High-density speckle contrast optical tomography (SCOT) for three dimensional tomographic imaging of the small animal brain.

High-density speckle contrast optical tomography (SCOT) utilizing tens of thousands of source-detector pairs, was developed for in vivo imaging of blood flow in small animals. The reduction in cerebral blood flow (CBF) due to local ischemic stroke in a mouse brain was transcanially imaged and reconstructed in three dimensions. The reconstructed volume was then compared with corresponding magnetic resonance images demonstrating that the volume of reduced CBF agrees with the infarct zone at twenty-four hours.

Video-rate all-optical ultrasound imaging.

All-optical ultrasound imaging, where ultrasound is generated and detected using light, has recently been demonstrated as a viable modality that is inherently insensitive to electromagnetic interference and exhibits wide bandwidths. High-quality 2D and 3D all-optical ultrasound images of tissues have previously been presented; however, to date, long acquisition times (ranging from minutes to hours) have hindered clinical application. Here, we present the first all-optical ultrasound imaging system capable of video-rate, real-time two-dimensional imaging of biological tissue. This was achieved using a spatially extended nano-composite optical ultrasound generator, a highly sensitive fibre-optic acoustic receiver, and eccentric illumination resulting in an acoustic source exhibiting optimal directivity. This source was scanned across a one-dimensional source aperture using a fast galvo mirror, thus enabling the dynamic synthesis of source arrays comprising spatially overlapping sources at non-uniform source separation distances. The resulting system achieved a sustained frame rate of 15 Hz, a dynamic range of 30 dB, a penetration depth of at least 6 mm, a resolution of 75 µm (axial) by 100 µm (lateral), and enabled the dynamics of a pulsating ex vivo carotid artery to be captured.

Comparing the effective attenuation lengths for long wavelength in vivo imaging of the mouse brain.

Light attenuation in thick biological tissues, caused by a combination of absorption and scattering, limits the penetration depth in multiphoton microscopy (MPM). Both tissue scattering and absorption are dependent on wavelengths, which makes it essential to choose the excitation wavelength with minimum attenuation for deep imaging. Although theoretical models have been established to predict the wavelength dependence of light attenuation in brain tissues, the accuracy of these models in experimental settings needs to be verified. Furthermore, the water absorption contribution to the tissue attenuation, especially at 1450 nm where strong water absorption is predicted to be the dominant contributor in light attenuation, has not been confirmed. Here we performed a systematic study of in vivo three-photon imaging at different excitation wavelengths, 1300 nm, 1450 nm, 1500 nm, 1550 nm, and 1700 nm, and quantified the tissue attenuation by calculating the effective attenuation length at each wavelength. The experimental data show that the effective attenuation length at 1450 nm is significantly shorter than that at 1300 nm or 1700 nm. Our results provide unequivocal validation of the theoretical estimations based on water absorption and tissue scattering in predicting the effective attenuation lengths for long wavelength in vivo imaging.

Full video pulse extraction.

This paper introduces a new method to automate heart-rate detection using remote photoplethysmography (rPPG). The method replaces the commonly used region of interest (RoI) detection and tracking, and does not require initialization. Instead, it combines a number of candidate pulse-signals computed in the parallel, each biased towards differently colored objects in the scene. The method is based on the observation that the temporally averaged colors of video objects (skin and background) are usually quite stable over time in typical application-driven scenarios, such as the monitoring of a subject sleeping in bed, or an infant in an incubator. The resulting system, called full video pulse extraction (FVP), allows the direct use of raw video streams for pulse extraction. Our benchmark set of diverse videos shows that FVP enables long-term sleep monitoring in visible light and in infrared, and works for adults and neonates. Although we only demonstrate the concept for heart-rate monitoring, we foresee the adaptation to a range of vital signs, thus benefiting the larger video health monitoring field.

Reflection artifact identification in photoacoustic imaging using multi-wavelength excitation.

Photoacoustic imaging has been a focus of research for clinical applications owing to its ability for deep visualization with optical absorption contrast. However, there are various technical challenges remaining for this technique to find its place in clinics. One of the challenges is the occurrence of reflection artifacts. The reflection artifacts may lead to image misinterpretation. Here we propose a new method using multiple wavelengths for identifying and removing the reflection artifacts. By imaging the sample with multiple wavelengths, the spectral response of the features in the photoacoustic image is obtained. We assume that the spectral response of the reflection artifact is better correlated with the proper image feature of its corresponding absorber than with other features in the image. Based on this, the reflection artifacts can be identified and removed. Here, we experimentally demonstrated the potential of this method for real-time identification and correction of reflection artifacts in photoacoustic images in phantoms as well as in vivo using a handheld photoacoustic imaging probe.

