Kirill V. Larin

Recent progress in tissue optical clearing

Tissue optical clearing technique provides a prospective solution for the application of advanced optical methods in life sciences. This paper gives a review of recent developments in tissue optical clearing techniques. The physical, molecular and physiological mechanisms of tissue optical clearing are overviewed and discussed. Various methods for enhancing penetration of optical-clearing agents into tissue, such as physical methods, chemical-penetration enhancers and combination of physical and chemical methods are introduced. Combining the tissue optical clearing technique with advanced microscopy image or labeling technique, applications for 3D microstructure of whole tissues such as brain and central nervous system with unprecedented resolution are demonstrated. Moreover, the difference in diffusion and/or clearing ability of selected agents in healthy versus pathological tissues can provide a highly sensitive indicator of the tissue health/pathology condition. Finally, recent advances in optical clearing of soft or hard tissue for in vivo imaging and phototherapy are introduced.

Live Imaging of Early Developmental Processes in Mammalian Embryos with Optical Coherence Tomography.

Early embryonic imaging of cardiovascular development in mammalian models requires a method that can penetrate through and distinguish the many tissue layers with high spatial and temporal resolution. In this paper we evaluate the capability of Optical Coherence Tomography (OCT) technique for structural 3D embryonic imaging in mouse embryos at different stages of the developmental process ranging from 7.5 dpc up to 10.5 dpc. Obtained results suggest that the collected data is suitable for quantitative and qualitative measurements to assess cardiovascular function in mouse models, which is likely to expand our knowledge of the complexity of the embryonic heart, and its development into an adult heart.

Direct four-dimensional structural and functional imaging of cardiovascular dynamics in mouse embryos with 1.5 MHz optical coherence tomography

High-resolution three-dimensional (3D) imaging of cardiovascular dynamics in mouse embryos is greatly desired to study mammalian congenital cardiac defects. Here, we demonstrate direct four-dimensional (4D) imaging of the cardiovascular structure and function in live mouse embryos at a ∼43u2009u2009Hz volume rate using an optical coherence tomography (OCT) system with a ∼1.5u2009u2009MHz Fourier domain mode-locking swept laser source. Combining ultrafast OCT imaging with live mouse embryo culture protocols, 3D volumes of the embryo are directly and continuously acquired over time for a cardiodynamics analysis without the application of any synchronization algorithms. We present the time-resolved measurements of the heart wall motion based on the 4D structural data, report 4D speckle variance and Doppler imaging of the vascular system, and quantify spatially resolved blood flow velocity over time. These results indicate that the ultra-high-speed 4D imaging approach could be a useful tool for efficient cardiovascular phenotyping of mouse embryos.

Sequential Turning Acquisition and Reconstruction (STAR) method for four-dimensional imaging of cyclically moving structures

Optical coherence tomography allows for dynamic, three-dimensional (3D+T) imaging of the heart within animal embryos. However, direct 3D+T imaging frame rates remain insufficient for cardiodynamic analysis. Previously, this limitation has been addressed by reconstructing 3D+T representations of the beating heart based on sets of two-dimensional image sequences (2D+T) acquired sequentially at high frame rate and in fixed (and parallel) planes throughout the heart. These methods either require additional hardware to trigger the acquisition of each 2D+T series to the same phase of the cardiac cycle or accumulate registration errors as the slices are synchronized retrospectively by pairs, without a gating signal. Here, we present a sequential turning acquisition and reconstruction (STAR) method for 3D+T imaging of periodically moving structures, which does not require any additional gating signal and is not prone to registration error accumulation. Similarly to other sequential cardiac imaging methods, multiple fast image series are consecutively acquired for different sections but in between acquisitions, the imaging plane is rotated around the center line instead of shifted along the direction perpendicular to the slices. As the central lines of all image-sequences coincide and represent measurements of the same spatial position, they can be used to accurately synchronize all the slices to a single inherent reference signal. We characterized the accuracy of our method on a simulated dynamic phantom and successfully imaged a beating embryonic rat heart. Potentially, this method can be applied for structural or Doppler imaging approaches with any direct space imaging modality such as computed tomography, ultrasound, or light microscopy.

Increasing the field-of-view of dynamic cardiac OCT via post-acquisition mosaicing without affecting frame-rate or spatial resolution.

Optical coherence tomography (OCT) allows imaging dynamic structures and fluid flow within scattering tissue, such as the beating heart and blood flow in murine embryos. For any given system, the frame rate, spatial resolution, field-of-view (FOV), and signal-to-noise ratio (SNR) are interconnected: favoring one aspect limits at least one of the others due to optical, instrumentation, and software constraints. Here we describe a spatio-temporal mosaicing technique to reconstruct high-speed, high spatial-resolution, and large-field-of-view OCT sequences. The technique is applicable to imaging any cyclically moving structure and operates on multiple, spatially overlapping tiled image sequences (each sequence acquired sequentially at a given spatial location) and effectively decouples the (rigid) spatial alignment and (non-rigid) temporal registration problems. Using this approach we reconstructed full-frame OCT sequences of the beating embryonic rat heart (11.5 days post coitus) and compared it to direct imaging on the same system, demonstrating a six-fold improvement of the frame rate without compromising spatial resolution, FOV, or SNR.

Dynamic imaging and quantitative analysis of cranial neural tube closure in the mouse embryo using optical coherence tomography

Neural tube closure is a critical feature of central nervous system morphogenesis during embryonic development. Failure of this process leads to neural tube defects, one of the most common forms of human congenital defects. Although molecular and genetic studies in model organisms have provided insights into the genes and proteins that are required for normal neural tube development, complications associated with live imaging of neural tube closure in mammals limit efficient morphological analyses. Here, we report the use of optical coherence tomography (OCT) for dynamic imaging and quantitative assessment of cranial neural tube closure in live mouse embryos in culture. Through time-lapse imaging, we captured two neural tube closure mechanisms in different cranial regions, zipper-like closure of the hindbrain region and button-like closure of the midbrain region. We also used OCT imaging for phenotypic characterization of a neural tube defect in a mouse mutant. These results suggest that the described approach is a useful tool for live dynamic analysis of normal neural tube closure and neural tube defects in the mouse model.

