SignificanceColor differences between healthy and diseased tissue in the gastrointestinal (GI) tract are detected visually by clinicians during white light endoscopy; however, the earliest signs of cancer are often just a slightly different shade of pink compared to healthy tissue making it hard to detect. Improving contrast in endoscopy is important for early detection of disease in the GI tract during routine screening and surveillance.AimWe aim to target alternative colors for imaging to improve contrast using custom multispectral filter arrays (MSFAs) that could be deployed in an endoscopic “chip-on-tip” configuration.ApproachUsing an open-source toolbox, Opti-MSFA, we examined the optimal design of MSFAs for early cancer detection in the GI tract. The toolbox was first extended to use additional classification models (k-nearest neighbor, support vector machine, and spectral angle mapper). Using input spectral data from published clinical trials examining the esophagus and colon, we optimized the design of MSFAs with three to nine different bands.ResultsWe examined the variation of the spectral and spatial classification accuracies as a function of the number of bands. The MSFA configurations tested showed good classification accuracies when compared to the full hyperspectral data available from the clinical spectra used in these studies.ConclusionThe ability to retain good classification accuracies with a reduced number of spectral bands could enable the future deployment of multispectral imaging in an endoscopic chip-on-tip configuration using simplified MSFA hardware. Further studies using an expanded clinical dataset are needed to confirm these findings.
Rapid and accurate disease diagnosis is important to determine appropriate treatment methods in clinics. Optical properties of tissue, such as absorption and scattering coefficients, could be a useful marker as disease progression occurs structural and biochemical alterations of biological tissue, resulting in chances of its optical properties. In addition, optical imaging methods are usually cost-effective and safe compared to other medical imaging techniques; thus, optical imaging could be used as a versatile tool for disease diagnosis. In this study, we have developed a multimodal imaging system that combines hyperspectral imaging and spatial-frequency domain imaging techniques. The proposed optical system enables quantitative measurement of scattering and absorption coefficients in biological tissue. We demonstrated the capability of the proposed optical system in measuring optical properties by exploiting a tissue-mimicking phantom and color chart, indicating that it has potential as a useful tool for characterizing the optical properties of tissue.
The optimal design and development of multispectral cameras could potentially improve the diagnosis of esophageal and colon cancer during routine screening and surveillance programs. We used an open-source Python toolbox Opti-MSFA and spectral data from clinical studies to determine the optimal spectral bands for inclusion in a snap-shot multispectral imaging system based on multispectral filter arrays (MSFAs) atop a CMOS image sensor. We emulated the feasibility of the proposed optimization method via synthetic datasets, which shows that the optimized MSFAs allow the accurate spectral unmixing of blood oxygen levels. The next steps are to implement the spectral imaging system with optimized MSFAs and demonstrate that the selected spectral bands are versatile in clinical trials.
Hyperspectral imaging techniques measure spatial and spectral information, which enables discrimination between healthy tissue and lesions. Hyperspectral imaging applications in endoscopy for clinical diagnostic applications are still limited due to image distortion challenges that arise when working with a flexible endoscope, for example in the gastrointestinal tract. Here, we developed a hyperspectral endoscopy (HySE) system by exploiting a line-scanning spectrograph to measure spectral information at high spectral resolution and combining it with a CMOS camera that records wide-field images for hyperspectral image reconstruction. Moreover, we developed an image normalisation method using near-infrared light to obtain accurate hyperspectral signals by correcting uneven illumination conditions during clinical endoscopy. Our next step is to apply HySE to patients to identify abnormal features in the gastrointestinal tract in vivo.
Advanced optical endoscopic imaging techniques, including hyperspectral and holographic endoscopy, have shown promise in the improved diagnosis of the early stage of cancer. However, clinical applications of these imaging systems are still limited due to unclear diagnostic optical properties. Here, we developed a compact multimodal imaging system that enables hyperspectral imaging, spatial-frequency domain imaging, and 3D profilometric imaging to characterise the optimal optical features for the early detection of lesions. Optical properties of fresh specimens obtained from patients were measured within 30 minutes, and then histopathological assessment of specimens was performed to link extracted optical features to gold-standard diagnosis. With further sample collection and system refinements, this system can be used for high-throughput optical characterisation of fresh tissue specimens, allowing us to determine the optical signatures of early-stage disease.
Emerging clinical interest in combining standard white light endoscopy with targeted near-infrared (NIR) fluorescent contrast agents for improved early cancer detection has created demand for multimodal imaging endoscopes. We used two spectrally resolving detector arrays (SRDAs) to realize a bimodal endoscope capable of simultaneous reflectance-based imaging in the visible spectral region and multiplexed fluorescence-based imaging in the NIR. The visible SRDA was composed of 16 spectral bands, with peak wavelengths in the range of 463 to 648 nm and full-width at half-maximum (FWHM) between 9 and 26 nm. The NIR SRDA was composed of 25 spectral bands, with peak wavelengths in the range 659 to 891 nm and FWHM 7 to 15 nm. The spectral endoscope design was based on a “babyscope” model using a commercially available imaging fiber bundle. We developed a spectral transmission model to select optical components and provide reference endmembers for linear spectral unmixing of the recorded image data. The technical characterization of the spectral endoscope is presented, including evaluation of the angular field-of-view, barrel distortion, spatial resolution and spectral fidelity, which showed encouraging performance. An agarose phantom containing oxygenated and deoxygenated blood with three fluorescent dyes was then imaged. After spectral unmixing, the different chemical components of the phantom could be successfully identified via majority decision with high signal-to-background ratio (>3). Imaging performance was further assessed in an ex vivo porcine esophagus model. Our preliminary imaging results demonstrate the capability to simultaneously resolve multiple biological components using a compact spectral endoscopy system.