Imaging transparent intact cardiac tissue with single-cell resolution.

We developed a new method, SUT (Scheme Update on tissue Transparency), to view cardiac microstructures and unveil the molecular changes underlying cardiac diseases. SUT is an effective method to clear whole-hearts from different species. Over the course of 4 - 6 days we obtained transparent whole-layer left ventricular tissues from mice with only an approximate 1% protein loss. In addition, EAL (Electrophoretic Antibody Labeling) was used to achieve fast antibody labeling by electric force, which significantly reduced antibody incubation time from days to hours. SUT, together with EAL and modern imaging techniques, were successfully used to visualize three-dimensional spatial distribution of various molecules in cardiac tissue. We also observed changes in the number and phenotypes of fibroblasts during post-myocardial infarction in a stereoscopic pattern. We believe that our technique opens a new avenue to explore the mechanisms underlying cardiac diseases.

Large area laser scanning optical resolution photoacoustic microscopy using a fibre optic sensor.

A laser scanning optical resolution photoacoustic microscopy (LS OR-PAM) system based on a stationary fibre optic sensor is described. The sensor comprises an optically resonant interferometric polymer cavity formed on the tip of a rounded single mode optical fibre. It provides low noise equivalent pressure (NEP = 68.7 Pa over a 20 MHz measurement bandwidth), a broad bandwidth that extends to 80 MHz and a near omnidirectional response. The latter is a significant advantage, as it allows large areas (>1cm2) to be imaged without the need for translational mechanical scanning offering the potential for fast image acquisition. The system provides a lateral resolution of 8 µm, an axial resolution of 21 µm, and a field of view up to 10 mm × 10 mm. To demonstrate the system, in vivo 3D structural images of the microvasculature of a mouse ear were obtained, showing single capillaries overlaying larger vessels as well as functional images revealing blood oxygen saturation.

Incorporating reflection boundary conditions in the Neumann series radiative transport equation: application to photon propagation and reconstruction in diffuse optical imaging.

We propose a formalism to incorporate boundary conditions in a Neumann-series-based radiative transport equation. The formalism accurately models the reflection of photons at the tissue-external medium interface using Fresnel's equations. The formalism was used to develop a gradient descent-based image reconstruction technique. The proposed methods were implemented for 3D diffuse optical imaging. In computational studies, it was observed that the average root-mean-square error (RMSE) for the output images and the estimated absorption coefficients reduced by 38% and 84%, respectively, when the reflection boundary conditions were incorporated. These results demonstrate the importance of incorporating boundary conditions that model the reflection of photons at the tissue-external medium interface.

Reflective lens-free imaging on high-density silicon microelectrode arrays for monitoring and evaluation of in vitro cardiac contractility.

The high rate of drug attrition caused by cardiotoxicity is a major challenge for drug development. Here, we developed a reflective lens-free imaging (RLFI) approach to non-invasively record in vitro cell deformation in cardiac monolayers with high temporal (169 fps) and non-reconstructed spatial resolution (352 µm) over a field-of-view of maximally 57 mm2. The method is compatible with opaque surfaces and silicon-based devices. Further, we demonstrated that the system can detect the impairment of both contractility and fast excitation waves in cardiac monolayers. Additionally, the RLFI device was implemented on a CMOS-based microelectrode array to retrieve multi-parametric information of cardiac cells, thereby offering more in-depth analysis of drug-induced (cardiomyopathic) effects for preclinical cardiotoxicity screening applications.

Distinguishing between whole cells and cell debris using surface plasmon coupled emission.

Distinguishing between whole cells and cell debris is important in microscopy, e.g., in screening of pulmonary patients for infectious tuberculosis. We propose and theoretically demonstrate that whole cells and cell debris can be distinguished from the far-field pattern of surface plasmon coupled emission (SPCE) of a fluorescently-labeled sample placed on a thin metal layer. If fluorescently-labeled whole cells are placed on the metal film, SPCE takes place simultaneously at two or more different angles and creates two or more distinct rings in the far field. By contrast, if fluorescently-labeled cell debris are placed on the metal film, SPCE takes place at only one angle and creates one ring in the far-field. We find that the angular separation of the far-field rings is sufficiently distinct to use the presence of one or more rings to distinguish between whole cells and cell debris. The proposed technique has the potential for detection without the use of a microscope.

