Microscopy Image Analysis Research Articles

Pellet-Based Extrusion Additive Manufacturing of Lightweight Parts Using Inflatable Hollow Extrudates

Additive manufacturing (AM) has become a key element of Industry 4.0, particularly the extrusion AM (EAM) of thermoplastic materials, which is recognized as the most widely used technology. Fused Filament Fabrication (FFF), however, depends on expensive commercially available filaments, making pellet extruder-based EAM techniques more desirable. Large-format EAM systems could benefit from printing lightweight objects with reduced material use and lower power consumption by utilizing hollow rather than solid extrudates. In this study, a custom extruder head was designed and an EAM system capable of extruding inflatable hollow extrudates from a variety of materials was developed. By integrating a co-axial nozzle-needle system, a thermoplastic shell was extruded while creating a hollow core using pressurized nitrogen gas. This method allows for the production of objects with gradient part density and varied mechanical properties by controlling the inflation of the hollow extrudates. The effects of process parameters— such as extrusion temperature, extrusion speed, and gas pressure were investigated—using poly-lactic acid (PLA) and styrene-ethylene-butylene-styrene (SEBS) pellets. The preliminary tests identified the optimal range of these parameters for consistent hollow extrudates. We then varied the parameters to determine their impact on the dimensions of the extrudates, supported by analyses of microscopic images taken with an optical microscope. Our findings reveal that pressure is the most influential factor affecting extrudate dimensions. In contrast, variations in temperature and extrusion speed had a relatively minor impact, whereas changes in pressure led to significant alterations in the extrudate’s size and shape.

Rapid iPSC-derived neuromuscular junction model uncovers motor neuron dominance in amyotrophic lateral sclerosis cytopathy

The neuromuscular junction (NMJ) is essential for transmitting signals from motor neurons (MNs) to skeletal muscles (SKMs), and its dysfunction can lead to severe motor disorders. However, our understanding of the NMJ is limited by the absence of accurate human models. Although human induced pluripotent stem cell (iPSC)-derived models have advanced NMJ research, their application is constrained by challenges such as limited differentiation efficiency, lengthy generation times, and cryopreservation difficulties. To overcome these limitations, we developed a rapid human NMJ model using cryopreserved MNs and SKMs derived from iPSCs. Within 12 days of coculture, we successfully recreated NMJ-specific connectivity that closely mirrors in vivo synapse formation. Using this model, we investigated amyotrophic lateral sclerosis (ALS) and replicated ALS-specific NMJ cytopathies with SOD1 mutant and corrected isogenic iPSC lines. Quantitative analysis of 3D confocal microscopy images revealed a critical role of MNs in initiating ALS-related NMJ cytopathies, characterized by alterations in the volume, number, intensity, and distribution of acetylcholine receptors, ultimately leading to impaired muscle contractions. Our rapid and precise in vitro NMJ model offers significant potential for advancing research on NMJ physiology and pathology, as well as for developing treatments for NMJ-related diseases.

Insight into unusual complex thermodynamical behaviour of citric acid and ethanolamine solution

