Intracellular Structures Research Articles

Methods for Image Analysis of Intracellular Structures of Actin Labeled with Phalloidin

A cell is a complex three-dimensional system, which possesses a number of highly dynamic structures with extended, rugged, and uneven morphology. The actin cytoskeleton consists of fibrillar and globular actin, as well as auxiliary proteins that regulate organization. The shape and the rearrangements of actin cytoskeleton are closely related to functioning of the cell. The ability to characterize these changes allows scientists to confirm or refute any hypotheses in the research. Obtaining a numerical equivalent of the actin cytoskeleton organization could help compare actin structures in biological experiments (example: exposure to biologically active substances). The review summarizes methods for analyzing images of intracellular actin structures labeled with phalloidin using ImageJ. The methods considered make it possible to obtain a quantitative characteristic of the organization of actin structures for further evaluation and comparison of experimental results.

Integrins in the kidney - beyond the matrix.

The development and proper functioning of the kidney is dependent on the interaction of kidney cells with the surrounding extracellular matrix (ECM). These interactions are mediated by heterodimeric membrane-bound receptors called integrins, which bind to the ECM via their extracellular domain and via their cytoplasmic tail to intracellular adaptor proteins, to assemble large macromolecular adhesion complexes. These interactions enable integrins to control cellular functions such as intracellular signalling and organization of the actin cytoskeleton and are therefore crucial to organ function. The different nephron segments and the collecting duct system have unique morphologies, functions and ECM environments and are thus equipped with unique sets of integrins with distinct specificities for the ECM with which they interact. These cell-type-specific functions are facilitated by specific intracellular integrin binding proteins, which are critical in determining the integrin activation status, ligand-binding affinity and the type of ECM signals that are relayed to the intracellular structures. The spatiotemporal expression of integrins and their specific interactions with binding partners underlie the proper development, function and repair processes of the kidney. This Review summarizes our current understanding of how integrins, their binding partners and the actin cytoskeleton regulate kidney development, physiology and pathology.

Actin polymerization counteracts prewetting of N-WASP on supported lipid bilayers

Cortical condensates, transient punctate-like structures rich in actin and the actin nucleation pathway member Neural Wiskott-Aldrich syndrome protein (N-WASP), form during activation of the actin cortex in the Caenorhabditis elegans oocyte. Their emergence and spontaneous dissolution is linked to a phase separation process driven by chemical kinetics. However, the mechanisms that drive the onset of cortical condensate formation near membranes remain unexplored. Here, using a reconstituted phase separation assay of cortical condensate proteins, we demonstrate that the key component, N-WASP, can collectively undergo surface condensation on supported lipid bilayers via a prewetting transition. Actin partitions into the condensates, where it polymerizes and counteracts the N-WASP prewetting transition. Taken together, the dynamics of condensate-assisted cortex formation appear to be controlled by a balance between surface-assisted condensate formation and polymer-driven condensate dissolution. This opens perspectives for understanding how the formation of complex intracellular structures is affected and controlled by phase separation.

Study on the Effect of Phillyrin on Streptococcus suis In Vivo and In Vitro

As a zoonotic pathogen, S. suis serotype 2 (SS2) can cause severe diseases in both pigs and humans, and develop resistance to antibiotics. Plant natural compounds are regarded as promising alternatives to conventional antibiotics. Phillyrin is the major bioactive components of Chinese herbal medicine Forsythia suspensa. In this study, we explored the activity and action mechanism of phillyrin against SS2. The results showed that phillyrin could disrupt membrane integrity, destroy intracellular structures, and increase the exosmosis of DNA. Results of PCR revealed that phillyrin affected bacterial-virulence-related genes’ expression levels. Meanwhile, phillyrin significantly decreased the adhesion activity, inhibited lactate dehydrogenase (LDH) secretion, and reduced biofilm formation of SS2 in Newborn pig trachea epithelial (NPTr) cells. Furthermore, phillyrin protected tight junction protein of NPTr cells from SS2. We reported that phillyrin (0.1 mg/kg) treatment after bacterial challenge significantly improved the survival rate, ameliorated pulmonary inflammation, and inhibited the accumulation of multiple cytokines (IL-1, IL-6, IL-8, and TNF-α). Molecular docking showed that phillyrin had a good binding activity with the Ala88 and Asp111 of suilysin (SLY), one of the most important virulence factors of SS2. Collectively, phillyrin possesses antibacterial and anti-inflammatory activities, and is a promising candidate for preventing SS2 infection.

