Oufti: an integrated software package for high‐accuracy,high‐throughput quantitative microscopy analysis

With the realization that bacteria display phenotypic variability among cells and exhibit complex subcellular organization critical for cellular function and behavior, microscopy has re‐emerged as a primary tool in bacterial research during the last decade. However, the bottleneck in today's single‐cell studies is quantitative image analysis of cells and fluorescent signals. Here, we address current limitations through the development of Oufti, a stand‐alone, open‐source software package for automated measurements of microbial cells and fluorescence signals from microscopy images. Oufti provides computational solutions for tracking touching cells in confluent samples, handles various cell morphologies, offers algorithms for quantitative analysis of both diffraction and non‐diffraction‐limited fluorescence signals and is scalable for high‐throughput analysis of massive datasets, all with subpixel precision. All functionalities are integrated in a single package. The graphical user interface, which includes interactive modules for segmentation, image analysis and post‐processing analysis, makes the software broadly accessible to users irrespective of their computational skills.     

Early sequence of events triggered by the interaction of Neisseria meningitidis with endothelial cells

Neisseria meningitidis is a bacterium responsible for severe sepsis and meningitis. Following type IV pilus‐mediated adhesion to endothelial cells, bacteria proliferating on the cellular surface trigger a potent cellular response that enhances the ability of adhering bacteria to resist the mechanical forces generated by the blood flow. This response is characterized by the formation of numerous 100 nm wide membrane protrusions morphologically related to filopodia. Here, a high‐resolution quantitative live‐cell fluorescence microscopy procedure was designed and used to study this process. A farnesylated plasma membrane marker was first detected only a few seconds after bacterial contact, rapidly followed by actin cytoskeleton reorganization and bulk cytoplasm accumulation. The bacterial type IV pili‐associated minor pilin PilV is necessary for the initiation of this cascade. Plasma membrane composition is a key factor as cholesterol depletion with methyl‐β‐cyclodextrin completely blocks the initiation of the cellular response. In contrast membrane deformation does not require the actin cytoskeleton. Strikingly, plasma membrane remodelling undermicrocolonies is also independent of common intracellular signalling pathways as cellular ATP depletion is not inhibitory. This study shows that bacteria‐induced plasma membrane reorganization is a rapid event driven by a direct cross‐talk between type IV pili and the plasma membrane rather than by the activation of an intracellular signalling pathway that would lead to actin remodelling.     

Fluorometric identification of chromatin-packing level beyond resolution of light microscopy

We examined the dynamics of fluorescence the intensity, volume, and volume density of DAPI stained DNA and phosphorylated histone-H3 in the dividing cell nuclei of normal (Hikkone AW) and colchicines-treated third instar Drosophila melanogaster wing imaginal discs. A quantitative analysis of DAPI fluorescence intensity in cells at the stages of prophase, prometaphase and metaphase revealed two levels of DNA structural package in normal mitotic cells, i.e., one in prometaphase and another at the end of metaphase. This pattern of chromatin package was not visible in colchicine-treated mitoses. We concluded that fluorometric measurements were able to detect a level in the chromatin package beyond the resolution of light microscopy.     

TiLIA: a software package for image analysis of firefly flash patterns

As flash signaling patterns of fireflies are species specific, signal‐pattern analysis is important for understanding this system of communication. Here, we present time‐lapse image analysis (TiLIA), a free open‐source software package for signal and flight pattern analyses of fireflies that uses video‐recorded image data. TiLIA enables flight path tracing of individual fireflies and provides frame‐by‐frame coordinates and light intensity data. As an example of TiLIA capabilities, we demonstrate flash pattern analysis of the fireflies Luciola cruciata and L. lateralis during courtship behavior.     