Inverse freezing fluids (IFF) are the new field of science that thermodynamically acts opposite to conventional fluids. These IFFs are novel artificial materials that work as typical thermal metamaterials. This paper reports the IFF phenomenon with basic chemicals such as citric acid and ethanolamine subjected to different conditions. The amine molecule within the precursor compound is crucial in attaining this phenomenon. Its respective inter and intramolecular ‘hydrogen’ atoms remain the key reason for this effect. Results of rheometry, Raman, proton nuclear magnetic resonance (1HNMR), photoluminescence (PL) spectroscopy, and thermogravimetric analysis (TGA) of this fluid confirmed this phenomena, collectively. Experimental findings confirm the reversible phase transformation which is contrary to the conventional fluids. It will open the interest in finding new thermal metamaterials. This study can be considered the first attempt at synthesizing IFFs through a new preparation method and analysed in detail. Many challenges were faced during synthesis and characterizations, so this study will help to resolve the complexity in this domain. Further, the sample was sintered and found to be multi-layered reduced graphene oxide (rGO) powder. This rGO sample’s structural, chemical nature, and morphology are studied through x-ray diffraction (XRD), Fourier transformed infra-red (FTIR), Raman spectroscopy, scanning electron microscope (SEM), transmission electron microscope imaging and energy dispersive analysis of x-rays (EDAX). XRD pattern confirmed the presence of the main peak at 26° (2θ) corresponding to the basal reflection (002) of rGO phase. It was further evidenced by its D and G bands of Raman spectra at 1357 and 1574 cm−1, respectively. FTIR analysis also revealed a reduced OH- band whereas EDAX analysis confirms the purity of rGO by the presence of carbon and oxygen peaks only. Multilayer formation of rGO was confirmed by SEM and TEM observations and found that the measured thickness of the sheet was nearly 1 nm.Graphical

Expanding the Diversity of Actinobacterial Tectiviridae: A Novel Genus from Microbacterium.

Six novel Microbacterium phages belonging to the Tectiviridae family were isolated using Microbacterium testaceum as a host. Phages MuffinTheCat, Badulia, DesireeRose, Bee17, SCoupsA, and LuzDeMundo were purified from environmental samples by students participating in the Science Education Alliance Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES) program at Alliance University, New York. The phages have linear dsDNA genomes 15,438-15,636 bp with 112-120 bp inverted terminal repeats. Transmission electron microscopy (TEM) imaging analysis revealed that the six novel phages have six-sided icosahedral double-layered capsids with an internal lipid membrane that occasionally forms protruding nanotubules. Annotation analysis determined that the novel Microbacterium phages all have 32-34 protein-coding genes and no tRNAs. Like other Tectiviridae, the phage genomes are arranged into two segments and include three highly conserved family genes that encode a DNA polymerase, double jelly-roll major capsid protein, and packaging ATPase. Although the novel bacteriophages have 91.6 to 97.5% nucleotide sequence similarity to each other, they are at most 58% similar to previously characterized Tectiviridae genera. Consequently, these novel Microbacterium phages expand the diversity of the Tectiviridae family, and we propose they form the sixth genus, Zetatectivirus.

Outlier removal in cryo-EM via radial profiles.

The process of particle picking, a crucial step in cryo-electron microscopy (cryo-EM) image analysis, often encounters challenges due to outliers, leading to inaccuracies in downstream processing. In response to this challenge, this research introduces an additional automated step to reduce the number of outliers identified by the particle picker. The proposed method enhances both the accuracy and efficiency of particle picking, thereby reducing the overall running time and the necessity for expert intervention in the process. Experimental results demonstrate the effectiveness of the proposed approach in mitigating outlier inclusion and its potential to enhance cryo-EM data analysis pipelines significantly. This work contributes to the ongoing advancement of automated cryo-EM image processing methods, offering novel insights and solutions to challenges in structural biology research.

Microfluidics to Follow Spatiotemporal Dynamics at the Nucleo-Cytoplasmic Interface During Plant Root Growth.

Nuclear dynamics refers to global/local changes in the molecular and spatial organization of genomic DNA that can occur during development or in response to environmental stress signals and eventually impact genomic functions. In plants, nuclear dynamics relies notably on the connection of the nucleus with the cytoskeleton during development. It orchestrates genomic functions in response to developmental and environmental cues. This is particularly true in the plant root system, which is constantly exposed to a wide range of internal and external stimuli. Currently, studying nuclear dynamics in a growing root is challenging due to limitations regarding real-time imaging for quantitative analyses under controlled conditions. Microfluidic systems for plant cell studies are valuable analytical tools that provide precise control of culture conditions together with live-imaging capabilities at high temporal and spatial resolutions. Herein, we describe a microfluidic platform to unravel dynamically and noninvasively nuclear organization in the seedling root system exposed to various treatments. As exemplified here, our microfluidic platform can be conveniently used for real-time microscopy imaging and quantitative analysis of fine nuclear morphological changes upon modifying cytoskeleton dynamics. Importantly, our system can be applied to a wide variety of microscopic means including high-resolution microscopy to investigate diverse subcellular compartments or nuclear domains in Arabidopsis thaliana roots.

