Cellular Resolution Panretinal Imaging of Optogenetic Probes Using a Simple Funduscope

Bead encoding is a key problem central to all multiplexed bead-based microarrays. Most existing bead-based microarrays require complicated and costly bead fabrication and/or sophisticated bead decoding hardware to achieve high multiplexing. Recently, the development of spatial bead encoding techniques have opened up the possibility of using pattern matching and image processing to develop highly multiplexed, high-throughput bead-based arrays. In this thesis, we improve the existing spatial bead encoding scheme, and develop a pipeline of computational methods, which allows for automated spatial bead encoding. Six novel computational methods, that automate the proposed bead encoding scheme, are developed in this thesis. The proposed scheme improves the previously reported spatial encoding schemes by making them better suited for use in real world scenarios. In the spatial bead encoding scheme, a sequential bead deposition method is used to capture the identities of the beads in bright or dark field images of the array. While, a second fluorescence image of the array is used to quantify the target analyte concentrations associated with the beads. By aligning the two images using the patterns formed by the beads, both the bead identities (i.e. the target analyte associate with the bead) and the analyte concentrations are decoded. The six computational methods developed in this thesis, are grouped into three categories: bright and dark field image processing, fluorescence image processing, and bead pattern matching. These methods are developed such that they require minimal parameter tuning, and are able to deal with noisy images acquired in uncontrolled environments As part of the bright and dark field image processing we have developed two novel methods: a fully automatic method for detecting the underlying micro-well grid structure in the images, and an unsupervised learning based method to classify the micro-wells as either empty or containing a bead. We show the ability of the proposed methods to deal with extreme amounts of noise and distortions using multiple datasets. While, microarray image gridding is a well-studied problem in the context of fluorescence images of planar microarrays, to the best of our knowledge no methods have yet been developed for processing either bright or dark field images of bead-based microarrays. Unlike in bright field images, where all the beads are visible, in fluorescence images only those beads that are expressed in the sample are visible. Therefore, in fluorescence image analysis the first step is to detect the beads in the image. To this end, we have developed an unsupervised, simultaneous bead detection and segmentation method which uses the coherence in shape and size of the beads to circumvent the need for parameter optimization. The main challenge in this segmentation is the large dynamic range across which the beads are expressed. We have also developed a probabilistic non-linear gridding method that attempts to establish the micro-well grid structure from the detected beads. While similar gridding techniques have been developed in the past, we make two contributions in this regard: first, we put the gridding method in a probabilistic framework, and second, we show that in certain cases it is impossible to ascertain if the correct grid has been detected without using additional information about the physical properties of the array. The final step in the proposed bead encoding scheme is to establishing one-to-one correspondences between the beads detected in the bright field and fluorescence images. We have developed two methods for this matching. When the micro-well grid structure in the fluorescence image is successfully established, the grid information can be used to reduce the matching problem to a binary grid alignment problem. A grid matching method that is very fast and can handle a large number of beads (>50,000) has been developed for this purpose. Finally, to perform matching when the fluorescence image grid is not available, a novel point pattern matching method that is affine invariant and robust to small non-linear distortions has been developed. The point pattern matching method addresses the problem of matching large point sets in the presence of large amounts of outliers and occlusion. The proposed method is evaluated using several real and simulated datasets.

Rare-Earth Doping Graphitic Carbon Nitride Endows Distinctive Multiple Emissions with Large Stokes Shifts

