Live Cell Imaging Microscopy Research Articles

Genetically encoded epigenetic sensors for visualization of H3K9me3, H3K9ac and H3K4me1 histone modifications in living cells

Post-translational modifications of histones play a crucial role in chromatin structure maintenance and epigenetic regulation. The LiveMIEL (Live-cell Microscopic Imaging of Epigenetic Landscape) method represents a promising approach for tracking histone modifications. It involves visualization of epigenetic modifications using genetically encoded fluorescent sensors and further analysis of the obtained intranuclear patterns by multiparametric image analysis. In this study, we designed three new red fluorescent sensors—MPP8-Red, AF9-Red and DPF3-Red—for live-cell visualization of patterns of H3K9me3, H3K8ac and H3K4me1, respectively. The observed fluorescent patterns were visually distinguishable, and LiveMIEL analysis clearly classified them into three corresponding groups. We propose that these sensors can be used for live-cell dynamic analysis of changes in organization of three epigenetic types of chromatin.

Tracking induced pluripotent stem cell differentiation with a fluorescent genetically encoded epigenetic probe

Epigenetic modifications (methylation, acetylation, etc.) of core histones play a key role in regulation of gene expression. Thus, the epigenome changes strongly during various biological processes such as cell differentiation and dedifferentiation. Classical methods of analysis of epigenetic modifications such as mass-spectrometry and chromatin immuno-precipitation, work with fixed cells only. Here we present a genetically encoded fluorescent probe, MPP8-Green, for detecting H3K9me3, a histone modification associated with inactive chromatin. This probe, based on the chromodomain of MPP8, allows for visualization of H3K9me3 epigenetic landscapes in single living cells. We used this probe to track changes in H3K9me3 landscapes during the differentiation of induced pluripotent stem cells (iPSCs) into induced neurons. Our findings revealed two major waves of global H3K9me3 reorganization during 4-day differentiation, namely on the first and third days, whereas nearly no changes occurred on the second and fourth days. The proposed method LiveMIEL (Live-cell Microscopic Imaging of Epigenetic Landscapes), which combines genetically encoded epigenetic probes and machine learning approaches, enables classification of multiparametric epigenetic signatures of single cells during stem cell differentiation and potentially in other biological models.

Extremely high spatiotemporal resolution microscopy for live cell imaging by single photon counting, noise elimination, and a novel restoration algorithm based on probability calculation.

Optical microscopy is essential for direct observation of dynamic phenomena in living cells. According to the classic optical theories, the images obtained through light microscopes are blurred for about half the wavelength of light, and therefore small structures below this "diffraction limit" were thought unresolvable by conventional optical microscopy. In reality, accurately obtained optical images contain complete information about the observed objects. Temporal resolution is also important for the observation of dynamic phenomena. A challenge exists here to overcome the trade-off between the time required for measurement and the accuracy of the measurement. The present paper describes a concrete methodology for reconstructing the structure of an observed object, based on the information contained in the image obtained by optical microscopy. It is realized by accurate single photon counting, complete noise elimination, and a novel restoration algorithm based on probability calculation. This method has been implemented in the Super-resolution Confocal Live Imaging Microscopy (SCLIM) we developed. The new system named SCLIM2M achieves unprecedented high spatiotemporal resolution. We have succeeded in capturing sub-diffraction-limit structures with millisecond-level dynamics of organelles and vesicles in living cells, which were never observed by conventional optical microscopy. Actual examples of the high-speed and high-resolution 4D observation of living cells are presented.

Abstract A019: Inhibition of nicotinamide adenine dinucleotide (NAD) production is a potent therapeutic strategy to inactivate homologous recombination in cancer cells

