Search Results (23)

Telomeres play a key role in maintaining chromosome stability and cellular aging. They consist of repetitive DNA sequences that protect chromosome ends and regulate cell division. Telomerase is a reverse transcriptase enzyme counteracts the natural shortening of telomeres during cell division by extending them. Its activity is pivotal in stem cells and cancer cells but absent in most normal somatic cells. Recent advances in biosensor technologies have facilitated the in situ detection of telomerase activity, which is essential for understanding its role in aging and cancer. Techniques such as fluorescence, electrochemistry, and DNA nanotechnology are now being employed to monitor telomerase activity in living cells, providing real-time insights into cellular processes. DNA-based biosensors, especially those incorporating molecular beacons, DNA walkers, and logic gates, have shown promise for enhancing sensitivity and specificity in telomerase imaging. These approaches also facilitate the simultaneous analysis of related cellular pathways, offering potential applications in early cancer detection and precision therapies. This review explores recent developments in intracellular telomerase imaging, highlighting innovative approaches such as DNA-functionalized nanoparticles and multi-channel logic systems, which offer non-invasive, real-time detection of telomerase activity in complex cellular environments. Full article

‘Organ-on-a-chip’ technology is a promising and rapidly evolving model in biological research. This innovative microfluidic cell culture device was created using a microchip with continuously perfused chambers, populated by living cells arranged to replicate physiological processes at the tissue and organ levels. By consolidating multicellular structures, tissue–tissue interfaces, and physicochemical microenvironments, these microchips can replicate key organ functions. They also enable the high-resolution, real-time imaging and analysis of the biochemical, genetic, and metabolic activities of living cells in the functional tissue and organ contexts. This technology can accelerate research into tissue development, organ physiology and disease etiology, therapeutic approaches, and drug testing. It enables the replication of entire organ functions (e.g., liver-on-a-chip, hypothalamus–pituitary-on-a-chip) or the creation of disease models (e.g., amyotrophic lateral sclerosis-on-a-chip, Parkinson’s disease-on-a-chip) using specialized microchips and combining them into an integrated functional system. This technology allows for a significant reduction in the number of animals used in experiments, high reproducibility of results, and the possibility of simultaneous use of multiple cell types in a single model. However, its application requires specialized equipment, advanced expertise, and currently incurs high costs. Additionally, achieving the level of standardization needed for commercialization remains a challenge at this stage of development. Full article

Penetrating deep into the cells of the human body in real time has become increasingly possible with the implementation of modern technologies in medicine. Atomic force microscopy (AFM) enables the effective live imaging of cellular and molecular structures of biological samples (such as cells surfaces, components of biological membranes, cell nuclei, actin networks, proteins, and DNA) and provides three-dimensional surface visualization (in X-, Y-, and Z-planes). Furthermore, the AFM technique enables the study of the mechanical, electrical, and magnetic properties of cells and cell organelles and the measurements of interaction forces between biomolecules. The technique has found wide application in cancer research. With the use of AFM, it is not only possible to differentiate between healthy and cancerous cells, but also to distinguish between the stages of cancerous conditions. For many years, AFM has been an important tool for the study of neurodegenerative diseases associated with the deposition of peptide amyloid plaques. In recent years, a significant amount of research has been conducted on the application of AFM in the evaluation of connective tissue cell mechanics. This review aims to provide the spectrum of the most important applications of the AFM technique in medicine to date. Full article

Cryofixation by ultra-rapid freezing is widely regarded as the gold standard for preserving cell structure without artefacts for electron microscopy. However, conventional cryofixation technologies are not compatible with live imaging, making it difficult to capture dynamic cellular processes at a precise time. To overcome this limitation, we recently introduced a new technology, called microfluidic cryofixation. The principle is based on micro-hotplates counter-cooled with liquid nitrogen. While the power is on, the sample inside a foil-embedded microchannel on top of the micro-hotplate is kept warm. When the heater is turned off, the thermal energy is drained rapidly and the sample freezes. While this principle has been demonstrated experimentally with small samples (<0.5 mm²), there is an important trade-off between the attainable cooling rate, sample size, and heater power. Here, we elucidate these connections by theoretical modeling and by measurements. Our findings show that cooling rates of 10⁶ K s⁻¹, which are required for the vitrification of pure water, can theoretically be attained in samples up to ∼1 mm wide and 5 μm thick by using diamond substrates. If a heat sink made of silicon or copper is used, the maximum thickness for the same cooling rate is reduced to ∼3 μm. Importantly, cooling rates of 10⁴ K s⁻¹ to 10⁵ K s⁻¹ can theoretically be attained for samples of arbitrary area. Such rates are sufficient for many real biological samples due to the natural cryoprotective effect of the cytosol. Thus, we expect that the vitrification of millimeter-scale specimens with thicknesses in the 10 μm range should be possible using micro-hotplate-based microfluidic cryofixation technology. Full article