A three-dimensional solution for laser-induced thermoelastic deformation of the layered medium

We have derived an axially symmetric three-dimensional analytical solution for thermoelastic deformations and stresses in a layered medium irradiated by a laser beam. The solution was obtained for Gaussian radial temperature profile on the upper surface of the elastic layer, in the assumption that temperature decreases exponentially with depth. The developed theoretical model was used to calculate distributions of laser-induced deformations and displacements in a medium containing single layer over a half-space. The influence of the shear elastic properties of the layer and half-space on the stress and strain distributions was evaluated. It was shown that tissue response depends significantly on the elastic contrast between the layer and the half-space. The proposed solution could be used in photomechanical models of laser ablation of inhomogeneous materials and tissues.

Optical coherent elastography method for stiffness assessment of heart muscle tissues (Conference Presentation)

Many complex diseases such as diastolic dysfunction and some types of cardiomyopathy are often characterized by an increased stiffness of heart muscles which can potentially cause heart failure. While changes of heart muscle’s geometry could be detected by various imaging methods, non-invasive measurements of stiffness of the heart muscle are desired to assess such areas of the heart tissues without invasive surgery. nA novel minimally-invasive method of stiffness assessment of heart muscle – optical coherent elastography (OCE) – is based on a combination of applied acoustic radiation force for mechanical excitation of tissue with subsequent phase-sensitive optical coherence tomography (psOCT) measurements of spatio-temporal response of tissue. A minimally invasive probe comprising a small, 2x2 mm size, low-frequency (<5MHz) ultrasound transducer and a clinically approved psOCT imaging fiber was incorporated into a single housing such that psOCT beam and acoustic excitation beam were parallel. Acoustic radiation pressure pulse was applied to initiate tissue displacement and propagation of shear waves that were detected by psOCT. Given the known offset between ultrasound and psOCT beams, the speed of shear waves was measured and shear elastic modulus of the heart tissues can be reconstructed. nThe initial results demonstrate that our OCE probe can produce and measure the displacements on the order of several ten nanometers in heart tissue-mimicking phantoms. The results indicate that translate-rotate scanning of OCE probe can simultaneously image the tissue and map its shear elastic modulus.

Comparison between thermoelastic and ablative induced elastic waves in soft media using ultra-fast line-field low coherent holography

Laser induced elastic waves in soft media have great potential to characterize tissue biomechanical properties. The instantaneous increase in local temperature caused by absorption of laser energy leads to a mechanical perturbation in the sample, which can then propagate as a pressure (or an elastic) wave. The generation of the elastic wave can be via thermoelastic or ablative processes depending on the absorption coefficient of the sample and incident laser fluence. It is critical to differentiate between these regimes because only the thermoelastic regime is useful for nondestructive analysis of tissues. To investigate the transition point between these two different regimes, we induced elastic waves in tissue mimicking agar phantoms mixed with different concentrations of graphite powder. The elastic waves were excited by a 532nm pulsed laser with a pulse duration of 6 ns. The fluence of the pulsed laser was tuned from 0.08 J/cm2 to 3.19 J/cm2 , and the elastic wave was captured by ultra-fast line-field low coherent holography system capable of single-shot elastic wave imaging with nanometer-scale displacement sensitivity. Different concentrations of graphite powder enabled excitation in sample with controlled and variable attenuation coefficient, enabling measurement of the transition between the thermoelastic and ablative regimes. The results show that the transition from thermoelastic to ablative generated waves was 0.75 J/cm2 and 1.84 J/cm2 for phantoms with optical attenuation coefficients of 6.64±0.32 mm-1 and 26.19±1.70 mm-1, respectively. Our results show promise for all optical biomechanical characterization of tissues.

Ultra-high speed OCT allows measurement of intraocular pressure, corneal geometry, and corneal stiffness using a single instrument

Screening for ocular diseases, such as glaucoma and keratoconus, includes measuring the eye-globe intraocular pressure (IOP) and corneal biomechanical properties. However, currently available clinical tools cannot quantify corneal tissue material parameters, which can provide critical information for detecting diseases and evaluating therapeutic outcomes. Here, we demonstrate measurement of eye-globe IOP, corneal elasticity, and corneal geometry of in situ porcine corneas with a technique termed applanation optical coherence elastography (Appl-OCE) with single instrument. We utilize an ultrafast phase-sensitive optical coherence tomography system comprised of a 4X buffered Fourier domain mode-locked swept source laser with an Ascan rate of ~1.5 MHz and a 7.3 kHz resonant scanner. The IOP was measured by imaging the response of in situ porcine corneas to a large force air-puff. As with other noncontact tonometers, the time when the cornea was applanated during the inwards and outwards motion was correlated to a measure air-pressure temporal profile. The IOP was also measured with a commercially available rebound tonometer for comparison. The stiffness of the corneas was assessed by directly imaging and analyzing the propagation of a focused micro air-pulse induced elastic wave, and the corneal geometry was obtained from the OCT structural image. Our results show that corneal thickness decreased as IOP increased, and that corneal stiffness increased with IOP. Moreover, the IOP measurements made by Appl-OCE were more closely correlated with the artificially set IOP than the rebound tonometer, demonstrating the capabilities of Appl-OCE to measure corneal stiffness, eye-globe IOP, and corneal geometry with a single instrument.