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Lasers are widely applied in assisted reproductive technologies, including sperm fixation, sperm selection and intracytoplasmic sperm injections, to reduce procedure time and improve consistency and reproducibility. However, quantitative studies on laser-induced photodamage of sperm are lacking. In this study, we demonstrated that, by using optical tweezers, the kinematic parameters of freely swimming sperm are correlated with the frequency as well as the percentage of pausing duration of longitudinal rolling of the same sperm head in the optical trap. Furthermore, by trapping individual sperm cells using 1064-nm optical tweezers, we quantitatively characterized the time-dependence of longitudinal rolling frequency and percentage of pausing duration of sperm under different laser powers. Our study revealed that, as trapping time and the laser power time increase, the longitudinal rolling frequency of the optically trapped sperm decreases with an increasing percentage of pausing duration, which characterizes the effect of laser power and duration on the photodamage of individual sperm cells. Our study provides experimental basis for the optimization of laser application in assisted reproductive technology, which may reduce the photodamage-induced biosafety risk in the future.
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Optofluidic regulation of blood microflow in vivo represents a significant method for investigating illnesses linked to abnormal changes in blood circulation. Currently, non-invasive strategies are limited to regulation within capillaries of approximately 10 µm in diameter because the adaption to blood pressure levels in the order of several hundred pascals poses a significant challenge in larger microvessels. In this study, using laser-induced microbubble formation within microvessels of the mouse auricle, we regulate blood microflow in small vessels with diameters in the tens of micrometers. By controlling the laser power, we can control the growth and stability of microbubbles in vivo. This controlled approach enables the achievement of prolonged ischemia and subsequent reperfusion of blood flow, and it can also regulate the microbubbles to function as micro-pumps for reverse blood pumping. Furthermore, by controlling the microbubble, narrow microflow channels can be formed between the microbubbles and microvessels for assessing the apparent viscosity of leukocytes, which is 76.9 ± 11.8 Pa·s in the in vivo blood environment. The proposed design of in vivo microbubble valves opens new avenues for constructing real-time blood regulation and exploring cellular mechanics within living organisms.
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Rayos Láser , Microburbujas , Microvasos , Animales , Ratones , Microvasos/fisiologíaRESUMEN
Point-of-care testing (POCT) plays an increasingly important role in biomedical research and health care. Quantitative phase microscopes (QPMs) with good contrast, no invasion, no labeling, high speed and automation could be effectively applied for POCT. However, most QPMs are fixed on the optical platform with bulky size, lack of timeliness, which remained challenging in POCT solutions. In this paper, we proposed a plug-and-play QPM with multimode imaging based on the quantitative differential phase contrast (qDPC) method. The system employs a programmable LED array as the light source and uses the GPU to accelerate the calculation, which can realize multi-contrast imaging with six modes. Accurate phase measurement and real-time phase imaging are implemented by the proposed qDPC algorithms for quantitative phase targets and biomedical samples. A 3D electric control platform is designed for mechanical control of field of view and focusing without manual operations. The experimental results verify the robustness and high performance of the setup. Even a rookie could finish the POCT scheme for biomedical applications at the scene using the QPM with a compact size of 140 × 165 × 250 mm3.
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It is important to measure the deformability of red blood cells (RBCs) before transfusion, which is a key factor in the gas transport ability of RBCs and changes during storage of RBCs in vitro. Moreover, the morphology of RBCs also changes during storage. It is proposed that the change in morphology is related to the change in deformability. However, the efficiency of typical methods that use particles as handles is low, especially in the deformability measurement of echinocyte and spherocytes. Therefore, the deformability of RBCs with different morphologies is hard to be measured and compared in the same experiment. In this study, we developed a cost-effective and efficient rotating-glass-plate-based scanning optical tweezers device for the measurement of deformability of RBCs. The performance of this device was evaluated, and the deformability of three types of RBCs was measured using this device. Our results clearly show that the change of erythrocyte morphology from discocyte to echinocyte and spherocyte during storage in vitro is accompanied by a decrease in deformability.
