Confocal Spinning Disk Research Articles

Unlocking the Potential of the CICERO Spinning Disk System: Exploring Diverse Applications

Abstract CrestOptics aims to make their products accessible to researchers, enabling a variety of applications, and to create products that are both efficient and user-friendly. The CICERO confocal spinning disk system embodies these principles, designed for easy integration into any imaging setup. Its compactness and flexibility make advanced fluorescence microscopy accessible to laboratories worldwide. This article will focus on the great versatility and strengths of the CICERO system, which is a complete widefield (WF) and spinning disk confocal (CF) solution. It will demonstrate its key features, namely its use on both inverted and upright microscopy configurations, its wide spectral range, the high resolution and sectioning power, and its large field of view (FOV). Microscopy Today has awarded CICERO a 2024 Innovation Award for these features.

A novel multiplex fluorescent-labeling method for the visualization of mixed-species biofilms in vitro.

In nature, bacteria usually exist as mixed-species biofilms, where they engage in a range of synergistic and antagonistic interactions that increase their resistance to environmental challenges. Biofilms are a major cause of persistent infections, and dispersal from initial foci can cause new infections at distal sites thus warranting further investigation. Studies of development and spatial interactions in mixed-species biofilms can be challenging due to difficulties in identifying the different bacterial species in situ. Here, we apply CellTrace dyes to studies of biofilm bacteria and present a novel application for multiplex labeling, allowing identification of different bacteria in mixed-species, in vitro biofilm models. Oral bacteria labeled with CellTrace dyes (far red, yellow, violet, and CFSE [green]) were used to create single- and mixed-species biofilms, which were analyzed with confocal spinning disk microscopy (CSDM). Biofilm supernatants were studied with flow cytometry (FC). Both Gram-positive and Gram-negative bacteria were well labeled and CSDM revealed biofilms with clear morphology and stable staining for up to 4 days. Analysis of CellTrace labeled cells in supernatants using FC showed differences in the biofilm dispersal between bacterial species. Multiplexing with different colored dyes allowed visualization of spatial relationships between bacteria in mixed-species biofilms and relative coverage by the different species was revealed through segmentation of the CSDM images. This novel application, thus, offers a powerful tool for studying structure and composition of mixed-species biofilms in vitro.IMPORTANCEAlthough most chronic infections are caused by mixed-species biofilms, much of our knowledge still comes from planktonic cultures of single bacterial species. Studies of formation and development of mixed-species biofilms are, therefore, required. This work describes a method applicable to labeling of bacteria for in vitro studies of biofilm structure and dispersal. Critically, labeled bacteria can be multiplexed for identification of different species in mixed-species biofilms using confocal spinning disk microscopy, facilitating investigation of biofilm development and spatial interactions under different environmental conditions. The study is an important step in increasing the tools available for such complex and challenging studies.

Programmable comprehensive controller for multi-color 3D confocal spinning-disk image scanning microscope

This paper introduces a compact, portable, and highly accurate triggering control system for a 3D confocal spinning-disk image scanning microscope (CSD-ISM). Building upon on our previously published research, we expanded the hardware of the controller and synchronized it with a sub-micron translator which scans the object in the z-direction. As well as expanding the hardware, the software also was extended from previously published work similarly as it is stated for hardware while allowing full control over the 3D movement. We showed a clear and smooth 3D image made up of a collection of 2D images at different heights.

Portable low-cost and highly accurate programmable triggering controller for confocal spinning disk image scanning microscope

This work presents a very low-cost and highly accurate alternative way of implementing triggering control for a confocal spinning-disk image scanning microscope (CSD-ISM). Instead of using the previously reported hig-cost field programmable gate array (FPGA), that connects to a computer by an internal PCIe bus, we implemented the controller using a simple, low-cost real-time digital signal controller (DSC) development kit that connects to a computer via an external USB channel. The overall time resolution of the controller is better than 10 nanoseconds. The DSC programming is implemented using the free software included in the development kit and no additional commercial software is required. The operation of the CSD-ISM is performed by a 32-bit 150 MHz processor using the software environment Micro-Manager, a popular open-source platform for microscopy. Since software is used to calculate all the necessary times, the output signal is almost limitless.

Improving two-photon excitation microscopy for sharper and faster biological imaging.