WIS‐neuromath enables versatile high throughput analyses of neuronal processes

Automated analyses of neuronal morphology are important for quantifying connectivity and circuitry in vivo, as well as in high content imaging of primary neuron cultures. The currently available tools for quantification of neuronal morphology either are highly expensive commercial packages or cannot provide automated image quantifications at single cell resolution. Here, we describe a new software package called WIS‐NeuroMath, which fills this gap and provides solutions for automated measurement of neuronal processes in both in vivo and in vitro preparations. Diverse image types can be analyzed without any preprocessing, enabling automated and accurate detection of neurites followed by their quantification in a number of application modules. A cell morphology module detects cell bodies and attached neurites, providing information on neurite length, number of branches, cell body area, and other parameters for each cell. A neurite length module provides a solution for images lacking cell bodies, such as tissue sections. Finally, a ganglion explant module quantifies outgrowth by identifying neurites at different distances from the ganglion. Quantification of a diverse series of preparations with WIS‐NeuroMath provided data that were well matched with parallel analyses of the same preparations in established software packages such as MetaXpress or NeuronJ. The capabilities of WIS‐NeuroMath are demonstrated in a range of applications, including in dissociated and explant cultures and histological analyses on thin and whole‐mount sections. WIS‐NeuroMath is freely available to academic users, providing a versatile and cost‐effective range of solutions for quantifying neurite growth, branching, regeneration, or degeneration under different experimental paradigms. © 2012 Wiley Periodicals, Inc. Develop Neurobiol, 2013     

Systems microscopy: An emerging strategy for the life sciences

Dynamic cellular processes occurring in time and space are fundamental to all physiology and disease. To understand complex and dynamic cellular processes therefore demands the capacity to record and integrate quantitative multiparametric data from the four spatiotemporal dimensions within which living cells self-organize, and to subsequently use these data for the mathematical modeling of cellular systems. To this end, a raft of complementary developments in automated fluorescence microscopy, cell microarray platforms, quantitative image analysis and data mining, combined with multivariate statistics and computational modeling, now coalesce to produce a new research strategy, “systems microscopy”, which facilitates systems biology analyses of living cells. Systems microscopy provides the crucial capacities to simultaneously extract and interrogate multiparametric quantitative data at resolution levels ranging from the molecular to the cellular, thereby elucidating a more comprehensive and richly integrated understanding of complex and dynamic cellular systems. The unique capacities of systems microscopy suggest that it will become a vital cornerstone of systems biology, and here we describe the current status and future prospects of this emerging field, as well as outlining some of the key challenges that remain to be overcome.     

Multi‐color FISH facilitates analysis of cell‐type diversification and developmental gene regulation in the Parasteatoda spider embryo

The simultaneous and quantitative analysis of the expression of multiple genes helps to shed light on gene regulatory networks. We established a method for multi‐color fluorescence in situ hybridization (mFISH) for the analysis of cell‐type diversification and developmental gene regulation in the embryo of the spider Parasteatoda tepidariorum. This mFISH technique allowed quadruple staining using four types of labels for RNA probes, digoxigenin, fluorescein, biotin, and dinitrophenyl, together with different fluorescent tyramides. To validate the usability of mFISH, we conducted four experiments. First, we distinguished similar gene expression patterns with mFISH, which showed overlaps and differences in the expression domains of anterior patterning hedgehog (hh), orthodenticle (otd), and labial genes at a cellular resolution. Second, we used mFISH to identify early cell types that are internalized on the anterior side. We found that fork head‐positive cells were subdivided into two cell types, 012_A08‐positive endoderm cells and twist‐positive mesoderm cells. Third, we quantified the ratio of expression levels of the odd‐paired (opa) gene in the chelicera and pedipalp segments based on the intensity of mFISH signals. Finally, we combined mFISH with embryonic RNA interference. It was possible to identify opa knockdown cell clones and detect the specific reduction of opa and the upregulation of otd and hh expression levels in the same cell clone that formed in the head region. This study proposes that mFISH is a powerful tool for the cell‐level analysis of gene regulation and quantification in the spider model.     

A Quantitative Image Cytometry Technique for Time Series or Population Analyses of Signaling Networks

Background

Modeling of cellular functions on the basis of experimental observation is increasingly common in the field of cellular signaling. However, such modeling requires a large amount of quantitative data of signaling events with high spatio-temporal resolution. A novel technique which allows us to obtain such data is needed for systems biology of cellular signaling.

