This paper presents an application of digital Fourier holography for selective imaging of scatterers with different sizes in turbid media such as biological tissues. A combination of Fourier holography and high-resolution digital recording, digital Fourier microscopy (DFM) permits crucial flexibility in applying filtering to highlight scatterers of interest in the tissue. The high-resolution digital hologram is a result of the collation of Fourier holographic frames to form a large-size composite hologram. It is expected that DFM has an improved signal-to-noise ratio as compared to conventional direct digital imaging, e.g. phase microscopy, as applied to imaging of small-size objects. The demonstration of the Fourier filtering capacity of DFM using a biological phantom represents the main focus of this paper.
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 medicine and industry. Herein, we report a fully flexible ultrathin fibre endoscope taking 3D holographic images of unstained tissues with 0.87-μm spatial resolution. Using a bare fibre bundle as thin as 200-μm diameter, we design a lensless Fourier holographic imaging configuration to selectively detec…
Optical metasurfaces are perfect candidates for the phase and amplitude modulation of light, featuring an excellent basis for holographic applications. In this work, we present a dual amplitude holographic scheme based on the photon sieve principle, which is then combined with a phase hologram by utilizing the Pancharatnam-Berry phase. We demonstrate that two types of apertures, rectangular and square shapes in a gold film filled with silicon nanoantennas are sufficient to create two amplitude holograms at two different wavelengths in the visible, multiplexed with an additional phase-only holo…
Holographic microscopy presents challenges for color reproduction due to the usage of narrow-band illumination sources, which especially impacts the imaging of stained pathology slides for clinical diagnoses. Here, an accurate color holographic microscopy framework using absorbance spectrum estimation is presented. This method uses multispectral holographic images acquired and reconstructed at a small number (e.g., three to six) of wavelengths, estimates the absorbance spectrum of the sample, and projects it onto a color tristimulus. Using this method, the wavelength selection is optimized to …
Common-path digital in-line holography is considered as a valuable 3D diagnostic techniques for a wide range of applications. This configuration is cost effective and relatively immune to variation in the experimental environment. Nevertheless, due to its common-path geometry, the signal to noise-ratio of the acquired hologram is weak as most of the detector (i.e. CCD/CMOS sensor) dynamics is occupied by the reference field signal, whose energy is orders of magnitude higher than the field scattered by the imaged object. As it is intrinsically impossible to modify the ratio of energy of referen…
In this chapter, we introduce digital holographic microscopy (DHM) as a marker-free method to determine the refractive index of single, spherical cells in suspension. The refractive index is a conclusive measure in a biological context. Cell conditions, such as differentiation or infection, are known to yield significant changes in the refractive index. Furthermore, the refractive index of biological tissue determines the way it interacts with light. Besides the biological relevance of this interaction in the retina, a lot of methods used in biology, including microscopy, rely on light-tissue …
Recovering molecular information remains a grand challenge in the widely used holographic and computational imaging technologies. To address this challenge, we developed a computational mid-infrared photothermal microscope, termed Bond-selective Intensity Diffraction Tomography (BS-IDT). Based on a low-cost brightfield microscope with an add-on pulsed light source, BS-IDT recovers both infrared spectra and bond-selective 3D refractive index maps from intensity-only measurements. High-fidelity infrared fingerprint spectra extraction is validated. Volumetric chemical imaging of biological cells …
We report on wide-field imaging of pulsatile microvascular blood flow in the exposed cerebral cortex of a mouse by holographic interferometry. We recorded interferograms of laser light backscattered by the tissue, beating against an off-axis reference beam with a 50 kHz framerate camera. Videos of local Doppler contrasts were rendered numerically by Fresnel transformation and short-time Fourier transform analysis. This approach enabled instantaneous imaging of pulsatile blood flow contrasts in superficial blood vessels over 256 x 256 pixels with a spatial resolution of 10 microns and a tempora…
In most biological tissues, light scattering due to small differences in refractive index limits the depth of optical imaging systems. Two-photon microscopy (2PM), which significantly reduces the scattering of the excitation light, has emerged as the most common method to image deep within scattering biological tissue. This technique, however, requires high-power pulsed lasers that are both expensive and difficult to integrate into compact portable systems. In this paper, using a combination of theoretical and experimental techniques, we show that Selective Plane Illumination Microscopy (SPIM)…