GPR180 is a new member of the Golgi-dynamics domain seven-transmembrane helix protein family

GOLD domain seven-transmembrane helix (GOST) proteins form a new protein family involved in trafficking of membrane-associated cargo. They share a characteristic extracellular/luminal Golgi-dynamics (GOLD) domain, possibly responsible for ligand recognition. Based on structural homology, GPR180 is a new member of this protein family, but little is known about the cellular role of GPR180. Here we show the X-ray structure of the N-terminal domain of GPR180 (1.9 Å) and can confirm the homology to GOLD domains. Using cellular imaging we show the localization of GPR180 in intracellular vesicular structures implying its exposure to acidic pH environments. With Hydrogen/Deuterium Exchange-Mass Spectrometry (HDX-MS) we identify pH-dependent conformational changes, which can be mapped to a putative ligand binding site in the transmembrane region. The results reveal GPR180’s role in intracellular vesicles and offer insights into the pH-dependent function of this conserved GOST protein.

Mitigating methylglyoxal-induced glycation stress: the protective role of iron, copper, and manganese coordination compounds in Saccharomyces cerevisiae.

Glycation-induced stress (G-iS) is a physiological phenomenon that leads to the formation of advanced glycation end-products, triggering detrimental effects such as oxidative stress, inflammation, and damage to intracellular structures, tissues, and organs. This process is particularly relevant because it has been associated with various human pathologies, including cancer, neurodegenerative diseases, and diabetes. As therapeutic alternatives, coordination compounds with antioxidant activity show promising potential due to their versatility in attenuating oxidative stress and inflammation. Herein, we investigated the antioxidant-related protective potential of a series of complexes: [Cu(II)(BMPA)Cl2] (1), [Fe(III)(BMPA)Cl3] (2), and [Cl(BMPA)MnII-(μ-Cl)2-MnII(BMPA)-(μ-Cl)- MnII(BMPA)(Cl)2]•5H2O (3), all synthesized with the ligand bis-(2-pyridylmethyl)amine (BMPA) in Saccharomyces cerevisiae exposed to G-iS caused by methylglyoxal (MG). Pre- treatment with complexes 1-3 proved highly effective, increasing yeast tolerance to G-iS and attenuating mitochondrial dysfunction. This observed phenotype appears to result from a reduction in intracellular oxidation, lipid peroxidation levels, and glycation. Additionally, an increase in the activity of the antioxidant enzymes superoxide dismutase and catalase was observed following treatment with complexes 1-3. Notably, although complexes 1-3 provided significant protection against oxidative stress induced by H2O2 and menadione, their protective role was more effective against MG-induced glycation stress. Our results indicate that these complexes possess both antiglycation and antioxidant properties, warranting further investigation as potential interventions for mitigating glycation and oxidative stress-related pathologies.

Optimal Paradigms for Quantitative Modeling in Systems Biology Demonstrated for Spinal Motor Neuron Synthesis

Since the 1990s, Petri nets have been used in systems biology for quantitative modeling. Despite the increasing number of models developed during this period, doubts remain about their biological relevance. Although biological systems predominantly exhibit intracellular or cellular structures, the models rely largely on deterministic predictions, failing to capture the inherent randomness and uncertainties of such systems. The question arises whether these models accurately describe the dynamic behavior of biological systems. This paper introduces a methodology for selecting the appropriate modeling paradigms in systems biology. Initially, we construct a Petri net model and perform deterministic, stochastic, and fuzzy stochastic simulations. Then we perform various statistical tests to measure the discrepancies between the simulation results. Based on scale-density analysis, we determine the modeling approach that best approximates the biological system. Finally, we compare the results of the statistical tests and the scale-density analysis to identify the optimal modeling approach. We applied the proposed methodology to the synthesis of spinal motor neuron protein from the spinal motor neuron-2 gene. Analysis revealed significant discrepancies between the simulation results of different modeling paradigms. Due to the sparse nature of the underlying drug-disease network, we conclude that the fuzzy stochastic paradigm provides the most biologically relevant results. We predict drug combinations that could lead to an up to 149-fold increase in spinal motor neuron protein levels, indicating a promising treatment for the disease. This methodology has the potential for application to other gene-drug-disease networks and broader biological systems.

Chemical Landscape of Adipocytes Derived from 3T3-L1 Cells Investigated by Fourier Transform Infrared and Raman Spectroscopies.