Two-photon excitation laser scanning microscopy (TPLSM) has provided many insights into the life sciences, especially for thick biological specimens, because of its superior penetration depth and less invasiveness owing to the near-infrared wavelength of its excitation laser light. This paper introduces our four kinds of studies to improve TPLSM by utilizing several optical technologies as follows: (1) A high numerical aperture objective lens significantly deteriorates the focal spot size in deeper regions of specimens. Thus, approaches to adaptive optics were proposed to compensate for optical aberrations for deeper and sharper intravital brain imaging. (2) TPLSM spatial resolution has been improved by applying super-resolution microscopic techniques. We also developed a compact stimulated emission depletion (STED) TPLSM that utilizes electrically controllable components, transmissive liquid crystal devices, and laser diode-based light sources. The spatial resolution of the developed system was five times higher than conventional TPLSM. (3) Most TPLSM systems adopt moving mirrors for single-point laser beam scanning, resulting in the temporal resolution caused by the limited physical speed of these mirrors. For high-speed TPLSM imaging, a confocal spinning-disk scanner and newly-developed high-peak-power laser light sources enabled approximately 200 foci scanning. (4) Several researchers have proposed various volumetric imaging technologies. However, most technologies require large-scale and complicated optical setups based on deep expertise for microscopic technologies, resulting in a high threshold for biologists. Recently, an easy-to-use light-needle-creating device was proposed for conventional TPLSM systems to achieve one-touch volumetric imaging.

Multi-view confocal microscopy enables multiple organ and whole organism live-imaging.

Understanding how development is coordinated in multiple tissues and gives rise to fully functional organs or whole organisms necessitates microscopy tools. Over the last decade numerous advances have been made in live-imaging, enabling high resolution imaging of whole organisms at cellular resolution. Yet, these advances mainly rely on mounting the specimen in agarose or aqueous solutions, precluding imaging of organisms whose oxygen uptake depends on ventilation. Here, we implemented a multi-view multi-scale microscopy strategy based on confocal spinning disk microscopy, called Multi-View confocal microScopy (MuViScopy). MuViScopy enables live-imaging of multiple organs with cellular resolution using sample rotation and confocal imaging without the need of sample embedding. We illustrate the capacity of MuViScopy by live-imaging Drosophila melanogaster pupal development throughout metamorphosis, highlighting how internal organs are formed and multiple organ development is coordinated. We foresee that MuViScopy will open the path to better understand developmental processes at the whole organism scale in living systems that require gas exchange by ventilation.

二光子顕微鏡の高分解能化による生体内微細構造イメージング

Two-photon excitation laser scanning fluorescence microscopy (TPLSM) is a powerful tool to visualize intravital microstructures. This is because of its superior penetration depth and less-invasiveness in specimens owing to its near-infrared excitation laser wavelength compared with the single-photon excitation. In this presentation, our studies to improve spatial and temporal resolution of TPLSM by utilizing some of novel optical technologies are introduced. To improve the spatial resolution, we applied stimulated emission depletion (STED) technologies utilizing electrically controllable components, transimissive liquid crystal devises and laser diode-based light sources [Opt. Express 2014; Biomed Opt. Express 2018], resulting in 5-times hihger spatial resolution than conventional [Biomed Opt. Express 2019]. In addition, we recently adopted other technologies such as adaptive optics [PLOS One 2020; ACS Omega 2021] or nano-sheet based observation methods [iSci. 2020; Star Protocol's. 2021] for sharper intravital imaging. For higher temporal resolution, a confocal spinning disk scanning unit and high-peak-power laser light sources were utilized for TPLSM imaging [Anal. Sci. 2015; Front. Physical. 2019; BBRC 2020]. The developed system achieved large field of view, fine spatial resolution and penetration depth, and has been used for several biological applications.

The Association between α-Synuclein and α-Tubulin in Brain Synapses.

α-synuclein is a small protein that is mainly expressed in the synaptic terminals of nervous tissue. Although its implication in neurodegeneration is well established, the physiological role of α-synuclein remains elusive. Given its involvement in the modulation of synaptic transmission and the emerging role of microtubules at the synapse, the current study aimed at investigating whether α-synuclein becomes involved with this cytoskeletal component at the presynapse. We first analyzed the expression of α-synuclein and its colocalization with α-tubulin in murine brain. Differences were found between cortical and striatal/midbrain areas, with substantia nigra pars compacta and corpus striatum showing the lowest levels of colocalization. Using a proximity ligation assay, we revealed the direct interaction of α-synuclein with α-tubulin in murine and in human brain. Finally, the previously unexplored interaction of the two proteins in vivo at the synapse was disclosed in murine striatal presynaptic boutons through multiple approaches, from confocal spinning disk to electron microscopy. Collectively, our data strongly suggest that the association with tubulin/microtubules might actually be an important physiological function for α-synuclein in the synapse, thus suggesting its potential role in a neuropathological context.