We report on local superficial blood flow monitoring in biological tissue from laser Doppler holographic imaging. In time averaging recording conditions, holography acts as a narrowband bandpass filter, which, combined with a frequency shifted reference beam, permits frequency selective imaging in the radiofrequency range. These Doppler images are acquired with an off axis Mach Zehnder interferometer. Microvascular hemodynamic components mapping is performed in the cerebral cortex of the mouse and the eye fundus of the rat with near-infrared laser light without any exogenous marker. These meas…
University of California, Los Angeles（加州大学洛杉矶分校）；California NanoSystems Institute（加州纳米系统研究所）
The application of deep learning techniques has greatly enhanced holographic imaging capabilities, leading to improved phase recovery and image reconstruction. Here, we introduce a deep neural network termed enhanced Fourier Imager Network (eFIN) as a highly generalizable framework for hologram reconstruction with pixel super-resolution and image autofocusing. Through holographic microscopy experiments involving lung, prostate and salivary gland tissue sections and Papanicolau (Pap) smears, we demonstrate that eFIN has a superior image reconstruction quality and exhibits external generalizatio…
University of California, Los Angeles（加州大学洛杉矶分校）；David Geffen School of Medicine, University of California, Los Angeles（加州大学洛杉矶分校大卫·格芬医学院）
We report a framework based on a generative adversarial network (GAN) that performs high-fidelity color image reconstruction using a single hologram of a sample that is illuminated simultaneously by light at three different wavelengths. The trained network learns to eliminate missing-phase-related artifacts, and generates an accurate color transformation for the reconstructed image. Our framework is experimentally demonstrated using lung and prostate tissue sections that are labeled with different histological stains. This framework is envisaged to be applicable to point-of-care histopathology…
University of California, Los Angeles（加利福尼亚大学洛杉矶分校）；California Nano Systems Institute (CNSI)（加州纳米系统研究所）；David Geffen School of Medicine, University of California, Los Angeles（加利福尼亚大学洛杉矶分校大卫·格芬医学院）
Digital holography is one of the most widely used label-free microscopy techniques in biomedical imaging. Recovery of the missing phase information of a hologram is an important step in holographic image reconstruction. Here we demonstrate a convolutional recurrent neural network (RNN) based phase recovery approach that uses multiple holograms, captured at different sample-to-sensor distances to rapidly reconstruct the phase and amplitude information of a sample, while also performing autofocusing through the same network. We demonstrated the success of this deep learning-enabled holography me…
University of California, Los Angeles（加州大学洛杉矶分校）；California NanoSystems Institute（加利福尼亚纳米系统研究所）；David Geffen School of Medicine, University of California, Los Angeles（加州大学洛杉矶分校大卫·格芬医学院）
Phase recovery from intensity-only measurements forms the heart of coherent imaging techniques and holography. Here we demonstrate that a neural network can learn to perform phase recovery and holographic image reconstruction after appropriate training. This deep learning-based approach provides an entirely new framework to conduct holographic imaging by rapidly eliminating twin-image and self-interference related spatial artifacts. Compared to existing approaches, this neural network based method is significantly faster to compute, and reconstructs improved phase and amplitude images of the o…
Fiber-based micro-endoscopes are a critically important tool for minimally-invasive deep-tissue imaging. However, the state-of-the-art micro-endoscopes cannot perform three-dimensional imaging through dynamically-bent fibers without the use of bulky optical elements such as lenses and scanners at the distal end, increasing the footprint and tissue-damage. While great efforts have been invested in developing approaches that avoid distal bulky optical elements, the fundamental barrier of dynamic optical wavefront-distortions in propagation through flexible fibers, limits current approaches to ne…
Metasurface holography has the advantage of realizing complex wavefront modulation by thin layers together with the progressive technique of computer-generated holographic imaging. Despite the well-known light parameters, like amplitude, phase, polarization and frequency, the orbital angular momentum (OAM) of a beam can be regarded as another degree of freedom. Here, we propose and demonstrate orbital angular momentum multiplexing at different polarization channels using a birefringent metasurface for holographic encryption. The OAM selective holographic information can only be reconstructed w…