Methodology/Principal Findings

We developed a fully automatable assay technique, termed quantitative image cytometry (QIC), which integrates a quantitative immunostaining technique and a high precision image-processing algorithm for cell identification. With the aid of an automated sample preparation system, this device can quantify protein expression, phosphorylation and localization with subcellular resolution at one-minute intervals. The signaling activities quantified by the assay system showed good correlation with, as well as comparable reproducibility to, western blot analysis. Taking advantage of the high spatio-temporal resolution, we investigated the signaling dynamics of the ERK pathway in PC12 cells.

Conclusions/Significance

The QIC technique appears as a highly quantitative and versatile technique, which can be a convenient replacement for the most conventional techniques including western blot, flow cytometry and live cell imaging. Thus, the QIC technique can be a powerful tool for investigating the systems biology of cellular signaling.     

Automated microscopy as a quantitative method to measure differences in adipogenic differentiation in preparations of human mesenchymal stromal cells

Background aimsMultipotent stromal cells, also called mesenchymal stromal cells (MSCs), are potentially valuable as a cellular therapy because of their differentiation and immunosuppressive properties. As the result of extensive heterogeneity of MSCs, quantitative approaches to measure differentiation capacity between donors and passages on a per-cell basis are needed.MethodsHuman bone marrow-derived MSCs were expanded to passages P3, P5 and P7 from eight different donors and were analyzed for colony-forming unit capacity (CFU), cell size, surface marker expression and forward/side-scatter analysis by flow cytometry. Adipogenic differentiation potential was quantified with the use of automated microscopy. Percentage of adipogenesis was determined by quantifying nuclei and Nile red–positive adipocytes after automated image acquisition.ResultsMSCs varied in expansion capacity and increased in average cell diameter with passage. CFU capacity decreased with passage and varied among cell lines within the same passage. The number of adipogenic precursors varied between cell lines, ranging from 0.5% to 13.6% differentiation at P3. Adipogenic capacity decreased significantly with increasing passage. MSC cell surface marker analysis revealed no changes caused by passaging or donor differences.ConclusionsWe measured adipogenic differentiation on a per-cell basis with high precision and accuracy with the use of automated fluorescence microscopy. We correlated these findings with other quantitative bioassays to better understand the role of donor variability and passaging on CFU, cell size and adipogenic differentiation capacity in vitro. These quantitative approaches provide valuable tools to measure MSC quality and measure functional biological differences between donors and cell passages that are not revealed by conventional MSC cell surface marker analysis.     

In situ visualization of newly synthesized proteins in environmental microbes using amino acid tagging and click chemistry

Here we describe the application of a new click chemistry method for fluorescent tracking of protein synthesis in individual microorganisms within environmental samples. This technique, termed bioorthogonal non‐canonical amino acid tagging (BONCAT), is based on the in vivo incorporation of the non‐canonical amino acid L‐azidohomoalanine (AHA), a surrogate for l ‐methionine, followed by fluorescent labelling of AHA‐containing cellular proteins by azide‐alkyne click chemistry. BONCAT was evaluated with a range of phylogenetically and physiologically diverse archaeal and bacterial pure cultures and enrichments, and used to visualize translationally active cells within complex environmental samples including an oral biofilm, freshwater and anoxic sediment. We also developed combined assays that couple BONCAT with ribosomal RNA (rRNA)‐targeted fluorescence in situ hybridization (FISH), enabling a direct link between taxonomic identity and translational activity. Using a methanotrophic enrichment culture incubated under different conditions, we demonstrate the potential of BONCAT‐FISH to study microbial physiology in situ. A direct comparison of anabolic activity using BONCAT and stable isotope labelling by nano‐scale secondary ion mass spectrometry (¹⁵NH₃ assimilation) for individual cells within a sediment‐sourced enrichment culture showed concordance between AHA‐positive cells and ¹⁵N enrichment. BONCAT‐FISH offers a fast, inexpensive and straightforward fluorescence microscopy method for studying the in situ activity of environmental microbes on a single‐cell level.     

