Wide-field Fluorescence Microscopy Research Articles

4D Widefield Fluorescence Imaging of Appressorium Morphogenesis by Magnaporthe oryzae.

Fluorescence microscopy has become a widely used and indispensable tool for the M. oryzae research community, providing unique insight into appressorium formation and function. A common practice within the field is to acquire and present images of a number of different conidia, expressing a fluorescent fusion protein of interest, at various stages of infectious development, therein providing a representative "snapshot" of the population at a given point in time. Furthermore, these images typically show only a single focal plane through the specimen (2D) and therefore lack, often valuable, volumetric information. While this approach has its advantages, the continuous imaging of (multiple) single conidia in three dimensions (3D), and over time (4D), can provide additional insight into the spatial and temporal dynamics of fluorescent fusion proteins, and the subcellular structures and compartments they label, in living cells. Here we describe our typical workflow for the 4D live-cell imaging of appressorium morphogenesis in vitro using two-color widefield fluorescence microscopy and briefly outline some important considerations for strain construction, and downstream image processing and visualization.

Highly Efficient and Scalable Framework for High-Speed Super-Resolution Microscopy

The multiple signal classification algorithm (MUSICAL) is a statistical super-resolution technique for wide-field fluorescence microscopy. Although MUSICAL has several advantages, such as its high resolution, its low computational performance has limited its exploitation. This paper aims to analyze the performance and scalability of MUSICAL for improving its low computational performance. We first optimize MUSICAL for performance analysis by using the latest high-performance computing libraries and parallel programming techniques. Thereafter, we provide insights into MUSICAL's performance bottlenecks. Based on the insights, we develop a new parallel MUSICAL in C++ using Intel Threading Building Blocks and the Intel Math Kernel Library. Our experimental results show that our new parallel MUSICAL achieves a speed-up of up to 30.36x on a commodity machine with 32 cores with an efficiency of 94.88%. The experimental results also show that our new parallel MUSICAL outperforms the previous versions of MUSICAL in Matlab, Java, and Python by 30.43x, 2.63x, and 1.69x, respectively, on commodity machines.

Neural Stimulation In Vitro and In Vivo by Photoacoustic Nanotransducers

Summary Neuromodulation is an invaluable approach for the study of neural circuits and clinical treatment of neurological diseases. Here, we report semiconducting polymer nanoparticles based photoacoustic nanotransducers (PANs) for neural stimulation in vitro and in vivo. Our PANs strongly absorb the nanosecond pulsed laser in the near-infrared second window (NIR-II) and generate localized acoustic waves. PANs are shown to be surface modified and selectively bind onto neurons. PAN-mediated activation of primary neurons in vitro is achieved with ten 3-ns laser pulses at 1,030 nm over a 3-ms duration. In vivo neural modulation of mouse brain activities and motor activities is demonstrated by PANs directly injected into brain cortex. With submillimeter spatial resolution and negligible heat deposition, PAN stimulation is a new non-genetic method for precise control of neuronal activities, opening up potentials in non-invasive brain modulation.

<p><strong>A close affinity of the enigmatic genus <em>Stegocoleus</em> with <em>Lepidomma</em> revealed by new fossil evidence (Coleoptera: Archostemata: Ommatidae)</strong></p>

Stegocoleus Jarzembowski & Wang is an enigmatic genus in the family Ommatidae, known to date only from Burmese amber. This genus possesses a unique combination of characters, including antennal grooves on the ventral side of head, well-developed epipleural rims, and presence of separated procoxae, which makes its systematic position unclear. Here we report two new species of Lepidomma from mid-Cretaceous Burmese amber, L. longisquama sp. nov. and L. jarzembowskii sp. nov. Based on these well-preserved specimens and new morphological details revealed by widefield fluorescence and confocal microscopy, we discuss the morphological similarities between Lepidomma and the enigmatic Stegocoleus. We suggest that Stegocoleus is not a basal ommatid, but a highly derived form of ommatid beetles.