Digital holography (DH) is a novel, real-time, non-destructive, and quantitative phase-contrast imaging method that is particularly suitable for label-free live biological cell imaging and real-time dynamic monitoring. It is currently a research hotspot in the interdisciplinary field of optics and biomedical sciences, both domestically and internationally. This article proposes an improved angle spectrum algorithm based on holographic technology, which reconstructs a cellular hologram based on phase information. Optical images and chromosome cell images, reconstructed using holographic technology at different diffraction distances under the improved angle spectrum algorithm, were analyzed and compared. The optimal diffraction distance for reconstructing chromosome cell images was selected, and chromosome cell images reproduced using traditional angle spectrum algorithms, angle spectrum algorithms combined with GS, and improved angle spectrum algorithms were compared. Comparative experiments with the different models show that the proposed algorithm is superior to traditional angle spectrum algorithms in reconstructing cell images based on phase information. Furthermore, experiments have shown that images reconstructed using the improved algorithm can resolve high signal-to-noise ratio information. This algorithmic improvement provides new applications for cellular detection in clinical diagnostics and is more suitable for cell phase reconstruction in practical applications. Full article

Carbon monoxide (CO) is a vital endogenous gaseous transmitter molecule involved in the regulation of various physiological and pathological processes in living biosystems. In order to investigate the biological function of CO, many technologies have been developed to monitor the level of endogenous CO in biosystems. Among them, the fluorescence detection technology based on the fluorescent probe has the advantages of high sensitivity, excellent selectivity, simple operation, especially non-invasive damage to biological samples, and the possibility of real-time in situ detection, etc., which is considered to be one of the most effective and applicable detection techniques. Therefore, in the last few years, a lot of work has been carried out on the design, synthesis and in vivo fluorescence imaging studies of CO fluorescent probes. Furthermore, using fluorescent probes to detect the changes in CO concentrations in living cells and tissues as well as in organisms has been one of the hot research topics in recent years. However, it is still a challenge to rationally design CO fluorescent probe with excellent optical performance, structural stability, low background interference, good biocompatibility, and excellent water solubility. Therefore, this review focuses on the research progress of CO fluorescent probes in the detection mechanism and biological applications in recent years. However, this popular and leading topic has rarely been summarized comprehensively to date. Thus, the research progress of CO fluorescent probes in recent years is reviewed in terms of their design concept, detection mechanism, and their biological applications. In addition, the relationship between the structure and performance of the probes was also discussed. More significantly, we hope that more excellent optical properties fluorescent probes for gaseous transmitter molecule CO detection and imaging will overcome the current problems of high biotoxicity and limited water solubility in future. Full article

Mesenchymal stem cells (MSC) make up less than 1% of the bone marrow (BM). Several methods are used for their isolation such as gradient separation or centrifugation, but these methodologies are not direct and, thus, plastic adherence outgrowth or magnetic/fluorescent-activated sorting is required. To overcome this limitation, we investigated the use of a new separative technology to isolate MSCs from BM; it label-free separates cells based solely on their physical characteristics, preserving their native physical properties, and allows real-time visualization of cells. BM obtained from patients operated for osteochondral defects was directly concentrated in the operatory room and then analyzed using the new technology. Based on cell live-imaging and the sample profile, it was possible to highlight three fractions (F1, F2, F3), and the collected cells were evaluated in terms of their morphology, phenotype, CFU-F, and differentiation potential. Multipotent MSCs were found in F1: higher CFU-F activity and differentiation potential towards mesenchymal lineages compared to the other fractions. In addition, the technology depletes dead cells, removing unwanted red blood cells and non-progenitor stromal cells from the biological sample. This new technology provides an effective method to separate MSCs from fresh BM, maintaining their native characteristics and avoiding cell manipulation. This allows selective cell identification with a potential impact on regenerative medicine approaches in the orthopedic field and clinical applications. Full article

