Path sampling and integration method to calculate speckle patterns.

A stable speckle pattern is generated when a coherent beam illuminates a stationary scattering medium that contains numerous scatterers with fixed positions. To date, there has been no valid method to the best of our knowledge for calculating the speckle pattern of a macro medium with a large number of scatterers. Here, a new method based on possible path sampling with corresponding weights and coherent superposition is presented for the simulation of optical field propagation in a scattering medium and output speckle patterns. In this method, a photon is launched onto a medium with fixed scatterers. It propagates in one direction; upon collision with a scatterer, its direction is updated. The procedure is repeated until it exits the medium. A sampled path is obtained in this manner. By repeatedly launching photons, numerous independent optical paths can be sampled. A speckle pattern, corresponding to the probability density of the photon, is formed by the coherent superposition of sufficiently sampled path lengths ending on a receiving screen. This method can be used in sophisticated studies of the influences of medium parameters, motion of scatterers, sample distortions on speckle distributions, and morphological appearances. It can be used for micro-examination of optical fields in scattering media and may inspire new methods and techniques for non-invasive precision detection and diagnosis of scattering media.

Toward photoswitchable electronic pre-resonance stimulated Raman probes.

Reversibly photoswitchable probes allow for a wide variety of optical imaging applications. In particular, photoswitchable fluorescent probes have significantly facilitated the development of super-resolution microscopy. Recently, stimulated Raman scattering (SRS) imaging, a sensitive and chemical-specific optical microscopy, has proven to be a powerful live-cell imaging strategy. Driven by the advances of newly developed Raman probes, in particular the pre-resonance enhanced narrow-band vibrational probes, electronic pre-resonance SRS (epr-SRS) has achieved super-multiplex imaging with sensitivity down to 250 nM and multiplexity up to 24 colors. However, despite the high demand, photoswitchable Raman probes have yet to be developed. Here, we propose a general strategy for devising photoswitchable epr-SRS probes. Toward this goal, we exploit the molecular electronic and vibrational coupling, in which we switch the electronic states of the molecules to four different states to turn their ground-state epr-SRS signals on and off. First, we showed that inducing transitions to both the electronic excited state and triplet state can effectively diminish the SRS peaks. Second, we revealed that the epr-SRS signals can be effectively switched off in red-absorbing organic molecules through light-facilitated transitions to a reduced state. Third, we identified that photoswitchable proteins with near-infrared photoswitchable absorbance, whose states are modulable with their electronic resonances detunable toward and away from the pump photon energy, can function as the photoswitchable epr-SRS probes with desirable sensitivity (<1 µM) and low photofatigue (>40 cycles). These photophysical characterizations and proof-of-concept demonstrations should advance the development of novel photoswitchable Raman probes and open up the unexplored Raman imaging capabilities.

Photoacoustic Imaging.

Photoacoustic imaging (PAI) is an emerging imaging modality that shows great potential for preclinical research and clinical practice. As a hybrid technique, PAI uniquely combines the advantages of optical excitation and of acoustic detection. Optical excitation provides a rich contrast mechanism from either endogenous or exogenous chromophores, allowing PAI to perform biochemical, functional, and molecular imaging. Acoustic detection benefits from the low scattering of ultrasound in biological tissue, enabling PAI to generate high-resolution images in both the optical ballistic and diffusive regimes. Accordingly, this hybrid imaging modality features high sensitivity to optical absorption and wide scalability of spatial resolution with the desired imaging depth. Over the past two decades, the photoacoustic technique has led to a variety of exciting discoveries and applications from laboratory research to clinical patient care. In biological research, PAI has become an irreplaceable tool, providing functional optical contrast with high spatiotemporal resolution. Translational PAI also attracted growing interest in clinical applications including tumor margin examination, internal organ imaging, breast cancer screening, and sentinel lymph node mapping, among others.

Intelligently optimized digital optical phase conjugation with particle swarm optimization.

Wavefront shaping (WFS) based on digital optical phase conjugation (DOPC) has gained major interest in focusing light through or inside scattering media. However, the quality of DOPC is greatly limited by imperfections of the system in a complicated and coupled way. In this Letter, we incorporate the concept of global optimization to solve this problem comprehensively for the first time, to the best of our knowledge. An automatic and intelligent optimization framework for DOPC techniques is proposed, leveraging the global optimization ability of particle swarm optimization (PSO). We demonstrate the general and powerful ability of the proposed approach in a series of DOPC-related experiments for focusing through and inside scattering media. This novel work can improve the OPC quality greatly and simplify the development of a high-performance DOPC system, which may open up a new avenue for the general scientific community to benefit from DOPC-based WFS in their potential applications.