Abstract Homologous recombination (HR) is the most faithful DNA double-strand breaks (DSBs) repairing pathway. HR deficiency (HRD) can result from mutations in BRCA1, BRCA2, and PALB2, which are associated with genomic instability and cancer. HR deficient cells can survive through the use of alternative DNA repair pathways. Therefore, inhibiting these repair pathways in HRD cells trigger cancer cell death. This phenomenon is called synthetic lethality. The best-known example of synthetic lethality is that involving BRCA1/BRCA2/PALB2 deficiency with PARP-1 inhibition by Olaparib. However, ∼40% of patients will develop resistance to PARP inhibition because of several mechanisms, including a second mutation in BRCA1/2, restoring their full-length expression or RAD51 overexpression. RAD51 is one of the most important mediators of HR as it promotes the invasion of a sister chromatid allowing faithful DSB repair. There are many pieces of evidence which show that RAD51 overexpression is correlated with resistance and worse overall survival. Our hypothesis is that targeting RAD51 can trigger HR deficiency, particularly in cancer cells resistant to standard treatments. We thus sought to find inhibitors of the RAD51 recombinase. We have developed an in cellulo screening technique to test the direct effect of chemical compounds on RAD51 foci formation following irradiation. Using this technique, we identified , among 1381 chemical molecules, FK866 (Daporinad) which is a Nicotinamide Phosphoribosyltransferase Inhibitor (NAMPTi). NAMPT is involved in producing cellular Nicotinamide Adenine Dinucleotide (NAD+), which plays a crucial role in various metabolic processes within the cell, primarily in energy production and DNA repair. Remarkably, treatment of Daporinad reduced RAD51 foci formation and triggered a destabilization of the RAD51 protein by the proteasome. This destabilization seemed to be specific for RAD51, as NHEJ factors were not affected by NAMPTi. We will present data related to our objectives: i) Monitoring the effect of Daporinad on different cancer cell lines originated from cancer types which has shown resistance and worse overall survival because of RAD51 overexpression. Western blot and qPCR techniques will be used to investigate the effect of Daporinad on RAD51 protein and mRNA level and using Zeiss Celldiscoverer 7 system and Incucyte live cell imaging microscopy to observe the effect of Daporinad on cell survival; ii) To confirm the specific targeting of HR by Daporinad, we will monitor the effect of Daporinad on HR and other DNA DSB repair pathways using in cellulo reporter systems; iii) In the long term, we aim to evaluate the effect of Daporinad in vivo on tumor cell growth in pre-clinical models, such as mouse xenografts. In conclusion, small molecules affecting HR provide a source of new cancer therapies targeting RAD51 overexpression or using synthetic lethality. Citation Format: Sadaf Valeh Sheida, Thibaut Peterlini, Mélissa Thomas, Guy Poirier, Jean-Yves Masson. Inhibition of nicotinamide adenine dinucleotide (NAD) production is a potent therapeutic strategy to inactivate homologous recombination in cancer cells [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr A019.

Tau fibrils induce nanoscale membrane damage and nucleate cytosolic tau at lysosomes

The prion-like spread of protein aggregates is a leading hypothesis for the propagation of neurofibrillary lesions in the brain, including the spread of tau inclusions associated with Alzheimer's disease. The mechanisms of cellular uptake of tau seeds and subsequent nucleated polymerization of cytosolic tau are major questions in the field, and the potential for coupling between the entry and nucleation mechanisms has been little explored. We found that in primary astrocytes and neurons, endocytosis of tau seeds leads to their accumulation in lysosomes. This in turn leads to lysosomal swelling, deacidification, and recruitment of ESCRT proteins, but not Galectin-3, to the lysosomal membrane. These observations are consistent with nanoscale damage of the lysosomal membrane. Live cell imaging and STORM superresolution microscopy further show that the nucleation of cytosolic tau occurs primarily at the lysosome membrane under these conditions. These data suggest that tau seeds escape from lysosomes via nanoscale damage rather than wholesale rupture and that nucleation of cytosolic tau commences as soon as tau fibril ends emerge from the lysosomal membrane.

C-Src Facilitates ER-to-Mitochondria Ca2+ Transport and Activates Cardiac Fibroblasts under Pulmonary Arterial Hypertension

