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Register a new userHigh-resolution label-free imaging of tissue morphology with confocal phase microscopy
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Label-free imaging approaches seek to simplify and augment histopathologic assessment by replacing the current practice of staining by dyes to visualize tissue morphology with quantitative optical measurements. Quantitative phase imaging (QPI) operates with visible/UV light and thus provides a resolution matched to current practice. Here we introduce and demonstrate confocal QPI for label-free imaging of tissue sections and assess its utility for manual histopathologic inspection. Imaging cancerous and normal adjacent human breast and prostate, we show that tissue structural organization can be resolved with high spatial detail comparable to conventional H&E stains. Our confocal QPI images are found to be free of halo, solving this common problem in QPI. We further describe and apply a virtual imaging system based on Finite-Difference Time-Domain (FDTD) calculations to quantitatively compare confocal with wide-field QPI methods and explore performance limits using numerical tissue phantoms.
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We present a technically simple implementation of quantitative phase imaging in confocal microscopy based on synthetic optical holography with sinusoidal-phase reference waves. Using a Mirau interference objective and low-amplitude vertical sample vibration with a piezo-controlled stage, we record synthetic holograms on commercial confocal microscopes (Nikon, model: A1R; Zeiss: model: LSM-880), from which quantitative phase images are reconstructed. We demonstrate our technique by stain-free imaging of cervical (HeLa) and ovarian (ES-2) cancer cells and stem cell (mHAT9a) samples. Our technique has the potential to extend fluorescence imaging applications in confocal microscopy by providing label-free cell finding, monitoring cell morphology, as well as non-perturbing long-time observation of live cells based on quantitative phase contrast.
Acoustic impedance mismatches between soft tissues and bones are known to result in strong aberrations in optoacoustic and ultrasound images. Of particular importance are the severe distortions introduced by the human skull, impeding transcranial brain imaging with these modalities. While modelling of ultrasound propagation through the skull may in principle help correcting for some of the skull-induced aberrations, these approaches are commonly challenged by the highly heterogeneous and dispersive acoustic properties of the skull and lack of exact knowledge on its geometry and internal structure. Here we demonstrate that the spatio-temporal properties of the acoustic distortions induced by the skull are preserved for signal sources generated at neighboring intracranial locations by means of optoacoustic excitation. This optoacoustic memory effect is exploited for building a three-dimensional model accurately describing the generation, propagation and detection of time-resolved broadband optoacoustic waveforms traversing the skull. The memory-based model-based inversion is then shown to accurately recover the optical absorption distribution inside the skull with spatial resolution and image quality comparable to those attained in skull-free medium.
Over the past few decades, researchers have developed several approaches such as the Reference Phantom Method (RPM) to estimate ultrasound attenuation coefficient (AC) and backscatter coefficient (BSC). AC and BSC can help to discriminate pathology from normal tissue during in-vivo imaging. In this paper, we propose a new RPM model to simultaneously compute AC and BSC for harmonic imaging and a normalized score that combines the two parameters as a measure of disease progression. The model utilizes the spectral difference between two regions of interest, the first, a proximal, close to the probe and second, a distal, away from the probe. We have implemented an algorithm based on the model and shown that it provides accurate and stable estimates to within 5% of AC and BSC for simulated received echo from post-focal depths of a homogeneous liver-like medium. For practical applications with time gain and time frequency compensated in-phase and quadrature (IQ) data from ultrasound scanner, the method has been approximated and generalized to estimate AC and BSC for tissue layer underlying a more attenuative subcutaneous layer. The angular spectrum approach for ultrasound propagation in biological tissue is employed as a virtual Reference Phantom (VRP). The VRP is calibrated with a fixed probe and scanning protocol for application to liver tissue. In a feasibility study with 16 subjects, the method is able to separate 9/11 cases of progressive non-alcoholic fatty liver disease from 5 normal. In particular, it is able to separate 4/5 cases of non-alcoholic steato-hepatitis and early fibrosis (F<=2) from normal tissue. More extensive clinical studies are needed to assess the full capability of this model for screening and monitoring disease progression in liver and other tissues.
We present the first label-free, non-contact, in-vivo imaging of the ocular vasculature using photoacoustic remote sensing (PARS) microscopy. Both anterior and posterior segments mouse eye were imaged. Vasculature of iris, sclera and retina tissues were clearly resolved. To best of our knowledge this the first study showing non-contact photoacoustic imaging conducted on in-vivo ocular tissue. We believe that PARS microscopy has the potential to advance the diagnosis and treatment of ocular diseases.
High-resolution optical microscopy suffers from a low contrast in scattering media where a multiply scattered wave obscures a ballistic wave used for image formation. To extend the imaging depth, various gating operations - confocal, coherence, and polarization gating - have been devised to filter out the multiply scattered wave. However, these gating methods are imperfect as they all act on the detection plane located outside a scattering medium. Here, we present a new gating scheme, called space gating, that rejects the multiply scattered wave directly at the object plane inside a scattering medium. Specifically, we introduced a 30 $mu$m-wide acoustic focus to the object plane and reconstructed a coherent image only with the ballistic wave modulated by acousto-optic interaction. This method allows us to reject the multiply scattered wave that the existing gating methods cannot filter out and improves the ratio of the ballistic wave to the multiply scattered wave by more than 100 times for a scattering medium more than 20 times thicker than its scattering mean free path. Using the coherent imaging technique based on space gating, we demonstrate the unprecedented imaging capability - phase imaging of optically transparent biological cells fully embedded within a scattering medium - with a spatial resolution of 1.5 $mu$m.
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