Prostate cancer is the leading cancer diagnosed among men worldwide. The prostate cancer-associated mortality is due to the development of aggressive neuroendocrine prostate cancer (NEPC). Unfortunately, there is no effective treatment available for NEPC at present. This is due to the lack of proper understanding of the molecular switch that triggers neuroendocrine changes in prostate adenocarcinoma cells. Recent research demonstrated that peripheral nerves promote prostate cancer progression and metastasis. However, there is no clear understanding of the involvement of sympathetic nerves or their signaling in facilitating NEPC. In this work, we examined the effect of the sympathetic neurotransmitter norepinephrine (NE) in inducing neuroendocrine differentiation (NED) of prostate cancer cells in vitro. We used androgen-sensitive LNCaP and androgen-insensitive DU145 cells for the study. We examined the dose and time-dependent effect of NE in inducing NED-related phenotypic alterations in these cells using bright field and fluorescence imaging. NED-related molecular alterations were assessed using real-time PCR, western bot and immunocytochemistry. We found that NE induces characteristic NEPC morphological changes, such as compact cell bodies and neurite-like extensions, in both DU145 and LNCaP cells. At the mRNA level, NE induced the expression of the NED markers chromogranin B, synaptophysin and βIII tubulin in both cell types. Further immunoblot and immunostaining showed the upregulation of chromogranin B in these cells. We repeated the experiments in growth factor deprived conditions by avoiding fetal bovine serum in the cultures to mimic growth deprivation to prostate cancer cells, which they experience during androgen deprivation therapy. Surprisingly, the NE induced NED changes were more pronounced in the growth factor deprived conditions. Overall, our result indicates the potential of NE in inducing NED in prostate cancer cells and point towards a critical role for this sympathetic neurotransmitter in facilitating the development of NEPC. Citation Format: Anand Krishnan, Shubham Dwivedi, Maricris Bautista, Tauqeer Iftikhar. Adrenergic signaling induces neuroendocrine differentiation of prostate adenocarcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB077.

Single cell sorting is commonly used for ensuring monoclonality and producing homogenous target cell populations. Current single cell verification methods involve manually confirming the existence of single cells or colonies in a well using a standard light microscope. However, the manual verification method is time-consuming and highly tedious, which prompts a need for an accurate and rapid detection method for verifying single cell sorting capability. Here, we demonstrate a rapid single cell sorting verification method using the Celigo Image Cytometer. Calcein AM-stained Jurkat cells and fluorescent beads are sorted into 96-well half area microplates using the MoFlo Astrios EQ. Whole well bright field and fluorescent images are acquired and analyzed using the image cytometer in less than 8 min. The proposed single cell verification detection method in multi-well microplates can allow for quick optimization of FACS instruments at flow core laboratories, as well as improvement of downstream biological assays by accurately confirming the presence of single cells in each well.

PurposeOphthalmic disorders are among the most prevalent Down syndrome (DS) comorbidities. Therefore, when studying mouse models of DS, ignoring how vision is affected can lead to misinterpretation of results from assessments dependent on the integrity of the visual system. Here, we used imaging and electroretinography (ERG) to study eye structure and function in two important mouse models of DS: Ts65Dn and Dp(16)1Yey/+.MethodsCornea and anterior segment were examined with a slit-lamp. Thickness of retinal layers was quantified by optical coherence tomography (OCT). Eye and lens dimensions were measured by magnetic resonance imaging (MRI). Retinal vasculature parameters were assessed by bright field and fluorescent imaging, and by retinal flat-mount preparations. Ganzfeld ERG responses to flash stimuli were used to assess retinal function in adult mice.ResultsTotal retinal thickness is significantly increased in Ts65Dn and Dp(16)1Yey/+ compared with control mice, because of increased thickness of inner retinal layers, including the inner nuclear layer (INL). Increased retinal vessel caliber was found in both chromosomally altered mice when compared with controls. ERG responses in Ts65Dn and Dp(16)1Yey/+ mice showed subtle alterations compared with controls. These, however, seemed to be unrelated to the thickness of the INL, but instead dependent on the anesthetic agent used (ketamine, tribromoethanol, or urethane).ConclusionsWe provide evidence of retinal alterations in Ts65Dn and Dp(16)1Yey/+ mice that are similar to those reported in persons with DS. Our ERG results are also a reminder that consideration should be given to the choice of anesthetic agents in such experiments.

