Single Molecular Imaging Research Articles

PH-responsive albumin-mimetic synthetic nanoprobes for magnetic resonance/fluorescence imaging of thyroid cancer.

The combination of magnetic resonance and fluorescence imaging in dual-modality imaging not only resolves the limitations of conventional single molecular imaging techniques in terms of specificity, sensitivity, and resolution but also expands the possibilities of molecular imaging techniques in diagnostics and therapeutic monitoring. Herein, a novel pH-responsive magnetic resonance/near-infrared fluorescence (MR/NIRF) nanoprobe (MnO2@BSA-Cy5.5) was successfully prepared by biomineralizing manganese dioxide (MnO2) with bovine serum albumin (BSA) while coupling fluorescent dye Cy5.5 for precise tumor detection and visualization. The synthesized MnO2@BSA-Cy5.5 nanoprobes were spherical particles of approximately 22.62 ± 3.31 nm in size, and their relaxation rates and T1 imaging signals were activated-enhanced in an acidic environment. Cytotoxicity assay and hematoxylin and eosin staining demonstrated that MnO2@BSA-Cy5.5 had low cytotoxicity and good biocompatibility. More importantly, active targeting via solid tumor albumin-binding protein receptor and enhanced permeability and retention effect, the probe can be specifically aggregated to the tumor site of the 8305C tumor model and exhibit excellent MR/NIRF imaging properties. Our results show that MnO2@BSA-Cy5.5 has high resolution and sensitivity in tumor imaging and is expected to be applied as an MR/NIRF contrast agent for accurate diagnosis of thyroid cancer.

Abstract 6087: Cytomics-on-chip and AI-driven predictive analysis platform for early detection of epithelial cancers

Abstract Cancer is the 2nd leading cause of death (over 605,000 people) in the US, at an expense of over $200B, with 1 in 3 people projected to have cancer during their lifetime per CDC. Despite the significant impact of early detection and screening on prognosis, only some cancers are diagnosed at an early stage. Carcinomas, comprising >80% of cancer incidence, allow ease in cytology sample access chairside, due to the lesions’ epithelial presentation. This presents a unique opportunity for early detection and screening in epithelial cancers. In low-resource healthcare settings, from clinical examination to the long, tedious and expensive diagnostic journey for cancers & pre-cancerous lesions, can lead to missed, delayed or over diagnosis scenarios. This affects treatment initiation and potentially outcome. To facilitate early intervention, there is compelling need to develop accurate and effective minimally invasive screening platforms. Recent advances in the -omics disciplines, microfluidics and AI tools are starting to reveal promising signatures of early disease detection, with potential to drastically improve screening and diagnostic systems. We are developing a novel application of the cytomics-on-chip platform, for enabling chairside, quantitative screening of suspicious epithelial lesions. The biosensor module involves 1. a single use, cytomics platform employing a cartridge with cellular array and high specificity biomarker reagents, that allows single cell molecular imaging to be completed in a portable analyzer. 2. a microfluidics module that allows cytomorphometric measurements to be completed. 3. The results generated are utilized to train machine learning algorithms to detect cyto-signatures and provide an intuitive result that may be utilized by health care practitioners in clinical-decision making. The first cell-based point-of-care oncology tool has recently been validated with high accuracy (99.3%), sensitivity and specificity, in a multi-site prospective clinical study. Here we demonstrate a pilot study towards development of a smart single cell cytomics-on-chip platform for prompt cytomorphometric and biomarker characterization towards diagnosis of urothelial, anal and cervical cancers, and dysplasia lesions, utilizing brush/pap and fresh urine samples. This has potential for continuous quantitative indexing, for disease categorization. As cancers become more pervasive, improved early detection/screening methods that are accurate, cost effective, easy to implement during routine clinical practice, and providing minimal discomfort to the patient, are urgently needed, improving confidence in clinicians’ decisions. Citation Format: Kritika Srinivasan Rajsri, Michael P. McRae, Nicolaos J. Christodoulides, Khaled Algashaamy, Monica T. Garcia-Buitrago, Fei Chen, Fang-Ming Deng, Jennifer S. Smith, John T. McDevitt. Cytomics-on-chip and AI-driven predictive analysis platform for early detection of epithelial cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6087.