Adipocytes derived from 3T3-L1 cells are a gold standard for analyses of adipogenesis processes and the metabolism of fat cells. A widely used histological and immunohistochemical staining and mass spectrometry lipidomics are mainly aimed for examining lipid droplets (LDs). Visualizing other cellular compartments contributing to the cellular machinery requires additional cell culturing for multiple labeling. Here, we present the localization of the intracellular structure of the 3T3-L1-derived adipocytes utilizing vibrational spectromicroscopy, which simultaneously illustrates the cellular compartments and provides chemical composition without extensive sample preparation and in the naïve state. Both vibrational spectra (FTIR-Fourier transform infrared and RS-Raman scattering spectroscopy) extended the gathered chemical information. We proved that both IR and RS spectra provide distinct chemical information about lipid content and their structure. Despite the expected presence of triacylglycerols and cholesteryl esters in lipid droplets, we also estimated the length and unsaturation degree of the fatty acid acyl chains that were congruent with known MS lipidomics of these cells. In addition, the clustering of spectral images revealed that the direct surroundings around LDs attributed to lipid-associated proteins and a high abundance of mitochondria. Finally, by using quantified markers of biomolecules, we showed that the fixative agents, paraformaldehyde and glutaraldehyde, affected the cellular compartment differently. We concluded that PFA preserves LDs better, while GA fixation is better for cytochromes and unsaturated lipid analysis. The proposed analysis of the spectral images constitutes a complementary tool for investigations into the structural and molecular features of fat cells.

Highly Tunable, Nanomaterial-Functionalized Structural Templating of Intracellular Protein Structures Within Biological Species.

Inside living organisms, proteins are self-assembled into diverse 3D structures optimized for specific functions. This structure-function relationship can be exploited to synthesize functional materials through biotemplating and depositing functional materials onto protein structures. However, conventional biotemplating faces limitations due to the predominantly intracellular existence of proteins and associated challenges in achieving tunability while preserving functionality. In this study, Conversion to Advanced Materials via labeled Biostructures (CamBio), an integrated biotemplating platform that involves labeling target protein structures with antibodies followed by the growth of functional materials, ensuring outstanding nanostructure tunability is proposed. Protein-derived plasmonic nanostructures created by CamBio can serve as precise quantitative tools for assessing target species is demonstrated. The assessment is achieved through highly tunable and efficient surface-enhanced Raman spectroscopy (SERS). CamBio enables the formation of dense nanogap hot spots among metal nanoparticles, templated by diverse fibrous proteins comprising densely repeated monomers. Furthermore, iterative antibody labeling strategies to adjust the antibody density surrounding targets, amplifying the number of nanogaps and consequently improving SERS performance are employed. Finally, cell-patterned substrates and whole meat sections as SERS substrates, confirming their easily accessible, cost-effective, scalable preparation capabilities and dimensional tunability are incorporated.

Intracellular Redox Environment Determines Cancer-normal Cell Selectivity of Selenium Nanoclusters.

Elucidating the chemical structure and intracellular action mechanisms is still the critical limit for the clinical translation of nanomedicines. Intracellular redox environments originating from cell metabolism are key factors affecting internalized drug efficacy. Herein, we engineer Se-Se/Se-S bond to assemble selenium (Se) nanoclusters (SeClus) with intracellular redox environment-driven selective structure. Chemical structure analysis reveals that, the bonding of sulfur atom in intermediates to the two neighboring or interposition Se atoms in Se rings is the key internal driving force for SeClus formation. This nanocluster can be predominantly transformed to selenocysteine to facilitate selenoproteins synthesis in normal cells, while metabolize to cytotoxic SeO3 2- based on the oxidative intracellular redox environment of cancer cells. Resultantly, SeClus exhibit significant cell proliferation inhibition ability to cancer cells and impressive safety to normal cells. Taken together, this study not only clarifies the chemical nature of the atom engineering of SeClus, but also elucidates its intracellular redox environment-oriented anticancer mechanism.

