Wide-field Fluorescence Imaging Research Articles

On-chip lens-less holographic trichoscopy / trichometry technologies: microinterferometric, Fourier spectral and correlographic techniques for clinical trichology.

The novel principle of trichomorphological analysis is provided. This principle, which may be interpreted as a transition between trichoscopy and trichometry, was developed using methods and techniques, explicated from the lens-less holographic microscopy, lens-lens tomography and filtering / decomposition of microscopic images based on orthogonal transformations, such as the FFTW-library-assisted DFT using Bluestein's FFT algorithm (expresses the Chirp Z-transform as a convolution and implements it efficiently using FFT/IFFT), Rader's FFT algorithm (from MIT Lincoln Laboratory) and Cooley–Tukey FFT algorithm (corresponding to different factorizations and / or different memory-access patterns). It is possible, that such methods can be applicable not only for clinical diagnostics, but also for some specialized trichological investigations, including fast and strong determination of different descriptors for ethnomedicine, gender and hormonal or endocrinology-relevant differences, prenatal and neonatal trichology, bio-archaeometric analysis, including palaeotrichology, and criminological trichomorphology, analysis of efficiency of some pharmaceutical and toxicological agents and events (including microelement status) for different psychosomatic cases, etc. The principal schemes of the laser holographic trichometry setups also may be based on a principles of the combined reflection and transmission lens-less microscopes, on-chip differential interference contrast microscopy, lensless wide-field fluorescent imaging on a chip, off-axis holography and lensless incoherent holography, ulttaviolet microscopy, including deep-UV spectral range.

Epigallocatechin-3-Gallate suppresses neutrophil accumulation and the IL-1 beta pathway in a transgenic zebrafish model of inflammation

Abstract Background The zebrafish represents the simplest and most convenient vertebrate model for undertaking large screens because of its small size, rapid reproduction time and transparency of the body. Here, we adopt a well-established model of inflammation in zebrafish to assess the impact of Epigallocatechin-3-Gallate (EGCG), the main catechin in green tea. Method Tg (MPX:GFP) or Tg (MPX:mCHERRY) transgenic zebrafish were used, bearing GFP- or mCherry-tagged neutrophils, respectively. 4dpf embryos were anesthetized, and inflammation was induced by sterile tailfin-resection. The inflamed groups were separated into those that were non-treated or exposed to EGCG from 0 – 600 uM for 6 hours. 10 fish per group were then mounted alive in methyl cellulose for wide-field fluorescence imaging and analysis was done using ImageJ. Animation of neutrophil migration or accumulation was recorded using an inverted fluorescence confocal microscope till 6-hours after resection. To examine the mechanistic basis of EGCG’s effect, 37 zebrafish inflammatory genes were studied using qPCR. Results The static accumulation of neutrophils at the resection site was highly elevated after wounding (n=32, p<0.0001), and this accumulation was reduced significantly by 31% and 43%, respectively, after exposure to EGCG 300 uM (p<0.05) and 600 uM (p<0.01). Dynamic neutrophil tracking studies are in progress. qPCR studies revealed 6 signature genes in the IL-1 beta pathway to be significantly down-regulated at least 2-fold after EGCG treatment. Conclusion EGCG suppresses inflammation in zebrafish through similar biochemical pathways as in other species. This novel animal model allows facile screening of inflammatory modulators in real-time.

Multiple optical trapping assisted bead-array based fluorescence assay of free and total prostate-specific antigen in serum

Although suspension bead-based assay technology has been widely used owing to its advantages of high-throughput and microvolume detection, its sensitivity is greatly limited because it detects the fluorescence signal emitted by microbeads for a short time in the flowing fluid. In this work, we present the approach for prostate-specific antigen (PSA) detection of both free PSA (fPSA) and total PSA (tPSA) based on bead-array based fluorescence imaging by combining multiple optical trapping and bead-based bioassays. The polystyrene beads were employed to enrich the targets using the classic sandwich immuno-binding and tagged with fluorescent quantum dots (QDs), and the QDs-tagged beads in suspension were trapped array-by-array using multiple optical tweezers constructed with a diffraction optical element and excited with a 405 nm fiber laser for wide-field fluorescence imaging. The distinctive size information from the image of the trapped beads enabled the sorting of different targets. Moreover, the limits of detection for fPSA and tPSA are 3.8 pg/mL and 2.5 pg/mL respectively with good specificity. More importantly, this strategy was successfully used to detect fPSA and tPSA simultaneously in real serum samples. The high sensitivity, good selectivity, and tiny sample volume make this strategy a promising method for life sciences and clinical applications.