Folic acid functionalized gadolinium-doped carbon dots as fluorescence / magnetic resonance imaging contrast agent for targeted imaging of liver cancer

Gadolinium-doped carbon dots (Gd-CDs), as a new class of nanomaterial, has a wide application prospect in targeted imaging and monitoring diagnosis and treatment of liver cancer because of their good fluorescence (FL)-magnetic resonance (MR) imaging properties. First, Gd-CDs were synthesized by hydrothermal method with gadodiamide as gadolinium source, citric acid as carbon source and silane coupling agent (KH-792) as coupling agent with FL quantum yield (QY) of 48.2%. Then, folic acid (FA), which is highly expressed in liver cancer, was used as a targeting component to modify Gd-CDs to obtain targeted imaging agent (Gd-CDs-FA). The results showed that Gd-CDs and Gd-CDs-FA have low cytotoxicity and good biocompatibility, and the targeting and selectivity of Gd-CDs-FA to HepG2 cells could be observed under confocal laser scanning microscope (CLSM). The T1 longitudinal relaxation rates (r1) of Gd-CDs and Gd-CDs-FA are 15.92 mM−1s−1 and 13.56 mM−1s−1, respectively. They showed good MR imaging ability in vitro and in vivo, and MR imaging in nude mice further proved the targeting imaging performance of Gd-CDs-FA. Therefore, Gd-CDs-FA with higher QY showed good FL-MR targeting imaging ability of liver cancer, which broke through the limitations of single molecular imaging probe in sensitivity and soft tissue resolution. This study provides a new idea for the application of Gd-CDs in FL and MR targeting imaging of liver cancer.

TMIC-83. THE MOLECULAR AND SPATIAL ARCHITECTURE OF AN IMMUNE-RICH GLIOMA SUBTYPE

Abstract Diffuse glioma exhibit an immunosuppressive microenvironment, yet glioma subsets driven by RAF-alterations exhibit an increased immune signature. In pleomorphic xanthoastrocytoma (PXA), we identified CXCL14 as an important factor promoting a cytotoxic T cell response. The molecular and spatial relationships within this tumor type, however, are unknown. To address this, we use single nucleus RNA sequencing (snRNAseq), spatial transcriptomics, and spatial molecular imaging to construct a high-resolution molecular landscape of PXA. SnRNAseq was performed on 36,781 cells from 10 tumors, including initial and recurrent pairs, spatial transcriptomes were performed on a total of 91 FOVs comprised of 17 PXAs, and spatial molecular imaging (SMI) of 1,000 RNA probes at subcellular resolution was performed on 48,518 cells from 6 tumors, including two PXA, two pilocytic astrocytoma, and two ganglioglioma. For snRNAseq, neoplastic cells were separated from non-malignant cells based on analysis of inferred copy number alterations. CellChat was used to infer cell-cell communication. For spatial transcriptomics, data from 200-300µm FOVs were normalized and divided into CD3-high versus CD3-low groups. For SMI, cells with fewer than 20 transcripts were eliminated, data were normalized using simple total counts, cells were segmented using the Nanostring pipeline, and semi-supervised clustering was performed. Dual CXCL14 RNAscope and Iba1/CD3 immunostaining confirmed spatial relationships within the tumor. In PXA with high-CD3, differential gene expression analysis demonstrated broad immune-enrichment and identified upregulation of several ligand-receptor pairs. Integrating these data with snRNAseq and cell-cell communication analysis inferred paracrine interactions between tumor cells and T cells. One such factor, CXCL14, was expressed widely by tumor cells but spatial analysis revealed cohesive clusters of CXCL14-high expressing tumor cells. In anaplastic (CNS WHO grade 3) tumors, however, both CXCL14 expression and microglia decreased. These high-resolution molecular analyses of PXA identify dynamic tumor-immune crosstalk regulating the immune microenvironment.