Impact of photobleaching on quantitative, spatio-temporal, super-resolution imaging of mitochondria in live C. elegans larvae

Super-resolution (SR) 3D rendering allows superior quantitative analysis of intracellular structures but has largely been limited to fixed or ex vivo samples. Here we developed a method to perform SR live imaging of mitochondria during post-embryonic development of C. elegans larvae. Our workflow includes the drug-free mechanical immobilisation of animals using polystyrene nanobeads, which has previously not been used for in vivo SR imaging. Based on the alignment of moving objects and global threshold-based image segmentation, our method enables an efficient 3D reconstruction of individual mitochondria. We demonstrate for the first time that the frequency distribution of fluorescence intensities is not affected by photobleaching, and that global thresholding alone enables the quantitative comparison of mitochondria along timeseries. Our composite approach significantly improves the study of biological structures and processes in SR during C. elegans post-embryonic development. Furthermore, the discovery that image segmentation does not require any prior correction against photobleaching, a fundamental problem in fluorescence microscopy, will impact experimental strategies aimed at quantitatively studying the dynamics of organelles and other intracellular compartments in any biological system.

Label-Free Optical Transmission Tomography for Direct Mycological Examination and Monitoring of Intracellular Dynamics.

Live-cell imaging generally requires pretreatment with fluorophores to either monitor cellular functions or the dynamics of intracellular processes and structures. We have recently introduced full-field optical coherence tomography for the label-free live-cell imaging of fungi with potential clinical applications for the diagnosis of invasive fungal mold infections. While both the spatial resolution and technical set up of this technology are more likely designed for the histopathological analysis of tissue biopsies, there is to our knowledge no previous work reporting the use of a light interference-based optical technique for direct mycological examination and monitoring of intracellular processes. We describe the first application of dynamic full-field optical transmission tomography (D-FF-OTT) to achieve both high-resolution and live-cell imaging of fungi. First, D-FF-OTT allowed for the precise examination and identification of several elementary structures within a selection of fungal species commonly known to be responsible for invasive fungal infections such as Candida albicans, Aspergillus fumigatus, or Rhizopus arrhizus. Furthermore, D-FF-OTT revealed the intracellular trafficking of organelles and vesicles related to metabolic processes of living fungi, thus opening new perspectives in fast fungal infection diagnostics.

Auer rod-positive acute leukemia with predominantly lymphoid immunophenotype: Report on 11 cases and review of literature.

Auer rods (AuRs) are prominent intracellular structures found almost exclusively in myeloid cell malignancies, such as acute myeloid leukemia (AML), chronic and juvenile myelomonocytic leukemia and myelodysplastic syndrome. Extremely rare AuRs have been reported in patients with acute lymphoblastic leukemia (ALL) or among ambiguous lineage leukemia patients with a dominantly lymphoblastic immunophenotype. We report diagnostic and follow-up data of an international cohort of 11 children suffering from leukemias with AuRs and with significant presence of T and myeloid markers, majority of whom categorized as early T-cell precursor (ETP, n=7); or T-ALL (ETP status unknown, n=2), ALAL (acute leukemia of ambiguous lineage, n=1), and AML reclassified from ALAL (n=1). We described other diagnostic details and treatment types and responses. Moreover, we summarize previously published data. Among the four patients who started and remained on ALL-type therapy, all were in the first complete remission, whereas both patients who started and remained on AML-type therapy relapsed and died. Of the patients who followed either a combined ALL/AML protocol (Interfant 06) or who switched from one of the two types of therapy to the other, one patient died, and the remaining four were in first complete remission at the most recent follow-up. We also searched for similar cases in the literature and found only three additional children with nonmyeloid leukemia and AuRs and 10 adults with this type of leukemia. Briefly, ALL- or combined ALL/AML-type therapy may be effective for treating AuR-positive leukemia patients with a lymphoid immunophenotype.

Multi-Channel Signals in Dynamic Force-Clamp Mode of Microcantilever Sensors for Detecting Cellular Peripheral Brush.

The development of numerous diseases, such as renal cyst, cancer, and viral infection, is closely associated with the pathological changes and defects in the cellular peripheral brush. Therefore, it is necessary to develop a potential new method to detect lesions of cellular peripheral brush. Here, a piecewise linear viscoelastic constitutive model of cell is established considering the joint contribution of the peripheral brush and intra-cellular structure. By combining the Laplace transformation and its inverse transformation, and the differential method in the temporal domain and differential quadrature method (DQM) in the spatial domain, the signal interpretation models for quasi-static and dynamic signals of microcantilever are solved. The influence mechanisms of the peripheral brush on the viscoelastic properties of cells and quasi-static/dynamic signals of microcantilever are clarified. The results not only reveal that the peripheral brush has significant effects on the complex modulus of the cell and multi-channel signals of the microcantilever, but also suggest that an alternative mapping method by collecting multi-channel signals including quasi-static and higher frequency signals with more brush indexes could be potentially used to identify cancerous cells.

