Automated filtering of intrinsic movement artifacts during two-photon intravital microscopy.
ABSTRACT: In vivo imaging using two-photon microscopy is an essential tool to explore the dynamic of physiological events deep within biological tissues for short or extended periods of time. The new capabilities offered by this technology (e.g. high tissue penetrance, low toxicity) have opened a whole new era of investigations in modern biomedical research. However, the potential of using this promising technique in tissues of living animals is greatly limited by the intrinsic irregular movements that are caused by cardiac and respiratory cycles and muscular and vascular tone. Here, we show real-time imaging of the brain, spinal cord, sciatic nerve and myenteric plexus of living mice using a new automated program, named Intravital_Microscopy_Toolbox, that removes frames corrupted with motion artifacts from time-lapse videos. Our approach involves generating a dissimilarity score against precalculated reference frames in a specific reference channel, thus allowing the gating of distorted, out-of-focus or translated frames. Since the algorithm detects the uneven peaks of image distortion caused by irregular animal movements, the macro allows a fast and efficient filtering of the image sequence. In addition, extra features have been implemented in the macro, such as XY registration, channel subtraction, extended field of view with maximum intensity projection, noise reduction with average intensity projections, and automated timestamp and scale bar overlay. Thus, the Intravital_Microscopy_Toolbox macro for ImageJ provides convenient tools for biologists who are performing in vivo two-photon imaging in tissues prone to motion artifacts.
Project description:In vivo non-linear optical microscopy has been essential to advance our knowledge of how intact biological systems work. It has been particularly enabling to decipher fast spatiotemporal cellular dynamics in neural networks. The power of the technique stems from its optical sectioning capability that in turn also limits its application to essentially immobile tissue. Only tissue not affected by movement or in which movement can be physically constrained can be imaged fast enough to conduct functional studies at high temporal resolution. Here, we show dynamic two-photon Ca(2+) imaging in the spinal cord of a living rat at millisecond time scale, free of motion artifacts using an optical stabilization system. We describe a fast, non-contact adaptive movement compensation approach, applicable to rough and weakly reflective surfaces, allowing real-time functional imaging from intrinsically moving tissue in live animals. The strategy involves enslaving the position of the microscope objective to that of the tissue surface in real-time through optical monitoring and a closed feedback loop. The performance of the system allows for efficient image locking even in conditions of random or irregular movements.
Project description:Two-photon microscopy is widely used to investigate brain function across multiple spatial scales. However, measurements of neural activity are compromised by brain movement in behaving animals. Brain motion-induced artifacts are typically corrected using post hoc processing of two-dimensional images, but this approach is slow and does not correct for axial movements. Moreover, the deleterious effects of brain movement on high-speed imaging of small regions of interest and photostimulation cannot be corrected post hoc. To address this problem, we combined random-access three-dimensional (3D) laser scanning using an acousto-optic lens and rapid closed-loop field programmable gate array processing to track 3D brain movement and correct motion artifacts in real time at up to 1?kHz. Our recordings from synapses, dendrites and large neuronal populations in behaving mice and zebrafish demonstrate real-time movement-corrected 3D two-photon imaging with submicrometer precision.
Project description:Two-photon imaging of endogenous fluorescence can provide physiological and metabolic information from intact tissues. However, simultaneous imaging of multiple intrinsic fluorophores, such as nicotinamide adenine dinucleotide(phosphate) (NAD(P)H), flavin adenine dinucleotide (FAD) and retinoids in living systems is generally hampered by sequential multi-wavelength excitation resulting in motion artifacts. Here, we report on efficient and simultaneous multicolor two-photon excitation of endogenous fluorophores with absorption spectra spanning the 750-1040?nm range, using wavelength mixing. By using two synchronized pulse trains at 760 and 1041?nm, an additional equivalent two-photon excitation wavelength at 879?nm is generated, and achieves simultaneous excitation of blue, green and red intrinsic fluorophores. This method permits an efficient simultaneous imaging of the metabolic coenzymes NADH and FAD to be implemented with perfect image co-registration, overcoming the difficulties associated with differences in absorption spectra and disparity in concentration. We demonstrate ratiometric redox imaging free of motion artifacts and simultaneous two-photon fluorescence lifetime imaging (FLIM) of NADH and FAD in living tissues. The lifetime gradients of NADH and FAD associated with different cellular metabolic and differentiation states in reconstructed human skin and in the germline of live C. Elegans are thus simultaneously measured. Finally, we present multicolor imaging of endogenous fluorophores and second harmonic generation (SHG) signals during the early stages of Zebrafish embryo development, evidencing fluorescence spectral changes associated with development.