Abstract 785: A smart cytopathology risk-assessment platform for oral potentially malignant disorders and oral squamous cell carcinoma at the point-of-care

Abstract Background & Objective: Oral squamous cell carcinoma (OSCC) affects over 400,000 individuals globally, every year. When diagnosed early, the 5-year survival rate for OSCC is 64%, but two-third of these lesions are diagnosed in later stages, leading to lower survival rates (<40%). Clinical diagnosis of these lesions is complicated by benign and Oral-Potentially-Malignant-Disorders (OPMD) like oral lichenoid conditions (OLC), mimicking OSCC in clinical presentation. Studies in literature demonstrate high rates of incorrect chair-side diagnosis for OSCC and OPMD, making their early and accurate detection challenging. This clinical gap prompts a strong need for clinical chair-side point-of-care (POC) platforms, to aid accurate screening of these lesions and prevent diagnostic delays. Methods: In this work we present a ‘smart diagnostic approach’ that combines three key capabilities into an integrated sensing modality as follows: i) powerful microfluidic engine that allows for cytology measurements to be completed outside of a sophisticated lab infrastructure, ii) cytomics platform that allows for single cell molecular imaging to be completed in a portable analyzer, iii) AI-linked diagnostic models for early disease detection using cyto-signatures. This platform has been clinically validated across a multi-site prospective clinical study. Results: Multiple parameters including cellular phenotypes, nuclear parameters, biomarker expressions were indexed. Further, combining these features allowed discrimination and stratification of these lesions with high accuracy (99.3%) and significance. Further examination using logistic regression and receiver operating characteristic curve analyses yielded significant lesion identifiers and AUC values towards positive discrimination of OLC and OSCC (0.824 and 0.95, respectively vs benign lesions), with high sensitivity and specificity. Conclusion: This rapid (<30 minutes) cytopathology POC solution has the potential to impact OSCC and OPMD screening accurately, characterizing subtle cellular changes to aid long-term monitoring of these lesions. Additionally, this platform has the capability to uncover new parameters that can further aid these assessments and improve confidence in clinicians’ decisions. Citation Format: Kritika Srinivasan Rajsri, Michael P. McRae, Glennon W. Simmons, Alexander Ross Kerr, Nadarajah Vigneswaran, Spencer W. Redding, Malvin Janal, Stella Kang, Leena Paloma, Nicolaos J. Christodoulides, John T. McDevitt. A smart cytopathology risk-assessment platform for oral potentially malignant disorders and oral squamous cell carcinoma at the point-of-care [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 785.

Direct Observation of Archaellar Motor Rotation by Single-Molecular Imaging Techniques.

Single-molecular techniques have characterized dynamics of molecular motors such as flagellum in bacteria and myosin, kinesin, and dynein in eukaryotes. We can apply these techniques to a motility machine of archaea, namely, the archaellum, composed of a thin helical filament and a rotary motor. Although the size of the motor hinders the characterization of its motor function under a conventional optical microscope, fluorescence-labeling techniques allow us to visualize the architecture and function of the archaellar filaments in real time. Furthermore, a tiny polystyrene bead attached to the filament enables the visualization of motor rotation through the bead rotation and quantification of biophysical properties such as speed and torque produced by the rotary motor imbedded in the cell membrane. In this chapter, I describe the details of the above biophysical method based on an optical microscope.