Considerations for Exploring Nanosecond Pulsed Electric Fields (nsPEFs) for Treatments of Cancer, Benign Skin Diseases, Atrial Fibrillation, and for New Mechanistic Understandings

Pulsed power includes acquiring electrical energy, compressing it, and releasing it in instantaneous bursts that are low in energy but very high in power. When the pulse duration is near the plasma membrane charging time constant, which is the time during which the cell interior is exposed to the applied pulsed electric field, it affects intracellular structures and functions. The technology is called nanosecond Pulsed Electric Fields (nsPEFs), nanosecond electric pulses (nsEP), or Nanopulse Stimulation (NPSTM) according to Pulse Biosciences, Inc., a company taking the technology to the market. Initial studies showed the elimination of tumor cells in vitro by apoptosis, and other regulated cell death mechanisms, elimination of rodent and canine osteosarcoma, and a basal cell carcinoma clinical trial. In the rat liver and mouse breast cancers, tumor-free animals were in situ vaccinated (ISV), preventing the recurrence of the treated cancers. The technology has also focused on treating benign skin diseases, with some advantages over cryoablation. More recently, the same technology called nanosecond pulsed-field ablation (nsPFA) has been used to treat cardiac arrhythmias like Atrial Fabulation (AFib) with catheters in humans. In pre-clinical studies and now in humans, this technology is showing advantages over radiofrequency ablation and cryoablation. On a new mechanistic landscape, nonlethal nsPEFs modulation of electron transport in the plasma membrane and the mitochondria show potential for controlling redox homeostasis and metabolism. Furthermore, different nsPEF waveforms have different effects on cells; waveforms can differ by pulse duration, rise time, electric field, and/or post-pulse features. So, for nsPEFs, there is a lethal side used for ablation as with NPS and nsPFA and a more recently recognized nonlethal side indicating new possibilities to differentially modify cell physiology depending on the different nsPEF waveforms.

Induced Pluripotent Stem Cell-Derived Parathyroid Organoids Resemble Parathyroid Morphology and Function.

The primary role of the parathyroid glands is to maintain calcium homeostasis through the secretion of parathyroid hormone (PTH). The limited proliferative capacity and differentiation of parathyroid cells hinder the generation of cell therapy options. In this study, parathyroid organoids are successfully generated from human-induced pluripotent stem cells (hiPSCs). At the end of the 20 days of differentiation, the parathyroid organoids exhibited distinct parathyroid morphology. Stereomicroscope, scanning electron microscopy (SEM), and transmission electron microscopy (TEM) analysis demonstrated the 3D arrangement of the cell layers in which intracellular structures of parathyroid cells resemble human parathyroid cellular morphology. Comprehensive molecular analyses, including RNA sequencing (RNA-Seq) and liquid chromatography/mass spectrometry (LC-MS/MS), confirmed the expression of key parathyroid-related markers. Protein expression of CasR, CxCr4, Gcm2, and PTH are observed in parathyroid organoids. Parathyroid organoids secrete PTH, demonstrate active intercellular calcium signaling, and induce osteogenic differentiation via their secretome. The tissue integration potential of parathyroid organoids is determined by transplantation into parathyroidectomized rats. The organoid transplanted animals showed significant elevations in PTH-related markers (CasR, CxCr4, Foxn1, Gcm2, and PTH). PTH secretion is detected in organoid-transplanted animals. The findings represent a significant advancement in parathyroid organoid culture and may offer a cellular therapy for treating PTH-related diseases, including hypoparathyroidism.

Dedifferentiation- and aging-induced loss of mechanical contractility and polarity in vascular smooth muscle cells: Heterogeneous changes in macroscopic and microscopic behavior of cells in serial passage culture