Artifact analysis and fast image reconstruction for spinning disk confocal Image Scanning Microscopy

Image Scanning Microscopy is a linear super-resolution fluorescence microscopy, which does not involve very complex optical system design, but is based on existing setup and capable of taking the advantage of computing and image processing methods to achieve super-resolution imaging. As ISM image acquisition and image reconstruction are actually based on discrete space, some factors, such as the pattern of scanning path, reconstruction accuracy, and background could result in artifacts, which could affect the image-quality of the final reconstructed ISM image seriously and has to be taken into consideration in practice. In this article, we analyzed the artifact problem frequently emerged in the spinning disk confocal (SDC) ISM systems, based on which a modified ISM reconstruction technique is proposed to accelerate the image reconstruction and avoid potential artifacts. The experimental result shows that the proposed ISM reconstruction method can be 10 times faster than the existing method and it validates that artifacts can be caused by multiple factors, i.e. the inhomogeneity of sampling-interval for scanning, pixels reassignment accuracy and the background. It also shows that these artifacts can be significantly reduced by these steps: remove image background from the raw image, calculate ISM image with accurate reconstruction and apply appropriate deconvolution to the accurately reconstructed ISM image. The proposed ISM image reconstruction and the artifacts removal technique improves the image reconstruction significantly and enable SDC-ISM system to guarantee high image-quality output.

Doubling the resolution of a confocal spinning-disk microscope using image scanning microscopy

Fluorescence microscopy has become an indispensable tool for cell biology. Recently, super-resolution methods have been developed to overcome the diffraction limit of light and have shown living cells in unprecedented detail. Often, these methods come at a high cost and with complexity in terms of instrumentation and sample preparation, thus calling for the development of low-cost, more accessible methods. We previously developed image scanning microscopy (ISM), which uses structured illumination to double the resolution and quadruple the contrast of a confocal microscope. Implementing this technique into a confocal spinning-disk (CSD) microscope allows recording ISM images with up to ~1 frame per second, making it ideal for imaging dynamic biological processes. Here we present a step-by-step protocol describing how to convert any existing commercial CSD microscope into a CSD-ISM, with only moderate changes to the hardware and at low cost. Operation of the CSD-ISM is realized with a field programmable gate array using the software environment Micro-Manager, a popular open-source platform for microscopy. The provided software ( https://projects.gwdg.de/projects/csdism-2020 ) takes care of all algorithmic complexities and numerical workload of the CSD-ISM, including hardware synchronization and image reconstruction. The hardware modifications described here result in a theoretical maximum increase in resolution of √2 ≈ 1.41, which can be further improved by deconvolution to obtain a theoretical maximum twofold increase. An existing CSD setup can be upgraded in ~3 d by anyone with basic knowledge in optics, electronics and microscopy software.

Subcellular Localization and Mitotic Interactome Analyses Identify SIRT4 as a Centrosomally Localized and Microtubule Associated Protein.

The stress-inducible and senescence-associated tumor suppressor SIRT4, a member of the family of mitochondrial sirtuins (SIRT3, SIRT4, and SIRT5), regulates bioenergetics and metabolism via NAD+-dependent enzymatic activities. Next to the known mitochondrial location, we found that a fraction of endogenous or ectopically expressed SIRT4, but not SIRT3, is present in the cytosol and predominantly localizes to centrosomes. Confocal spinning disk microscopy revealed that SIRT4 is found during the cell cycle dynamically at centrosomes with an intensity peak in G2 and early mitosis. Moreover, SIRT4 precipitates with microtubules and interacts with structural (α,β-tubulin, γ-tubulin, TUBGCP2, TUBGCP3) and regulatory (HDAC6) microtubule components as detected by co-immunoprecipitation and mass spectrometric analyses of the mitotic SIRT4 interactome. Overexpression of SIRT4 resulted in a pronounced decrease of acetylated α-tubulin (K40) associated with altered microtubule dynamics in mitotic cells. SIRT4 or the N-terminally truncated variant SIRT4(ΔN28), which is unable to translocate into mitochondria, delayed mitotic progression and reduced cell proliferation. This study extends the functional roles of SIRT4 beyond mitochondrial metabolism and provides the first evidence that SIRT4 acts as a novel centrosomal/microtubule-associated protein in the regulation of cell cycle progression. Thus, stress-induced SIRT4 may exert its role as tumor suppressor through mitochondrial as well as extramitochondrial functions, the latter associated with its localization at the mitotic spindle apparatus.

