Traditional stimulation-evoked cortical activity analysis mainly relies on the manual detection of the bright spots in a specific size regarded as active cells. However, there is limited research on the automatic detection of the cortical active cell in optical imaging of in vivo experiments which is much noisy. To address the laborious and hard annotation work, we propose a novel weakly supervised approach for active cell detection along the temporal frames. Compared to prevalent detection methods on common datasets, we formulate cell activation detection as a classification problem. We combine the techniques of clustering and deep neural network with little user indication of the Maximum Intensity Projection (MIP) of the time-lapse optical image sequence to realize the unsupervised classification model. The proposed approach achieves comparable performance on our optical image sequence with instant activation changing at each frame, which marks the cells using the fluorescent indicators. Although much noise is introduced during in vivo imaging, our algorithm is designed to accurately and effectively generate statistics on cell activation without requiring any prior training data preparation. This feature makes it particularly valuable for analyzing cell responses to psychopharmacological stimulation in subsequent analyses.
As transient intracellular Ca2+ changes play an important role in many essential processes including neuronal and astrocytic plasticity, tracking brain activity via Ca2+ is crucial. Unlike hemodynamics, Ca2+ change must be measured optically using an ionic fluorescent Ca2+ indicator. Here, we combine our highly sensitive multimodality optical imaging platform with genetically encoded Ca2+ indicator (GCaMP6f) expressed in neurons or astrocytes in somatosensory cortex, which enables simultaneous tracking of single-stimulation-evoked neuronal, astrocytic Ca2+ transients along with the corresponding hemodynamic responses at high spatiotemporal resolutions. We imaged neuronal and astrocytic Ca2+ transients from mouse cortex in response to a single electrical pulse (3mA, 0.3ms). Our results show that the neuronal Ca2+ responses were strong (ΔF/FN=6.4±0.29%), fast (latency τN=6±2.7ms) and of short duration (ΔtN=537±34ms) whereas the astrocyte responses were weak, slow and long-lasting (i.e., ΔF/FA=1.7±0.1%, τA=313±65ms, ΔtA =993±48ms). The synchronized activities among astrocytes were temporally less correlated than those among neurons. These results demonstrate the capability of optical detection of cell-specific Ca2+ activities from synchronized neuronal, astrocyte ensembles concurrently with the hemodynamic responses within the neuro-glio-vascular network, which can facilitate the study of the roles of astrocytes in the neurovascular coupling process. We also report time-lapse image results to analyze the interactions between stimuli-evoked neurovascular response versus the spontaneous cortical slow oscillations for brain functional studies
Optical coherence tomography angiography (OCTA) is a promising tool for imaging subsurface microvascular networks owing to its micron-level resolution and high sensitivity. However, it is not uncommon that OCTA imaging tend to suffer from strip artifacts induced by tissue motion. Although various algorithms for motion correction have been reported, a method that enables motion correction on a single en face OCTA image remains a challenge. In this study, we proposed a novel motion correction approach based on microvasculature detection and broken gap filling. Unlike previous methods using registration to restore disturbed vasculature during motion artifact removal, tensor voting is performed in individual projected image to connect the broken vasculature. Both simulation and in vivo 3D OCTA imaging of mouse bladder are performed to validate the effectiveness of this method. A comparison of in vivo images before and after motion correction shows that our method effectively corrects tissue motion artifacts while preserving continuity of vasculature network. Furthermore, in vivo results of this technique are presented to demonstrate the utility for imaging tumor angiogenesis in the mouse bladder.
Optical coherence tomography(OCT) imaging of bladder is gaining recognization due to the capability of noninvasive cross-sectional imaging of the bladder at the micron-level resolution and a relatively large field of view. Previous studies have shown the potential of OCT image to enhance detection of bladder transitional cell carcinoma(TCC). However, quantitative OCT image analysis for affirmative identification of bladder tumor remains a challenge[1]. Here, we report a novel method to enhance detection of TCC based on OCT images by analyzing anatomical and textural alteration of bladder. Specifically, OCT images are first processed with Dual Tree Complex Wavelet Transform denoising algorithms to reduce image speckle noise. Then, the layer segmentation method that mainly based on a dual path graph searching algorithm is performed on the denoised images to delineate three layers of bladder. The segmentation results show improved effectiveness and robustness in comparison to conventional graph theory based method. With layer segmentation, multiple measurements including layer thickness and texture can be quantified. The significant difference in quantified metrics between TCC and normal bladder indicate the potential use of those metrics for TCC identification. The proposed method provides valuable insights into TCC and has the potential to enhance the detection of tumor in the clinic.