Abstract 3817: A single-cell, spatial multiomics atlas and cellular interactome of all major skin cancer types

Abstract Skin cancer is by far the most common cancer, encompassing squamous cell carcinoma (SCC), basal cell carcinoma (BCC), and melanoma. The diversity of cell types and tissue organisation in skin cancer remains poorly understood yet is required to improve diagnosis and treatment. In this work, we integrated six imaging and sequencing technologies to build the first spatial single cell reference for all three major skin cancer types and create a comprehensive skin cancer interactome. Using single-cell RNA-Seq (RNA) of >100,000 cells from 11 paired patient biopsies, we identified 28 SCC cell types, including 10 immune cell types, and found core suites of 39 cancer genes and 222 healthy genes shared across ≥80% patient samples. Using independent Nanostring Digital Spatial Profiling (RNA, protein), we validated most immune cell types and gene markers at protein and RNA levels. The enrichment of an immune signalling signature in SCC was further revealed by spatial Nanostring Single Molecular Imaging - SMI (RNA). Strikingly, we found the high consistency in mapping cell types in scRNAseq data and the independent SMI data, for example, the distribution of the three keratinocyte layers (basal, cycling and differentiated). This observation suggested the power of combining scRNAseq data with spatial SMI data. Furthermore, we implemented three approaches to validate the spatial distribution and cell type co-localisation by both Visium Spatial Transcriptomics (RNA), SMI (RNA) and Opal Multiplex Polaris (protein). Finally, cell-cell interactions were inferred at the global level using scRNAseq data (no spatial information) and Visium data (with spatial dimension), which were then validated at high throughput (517 ligands/receptors) and single-cell resolution using SMI. These in situ interaction maps were built across all three cancer types to create a comprehensive spatial interaction atlas of skin cancer. We also used targeted approaches with Polaris (protein) and RNAScope (RNA) to confirm and visualise clinically-important ligand-receptor pairs, including checkpoint inhibitor drug targets PD-1 and PD-L1. By integrating six distinct yet complementary spatial and single cell technologies, this study highlights the power of a spatial multi-omics approach for understanding cell types and their activities in cancer tissues. Citation Format: Laura Grice, Guiyan Ni, Xinnan Jin, Minh Tran, Emily Killingbeck, Mark Gregory, Onkar Mulay, Siok-Min Teoh, Arutha Kulasinghe, Michael Leon, Sarah Murphy, Sarah Warren, Youngmi Kim, Quan Nguyen. A single-cell, spatial multiomics atlas and cellular interactome of all major skin cancer types [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3817.

Parallel optically detected magnetic resonance spectrometer for dozens of single nitrogen-vacancy centers using laser-spot lattice.

We develop a parallel optically detected magnetic resonance (PODMR) spectrometer to address, manipulate, and read out an array of single nitrogen-vacancy (NV) centers in diamond in parallel. In this spectrometer, we use an array of micro-lenses to generate a 20 × 20 laser-spot lattice (LSL) on the objective focal plane and then align the LSL with an array of single NV centers. The quantum states of NV centers are manipulated by a uniform microwave field from a Ω-shape coplanar coil. As an experimental demonstration, we observe 80 NV centers in the field of view. Among them, magnetic resonance (MR) spectra and Rabi oscillations of 18 NV centers along the external magnetic field are measured in parallel. These results can be directly used to realize parallel quantum sensing and multiple times speedup compared with the confocal technique. Regarding the nanoscale MR technique, PODMR will be crucial for a high throughput single molecular MR spectrum and imaging.

Photoacoustic Research Development in Industrial Era 4.0

Photoacoustic (PA) is the phenomenon of changing optical power into acoustics (sound). Photoacoustic research continues to grow and have broader field of application, especially after the development of laser technology and the development of laser diodes. In the industrial era 4.0, PA research is increasingly developing, especially in the PA tomography topics or PA imaging which is widely applied in medicine, as well as in single molecular imaging research and the 3D imaging of body tissue system.