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Optical tweezers are widely used to measure the mechanical properties of erythrocytes, which is crucial to the study of pathology and clinical diagnosis of disease. During the measurement, the blood sample is diluted and suspended in an exogenous physiological fluid, which may affect the elastic properties of the cells in vitro. Here, we investigate the effect of different diluents on the elastic properties of mouse erythrocytes by quantitatively evaluating their elastic constants using optical tweezers. The diluents are plasma extracted from mouse blood, veterinary blood diluent (V-52D), Dulbecco's modified Eagle's medium (DMEM), phosphate-buffered saline (PBS), and normal saline (NS). To create an environment that closely resembles in vivo conditions, the experiment is performed at 36.5 °C. The results show that the spring constant of mouse erythrocytes in plasma is 6.23 ± 0.41 µN m-1. The elasticity of mouse erythrocytes in V-52D and DMEM is 8.21 ± 0.91 and 6.95 ± 0.85 µN m-1, which are higher than that in plasma extracted from blood, whereas, the elasticity in PBS and NS is 4.23 ± 0.85 and 4.68 ± 0.79 µN m-1, which are less than that in plasma extracted from blood. At last, we observe the size and circularity of erythrocytes in different diluents, and consider that the erythrocyte diameter and circularity may affect cell deformability. Our results provide a reference of the diluent choice for measuring the mechanical properties of erythrocytes in vitro.
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Deformación Eritrocítica , Pinzas Ópticas , Animales , Ratones , Eritrocitos/fisiología , Elasticidad , PlasmaRESUMEN
Motility is one of the most critical features to evaluate sperm quality. As longitudinal rolling of human sperm has long been ignored until recently, its detailed dynamics and cellular biological mechanisms are still largely unknown. Here we report an optical-tweezers-based method to evaluate the chirality and frequency of sperm rotation. According to the intensity distribution patterns of off-focus micron-size particles, we established a method to judge the orientation of the sperm head along the optical axis in the optical trap. Together with the rotation direction of the projection of the sperm head, the chirality of longitudinal rolling of sperm can be measured without the application of three-dimensional tracking techniques or complex optical design. By video tracking optically trapped sperm cells from different patients, both rolling chirality and rolling frequency were analyzed. In this study, all the vertically trapped human sperm cells adopt a right-hand longitudinal rolling. The orientation and rolling frequency but not the rolling chirality of sperm in the optical trap are affected by the trap height. The rotation analysis method developed in this study may have clinical potential for sperm quality evaluation.
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Oblique illumination imaging can significantly improve the contrast of transparent thin samples. However, in traditional oblique illumination methods, either the condenser is offset or a block is added to the condenser, which makes it complicated and challenged to build a stable oblique illumination imaging. Herein, we present a method to measure the optimal shading ratio of oblique illumination in an inverted microscope, and develop an apparatus for stable high-speed high-contrast imaging with uniform brightness. At optimal shading ratio, the oblique illumination imaging has better imaging quality than differential interference contrast, which characteristic is independent on sample. In oblique illumination with low magnification objective, the images have uneven brightness. According to target brightness, we have developed a brightness unevenness correction algorithm to form uniform background brightness for oblique illumination. Integrating the algorithm with imaging acquisition, corrected oblique illumination microscopy is appropriate to observe living cells with high contrast.
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Iluminación , Microscopía , Iluminación/métodos , Microscopía/métodos , AlgoritmosRESUMEN
Distributive shock is considered to be a condition of microvascular hypoperfusion, which can be fatal in severe cases. However, traditional therapeutic methods to restore the macro blood flow are difficult to accurately control the blood perfusion of microvessels, and the currently developed manipulation techniques are inevitably incompatible with biological systems. In our approach, infrared optical tweezers are used to dynamically control the microvascular reperfusion within subdermal capillaries in the pinna of mice. Furthermore, we estimate the effect of different optical trap positions on reperfusion at branch and investigate the effect of the laser power on reperfusion. The results demonstrate the ability of optical tweezers to control microvascular reperfusion. This strategy allows near-noninvasive reperfusion of the microvascular hypoperfusion in vivo. Hence, our work is expected to provide unprecedented insights into the treatment of distributive shock.