The integrative role of cryo electron microscopy in molecular and cellular structural biology

After gradually moving away from preparation methods prone to artefacts such as plastic embedding and negative staining for cell sections and single particles, the field of cryo electron microscopy (cryo‐EM) is now heading off at unprecedented speed towards high‐resolution analysis of biological objects of various sizes. This ‘revolution in resolution’ is happening largely thanks to new developments of new‐generation cameras used for recording the images in the cryo electron microscope which have much increased sensitivity being based on complementary metal oxide semiconductor devices. Combined with advanced image processing and 3D reconstruction, the cryo‐EM analysis of nucleoprotein complexes can provide unprecedented insights at molecular and atomic levels and address regulatory mechanisms in the cell. These advances reinforce the integrative role of cryo‐EM in synergy with other methods such as X‐ray crystallography, fluorescence imaging or focussed‐ion beam milling as exemplified here by some recent studies from our laboratory on ribosomes, viruses, chromatin and nuclear receptors. Such multi‐scale and multi‐resolution approaches allow integrating molecular and cellular levels when applied to purified or in situ macromolecular complexes, thus illustrating the trend of the field towards cellular structural biology.     

A high‐throughput all‐optical laser‐scanning imaging flow cytometer with biomolecular specificity and subcellular resolution

Image‐based cellular assay advances approaches to dissect complex cellular characteristics through direct visualization of cellular functional structures. However, available technologies face a common challenge, especially when it comes to the unmet need for unraveling population heterogeneity at single‐cell precision: higher imaging resolution (and thus content) comes at the expense of lower throughput, or vice versa. To overcome this challenge, a new type of imaging flow cytometer based upon an all‐optical ultrafast laser‐scanning imaging technique, called free‐space angular‐chirp‐enhanced delay (FACED) is reported. It enables an imaging throughput (>20 000 cells s^?1) 1 to 2 orders of magnitude higher than the camera‐based imaging flow cytometers. It also has 2 critical advantages over optical time‐stretch imaging flow cytometry, which achieves a similar throughput: (1) it is widely compatible to the repertoire of biochemical contrast agents, favoring biomolecular‐specific cellular assay and (2) it enables high‐throughput visualization of functional morphology of individual cells with subcellular resolution. These capabilities enable multiparametric single‐cell image analysis which reveals cellular heterogeneity, for example, in the cell‐death processes demonstrated in this work—the information generally masked in non‐imaging flow cytometry. Therefore, this platform empowers not only efficient large‐scale single‐cell measurements, but also detailed mechanistic analysis of complex cellular processes.      

The iRoCS Toolbox – 3D analysis of the plant root apical meristem at cellular resolution

To achieve a detailed understanding of processes in biological systems, cellular features must be quantified in the three‐dimensional (3D) context of cells and organs. We described use of the intrinsic root coordinate system (iRoCS) as a reference model for the root apical meristem of plants. iRoCS enables direct and quantitative comparison between the root tips of plant populations at single‐cell resolution. The iRoCS Toolbox automatically fits standardized coordinates to raw 3D image data. It detects nuclei or segments cells, automatically fits the coordinate system, and groups the nuclei/cells into the root's tissue layers. The division status of each nucleus may also be determined. The only manual step required is to mark the quiescent centre. All intermediate outputs may be refined if necessary. The ability to learn the visual appearance of nuclei by example allows the iRoCS Toolbox to be easily adapted to various phenotypes. The iRoCS Toolbox is provided as an open‐source software package, licensed under the GNU General Public License, to make it accessible to a broad community. To demonstrate the power of the technique, we measured subtle changes in cell division patterns caused by modified auxin flux within the Arabidopsis thaliana root apical meristem.     