Rapid Morphological and Cytoskeletal Response to Microgravity in Human Primary Macrophages.

The FLUMIAS (Fluorescence-Microscopic Analyses System for Life-Cell-Imaging in Space) confocal laser spinning disk fluorescence microscope represents a new imaging capability for live cell imaging experiments on suborbital ballistic rocket missions. During the second pioneer mission of this microscope system on the TEXUS-54 suborbital rocket flight, we developed and performed a live imaging experiment with primary human macrophages. We simultaneously imaged four different cellular structures (nucleus, cytoplasm, lysosomes, actin cytoskeleton) by using four different live cell dyes (Nuclear Violet, Calcein, LysoBrite, SiR-actin) and laser wavelengths (405, 488, 561, and 642 nm), and investigated the cellular morphology in microgravity (10−4 to 10−5 g) over a period of about six minutes compared to 1 g controls. For live imaging of the cytoskeleton during spaceflight, we combined confocal laser microscopy with the SiR-actin probe, a fluorogenic silicon-rhodamine (SiR) conjugated jasplakinolide probe that binds to F-actin and displays minimal toxicity. We determined changes in 3D cell volume and surface, nuclear volume and in the actin cytoskeleton, which responded rapidly to the microgravity environment with a significant reduction of SiR-actin fluorescence after 4–19 s microgravity, and adapted subsequently until 126–151 s microgravity. We conclude that microgravity induces geometric cellular changes and rapid response and adaptation of the potential gravity-transducing cytoskeleton in primary human macrophages.

Anisotropy vs isotropy in living cell indentation with AFM

The measurement of local mechanical properties of living cells by nano/micro indentation relies on the foundational assumption of locally isotropic cellular deformation. As a consequence of assumed isotropy, the cell membrane and underlying cytoskeleton are expected to locally deform axisymmetrically when indented by a spherical tip. Here, we directly observe the local geometry of deformation of membrane and cytoskeleton of different living adherent cells during nanoindentation with the integrated Atomic Force (AFM) and spinning disk confocal (SDC) microscope. We show that the presence of the perinuclear actin cap (apical stress fibers), such as those encountered in cells subject to physiological forces, causes a strongly non-axisymmetric membrane deformation during indentation reflecting local mechanical anisotropy. In contrast, axisymmetric membrane deformation reflecting mechanical isotropy was found in cells without actin cap: cancerous cells MDA-MB-231, which naturally lack the actin cap, and NIH 3T3 cells in which the actin cap is disrupted by latrunculin A. Careful studies were undertaken to quantify the effect of the live cell fluorescent stains on the measured mechanical properties. Using finite element computations and the numerical analysis, we explored the capability of one of the simplest anisotropic models – transverse isotropy model with three local mechanical parameters (longitudinal and transverse modulus and planar shear modulus) – to capture the observed non-axisymmetric deformation. These results help identifying which cell types are likely to exhibit non-isotropic properties, how to measure and quantify cellular deformation during AFM indentation using live cell stains and SDC, and suggest modelling guidelines to recover quantitative estimates of the mechanical properties of living cells.

Tumour cell blebbing and extracellular vesicle shedding: key role of matrikines and ribosomal protein SA

BackgroundCarcinogenesis occurs in elastin-rich tissues and leads to local inflammation and elastolytic proteinase release. This contributes to bioactive matrix fragment (Matrikine) accumulation like elastin degradation products (EDP) stimulating tumour cell invasive and metastatic properties. We previously demonstrate that EDPs exert protumoural activities through Hsp90 secretion to stabilised extracellular proteinases.MethodsEDP influence on cancer cell blebbing and extracellular vesicle shedding were examined with a videomicroscope coupled with confocal Yokogawa spinning disk, by transmission electron microscopy, scanning electron microscopy and confocal microscopy. The ribosomal protein SA (RPSA) elastin receptor was identified after affinity chromatography by western blotting and cell immunolocalisation. mRNA expression was studied using real-time PCR. SiRNA were used to confirm the essential role of RPSA.ResultsWe demonstrate that extracellular matrix degradation products like EDPs induce tumour amoeboid phenotype with cell membrane blebbing and shedding of extracellular vesicle containing Hsp90 and proteinases in the extracellular space. EDPs influence intracellular calcium influx and cytoskeleton reorganisation. Among matrikines, VGVAPG and AGVPGLGVG peptides reproduced EDP effects through RPSA binding.ConclusionsOur data suggests that matrikines induce cancer cell blebbing and extracellular vesicle release through RPSA binding, favouring dissemination, cell-to-cell communication and growth of cancer cells in metastatic sites.