Electrodynamic characteristics of λ-DNA molecule translocating through the microfluidic channel port studied with single molecular fluorescence imaging technology

Manipulating a single DNA molecule and effectively introducing it into and exporting micro-nano-fluidic channels are prerequisites for the functional DNA biochips. And it is the key to the precise separation and screening of different DNA molecules by the micro-/nanochannel system that accurately understanding the movement characteristics and dynamic mechanism of DNA molecules moving near the channel port. In this paper, the electrodynamic characteristics of λ-DNA molecule entering into/leaving off a 50 μm channel port driven by the electric field force are systematically investigated and analyzed by the single molecule fluorescence microscopy. The experimental results indicated that there were the maximum (<i>E</i><sub>max</sub>) and minimum (<i>E</i><sub>min</sub>) thresholds of the applied electric field intensity, and only when the field intensity <i>E</i> meets <i>E</i><sub>min</sub> ≤ <i>E</i> ≤ <i>E</i><sub>max</sub>, the single λ-DNA molecule could successfully enter into the <i>trans</i> port and exit out of the <i>cis</i> port; when the electric field intensity was less than the minimum threshold, <i>E</i> ≤ <i>E</i><sub>min</sub>, λ-DNA molecules could not enter the <i>trans</i> port; when the electric field intensity was greater than the maximum threshold, <i>E</i><sub>max</sub> ≤ <i>E</i>, λ-DNA molecules could move into the microchannel through the <i>trans</i> port, but not exit out of the <i>cis</i> port. When λ-DNA molecule migrated toward the <i>cis</i> port along the channel, the movement state was changed, some new phenomena were observed, e.g. the translocation direction was reversed, reciprocated, or even rotated; moreover, the DNA molecules were easy to adhere to the channel wall. In addition, when the electric field intensity enhanced, the distance between the position where DNA molecular direction reversing and the <i>cis</i> port was increased. Based on the microfluidic electrodynamics, the physical mechanism of the velocities and translocation states of single λ-DNA molecule passing microchannel port was preliminarily analyzed. The results of this study have certain practical guiding significance for the development of gene chip laboratory and DNA molecular sensors based on the micro/nanochannel fluidic system.

Brain glucose uptake during transcranial direct current stimulation measured with functional [18F]FDG-PET

Previous evidence indicates that transcranial direct stimulation (tDCS) is a neuromodulatory brain stimulation technique. Easy applicability, low side-effects and negligible costs facilitated its wide–spread application in efforts to modulate brain function, however neuronal mechanisms of tDCS are insufficiently understood. Hence, we investigated the immediate impact of tDCS on the brain’s glucose consumption in a continuous infusion protocol with the radioligand 2-[18F]fluoro-2-deoxy-D-glucose ([18F]FDG) and positron emission tomography (PET). This novel functional PET (fPET) method is capable to reliably detect area-specific and dynamic absolute glucose demand related to neuronal activity in a single molecular imaging session. Fifteen healthy subjects underwent tDCS at 0.5, 1 and 2 mA (mA) at the bilateral dorsolateral prefrontal cortex (dlPFC, cathodal right) for 10 min during functional [18F]FDG-PET lasting 70 min. Active stimulation compared to sham did not yield significant changes in glucose consumption at any tested stimulation intensity in this paradigm. Exploratory investigation of aftereffects provided hints for increased glucose consumption with a delay of 5 min at 1 mA in the right posterior temporal cortex. This is the first study investigating changes of glucose consumption in the brain during tDCS. The lack of immediately increased glucose consumption indicates that energy demanding processes in the brain such as glutamatergic signaling might not be immediately increased by tDCS. However, our results implicate the need of fPET investigations for medium-term and long-term effects.