Quantitative insights into actin rearrangements and bacterial target site selection from Salmonella Typhimurium infection of micropatterned cells

Reorganization of the host cell actin cytoskeleton is crucial during pathogen invasion. We established micropatterned cells as a standardized infection model for cell invasion to quantitatively study actin rearrangements triggered by Salmonella Typhimurium (S. Tm). Micropatterns of extracellular matrix proteins force cells to adopt a reproducible shape avoiding strong cell‐to‐cell variations, a major limitation in classical cell culture conditions. S. Tm induced F‐actin‐rich ruffles and invaded micropatterned cells similar to unconstrained cells. Yet, standardized conditions allowed fast and unbiased comparison of cellular changes triggered by the SipA and SopE bacterial effector proteins. Intensity measurements in defined regions revealed that the content of pre‐existing F‐actin remained unchanged during infection, suggesting that newly polymerized F‐actin in bacteria‐triggered ruffles originates from the G‐actin pool. Analysing bacterial target sites, we found that bacteria did not show any preferences for the local actin cytoskeleton specificities. Rather, invasion was constrained to a specific ‘cell height’, due to flagella‐mediated near‐surface swimming. We found that invasion sites were similar to bacterial binding sites, indicating that S. Tm can induce a permissive invasion site wherever it binds. As micropatterned cells can be infected by many different pathogens they represent a valuable new tool for quantitative analysis of host–pathogen interactions.     

Volume scanning electron microscopy for imaging biological ultrastructure

Electron microscopy (EM) has been a key imaging method to investigate biological ultrastructure for over six decades. In recent years, novel volume EM techniques have significantly advanced nanometre‐scale imaging of cells and tissues in three dimensions. Previously, this had depended on the slow and error‐prone manual tasks of cutting and handling large numbers of sections, and imaging them one‐by‐one with transmission EM. Now, automated volume imaging methods mostly based on scanning EM (SEM) allow faster and more reliable acquisition of serial images through tissue volumes and achieve higher z‐resolution. Various software tools have been developed to manipulate the acquired image stacks and facilitate quantitative analysis. Here, we introduce three volume SEM methods: serial block‐face electron microscopy (SBEM), focused ion beam SEM (FIB‐SEM) and automated tape‐collecting ultramicrotome SEM (ATUM‐SEM). We discuss and compare their capabilities, provide an overview of the full volume SEM workflow for obtaining 3D datasets and showcase different applications for biological research.     

FIRST HARMFUL DINOPHYSIS (DINOPHYCEAE,DINOPHYSIALES) BLOOM IN THE U.S. IS REVEALED BY AUTOMATED IMAGING FLOW CYTOMETRY1

Imaging FlowCytobot (IFCB) combines video and flow cytometric technology to capture images of nano‐ and microplankton (～10 to >100 μm) and to measure the chlorophyll fluorescence associated with each image. The images are of sufficient resolution to identify many organisms to genus or even species level. IFCB has provided >200 million images since its installation at the entrance to the Mission‐Aransas estuary (Port Aransas, TX, USA) in September 2007. In early February 2008, Dinophysis cells (1–5 · mL^?1) were detected by manual inspection of images; by late February, abundance estimates exceeded 200 cells · mL^?1. Manual microscopy of water samples from the site confirmed that D. cf. ovum F. Schütt was the dominant species, with cell concentrations similar to those calculated from IFCB data, and toxin analyses showed that okadaic acid was present, which led to closing of shellfish harvesting. Analysis of the time series using automated image classification (extraction of image features and supervised machine learning algorithms) revealed a dynamic phytoplankton community composition. Before the Dinophysis bloom, Myrionecta rubra (a prey item of Dinophysis) was observed, and another potentially toxic dinoflagellate, Prorocentrum, was observed after the bloom. Dinophysis cell‐division rates, as estimated from the frequency of dividing cells, were the highest at the beginning of the bloom. Considered on a daily basis, cell concentration increased roughly exponentially up to the bloom peak, but closer inspection revealed that the increases generally occurred when the direction of water flow was into the estuary, suggesting the source of the bloom was offshore.     