Single-shot compressed ultrafast photography at one hundred billion frames per second.

The capture of transient scenes at high imaging speed has been long sought by photographers, with early examples being the well known recording in 1878 of a horse in motion and the 1887 photograph of a supersonic bullet. However, not until the late twentieth century were breakthroughs achieved in demonstrating ultrahigh-speed imaging (more than 10(5) frames per second). In particular, the introduction of electronic imaging sensors based on the charge-coupled device (CCD) or complementary metal-oxide-semiconductor (CMOS) technology revolutionized high-speed photography, enabling acquisition rates of up to 10(7) frames per second. Despite these sensors' widespread impact, further increasing frame rates using CCD or CMOS technology is fundamentally limited by their on-chip storage and electronic readout speed. Here we demonstrate a two-dimensional dynamic imaging technique, compressed ultrafast photography (CUP), which can capture non-repetitive time-evolving events at up to 10(11) frames per second. Compared with existing ultrafast imaging techniques, CUP has the prominent advantage of measuring an x-y-t (x, y, spatial coordinates; t, time) scene with a single camera snapshot, thereby allowing observation of transient events with temporal resolution as tens of picoseconds. Furthermore, akin to traditional photography, CUP is receive-only, and so does not need the specialized active illumination required by other single-shot ultrafast imagers. As a result, CUP can image a variety of luminescent--such as fluorescent or bioluminescent--objects. Using CUP, we visualize four fundamental physical phenomena with single laser shots only: laser pulse reflection and refraction, photon racing in two media, and faster-than-light propagation of non-information (that is, motion that appears faster than the speed of light but cannot convey information). Given CUP's capability, we expect it to find widespread applications in both fundamental and applied sciences, including biomedical research.

Graphics processing unit accelerating compressed sensing photoacoustic computed tomography with total variation.

Photoacoustic computed tomography with compressed sensing (CS-PACT) is a commonly used imaging strategy for sparse-sampling PACT. However, it is very time-consuming because of the iterative process involved in the image reconstruction. In this paper, we present a graphics processing unit (GPU)-based parallel computation framework for total-variation-based CS-PACT and adapted into a custom-made PACT system. Specifically, five compute-intensive operators are extracted from the iteration algorithm and are redesigned for parallel performance on a GPU. We achieved an image reconstruction speed 24-31 times faster than the CPU performance. We performed in vivo experiments on human hands to verify the feasibility of our developed method.

A practical guide to photoacoustic tomography in the life sciences.

The life sciences can benefit greatly from imaging technologies that connect microscopic discoveries with macroscopic observations. One technology uniquely positioned to provide such benefits is photoacoustic tomography (PAT), a sensitive modality for imaging optical absorption contrast over a range of spatial scales at high speed. In PAT, endogenous contrast reveals a tissue's anatomical, functional, metabolic, and histologic properties, and exogenous contrast provides molecular and cellular specificity. The spatial scale of PAT covers organelles, cells, tissues, organs, and small animals. Consequently, PAT is complementary to other imaging modalities in contrast mechanism, penetration, spatial resolution, and temporal resolution. We review the fundamentals of PAT and provide practical guidelines for matching PAT systems with research needs. We also summarize the most promising biomedical applications of PAT, discuss related challenges, and envision PAT's potential to lead to further breakthroughs.

Multiscale photoacoustic tomography using reversibly switchable bacterial phytochrome as a near-infrared photochromic probe.

Photoacoustic tomography (PAT) of genetically encoded probes allows for imaging of targeted biological processes deep in tissues with high spatial resolution; however, high background signals from blood can limit the achievable detection sensitivity. Here we describe a reversibly switchable nonfluorescent bacterial phytochrome for use in multiscale photoacoustic imaging, BphP1, with the most red-shifted absorption among genetically encoded probes. BphP1 binds a heme-derived biliverdin chromophore and is reversibly photoconvertible between red and near-infrared light-absorption states. We combined single-wavelength PAT with efficient BphP1 photoswitching, which enabled differential imaging with substantially decreased background signals, enhanced detection sensitivity, increased penetration depth and improved spatial resolution. We monitored tumor growth and metastasis with ∼ 100-µm resolution at depths approaching 10 mm using photoacoustic computed tomography, and we imaged individual cancer cells with a suboptical-diffraction resolution of ∼ 140 nm using photoacoustic microscopy. This technology is promising for biomedical studies at several scales.