Decoding cortical brain states from widefield calcium imaging data using visibility graph.

Widefield optical imaging of neuronal populations over large portions of the cerebral cortex in awake behaving animals provides a unique opportunity for investigating the relationship between brain function and behavior. In this paper, we demonstrate that the temporal characteristics of calcium dynamics obtained through widefield imaging can be utilized to infer the corresponding behavior. Cortical activity in transgenic calcium reporter mice (n=6) expressing GCaMP6f in neocortical pyramidal neurons is recorded during active whisking (AW) and no whisking (NW). To extract features related to the temporal characteristics of calcium recordings, a method based on visibility graph (VG) is introduced. An extensive study considering different choices of features and classifiers is conducted to find the best model capable of predicting AW and NW from calcium recordings. Our experimental results show that temporal characteristics of calcium recordings identified by the proposed method carry discriminatory information that are powerful enough for decoding behavior.

Cellular in vivo 3D imaging of the cornea by confocal laser scanning microscopy.

We present an in vivo confocal laser scanning microscopy based method for large 3D reconstruction of the cornea on a cellular level with cropped volume sizes up to 266 x 286 x 396 µm3. The microscope objective used is equipped with a piezo actuator for automated, fast and precise closed-loop focal plane control. Furthermore, we present a novel concave surface contact cap, which significantly reduces eye movements by up to 87%, hence increasing the overlapping image area of the whole stack. This increases the cuboid volume of the generated 3D reconstruction significantly. The possibility to generate oblique sections using isotropic volume stacks opens the window to slit lamp microscopy on a cellular level.

Optimization of wavelength selection for multispectral image acquisition: a case study of atrial ablation lesions.

In vivo autofluorescence hyperspectral imaging of moving objects can be challenging due to motion artifacts and to the limited amount of acquired photons. To address both limitations, we selectively reduced the number of spectral bands while maintaining accurate target identification. Several downsampling approaches were applied to data obtained from the atrial tissue of adult pigs with sites of radiofrequency ablation lesions. Standard image qualifiers such as the mean square error, the peak signal-to-noise ratio, the structural similarity index map, and an accuracy index of lesion component images were used to quantify the effects of spectral binning, an increased spectral distance between individual bands, as well as random combinations of spectral bands. Results point to several quantitative strategies for deriving combinations of a small number of spectral bands that can successfully detect target tissue. Insights from our studies can be applied to a wide range of applications.

Comparative study of the imaging contrasts of Mueller matrix derived parameters between transmission and backscattering polarimetry.

Mueller matrix polarimetry is a potentially powerful tool for biomedical diagnosis. Recently, the transmission Mueller matrix microscope and backscattering Mueller matrix endoscope were developed and applied to various pathological samples. However, a comparative study of imaging contrasts of Mueller matrix derived parameters between transmission and backscattering measurements is still needed to help decide which information obtained from transmission Mueller matrix microscope can be directly applied to in vivo Mueller matrix imaging. Here, to compare the imaging contrasts of Mueller matrix derived parameters between transmission and backscattering polarimetry, we measure porcine liver tissue samples and human breast carcinoma tissue specimens. The experiments and corresponding Monte Carlo stimulation results demonstrate that the backscattering and transmission retardance-related Mueller matrix parameters have very similar contrasts to characterize the anisotropic and isotropic structures of pathological tissues, meaning that the conclusions made from Mueller matrix microscopic imaging based on retardance can also be helpful to guide the in situ backscattering Mueller matrix polarimetric diagnosis. However, the values and contrasts of depolarization-related Mueller matrix parameters have some differences between transmission and backscattering polarimetry.

In vivo detection of UV-induced acute skin effects using optical coherence tomography.

Ultraviolet (UV) rays have been identified as a carcinogen with long-term irradiation and are an important risk factor for skin cancer. Here, we report the use of optical coherence tomography/optical coherence tomography angiography (OCT/OCTA) to study acute UV-induced effects on skin in vivo. To understand the relationship between the acute effects and irradiated UV power density, three groups were irradiated with different power densities in our experiments. Furthermore, the same skin area was repeatedly scanned with OCT during UV irradiation to investigate the progress of the induced acute effects and after irradiation for observation of skin recovery. Subsequently, the OCT/OCTA results were quantitatively analyzed to acquire skin thickness and blood-vessel density for comparison. UV-induced acute effects on morphology and microcirculation can be identified from OCT/OCTA results, which showed the increases in the skin thickness and blood-vessel density and even severe damage types such as blisters. The results of quantitative analyses also illustrated that the severity of damage induced by UV irradiation can be distinguished and the skin recovery can be monitored with OCT. Our results indicate that OCT can be a promising tool for early detection of UV-induced acute skin damage.