Introduction: Similar to left ventricular (LV) fibrosis, growing evidence suggests a potential link between right ventricular (RV) fibrosis, poor function of the pressure-overloaded RV, and mortality in patients with pulmonary arterial hypertension (PAH). However, no therapies are currently available that specifically target RV fibrosis. Although the pathological roles of an oxidative stress-sensitive tyrosine kinase c-Src in LV hypertrophy and failure are well characterized, it remains unclear whether c-Src is involved in RV pathology under PAH. Using heterologous expression systems, we previously showed that mitofusin 2 (Mfn2), a key component of the tethering structures between the endoplasmic reticulum (ER) and mitochondria (Mito) is a novel c-Src substrate in mitochondria; and c-Src-dependent tyrosine phosphorylation (P-Tyr) of Mfn2 at the outer mitochondrial membrane (OMM) decreases the ER-Mito distance, possibly through conformational changes of Mfn2, which facilitates ER-to-Mito Ca2+ transfer and increases reactive oxygen species (ROS). Hypothesis: c-Src signaling alters the size of ER-Mito microdomains and enhances oxidative stress in the RV during PAH. Methods: Primary adult cardiac fibroblasts (CFs) from human ventricles and a preclinical rat PAH model induced by Su gen 5416 injection and 3-week h ypo x ia followed by 4-week normoxia (SuHx) were used for in vitro and in vivo assays, respectively. RV tissues from pulmonary hypertension (PH) patients and sex-/aged-matched controls were used for biochemical and cell biological assays. Results: c-Src activation occurred specifically in CFs in the RV, but not in RV myocytes in a rat SuHx PAH model and human PH-RVs as assessed by immunohistochemistry, which is notably different from the reports on LV hypertrophy and failure. P-Tyr of Mfn2 was detectable from human PH-RVs by Western Blots. RV-CFs from PAH rats exhibited increased c-Src activity, decreased ER-Mito distance, increased ROS, and proliferative signaling activation compared to RV-CFs from control rats as assessed by live cell imaging, immunocytochemistry, and transmission electron microscopy. All of these changes in rat CFs from PAH-RVs were recapitulated by overexpressing c-Src in human CFs in vitro. In addition, c-Src significantly increased the speed and amount of mitochondrial Ca2+ uptake without changing cytosolic Ca2+ dynamics in human CFs, suggesting a potential link between c-Src activation, the size of ER-Mito microdomains, and RV fibrosis under PAH. To further dissect the role of c-Src activity in CFs especially in the mitochondria, we specifically inhibited c-Src activity at the OMM, where Mfn2 is located, using an OMM-targeted dominant-negative c-Src (termed mt-c-Src-DN) that was generated by the addition of an OMM-targeting sequence (amino acid 1-33 from human TOM20) to the N-terminus of kinase-dead c-Src-K295R/Y527F. The introduction of mt-c-Src-DN increased the ER-Mito distance and reduced the speed of ER-to-Mito Ca2+ transport in human CFs. Conclusion: c-Src-dependent P-Tyr of Mfn2 alters the ER-Mito tethering structure, facilitates ER-to-Mito Ca2+ transport, and increases mitochondrial ROS generation, which promotes CF activation in PAH. Therefore, CF- and mitochondria-specific inhibition of c-Src may provide an additional therapeutic strategy to attenuate RV fibrosis and failure in response to PAH. NIH R01HL136757 (to J.O.-U.), R01HL160699 (to B.S.J.), P30GM1114750 (Targeted project to J.O.-U.), P30GM110759 (Targeted project to J.O.-U.), U54GM115677 (Targeted project to B.S.J.), and American Heart Association (AHA) 18CDA34110091 (to B.S.J.). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Antibiofilm activity of lawsone against polymicrobial enterohemorrhagic Escherichia coli O157:H7 and Candida albicans by suppression of curli production and hyphal growth