Doppler optical coherence tomography (DOCT) is a promising functional imaging modality for quantitative blood flow measurement. A major limitation of DOCT is that only the axial component of flow can be measured, which is strongly influenced by the geometry of the vasculature. To overcome this challenge, we proposed a new method to retrieve absolute blood flow velocity of the vascular networks with fully restored topology. The application of open active snake can detect the skeleton of vasculature network without the need of vasculature segmentation, allowing Doppler angle to be calculated. The discontinuity of vasculature induced by Doppler angle and the limited dynamic range is corrected by a tensor voting based method, enabling the measurement of absolute blood flow velocity along each fully connected vessel branch. We present the results of in vivo cerebral blood flow (CBF) networks to demonstrate the efficacy of the proposed method.
Cocaine abuse can lead to cerebral strokes and hemorrhages secondary to cocaine’s cerebrovascular effects, which are poorly understood. We assessed cocaine’s effects on cerebrovascular anatomy and function in the somatosensory cortex of the rat’s brain. Optical coherence tomography was used for in vivo imaging of three-dimensional cerebral blood flow (CBF) networks and to quantify CBF velocities (CBFv), and multiwavelength laser-speckle-imaging was used to simultaneously measure changes in CBFv, oxygenated (Δ[HbO2]) and deoxygenated hemoglobin (Δ[HbR]) concentrations prior to and after an acute cocaine challenge in chronically cocaine exposed rats. Immunofluorescence techniques on brain slices were used to quantify microvasculature density and levels of vascular endothelial growth factor (VEGF). After chronic cocaine (2 and 4 weeks), CBFv in small vessels decreased, whereas vasculature density and VEGF levels increased. Acute cocaine further reduced CBFv and decreased Δ[HbO2] and this decline was larger and longer lasting in 4 weeks than 2 weeks cocaine-exposed rats, which indicates that risk for ischemia is heightened during intoxication and that it increases with chronic exposures. These results provide evidence of cocaine-induced angiogenesis in cortex. The CBF reduction after chronic cocaine exposure, despite the increases in vessel density, indicate that angiogenesis was insufficient to compensate for cocaine-induced disruption of cerebrovascular function.
Simultaneous measurement of hemodynamics is of great importance to evaluate the brain functional changes induced by brain diseases such as drug addiction. Previously, we developed a multimodal-imaging platform (OFI) which combined laser speckle contrast imaging with multi-wavelength imaging to simultaneously characterize the changes in cerebral blood flow (CBF), oxygenated- and deoxygenated- hemoglobin (HbO and HbR) from animal brain. Recently, we upgraded our OFI system that enables detection of hemodynamic changes in response to forepaw electrical stimulation to study potential brain activity changes elicited by cocaine. The improvement includes 1) high sensitivity to detect the cortical response to single forepaw electrical stimulation; 2) high temporal resolution (i.e., 16Hz/channel) to resolve dynamic variations in drug-delivery study; 3) high spatial resolution to separate the stimulation-evoked hemodynamic changes in vascular compartments from those in tissue. The system was validated by imaging the hemodynamic responses to the forepaw-stimulations in the somatosensory cortex of cocaine-treated rats. The stimulations and acquisitions were conducted every 2min over 40min, i.e., from 10min before (baseline) to 30min after cocaine challenge. Our results show that the HbO response decreased first (at ~4min) followed by the decrease of HbR response (at ~6min) after cocaine, and both did not fully recovered for over 30min. Interestingly, while CBF decreased at 4min, it partially recovered at 18min after cocaine administration. The results indicate the heterogeneity of cocaine’s effects on vasculature and tissue metabolism, demonstrating the unique capability of optical imaging for brain functional studies.