In addition to the horizontal bands of myelinated axons that produce the line of Gennari and the inner band of Baillarger, the macaque primary visual cortex contains prominent vertical bundles of myelinated axons. In tangential sections through layer IVC, these axon bundles are regularly arranged. They have a mean center-to-center spacing of about 23 microns, and each one contains an average of 34 (S.D. +/- 13) myelinated axons. These bundles seem to be largely composed of efferent fibers, because in material in which pyramidal cells have been labelled in layer II/III and in layers IVA and IVB the axons of these neurons descend towards the white matter in bundles. However, it is doubtful whether all of the descending myelinated axons from the superficial layers emerge from the cortex, since counts show that the bundles contain maximum numbers of myelinated axons at the level of layer IVC, and that in layers V and VI their number is reduced by about 30%. Perhaps some of the axons enter the line of Baillarger, in layer V. When the bundles of myelinated axons and the clusters of apical dendrites of the layer V pyramidal cells are visualized simultaneously within layer IVC in electron microscopic preparations, it is apparent that their center-to-center spacing is similar, namely, about 23 microns and that a bundle of axons has a cluster of apical dendrites lying adjacent to it. Because of this association, and because axons from layer III pyramidal cells have been shown to enter the bundles, it is suggested that the myelinated axon bundles contain the efferent axons from the projection neurons in the individual pyramidal cell modules. However, in addition to the myelinated axons, the bundles contain unmyelinated axons, so that they also probably serve as the conduits for axons forming connections between layers. It is proposed that the pyramidal cell modules are the basic, functional neuronal units of the visual cortex, and since the neurons within a particular module can be expected to have slightly different inputs and response properties from those in neighboring modules, the individual axon bundles that emerge from each module would be expected to carry a unique set of efferent information.

For the effectiveness of a computer-aided diagnosis system, the quality of whole-slide image (WSI) is the foundation, and a useful autofocus method is an important part of ensuring the quality of WSI. The existing autofocus methods need to balance focusing speed and focusing accuracy, and need to be optimized separately for different samples or scenes. In this paper, a robust autofocus method based on fiber bundle illumination and image normalization analysis is proposed. For various application scenes, it meets the requirements of autofocusing through active illumination, such as bright field imaging and fluorescence imaging. For different structures on samples, it ensures the autofocusing accuracy through image analysis. The experimental results imply that the autofocusing method in this paper can effectively track the change of the distance from the sample to the focal plane and significantly improve the WSI quality.

Culturing cells in three-dimensions (3D) has become a well established approach as it is more representative of the in vivo environment than traditional two-dimensional (2D) cultures. Allowing cells to interact with each other in a spheroid creates a micro-environment which mimics in vivo tissue and is a better model for examining the effect of drugs in cancer. Developing uniform spheroids becomes especially important as it forms the basis for robust and reliable assays. S-BIO PrimeSurface® cultureware are ultra low attachment (ULA) dishes and plates that promote scaffold free, self assembly spheroid formation. The plates are pre-coated with a proprietary hydrophilic polymer that enables spontaneous spheroid formation of uniform size. PrimeSurface 96 and 384 ULA plates have good optical clarity making them highly suitable for brightfield and fluorescent imaging. Imaging technologies such as the BioTek Cytation™ 5 allows researchers to study not only spheroid proliferation through brightfield imaging, but also phenotypic events such as hypoxia, apoptosis, or necrosis induction through the use of fluorescent probes and fluorescence imaging. Incorporation of z-stacking and projection techniques in the Gen5™ Microplate Reader and Imager Software create in-focus images of spheroidal cells, allowing accurate, robust, and repeatable determination of the effect of test molecules or conditions. In this app note, we present data generated with BioTek Cytation 5 using PrimeSurface ULA plates to develop simple robust spheroid assays for brightfield and fluorescence imaging.

Trichrome, Congo red, and thioflavin S stains are comparable for the diagnosis of amyloid deposits in endomyocardial biopsies.

