Field Of Cellular Imaging Research Articles

Iterative Immunostaining and NEDD Denoising for Improved Signal-To-Noise Ratio in ExM-LSCM.

Expansion microscopy (ExM) has significantly reformed the field of super-resolution imaging, emerging as a powerful tool for visualizing complex cellular structures with nanoscale precision. Despite its capabilities, the epitope accessibility, labeling density, and precision of individual molecule detection pose challenges. We recently developed an iterative indirect immunofluorescence (IT-IF) method to improve the epitope labeling density, improving the signal and total intensity. In our protocol, we iteratively apply immunostaining steps before the expansion and exploit signal processing through noise estimation, denoising, and deblurring (NEDD) to aid in quantitative image analyses. Herein, we describe the steps of the iterative staining procedure and provide instructions on how to perform NEDD-based signal processing. Overall, IT-IF in ExM-laser scanning confocal microscopy (LSCM) represents a significant advancement in the field of cellular imaging, offering researchers a versatile tool for unraveling the structural complexity of biological systems at the molecular level with an increased signal-to-noise ratio and fluorescence intensity. Key features • Builds upon the method developed by Mäntylä et al. [1] and introduces the IT-IF method and signal-processing platform for several nanoscopy imaging applications. • Retains signal-to-noise ratio and significantly enhances the fluorescence intensity of ExM-LSCM data. • Automatic estimation of noise, signal reconstruction, denoising, and deblurring for increased reliability in image quantifications. • Requires at least seven days to complete.

Effect of Alkyl Chain Length on Room-Temperature Phosphorescent Probes for Mitochondria-Targeted Imaging.

Room temperature phosphorescent (RTP) probes have significant advantages in the field of cellular imaging, as their long lifetimes can prevent interference from the spontaneous fluorescence of organisms. Persulfurated arenes are a typical RTP molecular parent nucleus. However, most of the applied research on them is concentrated in anti-counterfeiting, and relatively few are applied in bioimaging. The molecular structure and structure-property relationship of them applied in bioimaging are still in the exploration stage. In this work, we have designed and synthesized a series of RTP probes with long alkyl chains, all of which can be targeted to mitochondria with good water solubility for mitochondria-targeted imaging. Further, we investigated the effect of alkyl chains on the luminescence properties of these probes, and found that the moderate length of alkyl chains can realize the enhancement of phosphorescence intensity. We believe this finding is of guiding significance for the design of molecular structures in the field of RTP probes.

Electrochemiluminescence-Based Single-Particle Tracking of the Biomolecules Moving along Intercellular Membrane Nanotubes between Live Cells.

Electrochemiluminescence (ECL) imaging, a rapidly evolving technology, has attracted significant attention in the field of cellular imaging. However, its primary limitation lies in its inability to analyze the motion behaviors of individual particles in live cellular environments. In this study, we leveraged the exceptional ECL properties of quantum dots (QDs) and the excellent electrochemical properties of carbon dots (CDs) to develop a high-brightness ECL nanoprobe (CDs-QDs) for real-time ECL imaging between living cells. This nanoprobe has excellent signal-to-noise ratio imaging capabilities for the single-particle tracking (SPT) of biomolecules. Our finding elucidated the enhanced ECL mechanism of CDs-QDs in the presence of reactive oxygen species through photoluminescence, electrochemistry, and ECL techniques. We further tracked the movement of single particles on membrane nanotubes between live cells and confirmed that the ECL-based SPT technique using CD-QD nanoparticles is an effective approach for monitoring the transport behaviors of biomolecules on membrane nanotubes between live cells. This opens a promising avenue for the advancement of ECL-based single-particle detection and the dynamic quantitative imaging of biomolecules.

Room-temperature synthesized carbon quantum dots and potential applications to cell imaging

Carbon quantum dots (CQDs) with excellent properties have attracted attention owing to wide applications in many fields. In this paper, we report a method to synthesize CQDs at room temperature without any external energy supply and energy-catalyzing reagents. The characterization results indicate that the CQDs have good dispersion and water solubility, the averaged dimension is around 5.37 nm, consisting mainly of C, O, N, and S elements, the fluorescence quantum yield was 8.72%. In addition, the experimental results show that CQDs have excellent optical stability and good biocompatibility, which can be used in the field of cellular imaging

31P ParaCEST: 31P MRI-CEST Imaging Based on the Formation of a Ternary Adduct between Inorganic Phosphate and Eu-DO3A.