The Enlightened Brain: Novel Imaging Methods Focus on Epileptic Networks at Multiple Scales.

Epilepsy research is rapidly adopting novel fluorescence optical imaging methods to tackle unresolved questions on the cellular and circuit mechanisms of seizure generation and evolution. State of the art two-photon microscopy and wide-field fluorescence imaging can record the activity in epileptic networks at multiple scales, from neuronal microcircuits to brain-wide networks. These approaches exploit transgenic and viral technologies to target genetically encoded calcium and voltage sensitive indicators to subclasses of neurons, and achieve genetic specificity, spatial resolution and scalability that can complement electrophysiological recordings from awake animal models of epilepsy. Two-photon microscopy is well suited to study single neuron dynamics during interictal and ictal events, and highlight the differences between the activity of excitatory and inhibitory neuronal classes in the focus and propagation zone. In contrast, wide-field fluorescence imaging provides mesoscopic recordings from the entire cortical surface, necessary to investigate seizure propagation pathways, and how the unfolding of epileptic events depends on the topology of brain-wide functional connectivity. Answering these questions will inform pre-clinical studies attempting to suppress seizures with gene therapy, optogenetic or chemogenetic strategies. Dissecting which network nodes outside the seizure onset zone are important for seizure generation, propagation and termination can be used to optimize current and future evaluation methods to identify an optimal surgical strategy.

Development of a novel carboplatin like cytoplasmic trackable near infrared fluorophore conjugate via strain-promoted azide alkyne cycloaddition

The successful design and pre-clicked synthesis of a non-toxic and cytosol trackable carboplatin-like near infrared fluorophore conjugate via strain-promoted azide alkyne cycloaddition (SPAAC) is reported. Reaction of cis-[Pt(2-azidopropane-1,3-diamine)(cbdca)] (cbdca = cyclobutane-1,1-dicarboxylato) and a bicyclo[6.1.0]non-4-yne near-infrared (NIR) azadipyrromethene fluorophore gave the corresponding clicked Pt-Fluorophore conjugate. The X-ray crystal structure of cis-[Pt(2-azidopropane-1,3-diamine)(cbdca)] was determined featuring the azide on the bidentate 1,3-diaminopropane ligand. The Pt-fluorophore conjugate is the first example of a Pt conjugate clicked via strain-promoted azide alkyne cycloaddition (SPAAC) where the reactive azide handle is on the amine carrier ligand. The in vitro cytotoxicity and widefield fluorescence imaging of the Pt-Fluorophore conjugate in A2780P and A2780cisR cells are described.

Accurate estimation of the illumination pattern's orientation and wavelength in sinusoidal structured illumination microscopy.

Structured illumination microscopy is able to improve the spatial resolution of wide-field fluorescence imaging by applying sinusoidal stripe pattern illumination to the sample. The corresponding computational image reconstruction requires precise knowledge of the pattern's parameters, which are its phase (ϕ) and wave vector (p). Here, a computationally inexpensive method for estimation of p from the raw data is proposed and illustrated with simulations. The method estimates p through a selective discrete Fourier transform at tunable subpixel precision. This results in an accurate p estimation for all the illumination patterns and subsequently improves the superresolution image recovery by a factor of 10 around sharp edges as compared to an integer pixel approach. The technique as presented here is of major interest to the large variety of custom-build systems that are used. The feasibility of the presented method is proven in comparison with published data.

