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Journal of Visualized Experiments : JoVE Mar 2024High-speed atomic force microscopy (HS-AFM) is a popular molecular imaging technique for visualizing single-molecule biological processes in real-time due to its ability...
High-speed atomic force microscopy (HS-AFM) is a popular molecular imaging technique for visualizing single-molecule biological processes in real-time due to its ability to image under physiological conditions in liquid environments. The photothermal off-resonance tapping (PORT) mode uses a drive laser to oscillate the cantilever in a controlled manner. This direct cantilever actuation is effective in the MHz range. Combined with operating the feedback loop on the time domain force curve rather than the resonant amplitude, PORT enables high-speed imaging at up to ten frames per second with direct control over tip-sample forces. PORT has been shown to enable imaging of delicate assembly dynamics and precise monitoring of patterns formed by biomolecules. Thus far, the technique has been used for a variety of dynamic in vitro studies, including the DNA 3-point-star motif assembly patterns shown in this work. Through a series of experiments, this protocol systematically identifies the optimal imaging parameter settings and ultimate limits of the HS-PORT AFM imaging system and how they affect biomolecular assembly processes. Additionally, it investigates potential undesired thermal effects induced by the drive laser on the sample and surrounding liquid, particularly when the scanning is limited to small areas. These findings provide valuable insights that will drive the advancement of PORT mode's application in studying complex biological systems.
Topics: Microscopy, Atomic Force; Nanotechnology; Mechanical Phenomena; Molecular Imaging; DNA
PubMed: 38587397
DOI: 10.3791/66470 -
Journal of Visualized Experiments : JoVE Mar 2024The lymphatic vasculature, now often referred to as "the third circulation," is located in many vital organ systems. A principal mechanical function of the lymphatic...
The lymphatic vasculature, now often referred to as "the third circulation," is located in many vital organ systems. A principal mechanical function of the lymphatic vasculature is to return fluid from extracellular spaces back to the central venous ducts. Lymph transport is mediated by spontaneous rhythmic contractions of lymph vessels (LVs). LV contractions are largely regulated by the cyclic rise and fall of cytosolic, free calcium ([Ca]i). This paper presents a method to concurrently calculate changes in absolute concentrations of [Ca]i and vessel contractility/rhythmicity in real time in isolated, pressurized LVs. Using isolated rat mesenteric LVs, we studied changes in [Ca]i and contractility/rhythmicity in response to drug addition. Isolated LVs were loaded with the ratiometric Ca-sensing indicator Fura-2AM, and video microscopy coupled with edge-detection software was used to capture [Ca]i and diameter measurements continuously in real time. The Fura-2AM signal from each LV was calibrated to the minimum and maximum signal for each vessel and used to calculate absolute [Ca]i. Diameter measurements were used to calculate contractile parameters (amplitude, end diastolic diameter, end systolic diameter, calculated flow) and rhythmicity (frequency, contraction time, relaxation time) and correlated with absolute [Ca]i measurements.
Topics: Rats; Animals; Calcium; Lymphatic Vessels; Lymph; Muscle Contraction
PubMed: 38587372
DOI: 10.3791/66535 -
Journal of Biomedical Optics Jun 2024Full-field optical coherence microscopy (FF-OCM) is a prevalent technique for backscattering and phase imaging with epi-detection. Traditional methods have two...
SIGNIFICANCE
Full-field optical coherence microscopy (FF-OCM) is a prevalent technique for backscattering and phase imaging with epi-detection. Traditional methods have two limitations: suboptimal utilization of functional information about the sample and complicated optical design with several moving parts for phase contrast.
AIM
We report an OCM setup capable of generating dynamic intensity, phase, and pseudo-spectroscopic contrast with single-shot full-field video-rate imaging called bichromatic tetraphasic (BiTe) full-field OCM with no moving parts.
APPROACH
BiTe OCM resourcefully uses the phase-shifting properties of anti-reflection (AR) coatings outside the rated bandwidths to create four unique phase shifts, which are detected with two emission filters for spectroscopic contrast.
RESULTS
BiTe OCM overcomes the disadvantages of previous FF-OCM setup techniques by capturing both the intensity and phase profiles without any artifacts or speckle noise for imaging scattering samples in three-dimensional (3D). BiTe OCM also utilizes the raw data effectively to generate three complementary contrasts: intensity, phase, and color. We demonstrate BiTe OCM to observe cellular dynamics, image live, and moving micro-animals in 3D, capture the spectroscopic hemodynamics of scattering tissues along with dynamic intensity and phase profiles, and image the microstructure of fall foliage with two different colors.