Brain functional connectivity is mapped using spontaneous low-frequency oscillations (LFOs) in blood-oxygen-leveldependent (BOLD) signals using fMRI. However, the origin of spontaneous BOLD oscillations remains elusive. Specifically, the coupling of regional hemodynamic LFOs to neuronal activity in a resting brain is rarely examined directly. Here we present a method based on instantaneous-frequency (IF) analysis to detect regional LFOs of cerebral blood flow (CBF) along with local-field potential (LFP) changes of neurons in resting state to study neurovascular coupling. CBF and LFP were simultaneously acquired using laser Doppler flowmetry (LDF) and electroencephalography in the rat’s somatosensory cortex with high temporal resolution (i.e., 20Hz for CBF and 2kHz for LDF, respectively). Instead of fast Fourier transform analysis, a peak-detection algorithm was used to define the LFP activities and CBF spontaneous oscillations in the time domain and the time lapses were used to calculate the IFs of hemodynamic (i.e., CBF) oscillations and neuronal (i.e., LFP) activities. Our results showed that the CBF mostly oscillated at ~0.1Hz with a full-half-bandwidth of [0.08Hz, 0.15Hz]. In addition, the maximal frequency of LFP firings was also approximately at 0.1Hz, which collaborated with to the frequency of CBF oscillations. Interestingly, CBF increased linearly with the LFP activity up to 0.15Hz (r=0.93), and both signals then decreased rapidly as a function of activity frequency. This indicates the spontaneous hemodynamic LFOs were associated with neuronal activities, thus confirming the neuronal origin of the hemodynamic oscillations.
Ultra-high resolution optical Doppler coherence tomography (μODT) is a promising tool for brain functional imaging. However, its sensitivity for detecting slow flows in capillary beds may limit its utility in visualizing and quantifying subtle changes in brain microcirculation. To address this limitation, we developed a novel method called contrast-enhanced μODT (c-μODT) in which intralipid is injected into mouse tail vein to enhance μODT detection sensitivity. We demonstrate that after intralipid injection, the flow detection sensitivity of μODT is dramatically enhanced by 230% as quantified by the fill factor (FF) of microvasculature. More importantly, we show that c-μODT preserves the quantitative properties for flow imaging, i.e., showing a comparable change ratio of hypercapnia-induced flow increase in the capillary network before and after injecting intralipid.
Deficits in prefrontal function play a crucial role in compulsive cocaine use, which is a hallmark of addiction.
Dysfunction of the prefrontal cortex might result from effects of cocaine on neurons as well as from disruption of
cerebral blood vessels. However, the mechanisms underlying cocaine’s neurotoxic effects are not fully understood,
partially due to technical limitations of current imaging techniques (e.g., PET, fMRI) to differentiate vascular from
neuronal effects at sufficiently high temporal and spatial resolutions. We have recently developed a multimodal imaging
platform which can simultaneously characterize the changes in cerebrovascular hemodynamics, hemoglobin oxygenation
and intracellular calcium fluorescence for monitoring the effects of cocaine on the brain. Such a multimodality imaging
technique (OFI) provides several uniquely important merits, including: 1) a large field-of-view, 2) high spatiotemporal
resolutions, 3) quantitative 3D imaging of the cerebral blood flow (CBF) networks, 4) label-free imaging of
hemodynamic changes, 5) separation of vascular compartments (e.g., arterial and venous vessels) and monitoring of
cortical brain metabolic changes, 6) discrimination of cellular (neuronal) from vascular responses. These imaging
features have been further advanced in combination with microprobes to form micro-OFI that allows quantification of
drug effects on subcortical brain. In addition, our ultrahigh-resolution ODT (μODT) enables 3D microangiography and
quantitative imaging of capillary CBF networks. These optical strategies have been used to investigate the effects of
cocaine on brain physiology to facilitate the studies of brain functional changes induced by addictive substance to
provide new insights into neurobiological effects of the drug on the brain.
