Comment on ��Measurement and correction of transverse chromatic offsets for multi-wavelength retinal microscopy in the living eye��

An interesting method to measure and correct chromatic magnification offsets in a multi-wavelength retinal imaging microscope was recently reported [Harmening et al., Biomed. Opt. Express 3, 2066 (2012)]. These values were in part related to the ocular transverse chromatic aberration (TCA). This method could be potentially used in the future to overcome the fundamental limitation to estimate the eye’s TCA with ophthalmoscopic (double-pass) configurations.OCIS codes: (170.4460) Ophthalmic optics and devices, (330.5370) Physiological optics     

"Bull’s eye” appearance of hepatocellular adenomas in patients with glycogen storage disease type I — atypical magnetic resonance imaging findings: Two case reports

BACKGROUNDHepatocellular adenomas are rare tumors that can occur in patients with glycogen storage disease type I.CASE SUMMARYWe herein report two cases of histologically proven hepatocellular adenomas in patients with glycogen storage disease type I. Magnetic resonance imaging (MRI) was performed after bolus injection of gadoxetate disodium, a liver-specific gadolinium-based MRI contrast agent. In the present cases, some of the hepatocellular adenomas showed unexpectedly a “bull’s eye” appearance on T2-weighted and post-contrast images, which was not previously described as imaging findings of hepatocellular adenomas in glycogen storage disease. A bull’s eye appearance on T2-weighted images can be encountered in both benign (i.e., abscess) or malignant (i.e., epithelioid hemangioendothelioma, cholangio-carcinoma, and metastases) hepatic lesions.CONCLUSIONWe present two cases of hepatocellular adenomas in patients with glycogen storage disease type 1, in which gadoxetate disodium-MRI showed atypical imaging findings for hepatocellular adenomas. At present there is no systematic study evaluating MRI findings of hepatocellular adenomas in patients with glycogen storage disease, further studies are needed to specifically investigate this issue.     

Design of high-performance adaptive objective lens with large optical depth scanning range for ultrabroad near infrared microscopic imaging

We report on the theory and design of adaptive objective lens for ultra broadband near infrared light imaging with large dynamic optical depth scanning range by using an embedded tunable lens, which can find wide applications in deep tissue biomedical imaging systems, such as confocal microscope, optical coherence tomography (OCT), two-photon microscopy, etc., both in vivo and ex vivo. This design is based on, but not limited to, a home-made prototype of liquid-filled membrane lens with a clear aperture of 8mm and the thickness of 2.55mm ~3.18mm. It is beneficial to have an adaptive objective lens which allows an extended depth scanning range larger than the focal length zoom range, since this will keep the magnification of the whole system, numerical aperture (NA), field of view (FOV), and resolution more consistent. To achieve this goal, a systematic theory is presented, for the first time to our acknowledgment, by inserting the varifocal lens in between a front and a back solid lens group. The designed objective has a compact size (10mm-diameter and 15mm-length), ultrabroad working bandwidth (760nm - 920nm), a large depth scanning range (7.36mm in air) — 1.533 times of focal length zoom range (4.8mm in air), and a FOV around 1mm × 1mm. Diffraction-limited performance can be achieved within this ultrabroad bandwidth through all the scanning depth (the resolution is 2.22 μm - 2.81 μm, calculated at the wavelength of 800nm with the NA of 0.214 - 0.171). The chromatic focal shift value is within the depth of focus (field). The chromatic difference in distortion is nearly zero and the maximum distortion is less than 0.05%.OCIS codes: (220.0220) Optical design and fabrication, (220.1080) Active or adaptive optics, (080.2468) First-order optics, (080.2740) Geometric optical design, (170.3890) Medical optics instrumentation, (170.6900) Three-dimensional microscopy, (170.1790) Confocal microscopy, (170.4500) Optical coherence tomography     

Investigation of different wavelengths for scattering-based light sheet microscopy

Scattering-based light sheet microscopy (sLSM) is a microscopy technique that can visualize cellular morphologic details based on the scattering signal. While sLSM was previously shown to image animal tissues ex vivo at a cellular resolution, the wavelength used was chosen based on other in vivo microscopy technologies rather than through a comparison of the sLSM imaging performance between different wavelengths. In this paper, we report the development of a multi-wavelength sLSM setup that facilitates the investigation of different wavelengths for sLSM imaging. Preliminary results of imaging human anal tissues ex vivo showed that the sLSM setup allowed for comparisons of the cellular imaging performance at the same tissue location between different wavelengths. Both the quantitative analysis of the image contrast and the visual assessment by a pathologist showed that the imaging depth increased with wavelength, and the imaging depth increase was most notable around 600 nm. The preliminary results showed that the multi-wavelength sLSM setup could be useful in identifying the optimal wavelength for the specific tissue type.     