CONCLUSIONS
BiTe OCM can maximize the information efficiency of FF-OCM while maintaining overall simplicity in design for quantitative, dynamic, and spectroscopic characterization of biological samples.
Topics: Animals; Microscopy; Tomography, Optical Coherence; Microscopy, Phase-Contrast
PubMed: 38584966
DOI: 10.1117/1.JBO.29.S2.S22704 -
Journal of Visualized Experiments : JoVE Mar 2024This protocol describes the synthesis of Au nanoparticle seeds and the subsequent formation of Au-Sn bimetallic nanoparticles. These nanoparticles have potential...
This protocol describes the synthesis of Au nanoparticle seeds and the subsequent formation of Au-Sn bimetallic nanoparticles. These nanoparticles have potential applications in catalysis, optoelectronics, imaging, and drug delivery. Previously, methods for producing alloy nanoparticles have been time-consuming, require complex reaction conditions, and can have inconsistent results. The outlined protocol first describes the synthesis of approximately 13 nm Au nanoparticle seeds using the Turkevich method. The protocol next describes the reduction of Sn and its incorporation into the Au seeds to generate Au-Sn alloy nanoparticles. The optical and structural characterization of these nanoparticles is described. Optically, prominent localized surface plasmon resonances (LSPRs) are apparent using UV-visible spectroscopy. Structurally, powder X-ray diffraction (XRD) reflects all particles to be less than 20 nm and shows patterns for Au, Sn, and multiple Au-Sn intermetallic phases. Spherical morphology and size distribution are obtained from transmission electron microscopy (TEM) imaging. TEM reveals that after Sn incorporation, the nanoparticles grow to approximately 15 nm in diameter.
Topics: Gold Alloys; Silver; Gold; Tin; Metal Nanoparticles; Alloys
PubMed: 38557764
DOI: 10.3791/66628 -
Computational and Structural... Dec 2024Early identification of human pathogens is crucial for the effective treatment of bloodstream infections to prevent sepsis. Since pathogens that are present in small...
Early identification of human pathogens is crucial for the effective treatment of bloodstream infections to prevent sepsis. Since pathogens that are present in small numbers are usually difficult to detect directly, we hypothesize that the behavior of the immune cells that are present in large numbers may provide indirect evidence about the causative pathogen of the infection. We previously applied time-lapse microscopy to observe that neutrophils isolated from human whole-blood samples, which had been infected with the human-pathogenic fungus or , indeed exhibited a characteristic morphodynamic behavior. Tracking the neutrophil movement and shape dynamics over time, combined with machine learning approach, the accuracy for the differentiation between the two species was about 75%. In this study, the focus is on improving the classification accuracy of the species using advanced deep learning methods. We implemented (i) gated recurrent unit (GRU) networks and transformer-based networks for video data, and (ii) convolutional neural networks (CNNs) for individual frames of the time-lapse microscopy data. While the GRU and transformer-based approaches yielded promising results with 96% and 100% accuracy, respectively, the classification based on videos proved to be very time-consuming and required several hours. In contrast, the CNN model for individual microscopy frames yielded results within minutes, and, utilizing a majority-vote technique, achieved 100% accuracy both in identifying the pathogen-free blood samples and in distinguishing between the species. The applied CNN demonstrates the potential for automatically differentiating bloodstream infections with high accuracy and efficiency. We further analysed the results of the CNN using explainable artificial intelligence (XAI) techniques to understand the critical features and patterns, thereby shedding light on potential key morphodynamic characteristics of neutrophils in response to different species. This approach could provide new insights into host-pathogen interactions and may facilitate the development of rapid, automated diagnostic tools for differentiating fungal species in blood samples.
PubMed: 38550973
DOI: 10.1016/j.csbj.2024.03.006 -
Single-cell mechanical assay unveils viscoelastic similarities in normal and neoplastic brain cells.Biophysical Journal May 2024Understanding cancer cell mechanics allows for the identification of novel disease mechanisms, diagnostic biomarkers, and targeted therapies. In this study, we utilized...