Versatile platforms of mussel-inspired agarose scaffold for cell cultured meat.

Biomaterial scaffolds are critical for cell cultured meat production. polysaccharide scaffolds lack essential animal cell adhesion receptors, leading to significant challenges in cell proliferation and myogenic differentiation. Thus, enhancing cell adhesion and growth on polysaccharide scaffolds is strongly required to supply the gaps in cell-cultured meat production. This study aims to develop a multifunctional cell-responsive hydrogel scaffold for the in vitro production of myofibers and structured cell cultured meat through a "cell adhesion-proliferation-differentiation" strategy. A polydopamine coating was applied to agarose hydrogel scaffolds using a dipping technique. The capability of scaffolds for myofiber preparation was assessed by evaluating cell adhesion, proliferation, and myogenic differentiation. Utilizing isolated porcine skeletal muscle satellite cells (PSMSCs), the feasibility of structured cell cultured pork tissue supported by agarose hydrogel film scaffolds was further investigated through three-dimensional imaging and scanning electron microscopy analysis. The physicochemical properties of the structured cell cultured pork tissue were evaluated through staining and texture analysis. The incorporation of a polydopamine coating facilitated a remarkable 100% cell adhesion rate on agarose hydrogel scaffolds, which also demonstrated reusability. The agarose hydrogel scaffolds retained adequate mechanical properties, enabling the adhered cells to proliferate effectively and differentiate into myofiber. Moreover, isolated PSMSCs maintained growth potential on the agarose hydrogel scaffolds, thereby imparting the scaffolds with the ability to generate substantial quantities of multinucleated myofibers. Furthermore, we established a structured cell culture pork meat model, characterized by high-density myofibers and agarose hydrogel film scaffolds, which exhibited the texture and color typical of real pork. The innovative agarose/polydopamine scaffold functions as a multifunctional platform for cell culture, offering novel avenues for the diversification and scalable production of cultured meat, and promising significant reductions in production costs for cell cultured meat.

Synergistic effect and mechanism of ferric ion modified citric acid depressant in the flotation separation of scheelite from cassiterite

Scheelite and cassiterite often coexist in skarn deposits, which are prone to excessive crushing during dissociation, and gravity separation faces a challenge. However, research on flotation separation is limited. This study aimed to investigate the selective depression behavior and depression mechanism of a combined depressant containing ferric chloride and citric acid on cassiterite to successfully achieve the flotation separation of scheelite and cassiterite. Experimental studies on single minerals and artificially mixed minerals indicated that the individual ferric chloride or citric acid exhibited weak depressive abilities and poor selectivity toward cassiterite. However, a combination of ferric chloride and citric acid in a mass ratio of 3:7 achieved better separation results. At a pH of 8, with a concentration of 1 × 10–3 mol/L mixed depressant, and a sodium oleate concentration of 5 × 10–5 mol/L, scheelite and cassiterite exhibited recovery of 73.35 % and 19.98 %, with grades of 61.76 % and 16.18 %, respectively. Fourier–transform infrared spectroscopy and X–ray photoelectron spectroscopy analyses revealed that the mixed depressant strongly chemically adsorbed on the cassiterite surface, forming FeOOH at ferric species sites. Contact angle measurements indicated a significant reduction in surface hydrophobicity of the cassiterite surface due to mixed depressant components. Atomic force microscopy imaging analysis elucidated the adsorption morphology of the mixed depressant depressive component FeOOH on the cassiterite surface in both 2D and 3D imaging, indicating abundant high–intensity reagent peak adsorption. Therefore, the mixed depressant enhanced the selectivity of the depressant, facilitating the effective separation of scheelite and cassiterite.