Extending Live-Cell Fluorescence Imaging to Anaerobes of the GUT Microbiome

The gut microbiome supplements human metabolism and immunity by catabolizing host-indigestible glycans. The microbiome operates as a community of trillions of bacteria, and we are beginning to understand the contribution of individual species to glycan digestion. This work extends wide-field and super-resolution fluorescence imaging to the complexity of the human gut microbiome. Past in vitro studies have been limited by mono-culture approaches, and previous live-cell fluorescence microscopy has been limited by the oxygen-dependence of traditional fluorescent proteins like GFP. To address these limitations, we have implemented anaerobic live-cell imaging based on the fatty-acid binding protein, UnaG, in the gut bacterium Bacteroides thetaiotaomicron (B. theta). We have also extended our previous work on B. theta starch utilization system (Sus) enzymes to mixed cultures of B. theta and Ruminococcus bromii, a key species in resistant starch degradation, to understand the relationship between the two species in a microbiome. Finally, we are studying the xyloglucan locus in Bacteroidesovatus to compare polysaccharide utilization loci across species.

Acoustical structured illumination for super-resolution ultrasound imaging

Structured illumination microscopy is an optical method to increase the spatial resolution of wide-field fluorescence imaging beyond the diffraction limit by applying a spatially structured illumination light. Here, we extend this concept to facilitate super-resolution ultrasound imaging by manipulating the transmitted sound field to encode the high spatial frequencies into the observed image through aliasing. Post processing is applied to precisely shift the spectral components to their proper positions in k-space and effectively double the spatial resolution of the reconstructed image compared to one-way focusing. The method has broad application, including the detection of small lesions for early cancer diagnosis, improving the detection of the borders of organs and tumors, and enhancing visualization of vascular features. The method can be implemented with conventional ultrasound systems, without the need for additional components. The resulting image enhancement is demonstrated with both test objects and ex vivo rat metacarpals and phalanges.

Thermal nanoimprint lithography for drift correction in super-resolution fluorescence microscopy.

Localization-based super-resolution microscopy enables imaging of biological structures with sub-diffraction-limited accuracy, but generally requires extended acquisition time. Consequently, stage drift often limits the spatial precision. Previously, we reported a simple method to correct for this by creating an array of 1 μm3 fiducial markers, every ~8 μm, on the coverslip, using UV-nanoimprint lithography (UV-NIL). While this allowed reliable and accurate 3D drift correction, it suffered high autofluorescence background with shorter wavelength illumination, unstable adsorption to the substrate glass surface, and suboptimal biocompatibility. Here, we present an improved fiducial micro-pattern prepared by thermal nanoimprint lithography (T-NIL). The new pattern is made of a thermal plastic material with low fluorescence backgrounds across the wide excitation range, particularly in the blue-region; robust structural stability under cell culturing condition; and a high bio-compatibility in terms of cell viability and adhesion. We demonstrate drift precision to 1.5 nm for lateral (x, y) and 6.1 nm axial (z) axes every 0.2 seconds for a total of 1 min long image acquisition. As a proof of principle, we acquired 4-color wide-field fluorescence images of live mammalian cells; we also acquired super-resolution images of fixed hippocampal neurons, and super-resolution images of live glutamate receptors and postsynaptic density proteins.

