes as a plasmonic lens with high flexibility in the far-field control.

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.

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.

Overcoming retinoic acid resistance in HER2-enriched breast cancers: role of MYC.

HER2-enriched (HER2+) breast cancers express high levels of the growth-promoting HER2 protein. Although these cancers are treated with the HER2-targeted drug, trastuzumab, resistance to treatment is common. Retinoic acid (RA) is an anti-cancer agent that has been successfully used for the treatment of leukemia and holds promise for the treatment of solid cancers, including breast cancer. The HER2 gene is frequently co-amplified with RARA, a key determinant of RA sensitivity in breast cancers. It seems surprising, therefore, that HER2+ breast cancers are refractory to RA treatment. Here, we show that MYC mediates RA resistance by suppressing the expression of cellular retinoic acid binding protein 2 (CRABP2), resulting in RARα inactivation. CRABP2 is an intracellular RA transporter that delivers RA to the nuclear receptor RARα for its activation. Our results indicate that response to RA is enhanced by MYC depletion in HER2+ breast cancer cells and that RA treatment enhances trastuzumab responsiveness. Our findings support the use of RA and trastuzumab for the treatment of subsets of patients with breast cancers that are HER2-RARα co-amplified and have low levels of MYC.