Insights into the origin of the high energy-conversion efficiency of F1-ATPase

Our understanding of the rotary-coupling mechanism of F1-ATPase has been greatly enhanced in the last decade by advances in X-ray crystallography, single-molecular imaging, and theoretical models. Recently, Volkán-Kacsó and Marcus [S. Volkán-Kacsó, R. A. Marcus, Proc. Natl. Acad. Sci. U.S.A. 112, 14230 (2015)] presented an insightful thermodynamic model based on the Marcus reaction theory coupled with an elastic structural deformation term to explain the observed γ-rotation angle dependence of the adenosine triphosphate (ATP)/adenosine diphosphate (ADP) exchange rates of F1-ATPase. Although the model is successful in correlating single-molecule data, it is not in agreement with the available theoretical results. We describe a revision of the model, which leads to consistency with the simulation results and other experimental data on the F1-ATPase rotor compliance. Although the free energy liberated on ATP hydrolysis by F1-ATPase is rapidly dissipated as heat and so cannot contribute directly to the rotation, we show how, nevertheless, F1-ATPase functions near the maximum possible efficiency. This surprising result is a consequence of the differential binding of ATP and its hydrolysis products ADP and Pi along a well-defined pathway.

Genetically encoded orientation probes for F-actin for fluorescence polarization microscopy.

Fluorescence polarization microscopy, which can visualize both position and orientation of fluorescent molecules, is useful for analyzing architectural dynamics of proteins in vivo, especially that of cytoskeletal proteins such as actin. Fluorescent phalloidin conjugates and SiR-actin can be used as F-actin orientation probes for fluorescence polarization microscopy, but a lack of appropriate methods for their introduction to living specimens especially to tissues, embryos, and whole animals hampers their applications to image the orientation of F-actin. To solve this problem, we have developed genetically encoded F-actin orientation probes for fluorescence polarization microscopy. We rigidly connected circular permutated green fluorescent protein (GFP) to the N-terminal α-helix of actin-binding protein Lifeact or utrophin calponin homology domain (UtrCH), and normal mEGFP to the C-terminal α-helix of UtrCH. After evaluation of ensemble and single particle fluorescence polarization with the instantaneous FluoPolScope, one of the constructs turned out to be suitable for practical usage in live cell imaging. Our new, genetically encoded F-actin orientation probe, which has a similar property of an F-actin probe to conventional GFP-UtrCH, is expected to report the 3D architecture of the actin cytoskeleton with fluorescence polarization microscopy, paving the way for both the single molecular orientation imaging in cultured cells and the sub-optical resolution architectural analysis of F-actin networks analysis of F-actin in various living systems.

Light-driven Molecular Motors on Surfaces for Single Molecular Imaging

Light-driven Molecular Motors on Surfaces for Single Molecular Imaging

Light-driven Molecular Motors on Surfaces for Single Molecular Imaging.

The design and synthesis of a synthetic system that aims for the direct visualization of a synthetic rotary motor at the single molecule level on surfaces are demonstrated. This work requires careful design, considerable synthetic effort, and proper analysis. The rotary motion of the molecular motor in solution is shown by 1H NMR and UV-vis absorption spectroscopy techniques. In addition, the method to graft the motor onto an amine-coated quartz is described. This method helps to gain more insight into molecular machines.

Design, Synthesis, and Isomerization Studies of Light-Driven Molecular Motors for Single Molecular Imaging.

The design of a multicomponent system that aims at the direct visualization of a synthetic rotary motor at the single molecule level on surfaces is presented. The synthesis of two functional motors enabling photochemical rotation and fluorescent detection is described. The light-driven molecular motor is found to operate in the presence of a fluorescent tag if a rigid long rod (32 Å) is installed between both photoactive moieties. The photochemical isomerization and subsequent thermal helix inversion steps are confirmed by 1H NMR and UV–vis absorption spectroscopies. In addition, the tetra-acid functioned motor can be successfully grafted onto amine-coated quartz and it is shown that the light responsive rotary motion on surfaces is preserved.

Lysyl oxidase activates cancer stromal cells and promotes gastric cancer progression: quantum dot-based identification of biomarkers in cancer stromal cells.