Bloch Surface Wave Assisted Structured Illumination Microscopy for Sub-100 nm Resolution

Structured illumination microscopy (SIM) is one of the most powerful and versatile super-resolution methods due to its live-cell imaging ability of subcellular structures with high speed. In this paper, we propose an alternative SIM assisted by Bloch surface wave (BSW). Through replacing the conventional laser interference fringes with sub-diffraction BSW counterparts with higher spatial frequency, the imaging resolution of SIM could be enhanced greatly and the super-resolution imaging capability down to 80 nm could be achieved. Compared with traditional wide-field fluorescence microscopy, this BSW SIM demonstrates 2.78 times enhancement in imaging resolution, which surpasses general SIM with only 2 times improvement. Moreover, the structured illumination intensity could be boosted drastically, which is beneficial to nonlinear super-resolution imaging techniques, such as saturated SIM. This BSW SIM would provide a super-resolution, wide field of view approach for surface super-resolution fluorescent imaging while maintaining high imaging speed and good bio-compatibility.

Microscopy automation on a budget: capturing high-speed multi-labelled events with LED Illumination Systems.

Capturing fast cellular processes with widefield fluorescence microscopy and multiple fluorophores has historically required the addition of motorised components. The light source has often been overlooked as an affordable means of achieving multi-wavelength imaging, but LEDs have changed the game. Here we compare typical configurations for automated illumination systems in terms of speed, cost and signal-to-noise ratio (i.e. image contrast). We also explore how to transform a manual microscope into a powerful automated imaging system by capitalising on LED switching speeds, with lower cost filters and minimal requirement for expensive motorised components.

Preventing Photomorbidity in Long-Term Multi-color Fluorescence Imaging of Saccharomyces cerevisiae and S. pombe.

Time-lapse imaging of live cells using multiple fluorescent reporters is an essential tool to study molecular processes in single cells. However, exposure to even moderate doses of visible excitation light can disturb cellular physiology and alter the quantitative behavior of the cells under study. Here, we set out to develop guidelines to avoid the confounding effects of excitation light in multi-color long-term imaging. We use widefield fluorescence microscopy to measure the effect of the administered excitation light on growth rate (here called photomorbidity) in yeast. We find that photomorbidity is determined by the cumulative light dose at each wavelength, but independent of the way excitation light is applied. Importantly, photomorbidity possesses a threshold light dose below which no effect is detectable (NOEL). We found, that the suitability of fluorescent proteins for live-cell imaging at the respective excitation light NOEL is equally determined by the cellular autofluorescence and the fluorescent protein brightness. Last, we show that photomorbidity of multiple wavelengths is additive and imaging conditions absent of photomorbidity can be predicted. Our findings enable researchers to find imaging conditions with minimal impact on physiology and can provide framework for how to approach photomorbidity in other organisms.

Evaluating the Effects of Disinfectants on Bacterial Biofilms Using a Microfluidics Flow Cell and Time-Lapse Fluorescence Microscopy.

A commercially available microfluidics flow cell was utilized together with widefield fluorescence microscopy to evaluate the effects of disinfectants on bacterial strains. The flow cell’s inner surface supports the formation of biofilms of numerous bacterial species. The modular setup of the flow cell accessories allows connection to syringes, pumps and collection vials, facilitating aseptic experiments in a controlled fluidics environment which can be documented with precisely timed microscopy imaging. The flow cell is inoculated with a suspension of bacteria in a nutrient medium and incubated for several days allowing bacterial cells to form a biofilm. Shortly before performing an assay, the biofilm is labelled with a dual-fluorescent DNA probe which distinguishes unharmed and damaged bacteria. Then a disinfectant sample (or control) is gently injected and time-lapse imaging is used for quantifying the course of bacterial biomass response. We use a simplified widefield microscopy method that allows intensive recording and quantification of time series of two-dimensional frames for tracking the course of disinfectant action on a variety of microbial strains. This procedure has potential for the rapid evaluation of novel products.