PO-269 Development of a fluorescence in situ hybridization (FISH) method for detection of intra-tumour bacteria involved in pancreatic cancer chemoresistance

IntroductionPancreatic cancer (PC) is projected to become the second leading cause of cancer-related deaths in 2020. Recent studies demonstrate that bacteria are a component of pancreatic tumour microenvironment, and they may also play a critical role in mediating resistance to chemotherapy.1 Remarkably, a prospective study showed that the presence of two oral pathogens, Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans, was associated with an elevated risk of developing PC.2Material and methodsThis study aimed at developing a novel fluorescence in situ hybridization (FISH) based method to specifically detect oral bacteria in formalin-fixed paraffin-embedded (FFPE) sections from PC patients. Multi-wavelength confocal spinning disk microscopy was used to discriminate with high spatial resolution and sensitivity the tissue high autofluorescence signal from even low number of emitters/bacteria. Bacterial FISH probes were primers for the 16 s rRNA molecule.Results and discussionsFISH allowed the in situ localisation and the study of spatial organisation of bacterial cells as they occur in PC. FISH results were always definitive and well-detectable both in in 4 and 20 µm FFPE sections. The intensity of the fluorescence was a direct measure for the activity of the bacterial cells, whereas inactive cells were recognised by their low intensity fluorescence. Thus, our method gives a optimal sensitivity of fluorescence and low background by subtracting autofluorescence components.Our FISH preliminary data confirmed the presence of bacteria in the pancreatic tumour microenvironment and support further studies aimed at elucidating how oral bacteria are involved in PC aetiology.It is well-known that the chemoresistance (intrinsic and acquired) has been a major problem for PC treatment3 and a recent study showed the role of bacteria in resistance to gemcitabine.1 To this end, the establishment of our reliable FISH protocol allows us to further investigate the potential role of oral bacteria in the failure of standard treatments for PC.ConclusionOur new robust methodology will contribute to obtain meaningful data and to unravel the complex interaction between oral bacteria and pancreatic cancer chemoresistance in clinically well-annotated tissues, ultimately identifying specific bacteria as potential biomarkers for early detection/treatment.References. Geller, et al. Science2017;357:1156–1160.. Farrell JJ, et al. Gut2012; 61:582–588.. Coppola, et al. Drug Resist. Updates 2017;31:43–51.

Phenotype analysis of Myo10 knockout mice lacking the motorized full‐length, but not the brain‐specific headless isoform

Unconventional myosin‐X (Myo10) is a MyTH4‐FERM and PH domain containing actin‐based motor protein which is widely expressed among vertebrate tissues. MyTH4‐FERM myosins are associated with protrusions enriched in bundled actin filaments. Full‐length (fl) Myo10 is best known for its localization to the tips of filopodia and for its ability to induce and regulate the formation of these dynamic finger‐like structures. At the cellular level, Myo10 has been implicated in diverse functions, including oriented cell migration, axon guidance, melanosome transfer, spindle assembly and phagocytosis. However, the physiological functions at the organismal level of this filopodial cargo carrier are not clear. We studied the phenotypes of Myo10 reporter knockout mice (Myo10tm2), to elucidate Myo10 function in vivo. Using Western blot analysis, we showed that in homozygous knockout Myo10tm2/tm2 mice, the expression of motorized fl Myo10 was deleted, but the brain‐specific headless (hdl) isoform was not abolished. Macroscopic examination of Myo10‐deficient mice revealed ventral coat pigmentation defects (100% penetrance) and a limb malformation, syndactyly, with a high penetrance of >95%. 24% of homozygous KO embryos developed exencephaly, a severe neural tube closure defect that is lethal. Magnetic resonance imaging (MRI) of isolated brains revealed, in contrast to mice lacking the Myo10 cargo protein DCC, intact cerebral commissures. Furthermore, Myo10 KO mice consistently displayed bilateral persistence of the hyaloid vasculature, visualized by MRI and retinal whole mount preparations, whereas retinal vascularization, phagocytosis and chemotactic behavior were not impaired. Confocal spinning disk microscopy confirmed that the hdl isoform of Myo10 does not induce filopodia and we found out that hdl Myo10 strongly localizes to the plasma membrane in dependency of its PH domains, supporting the auto‐inhibition model in which head‐tail interactions of fl Myo10 abolishes inactive monomer binding to PIP3‐rich membranes. In conclusion, Myo10 is important for normal vertebrate development, both prenatal (neural tube closure and digit separation) and postnatal (hyaloid regression).Support or Funding InformationDeutsche Forschungsgemeinschaft (DFG)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Anisotropic Mechanical Properties of Living Cells Revealed by Integrated Spinning Disk Confocal and Atomic Force Microscopy