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The flow-cell based single-molecule manipulation technique has found many applications in the study of DNA mechanics and protein-DNA interactions. However, the force in these experiments has not been fully characterized and is usually limited to a moderate force regime (<25 pN). In this work, using the "tethered-bead" assay, the hydrodynamic drag of DNA has been quantitatively evaluated based on a "bead-spring chain" model. The force derived from the Brownian motion of the bead thus contains both contributions from this equivalent hydrodynamic drag of DNA and the pulling force from the tethered bead. Next, using flow-cell based DNA pulling experiments, the linear relationship between the flow rate and total hydrodynamic force on the bead-DNA system has been demonstrated to be valid over a wide force range (0-110 pN). Consequently, the force can be directly converted from the flow rate by a linear factor that can be calibrated either by the bead's Brownian motion at low flow rates or using DNA overstretching transition. Furthermore, the hydrodynamic force and torque due to the shear flow on the bead as well as the equivalent stretching force on DNA are calculated based on theoretical models with the hydrodynamic drag on DNA also considered. The calculated force-extension curves show a good agreement with the measured ones. These results offer important insights into the force in flow-cell based single-molecule stretching experiments and provide a foundation for establishing flow-cells as a simple, low-cost, yet flexible and precise tool for single-molecule force measurements over a wide force range.
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ADN , Fenómenos Mecánicos , Hidrodinámica , Modelos Teóricos , Movimiento (Física)RESUMEN
The surface-coupled optical tweezers are widely used to resolve small units of motion in biology. However, such motions could readily be interfered by the drift between the trap and surface. We present a simple and low-cost method to correct the drift both actively and passively based on video tracking the distance between the laser reflection pattern and the reference bead. As a result, we achieved sub-nanometer resolution and stability for the stuck bead over a broad range of averaging time (0.002-100 s) as demonstrated by the Allan deviation analysis. The sub-nanometer resolution was further manifested with step measurement. Finally, in double-stranded DNA and DNA hairpin stretching experiments, an extension resolution of 1-2 nm with the stability over 120 s has been demonstrated under a constant force. This work thus provides an easy way to bring the benefit of nanometer resolution and long-term stability to the surface-coupled optical tweezers.
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Rayos Láser/normas , Pinzas Ópticas/normas , HumanosRESUMEN
A click chemistry assay based on a newly discovered DNAzyme, CLICK-17, with azide modified gold nanoparticles (azide-AuNPs) and alkyne capped dsDNA (alkyne-linker DNA) was employed for novel and selective detection of Cu2+ visually. The strategy involved using CLICK-17 to mediate a catalytic reaction for triazole formation between azide-AuNPs and alkyne-linker DNA under the help of Cu2+ (without sodium ascorbate) or Cu+, which eventually led to the aggregation of AuNPs. The obvious color change from ruby red to bluish purple was then observed by the naked eye and the absorbance peak shifted from 525 to 570 nm. Interestingly, CLICK-17 and Cu+-catalyzed click reaction had the best performance compared to either Cu+ alone or CLICK-17 and Cu2+-mediated reaction in terms of the reaction time and sensitivity. This system has been demonstrated to allow quantitative measurement of Cu2+ with a detection limit as low as 26.8 nM and also has high specificity that can distinguish Cu2+ from other metal ions. Further, the method was tested with a real mineral water sample for Cu2+ concentration determination. Satisfactory recoveries of 90.8% and 99.8% were achieved.