Dedifferentiation and aging of vascular smooth muscle cells (VSMCs) are associated with serious vascular diseases, such as arteriosclerosis and aneurysm. However, how cell dedifferentiation and aging affect cellular mechanical behaviors at the single-cell and intracellular structure levels remains unclear. An in-depth understanding of these interactions is extremely important for understanding the mechanism underlying VSMC mechanical integrity and homeostatic regulation of vascular walls. Herein, we systematically investigated changes in VSMC morphology, structure, contractility, and motility during dedifferentiation and aging induced by serial passage culture using traction force microscopy with elastic micropillar substrates, laser nanodissection of cytoskeletons, confocal fluorescence microscopy, and atomic force microscopy. We found that VSMC dedifferentiation started in the middle stage of serial passage culture, accompanied by a transient cell spreading in the cell width and decrease in contractile protein expression. Dedifferentiated VSMCs showed a significant decrease in the contraction and stiffness of individual actin stress fibers; however, their overall cell traction forces were maintained. Simultaneously, a significant increase in cell motility and the number of actin fibers was observed in dedifferentiated VSMCs, which may be associated with the enhancement of cell migration and disruption of cell/tissue integrity during the early stage of vascular diseases. As cell senescence progressed in the later stage of serial passage culture, VSMCs displayed reduced cell spreading and migration with decrease in the overall cell traction forces and drastic reduction in mechanical polarity of cell structures and forces. These results suggested that cell senescence causes loss of mechanical contractility and polarity in VSMCs, which may be an important factor in vascular disease progression. The experimental systems established in this study can be powerful tools for understanding the mechanisms underlying cellular dedifferentiation and aging from a biomechanical perspective.

Mild Heat Stress Alters the Physical State and Structure of Membranes in Triacylglycerol-Deficient Fission Yeast, Schizosaccharomyces pombe.

We investigated whether the elimination of two major enzymes responsible for triacylglycerol synthesis altered the structure and physical state of organelle membranes under mild heat shock conditions in the fission yeast, Schizosaccharomyces pombe. Our study revealed that key intracellular membrane structures, lipid droplets, vacuoles, the mitochondrial network, and the cortical endoplasmic reticulum were all affected in mutant fission yeast cells under mild heat shock but not under normal growth conditions. We also obtained direct evidence that triacylglycerol-deficient cells were less capable than wild-type cells of adjusting their membrane physical properties during thermal stress. The production of thermoprotective molecules, such as HSP16 and trehalose, was reduced in the mutant strain. These findings suggest that an intact system of triacylglycerol metabolism significantly contributes to membrane protection during heat stress.

µMap proximity labeling in living cells reveals stress granule disassembly mechanisms.

Phase-separated condensates are membrane-less intracellular structures comprising dynamic protein interactions that organize essential biological processes. Understanding the composition and dynamics of these organelles advances our knowledge of cellular behaviors and disease pathologies related to granule dysregulation. In this study, we apply microenvironment mapping with a HaloTag-based platform (HaloMap) to characterize intracellular stress granule dynamics in living cells. After validating the robustness and sensitivity of this approach, we then profile the stress granule proteome throughout the formation and disassembly and under pharmacological perturbation. These experiments reveal several ubiquitin-related modulators, including the HECT (homologous to E6AP C terminus) E3 ligases ITCH and NEDD4L, as well as the ubiquitin receptor toll-interacting protein TOLLIP, as key mediators of granule disassembly. In addition, we identify an autophagy-related pathway that promotes granule clearance. Collectively, this work establishes a general photoproximity labeling approach for unraveling intracellular protein interactomes and uncovers previously unexplored regulatory mechanisms of stress granule dynamics.

Full-field optical coherence microscopy enables high-resolution label-free imaging of the dynamics of live mouse oocytes and early embryos

High quality label-free imaging of oocytes and early embryos is essential for accurate assessment of their developmental potential, a key element of assisted reproduction procedures. To achieve this goal, we propose full-field optical coherence microscopy (FF-OCM), constructed as a compact module fully integrated with a commercial wide-field fluorescence microscope. Our system achieves optical sectioning in wide-field, high in-plane resolution of 0.5 µm, and high sensitivity to backscattered light. To demonstrate its imaging capabilities, we study live mouse oocytes and embryos at all important stages of meiotic maturation and early embryogenesis. Our system enables visualization of intracellular structures, which are not visible in common bright-field microscopy, i.e., internal structure of nuclear apparatus, cytoskeletal filaments, cellular cortex, cytoplasmic protrusions, or zona pellucida features. Additionally, we visualize and quantify intracellular dynamics like cytoplasmic stirring motion, nuclear envelope fluctuations and nucleolus mobility. Altogether, we demonstrate that FF-OCM is a powerful tool for research in developmental biology that also holds great potential for non-invasive time-lapse monitoring of oocyte and embryo quality in assisted reproduction.