The development of cell models of human diseases based on induced pluripotent stem cells (iPSCs) and a cell therapy approach based on differentiated iPSC derivatives has provided a powerful stimulus in modern biomedical research development. Moreover, it led to the creation of personalized regenerative medicine. Due to this, in the last decade, the pathological mechanisms of many monogenic diseases at the cell level have been revealed, and clinical trials of various cell products derived from iPSCs have begun. However, it is necessary to reach a qualitatively new level of research with cell models of diseases based on iPSCs for more efficient searching and testing of drugs. Biosensor technology has a great application prospect together with iPSCs. Biosensors enable researchers to monitor ions, molecules, enzyme activities, and channel conformation in live cells and use them in live imaging and drug screening. These probes facilitate the measurement of steady-state concentrations or activity levels and the observation and quantification of in vivo flux and kinetics. Real-time monitoring of drug action in a specific cellular compartment, organ, or tissue type; the ability to screen at the single-cell resolution; and the elimination of the false-positive results caused by low drug bioavailability that is not detected by in vitro testing methods are a few of the benefits of using biosensors in drug screening. Here, we discuss the possibilities of using biosensor technology in combination with cell models based on human iPSCs and gene editing systems. Furthermore, we focus on the current achievements and problems of using these methods. Full article

Conventional plaque assays rely on the use of overlays to restrict viral infection allowing the formation of distinct foci that grow in time as the replication cycle continues leading to countable plaques that are visualized with standard techniques such as crystal violet, neutral red, or immunolabeling. This classical approach takes several days until large enough plaques can be visualized and counted with some variation due to subjectivity in plaque recognition. Since plaques are clonal lesions produced by virus-induced cytopathic effect, we applied DNA fluorescent dyes with differential cell permeability to visualize them by live-cell imaging. We could observe different stages of that cytopathic effect corresponding to an early wave of cells with chromatin-condensation followed by a wave of dead cells with membrane permeabilization within plaques generated by different animal viruses. This approach enables an automated plaque identification using image analysis to increase single plaque resolution compared to crystal violet counterstaining and allows its application to plaque tracking and plaque reduction assays to test compounds for both antiviral and cytotoxic activities. This fluorescent real-time plaque assay sums to those next-generation technologies by combining this robust classical method with modern fluorescence microscopy and image analysis approaches for future applications in virology. Full article

Autophagy is an essential cellular process of self-degradation for dysfunctional or unnecessary cytosolic constituents and organelles. Dysregulation of autophagy is thus involved in various diseases such as neurodegenerative diseases. To investigate the complex process of autophagy, various biochemical, chemical assays, and imaging methods have been developed. Here we introduce various methods to study autophagy, in particular focusing on the review of designs, principles, and limitations of the fluorescent protein (FP)-based autophagy biosensors. Different physicochemical properties of FPs, such as pH-sensitivity, stability, brightness, spectral profile, and fluorescence resonance energy transfer (FRET), are considered to design autophagy biosensors. These FP-based biosensors allow for sensitive detection and real-time monitoring of autophagy progression in live cells with high spatiotemporal resolution. We also discuss future directions utilizing an optobiochemical strategy to investigate the in-depth mechanisms of autophagy. These cutting-edge technologies will further help us to develop the treatment strategies of autophagy-related diseases. Full article

Polydimethylsiloxane (PDMS) has been used in microfluidic systems for years, as it can be easily structured and its flexibility makes it easy to integrate actuators including pneumatic pumps. In addition, the good optical properties of the material are well suited for analytical systems. In addition to its positive aspects, PDMS is well known to adsorb small molecules, which limits its usability when it comes to drug testing, e.g., in organ-on-a-chip (OoC) systems. Therefore, alternatives to PDMS are in high demand. In this study, we use thermoplastic elastomer (TPE) films thermally bonded to laser-cut poly(methyl methacrylate) (PMMA) sheets to build up multilayered microfluidic devices with integrated pneumatic micro-pumps. We present a low-cost manufacturing technology based on a conventional CO₂ laser cutter for structuring, a spin-coating process for TPE film fabrication, and a thermal bonding process using a pneumatic hot-press. UV treatment with an Excimer lamp prior to bonding drastically improves the bonding process. Optimized bonding parameters were characterized by measuring the burst load upon applying pressure and via profilometer-based measurement of channel deformation. Next, flow and long-term stability of the chip layout were measured using microparticle Image Velocimetry (uPIV). Finally, human endothelial cells were seeded in the microchannels to check biocompatibility and flow-directed cell alignment. The presented device is compatible with a real-time live-cell analysis system. Full article