Development of the field of magnetic resonance imaging (MRI) chemical exchange saturation transfer (CEST) contrast agents is hampered by the limited sensitivity of the technique. In water, the high proton concentration allows for an enormous amplification of the exchanging proton pool. However, the 1H CEST in water implies that the number of nuclear spins of the CEST-generating species has to be in the millimolar range. The use of nuclei other than a proton allows exploitation of signals different from that of water, thus lowering the concentration of the exchanging pool as the source of the CEST effect. In this work, we report on the detection of a 31P signal from endogenous inorganic phosphate (Pifree) as the source of CEST contrast by promoting its exchange with the Pi bound to the exogenous complex 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (Pibound). The herein-reported results demonstrate that this approach can improve the detectability threshold by 3 orders of magnitude with respect to the conventional 1H CEST detection (considered per single proton). This achievement reflects the decrease of the bulk concentration of the detected signal from 111.2 M (water) to 10 mM (Pi). This method paves the way to a number of biological studies and clinically translatable applications, herein addressed with a proof-of-concept in the field of cellular imaging.

Facile and Green Synthesis of Highly Fluorescent Carbon Quantum Dots from Water Hyacinth for the Detection of Ferric Iron and Cellular Imaging.

Natural biomass is used for facile synthesis of carbon quantum dots (CQDs) with high fluorescence, owing to its abundance, low cost, and eco-friendliness. In this study, a bottom-up hydrothermal method was used to prepare CQDs from water hyacinth (wh) at a constant temperature of 180 °C for 12 h. The synthesized wh-CQDs had uniform size, amorphous graphite structure, high water solubility (containing multiple hydroxyl and carboxyl groups on the surface), excitation light-dependent characteristics, and high photostability. The results showed that the aqueous solution of CQDs could detect Fe3+ rapidly, sensitively, and highly selectively with a detection limit of 0.084 μM in the linear range of 0–330 μM, which is much lower than the detection limit of 0.77 μM specified by the World Health Organization. More importantly, because the wh-CQDs were synthesized without any additives, they exhibited low toxicity to Klebsiella sp. cells even at high concentrations. Moreover, wh-CQDs emitted bright blue fluorescence in Klebsiella sp. cells, indicating its strong penetrating ability. Correspondingly, the fluorescent cell sorting results also revealed that the proportion of cell internalization reached 41.78%. In this study, wh-CQDs derived from natural biomass were used as high-performance fluorescent probes for Fe3+ detection and Klebsiella sp. imaging. This study is expected to have great significance for the application of biomass carbon spots in the field of cellular imaging and biology.

Nitrogen-doped carbon dots for wash-free imaging of nucleolus orientation.

Carbon dots (CDs) are a rising star in the field of cellular imaging, especially cytoplasmic imaging, attributing to the super-stable optical performance and ultra-low biological toxicity. Nucleolus can accurately reflect the expression state of a cell and is strongly linked to the occurrence and development of many diseases, so exploring bran-new CDs for nucleolus-orientation imaging with no-wash technology has important theoretical value and practical significance. Herein, nitrogen-doped carbon dots (N-CDs) with green fluorescence (the relative fluorescence quantum yield of 24.4%) was fabricated by the hydrothermal treatment of m-phenylenediamine and p-aminobenzoic acid. The N-CDs possess small size, bright green fluorescence, abundant surface functional groups, excellent fluorescence stability and good biocompatibility, facilitating that the N-CDs are an excellent imaging reagent for cellular imaging. N-CDs can particularly bind to RNA in nucleoli to enhance their fluorescence, which ensures that the N-CDs can be used in nucleolus-orientation imaging with high specificity and wash-free technique. This study demonstrates that the N-CDs have a significant feasibility to be used for nucleolus-orientation imaging in biomedical analysis and clinical diagnostic applications.