Efficient two-photon excitation by photonic dimers.

In this Letter, we show that photonic dimers, the quantum mechanical bound states of two photons, enable efficient nonlinear two-photon excitation, primarily due to the Lorentzian energy anti-correlation and to the temporal proximity between the constituent photons. We analytically and numerically demonstrate the order-of-magnitude improvement of the excitation efficiency per photon by the photonic dimers over the ultrashort pulses from a laser. We further show that, owing to the high excitation efficiency, the two-photon transition rate by photonic dimers deviates from the well-known linear intensity dependence at low intensities. Possible approaches for generating the photonic dimers in semiconductor platforms are also investigated.

High-speed label-free functional photoacoustic microscopy of mouse brain in action.

We present fast functional photoacoustic microscopy (PAM) for three-dimensional high-resolution, high-speed imaging of the mouse brain, complementary to other imaging modalities. We implemented a single-wavelength pulse-width-based method with a one-dimensional imaging rate of 100 kHz to image blood oxygenation with capillary-level resolution. We applied PAM to image the vascular morphology, blood oxygenation, blood flow and oxygen metabolism in both resting and stimulated states in the mouse brain.

Label-free cell nuclear imaging by Grüneisen relaxation photoacoustic microscopy.

Photoacoustic microscopy (PAM) with ultraviolet (UV) laser illumination has recently been demonstrated as a promising tool that provides fast, label-free, and multilayered histologic imaging of human breast tissue. Thus far, the axial resolution has been determined ultrasonically. To enable optically defined axial resolution, we exploit the Grüneisen relaxation (GR) effect. By imaging mouse brain slices, we show that GRUV-PAM reveals detailed information about three-dimensional cell nuclear distributions and internal structures, which are important diagnostic features for cancers. Due to the nonlinear effect, GRUV-PAM also provides better contrast in images of cell nuclei.

Dual-view photoacoustic microscopy for quantitative cell nuclear imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging imaging modality for studying biological tissues. However, in conventional single-view OR-PAM, the lateral and axial resolutions-determined optically and acoustically, respectively-are highly anisotropic. In this Letter, we introduce dual-view OR-PAM to improve axial resolution, achieving three-dimensional (3D) resolution isotropy. We first use 0.5 µm polystyrene beads and carbon fibers to validate the resolution isotropy improvement. Imaging of mouse brain slices further demonstrates the improved resolution isotropy, revealing the 3D structure of cell nuclei in detail, which facilitates quantitative cell nuclear analysis.

High-resolution photoacoustic tomography of resting-state functional connectivity in the mouse brain.

The increasing use of mouse models for human brain disease studies presents an emerging need for a new functional imaging modality. Using optical excitation and acoustic detection, we developed a functional connectivity photoacoustic tomography system, which allows noninvasive imaging of resting-state functional connectivity in the mouse brain, with a large field of view and a high spatial resolution. Bilateral correlations were observed in eight functional regions, including the olfactory bulb, limbic, parietal, somatosensory, retrosplenial, visual, motor, and temporal regions, as well as in several subregions. The borders and locations of these regions agreed well with the Paxinos mouse brain atlas. By subjecting the mouse to alternating hyperoxic and hypoxic conditions, strong and weak functional connectivities were observed, respectively. In addition to connectivity images, vascular images were simultaneously acquired. These studies show that functional connectivity photoacoustic tomography is a promising, noninvasive technique for functional imaging of the mouse brain.

A review of snapshot multidimensional optical imaging: measuring photon tags in parallel.

Multidimensional optical imaging has seen remarkable growth in the past decade. Rather than measuring only the two-dimensional spatial distribution of light, as in conventional photography, multidimensional optical imaging captures light in up to nine dimensions, providing unprecedented information about incident photons' spatial coordinates, emittance angles, wavelength, time, and polarization. Multidimensional optical imaging can be accomplished either by scanning or parallel acquisition. Compared with scanning-based imagers, parallel acquisition-also dubbed snapshot imaging-has a prominent advantage in maximizing optical throughput, particularly when measuring a datacube of high dimensions. Here, we first categorize snapshot multidimensional imagers based on their acquisition and image reconstruction strategies, then highlight the snapshot advantage in the context of optical throughput, and finally we discuss their state-of-the-art implementations and applications.