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Minor-groove base triples formed between stem 1 and loop 2 of the simian retrovirus type 1 (SRV-1) mRNA frameshifting pseudoknot are essential in stimulating -1 ribosomal frameshifting. How tertiary base triple formation affects the local stabilities of secondary structures (stem 1 and stem 2) and thus ribosomal frameshifting efficiency is not well understood. We made a short peptide nucleic acid (PNA) that is expected to invade stem 1 of the SRV-1 pseudoknot by PNA-RNA duplex formation to mimic the stem 1 unwinding process by a translating ribosome. In addition, we used a PNA for invading stem 2 in the SRV-1 pseudoknot. Our nondenaturing polyacrylamide gel electrophoresis data for the binding of PNA to the SRV-1 pseudoknot and mutants reveal that mutations in loop 2 disrupting base triple formation between loop 2 and stem 1 in the SRV-1 pseudoknot result in enhanced invasion by both PNAs. Our data suggest that tertiary stem 1-loop 2 base triple interactions in the SRV-1 pseudoknot can stabilize both of the secondary structural components, stem 1 and stem 2. Stem 2 stability is thus coupled to the structural stability of stem 1-loop 2 base triples, mediated through a long-range effect. The apparent dissociation constants of both PNAs are positively correlated with the pseudoknot mechanical stabilities and frameshifting efficiencies. The relatively simple PNA local invasion experiment may be used to characterize the energetic contribution of tertiary interactions and ligand binding in many other RNA and DNA structures.
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Sistema de Lectura Ribosómico , Virus del Mono Mason-Pfizer/genética , ARN Viral/química , Electroforesis en Gel de Poliacrilamida Nativa , Conformación de Ácido Nucleico , Ácidos Nucleicos de Péptidos/química , Ácidos Nucleicos de Péptidos/genética , Ribosomas/genética , Ribosomas/virologíaRESUMEN
DNA:RNA hybrid duplex plays important roles in various biological processes. Both its structural stability and interactions with proteins are highly sequence dependent. In this study, we utilize homebuilt optical tweezers to investigate how GC contents in the sequence influence the structural and mechanical properties of DNA:RNA hybrid by measuring its contour length, elasticities, and overstretching dynamics. Our results support that the DNA:RNA hybrid adopts a conformation between the A- and B-form helix, and the GC content does not affect its structural and elastic parameters obviously when varying from 40 to 60% before the overstretching transition of DNA:RNA hybrid occurs. In the overstretching transition, however, our study unravels significant heterogeneity and strong sequence dependence, suggesting the presence of a highly dynamic competition between the two processes, namely the S-form duplex formation (nonhysteretic) and the unpeeling (hysteretic). Analyzing the components left in DNA:RNA hybrid after the overstretching transition suggests that the RNA strand is more easily unpeeled than the DNA strand, whereas an increase in the GC content from 40 to 60% can significantly reduce unpeeling. Large hysteresis is observed between the stretching and relaxation processes, which is also quantitatively correlated with the percentage of unpeeling in the DNA:RNA duplex. Increasing in both the salt concentration and GC content can effectively reduce the hysteresis with the latter being more significant. Together, our study reveals that the mechanical properties of DNA:RNA hybrid duplexes are significantly different from double-stranded DNA and double-stranded RNA, and its overstretching behavior is highly sequence dependent. These results should be taken into account in the future studies on DNA:RNA-hybrid-related functional structures and motor proteins.
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ADN , Pinzas Ópticas , Composición de Base , Elasticidad , Conformación de Ácido NucleicoRESUMEN
The fluorescence enhancement in an over-etched gold zero-mode waveguide (ZMW) was investigated through both numerical simulation and experiments. Using Cy3 and Cy5 as the fluorescent probes, the simulation showed that the undercut not only enhances the fluorescence signals of both fluorophores, but also greatly improves the radial uniformity of the excitation fields in the ZMW. Furthermore, using a focused-ion-beam tool, we fabricated Au-ZMW arrays with different radius and undercut. The fluorescence enhancement per molecule and the effective excitation volume of the Au-ZMW were then measured as functions of its radial size and over-etching depth by using fluorescence correlation spectroscopy. It was found that the undercut can significantly enhance the fluorescence signal per molecule in the ZMW, but it also slightly increased the excitation volume. Decreasing the radial size of the ZMW can efficiently reduce the excitation volume and also further enhance the fluorescence per molecule. These results together indicate that combining the undercut and reduction of radius of the ZMW can serve as a simple and effective way to essentially improve the performance of an Au-ZMW for single molecule fluorescence detection.