Characterization of cerebral hemodynamic and oxygenation metabolic changes, as well neuronal function is of great
importance to study of brain functions and the relevant brain disorders such as drug addiction. Compared with other
neuroimaging modalities, optical imaging techniques have the potential for high spatiotemporal resolution and dissection
of the changes in cerebral blood flow (CBF), blood volume (CBV), and hemoglobing oxygenation and intracellular Ca
([Ca2+]i), which serves as markers of vascular function, tissue metabolism and neuronal activity, respectively. Recently,
we developed a multiwavelength imaging system and integrated it into a surgical microscope. Three LEDs of λ1=530nm,
λ2=570nm and λ3=630nm were used for exciting [Ca2+]i fluorescence labeled by Rhod2 (AM) and sensitizing total
hemoglobin (i.e., CBV), and deoxygenated-hemoglobin, whereas one LD of λ1=830nm was used for laser speckle
imaging to form a CBF mapping of the brain. These light sources were time-sharing for illumination on the brain and
synchronized with the exposure of CCD camera for multichannel images of the brain. Our animal studies indicated that
this optical approach enabled simultaneous mapping of cocaine-induced changes in CBF, CBV and oxygenated- and
deoxygenated hemoglobin as well as [Ca2+]i in the cortical brain. Its high spatiotemporal resolution (30μm, 10Hz) and
large field of view (4x5 mm2) are advanced as a neuroimaging tool for brain functional study.
Ca2+ plays a vital role as second messenger in signal transduction and the intracellular Ca2+ ([Ca2+]i) change is an
important indicator of neuronal activity in the brain, including both cortical and subcortical brain regions. Due to the
highly scattering and absorption of brain tissue, it is challenging to optically access the deep brain regions (e.g., striatum
at >3mm under the brain surface) and image [Ca2+]i changes with cellular resolutions. Here, we present two micro-probe
approaches (i.e., microlens, and micro-prism) integrated with a fluorescence microscope modified to permit imaging of
neuronal [Ca2+]i signaling in the striatum using a calcium indicator Rhod2(AM). While a micro-prism probe provides a
larger field of view to image neuronal network from cortex to striatum, a microlens probe enables us to track [Ca2+]i
dynamic change in individual neurons within the brain. Both techniques are validated by imaging neuronal [Ca2+]i
changes in transgenic mice with dopamine receptors (D1R, D2R) expressing EGFP. Our results show that micro-prism
images can map the distribution of D1R- and D2R-expressing neurons in various brain regions and characterize their
different mean [Ca2+]i changes induced by an intervention (e.g., cocaine administration, 8mg/kg., i.p). In addition,
microlens images can characterize the different [Ca2+]i dynamics of D1 and D2 neurons in response to cocaine, including
new mechanisms of these two types of neurons in striatum. These findings highlight the power of the optical
micro-probe imaging for dissecting the complex cellular and molecular insights of cocaine in vivo.
Because of its high spatial resolution and noninvasive imaging capabilities, optical coherence tomography has been used
to characterize the morphological details of various biological tissues including urinary bladder and to diagnose their
alternations (e.g., cancers). In addition to static morphology, the dynamic features of tissue morphology can provide
important information that can be used to diagnose the physiological and functional characteristics of biological tissues.
Here, we present the imaging studies based on optical coherence tomography to characterize motion related physiology
and functions of rat bladder detrusor muscles and compared the results with traditional biomechanical measurements.
Our results suggest that optical coherence tomography is capable of providing quantitative evaluation of contractile
functions of intact bladder (without removing bladder epithelium and connective tissue), which is potentially of more
clinical relevance for future clinical diagnosis - if incorporated with cystoscopic optical coherence tomography.