Photobleaching imprinting microscopy: seeing clearer and deeper.

We present a generic sub-diffraction-limited imaging method - photobleaching imprinting microscopy (PIM) - for biological fluorescence imaging. A lateral resolution of 110 nm was measured, more than a twofold improvement over the optical diffraction limit. Unlike other super-resolution imaging techniques, PIM does not require complicated illumination modules or specific fluorescent dyes. PIM is expected to facilitate the conversion of super-resolution imaging into a routine lab tool, making it accessible to a much broader biological research community. Moreover, we show that PIM can increase the image contrast of biological tissue, effectively extending the fundamental depth limit of multi-photon fluorescence microscopy.

Focusing light through scattering media by full-polarization digital optical phase conjugation.

Digital optical phase conjugation (DOPC) is an emerging technique for focusing light through or within scattering media such as biological tissue. Since DOPC systems are based on time reversal, they benefit from collecting as much information about the scattered light as possible. However, existing DOPC techniques record and subsequently phase-conjugate the scattered light in only a single-polarization state, limited by the operating principle of spatial light modulators. Here, we develop the first, to the best of our knowledge, full-polarization DOPC system that records and phase-conjugates scattered light along two orthogonal polarizations. When focusing light through thick scattering media, such as 2 mm and 4 mm-thick chicken breast tissue, our full-polarization DOPC system on average doubles the focal peak-to-background ratio achieved by single-polarization DOPC systems and improves the phase-conjugation fidelity.

Bit-efficient, sub-millisecond wavefront measurement using a lock-in camera for time-reversal based optical focusing inside scattering media.

Time-reversed ultrasonically encoded optical focusing measures the wavefront of ultrasonically tagged light, and then phase conjugates the tagged light back to the ultrasonic focus, thus focusing light deep inside the scattering media. In previous works, the speed of wavefront measurement was limited by the low frame rates of conventional cameras. In addition, these cameras used most of their bits to represent an informationless background when the signal-to-background ratio was low, resulting in extremely low efficiencies in the use of bits. Here, using a lock-in camera, we increase the bit efficiency and reduce the data transfer load by digitizing only the signal after rejecting the background. With this camera, we obtained the wavefront of ultrasonically tagged light after a single frame of measurement taken within 0.3 ms, and focused light in between two diffusers. The phase sensitivity has reached 0.51 rad even when the SBR is 6×10-4.

Photoacoustic elastography.

Elastography can noninvasively map the elasticity distribution in biological tissue, which can potentially be used to reveal disease conditions. In this Letter, we have demonstrated photoacoustic elastography by using a linear-array photoacoustic computed tomography system. The feasibility of photoacoustic elastography was first demonstrated by imaging the strains of single-layer and bilayer gelatin phantoms with various stiffness values. The measured strains agreed well with theoretical values, with an average error of less than 5.2%. Next, in vivo photoacoustic elastography was demonstrated on a mouse leg, where the fat and muscle distribution was mapped based on the elasticity contrast. We confirmed the photoacoustic elastography results by ultrasound elastography performed simultaneously.

Single-cell label-free photoacoustic flowoxigraphy in vivo.

Label-free functional imaging of single red blood cells (RBCs) in vivo holds the key to uncovering the fundamental mechanism of oxygen metabolism in cells. To this end, we developed single-RBC photoacoustic flowoxigraphy (FOG), which can image oxygen delivery from single flowing RBCs in vivo with millisecond-scale temporal resolution and micrometer-scale spatial resolution. Using intrinsic optical absorption contrast from oxyhemoglobin (HbO2) and deoxyhemoglobin (HbR), FOG allows label-free imaging. Multiple single-RBC functional parameters, including total hemoglobin concentration (C(Hb)), oxygen saturation (sO2), sO2 gradient (VsO2), flow speed (v(f)), and oxygen release rate (rO2), have been quantified simultaneously in real time. Working in reflection instead of transmission mode, the system allows minimally invasive imaging at more anatomical sites. We showed the capability to measure relationships among sO2, VsO2, v(f), and rO2 in a living mouse brain. We also demonstrated that single-RBC oxygen delivery was modulated by changing either the inhalation gas or blood glucose. Furthermore, we showed that the coupling between neural activity and oxygen delivery could be imaged at the single-RBC level in the brain. The single-RBC functional imaging capability of FOG enables numerous biomedical studies and clinical applications.