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Alternative splicing of MAPT cassette exon 10 produces tau isoforms with four microtubule-binding repeat domains (4R) upon exon inclusion or three repeats (3R) upon exon skipping. In human neurons, deviations from the â¼1:1 physiological 4R:3R ratio lead to frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17). Certain FTDP-17-associated mutations affect a regulatory hairpin that sequesters the exon 10 5' splice site (5'ss, located at the exon 10-intron 10 junction). These mutations tend to increase the 4R:3R ratio by destabilizing the hairpin, thereby improving 5'ss recognition by U1 snRNP. Interestingly, a single C-to-G mutation at the 19th nucleotide in intron 10 (C19G or +19G) decreases the level of exon 10 inclusion significantly from 56% to 1%, despite the disruption of a G-C base pair in the bottom stem of the hairpin. Here, we show by biophysical characterization, including thermal melting, fluorescence, and single-molecule mechanical unfolding using optical tweezers, that the +19G mutation alters the structure of the bottom stem, resulting in the formation of a new bottom stem with enhanced stability. The cell culture alternative splicing patterns of a series of minigenes reveal that the splicing activities of the mutants with destabilizing mutations on the top stem can be compensated in a position-dependent manner by the +19G mutation in the bottom stem. We observed an excellent correlation between the level of exon 10 inclusion and the rate of mechanical unfolding at 10 pN, indicating that the unfolding of the splice site hairpins (to facilitate subsequent binding of U1 snRNA) may be aided by helicases or other proteins.
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Exones , Empalme del ARN , ARN/química , Proteínas tau/genética , Secuencia de Bases , Células HEK293 , Humanos , Secuencias Invertidas Repetidas , Mutación Puntual , ARN/genética , Pliegue del ARN , Temperatura de TransiciónRESUMEN
A wobble A·C pair can be protonated at near physiological pH to form a more stable wobble A+·C pair. Here, we constructed an RNA hairpin (rHP) and three mutants with one A-U base pair substituted with an A·C mismatch on the top (near the loop, U22C), middle (U25C), and bottom (U29C) positions of the stem, respectively. Our results on single-molecule mechanical (un)folding using optical tweezers reveal the destabilization effect of A-U to A·C pair substitution and protonation-dependent enhancement of mechanical stability facilitated through an increased folding rate, or decreased unfolding rate, or both. Our data show that protonation may occur rapidly upon the formation of an apparent mechanical folding transition state. Furthermore, we measured the bulk -1 ribosomal frameshifting efficiencies of the hairpins by a cell-free translation assay. For the mRNA hairpins studied, -1 frameshifting efficiency correlates with mechanical unfolding force at equilibrium and folding rate at around 15 pN. U29C has a frameshifting efficiency similar to that of rHP (â¼2%). Accordingly, the bottom 2-4 base pairs of U29C may not form under a stretching force at pH 7.3, which is consistent with the fact that the bottom base pairs of the hairpins may be disrupted by ribosome at the slippery site. U22C and U25C have a similar frameshifting efficiency (â¼1%), indicating that both unfolding and folding rates of an mRNA hairpin in a crowded environment may affect frameshifting. Our data indicate that mechanical (un)folding of RNA hairpins may mimic how mRNAs unfold and fold in the presence of translating ribosomes.