Introduction: Bone morphogenetic protein 7 (BMP-7), a protein with BMP-activating and TGF-β inactivating properties, induces the proliferation of a unique population of progenitor-like cells characterized by the expression of PDX1 and the BMP receptor activin-like kinase 3 (ALK3/BMPR1A). These cells can be sorted using ALK3 and the purinergic receptor P2Y1 (P2RY1) as a novel surrogate surface marker for PDX1. Sorted P2RY1+/ALK3bright+ cells form BMP-7 expandable colonies. Upon BMP-7 withdrawal, they differentiate into cells of all three lineages (endocrine, acinar and ductal) of the adult pancreas. PDX1+/ALK3+ cells absent from islets are represented in the major pancreatic ducts and pancreatic duct glands. Preliminary evidence indicates that these cells are also present in the murine pancreas. We hypothesized that islet β-cell regeneration could be studied in real time using live murine pancreatic slices transduced with a loxP-dsRed-loxP-EGFP reporter + insulin promoter-Cre adenovirus. If new β-cells arise from preexisting insulin-expressing-cells they would remain green. However, if neogenesis occurs from non-β-cells, there is a window of time during which the degrading red and the new green protein co-localize in the cell. Allowing us to discriminate between neogenesis and β-cell proliferation. Methods: Low melting point (LMP) agarose-embedded, vibratome-sectioned live pancreatic slices were generated from CD-1 mice. Transduction with a loxP-dsRed-loxP-EGFP reporter + insulin promoter-Cre adenovirus was done in slices cultured atop optically clear membranes. Slices were treated for 4 days with an ALK3 agonist (THR-123) or saline at the same time of the co-transduction. Bright field and fluorescence images of them were taken every 24h for 7 days. Conclusions: This is the first report on the use of live pancreatic slices as a novel tool for the study of pancreatic regeneration. Our preliminary results suggest that there are quantifiable β-cell neogenic events in response to BMP-7-like agents. Disclosure F. Qadir: None. J. Weitz: None. S. Alvarez-Cubela: None. D. Klein: None. G. Lanzoni: None. C.A. Garcia-Santana: Employee; Self; Ophysio Incorporated. A. Montalvo: None. C. Ricordi: None. L. Inverardi: None. A. Caicedo: None. R. Pastori: None. J. Domínguez-Bendala: Stock/Shareholder; Self; Ophysio.

Electrochemical reactions are involved in many natural phenomena, and are responsible for various applications, including energy conversion and storage, material processing and protection, and chemical detection and analysis. An electrochemical reaction is accompanied by electron transfer between a chemical species and an electrode. For this reason, it has been studied by measuring current, charge, or related electrical quantities. This approach has led to the development of various electrochemical methods, which have played an essential role in the understanding and applications of electrochemistry. While powerful, most of the traditional methods lack spatial and temporal resolutions desired for studying heterogeneous electrochemical reactions on electrode surfaces and in nanoscale materials. To overcome the limitations, scanning probe microscopes have been invented to map local electrochemical reactions with nanometer resolution. Examples include the scanning electrochemical microscope and scanning electrochemical cell microscope, which directly image local electrochemical reaction current using a scanning electrode or pipet. The use of a scanning probe in these microscopes provides high spatial resolution, but at the expense of temporal resolution and throughput. This Account discusses an alternative approach to study electrochemical reactions. Instead of measuring electron transfer electrically, it detects the accompanying changes in the reactant and product concentrations on the electrode surface optically via surface plasmon resonance (SPR). SPR is highly surface sensitive, and it provides quantitative information on the surface concentrations of reactants and products vs time and electrode potential, from which local reaction kinetics can be analyzed and quantified. The plasmonic approach allows imaging of local electrochemical reactions with high temporal resolution and sensitivity, making it attractive for studying electrochemical reactions in biological systems and nanoscale materials with high throughput. The plasmonic approach has two imaging modes: electrochemical current imaging and interfacial impedance imaging. The former images local electrochemical current associated with electrochemical reactions (faradic current), and the latter maps local interfacial impedance, including nonfaradic contributions (e.g., double layer charging). The plasmonic imaging technique can perform voltammetry (cyclic or square wave) in an analogous manner to the traditional electrochemical methods. It can also be integrated with bright field, dark field, and fluorescence imaging capabilities in one optical setup to provide additional capabilities. To date the plasmonic imaging technique has found various applications, including mapping of heterogeneous surface reactions, analysis of trace substances, detection of catalytic reactions, and measurement of graphene quantum capacitance. The plasmonic and other emerging optical imaging techniques (e.g., dark field and fluorescence microscopy), together with the scanning probe-based electrochemical imaging and single nanoparticle analysis techniques, provide new capabilities for one to study single nanoparticle electrochemistry with unprecedented spatial and temporal resolutions. In this Account, we focus on imaging of electrochemical reactions at single nanoparticles.