Project description:In this work, we report a biopsy-needle compatible rigid probe, capable of performing three-dimensional (3D) two-photon optical biopsy. The probe has a small outer diameter of 1.75?mm and fits inside a gauge-14 biopsy needle to reach internal organs. A carefully designed focus scanning mechanism has been implemented in the rigid probe, which, along with a rapid two-dimensional MEMS scanner, enables 3D imaging. Fast image acquisition up to 10 frames per second is possible, dramatically reducing motion artifacts during in vivo imaging. Equipped with a high-numerical aperture micro-objective, the miniature rigid probe offers a high two-photon resolution (0.833?×?6.11??m, lateral × axial), a lateral field of view of 120??m, and an axial focus tuning range of 200??m. In addition to imaging of mouse internal organs and subcutaneous tumor in vivo, first-of-its-kind depth-resolved two-photon optical biopsy of an internal organ has been successfully demonstrated on mouse kidney in vivo and in situ.
Project description:Digital-scanned light-sheet microscopy (DSLM) illuminates a sample in a plane and captures single-photon-excitation fluorescence images with a camera from a direction perpendicular to the light sheet. This method is potentially useful for observing biological specimens, because image acquisition is relatively fast, resulting in reduction of phototoxicity. However, DSLM cannot be effectively applied to high-scattering materials due to the image blur resulting from thickening of the light sheet by scattered photons. However, two-photon-excitation DSLM (2p-DSLM) enables collection of high-contrast image with near infrared (NIR) excitation. In conventional 2p-DSLM, the minimal excitation volume for two-photon excitation restricts the field of view. In this study, we achieved wide-field 2p-DSLM by using a high-pulse energy fiber laser, and then used this technique to perform intravital imaging of a small model fish species, medaka (Oryzias latipes). Wide fields of view (>700 μm) were achieved by using a low-numerical aperture (NA) objective lens and high-peak energy NIR excitation at 1040 nm. We also performed high-speed imaging at near-video rate and successfully captured the heartbeat movements of a living medaka fish at 20 frames/sec.
Project description:The electroencephalogram (EEG) constitutes a relevant tool to study neural dynamics and to develop brain-machine interfaces (BMI) for rehabilitation of patients with paralysis due to stroke. However, the EEG is easily contaminated by artifacts of physiological origin, which can pollute the measured cortical activity and bias the interpretations of such data. This is especially relevant when recording EEG of stroke patients while they try to move their paretic limbs, since they generate more artifacts due to compensatory activity. In this paper, we study how physiological artifacts (i.e., eye movements, motion artifacts, muscle artifacts and compensatory movements with the other limb) can affect EEG activity of stroke patients. Data from 31 severely paralyzed stroke patients performing/attempting grasping movements with their healthy/paralyzed hand were analyzed offline. We estimated the cortical activation as the event-related desynchronization (ERD) of sensorimotor rhythms and used it to detect the movements with a pseudo-online simulated BMI. Automated state-of-the-art methods (linear regression to remove ocular contaminations and statistical thresholding to reject the other types of artifacts) were used to minimize the influence of artifacts. The effect of artifact reduction was quantified in terms of ERD and BMI performance. The results reveal a significant contamination affecting the EEG, being involuntary muscle activity the main source of artifacts. Artifact reduction helped extracting the oscillatory signatures of motor tasks, isolating relevant information from noise and revealing a more prominent ERD activity. Lower BMI performances were obtained when artifacts were eliminated from the training datasets. This suggests that artifacts produce an optimistic bias that improves theoretical accuracy but may result in a poor link between task-related oscillatory activity and BMI peripheral feedback. With a clinically relevant dataset of stroke patients, we evidence the need of appropriate methodologies to remove artifacts from EEG datasets to obtain accurate estimations of the motor brain activity.
Project description:Background:Optical coherence tomography (OCT) is an innovative imaging technique that generates high-resolution intracoronary images. In the last few years, the need for more precise analysis regarding coronary artery disease to achieve optimal treatment has made intravascular imaging an area of primary importance in interventional cardiology. One of the main challenges in OCT image analysis is the accurate detection of lumen which is significant for the further prognosis. Method:In this research, we present a new approach to the segmentation of lumen in OCT images. The proposed work is focused on designing an efficient automatic algorithm containing the following steps: preprocessing (artifacts removal: speckle noise, circular rings, and guide wire), conversion between polar and Cartesian coordinates, and segmentation algorithm. Results:The implemented method was tasted on 667 OCT frames. The lumen border was extracted with a high correlation compared to the ground truth: 0.97 ICC (0.97-0.98). Conclusions:Proposed algorithm allows for fully automated lumen segmentation on optical coherence tomography images. This tool may be applied to automated quantitative lumen analysis.