Most bacteria and fungi form biofilms that attach to living or abiotic surfaces. These biofilms diminish the efficacy of antimicrobial agents and contribute to chronic infections. Furthermore, multispecies biofilms composed of bacteria and fungi are often found at chronic infection sites. In this study, lawsone (2‑hydroxy-1,4-naphthoquinone) and its parent 1,4-naphthoquinone were studied for antimicrobial and antibiofilm activities against single-species and multispecies biofilms of enterohemorrhagic Escherichia coli O157:H7 (EHEC) and Candida albicans. Biofilm formation assays, biofilm eradication assays, antimicrobial assays, live cell imaging microscopy, confocal laser scanning microscopy (CLSM), scanning electron microscopy (SEM), extracellular polymeric substances and indole production, cell surface hydrophilicity assay, cell motility, cell aggregation, hyphal growth, dual species biofilm formation, quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR), and toxicity assays on plant seed germination and nematode model were utilized to investigate how lawsone affect biofilm development. Sub-inhibitory concentrations of lawsone (35µg/ml) significantly inhibited single-and multispecies biofilm development. Lawsone reduced the production of curli and indole, and the swarming motility of EHEC, efficiently inhibited C. albicans cell aggregation and hyphal formation, and increased the cell surface hydrophilicity of C. albicans. Transcriptomic analysis showed that lawsone suppressed the expression of the curli-related genes csgA and csgB in EHEC, and the expression of several hypha- and biofilm-related genes (ALS3, ECE1, HWP1, and UME6) in C. albicans. In addition, lawsone up to 100µg/ml was nontoxic to the nematode Caenorhabditis elegans and to the seed growth of Brassica rapa and Triticum aestivum. These results show that lawsone inhibits dual biofilm development and suggest that it might be useful for controlling bacterial or fungal infections and multispecies biofilms.

CNN-BASED UNSUPERVISED REGISTRATION OF TIME-LAPSE MICROSCOPY IMAGE SEQUENCES

Abstract. Image registration is widely used in live cell microscopy image analysis to compensate for the cell motion. It is a challenging task as the cell is not only moving (which causes rotation and translation), but also changes its form in time making the motion non-rigid. To address this, we propose a CNN-based unsupervised method for non-rigid registration of live cell image sequences. Our network predicts both the deformation field between a pair of images of the sequence and an affine transformation matrix for the cell motion compensation. The method can be used alone or in combination with other approaches. The proposed approach was successfully applied to real live cell microscopy image sequences.We conducted an experimental comparison with existing methods including contour-based, intensity-based and a deep learning based joint denoising and registration method. In addition, we analyzed different deformation regularizers and their impact on the alignment results. In combination with contour-based method we outperformed the existing approaches in average registration accuracy for two metrics on the standard evaluation dataset.

Large-scale in vitro microdosimetry via live cell microscopy imaging: implications for radiosensitivity and RBE evaluations in alpha-emitter radiopharmaceutical therapy

BackgroundAlpha-emitter radiopharmaceutical therapy (αRPT) has shown promising outcomes in metastatic disease. However, the short range of the alpha particles necessitates dosimetry on a near-cellular spatial scale. Current knowledge on cellular dosimetry is primarily based on in vitro experiments using cell monolayers. The goal of such experiments is to establish cell sensitivity to absorbed dose (AD). However, AD cannot be measured directly and needs to be modeled. Current models, often idealize cells as spheroids in a regular grid (geometric model), simplify binding kinetics and ignore the stochastic nature of radioactive decay. It is unclear what the impact of such simplifications is, but oversimplification results in inaccurate and non-generalizable results, which hampers the rigorous study of the underlying radiobiology.MethodsWe systematically mapped out 3D cell geometries, clustering behavior, agent binding, internalization, and subcellular trafficking kinetics for a large cohort of live cells under representative experimental conditions using confocal microscopy. This allowed for realistic Monte Carlo-based (micro)dosimetry. Experimentally established surviving fractions of the HER2 + breast cancer cell line treated with a 212Pb-labelled anti-HER2 conjugate or external beam radiotherapy, anchored a rigorous statistical approach to cell sensitivity and relative biological effectiveness (RBE) estimation. All outcomes were compared to a reference geometric model, which allowed us to determine which aspects are crucial model components for the proper study of the underlying radiobiology.ResultsIn total, 567 cells were measured up to 26 h post-incubation. Realistic cell clustering had a large (2x), and cell geometry a small (16.4% difference) impact on AD, compared to the geometric model. Microdosimetry revealed that more than half of the cells do not receive any dose for most of the tested conditions, greatly impacting cell sensitivity estimates. Including these stochastic effects in the model, resulted in significantly more accurate predictions of surviving fraction and RBE (permutation test; p < .01).ConclusionsThis comprehensive integration of the biological and physical aspects resulted in a more accurate method of cell survival modelling in αRPT experiments. Specifically, including realistic stochastic radiation effects and cell clustering behavior is crucial to obtaining generalizable radiobiological parameters.