Recent Progress in Synthesis and Applications of Tunable Materials and Nanomaterials Based on Organic Salts

AbstractGUMBOS (group of uniform materials based on organic salts) are a new class of solid organic ionic materials that shared similar properties as that of ionic liquids, but have a wider range of melting points from 25 °C to 250 °C. A large number of combinations have been made to design and synthesize multifunctional GUMBOS with tuned properties that are encouraged to explore these organic materials for various applications. Further, nanomaterials can be fabricated from GUMBOS through various reliable synthetic approaches. These nanomaterials have known as ‚nanoGUMBOS‘ that possessed advantageous properties at the nanoscale and can be used for target applications. Besides, the size, shape, and uniformity of nanomaterials can be tuned by varying different experimental parameters, and synthetic routes.This review is divided into two parts and presents all the published data regarding GUMBOS and nanoGUMBOS. The first part enclosed the synthesis of GUMBOS as well as their potential applications in the field of analytical, optoelectronics, biology, and sensor technology. In the second part, the synthesis of nanoGUMBOS using different methods and their applications in the field of cellular imaging, optoelectronics, catalysis, enantiorecognition, and sensor technology will be highlighted.

Get in Touch With Dendritic Epithelial T Cells!

Innate and adaptive immune systems continuously interchange information and orchestrate their immune responses to protect the host. γδT cells play crucial roles, as they incorporate both innate and adaptive immune characteristics. Dendritic epidermal T cells (DETC) are specialized γδT cells, which are uniquely positioned to rapidly respond to skin wounds and infections. Their elongated dendrite morphology allows them to be in continuous contact with multiple neighboring keratinocytes and Langerhans cells. Cellular interactions are fundamental to the formation, activation and maintenance of immune cell functions during steady state and pathology. Recent technological advances, especially in the field of cellular imaging, have contributed greatly to the characterization of complex cellular interactions in a spatiotemporally resolved manner. In this review, we will highlight the often-underappreciated function of DETC and other γδT cells during steady state and an ongoing immune response. More specifically, we discuss how DETC-precursors are shaped in the fetal thymus during embryogenesis as well as how direct cell-cell interactions of DETC with neighboring epidermal cells shape skin homeostasis and effector functions. Furthermore, we will discuss seminal work and recent discoveries made in the γδT cell field, which have highlighted the importance of γδT cells in the skin, both in humans and mice.

Cellular Imaging Using Stimulated Raman Scattering Microscopy.

Cellular imaging is an active area of research that enables researchers to monitor cellular dynamics, as well as responses to various external stimuli (physiological stress, exogenous compounds, etc.). Stimulated Raman scattering (SRS) microscopy is one popular experimental tool used to image cells, largely because of its chemical specificity, high spatial resolution, and high image acquisition speed. In this Perspective, the theoretical background and experimental implementation of SRS microscopy are discussed and recent developments in the field of cellular imaging with SRS are highlighted and summarized.

Investigation of the phosphatidylserine binding properties of the lipid biosensor, Lactadherin C2 (LactC2), in different membrane environments.

Lipid biosensors are robust tools used in both in vitro and in vivo applications of lipid imaging and lipid detection. Lactadherin C2 (LactC2) was described in 2000 as being a potent and specific sensor for phosphatidylserine (PS) (Andersen et al. Biochemistry 39:6200-6206, 2000). PS is an anionic phospholipid enriched in the inner leaflet of the plasma membrane and has paramount roles in apoptosis, cells signaling, and autophagy. The myriad roles PS plays in membrane dynamics make monitoring PS levels and function an important endeavor. LactC2 has functioned as a tantamount PS biosensor namely in the field of cellular imaging. While PS specificity and high affinity of LactC2 for PS containing membranes has been well established, much less is known regarding LactC2 selectivity for subcellular pools of PS or PS within different membrane environments (e.g., in the presence of cholesterol). Thus, there has been a lack of studies that have compared LactC2 PS sensitivity based upon the acyl chain length and saturation or the presence of other host lipids such as cholesterol. Here, we use surface plasmon resonance as a label-free method to quantitatively assess the apparent binding affinity of LactC2 for membranes containing PS with different acyl chains, different fluidity, as well as representative lipid vesicle mimetics of cellular membranes. Results demonstrate that LactC2 is an unbiased sensor for PS, and can sensitively interact with membranes containing PS with different acyl chain saturation and interact with PS species in a cholesterol-independent manner.

Cascades Neural Network based Segmentation of Fluorescence Microscopy Cell Nuclei

The visual extraction of cellular, nuclear and tissue components from medical images is very vital in the diagnosis routine of different health related abnormalities and diseases. The objective of this work is to modify and efficiently combine different image processing methods supported by cascaded artificial neural networks in an automated system to perform segmentation analysis of medical microscopy images to extract nuclei located in either simple or complex clusters. The proposed system is applied on a publicly available data sets of microscopy nuclei cells. A GUI is designed and presented in this work to ease the analysis and screening of these images. The proposed system shows promising performance and reduced computational time cost. It is hoped that thus system and the corresponding GUI will construct platform base for several biomedical studies in the field of cellular imaging where further complex investigations and modelling of microscopy images could take place.