Sources of widefield fluorescence from the brain.

Widefield fluorescence microscopy is used to monitor the spiking of populations of neurons in the brain. Widefield fluorescence can originate from indicator molecules at all depths in cortex and the relative contributions from somata, dendrites, and axons are often unknown. Here, I simulate widefield illumination and fluorescence collection and determine the main sources of fluorescence for several GCaMP mouse lines. Scattering strongly affects illumination and collection. One consequence is that illumination intensity is greatest ~300-400 µm below the pia, not at the brain surface. Another is that fluorescence from a source deep in cortex may extend across a diameter of 3-4 mm at the brain surface, severely limiting lateral resolution. In many mouse lines, the volume of tissue contributing to fluorescence extends through the full depth of cortex and fluorescence at most surface locations is a weighted average across multiple cortical columns and often more than one cortical area.

Lab on a rod: Size-based particle separation and sorting in a helical channel.

Size-based particle separation using inertial microfluidics in spiral channels has been well studied over the past decade. Though these devices can effectively separate particles, they require a relatively large device footprint with a typical outer channel radius of approximately 15 mm. In this paper, we describe a microfluidic device with a footprint diameter of 5.5 mm, containing a helical channel capable of inertial particle separation fabricated using abrasive jet micromachining. The separation of particles in several channel geometries was studied using wide-field fluorescence microscopy. A maximum separation efficiency of approximately 90% was achieved at a flow rate of 1.5 ml/min with a purity of approximately 95% at the outlet, where large particles were collected. An accompanying computational fluid dynamics model was developed to allow researchers to quickly assess the separation capability of their helical or spiral devices.

Imaging adult C. elegans live using light-sheet microscopy.

SummaryLive observation of biological phenomena in the context of living organisms can provide important insights in the mechanisms of these phenomena. However, the spatially complex and dynamic physiology of multicellular organisms can be a challenging environment to make observations with fluorescence microscopy. Due to the illumination of out‐of‐focus planes, confocal and particularly widefield fluorescence microscopy suffer from low signal‐to‐background ratio (SBR), photo toxicity and bleaching of fluorescent probes. In light‐sheet microscopy (LSM), solely the focal plane of the detection objective is illuminated, minimising out‐of‐focus fluorescence and photobleaching, thereby enhancing SBR, allowing for low laser intensities and longer acquisition periods. Here we present a straightforward light‐sheet microscope with a 1.0‐NA detection objective and a fast sample‐positioning stage that allows for four degrees of freedom. By imaging the sensory cilia and nervous system of living young adult C. elegans, we demonstrate that the instrument is well suited for relatively fast, volumetric imaging of larger (hundreds of micrometres cubed) living samples. These experiments demonstrate that such an instrument provides a valuable addition to commonly used widefield and confocal fluorescence microscopes.Lay descriptionIn fluorescence microscopy, sharp images can only be obtained when the light obtained from the section of the image that is in focus is not overwhelmed by light emerging from elsewhere. In this paper, we present a light‐sheet fluorescence microscope, based on the OpenSPIM initiative, with a magnification of 90× and a sensitive sample positioning stage that allows for fast controlled linear movement and rotation. In a light‐sheet microscope (LSM), the sample is illuminated from the side, compared to the direction of detection, limiting illumination only to the part of the sample that is imaged in the focal plane (general resources: Wikipedia or MicroscopyU). This does not only limit background noise, but also reduces damage to the sample due to phototoxicity. This makes a LSM particularly suitable for imaging living samples at high resolution, in three dimensions, over long periods of time.Our instrument was specifically designed for imaging adult C. elegans nematodes. We show here how the instrument compares to a standard epifluorescence microscope, imaging neuronal structures in the animals. The instrument proved well suited for fast volumetric imaging of larger cellular structures such as C. elegans neuronal cell bodies. Our experiments show that the instrument provides a valuable addition to widefield and confocal fluorescence microscopes commonly used to image adult C. elegans.