Project description:The breakthrough capacity of optoacoustics for three-dimensional visualization of dynamic events in real time has been recently showcased. Yet, efficient spectral unmixing for functional imaging of entire volumetric regions is significantly challenged by motion artifacts in concurrent acquisitions at multiple wavelengths. Here, we introduce a method for simultaneous acquisition of multispectral volumetric datasets by introducing a microsecond-level delay between excitation laser pulses at different wavelengths. Robust performance is demonstrated by real-time volumetric visualization of functional blood parametrers in human vasculature with a handheld matrix array optoacoustic probe. This approach can avert image artifacts imposed by velocities greater than 2?m/s, thus, does not only facilitate imaging influenced by respiratory, cardiac or other intrinsic fast movements in living tissues, but can achieve artifact-free imaging in the presence of more significant motion, e.g. abrupt displacements during handheld-mode operation in a clinical environment.
Project description:This document describes the collection and organization of a dataset that consists of raw videos and extracted sub-images from video frames of a promising additive manufacturing technique called Two-Photon Lithography (TPL). Four unprocessed videos were collected, with each video capturing the printing process of different combinations of 3D parts on different photoresists at varying light dosages. These videos were further trimmed to obtain short clips that are organized by experimental parameters. Additionally, this dataset also contains a python script to reproduce an organized directory of cropped video frames extracted from the trimmed videos. These cropped frames focus on a region of interest around the parts being printed. We envision that the raw videos and cropped frames provided in this dataset will be used to train various computer vision and machine learning algorithms for applications such as object segmentation and localization applications. The cropped video frames were manually labelled by an expert to determine the quality of the printed part and for printing parameter optimization as presented in "Automated Detection of Part Quality during Two-Photon Lithography via Deep Learning" .
Project description:BACKGROUND:A low excitation flip angle (??<?10°) steady-state free precession (SSFP) proton-density (PD) reference scan is often used to estimate the B1-field inhomogeneity for surface coil intensity correction (SCIC) of the saturation-recovery (SR) prepared high flip angle (??=?40-50°) SSFP myocardial perfusion images. The different SSFP off-resonance response for these two flip angles might lead to suboptimal SCIC when there is a spatial variation in the background B0-field. The low flip angle SSFP-PD frames are more prone to parallel imaging banding artifacts in the presence of off-resonance. The use of FLASH-PD frames would eliminate both the banding artifacts and the uneven frequency response in the presence of off-resonance in the surface coil inhomogeneity estimate and improve homogeneity of semi-quantitative and quantitative perfusion measurements. METHODS:B0-field maps, SSFP and FLASH-PD frames were acquired in 10 healthy volunteers to analyze the SSFP off-resonance response. Furthermore, perfusion scans preceded by both FLASH and SSFP-PD frames from 10 patients with no myocardial infarction were analyzed semi-quantitatively and quantitatively (rest n?=?10 and stress n?=?1). Intra-subject myocardial blood flow (MBF) coefficient of variation (CoV) over the whole left ventricle (LV), as well as intra-subject peak contrast (CE) and upslope (SLP) standard deviation (SD) over 6 LV sectors were investigated. RESULTS:In the 6 out of 10 cases where artifacts were apparent in the LV ROI of the SSFP-PD images, all three variability metrics were statistically significantly lower when using the FLASH-PD frames as input for the SCIC (CoVMBF-FLASH?=?0.3?±?0.1, CoVMBF-SSFP?=?0.4?±?0.1, p?=?0.03; SDCE-FLASH?=?10?±?2, SDCE-SSFP?=?32?±?7, p?=?0.01; SDSLP-FLASH?=?0.02?±?0.01, SDSLP-SSFP?=?0.06?±?0.02, p?=?0.03). Example rest and stress data sets from the patient pool demonstrate that the low flip angle SSFP protocol can exhibit severe ghosting artifacts originating from off-resonance banding artifacts at the edges of the field of view that parallel imaging is not able to unfold. These artifacts lead to errors in the quantitative perfusion maps and the semi-quantitative perfusion indexes, such as false positives. It is shown that this can be avoided by using FLASH-PD frames as input for the SCIC. CONCLUSIONS:FLASH-PD images are recommended as input for SCIC of SSFP perfusion images instead of low flip angle SSFP-PD images.