Recipient UvrD helicase is involved in single- to double-stranded DNA conversion during conjugative plasmid transfer.

Dissemination of antibiotic resistance, a current societal challenge, is often driven by horizontal gene transfer through bacterial conjugation. During conjugative plasmid transfer, single-stranded (ss) DNA is transferred from the donor to the recipient cell. Subsequently, a complete double-stranded (ds) plasmid molecule is generated and plasmid-encoded genes are expressed, allowing successful establishment of the transconjugant cell. Such dynamics of transmission can be modulated by host- or plasmid-encoded factors, either in the donor or in the recipient cell. We applied transposon insertion sequencing to identify host-encoded factors that affect conjugative transfer frequency in Escherichia coli. Disruption of the recipient uvrD gene decreased the acquisition frequency of conjugative plasmids belonging to different incompatibility groups. Results from various UvrD mutants suggested that dsDNA binding activity and interaction with RNA polymerase are dispensable, but ATPase activity is required for successful plasmid establishment of transconjugant cells. Live-cell microscopic imaging showed that the newly transferred ssDNA within a uvrD- recipient often failed to be converted to dsDNA. Our work suggested that in addition to its role in maintaining genome integrity, UvrD is also key for the establishment of horizontally acquired plasmid DNA that drives genome diversity and evolution.

Automated cell segmentation for reproducibility in bioimage analysis.

Live-cell imaging is extremely common in synthetic biology research, but its ability to be applied reproducibly across laboratories can be hindered by a lack of standardized image analysis. Here, we introduce a novel cell segmentation method developed as part of a broader Independent Verification & Validation (IV&V) program aimed at characterizing engineered Dictyostelium cells. Standardizing image analysis was found to be highly challenging: the amount of human judgment required for parameter optimization, algorithm tweaking, training and data pre-processing steps forms serious challenges for reproducibility. To bring automation and help remove bias from live-cell image analysis, we developed a self-supervised learning (SSL) method that recursively trains itself directly from motion in live-cell microscopy images without any end-user input, thus providing objective cell segmentation. Here, we highlight this SSL method applied to characterizing the engineered Dictyostelium cells of the original IV&V program. This approach is highly generalizable, accepting images from any cell type or optical modality without the need for manual training or parameter optimization. This method represents an important step toward automated bioimage analysis software and reflects broader efforts to design accessible measurement technologies to enhance reproducibility in synthetic biology research.

Substituent directed cellular imaging in the 800-850 nm range with BF2-azadipyrromethene fluorophores.

Three bis(anilino)-substituted NIR-AZA fluorophores have been designed, synthesized and tested to bridge the availability gap of molecular fluorophores for live-cell microscopy imaging in the 800-850 nm spectral range. The concise synthetic route allows for the later stage introduction of three tailored peripheral substituents which guides the sub-cellular localization and imaging. Live-cell fluorescence imaging of lipid droplets, plasma membrane and cytosolic vacuoles was successfully achieved. Photophysical and internal charge transfer (ICT) properties of each fluorophore were examined through solvent studies and analyte responses.

Label-free third harmonic generation imaging and quantification of lipid droplets in live filamentous fungi

We report the utilization of Third-Harmonic Generation microscopy for label-free live cell imaging of lipid droplets in the hypha of filamentous fungus Phycomyces blakesleeanus. THG microscopy images showed bright spherical features dispersed throughout the hypha cytoplasm in control conditions and a transient increase in the number of bright features after complete nitrogen starvation. Colocalization analysis of THG and lipid-counterstained images disclosed that the cytoplasmic particles were lipid droplets. Particle Size Analysis and Image Correlation Spectroscopy were used to quantify the number density and size of lipid droplets. The two analysis methods both revealed an increase from 16 × 10−3 to 23 × 10−3 lipid droplets/µm2 after nitrogen starvation and a decrease in the average size of the droplets (range: 0.5–0.8 µm diameter). In conclusion, THG imaging, followed by PSA and ICS, can be reliably used for filamentous fungi for the in vivo quantification of lipid droplets without the need for labeling and/or fixation. In addition, it has been demonstrated that ICS is suitable for THG microscopy.