Moxifloxacin based fluorescence imaging of intestinal goblet cells.

Goblet cells (GCs) in the intestine are specialized epithelial cells that secrete mucins to form the protective mucous layer. GCs are important in maintaining intestinal homeostasis, and the alteration of GCs is observed in inflammatory bowel diseases (IBDs) and neoplastic lesions. In the Barrett's esophagus, the presence of GCs is used as a marker of specialized intestinal metaplasia. Various endomicroscopic imaging methods have been used for imaging intestinal GCs, but high-speed and high-contrast GC imaging has been still difficult. In this study, we developed a high-contrast endoscopic GC imaging method: fluorescence endomicroscopy using moxifloxacin as a GC labeling agent. Moxifloxacin based fluorescence imaging of GCs was verified by using two-photon microscopy (TPM) in the normal mouse colon. Label-free TPM, which could visualize GCs in a negative contrast, was used as the reference. High-speed GC imaging was demonstrated by using confocal microscopy and endomicroscopy in the normal mouse colon. Confocal microscopy was applied to dextran sulfate sodium (DSS) induced colitis mouse models for the detection of GC depletion. Moxifloxacin based GC imaging was demonstrated not only by 3D microscopies but also by wide-field fluorescence microscopy, and intestinal GCs in the superficial region were imaged. Moxifloxacin based endomicroscopy has a potential for the application to human subjects by using FDA approved moxifloxacin.

Quantitative analysis of fluorescent ligand binding to dopamine D3 receptors using live-cell microscopy.

Dopamine receptors are G protein-coupled receptors that have several essential functions in the central nervous system. A better understanding of the regulatory mechanisms of ligand binding to the receptor may open new possibilities to affect the downstream signal transduction pathways. The majority of the available ligand binding assays use either membrane preparations, cell suspensions, or genetically modified receptors, which may give at least partially incorrect understanding of ligand binding. In this study, we implemented an assay combining fluorescence and bright-field microscopy to measure ligand binding to dopamine D3 receptors in live mammalian cells. For membrane fluorescence intensity quantification from microscopy images, we developed a machine learning-based user-friendly software membrane tools and incorporated it into a data management software aparecium that has been previously developed in our workgroup. For the experiments, a fluorescent ligand NAPS-Cy3B was synthesized by conjugating a dopaminergic antagonist N-(p-aminophenethyl)spiperone with a fluorophore Cy3B. The subnanomolar affinity of NAPS-Cy3B makes it a suitable ligand for the characterization of D3 receptors in live HEK293 cells. Using a microplate compatible automated widefield fluorescence microscope, together with the membrane tools software, enables the detection and quantification of ligand binding with a high-throughput. The live cell assay is suitable for the characterization of fluorescent ligand binding and also in the competition experiments for the screening of novel unlabeled dopaminergic ligands. We propose that this simple yet more native-like approach is feasible in GPCR research, as it enables the detection of ligand binding in an environment containing more components involved in the signal transduction cascade.

Lissajous scanning structured illumination microscopy.

