-
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 -
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 -
Journal of Neurosurgery. Case Lessons Mar 2024Local vasogenic edema induced after direct revascularization in moyamoya disease (MMD) is associated with blood-brain barrier dysfunction, potentially leading to...
Impact of intraoperative cortical indocyanine green extravasation on local vasogenic edema immediately after direct revascularization in an adult with moyamoya disease: illustrative case.
BACKGROUND
Local vasogenic edema induced after direct revascularization in moyamoya disease (MMD) is associated with blood-brain barrier dysfunction, potentially leading to postoperative cerebral hyperperfusion (CHP) or delayed intracerebral hemorrhage. This phenomenon allows the leakage of fluids, proteins, and other substances from the blood vessels into the extracellular compartment. Typically, such edema is observed postoperatively rather than intraoperatively.
OBSERVATIONS
A 48-year-old female with ischemic-onset MMD underwent revascularization on her left hemisphere with Suzuki's angiographic stage III. Direct bypass was successfully performed, as confirmed by intravenous indocyanine green (ICG) video angiography. Subsequently, ICG extravasation was observed near the anastomosis site, despite the absence of cortical injury or bleeding under white light microscopy. Postoperative radiological imaging showed reversible pure vasogenic edema in the corresponding area, with no evidence of CHP. The patient did not exhibit neurological deterioration and was discharged home on postoperative day 16.
LESSONS
ICG, characterized by low molecular weight, water solubility, and high affinity with plasma proteins, can extravasate, serving as a direct indication of local vasogenic edema induced by direct revascularization in MMD. To enhance comprehension of the vulnerability of the blood-brain barrier in MMD, it is advisable to gather cases with prolonged observations of ICG video angiography after direct revascularization.
PubMed: 38531081
DOI: 10.3171/CASE2465 -
Frontiers in Microbiology 2024We present a novel optical nanomotion-based rapid antibiotic and antifungal susceptibility test. The technique consisted of studying the effects of antibiotics or...
We present a novel optical nanomotion-based rapid antibiotic and antifungal susceptibility test. The technique consisted of studying the effects of antibiotics or antifungals on the nanometric scale displacements of bacteria or yeasts to assess their sensitivity or resistance to drugs. The technique relies on a traditional optical microscope, a video camera, and custom-made image analysis software. It provides reliable results in a time frame of 2-4 h and can be applied to motile, non-motile, fast, and slowly growing microorganisms. Due to its extreme simplicity and low cost, the technique can be easily implemented in laboratories and medical centers in developing countries.
PubMed: 38516011
DOI: 10.3389/fmicb.2024.1328923 -
Biological Imaging 2023In vivo fluorescence microscopy is a powerful tool to image the beating heart in its early development stages. A high acquisition frame rate is necessary to study its...
In vivo fluorescence microscopy is a powerful tool to image the beating heart in its early development stages. A high acquisition frame rate is necessary to study its fast contractions, but the limited fluorescence intensity requires sensitive cameras that are often too slow. Moreover, the problem is even more complex when imaging distinct tissues in the same sample using different fluorophores. We present Paired Alternating AcQuisitions, a method to image cyclic processes in multiple channels, which requires only a single (possibly slow) camera. We generate variable temporal illumination patterns in each frame, alternating between channel-specific illuminations (fluorescence) in odd frames and a motion-encoding brightfield pattern as a common reference in even frames. Starting from the image pairs, we find the position of each reference frame in the cardiac cycle through a combination of image-based sorting and regularized curve fitting. Thanks to these estimated reference positions, we assemble multichannel videos whose frame rate is virtually increased. We characterize our method on synthetic and experimental images collected in zebrafish embryos, showing quantitative and visual improvements in the reconstructed videos over existing nongated sorting-based alternatives. Using a 15 Hz camera, we showcase a reconstructed video containing two fluorescence channels at 100 fps.
PubMed: 38510170
DOI: 10.1017/S2633903X23000223 -
Biophysical Reports Jun 2024We present a method for tracking densely clustered, high-velocity, indistinguishable objects being spawned at a high rate and moving in a directed force field using only...
We present a method for tracking densely clustered, high-velocity, indistinguishable objects being spawned at a high rate and moving in a directed force field using only object centroids as inputs and no other image information. The algorithm places minimal restrictions on the velocities or accelerations of the objects being tracked and uses a methodology based on a scoring function and a backtracking refinement process. This combination leads to successful tracking of hundreds of particles in challenging environments even when the displacement of the individual objects at successive times approaches the separation between neighboring objects in any one frame. We note that these cases can be particularly difficult to handle by existing methods. The performance of the algorithm is methodically examined by comparison to simulated trajectories, which vary the temporal and spatial densities, velocities, and accelerations of the objects in motion, as well as the signal/noise ratio. Also, we demonstrate its capability by analyzing data from experiments with superparamagnetic microspheres moving in an inhomogeneous magnetic field in aqueous buffer at room temperature. Our method should be widely applicable since trajectory determination problems are ubiquitous in video microscopy applications in biology, materials science, physics, and engineering.
PubMed: 38505834
DOI: 10.1016/j.bpr.2024.100148 -
Photodiagnosis and Photodynamic Therapy Apr 2024Hypoxia is a characteristic feature of many tumors. It promotes tumor proliferation, metastasis, and invasion and can reduce the effectiveness of many types of cancer...
BACKGROUND
Hypoxia is a characteristic feature of many tumors. It promotes tumor proliferation, metastasis, and invasion and can reduce the effectiveness of many types of cancer treatment.
OBJECTIVE
The aim of this study was to investigate the pharmacokinetics of methylene blue (MB) and its impact on the tumor oxygenation level at mouse Lewis lung carcinoma (LLC) model using spectroscopic methods.
APPROACH
The pharmacokinetics of MB were studied qualitatively and quantitatively using video fluorescence imaging and fluorescence spectroscopy. The degree of hemoglobin oxygenation in vivo was examined by calculating hemoglobin optical absorption from the measured diffuse reflectance spectra. The distribution of MB fluorescence and the lifetime of NADH were analyzed using laser scanning microscopy and fluorescence lifetime imaging microscopy (FLIM) to assess cellular metabolism.
RESULTS
After intravenous administration of MB at 10-20 mg/kg, it quickly transitioned in the tumor to a colorless leucomethylene blue, with maximum accumulation in the tumor occurring after 5-10 min. A concentration of 10 mg/kg resulted in a relative increase of the tumor oxygenation level for small tumors (volume 50-75 mm) and normal tissue 120 min after the introduction of MB. A shift in tumor metabolism towards oxidative phosphorylation (according to the lifetime of the NADH coenzyme) was measured using FLIM method after intravenous administration of 10 mg/kg of MB. Intravenous administration of MB at 20 mg/kg results in a long-term decrease in oxygenation, which persisted for at least 120 min after the administration and did not return to its initial level.
CONCLUSIONS
Administration of MB at 10 mg/kg shown to increase tumor oxygenation level, potentially leading to more effective antitumor therapy. However, at higher doses (20 mg/kg), MB may cause long-term decrease in oxygenation.
Topics: Methylene Blue; Animals; Mice; Carcinoma, Lewis Lung; Oxygen; Photosensitizing Agents; Mice, Inbred C57BL; Dose-Response Relationship, Drug; Photochemotherapy; Cell Line, Tumor; Spectrometry, Fluorescence
PubMed: 38503388
DOI: 10.1016/j.pdpdt.2024.104047