<p>Overlay of bright field and fluorescent images from live cell imaging experiment demonstrated uptake of blue labeled chronic lymphocytic leukemia (CLL) cells by red labeled human macrophages in the presence of the anti-CD20 mAb rituximab. Time is indicated in minutes. Scale bar = 20 μm.</p>

Development of a high-throughput image cytometric screening method as a research tool for immunophenotypic characterization of patient samples from clinical studies

An automatic microscope image acquisition, evaluation, and recognition system was developed for the analysis of Utermöhl plankton chambers in terms of taxonomic algae recognition. The system called PLASA (Plankton Structure Analysis) comprises (1) fully automatic archiving (optical fixation) of aqueous specimens as digital bright field and fluorescence images, (2) phytoplankton analysis and recognition, and (3) training facilities for new taxa. It enables characterization of aqueous specimens by their populations. The system is described in detail with emphasis on image analytical aspects. Plankton chambers are scanned by sizable grids, divers objective(s), and up to four fluorescence spectral bands. Acquisition positions are focused and digitized by a TV camera and archived on disk. The image data sets are evaluated by a large set of quantitative features. Automatic classifications for a number of organisms are developed and embedded in the program. Interactive programs for the design of training sets were additionally implemented. A long-term sampling period of 23 weeks from two ponds at two different locations each was performed to generate a reliable data set for training and testing purposes. These data were used to present this system's results for phytoplankton structure characterization. PLASA represents an automatic system, comprising all steps from specimen processing to algae identification up to species level and quantification.

Since the FDA approval of two CAR T cell therapies, Kymriah (Tisagenlecleucel, Novartis) and Yescarta (Axicabtagene ciloleucel, Kite) for CD19+ malignancies, there has been a significant interest for developing new CARs targeting other disease indications. As such, the ability to simultaneously monitor manufacturing criteria and functional characteristics of multiple CAR T cell products would likely be critical to accelerate candidate therapies into clinical trials. Here, we demonstrate that image-based cytometry using the Celigo Image Cytometer (Nexcelom Bioscience LLC) yields high-throughput measurements of CAR T cell proliferation and size throughout the cell-manufacturing process, and captures the kinetics of antigen-specific CAR T cell-mediated killing in vitro. The data acquired and analyzed by the Celigo Image Cytometer were congruent with results from conventional instruments when tested contemporaneously, such as the Multisizer 3 (Beckman Coulter) which monitored the properties of CAR T cell expansion, and a luciferase-based assay which measured the cytotoxic potential of CAR T cells. More importantly, the use of bright field and fluorescent imaging by the Celigo Image Cytometer provided kinetic measurements and rapid data acquisition and analysis which were direct advantages over the conventional technologies described herein. Together, the Celigo Image Cytometer enabled fast, reproducible, and high-throughput assessment of CAR T cell manufacturing criteria and effector function, which could greatly facilitate the identification and evaluation of novel CARs with therapeutic potential. Citation Format: Leo Li-Ying Chan, Colby Maldini, Andrea Love, Kevin Tosh, Kevin Gayout, Tim Smith, James Riley. A high-throughput in vitro characterization method of CAR T cells using image cytometry [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6288.