PurposeSemiconductor quantum dots (QDs) are a promising alternative to organic fluorescent dyes for multiplexed molecular imaging of cancer stroma, which have great advantages in holistically analyzing the complex interactions among cancer stromal components in situ.Patients and methodsA QD probe-based multiplexed spectral molecular imaging method was established for simultaneous imaging. Three tissue microarrays (TMAs) including 184 gastric cancer (GC) tissues were constructed for the study. Multispectral analyses were performed for quantifying stromal biomarkers, such as lysyl oxidase (LOX). The stromal status including infiltrating of immune cells (high density of macrophages), angiogenesis (high density of microvessel density [MVD], low neovessel maturation) and extracellular matrix (ECM) remodeling (low density of type IV collagen, intense expression of matrix metalloproteinase 9 [MMP-9]) was evaluated.ResultsThis study compared the imaging features of the QD probe-based single molecular imaging method, immunohistochemistry, and organic dye-based immunofluorescent methods, and showed the advantages of the QD probe-based multiple molecular imaging method for simultaneously visualizing complex components of cancer stroma. The risk of macrophages in high density, high MVD, low neomicrovessel maturation, MMP-9 expression and low type IV collagen was significantly increased for the expression of LOX. With the advantages of the established QD probe-based multiplexed molecular imaging method, the spatial relationship between LOX and stromal essential events could be simultaneously evaluated histologically. Stromal activation was defined and then evaluated. Survival analysis showed that the stromal activation was correlated with overall survival and disease-free survival (P<0.001 for all). The expression of LOX was significantly increased in the intense activation subgroup (P<0.001).ConclusionQuantifying assessment of the stroma indicates that the LOX may be a stromal marker for GC and stromal activation, which is not only responsible for the ECM remodeling morphologically, but also for the formation of invasive properties and recurrence. These results support the possibility to integrate morphological and molecular biomarker information for cancer research by the biomedical application of QDs.

Heterodimerization of two pore domain K+ channel TASK1 and TALK2 in living heterologous expression systems.

Two-pore-domain K+ (K2P) channels sense a wide variety of stimuli such as mechanical stress, inhalational anesthetics, and changes in extracellular pH or temperature. The K2P channel activity forms a background K+ current and, thereby, contributes to resting membrane potentials. Six subfamilies including fifteen subtypes of K2P channels have been identified. Each K2P channel molecule with two pores consists of a homodimer of each subtype. In addition, a few heterodimers mainly within the same subfamilies have been found recently. In the present study, the possibility of heterodimerization between TASK1 (TWIK-Related Acid-Sensitive K+ channel) and TALK2 (TWIK-Related Alkaline pH-Activated K+ channel) was examined. These channels belong to separate subfamilies and show extremely different channel properties. Surprisingly, single molecular imaging analyses in this study using a total internal reflection microscope suggested the heterodimerization of TASK1 and TALK2 in a pancreatic cell line, QGP-1. This heterodimer was also detected using a bimolecular fluorescence complementation assay in a HEK293 heterologous expression system. Fluorescence resonance energy transfer analyses showed that the affinity between TASK1 and TALK2 appeared to be close to those of homodimers. Whole-cell patch-clamp recordings revealed that TASK1 currents in HEK293 cells were significantly attenuated by co-expression of a dominant-negative form of TALK2 in comparison with that of wild-type TALK2. The sensitivities of TASK1-TALK2 tandem constructs to extracellular pH and halothane were characterized as a unique hybrid of TASK1 and TALK2. These results suggested that heterodimerization of TASK1 and TALK2 provides cells with the ability to make multiple responses to a variety of physiological and pharmacological stimuli.