Accelerating the Discovery of Abiotic Vesicles with AI-Guided Automated Experimentation.

The first protocells are speculated to have arisen from the self-assembly of simple abiotic carboxylic acids, alcohols, and other amphiphiles into vesicles. To study the complex process of vesicle formation, we combined laboratory automation with AI-guided experimentation to accelerate the discovery of specific compositions and underlying principles governing vesicle formation. Using a low-cost commercial liquid handling robot, we automated experimental procedures, enabling high-throughput testing of various reaction conditions for mixtures of seven (7) amphiphiles. Multitemplate multiscale template matching (MMTM) was used to automate confocal microscopy image analysis, enabling us to quantify vesicle formation without tedious manual counting. The results were used to create a Gaussian process surrogate model, and then active learning was used to iteratively direct the laboratory experiments to reduce model uncertainty. Mixtures containing primarily trimethyl decylammonium and decylsulfate in equal amounts formed vesicles at submillimolar critical vesicle concentrations, and more than 20% glycerol monodecanoate prevented vesicles from forming even at high total amphiphile concentrations.

Enabling Electric Field Model of Microscopically Realistic Brain.

Modeling brain stimulation at the microscopic scale may reveal new paradigms for various stimulation modalities. We present the largest map to date of extracellular electric field distributions within a layer L2/L3 mouse primary visual cortex brain sample. This was enabled by the automated analysis of serial section electron microscopy images with improved handling of image defects, covering a volume of 250 × 140 × 90 μm³. The map was obtained by applying a uniform brain stimulation electric field at three different polarizations and accurately computing microscopic field perturbations using the boundary element fast multipole method. We used the map to identify the effect of microscopic field perturbations on the activation thresholds of individual neurons. Previous relevant studies modeled a macroscopically homogeneous cortical volume. Our result shows that the microscopic field perturbations - an 'electric field spatial noise' with a mean value of zero - only modestly influence the macroscopically predicted stimulation field strengths necessary for neuronal activation. The thresholds do not change by more than 10% on average. Under the stated limitations and assumptions of our method, this result essentially justifies the conventional theory of "invisible" neurons embedded in a macroscopic brain model for transcranial magnetic and transcranial electrical stimulation. However, our result is solely sample-specific and is only relevant to this relatively small sample with 396 neurons. It largely neglects the effect of the microcapillary network. Furthermore, we only considered the uniform impressed field and a single-pulse stimulation time course.

Engineering Adhesion of the Probiotic Strain Escherichia coli Nissle to the Fungal Pathogen Candida albicans.

Engineering live biotherapeutic products against fungal pathogens such as Candida albicans has been suggested as a means to tackle the increasing threat of fungal infections and the development of resistance to classical antifungal treatments. One important challenge in the design of live therapeutics is to control their localization inside the human body. The specific binding capability to target organisms or tissues would greatly increase their effectiveness by increasing the local concentration of effector molecules at the site of infection. In this study, we utilized surface display of carbohydrate binding domains to enable the probiotic E. coli Nissle 1917 to adhere specifically to the pathogenic yeast Candida albicans. Binding was quantified using a newly developed method based on the automated analysis of microscopic images. In addition to a rationally selected chitin binding domain, a synthetic peptide of identical length but distinct sequence also conferred binding. Efficient binding was specific to fungal hyphae, the invasive form of C. albicans, while the yeast form, as well as abiotic cellulose and PET particles, was only weakly recognized.

Oral Curcumin through Mesoporous Silica Nanomaterials with Distinct Morphologies: Synthesis, Characterization, Biosafety Evaluation, and Antioxidant Activity In Vivo.