Imaging brain tissues is an essential part of neuroscience because understanding brain structure provides relevant information about brain functions and alterations associated with diseases. Magnetic resonance imaging and positron emission tomography exemplify conventional brain imaging tools, but these techniques suffer from low spatial resolution around 100 μm. As a complementary method, histopathology has been utilized with the development of optical microscopy. The traditional method provides the structural information about biological tissues to cellular scales, but relies on labor-intensive staining procedures. With the advances of illumination sources, label-free imaging techniques based on nonlinear interactions, such as multiphoton excitations and Raman scattering, have been applied to molecule-specific histopathology. Nevertheless, these techniques provide limited qualitative information and require a pulsed laser, which is difficult to use for pathologists with no laser training.
Here, we present a label-free optical imaging of mouse brain tissues for addressing structural alteration in Alzheimer’s disease. To achieve the mesoscopic, unlabeled tissue images with high contrast and sub-micrometer lateral resolution, we employed holographic microscopy and an automated scanning platform. From the acquired hologram of the brain tissues, we could retrieve scattering coefficients and anisotropies according to the modified scattering-phase theorem. This label-free imaging technique enabled direct access to structural information throughout the tissues with a sub-micrometer lateral resolution and presented a unique means to investigate the structural changes in the optical properties of biological tissues.
Parkinson’s disease (PD) is a common neurodegenerative disease that causes symptoms of postural instability and slowness of movement. Neurodegeneration in dopaminergic neurons at the substantia nigra has been reported as pathologic features, however, detailed mechanisms underlying neurodegeneration are still remain unclear. To investigate a neurodegenerative process, various imaging tools including phase contrast microscopy, electron microscopy, and fluorescence microscopy are utilized. However, these imaging methods provide qualitative information and require invasive approaches such as the use of fluorescence agents or chemical fixation procedures that disturb normal physiological conditions of neuron cells.
In order to quantify the neurodegenerative process in a non-invasive manner, we exploited optical diffraction tomography (ODT). ODT is a 3D quantitative phase imaging method that measures 3D refractive index (RI) distributions of a sample which provide quantitative structural (volume, surface area, sphericity) and biochemical (protein concentration, total cellular dry mass) information. We investigated neurotoxic effects of MPP+ on SH-SY5Y cells by using quantitative information obtained from 3D RI distributions. We also performed temporal measurements of 3D RI distributions of an individual SH-SY5Y cell to analyze neurotoxic effects on intracellular vesicle dynamics.
White blood cells (WBC) have crucial roles in immune systems which defend the host against from disease conditions and harmful invaders. Various WBC subsets have been characterized and reported to be involved in many pathophysiologic conditions. It is crucial to isolate a specific WBC subset to study its pathophysiological roles in diseases. Identification methods for a specific WBC population are rely on invasive approaches, including Wright-Gimesa staining for observing cellular morphologies and fluorescence staining for specific protein markers. While these methods enable precise classification of WBC populations, they could disturb cellular viability or functions.
In order to classify WBC populations in a non-invasive manner, we exploited optical diffraction tomography (ODT). ODT is a three-dimensional (3-D) quantitative phase imaging technique that measures 3-D refractive index (RI) distributions of individual WBCs. To test feasibility of label-free classification of WBC populations using ODT, we measured four subtypes of WBCs, including B cell, CD4 T cell, CD8 T cell, and natural killer (NK) cell. From measured 3-D RI tomograms of WBCs, we obtain quantitative structural and biochemical information and classify each WBC population using a machine learning algorithm.
Optical diffraction tomography (ODT) is an interferometric microscopy technique capable of measuring 3-D refractive index (RI) distribution of transparent samples. Multiple 2-D holograms of a sample illuminated with various angles are measured, from which 3-D RI map of the sample is reconstructed via the diffraction theory. ODT has been proved as a powerful tool for the study of biological cells, due to its non-invasiveness, label-free and quantitative imaging capability. Recently, our group has demonstrated that a digital micromirror device (DMD) can be exploited for fast and precise control of illumination beams for ODT. In this work, we systematically study the precision and stability of the ODT system equipped with a DMD and present measurements of 3-D and 4-D RI maps of various types of live cells including human red blood cells, white blood cells, hepatocytes, and HeLa cells. Furthermore, we also demonstrate the effective visualization of 3-D RI maps of live cells utilizing the measured information about the values and gradient of RI tomograms.
