Optoacoustic Microscopy Research Articles

Selective plane illumination optical and optoacoustic microscopy for postembryonic imaging

AbstractIntravital imaging of large specimens is intrinsically challenging for postembryonic studies. Selective plane illumination microscopy (SPIM) has been introduced to volumetrically visualize organisms used in developmental biology and experimental genetics. Ideally suited for imaging transparent samples, SPIM can offer high frame rate imaging with optical microscopy resolutions and low phototoxicity. However, its performance quickly deteriorates when applied to opaque tissues. To overcome this limitation, SPIM optics were merged with optical and optoacoustic (photoacoustic) readouts. The performance of this hybrid imaging system was characterized using various phantoms and by imaging a highly scattering ex vivo juvenile zebrafish. The results revealed the system's enhanced capability over that of conventional SPIM for high‐resolution imaging over extended depths of scattering content. The approach described here may enable future visualization of organisms throughout their entire development, encompassing regimes in which the tissue may become opaque. image

Combining microscopy with mesoscopy using optical and optoacoustic label-free modes.

Biology requires observations at multiple geometrical scales, a feature that is not typically offered by a single imaging modality. We developed a hybrid optical system that not only provides different contrast modes but also offers imaging at different geometrical scales, achieving uniquely broad resolution and a 1000-fold volume sampling increase compared to volumes scanned by optical microscopy. The system combines optoacoustic mesoscopy, optoacoustic microscopy and two-photon microscopy, the latter integrating second and third harmonic generation modes. Label-free imaging of a mouse ear and zebrafish larva ex-vivo demonstrates the contrast and scale complementarity provided by the hybrid system. We showcase the superior anatomical orientation offered by the label-free capacity and hybrid operation, over fluorescence microscopy, and the dynamic selection between field of view and resolution achieved, leading to new possibilities in biological visualization.

Effects of the murine skull in optoacoustic brain microscopy.

Despite the great promise behind the recent introduction of optoacoustic technology into the arsenal of small-animal neuroimaging methods, a variety of acoustic and light-related effects introduced by adult murine skull severely compromise the performance of optoacoustics in transcranial imaging. As a result, high-resolution noninvasive optoacoustic microscopy studies are still limited to a thin layer of pial microvasculature, which can be effectively resolved by tight focusing of the excitation light. We examined a range of distortions introduced by an adult murine skull in transcranial optoacoustic imaging under both acoustically- and optically-determined resolution scenarios. It is shown that strong low-pass filtering characteristics of the skull may significantly deteriorate the achievable spatial resolution in deep brain imaging where no light focusing is possible. While only brain vasculature with a diameter larger than 60 µm was effectively resolved via transcranial measurements with acoustic resolution, significant improvements are seen through cranial windows and thinned skull experiments.

All-optical broadband ultrasonography of single cells.

Cell mechanics play a key role in several fundamental biological processes, such as migration, proliferation, differentiation and tissue morphogenesis. In addition, many diseased conditions of the cell are correlated with altered cell mechanics, as in the case of cancer progression. For this there is much interest in methods that can map mechanical properties with a sub-cell resolution. Here, we demonstrate an inverted pulsed opto-acoustic microscope (iPOM) that operates in the 10 to 100 GHz range. These frequencies allow mapping quantitatively cell structures as thin as 10 nm and resolving the fibrillar details of cells. Using this non-invasive all-optical system, we produce high-resolution images based on mechanical properties as the contrast mechanisms, and we can observe the stiffness and adhesion of single migrating stem cells. The technique should allow transferring the diagnostic and imaging abilities of ultrasonic imaging to the single-cell scale, thus opening new avenues for cell biology and biomaterial sciences.

Intravital Microscopy for Atherosclerosis Research.

Recruitment of leukocytes into arteries is a hallmark event throughout all stages of atherosclerosis and hence stands out as a primary therapeutic target. To understand the molecular mechanisms of arterial leukocyte subset infiltration, real-time visualization of recruitment processes of leukocyte subsets at high resolution is a prerequisite. In this review we provide a balanced overview of optical imaging modalities in the more commonly used experimental models for atherosclerosis (e.g., mouse models) allowing for in vivo display of recruitment processes in large arteries and further detail strategies to overcome hurdles inherent to arterial imaging. We further provide a synopsis of techniques allowing for non-toxic, photostable labeling of target structures. Finally, we deliver a short summary of ongoing developments including the emergence of novel labeling approaches, the use of superresolution microscopy, and the potentials of opto-acoustic microscopy and intravascular 2-dimensional near-infrared fluorescence microscopy.

Improved optoacoustic microscopy through three-dimensional spatial impulse response synthetic aperture focusing technique.