Antioxidant play a crucial role in the prevention and treatment of diseases associated with oxidative stress. Curcumin (CUR), as a natural antioxidant, exhibits numerous therapeutic properties, including antioxidant, anti-inflammatory, antibacterial, and antitumor activities. However, its limited bioavailability and poor water solubility hinder its application as an effective antioxidant. In this study, a series of mesoporous silica nanomaterials with distinct morphologies, i.e., mesoporous silica nanoparticles (MSN) and mesoporous silica nanorods (MSR) were synthesized by a template-sediment-etching method. CUR was selected as a model drug and encapsulated into these nanomaterials to improve its bioavailability in vivo. The morphology and size distribution of MSN and MSR were determined through transmission electron microscopy (TEM) imaging and Zetasizer analysis. Fourier transform infrared spectroscopy (FTIR) spectra confirmed the successful encapsulation of CUR within these nanomaterials. Furthermore, these CUR-loaded silica nanomaterials, denoted as CUR@MSN and CUR@MSR, exhibited excellent DPPH and ABTS free radical scavenging activity in vitro. Furthermore, CUR@MSN and CUR@MSR also exhibited obvious in vivo antioxidant activity. This study opens up new avenues for the development of enhanced antioxidants through the utilization of mesoporous silica nanomaterials.

Recent Technologies on 2D and 3D Imaging Flow Cytometry.

Imaging flow cytometry is a technology that performs microscopy image analysis of cells within flow cytometry and allows high-throughput, high-content cell analysis based on their intracellular molecular distribution and/or cellular morphology. While the technology has been available for a couple of decades, it has recently gained significant attention as technical limitations for higher throughput, sorting capability, and additional imaging dimensions have been overcome with various approaches. These evolutions have enabled imaging flow cytometry to offer a variety of solutions for life science and medicine that are not possible with conventional flow cytometry or microscopy-based screening. It is anticipated that the extent of applications will expand in the upcoming years as the technology becomes more accessible through dissemination. In this review, we will cover the technical advances that have led to this new generation of imaging flow cytometry, focusing on the advantages and limitations of each technique.

Behavior of PGS/apatite foam scaffolds during incubation in SBF, PBS, Ringer's solution, artificial saliva, and distilled water.

Poly(glycerol sebacate) is a polymeric material with potential biomedical application in the field of tissue engineering. In order to act as a biodegradable scaffold, its incubation study is vital to simulate its behavior. This study explores the degradation of porous poly(glycerol sebacate)/hydroxyapatite scaffolds subjected to incubation in various physiological solutions. The research involved monitoring pH and conductivity values over a 14-day period, as well as analyzing the swelling capacity and mass alterations of the scaffolds. In simulated body fluid (SBF) and phosphate-buffered saline (PBS), the pH levels remained relatively stable, whereas Ringer's solution caused a pH decrease. Conversely, artificial saliva demonstrated an increase in pH, and distilled water caused a slight decrease. The conductivity values remained stable in SBF and Ringer's solution, slightly decreased in PBS, increased in artificial saliva, and significantly increased in distilled water. The swelling capacity of the scaffolds varied depending on the solution used, with the lowest equilibrium swelling observed in SBF and PBS. The effect of the presence of ceramics on this parameter was also observed. The mass changes of the scaffolds indicated deposition of particles or salts from the incubation solutions, and subsequent rinsing in distilled water led to a decrease in mass. Scanning electron microscopy (SEM) imaging and elemental analysis confirmed the presence of crystallized salts on the scaffold surfaces after incubation in SBF. Surface roughness measurements revealed changes in roughness depending on the solution, with deposition of additional layers in SBF and degradation in artificial saliva. In summary, the scaffolds exhibited biodegradation in physiological solutions, with variations in pH, conductivity, swelling capacity, mass changes, and surface morphology depending on the specific solution and scaffold composition.

Assembly and Evaluation of a Confocal Microscopy Image Analysis Pipeline Useful in Revealing the Secrets of Plant-Fungal Interactions.