Development of the fluorescent imaging method of single-molecule sRNA SgrS &amp;lt;italic&amp;gt;in situ&amp;lt;/italic&amp;gt; in &amp;lt;italic&amp;gt;Escherichia coli&amp;lt;/italic&amp;gt;

Single-cell gene expression studies could not only provide the real-time information on intracellular amount and subcellular localization of RNA, but also could reveal the regulatory mechanism of bacterial variation, which cannot be obtained based on traditional view of bacterial populations. Owing to the application of green fluorescent protein (GFP) reporters, most of studies have mainly focused on proteins. Recently, the quantitative and localization of bacterial RNAs have been also implemented at single transcript level. All of the non-coding RNAs who function as crucial regulators of gene expression are called regulatory small RNAs (sRNA) in bacteria. Single molecular imaging techniques for labeling bacterial sRNA can be used to detect the activity and abundance of sRNA, which will help us to understand its regulatory mechanism and biological function. SgrS, a conservative sRNA in γ-proteobacteria, is highly expressed under glucose-phosphate stress and involve in the sugar metabolism in bacteria. We chose SgrS as a model to establish the single-molecule fluorescence in situ hybridization (smFISH) method of RNA detection at the single-molecule level in Escherichia coli . It will be helpful for further studies on sRNA subcellular localization and gene regulation. We used kanamycin resistance box replace sgrS to successful construct sgrS deletion mutant Δ sgrS and subsequently cured the helper plasmid pKD46. The SgrS overexpression strain Δ sgrS -pBAD-SgrS was generated by introducing SgrS-overexpressed plasmid into the Δ sgrS strains. E. coli MG1655, Δ sgrS and Δ sgrS -pBAD-SgrS, all E. coli K-12 derivatives, were used in this study. The smFISH method on SgrS in E. coli was established. The optimal conditions are briefly stated as follows. The cells were fixed in 3.7% formaldehyde at room temperature for 30 minutes. Cells were permeabilized at 4°C for overnight. Washed cells with 40% wash solution was resuspended in 40% hybridization buffer. The cells were centrifuged and resuspended in the hybridization buffer with 1 μM sRNAs probes that denatured at 98°C for 10 minutes. Hybridization reactions were incubated in the hybridization oven at 40°C for 3 hours. The cells were washed five times with 40% wash solution. During wash, cells were resuspended and incubated at 40°C for 30 minutes. Cells were pelleted by 600× g centrifugation for 3 minutes, and the supernatant was removed. Cells were resuspended with 100 μL of freshly prepared 5 μg mL− 1 Alexa-555 WGA in 1×PBS, and incubated at room temperature for 10 minutes. Cells were pelleted by 600× g centrifugation for 3 minutes, and the supernatant was removed. The cells were resuspended in 5 μL of 2×SSC. Then 1 μL of sample and 1 μL of SlowFade Diamond Antifade Mountant were pipetted to poly-L-lysine treated borosilicate chambered coverslip. A coverslip was placed on the sample. Super-resolution imaging was performed on Nikon inverted microscope with a 100× oil immersion objective. SgrS was indicated as green fluorescence granular with diffuse distribution in cytoplasm, while cellular wall images exhibited red fluorescence upon being stained with Alexa-555 WGA. The combination of smFISH method and super-resolution microscopy techniques provides the potential clues and technical reference for further studies on revealing sRNA localization and sRNA-mediated regulatory mechanism.

Coherent soft X-ray diffraction imaging of coliphage PR772 at the Linac coherent light source

Single-particle diffraction from X-ray Free Electron Lasers offers the potential for molecular structure determination without the need for crystallization. In an effort to further develop the technique, we present a dataset of coherent soft X-ray diffraction images of Coliphage PR772 virus, collected at the Atomic Molecular Optics (AMO) beamline with pnCCD detectors in the LAMP instrument at the Linac Coherent Light Source. The diameter of PR772 ranges from 65–70 nm, which is considerably smaller than the previously reported ~600 nm diameter Mimivirus. This reflects continued progress in XFEL-based single-particle imaging towards the single molecular imaging regime. The data set contains significantly more single particle hits than collected in previous experiments, enabling the development of improved statistical analysis, reconstruction algorithms, and quantitative metrics to determine resolution and self-consistency.