Efficient super‐resolution volumetric imaging by radial fluctuation Bayesian analysis light‐sheet microscopy

Various computational super‐resolution methods are available based on the analysis of fluorescence fluctuation behind acquired frames. However, dilemmas often exist in the balance of fluorophore characteristics, computation cost, and achievable resolution. Here we present an approach that uses a super‐resolution radial fluctuations (SRRF) image to guide the Bayesian analysis of fluorophore blinking and bleaching (3B) events, allowing greatly accelerated localization of overlapping fluorophores with high accuracy. This radial fluctuation Bayesian analysis (RFBA) approach is also extended to three dimensions for the first time and combined with light‐sheet fluorescence microscopy, to achieve super‐resolution volumetric imaging of thick samples densely labeled with common fluorophores. For example, a 700‐nm thin Bessel plane illumination is developed to optically section the Drosophila brain, providing a high‐contrast 3D image of rhythmic neurons. RFBA analyzes 30 serial volumes to reconstruct a super‐resolved 3D image at 4‐times higher resolutions (~70 and 170 nm), and precisely resolve the axon terminals. The computation is over 2‐orders faster than conventional 3B analysis microscopy. The capability of RFBA is also verified through dual‐color imaging of cell nucleus in live Drosophila brain. The spatial co‐localization patterns of the nuclear envelope and DNA in a neuron deep inside the brain can be precisely extracted by our approach.     

Kalman estimator- and general linear model-based on-line brain activation mapping by near-infrared spectroscopy

Background

Methods of manual cell localization and outlining are so onerous that automated tracking methods would seem mandatory for handling huge image sequences, nevertheless manual tracking is, astonishingly, still widely practiced in areas such as cell biology which are outside the influence of most image processing research. The goal of our research is to address this gap by developing automated methods of cell tracking, localization, and segmentation. Since even an optimal frame-to-frame association method cannot compensate and recover from poor detection, it is clear that the quality of cell tracking depends on the quality of cell detection within each frame.

Methods

Cell detection performs poorly where the background is not uniform and includes temporal illumination variations, spatial non-uniformities, and stationary objects such as well boundaries (which confine the cells under study). To improve cell detection, the signal to noise ratio of the input image can be increased via accurate background estimation. In this paper we investigate background estimation, for the purpose of cell detection. We propose a cell model and a method for background estimation, driven by the proposed cell model, such that well structure can be identified, and explicitly rejected, when estimating the background.

Results

The resulting background-removed images have fewer artifacts and allow cells to be localized and detected more reliably. The experimental results generated by applying the proposed method to different Hematopoietic Stem Cell (HSC) image sequences are quite promising.

Conclusion

The understanding of cell behavior relies on precise information about the temporal dynamics and spatial distribution of cells. Such information may play a key role in disease research and regenerative medicine, so automated methods for observation and measurement of cells from microscopic images are in high demand. The proposed method in this paper is capable of localizing single cells in microwells and can be adapted for the other cell types that may not have circular shape. This method can be potentially used for single cell analysis to study the temporal dynamics of cells.     

Quantitative FRET measurement based on spectral unmixing of donor,acceptor and spontaneous excitation‐emission spectra

The spontaneous excitation‐emission (ExEm) spectrum is introduced to the quantitative mExEm‐spFRET methodology we recently developed as a spectral unmixing component for quantitative fluorescence resonance energy transfer measurement, named as SPEES‐FRET method. The spectral fingerprints of both donor and acceptor were measured in HepG2 cells with low autofluorescence separately expressing donor and acceptor, and the spontaneous spectral fingerprint of HEK293 cells with strong autofluoresence was measured from blank cells. SPEES‐FRET was performed on improved spectrometer‐microscope system to measure the FRET efficiency (E) and concentration ratio (R _C) of acceptor to donor vales of FRET tandem plasmids in HEK293 cells, and obtained stable and consistent results with the expected values. Moreover, SPEES‐FRET always obtained stable results for the bright and dim cells coexpressing Cerulean and Venus or Cyan Fluorescent Protein (CFP)‐Bax and Yellow fluorescent protein (YFP)‐Bax, and the E values between CFP‐Bax and YFP‐Bax were 0.02 for healthy cells and 0.14 for the staurosporine (STS)‐treated apoptotic cells. Collectively, SPEES‐FRET has very strong robustness against cellular autofluorescence, and thus is applicable to quantitative evaluation on the protein‐protein interaction in living cells with strong autofluoresence.