Fluorescent hydroxyapatite‐loaded biodegradable polymer nanoparticles with folate decoration for targeted imaging

The preparation of the luminescent hydroxyapatite (HAP)‐loaded biocompatible nanoparticles (NPs) for targeted imaging of cancer cells is described. Currently, cellular imaging using fluorescent probes is an important technique for the early diagnosis of cancer. Compared with the quantum dots, luminescent HAP is a new fluorescent material with many advantages such as low toxicity, biocompatibility, thermal stability, resistance to erosion, and low prices. Thus, luminescent HAP has enormous potential to be used as biological fluorescent probes. However, luminescent HAP is water‐insoluble, low sensitivity, which limit its application in the field of cellular imaging. Surface modification of NPs with targeting molecule was carried out to achieve its target function. Thus, novel fluorescent NPs with low toxicity, high sensitivity, and good photostability were prepared to be used for targeted imaging of cancer cells. This study initially explored the applications of luminescent HAP in the field of targeted cellular imaging. This NPs platform will be a promising tool for molecular imaging and medical diagnostics, especially the detection of cancer at its early stage. © 2013 American Institute of Chemical Engineers AIChE J, 59: 4494–4501, 2013

Gold Nanoflower@Gelatin Core–Shell Nanoparticles Loaded with Conjugated Polymer Applied for Cellular Imaging

In the present work, a facile one-pot method is designed to fabricate a core-shell fluorescent nanoparticle (NP) for cellular imaging based on a new cationic conjugated polymer, poly[9,9'-bis(6,6'-(N,N,N-trimethylaminium)fluorene-2,7-ylenevinylene-co-alt-2,5-dicyano-1,4-phenylene] (PFVCN). Gold nanoflowers (AuNFs) are prepared by a seedless method, in which a gelatin layer formed through a sol-gel phase transition is deposited on the surface of each AuNF. The cationic PFVCN self-assembles onto the negative surface of the resultant (AuNF@Gelatin NPs) driven by electrostatic attraction. An obvious enhancement of fluorescence intensity is observed. The AuNF@Gelatin/PFVCN NPs exhibit excellent cytocompatibility, and their cellular imaging ability is demonstrated when cocultured with HeLa cells. AuNF@Gelatin/PFVCN hybrid NPs are expected to be a desirable material in the field of cellular imaging and biosensing.

Facile and mild preparation of fluorescent ZnO nanosheets and their bioimaging applications

A viscous tertiary amine and triol, triethanolamine, is engaged in this work as a structure-shaping agent to prepare the high-quality ZnO nanosheets under mild conditions, i.e., at nearly neutral pH and low temperature. The X-Ray diffraction and transmission electron microscopic results validated that the obtained ZnO nanosheets show the perfect hexagonal nanostructures, with an average edge length of 46.6 ± 8.5 nm. The triethanolamine molecule is deemed to act as the hydroxide anion generator as well as the surface modification agent, as evidenced by the FT-IR spectrum. The mechanism for the formation of ZnO nanosheets has been discussed. The simple strategy allows the efficient syntheses of fluorescent ZnO with the strong emission around 560 nm, which was attributed to the defect emission of the ZnO nanostructures. The predominant fluorescent properties have been successfully used in the field of cellular imaging, suggesting their promising biomedical applications.

Computational Framework for Simulating Fluorescence Microscope Images With Cell Populations

Fluorescence microscopy combined with digital imaging constructs a basic platform for numerous biomedical studies in the field of cellular imaging. As the studies relying on analysis of digital images have become popular, the validation of image processing methods used in automated image cytometry has become an important topic. Especially, the need for efficient validation has arisen from emerging high-throughput microscopy systems where manual validation is impractical. We present a simulation platform for generating synthetic images of fluorescence-stained cell populations with realistic properties. Moreover, we show that the synthetic images enable the validation of analysis methods for automated image cytometry and comparison of their performance. Finally, we suggest additional usage scenarios for the simulator. The presented simulation framework, with several user-controllable parameters, forms a versatile tool for many kinds of validation tasks, and is freely available at http://www.cs.tut.fi/sgn/csb/simcep.