Understanding cancer cell mechanics allows for the identification of novel disease mechanisms, diagnostic biomarkers, and targeted therapies. In this study, we utilized our previously established fluid shear stress assay to investigate and compare the viscoelastic properties of normal immortalized human astrocytes and invasive human glioblastoma (GBM) cells when subjected to physiological levels of shear stress that are present in the brain microenvironment. We used a parallel-flow microfluidic shear system and a camera-coupled optical microscope to expose single cells to fluid shear stress and monitor the resulting deformation in real time, respectively. From the video-rate imaging, we fed cell deformation information from digital image correlation into a three-parameter generalized Maxwell model to quantify the nuclear and cytoplasmic viscoelastic properties of single cells. We further quantified actin cytoskeleton density and alignment in immortalized human astrocytes and GBM cells via fluorescence microscopy and image analysis techniques. Results from our study show that contrary to the behavior of many extracranial cells, normal and cancerous brain cells do not exhibit significant differences in their viscoelastic properties. Moreover, we also found that the viscoelastic properties of the nucleus and cytoplasm as well as the actin cytoskeletal densities of both brain cell types are similar. Our work suggests that malignant GBM cells exhibit unique mechanical behaviors not seen in other cancer cell types. These results warrant future studies to elucidate the distinct biophysical characteristics of the brain and reveal novel mechanical attributes of GBM and other primary brain tumors.
Topics: Humans; Viscosity; Elasticity; Brain Neoplasms; Cell Line, Tumor; Single-Cell Analysis; Astrocytes; Glioblastoma; Biomechanical Phenomena; Brain; Cell Nucleus; Stress, Mechanical; Actin Cytoskeleton
PubMed: 38544410
DOI: 10.1016/j.bpj.2024.03.034 -
Scientific Reports Mar 2024A wide-field microscope with epi-fluorescence and selective plane illumination was combined with a single-photon avalanche diode (SPAD) array camera to enable live-cell...
A wide-field microscope with epi-fluorescence and selective plane illumination was combined with a single-photon avalanche diode (SPAD) array camera to enable live-cell fluorescence lifetime imaging (FLIM) using time-correlated single-photon counting (TCSPC). The camera sensor comprised of pixels, each integrating a single SPAD and a time-to-digital converter. Jointly, they produced a stream of single-photon images of photon arrival times with accuracy. The photon arrival times were subject to systematic delays and nonlinearities, which were corrected by a Monte-Carlo algorithm. The SPAD camera was then applied to FLIM where histogramming the resulting photon arrival times in each pixel resulted in decays compatible with common data processing pipelines for fluorescence lifetime analysis. The capabilities of the TCSPC camera-based FLIM microscope were demonstrated by imaging living unicellular photosynthetic algae and artificial lipid vesicles. Epi-fluorescence illumination enabled rapid fluorescence lifetime imaging of living cells and selective-plane illumination enabled 3-dimensional FLIM of stationary samples.
Topics: Microscopy, Fluorescence; Algorithms
PubMed: 38538638
DOI: 10.1038/s41598-024-56122-1 -
Brain : a Journal of Neurology Mar 2024Biallelic SORD mutations cause one of the most frequent forms of recessive hereditary neuropathy, estimated to affect approximately 10,000 patients in North America and...
Biallelic SORD mutations cause one of the most frequent forms of recessive hereditary neuropathy, estimated to affect approximately 10,000 patients in North America and Europe alone. Pathogenic SORD loss-of-function changes in the encoded enzyme sorbitol dehydrogenase result in abnormally high sorbitol levels in cells and serum. How sorbitol accumulation leads to peripheral neuropathy remains to be elucidated. A reproducible animal model for SORD neuropathy is essential to illuminate the pathogenesis of SORD deficiency and for preclinical studies of potential therapies. Therefore, we have generated a Sord knockout (KO), Sord-/-, Sprague Dawley rat, to model the human disease and to investigate the pathophysiology underlying SORD deficiency. We have characterized the phenotype in these rats with a battery of behavioral tests as well as biochemical, physiological, and comprehensive histological examinations. Sord-/- rats had remarkably increased levels of sorbitol in serum, cerebrospinal fluid (CSF), and peripheral nerve. Moreover, serum from Sord-/- rats contained significantly increased levels of neurofilament light chain, NfL, an established biomarker for axonal degeneration. Motor performance significantly declined in Sord-/- animals starting at ∼7 months of age. Gait analysis evaluated with video motion tracking confirmed abnormal gait patterns in the hindlimbs. Motor nerve conduction velocities of the tibial nerves were slowed. Light and electron microscopy of the peripheral nervous system revealed degenerating myelinated axons, de- and remyelinated axons, and a likely pathognomonic finding - enlarged "ballooned" myelin sheaths. These findings mainly affected myelinated motor axons; myelinated sensory axons were largely spared. In summary, Sord-/- rats develop a motor-predominant neuropathy that closely resembles the human phenotype. Our studies revealed novel significant aspects of SORD deficiency, and this model will lead to an improved understanding of the pathophysiology and the therapeutic options for SORD neuropathy.
