Multi-modal optical microscopy image analysis and matching techniques for spatially encoded bead-based microarrays

The methods used to characterize interfaces from diffraction contrast images are normally based on the contrast exhibited by the edge fringes in these images. In one approach to this problem, Amelinckx et al. have characterized the edge fringes due to a displacement at an interface as being α fringes and the edge fringes which are due to a difference in orientation across the boundary as being δ fringes. The general properties of so called symmetrical δ fringes can be summarized as follows: bright field images are asymmetrical (i.e. one edge fringe is dark while the other is bright) while dark field fringes are symmetrical. Of particular interest in the generation of δ fringes are situations in which the reflections, g and h in Fig. 1b (and therefore 0 and g - h in the same figure) are very close. Under these circumstances both bright and dark field images will be formed with two reflections (either 0 and g-h or g and h) passing through the objective aperture.

<p>Fig. S1. The binding specificity of consensus sequence CWCWR to MDA-MB-435 and MDA-MB-231 cells. Fig. S2. A, The schematic illustration of construction the focused library. B, Ten clones were randomly selected for sequencing to examine the diversity of the focused library. Fig. S3. Binding properties of peptides displayed on the bacteria surface. Fig. S4. The binding properties of TPP-1 and SPP-1 to PD-L1 were determined by the ELISA (n=3). Fig. S5. A, The PD-L1 expression of CHO-K1/PD-L1, MDA-MB-231 and MDA-MB-435 cell lines were determined by anti-PD-L1 antibody and flow cytometer. B, The binding properties of TPP-1 to MDA-MB-231 and MDA-MB-435 cell lines. Fig. S6. The bright field (A) and fluorescence microscopic image (B) of TPP-1 to CHO-K1. The bright field (C) and fluorescence microscopic image (D) of TPP-1 to CHO-K1/PD-L1. Fig. S7. A, The ED50 of PD-L1 to block the activated T cells (n=3). B, The TPP-1 peptide or PD-L1 plus TPP-1 peptide did not activate the T cells without the CD3 antibody. C, TPP-1 could effectively restore T cells proliferation which were inhibited by PD-L1 evaluated by T cell activation assay (n=3). Data were presented as mean {plus minus} SEM, *P<0.05, **P<0.01, unpaired t-test. Fig. S9. A, The bioluminescence signals of H460-Luc cells were determined by Cytation 3. B, The expression of PD-L1 on H460 was detected with flow cytometry. Fig. S8. A, The PD-L1 expression in matured DCs, the dotted line represented the isotype mAb and filled line represented the anti-PD-L1 mAb. B, The proliferation of T cells in MLR assay (n=3). Fig. S9. A, The bioluminescence signals of H460-Luc cells were determined by Cytation 3. B, The expression of PD-L1 on H460 was detected with flow cytometry. Fig. S10. A, In vivo fluorescence images of mice taken at different time points post s.c. injection of FITC labeled TPP-1. B, The statistical result of the relative fluorescence unit. Fig. S11. A, The location of CWCWR (yellow) in TPP-1 peptide. B, The possible binding location of TPP-1 (blue) peptide to PD-L1 (PDB ID of PD-L1: 3BIS). C, The crystal structures of PD-1 (light sea green) and PD-L1 (dark khaki) complex (PDB ID of the complex: 3BIK).</p>

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.

A new LEEM/PEEM apparatus has been designed for the study of surfaces and surface processes in Ultra High Vacuum (UHV). The design follows the general layout of the microscope operated succesfully by Prof. Bauer and coworkers, but differs significantly in a number of details. We designed a magnetic/electrostatic cathode lens with a resolution limit close to that of the uniform electrostatic accelerating field between sample and lens. The projection system consists of transfer lens, intermediate lens and projector lens. Total magnification is between 1,000x and 10,000x, with a field of view between 4 and 40 micrometer. The illuminating system features a continuously adjustable range of illumination, such that the loss of image intensity at high magnification can be balanced by a reduction of the illumiated area, without significant loss of electron beam intensity. Both bright field and dark field imaging are possible. The location of the diffraction aperture in the projection column allows for more flexible operation of the microscope in dark field mode. Care has been taken in the design of the microscope to integrate the stringent vacuum requirements with the geometric constraints of the electron optical components. A sample stage, featuring two orthogonal translations and two (eucentric) tilts, sample heating, as well as in situ sample exchange capability has been constructed. All motions are driven by remote control, UHV compatible piezoelectric actuators, eliminating the need for mechanical feedthroughs, and ensuring a short mechanical path between sample and cathode lens. In situ deposition, using e-beam evaporators and/or Knudsen cells will be possible. A sample preparation chamber contains standard surface cleaning and analytical equipment. We will report on the progress in the construction of this machine.