Amyloid β-protein (Aβ) is known as a key molecule related to the pathogenesis of Alzheimer’s disease (AD). Over time, the amyloid cascade disrupts essential function of mitochondria including Ca2+ homeostasis and reactive oxygen species (ROS) regulation, and eventually leads to neuronal cell death. However, there have been no methods that analyze and measure neuronal dysfuction in pathologic conditions quantitatively. Here, we suggest a cell-based optical assay to investigate neuronal function in AD using femtosecond-pulsed laser stimulation. We observed that laser stimulation on primary rat hippocampal neurons for a few microseconds induced intracellular Ca2+ level increases or produced intracellular ROS which was a primary cause of neuronal cell death depending on delivered energy. Although Aβ treatment alone had little effect on the neuronal morphologies and networks in a few hours, Aβ-treated neurons showed delayed Ca2+ increasing pattern and were more vulnerable to laser-induced cell death compared to normal neurons. Our results collectively indicate that femtosecond laser stimulation can be a useful tool to study neuronal dysfuction related to AD pathologies. We anticipate this optical method to enable studies in the early progression of neuronal impairments and the quantitative evaluation of drug effects on neurons in neurodegenerative diseases, including AD and Parkinson’s disease in a preclinical study.
Brain tumor, especially glioblastoma multiforme (GBM), is one of the most malignant tumors, which not only demands
perplexing treatment approaches but also requires potent and effective treatment modality to deal with recurrence of the tumor. Photodynamic therapy (PDT) is a treatment which has been recommended as a third-level treatment. We are
trying to investigate possibility of the PDT as an efficient adjuvant therapeutic modality for the treatment of brain tumor. Inhibition of tumor progression with photosensitizer was verified, in vitro. With micellar nanoscale drug delivery system, localization of the tumor was identified, in vivo, which is able to be referred as photodynamic diagnosis. With consequent results, we are suggesting photodynamic diagnosis and therapy is able to be performed simultaneously with our nanoscale drug delivery system.
Astrocyte, the most abundant cell type in the central nervous system, has been one of major topics in neuroscience. Even
though many tools have been developed for the analysis of astrocyte function, there has been no adequate tool that can
modulates astrocyte network without pharmaceutical or genetic interventions. Here we found that ultrashort pulsed laser
stimulation can induce label-free activation of astrocytes as well as apoptotic-like cell death in a dose-dependent manner.
Upon irradiation with high intensity pulsed lasers, the irradiated cells with short exposure time showed very rapid
mitochondria fragmentation, membrane blebbing and cytoskeletal retraction. We applied this technique to investigate in
vivo function of astrocyte network in the CNS: in the aspect of neurovascular coupling and blood-brain barrier. We
propose that this noninvasive technique can be widely applied for in vivo study of complex cellular network.
As the most abundant cell type in the central nervous system, astrocyte has been one of main research topics in neuroscience. Although various tools have been developed, at present, there is no tool that allows noninvasive activation of astrocyte in vivo without genetic or pharmacological perturbation. Here we report a noninvasive label-free optical method for physiological astrocyte activation in vivo using a femtosecond pulsed laser. We showed the laser stimulation robustly induced astrocytic calcium activation in vivo and further verified physiological relevance of the calcium increase by demonstrating astrocyte mediated vasodilation in the brain. This novel optical method will facilitate noninvasive physiological study on astrocyte function.
Even though catheterization or electric stimulation are used for treatment of neurogenic bladder, invasiveness and
inconvenience of these approaches prompt us to develop a new possible therapeutic method to control urination by using
optical stimulation. The optical method using femtosecond pulsed laser (FSPL) has advantages of focused and
subsurface stimulation. Irradiation of FSPL induced a rapid increase of intracellular calcium level followed by
contraction of primary cultured human bladder smooth muscle cells. Short exposure of bladder detrusor ex-vivo to FSPL
also induced a controlled contraction of detrusor. Collectively, we propose that FSPL can be considered as a potential
therapeutic approach for intractable neurogenic bladder.
Even though electrical stimulation is generally used for induction of smooth muscle cell contraction, it is very hard to
obtain fine control and also very invasive for inserting electrodes. Herein, we developed a new optical technology to
control smooth muscle cell contraction. This optical method using femtosecond pulsed laser (FSPL) has advantage of
focused stimulation and fine control of stimulation intensity. Upon brief exposure to FSPL, smooth muscle cells showed
a rapid increase of intracellular calcium levels followed by cell contraction. Collectively, we suggest that FSPL can be a
useful tool for control of smooth muscle cell contraction.
The diameters of blood vessels, especially in the brain, change dynamically over time to provide sufficient blood supply as needed. No existing technique allows noninvasive control of vascular diameter in vivo. We report that label-free irradiation with a femtosecond pulsed laser can trigger blood vessel contraction in vivo. In response to laser irradiation, cultured vascular smooth muscle cells showed a rapid increase in calcium concentration, followed by cell contraction. In a murine thinned skull window model, laser irradiation focused in the arterial vessel wall caused localized vascular contraction, followed by recovery. The nonlinear nature of the pulsed laser allowed highly specific targeting of subcortical vessels without affecting the surrounding region. We believe that femtosecond pulsed laser irradiation will become a useful experimental tool in the field of vascular biology.
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