Evaluation of a confocal WSI scanner for FISH slide imaging and image analysis

Background: Technological advances contribute to the maturation of digital pathology in clinical and research applications. However, there are only few reports on fluorescence scanning especially on confocal fluorescence imaging technology in digital pathology, which has superior depth resolution compared to wide-field fluorescence imaging. Here, we explored the features of a confocal WSI scanner for typical diagnostic and research imaging applications of fluorescence in situ hybridization (FISH) assay.Methods: Multi-layer stacking (Z-stack) which stores all image information from each layer, and extended focus which stores the optimal image information from all scanned layers, featured in the Pannoramic Confocal scanner (3DHISTECH Ltd., Budapest, Hungary) were employed in digitizing 14 FISH slides (ALK, EGFR, and multi-gene). The slides were scanned with a 40× water immersion objective producing a final image with pixel resolution of 0.1625 µm/pixel. Z-stack and extended focus were used for N=6, 13, and 26 multiple layers scanning at 1, 0.4, and 0.2 µm depth intervals respectively. Single-layer scanning was also done for comparison. Scanning time and resultant file size were recorded. The CaseViewer from 3DHISTECH was used to visualize images and export the annotated regions, and the exported images were further analyzed in Imaris (Bitplane, Zurich, Switzerland) for 3-dimensional reconstruction, nuclear segmentation, and the quantification and co-localization analysis of dots inside nuclei. Quantification data from Imaris were imported into Excel for statistic analysis.Results: Confocal fluorescence scanning of FISH slides enabled sharper image than wide-field scanning, although it required longer scanning time and larger file storage. More intra-nuclear dots were quantified from multi-layer Z-stack images than single-layer images, and the Z-stack increased scanning time and image file size. Furthermore, there were a reduced in the number of dots and an increased in the number of co-localized dots in extended-focus images compared to Z-stack. Dots in multiple channels were quantified and analyzed automatically, which supports clinical diagnosis of gene amplification, deletion, and translocation. Three-dimensional reconstruction of Z-stack produced precise measurement of spatial distance, which supports molecular research.Conclusion: Confocal provides sharper image than wide-field for FISH slide scanning. Extended focus reduces file size and storage, but could cause inaccurate analysis due to misinterpretation of overlapping information. Z-stack scanning provides high volume image information for spatial analysis. We foresee confocal multi-layer scanning as a digital pathology application tool for FISH imaging in both clinical and research in future.

Cellular Redox Profiling Using High-content Microscopy.

Reactive oxygen species (ROS) regulate essential cellular processes including gene expression, migration, differentiation and proliferation. However, excessive ROS levels induce a state of oxidative stress, which is accompanied by irreversible oxidative damage to DNA, lipids and proteins. Thus, quantification of ROS provides a direct proxy for cellular health condition. Since mitochondria are among the major cellular sources and targets of ROS, joint analysis of mitochondrial function and ROS production in the same cells is crucial for better understanding the interconnection in pathophysiological conditions. Therefore, a high-content microscopy-based strategy was developed for simultaneous quantification of intracellular ROS levels, mitochondrial membrane potential (ΔΨm) and mitochondrial morphology. It is based on automated widefield fluorescence microscopy and image analysis of living adherent cells, grown in multi-well plates, and stained with the cell-permeable fluorescent reporter molecules CM-H2DCFDA (ROS) and TMRM (ΔΨm and mitochondrial morphology). In contrast with fluorimetry or flow-cytometry, this strategy allows quantification of subcellular parameters at the level of the individual cell with high spatiotemporal resolution, both before and after experimental stimulation. Importantly, the image-based nature of the method allows extracting morphological parameters in addition to signal intensities. The combined feature set is used for explorative and statistical multivariate data analysis to detect differences between subpopulations, cell types and/or treatments. Here, a detailed description of the assay is provided, along with an example experiment that proves its potential for unambiguous discrimination between cellular states after chemical perturbation.

Cellular Redox Profiling Using High-content Microscopy

Reactive oxygen species (ROS) regulate essential cellular processes including gene expression, migration, differentiation and proliferation. However, excessive ROS levels induce a state of oxidative stress, which is accompanied by irreversible oxidative damage to DNA, lipids and proteins. Thus, quantification of ROS provides a direct proxy for cellular health condition. Since mitochondria are among the major cellular sources and targets of ROS, joint analysis of mitochondrial function and ROS production in the same cells is crucial for better understanding the interconnection in pathophysiological conditions. Therefore, a high-content microscopy-based strategy was developed for simultaneous quantification of intracellular ROS levels, mitochondrial membrane potential (ΔΨm) and mitochondrial morphology. It is based on automated widefield fluorescence microscopy and image analysis of living adherent cells, grown in multi-well plates, and stained with the cell-permeable fluorescent reporter molecules CM-H2DCFDA (ROS) and TMRM (ΔΨm and mitochondrial morphology). In contrast with fluorimetry or flow-cytometry, this strategy allows quantification of subcellular parameters at the level of the individual cell with high spatiotemporal resolution, both before and after experimental stimulation. Importantly, the image-based nature of the method allows extracting morphological parameters in addition to signal intensities. The combined feature set is used for explorative and statistical multivariate data analysis to detect differences between subpopulations, cell types and/or treatments. Here, a detailed description of the assay is provided, along with an example experiment that proves its potential for unambiguous discrimination between cellular states after chemical perturbation.