High-resolution fluorescent microscopic imaging techniques are in high demand to observe detailed structures or dynamic mechanisms of biological samples. Structured illumination microscopy (SIM) has grabbed much attention in super-resolution imaging due to simple configuration, high compatibility with common fluorescent molecules, and fast image acquisition. Here, we report Lissajous scanning SIM (LS-SIM) by using a high fill-factor Lissajous scanning micromirror and laser beam modulation. The LS-SIM was realized by a Lissajous scanned structured illumination module, relay optics, and a conventional fluorescent microscope. The micromirror comprises an inner mirror and an outer frame, which are scanned at pseudo-resonance with electrostatic actuation. The biaxial scanning frequencies are selected by the frequency selection rule for high fill-factor (> 80%) Lissajous scanning. Structured illumination (SI) was then realized by modulating the intensity of a laser beam at the least common multiple (LCM) of the scanning frequencies. A compact Lissajous scanned SI module containing a fiber-optic collimator and Lissajous micromirror has been fully packaged and coupled with relay optics and a fiber-based diode pumped solid state (DPSS) laser including acousto-optic-modulator (AOM). Various structured images were obtained by shifting the phase and orientation of the illumination patterns and finally mounted with a conventional fluorescent microscope. The LS-SIM has experimentally demonstrated high-resolution fluorescent microscopic imaging of reference targets and human lung cancer cell PC-9 cells. The LS-SIM exhibits the observable region in spatial frequency space over 2x, the line-edge sharpness over 1.5x, and the peak-to-valley (P-V) ratio over 2x, compared to widefield fluorescent microscopy. This method can provide a new route for advanced high-resolution fluorescent microscopic imaging.

Spatially and Temporally Resolved Heterogeneities in a Miscible Polymer Blend.

Mapping the spatial and temporal heterogeneities in miscible polymer blends is critical for understanding and further improving their material properties. However, a complete picture on the heterogeneous dynamics is often obscured in ensemble measurements. Herein, the spatial and temporal heterogeneities in fully miscible polystyrene/oligostyrene blend films are investigated by monitoring the rotational diffusion of embedded individual probe molecules using defocused wide-field fluorescence microscopy. In the same blend film, three significantly different types of dynamical behaviors (referred to as modes) of the probe molecules can be observed at the same time, namely, immobile, continuously rotating, and intermittently rotating probe molecules. This reveals a prominent spatial heterogeneity in local dynamics at the nanometer scale. In addition to that, temporal heterogeneity is uncovered by the nonexponential characteristic of the rotational autocorrelation functions of single-molecule probes. Moreover, the occurrence probabilities of these different modes strongly depend on the polystyrene: oligostyrene ratios in the blend films. Remarkably, some probe molecules switch between the continuous and intermittent rotational modes at elevated temperature, indicating a possible alteration in local dynamics that is triggered by the dynamic heterogeneity in the blends. Although some of these findings can be discussed by the self-concentration model and the results provided by ensemble averaging techniques (e.g., dielectric spectroscopy), there are implications that go beyond current models of blend dynamics.

BatchDeconvolution: a Fiji plugin for increasing deconvolution workflow

Abstract Deconvolution microscopy is a very useful, software-based technique allowing to deblur microscopy images and increase both lateral and axial resolutions. It can be used along with many of fluorescence microscopy imaging techniques. By increasing axial resolution, it also enables three-dimensional imaging using a basic wide-field fluorescence microscope. Unfortunately, commercially available deconvolution software is expensive, while freely available programs have limited capabilities of a batch file processing. In this work we present BatchDeconvolution, a Fiji plugin that bridges two programs that we used subsequently in an image deconvolution pipeline: PSF Generator and DeconvolutionLab2, both from Biomedical Imaging Group, EPFL. Our software provides a simple way to perform a batch processing of multiple microscopy files with minimal working time required from the user.

Inferring network structure and local dynamics from neuronal patterns with quenched disorder

Abstract An inverse procedure is proposed and tested which aims at recovering the a priori unknown functional and structural information from global signals of living brains activity. To this end, we consider a Leaky-Integrate and Fire (LIF) model with short term plasticity neurons, coupled via a directed network. Neurons are assigned a specific current value, which is heterogenous across the sample, and sets the firing regime in which the neuron is operating in. The aim of the method is to recover the distribution of incoming network degrees, as well as the distribution of the assigned currents, from global field measurements. The proposed approach to the inverse problem implements the reductionist Heterogenous Mean-Field approximation. This amounts in turn to organizing the neurons in different classes, depending on their associated degree and current. When tested against synthetic data, the method returns accurate estimates of the sought distributions, while managing to reproduce and interpolate almost exactly the time series of the supplied global field. Finally, we also applied the proposed technique to longitudinal wide-field fluorescence microscopy data of cortical functionality in awake Thy1-GCaMP6f mice. Mice are induced a photothrombotic stroke in the primary motor cortex and their recovery monitored in time. An all-to-all LIF model which accommodates for currents heterogeneity allows to adequately explain the recorded patterns of activation. Altered distributions in neuron excitability are in particular detected, compatible with the phenomenon of hyperexcitability in the penumbra region after stroke.