Synthetic aperture focusing technique (SAFT) is effective in restoring lateral resolution of ultrasonic images for scans with focusing-related distortions. Although successfully applied in pulse-echo ultrasonics, the physical nature of an optoacoustic modality requires a modified algorithm to return accurate results. The SIR-SAFT method reported here uses the spatial impulse response (SIR) of the transducer to weight the contributions to the SAFT and is tailored to provide significant resolution and signal gains for out-of-focus sources in scanning optoacoustic microscopy systems. Furthermore, the SIR-SAFT is implemented in full three dimensions, applicable to signals both far of and at the focus of the ultrasonic detector. The method has been further shown to outperform conventional SAFT algorithms for both simulated and experimental optoacoustic data.

Image Correction in Optoacoustic Microscopy. Numerical Simulation

Optoacoustic microscopy is an efficient method of three-dimensional biomedical visualization, which is based on using single-element focused ultrasonic antennas. Scanning of the studied medium by the focal constriction of an acoustic antenna allows one to retrieve the location of the sources of optoacoustic pulses without using reconstruction algorithms. However, the finite size of the focal constriction results in blurring of optoacoustic images. In this work, we demonstrate the algorithm which is based on calculating the Green’s function for an arbitrary acoustic antenna and allows one to correct optoacoustic images.

Hybrid multiphoton and optoacoustic microscope

We present a hybrid microscope combining multiphoton microscopy incorporating second-harmonic generation contrast and optical-resolution optoacoustic (photoacoustic) microscopy. We study the relative performance of the two systems and investigate the complementarity of contrast by demonstrating the label-free imaging capabilities of the hybrid microscope on zebrafish larvae ex vivo, concurrently visualizing the fish musculature and melanocytes. This implementation can prove useful in multiparametric microscopy studies, enabling broader information to be collected from biological specimens.

Real-time optoacoustic brain microscopy with hybrid optical and acoustic resolution

Conventional optoacoustic microscopy operates in two distinct modes of optical resolution, for visualization of superficial tissue layers, or acoustic resolution, intended for deep imaging in scattering tissues. Here we introduce a new microscope design with hybrid optical and acoustic resolution, which provides a smooth transition from optical resolution in superficial microscopic imaging to ultrasonic resolution when imaging at greater depths within intensely scattering tissue layers. Experimental validation of the new hybrid optoacoustic microscopy method was performed in phantoms and by means of transcranial mouse brain imaging in vivo.

High-resolution optoacoustic mesoscopy with a 24 MHz multidetector translate-rotate scanner

Optoacoustic (photoacoustic) mesoscopy aims at high-resolution optical imaging of anatomical, functional, and cellular parameters at depths that go well beyond those of optical-resolution optical or optoacoustic microscopy i.e., reaching several millimeters in depth. The approach utilizes tomography to achieve ultrasonic-diffraction resolution and operates at high-ultrasound frequencies (20 to 200 MHz) induced by few-nanosecond laser pulse excitation of tissues. We investigated here the performance of optoacoustic mesoscopy implemented at 24 MHz center frequency and its ability to resolve optical absorption contrast in the mouse kidney ex vivo. The developed system achieved better than 30 μm in-plane resolution and 110 μm elevation resolution over a cylindrical volume of 9-mm diameter and 9-mm height. This unprecedented combination of resolution and depth was achieved by implementing a translate-rotate detection geometry and by tomographic reconstruction. The approach yielded images of optically absorbing structures with a level of detail never-before visualized in an intact mouse kidney and allows insights into their unperturbed architecture. We discuss the ability to offer multispectral acquisitions and enable in vivo imaging.

Fast scanning coaxial optoacoustic microscopy

The hybrid nature of optoacoustic imaging might impose limitations on concurrent placement of optical and ultrasonic detection components, especially in high resolution microscopic applications that require dense arrangements and miniaturization of components. This hinders optimal deployment of the optical excitation and ultrasonic detection paths, leading to reduction of imaging speed and spatial resolution performance. We suggest a compact coaxial design for optoacoustic microscopy that allows optimizing both the light illumination and ultrasonic detection parameters of the imaging system. System performance is showcased in phantoms and in vivo imaging of microvasculature, achieving real time operation in two dimensions and penetration of 6 mm into optically dense human tissues.

Integrated optoacoustic-television methods for the nondestructive testing of solid nanomaterials

The problem of nondestructive testing is long standing for engineers all over the world. One method of solving this problem is the television and acoustic analysis of the state of nanomaterials. The structure and operational principle of a multifunctional intelligent optoacoustic microscope, as well as methods for the nondestructive testing (NDT) of nanomaterials, such as wavelet analysis and acoustic analysis, are considered.

Analysis of the structure of foam graphite by optoacoustic spectroscopy and scanning electron microscopy

A specimen of foam graphite foil is shown to have an anisotropic structure and consist of layers (stacks) of crystallite sheets weakly bound to one another.