The ability of laser scanning confocal microscopy to generate high-contrast 2D and 3D images has become essential in studying plant-fungal interactions. Techniques such as visualization of native fluorescence, fluorescent protein tagging of microbes, green fluorescent protein (GFP)/red fluorescent protein (RFP)-fusion proteins, and fluorescent labeling of plant and fungal proteins have been widely used to aid in these investigations. Use of fluorescent proteins has several pitfalls, including variability of expression in planta and the requirement of gene transformation. Here, we used the unlabeled pathogens Parastagonospora nodorum, Pyrenophora teres f. teres, and Cercospora beticola infecting wheat, barley, and sugar beet, respectively, to show the utility of a staining and imaging pipeline that uses propidium iodide (PI), which stains RNA and DNA, and wheat germ agglutinin labeled with fluorescein isothiocyanate (WGA-FITC), which stains chitin, to visualize fungal colonization of plants. This pipeline relies on the use of KOH to remove the cutin layer of the leaf, increasing its permeability, allowing the different stains to penetrate and effectively bind to their targets, resulting in a consistent visualization of cellular structures. To expand the utility of this pipeline, we used the staining techniques in conjunction with machine learning to analyze fungal biomass through volume analysis, as well as quantifying nuclear breakdown, an early indicator of programmed cell death (PCD). This pipeline is simple to use, robust, consistent across host and fungal species, and can be applied to most plant-fungal interactions. Therefore, this pipeline can be used to characterize model systems as well as nonmodel interactions where transformation is not routine. [Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 "No Rights Reserved" license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2024.

Enabling Electric Field Model of Microscopically Realistic Brain.

This study is arguably the first attempt to model brain stimulation at the microscopic scale, enabled by automated analysis of modern scanning electron microscopy images of the brain. It concentrates on modeling microscopic perturbations of the extracellular electric field caused by the physical cell structure and is applicable to any type of brain stimulation. Post-processed cell CAD models (383, stl format), microcapillary CAD models (34, stl format), post-processed neuron morphologies (267, swc format), extracellular electric field and potential distributions at different polarizations (267x3, MATLAB format), *.ses projects files for biophysical modeling with Neuron software (267x2, Neuron format), and computed neuron activating thresholds at different conditions (267x8, Excel tables, without the sample polarization correction from Section 2.8) are made available online through BossDB , a volumetric open-source database for 3D and 4D neuroscience data.

Weakly-Supervised Hair SEM Microscope Image Segmentation Using a Priori Structure Information

The analysis of microscopic images of hair has a wide range of applications in the domains of cosmetics, healthcare and forensics. The segmentation of the hair represents the initial step of automatic large-scale quantitative analysis of microscopic images of hair. It ensures that subsequent quantitative measurements are performed in an appropriate area corresponding to the hair, avoiding artifacts near the boundaries. This process can be time-consuming, tedious and susceptible to subjective errors when conducted by a human operator. Deep learning methods represent a promising solution; however, obtaining pixel-level accurate masks is a costly process. This paper presents a novel weakly supervised pipeline for the segmentation of hair SEM (Scanning Electron Microscope) microscopic images, which requires only simple image-level annotations for training. The proposed method incorporates the Radon transform, the Sobel operator and a novel Boundary Discrimination Module (BD-module) for the estimation of the presence of boundaries. The proposed pipeline was evaluated on a recently collected hair SEM dataset (429 images). Furthermore, it is benchmarked with methods including Unet and Segment Anything Model (SAM). The results demonstrated a mean Hausdorff Distance improvement of over 30% and a standard deviation improvement of over 50% in comparison with the Unet and SAM. Moreover, we proposed additional refinement modules to address boundary nonlinear cases and conducted Grad-CAM analysis to enhance the interpretability of the BD-module. Additionally, we proposed a novel quality estimation metric based on gradient map for self-quality assessment. The SEM hair dataset is accessible to the research community in an open-source format.