SOX2 Modulates the Nuclear Organization and Transcriptional Activity of the Glucocorticoid Receptor

Steroid receptors (SRs) are ligand-dependent transcription factors (TFs) relevant to key cellular processes in both physiology and pathology, including some types of cancer. SOX2 is a master TF of pluripotency and self-renewal of embryonic stem cells, and its dysregulation is also associated with various types of human cancers. A potential crosstalk between these TFs could be relevant in malignant cells yet, to the best of our knowledge, no formal study has been performed thus far. Here we show, by quantitative live-cell imaging microscopy, that ectopic expression of SOX2 disrupts the formation of hormone-dependent intranuclear condensates of many steroid receptors (SRs), including those formed by the glucocorticoid receptor (GR). SOX2 also reduces GR’s binding to specific DNA targets and modulates its transcriptional activity. SOX2-driven effects on GR condensates do not require the intrinsically disordered N-terminal domain of the receptor and, surprisingly, neither relies on GR/SOX2 interactions. SOX2 also alters the intranuclear dynamics and compartmentalization of the SR coactivator NCoA-2 and impairs GR/NCoA-2 interactions. These results suggest an indirect mechanism underlying SOX2-driven effects on SRs involving this coactivator. Together, these results highlight that the transcriptional program elicited by GR relies on its nuclear organization and is intimately linked to the distribution of other GR partners, such as the NCoA-2 coactivator. Abnormal expression of SOX2, commonly observed in many tumors, may alter the biological action of GR and, probably, other SRs as well. Understanding this crosstalk may help to improve steroid hormone-based therapies in cancers with elevated SOX2 expression.

Cell type-specific orientation and migration responses for a microgrooved surface with shallow grooves.

Directional cell migration due to mechanosensing for invivo microenvironment, such as microgrooved surfaces, is an essential process in tissue growth and repair in both normal and pathological states. Cell migration responses on the microgrooved surfaces might be reflected by the cell type difference, which is deeply involved in cellular physiological functions. Although the responses are implicated in focal adhesions (FAs) of cells, limited information is available about cell migration behavior on the microgrooved surfaces whose dimensions are comparable with the size of FAs. In the present study, we investigated the cell orientation and migration behavior of normal vascular smooth muscle cells (VSMCs) and cervical cancer HeLa cells on the microgrooved surface. The cells were cultured on the PDMS substrate comprising shallow grooves with 2-µm width and approximately 150-nm depth, which indicates the same order of magnitude as that of the horizontal and vertical size of FAs, respectively. The cell migration and intracellular structures were analyzed by live cell imaging and confocal fluorescence microscopy. The intracellular tension was also assessed using atomic force microscopy (AFM). VSMCs presenting well-aligned actin stress fibers with mature FAs revealed marked cell elongation and directional migration on the grooves; however, HeLa cells with nonoriented F-actin with smaller FAs did not. The internal force of the actin fibers was significantly higher in VSMCs than that in HeLa cells, and the increase or decrease in the cytoskeletal forces improved or diminished the sensing ability for shallow grooves, respectively. The results strongly indicated that directional cell migration should be modulated by cell type-specific cytoskeletal arrangements and intracellular traction forces. The differences in cell type-specific orientation and migration responses can be emphasized on the microgrooves as large as the horizontal and vertical size of FAs. The microgoove structure in the size range of the FA protein complex is a powerful tool to clarify subtle differences in the intracellular force-dependent substrate mechanosensing.

Mod3D: A low-cost, flexible modular system of live-cell microscopy chambers and holders

Live-cell microscopy imaging typically involves the use of high-quality glass-bottom chambers that allow cell culture, gaseous buffer exchange and optical properties suitable for microscopy applications. However, commercial sources of these chambers can add significant annual costs to cell biology laboratories. Consumer products in three-dimensional printing technology, for both Filament Deposition Modeling (FDM) and Masked Stereo Lithography (MSLA), have resulted in more biomedical research labs adopting the use of these devices for prototyping and manufacturing of lab plastic-based items, but rarely consumables. Here we describe a modular, live-cell chamber with multiple design options that can be mixed per experiment. Single reusable carriers and the use of biodegradable plastics, in a hybrid of FDM and MSLA manufacturing methods, reduce plastic waste. The system is easy to adapt to bespoke designs, with concept-to-prototype in a single day, offers significant cost savings to the users over commercial sources, and no loss in dimensional quality or reliability.