Fiber bundle-based integrated platform for wide-field fluorescence imaging and patterned optical stimulation for modulation of vasoconstriction in the deep brain of a living animal.

We report a fiber optics-based intravital fluorescence imaging platform that includes epi-fluorescence microscopy and laser patterned-light stimulation system. The platform can perform real-time fluorescence imaging with a lateral resolution of ~4.9 μm while directly inserted into the intact mouse brain, optically stimulate vasoconstriction during real-time imaging, and avoid vessel damage in the penetration path of imaging probe. Using 473-nm patterned-light stimulation, we successfully modulated the vasoconstriction of a single targeted 37-μm-diameter blood vessel located more than 4.7 mm below the brain surface of a live SM22-ChR2 mouse. This platform may permit the hemodynamic studies associated with deeper brain neurovascular disorders.

Fluorescence analysis of a tumor model in the chorioallantoic membrane used for the evaluation of different photosensitizers for photodynamic therapy

The development of a tumor in the chicken chorioallantoic membrane (CAM) enables a more individualized understanding of the dynamics of the photosensitizer (PS) interaction with the tumor blood vessels and cells. Photogem® and 5-aminolevulinic acid (ALA), a protoporphyrin IX (PpIX) precursor, were used as PS and their red fluorescence enabled the monitoring of PS dynamic distribution in the vessels and in the tumor. With a tumor model in CAM and fluorescence assessment, the aim of this study was to evaluate the PDT parameters comparing different photosensitezers. In this model, the topical application was chosen as the best way of drug delivery and widefield fluorescence images were at every 30min. The images were processed in a MATLAB® routine for a semi-quantitative analysis of the red fluorescence. PpIX formation in the blood vessels and in the tumor region was observed after 3h and 1.5h, respectively, whereas Photogem® was accumulated in the tumor region after 2h. The illumination was performed by a diode laser with emission centered at 635nm and irradiance of 80mW/cm2 for 10min. After PDT irradiation, the photobleaching for both compounds was observed. Photogem® showed a reduced photobleaching, however, both PS induced a destruction of the tumor mass and vascular network in the treated area.

Temporal focusing microscopy combined with three-dimensional structured illumination

Temporal focusing microscopy provides the optical sectioning capability in wide-field two-photon fluorescence imaging. Here, we demonstrate temporal focusing microscopy combined with three-dimensional structured illumination, which enables us to enhance the three-dimensional spatial resolution and reject the background fluorescence. Experimentally, the periodic pattern of the illumination was produced not only in the lateral direction but also in the axial direction by the interference between three temporal focusing pulses, which were easily generated using a digital micromirror device. The lateral resolution and optical sectioning capability were successfully enhanced by factors of 1.6 and 3.6, respectively, compared with those of temporal focusing microscopy. In the two-photon fluorescence imaging of a tissue-like phantom, the out-of-focus background fluorescence and the scattered background fluorescence could also be rejected.

Rapid imaging and detection of circulating tumor cells using a wide-field fluorescence imaging system

Circulating tumor cells (CTCs) provide potentially accessible in vivo sources of metastatic cancer cells. As such, considerable focus has been placed on analyzing the genetics of single-CTCs. Prior to these analyses, however, CTCs must first be detected within the blood samples of cancer patients. Current methods for detection of CTCs by fluorescence microscopy require the analysis of hundreds of images per blood sample, making this a time-consuming process that creates a bottleneck in CTC analysis. In this study, we therefore developed a wide-field fluorescence imaging system for rapid CTC detection. For these analyses, CTCs were first isolated using the microcavity array (MCA), a micro-sized filter for CTC recovery that separates cells based on differences in cell size and deformability. Notably, the proposed imaging system enabled rapid (∼10 s) visualization of all stained cells within the 6 mm × 6 mm MCA field via one-shot imaging. Furthermore, the morphology of the cells in the resulting images accurately reflected that observed by conventional microscopy. In total, isolation and detection of CTCs using the MCA combined with our novel wide-field fluorescence imaging system was achieved within 1 h. Thus, our proposed system will provide rapid CTC detection system.