Recent developments in fluorescent live-cell imaging and biophysical methods have significantly advanced our understanding of the dynamic biochemical and mechanical processes underlying cellular functions such as cell migration. One of most frequently used techniques for assessment of cell mechanical properties is the indentation experiments conducted with the atomic force microscope (AFM), due primarily to AFM's relative ease of operation, its high precision of force measurement, and high spatial resolution. We used the AFM in conjunction with a spinning disk confocal (SDC) microscope to directly visualize AFM indentation of living cells with high spatial and temporal resolution. With novel live cell imaging probes to fluorescently label F-actin, microtubules, and membrane, we were able to directly observe structural changes during the indentation process of a living cell (NIH 3T3 fibroblasts) with a spherical indenter. The most notable observation was a deformation of single apical stress fibers (ASF) under the action of the probe. The presence of ASF (also known as perinuclear actin cap) correlated with low cell height and high stiffness. Moreover, the presence of ASF caused an anisotropic indentation geometry as observed from surface displacement profiles calculated from the SDC images. Isotropic indentation profile was found in cells without ASF: cancerous cells MDA-MB-231 which are naturally lacking actin cap and NIH 3T3 cells in which ASF were disrupted by latrunculin A. Anisotropic indentation behavior suggests an anisotropic deformability and stiffness of the cell. We performed finite element simulations to extract anisotropic properties from the surface displacement data. Simulations suggest a very strong level of anisotropy in cells which role should be further elucidated.

Multiplexed 3D super-resolution imaging of whole cells using spinning disk confocal microscopy and DNA-PAINT

Single-molecule localization microscopy (SMLM) can visualize biological targets on the nanoscale, but complex hardware is required to perform SMLM in thick samples. Here, we combine 3D DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) with spinning disk confocal (SDC) hardware to overcome this limitation. We assay our achievable resolution with two- and three-dimensional DNA origami structures and demonstrate the general applicability by imaging a large variety of cellular targets including proteins, DNA and RNA deep in cells. We achieve multiplexed 3D super-resolution imaging at sample depths up to ~10 µm with up to 20 nm planar and 80 nm axial resolution, now enabling DNA-based super-resolution microscopy in whole cells using standard instrumentation.

Nano step height measurement using an optical method

This paper presents two optical methods for measuring nano step height standards and discusses the deviations in the measurements in detail. A white light interference (WLI) microscope and spinning disk confocal (SDCF) microscope were used in the experiments. The effect of illumination intensity and the standard’s surface reflectivity were studied and analyzed. The surface properties of the step height standard were ameliorated using secondary coating technology. The experimental results demonstrated that the secondary coating technology effectively improved the measurement accuracy of the SDCF microscope. The WLI microscope measured accurately both before and after a secondary coating was applied.

Imaging the Cell Cycle of Pathogen E. coli During Growth in Macrophage.

The study of the bacterial cell cycle at the single cell level can not only give insights on the fitness of the bacterial population but also reveal heterogeneous behavior. Typically, the DNA replication, the cell division, and the nucleoid conformation are appropriate representatives of the bacterial cell cycle. Because bacteria rapidly adapt their growth rate to environmental changes, the measure of cell cycle parameters gives valuable insights for the study of bacterial stress response or host-pathogen interactions. Here we describe methods to first introduce fluorescent fusion proteins and fluorescent tag within the chromosome of pathogenic bacteria to study these cell cycle steps; then to follow them within macrophages using a confocal spinning disk microscope.