Investigating the geomechanical behavior and mechanism of clay stabilization with natural zeolite and nano-magnetite under accelerated curing conditions (ACC)

Chemical stabilization is a commonly used technique for ground improvement. Traditional methods of chemical soil stabilization rely on additives such as cement and lime, which are relatively expensive and not environmentally friendly. Therefore, using accessible natural pozzolans, such as zeolite, in soil stabilization is an effective and economical option. This study investigated the effect of zeolite and nano-magnetite on the geomechanical behavior of fine-grained clayey soil. Unconfined compressive strength (UCS), ultrasonic pulse velocity (UPV), and electrical conductivity (EC) tests were performed on both stabilized and unstabilized samples. The samples were cured under standard curing conditions (SCC) and accelerated curing conditions (ACC). The UCS and UPV results for the clay sample containing 5% zeolite and 1% nano-magnetite showed significant increases of 85% and 48% under SCC and 174% and 59% under ACC, respectively, compared to untreated clayey soil. Additionally, the clay mixture containing 5% zeolite and 1% nano-magnetite, which achieved the highest UCS and UPV values under ACC, exhibited increases of 166 kPa and 100 m/s, respectively, compared to SCC. Moreover, the EC value decreased by approximately 51% and 60% for the samples with the highest strength in SCC and ACC, respectively. Based on Scanning Electron Microscope (SEM) imaging and Energy Dispersive Spectroscopy (EDS) analysis, the impact of zeolite and nano-magnetite on improving compaction and promoting hydrothermal reactions was evident, supporting the higher geomechanical results obtained. Overall, the findings suggest that using zeolite and nano-magnetite offers a cost-effective and eco-friendly solution for stabilizing clayey soil in civil engineering projects.

Measuring Metabolic Changes in Cancer Cells Using Two-Photon Fluorescence Lifetime Imaging Microscopy and Machine-Learning Analysis.

Two-photon (2P) fluorescence lifetime imaging microscopy (FLIM) was used to track cellular metabolism with drug treatment of auto-fluorescent coenzymes NAD(P)H and FAD in living cancer cells. Simultaneous excitation at 800 nm of both coenzymes was compared with traditional sequential 740/890 nm plus another alternative of 740/800 nm, before and after adding doxorubicin in an imaging time course. Changes of redox states at single cell resolution were compared by three analysis methods: our recently introduced fluorescence lifetime redox ratio (FLIRR: NAD(P)H-a2%/FAD-a1%), machine-learning (ML) algorithms using principal component (PCA) and non-linear multi-Feature autoencoder (AE) analysis. While all three led to similar biological conclusions (early drug response), the ML models provided statistically the most robust significant results. The advantage of the single 800 nm excitation of both coenzymes for metabolic imaging in above mentioned analysis is demonstrated.

MVE-Net: A label-free microscopic image visual enhancement network via mRetinex and nonreference loss guidance

Label-free microscopic cell image analysis (segmentation, detection, counting, e.g.) is elementary for unravelling the biological functions of cells and their organelles. However, low contrast, darker brightness, background inhomogeneous, and weak edges of cells cause challenges in subsequent cell image analysis processes. To address these challenges, a Microscopic Visual Enhancement Network (MVE-Net) is proposed to improve microscopic visual effects through pre-enhancement and enhancement processes. In the pre-enhancement stage, to overcome the difficulty of acquiring paired or unpaired images for training, the mRetinex block is proposed to guide the pre-enhancement network to image contrast, cell details, and structural features. Furthermore, a multi-scale extraction module is employed to extract and fuse cell texture and structural features from the pre-enhanced images at various scales, guiding the generator training. In the enhancement stage, a nonreference loss block is designed, incorporating spatial consistency, uneven illumination smoothness, and exposure adjustment loss terms, to further enhance the contrast between cells and the background, smooth the inhomogeneous background, and adjust overall image brightness, thereby guiding the generator's enhancement process and improving the visual effect of microscopic images. Experiments on the LIVECell and PNT1A datasets demonstrate that MVE-Net outperforms state-of-the-art image enhancement methods, significantly improving image contrast, brightness, cell detail, and structural features without the need for paired or unpaired reference standard images for training.