Scanning transmission microscopy with a 2 MeV alpha particle microbeam

The use of second-harmonic generation (SHG) microscopy in biomedical research is rapidly increasing. This is due in large part to the wide spread interest of using this imaging technique to examine the role of fibrillar collagen organization in diseases such as cancer. The co-examination of SHG images and traditional bright-field (BF) images of hematoxylin and eosin (H&E) stained tissue as a gold standard clinical validation is usually required. However, image registration of these two modalities has been mostly done by manually selecting corresponding landmarks which is labor intensive and error prone. We designed, implemented, and validated the first image intensity-based registration method capable of automatically aligning SHG images and BF images. In our algorithmic approach, a feature extractor is used to pre-process the BF image to block the content features not visible in SHG images and the output image is then aligned with the SHG image by maximizing the common image features. An alignment matrix maximizing the image mutual information is found by evolutionary optimization and the optimization is facilitated using a hierarchical multiresolution framework. The automatic registration results were compared to traditional manual registration to assess the performance of the algorithm. The proposed algorithm has been successfully used in several biomedical studies such as pancreatic and kidney cancer studies and shown great efficacy.

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.

The ultrastructure of ferritin macromolecules: I. Ultrahigh voltage electron microscopy (1–3 MeV)

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.

To acquire and characterize cellular-resolved in vivo fluorescence images of optogenetic probes expressed in rodent retinal ganglion cells, by adapting a low-cost and simple fundus system based on a topical endoscope. A custom endoscope-based fundus system was constructed (adapted from the design of Paques and colleagues). Bright field and fluorescence images were acquired from head-fixed transgenic mice expressing Channelrhodopsin2-eYFP, and Sprague Dawley rats virally transfected with the optogenetic probe GCaMP3. Images were compared to in vitro images of the same structures and were analyzed. The fundus system provides high-quality, high-resolution fluorescence images of the eye fundus that span the whole retina. The images allow resolving individual cells and axon bundles in the Channelrhodopsin2-eYFP mice and cellular-scale structures in the GCaMP3 expressing rats. The resolution in mouse eyes was estimated to be better than 20 μm (full width at half maximum) and is only marginally dependent on movement-related blurring. The fluorescence-endoscopy fundus system provides a powerful yet simple and widely accessible tool for obtaining cellular resolved fluorescent images of optogenetic and other fluorescent probes. The new system could prove to be a basic tool for non-invasive in vivo small animal retinal imaging in a wide array of translational vision applications, including the tracking of fluorescently tagged cells and the expression of gene-therapy and optogenetic vectors.

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.

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

<p>In vivo prototype dual bright-field and fluorescence bronchoscope imaging of rabbit lung tumor at 48 h post-injection, showing bright field image on the left and fluorescence image on the right.</p>

X-ray diffraction techniques, including both small- and wide-angle scattering, are valuable for scanning imaging when samples contain molecularly packed structures. These structures offer dark-field imaging contrasts, which are useful for visualizing various biological fibers such as collagens, axons, and muscles. Utilizing these contrasts, tomograms exceeding three dimensions can be reconstructed, exemplified by SAXS tensor tomography. Typically, dark-field imaging requires complementary bright-field imaging for a comprehensive understanding of the materials.Recently, we have developed a beamline, 12-ID-E, for simultaneous SAXS and ptychographic imaging. This instrument not only provides both dark and bright field imaging simultaneously but also captures multi-scale pictures of hierarchical structures. The beamline is equipped with two Pilatus detectors and a hexapod-based ptychography setup. In this presentation, we will detail the setup and showcase scientific examples involving the self-assembly of nanoobjects.