A fully-automated multiscale kernel graph cuts based particle localization scheme for temporal focusing two-photon microscopy.

The temporal focusing two-photon microscope (TFM) is developed to perform depth resolved wide field fluorescence imaging by capturing frames sequentially. However, due to strong nonignorable noises and diffraction rings surrounding particles, further researches are extremely formidable without a precise particle localization technique. In this paper, we developed a fully-automated scheme to locate particles positions with high noise tolerance. Our scheme includes the following procedures: noise reduction using a hybrid Kalman filter method, particle segmentation based on a multiscale kernel graph cuts global and local segmentation algorithm, and a kinematic estimation based particle tracking method. Both isolated and partial-overlapped particles can be accurately identified with removal of unrelated pixels. Based on our quantitative analysis, 96.22% isolated particles and 84.19% partial-overlapped particles were successfully detected.

Stationary bubble formation and Marangoni convection induced by CW laser heating of a single gold nanoparticle.

Gold nanoparticles (Au NPs) efficiently convert incident light into heat under the resonant conditions of localized surface plasmon. Controlling mass transfer through plasmonic heating of Au NPs has potential applications such as manipulation and fabrication within a small space. Here, we describe the formation of stationary microbubbles and subsequent fluid convection induced by CW laser heating of Au NPs in water. Stationary bubbles of about 1-20 μm in diameter were produced by irradiating individual Au NPs with a CW laser. Spatial profiles and velocity distribution of fluid convection around the microbubbles were visualized by the wide-field fluorescence imaging of tracer nanospheres. To evaluate the bubble-induced convection, numerical simulations were performed on the basis of general heat diffusion and Navier-Stokes equations. A comparison between the experimental and computational results revealed that a temperature derivative of surface tension at the bubble surface is a key factor to control the fluid convection. Temperature differences of a few Kelvin at the bubble surface resulted in convective velocities ranging from 102 to 103 μm s-1. The convective velocity gradually increased with increasing bubble diameter. This article covers both natural and Marangoni convection induced by plasmonic heating of Au NPs.

Automated algorithm for actinic cheilitis diagnosis by wide-field fluorescence imaging.

Actinic cheilitis (AC) is a disease caused by prolonged and cumulative sun exposure that mostly affects the lower lip, which can progress to a lip squamous cell carcinoma. Routine diagnosis relies on clinician experience and training. We investigated the diagnostic efficacy of wide-field fluorescence imaging coupled to an automated algorithm for AC recognition. Fluorescence images were acquired from 57 patients with confirmed AC and 46 normal volunteers. Three different algorithms were employed: two based on the emission characteristics of local heterogeneity, entropy and intensity range, and one based on the number of objects after K-mean clustering. A classification model was obtained using a fivefold cross correlation algorithm. Sensitivity and specificity rates were 86% and 89.1%, respectively.

Wide-field optical mapping of neural activity and brain haemodynamics: considerations and novel approaches.

Although modern techniques such as two-photon microscopy can now provide cellular-level three-dimensional imaging of the intact living brain, the speed and fields of view of these techniques remain limited. Conversely, two-dimensional wide-field optical mapping (WFOM), a simpler technique that uses a camera to observe large areas of the exposed cortex under visible light, can detect changes in both neural activity and haemodynamics at very high speeds. Although WFOM may not provide single-neuron or capillary-level resolution, it is an attractive and accessible approach to imaging large areas of the brain in awake, behaving mammals at speeds fast enough to observe widespread neural firing events, as well as their dynamic coupling to haemodynamics. Although such wide-field optical imaging techniques have a long history, the advent of genetically encoded fluorophores that can report neural activity with high sensitivity, as well as modern technologies such as light emitting diodes and sensitive and high-speed digital cameras have driven renewed interest in WFOM. To facilitate the wider adoption and standardization of WFOM approaches for neuroscience and neurovascular coupling research, we provide here an overview of the basic principles of WFOM, considerations for implementation of wide-field fluorescence imaging of neural activity, spectroscopic analysis and interpretation of results.This article is part of the themed issue ‘Interpreting BOLD: a dialogue between cognitive and cellular neuroscience’.