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Protones , ARN Mensajero/química , Ribosomas/química , Emparejamiento Base , Sitios de Unión , Conformación de Ácido Nucleico , Pinzas Ópticas , Pliegue del ARN , TemperaturaRESUMEN
The association of a plasmonic nano-antenna with single-molecule FRET technique presents new prospects to investigate the dynamics of biological molecules. However, the presence of a plasmonic nano-antenna significantly modifies the FRET rate and efficiency; this makes its applicability to the prevalent single-molecule FRET experiments unclear. Herein, using gold nanoparticle antennas of different sizes and DNA hairpins labelled with FRET pairs (Cy3 and Cy5) as the model system, we performed experiments to study the folding dynamics of single DNA hairpins at various salt concentrations. Our results indicate that gold nanoparticle antennas can enhance single-molecule fluorescence of Cy3 and Cy5 up to 3-5 folds, substantially reduce the FRET efficiency, and alter the obtained FRET efficiency histograms. However, the folding dynamics of DNA hairpins remains unaffected, and the correct kinetic and dynamic information can still be extracted from the seriously modified FRET efficiencies. Therefore, our experiments demonstrate the feasibility and compatibility for applying plasmonic nano-antennas to the mostly used single-molecule FRET assays, which provide a broad range of possibilities for the future applications of these nano-antennas in studying various essential biological processes.
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ADN/química , Transferencia Resonante de Energía de Fluorescencia , Oro , Nanopartículas del Metal , Ácidos Nucleicos Inmovilizados/química , CinéticaRESUMEN
Minus-one ribosomal frameshifting is a translational recoding mechanism widely utilized by many RNA viruses to generate accurate ratios of structural and catalytic proteins. An RNA pseudoknot structure located in the overlapping region of the gag and pro genes of Simian Retrovirus type 1 (SRV-1) stimulates frameshifting. However, the experimental characterization of SRV-1 pseudoknot (un)folding dynamics and the effect of the base triple formation is lacking. Here, we report the results of our single-molecule nanomanipulation using optical tweezers and theoretical simulation by steered molecular dynamics. Our results directly reveal that the energetic coupling between loop 2 and stem 1 via minor-groove base triple formation enhances the mechanical stability. The terminal base pair in stem 1 (directly in contact with a translating ribosome at the slippery site) also affects the mechanical stability of the pseudoknot. The -1 frameshifting efficiency is positively correlated with the cooperative one-step unfolding force and inversely correlated with the one-step mechanical unfolding rate at zero force. A significantly improved correlation was observed between -1 frameshifting efficiency and unfolding rate at forces of 15-35 pN, consistent with the fact that the ribosome is a force-generating molecular motor with helicase activity. No correlation was observed between thermal stability and -1 frameshifting efficiency.
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Sistema de Lectura Ribosómico , Productos del Gen gag/química , ARN Viral/genética , Retrovirus de los Simios , Ribosomas/química , Electroforesis en Gel de Poliacrilamida , Cinética , Espectroscopía de Resonancia Magnética , Simulación de Dinámica Molecular , Mutación , Conformación de Ácido Nucleico , Oligonucleótidos/química , Pinzas Ópticas , Desnaturalización Proteica , Pliegue de Proteína , ARN Helicasas/química , ARN Mensajero/metabolismo , TermodinámicaRESUMEN
Accurate recognition of splice sites is essential for pre-messenger RNA splicing. Mammalian 5' splice sites are mainly recognized by canonical base-pairing to the 5' end of U1 small nuclear RNA, yet we described multiple noncanonical base-pairing registers by shifting base-pair positions or allowing one-nucleotide bulges. By systematic mutational and suppressor U1 analyses, we prove three registers involving asymmetric loops and show that two-nucleotide bulges but not longer can form in this context. Importantly, we established that a noncanonical uridine-pseudouridine interaction in the 5' splice site/U1 helix contributes to the recognition of certain 5' splice sites. Thermal melting experiments support the formation of noncanonical registers and uridine-pseudouridine interactions. Overall, we experimentally validated or discarded the majority of predicted noncanonical registers, to derive a list of 5' splice sites using such alternative mechanisms that is much different from the original. This study allows not only the mechanistic understanding of the recognition of a wide diversity of mammalian 5' splice sites, but also the future development of better splice-site scoring methods that reliably predict the effects of disease-causing mutations at these sequences.