Widefield Fluorescence Microscopy in Preclinical Studies of Biomedical Materials, Scaffolds, and Biomedical Cell Products

We present the results of studies of the interaction of cells with various materials and products from them intended for biomedical use. Widefield fluorescence microscopy enables evaluating the cytotoxicity of samples, cell viability, morphology, and proliferative activity in the process of interaction with the test samples, as well as adhesion, migration, and cell distribution both on the surface and in the structure of scaffolds and biomedical cell products. The methods of fluorescence microscopy make possible intravital studies of the interaction of cells with materials without additional processing or destruction of samples.

A Compact High-Speed Image-Based Method for Measuring the Longitudinal Motion of Living Tissues.

Intraoperative imaging of living tissue at the cell level by endomicroscopy might help surgeons optimize surgical procedures and provide individualized treatments. However, the resolution of the microscopic image is limited by the motion of living tissue caused by heartbeat and respiration. An active motion compensation (AMC) strategy has been recognized as an effective way to reduce, or even eliminate, the influence of tissue movement for intravital fluorescence microscopy (IVM). To realize the AMC system, a high-speed sensor for measuring the motion of tissues is needed. At present, state-of-the-art commercialized displacement sensors are not suitable to apply in minimally invasive imaging instruments to measure the motion of living tissues because of the size problem, range of measurement or the update rate. In this study, a compact high-speed image-based method for measuring the longitudinal motion of living tissues is proposed. The complexity of the proposed method is the same as that of the traditional wide-field fluorescent microscopy (WFFM) system, which makes it easy to be miniaturized and integrated into a minimally invasive imaging instrument. Experimental results reveal that the maximum indication error, range of measurement and the sensitivity of the laboratory-built experimental prototype is 150 μm, 6 and −211.46 respectively. Experimental results indicate that the proposed optical method is expected to be used in minimally invasive imaging instruments to build an AMC system.

Moxifloxacin based axially swept wide-field fluorescence microscopy for high-speed imaging of conjunctival goblet cells.

Goblet cells (GCs) in the conjunctiva are specialized epithelial cells producing mucins on the ocular surface. GCs play important roles in maintaining homeostasis of the ocular surface, and GC dysfunction is associated with various complications including dry eye diseases. Current GC examination methods, which are conjunctival impression cytology and confocal reflection microscopy, have limitations for routine examination. Fluorescence microscopy using moxifloxacin was recently introduced as a non-invasive and high-contrast imaging method, but further development is needed to be used for GC examination. Here we developed a non-invasive high-speed high-contrast GC imaging method, called moxifloxacin based axially swept wide-field fluorescence microscopy (MBAS-WFFM). This method acquired multiple fluorescence images with the axial sweeping of the focal plane to capture moxifloxacin labeled GCs on the tilted conjunctival surface in focus and generated all-in-focus images by combining the acquired images. The imaging field of view and imaging speed were increased to 1.6 mm × 1.6 mm and 30 fps. An image processing method was developed for the analysis of GC density. MBAS-WFFM was applied to alkali burn mouse models and detected GC damage and recovery via longitudinal imaging. MBAS-WFFM could assess the status of GCs rapidly and non-invasively. We anticipate MBAS-WFFM to be a starting point for non-invasive GC examination and the diagnosis of GC associated diseases. For example, MBAS-WFFM could be used to classify dry eye diseases into detail categories for effective treatment.