PubMed: 38538210
DOI: 10.1093/brain/awae079 -
Trials Mar 2024Acute microcirculatory perfusion disturbances and organ edema are important factors leading to organ dysfunction during cardiac surgery with cardiopulmonary bypass...
Optimization of cardiopulmonary bypass prime fluid to preserve microcirculatory perfusion during on-pump coronary artery bypass graft surgery: PRIME study protocol for a double-blind randomized trial.
BACKGROUND
Acute microcirculatory perfusion disturbances and organ edema are important factors leading to organ dysfunction during cardiac surgery with cardiopulmonary bypass (CPB). Priming of the CPB system with crystalloid or colloid fluids, which inevitably leads to hemodilution, could contribute to this effect. However, there is yet no optimal evidence-based strategy for this type of priming. Hence, we will investigate different priming strategies to reduce hemodilution and preserve microcirculatory perfusion.
METHODS
The PRIME study is a single-center double-blind randomized trial. Patients undergoing elective coronary artery bypass graft surgery with CPB will be randomized into three groups of prime fluid strategy: (1) gelofusine with crystalloid, (2) albumin with crystalloid, or (3) crystalloid and retrograde autologous priming. We aim to include 30 patients, 10 patients in each arm. The primary outcome is the change in microcirculatory perfusion. Secondary outcomes include colloid oncotic pressure; albumin; hematocrit; electrolytes; fluid balance and requirements; transfusion rates; and endothelial-, glycocalyx-, inflammatory- and renal injury markers. Sublingual microcirculatory perfusion will be measured using non-invasive sidestream dark field video microscopy. Microcirculatory and blood measurements will be performed at five consecutive time points during surgery up to 24 h after admission to the intensive care unit.
DISCUSSION
PRIME is the first study to assess the effect of different prime fluid strategies on microcirculatory perfusion in cardiac surgery with CPB. If the results suggest that a specific crystalloid or colloid prime fluid strategy better preserves microcirculatory perfusion during on-pump cardiac surgery, the current study may help to find the optimal pump priming in cardiac surgery.
TRIAL REGISTRATION
ClinicalTrials.gov NCT05647057. Registered on 04/25/2023.
CLINICALTRIALS
gov PRS: Record Summary NCT05647057, all items can be found in the protocol.
Topics: Humans; Cardiopulmonary Bypass; Microcirculation; Coronary Artery Bypass; Crystalloid Solutions; Perfusion; Albumins; Colloids; Randomized Controlled Trials as Topic
PubMed: 38532434
DOI: 10.1186/s13063-024-08053-5 -
Scientific Reports Mar 2024Sepsis is accompanied by a less-known mismatch between hemodynamics and mitochondrial respiration. We aimed to characterize the relationship and time dependency of...
Sepsis is accompanied by a less-known mismatch between hemodynamics and mitochondrial respiration. We aimed to characterize the relationship and time dependency of microcirculatory and mitochondrial functions in a rodent model of intraabdominal sepsis. Fecal peritonitis was induced in rats, and multi-organ failure (MOF) was evaluated 12, 16, 20, 24 or 28 h later (n = 8/group, each) using rat-specific organ failure assessment (ROFA) scores. Ileal microcirculation (proportion of perfused microvessels (PPV), microvascular flow index (MFI) and heterogeneity index (HI)) was monitored by intravital video microscopy, and mitochondrial respiration (OxPhos) and outer membrane (mtOM) damage were measured with high-resolution respirometry. MOF progression was evidenced by increased ROFA scores; microcirculatory parameters followed a parallel time course from the 16th to 28th h. Mitochondrial dysfunction commenced with a 4-h time lag with signs of mtOM damage, which correlated significantly with PPV, while no correlation was found between HI and OxPhos. High diagnostic value was demonstrated for PPV, mtOM damage and lactate levels for predicting MOF. Our findings indicate insufficient splanchnic microcirculation to be a possible predictor for MOF that develops before the start of mitochondrial dysfunction. The adequate subcellular compensatory capacity suggests the presence of mitochondrial subpopulations with differing sensitivity to septic insults.
Topics: Rats; Animals; Microcirculation; Sepsis; Hemodynamics; Mitochondria; Multiple Organ Failure; Mitochondrial Diseases
PubMed: 38531957
DOI: 10.1038/s41598-024-57855-9