Non-invasive and single-shot holographic imaging through complex media is technically challenging due to random light scattering which significantly scrambles optical information. Recently, several methods have been presented to address this issue. However, they require complicated measurements of optical transmission matrices, or existing techniques do only retrieve intensity information. Here we propose and experimentally demonstrate a holographic approach for single-shot imaging through a scattering layer based on digital in-line holography in combination with the autocorrelation of the spe…
Scanning laser confocal holographic microscopy using a spatial heterodyne detection method is presented. Spatial heterodyne detection technique employs a Mach-Zehnder interferometer with the reference beam frequency shifted by two acousto-optic modulators (AOM) relative to the object beam frequency. Different from the traditional temporal heterodyne detection technique in which hundreds temporal samples are taken at each scanning point to achieve the complex signal, the spatial heterodyne detection technique generates spatial interference fringes by use of a linear tempo-spatial relation provi…
The imaging depth of deep-tissue optical microscopy is governed by the performance of the gating operation that suppresses the multiply scattered waves obscuring the ballistic waves. Although various gating operations based on confocal, time-resolved/coherence-gated, and polarization-selective detections have proven to be effective, each has its own limitation; certain types of multiply scattered waves can bypass the gating. Here, we propose a method, volumetric gating, that introduces ultrasound focus to confocal reflectance imaging to suppress the multiply scattered waves traveling outside t…
Fluorescent imaging plays a critical role in a myriad of scientific endeavors, particularly in the biological sciences. Three-dimensional imaging of fluorescent intensity often requires serial data acquisition, that is voxel-by-voxel collection of fluorescent light emitted throughout the specimen with a non-imaging single-element detector. While non-imaging fluorescence detection offers some measure of scattering robustness, the rate at which dynamic specimens can be imaged is severely limited. Other fluorescent imaging techniques utilize imaging detection to enhance collection rates. A notabl…
Generalizable Holographic Reconstruction via Amplitude-Only Diffusion Priors
Department of Bio and Brain Engineering（生物与脑工程系）；Department of Mechanical Engineering（机械工程系）；Kim Jaechul Graduate School of AI（Kim Jaechul人工智能研究生院）；KAIST Institute for Health Science and Technology（KAIST健康科学与技术研究所）
We performed non-invasive video imaging of retinal blood flow in a pigmented rat by holographic interferometry of near-infrared laser light backscattered by retinal tissue, beating against an off-axis reference beam sampled at a frame rate of 39 kHz with a high throughput camera. Local Doppler contrasts emerged from the envelopes of short-time Fourier transforms and the phase of autocorrelation functions of holograms rendered by Fresnel transformation. This approach permitted imaging of blood flow in large retinal vessels (30 microns diameter) over 400 by 400 pixels with a spatial resolution o…
Inferior soft-tissue contrast resolution is a major limitation of current CT scanners. The aim of the study is to improve the contrast resolution of CT scanners using dual-energy acquisition. Based on dual-energy material decomposition, the proposed method starts with extracting the outgoing energy spectrum by polychromatic forward projecting the material-selective images. The extracted spectrum is then reweighted to boost the soft-tissue contrast. A simulated water cylinder phantom with inserts that contain a series of six solutions of varying iodine concentration (range, 0-20 mg/mL) is used …
An approach reported recently by Alexandrov et al. on optical scatter imaging, termed digital Fourier microscopy (DFM), represents an adaptation of digital Fourier holography to selective imaging of biological matter. Holographic mode of recording of the sample optical scatter enables reconstruction of the sample image. Form-factor of the sample constituents provides a basis for discrimination of these constituents implemented via flexible digital Fourier filtering at the post processing stage. Like in the dark-field microscopy, the DFM image contrast appears to improve due to the suppressed o…
Advanced photonic tools may enable researchers and clinicians to visualize, track, control and manipulate biological processes at the single-cell level in space and time. Biological systems are complex and highly organized on both spatial and temporal levels. If we are to study, perturb, engineer or heal biological entities, we must be able to visualize key players in such systems and to track, control and manipulate them precisely and selectively. To achieve this goal, the engineering of non-destructive tools will allow us to interrogate and manipulate the function of proteins, pathways and c…