Real-time tissue differentiation based on optical emission spectroscopy for guided electrosurgical tumor resection

Complete surgical removal of cancer tissue with effective preservation of healthy tissue is one of the most important challenges in modern oncology. We present a method for real-time, in situ differentiation of tissue based on optical emission spectroscopy (OES) performed during electrosurgery not requiring any biomarkers, additional light sources or other excitation processes. The analysis of the optical emission spectra, enables the differentiation of healthy and tumorous tissue. By using multi-class support vector machine (SVM) algorithms, distinguishing between tumor types also seems to be possible. Due to its fast reaction time (0.05s) the method can be used for real-time navigation helping the surgeon achieve complete resection. The system’s easy realization has been proven by successful integration in a commercial electro surgical unit (ESU). In a first step the method was verified by using ex vivo tissue samples. The histological analysis confirmed, 95% of correctly classified tissue types.OCIS codes: (170.6510) Spectroscopy, tissue diagnostics; (170.1610) Clinical applications; (170.3890) Medical optics instrumentation     

Verification of the lack of correlation between age and longitudinal chromatic aberrations of the human eye from the visible to the infrared

Several researchers studied the longitudinal chromatic aberration (LCA) of the human eye and observed that it does not change due to age. We measured the LCA of 45 subjects’ normal right eyes at three distinct wavelengths (561, 690, and 840 nm) using a Hartmann–Shack wavefront aberrometer (HSWA) while consecutively switching between three light sources for wavefront sensing. We confirmed that the LCA of the human eye does not change due to age between 22 and 57 years.OCIS codes: (330.7325) Visual optics, metrology; (170.4460) Ophthalmic optics and devices; (330.5370) Physiological optics; (330.4875) Optics of physiological systems; (330.7326) Visual optics, modeling     

Images of photoreceptors in living primate eyes using adaptive optics two-photon ophthalmoscopy

In vivo two-photon imaging through the pupil of the primate eye has the potential to become a useful tool for functional imaging of the retina. Two-photon excited fluorescence images of the macaque cone mosaic were obtained using a fluorescence adaptive optics scanning laser ophthalmoscope, overcoming the challenges of a low numerical aperture, imperfect optics of the eye, high required light levels, and eye motion. Although the specific fluorophores are as yet unknown, strong in vivo intrinsic fluorescence allowed images of the cone mosaic. Imaging intact ex vivo retina revealed that the strongest two-photon excited fluorescence signal comes from the cone inner segments. The fluorescence response increased following light stimulation, which could provide a functional measure of the effects of light on photoreceptors.OCIS codes: (010.1080) adaptive optics, (330.4460) Ophthalmic optics and devices, (330.5310) Vision – photoreceptors, (330.7327) Visual optics, ophthalmic instrumentation     

Reflective mirror-based line-scan adaptive optics OCT for imaging retinal structure and function

Line-scan OCT incorporated with adaptive optics (AO) offers high resolution, speed, and sensitivity for imaging retinal structure and function in vivo. Here, we introduce its implementation with reflective mirror-based afocal telescopes, optimized for imaging light-induced retinal activity (optoretinography) and weak retinal reflections at the cellular scale. A non-planar optical design was followed based on previous recommendations with key differences specific to a line-scan geometry. The three beam paths fundamental to an OCT system –illumination/sample, detection, and reference– were modeled in Zemax optical design software to yield theoretically diffraction-limited performance over a 2.2 deg. field-of-view and 1.5 D vergence range at the eye’s pupil. The performance for imaging retinal structure was exemplified by cellular-scale visualization of retinal ganglion cells, macrophages, foveal cones, and rods in human observers. The performance for functional imaging was exemplified by resolving the light-evoked optical changes in foveal cone photoreceptors where the spatial resolution was sufficient for cone spectral classification at an eccentricity 0.3 deg. from the foveal center. This enabled the first in vivo demonstration of reduced S-cone (short-wavelength cone) density in the human foveola, thus far observed only in ex vivo histological preparations. Together, the feasibility for high resolution imaging of retinal structure and function demonstrated here holds significant potential for basic science and translational applications.     