A major obstacle in studying the interplay between cancer cells and the immune system has been the examination of proposed biological pathways and cell interactions in a dynamic, physiologically relevant system in vivo. Intravital imaging strategies are one of the few molecular imaging techniques that can follow biological processes at cellular resolution over long periods of time in the same individual. Bioluminescence imaging has become a standard preclinical in vivo optical imaging technique with ever-expanding versatility as a result of the development of new emission bioluminescent reporters, advances in genomic techniques, and technical improvements in bioluminescence imaging and processing methods. Herein, we describe an advance of technology with a molecular imaging window chamber platform that combines bioluminescent and fluorescent reporters with intravital macro-imaging techniques and bioluminescence spectral unmixing in real time applied to heterogeneous living systems in vivo for evaluating tumor signaling dynamics and immune cell enzyme activities concurrently. Full article

With an estimated three to five million human cases annually and the potential to infect domestic and wild animal populations, influenza viruses are one of the greatest health and economic burdens to our society, and pose an ongoing threat of large-scale pandemics. Despite our knowledge of many important aspects of influenza virus biology, there is still much to learn about how influenza viruses replicate in infected cells, for instance, how they use entry receptors or exploit host cell trafficking pathways. These gaps in our knowledge are due, in part, to the difficulty of directly observing viruses in living cells. In recent years, advances in light microscopy, including super-resolution microscopy and single-molecule imaging, have enabled many viral replication steps to be visualised dynamically in living cells. In particular, the ability to track single virions and their components, in real time, now allows specific pathways to be interrogated, providing new insights to various aspects of the virus-host cell interaction. In this review, we discuss how state-of-the-art imaging technologies, notably quantitative live-cell and super-resolution microscopy, are providing new nanoscale and molecular insights into influenza virus replication and revealing new opportunities for developing antiviral strategies. Full article

In biomedical research, there is an ongoing demand for new technologies to elucidate disease mechanisms and develop novel therapeutics. This requires comprehensive understanding of cellular processes and their pathophysiology based on reliable information on abundance, localization, post-translational modifications and dynamic interactions of cellular components. Traceable intracellular binding molecules provide new opportunities for real-time cellular diagnostics. Most prominently, intrabodies derived from antibody fragments of heavy-chain only antibodies of camelids (nanobodies) have emerged as highly versatile and attractive probes to study and manipulate antigens within the context of living cells. In this review, we provide an overview on the selection, delivery and usage of intrabodies to visualize and monitor cellular antigens in living cells and organisms. Additionally, we summarize recent advances in the development of intrabodies as cellular biosensors and their application to manipulate disease-related cellular processes. Finally, we highlight switchable intrabodies, which open entirely new possibilities for real-time cell-based diagnostics including live-cell imaging, target validation and generation of precisely controllable binding reagents for future therapeutic applications. Full article

From electronic devices to large-area electronics, from individual cells to skin substitutes, printing techniques are providing compelling applications in wide-ranging fields. Research has thus fueled the vision of a hybrid, printing platform to fabricate sensors/electronics and living engineered tissues simultaneously. Following this interest, we have fabricated interdigitated-electrode sensors (IDEs) by inkjet printing to monitor epithelial cell cultures. We have fabricated IDEs using flexible substrates with silver nanoparticles as a conductive element and SU-8 as the passivation layer. Our sensors are cytocompatible, have a topography that simulates microgrooves of 300 µm width and ~4 µm depth, and can be reused for cellular studies without detrimental in the electrical performance. To test the inkjet-printed sensors and demonstrate their potential use for monitoring laboratory-growth skin tissues, we have developed a real-time system and monitored label-free proliferation, migration, and detachment of keratinocytes by impedance spectroscopy. We have found that variations in the impedance correlate linearly to cell densities initially seeded and that the main component influencing the total impedance is the isolated effect of the cell membranes. Results obtained show that impedance can track cellular migration over the surface of the sensors, exhibiting a linear relationship with the standard method of image processing. Our results provide a useful approach for non-destructive in-situ monitoring of processes related to both in vitro epidermal models and wound healing with low-cost ink-jetted sensors. This type of flexible sensor as well as the impedance method are promising for the envisioned hybrid technology of 3D-bioprinted smart skin substitutes with built-in electronics. Full article