We present the first experimental result of direct delineation of the nuclei of living rat bladder epithelium with ultrahigh-resolution optical coherence tomography (uOCT). We demonstrate that the cellular details embedded in the speckle noise in a uOCT image can be uncovered by time-lapse frame averaging that takes advantage of the micromotion in living biological tissue. The uOCT measurement of the nuclear size (7.9±1.4 µm) closely matches the histological evaluation (7.2±0.8 µm). Unlike optical coherence microscopy (OCM), which requires a sophisticated high-NA microscopic objective, this approach uses a commercial-grade single achromatic lens (f/10 mm, NA/0.25) and provides a cross-sectional image over 0.6 mm of depth without focus tracking, thus holding great promise of endoscopic optical biopsy for diagnosis and grading of flat epithelial cancer such as carcinoma in situ in vivo.
To eliminate the influence of motion artifacts on measurements of intracellular calcium, a technique was developed using frequency domain analysis which filters motion artifact from the calcium transient signal arising from the perfused mouse heart using the calcium sensitive, fluorescent dye Rhod-2. The perfused mouse heart was stimulated at 8 Hz and placed in a water-jacketed chamber at 37 degree(s)C. After a washout period following Rhod-2 loading, ~6-fold increase in fluorescence above background was detected spectrofluorimetrically at 589nm when excited at 524 nm. Calcium dependent fluorescence transients mixed with motion artifacts and system noise were measured. Simultaneously, heart motion was monitored by recording the reflected excitation light from the heart. A Fourier transform was utilized to separate signals arising from the fluorescence transients and those resulting from motion in the frequency domain. Several major steps were adopted to implement the algorithm for elimination of motion as well as system instability from the transient signals. These included 1) extracting the fluorescence calcium transient signal from the raw data in the frequency domain by subtracting the motion recorded using the reflectance of excitation light, 2) digitally filtering out the random noise using multiple bandpass filters centralized at harmonic frequencies of the 8 Hz signal, and 3) extracting high frequency noise with the Kernel method. Comparing the processed signal of transients acquired with excessive motion artifact to transients acquired with minimal motion obtained by immobilizing the heart against the detection window demonstrated that the filtering techniques helped minimize the effects of motion.
We present a technique for optical detection of calcium transients in perfused mouse heart labeled with the calcium sensitive fluorescent dye Rhod-2. The isolated mouse-heart is placed in a water-jacketed chamber at 37 degrees Celsius, and is stimulated at 8 Hz, with 100 (mu) g of Rhod-2 bolused through the perfusate. After a 25-minute washout period, approximately 6 fold increase in fluorescence above background can be detected spectrofluorimetrically at 589 nm when excited at 524 nm. Both of these wavelengths are isobestic with regard to O2, thus minimizing interference due to changes in tissue oxygenation. Ca2+-dependent fluorescence transients are measured, as well as the corresponding left ventricular pressure signal. Our calcium transient signals represent 33 plus or minus 9% of diastolic fluorescence intensity. As the fluorescence emission is attenuated with the washout of Rhod-2 through the perfusate, the reflected absorbance between 524 nm (Rhod-2 sensitive) and 589 nm (Rhod- 2 insensitive) is used as a measure of dye concentration in tissue. The fluorescence-to-absorbance ratio measured from the perfused heart is verified to be insensitive to dye concentration, and thus can be used to determine the calcium concentration of the heart. Maximal calcium dependent fluorescence is calibrated in situ using high calcium and a SR Ca-ATPase inhibitor to tetanize the heart. The calculated cytosolic calcium concentrations for perfused mouse heart are 368 plus or minus 68 nM and 654 plus or minus 164 nM in diastole and systole, respectively. An effective method of minimizing changes in tissue scattering in the calcium quantification is also discussed.
KEYWORDS: Brain, Phase modulation, Spectroscopy, Near infrared spectroscopy, In vivo imaging, Blood, Hypoxia, Phase shift keying, Oximetry, In vitro testing
An approach of using phase modulation spectroscopy (PMS) system has been proposed which has single frequency, dual- wavelength with phase-only output for oximetry to minimize the influences of room light in the clinical environment. The availability of this method has been verified by laboratory experiments both in vitro and in vivo. In this study, a 200 Mhz PMS system has been used for measuring on piglet brain in vivo, to detect blood volume and saturation change during normoxia to hypoxia periods, and to monitor the brain depolarization in the hypoxic stress. Our experimental results show that the brain saturation estimated by PMS is approximately 84% and lies between arterial and venous blood values of 97% and 80% in normoxia, and tends to move close to the venous side in hypoxia. In addition, the excessive hypoxic stress triggers the brain to be bio-energetic deficits and finally introduced the neural depolarization, which can be effectively detected by recording the increment of tissue scattering.