In vivo imaging of human photoreceptor mosaic with wavefront sensorless adaptive optics optical coherence tomography

Wavefront sensorless adaptive optics optical coherence tomography (WSAO-OCT) is a novel imaging technique for in vivo high-resolution depth-resolved imaging that mitigates some of the challenges encountered with the use of sensor-based adaptive optics designs. This technique replaces the Hartmann Shack wavefront sensor used to measure aberrations with a depth-resolved image-driven optimization algorithm, with the metric based on the OCT volumes acquired in real-time. The custom-built ultrahigh-speed GPU processing platform and fast modal optimization algorithm presented in this paper was essential in enabling real-time, in vivo imaging of human retinas with wavefront sensorless AO correction. WSAO-OCT is especially advantageous for developing a clinical high-resolution retinal imaging system as it enables the use of a compact, low-cost and robust lens-based adaptive optics design. In this report, we describe our WSAO-OCT system for imaging the human photoreceptor mosaic in vivo. We validated our system performance by imaging the retina at several eccentricities, and demonstrated the improvement in photoreceptor visibility with WSAO compensation.OCIS codes: (110.4500) Optical coherence tomography, (010.1080) Active or adaptive optics, (220.1000) Aberration compensation, (170.0110) Imaging systems, (170.4470) Ophthalmology, (120.3890) Medical optics instrumentation     

Wavefront sensorless adaptive optics optical coherence tomography for in vivo retinal imaging in mice

We present wavefront sensorless adaptive optics (WSAO) Fourier domain optical coherence tomography (FD-OCT) for in vivo small animal retinal imaging. WSAO is attractive especially for mouse retinal imaging because it simplifies optical design and eliminates the need for wavefront sensing, which is difficult in the small animal eye. GPU accelerated processing of the OCT data permitted real-time extraction of image quality metrics (intensity) for arbitrarily selected retinal layers to be optimized. Modal control of a commercially available segmented deformable mirror (IrisAO Inc.) provided rapid convergence using a sequential search algorithm. Image quality improvements with WSAO OCT are presented for both pigmented and albino mouse retinal data, acquired in vivo.OCIS codes: (170.4460) Ophthalmic optics and devices, (110.1080) Active or adaptive optics, (110.4500) Optical coherence tomography     

Handheld multispectral fluorescence lifetime imaging system for in vivo applications

There is an increasing interest in the application of fluorescence lifetime imaging (FLIM) for medical diagnosis. Central to the clinical translation of FLIM technology is the development of compact and high-speed clinically compatible systems. We present a handheld probe design consisting of a small maneuverable box fitted with a rigid endoscope, capable of continuous lifetime imaging at multiple emission bands simultaneously. The system was characterized using standard fluorescent dyes. The performance was then further demonstrated by imaging a hamster cheek pouch in vivo, and oral mucosa tissue both ex vivo and in vivo, all using safe and permissible exposure levels. Such a design can greatly facilitate the evaluation of FLIM for oral cancer imaging in vivo.OCIS codes: (170.2520) Fluorescence microscopy, (170.3650) Lifetime-based sensing, (170.2150) Endoscopic imaging, (170.3890) Medical optics instrumentation     

Fast time-lens-based line-scan single-pixel camera with multi-wavelength source

A fast time-lens-based line-scan single-pixel camera with multi-wavelength source is proposed and experimentally demonstrated in this paper. A multi-wavelength laser instead of a mode-locked laser is used as the optical source. With a diffraction grating and dispersion compensating fibers, the spatial information of an object is converted into temporal waveforms which are then randomly encoded, temporally compressed and captured by a single-pixel photodetector. Two algorithms (the dictionary learning algorithm and the discrete cosine transform-based algorithm) for image reconstruction are employed, respectively. Results show that the dictionary learning algorithm has greater capability to reduce the number of compressive measurements than the DCT-based algorithm. The effective imaging frame rate increases from 200 kHz to 1 MHz, which shows a significant improvement in imaging speed over conventional single-pixel cameras.OCIS codes: (110.0110) Imaging systems, (060.2350) Fiber optics imaging, (260.2030) Dispersion     