Development of highspeed and super-resolution microscopy

Live cell imaging is a powerful tool to understand the molecular mechanisms of a wide range of cellular processes. However, it is difficult to detect very small objects such as transport vesicles (< 100 nm in diameter) by light microscopy because of the diffraction limit of light. To overcome this problem, several superresolution technologies such as STED, SIM, and STORM/PALM has been developed so far, but the time resolution of these methods is not sufficient to track the small vesicles undergoing fast movement inside the cell. In the present study, we developed a highspeed, superresolution, and multicolor optical microscope, which we call SCLIM2 (the second generation superresolution confocal live cell imaging microscopy). SCLIM2 consists of an inverted microscope equipped with piezo actuator for objective lens, a spinningdisk confocal scanner, an emission splitter unit, cooled image intensifiers, and ultrafast CMOS cameras (1000 frames/s). Using this system, we acquired fluorescence images of specimens at a single photon precision. We also developed a mathematical method for 4D (xyz and time) image reconstruction and novel deconvolution algorism based on the probability calculation. The resulting lateral resolution was less than 71 nm. We then applied SCLIM2 to observe a variety of membrane traffic events in live cells. We succeeded in visualizing a large number of small vesicles (< 100 nm in diameter) were moving around the Golgi apparatus.

Low-Power Two-Color Stimulated Emission Depletion Microscopy for Live Cell Imaging.

Stimulated emission depletion (STED) microscopy is a typical laser-scanning super-resolution imaging technology, the emergence of which has opened a new research window for studying the dynamic processes of live biological samples on a nanometer scale. According to the characteristics of STED, a high depletion power is required to obtain a high resolution. However, a high laser power can induce severe phototoxicity and photobleaching, which limits the applications for live cell imaging, especially in two-color STED super-resolution imaging. Therefore, we developed a low-power two-color STED super-resolution microscope with a single supercontinuum white-light laser. Using this system, we achieved low-power two-color super-resolution imaging based on digital enhancement technology. Lateral resolutions of 109 and 78 nm were obtained for mitochondria and microtubules in live cells, respectively, with 0.8 mW depletion power. These results highlight the great potential of the novel digitally enhanced two-color STED microscopy for long-term dynamic imaging of live cells.

Brain region specificity in reactive oxygen species production and maintenance of redox balance

The brain produces various reactive oxygen species in enzymatic and non-enzymatic reactions as a by-product of metabolism and/or for redox signaling. Effective antioxidant system in the brain cells maintains redox balance. However, neurons and glia from some brain regions are more vulnerable to oxidative stress in ischemia/reperfusion, epilepsy, and neurodegenerative disorders than the rest of the brain. Using fluorescent indicators in live cell imaging and confocal microscopy, we have measured the rate of cytosolic and mitochondrial reactive oxygen species production, lipid peroxidation, and glutathione levels in cortex, hippocampus, midbrain, brain stem and cerebellum in acute slices of rat brain. We have found that the basal rate of ROS production is at its highest in brain stem and cerebellum, and that it is mainly generated by glial cells. Activation of neurons and glia by glutamate and ATP led to maximal rates of ROS production in the midbrain compared to the rest of the brain. Mitochondrial ROS had only minor implication to the total ROS production with maximal values in the cortex and minimal in the midbrain. The basal rate of lipid peroxidation was higher in the midbrain and hippocampus, while the GSH level was similar in most brain regions with the lowest level in the midbrain. Thus, the rate of ROS production, lipid peroxidation and the level of GSH vary across brain regions.

27379 High resolution, dynamic imaging of collagen in skin cells using a bright reporter molecule

Collagen is the main component of the skin extracellular matrix and plays an integral role in supporting and maintaining the structure and biomechanical properties of the skin. There is increased interest to understand how collagen quality is controlled by the skin’s fibroblast cells, particularly in aging, and to quantify how product formulations improve the appearance of aging skin. In this work, we have developed a new in vitro tool to visualize collagen in skin fibroblasts at high speed and high resolution using live cell imaging and super resolution microscopy.