The noninvasive NIR detection technique can be used to detect the clinic-related parameter such as hemoglobin concentration and saturation. By now, many techniques have been demonstrated in both time-domain and frequency domain to obtain quantitative optical properties. In this paper, we demonstrate one kind of simple homodyne phase modulation system -- in- phase and quadrature phase (I&O) detection system, introducing its principle and constitution, presenting its performance through the tests of ink, intralipid and blood model.
Int his article, we analytically present the influence of the systematic error due to the theoretical assumptions on the blood oxygenation determination using a 'phase-only' dual-wavelength phase-modulation spectroscopy (PMS). To approach an accurate quantification of hemoglobin saturation, an empirical method to experimentally acquire the initial phase-shift value in oxygenation quantification form PMS data is investigated on blood-perfused phantom. Our results show that the quantification error of blood oxygenation determination can be substantially compensated by using the acquired (theta) 0(lambda ) method and is confirmed to be less than 10 percent by comparison with the measured results of the Pt electrode.
In order to quantify the chromophore components from in vivo blood NIRS, a blood- equivalent phantom has been developed which consists of properly diluted intralipid and ICG dye. The reflection and transmission near infrared spectroscopy (NIRS) of the phantoms with different scattering backgrounds and ICG concentrations are measured and analyzed by the spectral multicomponent analysis (MCA) method to extract ICG concentration. The experimental results show that the MCA method can be used to quantify absolute ICG concentrations in scattering media if the average path lengths are known. Moreover, it was found by the experiments that both the water absorption peak at 970 nm and the ICG absorption peak at 800 nm show similar behavior during the change of the scattering background. Thus the ratio of the MCA-estimated concentration factor of ICG to water is independent of the blood-phantom scattering.
On the basis of both homogeneous and layered skin models this paper analyzes the influences of multiple scattering of skin on the measurements of NIR spectroscopy. Emphasis is laid on the wavelengths of 660, 805, and 940 nm which are used in clinical monitoring systems. The results of Monte-Carlo simulation show that the overwhelming scattering of tissue leads to a nonlinearity in the Lambert-Beer's relation between optical density and chromophore concentration. The consequences of this effect shall be discussed using as an example the non invasive measurement of Indocyanine Green (ICG) in the blood. In this case the multiple scattering of skin causes substantial non-linear relation between the optical density at 805 nm and the concentration of an injected NIR dye in the blood if the concentration of ICG exceeds 10 mg/l. This leads to a significant distortion of the ICG clearance curve and in consequence to a systematic error in the determination of physiological parameters. For multi-wavelength spectroscopy the wavelength dependency of scattering coefficients has to be noticed. The consequence of this effect is demonstrated for blood oxygen saturation (SaO2) measurements.
The propagation properties of an ultrafast laser pulse through breast tissues are simulated in this paper. It has been shown that the use of time-gated imaging techniques could greatly improve the contrast of transmission image of the breast. Then several time-gated imaging techniques are discussed, and a new time-gated imaging technique -- the use of transient stimulated Raman scattering for both rapid time gating and amplification of the image -- is proposed.
KEYWORDS: Magnetism, Sensors, Thin films, Magnetic sensors, Fiber optics sensors, Fiber optics, Optical fibers, Particles, Birefringence, Signal to noise ratio
Based on the strong magnetically induced birefringence and the magneto-optical absorption characteristics of dense ferrofluid thin films, this paper deals with research of their applications for magnetic field and electrical current sensing, using fiber optics. Preliminary experiments show the sensitivity of the system for magnetic field measurement (delta) H is better than 0.1 Oe, and for current measurement (delta) I < 1.2 A with a minimum detectable current threshold 6.5 A, which shows a promising potential for practical applications.
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