Dual-slit confocal light sheet microscopy for in vivo whole-brain imaging of zebrafish

In vivo functional imaging at single-neuron resolution is an important approach to visualize biological processes in neuroscience. Light sheet microscopy (LSM) is a cutting edge in vivo imaging technique that provides micron-scale spatial resolution at high frame rate. Due to the scattering and absorption of tissue, however, conventional LSM is inadequate to resolve cells because of the attenuated signal to noise ratio (SNR). Using dual-beam illumination and confocal dual-slit detection, here a dual-slit confocal LSM is demonstrated to obtain the SNR enhanced images with frame rate twice as high as line confocal LSM method. Through theoretical calculations and experiments, the correlation between the slit’s width and SNR was determined to optimize the image quality. In vivo whole brain structural imaging stacks and the functional imaging sequences of single slice were obtained for analysis of calcium activities at single-cell resolution. A two-fold increase in imaging speed of conventional confocal LSM makes it possible to capture the sequence of the neurons’ activities and help reveal the potential functional connections in the whole zebrafish’s brain.OCIS codes: (180.2520) Fluorescence microscopy, (110.0110) Imaging systems, (170.3880) Medical and biological imaging, (170.2945) Illumination design, (180.1790) Confocal microscopy     

Connectivity-based deep learning approach for segmentation of the epithelium in in vivo human esophageal OCT images

Optical coherence tomography (OCT) is used for diagnosis of esophageal diseases such as Barrett’s esophagus. Given the large volume of OCT data acquired, automated analysis is needed. Here we propose a bilateral connectivity-based neural network for in vivo human esophageal OCT layer segmentation. Our method, connectivity-based CE-Net (Bicon-CE), defines layer segmentation as a combination of pixel connectivity modeling and pixel-wise tissue classification. Bicon-CE outperformed other widely used neural networks and reduced common topological prediction issues in tissues from healthy patients and from patients with Barrett’s esophagus. This is the first end-to-end learning method developed for automatic segmentation of the epithelium in in vivo human esophageal OCT images.     

Heartbeat OCT: in vivo intravascular megahertz-optical coherence tomography

Cardiac motion artifacts, non-uniform rotational distortion and undersampling affect the image quality and the diagnostic impact of intravascular optical coherence tomography (IV-OCT). In this study we demonstrate how these limitations of IV-OCT can be addressed by using an imaging system that we called “Heartbeat OCT”, combining a fast Fourier Domain Mode Locked laser, fast pullback, and a micromotor actuated catheter, designed to examine a coronary vessel in less than one cardiac cycle. We acquired in vivo data sets of two coronary arteries in a porcine heart with both Heartbeat OCT, working at 2.88 MHz A-line rate, 4000 frames/s and 100 mm/s pullback speed, and with a commercial system. The in vivo results show that Heartbeat OCT provides faithfully rendered, motion-artifact free, fully sampled vessel wall architecture, unlike the conventional IV-OCT data. We present the Heartbeat OCT system in full technical detail and discuss the steps needed for clinical translation of the technology.OCIS codes: (170.4500) Optical coherence tomography, (170.3880) Medical and biological imaging, (170.2150) Endoscopic imaging, (110.6880) Three-dimensional image acquisition, (110.2350) Fiber optics imaging, (120.5800) Scanners, (120.3890) Medical optics instrumentation     

In vivo imaging of retinal pigment epithelium cells in age related macular degeneration