Phase retrieval for arbitrary Fresnel-like linear shift-invariant imaging systems suitable for tomography.

We present a generalization of the non-iterative phase retrieval in X-ray phase contrast imaging applicable for an arbitrary linear shift-invariant (LSI) imaging system with a non-negligible amount of free space propagation (termed as Fresnel-like). Our novel approach poses no restrictions on the propagation distance between optical elements of the system. In turn, the requirements are only demanded for the transfer function of the optical elements, which should be approximable by second-order Taylor polynomials. Furthermore, we show that the method can be conveniently used as an initial guess for iterative phase retrieval, resulting in faster convergence. The proposed approach is tested on synthetic and experimentally measured holograms obtained using a Bragg magnifier microscope - a representative of Fresnel-like LSI imaging systems. Finally, the algorithm is applied to a whole micro-tomographic scan of a biological specimen of a tardigrade, revealing morphological details at the spatial resolution of 300 nm - limiting resolution of the actual imaging system.

Erratum: Visualization of laser tattoo removal treatment effects in a mouse model by two-photon microscopy: publisher's note.

[This corrects the article on p. 3735 in vol. 8, PMID: 28856046.].

Label-free assessment of carotid artery biochemical composition using fiber-based fluorescence lifetime imaging.

Novel diagnostic tools with the ability to monitor variations in biochemical composition and provide benchmark indicators of vascular tissue maturation are needed to create functional tissue replacements. We investigated the ability of fiber-based, label-free multispectral fluorescent lifetime imaging (FLIm) to quantify the anatomical variations in biochemical composition of native carotid arteries and validated these results against biochemical assays. FLIm-derived parameters in spectral band 415-455 nm correlated with tissue collagen content (R2 = 0.64) and cell number (R2 = 0.61) and in spectral band 465-553 nm strongly correlated with elastin content (R2 = 0.89). These results suggest that FLIm holds great potential for assessing vascular tissue maturation and functional properties based on tissue autofluorescence.

Dichroism-sensitive photoacoustic computed tomography.

Photoacoustic computed tomography (PACT), a fast-developing modality for deep tissue imaging, images the spatial distribution of optical absorption. PACT usually treats the absorption coefficient as a scalar. However, the absorption coefficients of many biological tissues exhibit an anisotropic property, known as dichroism or diattenuation, which depends on molecular conformation and structural alignment. Here we present a novel imaging method called dichroism-sensitive PACT (DS-PACT), which measures both the amplitude of tissue's dichroism and the orientation of the optic axis of uniaxial dichroic tissue. By modulating the polarization of linearly polarized light and measuring the alternating signals through lock-in detection, DS-PACT can boost dichroic signals from biological tissues. To validate the proposed approach, we experimentally demonstrated the performance of DS-PACT by imaging plastic polarizers and ex vivo bovine tendons deep inside scattering media. We successfully detected the orientation of the optic axis of uniaxial dichroic materials, even at a depth of 4.5 transport mean free paths. We anticipate that the proposed method will extend the capability of PACT to imaging tissue absorption anisotropy.

Real-time cancer detection with an integrated lensless fluorescence contact imager.

Microscopic tumor cell foci left in a patient after surgery significantly increase the chance of cancer recurrence. However, fluorescence microscopes used for intraoperative navigation lack the necessary sensitivity for imaging microscopic disease and are too bulky to maneuver within the resection cavity. We have developed a scalable chip-scale fluorescence contact imager for detecting microscopic cancer in vivo and in real-time. The imager has been characterized under simulated in vivo conditions using ex vivo samples, providing strong evidence that our device can be used in vivo. Angle-selective gratings enhance the resolution of the imager without impacting its physical size. We demonstrate detection of cancer cell clusters containing as few as 25 HCC1569 breast cancer cells and 400 LNCaP prostate cancer cells with integration times of only 50 ms and 70 ms, respectively. A cell cluster recognition algorithm is used to achieve both a sensitivity and specificity of 92 % for HCC1569 cell samples, indicating the reliability of the imager. The signal-to-noise ratio (SNR) degradation with increased separation is only 1.5 dB at 250 µm. Blood scattering and absorption reduce the SNR by less than 2 dB for typical concentrations. Moreover, HER2+ breast cancer tissue taken from a patient is distinguished from normal breast tissue with an integration time of only 75 ms.