Morgan and colleagues demonstrated that the RPE cell mosaic can be resolved in the living human eye non-invasively by imaging the short-wavelength autofluorescence using an adaptive optics (AO) ophthalmoscope. This method, based on the assumption that all subjects have the same longitudinal chromatic aberration (LCA) correction, has proved difficult to use in diseased eyes, and in particular those affected by age-related macular degeneration (AMD). In this work, we improve Morgan’s method by accounting for chromatic aberration variations by optimizing the confocal aperture axial and transverse placement through an automated iterative maximization of image intensity. The increase in image intensity after algorithmic aperture placement varied depending upon patient and aperture position prior to optimization but increases as large as a factor of 10 were observed. When using a confocal aperture of 3.4 Airy disks in diameter, images were obtained using retinal radiant exposures of less than 2.44 J/cm², which is ~22 times below the current ANSI maximum permissible exposure. RPE cell morphologies that were strikingly similar to those seen in postmortem histological studies were observed in AMD eyes, even in areas where the pattern of fluorescence appeared normal in commercial fundus autofluorescence (FAF) images. This new method can be used to study RPE morphology in AMD and other diseases, providing a powerful tool for understanding disease pathogenesis and progression, and offering a new means to assess the efficacy of treatments designed to restore RPE health.OCIS codes: (110.1080) Active or adaptive optics, (330.5310) Vision - photoreceptors, (170.1610) Clinical applications, (170.3880) Medical and biological imaging, (170.4470) Ophthalmology     

Wavefront sensorless adaptive optics fluorescence biomicroscope for in vivo retinal imaging in mice

Cellular-resolution in vivo fluorescence imaging is a valuable tool for longitudinal studies of retinal function in vision research. Wavefront sensorless adaptive optics (WSAO) is a developing technology that enables high-resolution imaging of the mouse retina. In place of the conventional method of using a Shack-Hartmann wavefront sensor to measure the aberrations directly, WSAO uses an image quality metric and a search algorithm to drive the shape of the adaptive element (i.e. deformable mirror). WSAO is a robust approach to AO and it is compatible with a compact, low-cost lens-based system. In this report, we demonstrated a hill-climbing algorithm for WSAO with a variable focus lens and deformable mirror for non-invasive in vivo imaging of EGFP (enhanced green fluorescent protein) labelled ganglion cells and microglia cells in the mouse retina.OCIS codes: (170.4460) Ophthalmic optics and devices, (010.1080) Active or adaptive optics, (170.0110) Imaging systems, (170.4470) Ophthalmology     

In vivo volumetric fluorescence sectioning microscopy with mechanical-scan-free hybrid illumination imaging

Optical sectioning microscopy in wide-field fashion has been widely used to obtain three-dimensional images of biological samples; however, it requires scanning in depth and considerable time to acquire multiple depth information of a volumetric sample. In this paper, in vivo optical sectioning microscopy with volumetric hybrid illumination, with no mechanical moving parts, is presented. The proposed system is configured such that the optical sectioning is provided by hybrid illumination using a digital micro-mirror device (DMD) for uniform and non-uniform pattern projection, while the depth of imaging planes is varied by using an electrically tunable-focus lens with invariant magnification and resolution. We present and characterize the design, implementation, and experimentally demonstrate the proposed system’s ability through 3D imaging of in vivo Canenorhabditis elegans’ growth cones.OCIS codes: (110.0110) Imaging systems, (180.2520) Fluorescence microscopy, (110.6880) Three-dimensional image acquisition     

Design and characterization of a time-domain optical tomography platform for mesoscopic lifetime imaging

We report on the system design and instrumental characteristics of a novel time-domain mesoscopic fluorescence molecular tomography (TD-MFMT) system for multiplexed molecular imaging in turbid media. The system is equipped with a supercontinuum pulsed laser for broad spectral excitation, based on a high-density descanned raster scanning intensity-based acquisition for 2D and 3D imaging and augmented with a high-dynamical range linear time-resolved single-photon avalanche diode (SPAD) array for lifetime quantification. We report on the system’s spatio-temporal and spectral characteristics and its sensitivity and specificity in controlled experimental settings. Also, a phantom study is undertaken to test the performance of the system to image deeply-seated fluorescence inclusions in tissue-like media. In addition, ex vivo tumor xenograft imaging is performed to validate the system’s applicability to the biological sample. The characterization results manifest the capability to sense small fluorescence concentrations (on the order of nanomolar) while quantifying fluorescence lifetimes and lifetime-based parameters at high resolution. The phantom results demonstrate the system’s potential to perform 3D multiplexed imaging thanks to spectral and lifetime contrast in the mesoscopic range (at millimeters depth). The ex vivo imaging exhibits the prospect of TD-MFMT to resolve intra-tumoral heterogeneity in a depth-dependent manner.