Super-multiplexed fluorescence microscopy via photostability contrast.

Fluorescence microscopy is widely used to observe and quantify the inner workings of the cell. Traditionally, multiple types of cellular structures or biomolecules are visualized simultaneously in a sample by using spectrally distinct fluorescent labels. The wide emission spectra of most fluorophores limits spectral multiplexing to four or five labels in a standard fluorescence microscope. Further multiplexing requires another dimension of contrast. Here, we show that photostability differences can be used to distinguish between fluorescent labels. By combining photobleaching characteristics with a novel unmixing algorithm, we resolve up to three fluorescent labels in a single spectral channel and unmix fluorescent labels with nearly identical emission spectra. We apply our technique to organic dyes, autofluorescent biomolecules and fluorescent proteins. Our approach has the potential to triple the multiplexing capabilities of any digital widefield or confocal fluorescence microscope with no additional hardware, making it readily accessible to a wide range of researchers.

Angiogenic potential of 3-nitro-4-hydroxy benzene arsonic acid (roxarsone).

BACKGROUND: Roxarsone (3-nitro-4-hydroxy benzene arsonic acid) is an arsenic compound widely used in the poultry industry as a feed additive to prevent coccidiosis, stimulate growth, and to improve tissue pigmentation. Little is known about the potential human health effects from roxarsone released into the environment from chicken waste or from residual compound in chicken products. OBJECTIVE: The growth potentiation and enhanced tissue pigmentation suggest that low levels of roxarsone exposure may have an angiogenic potential similar to that of inorganic arsenite (As(III)). The goal of this investigation was to test the hypothesis described above using cultured human aortic and lung microvascular endothelial cells in high-content imaging tube-forming assays and begin developing a molecular level understanding of the process. METHODS: We used a three-dimensional Matrigel assay for probing angiogenesis in cultured human endothelial cells, and a polymerase chain reaction (PCR) array to probe the gene changes as a function of roxarsone or As(III) treatment. In addition, we used Western blot analysis for changes in protein concentration and activation. RESULTS: Roxarsone was found to exhibit a higher angiogenic index than As(III) at lower concentrations. Increased endothelial nitric oxide synthase (eNOS) activity was observed for roxarsone but not for As(III)-induced angiogenesis. However, As(III) caused more rapid and pronounced phosphorylation of eNOS. Quantitative PCR array on select genes revealed that the two compounds have different and often opposite effects on angiogenic gene expression. CONCLUSIONS: The results demonstrate that roxarsone and As(III) promote angiogenic phenotype in human endothelial cells through distinctly different signaling mechanisms.

Characteristics and value of directed algorithms in high content screening.

High content screening requires image processing algorithms that can accurately and robustly analyze large image numbers without requiring human intervention. Thus, a suite of algorithms that are directed by an understanding of the biology being studied was developed for the optimized automated acquisition and quantitation of cellular images. Two categories of directed algorithms were developed: Developer Tools for assay development and Specific Algorithms for turnkey screening of specific biological situations. The same basic sequence of analysis steps are used in these directed algorithms: 1. Primary object identification. 2. Measurement of primary object properties. 3. Identification and measurements of associated targets. 4. Analysis of raw measurements for specific biological problems. The detailed application of these steps is guided by the biology being studied and the expected phenotypic changes. Most cell biological problems to be analyzed using high content screening can be categorized by either the phenotype of the problem or labeling pattern, or by a standard biological response behavior of the cells. This enables application of directed algorithms optimized for these categories. Examples of the use of directed algorithms for specific categories are discussed, as well as the detailed analysis steps for a specific directed algorithm.

Quantitative characterization of mitosis-blocked tetraploid cells using high content analysis.

A range of cellular evidence supporting a G1 tetraploidy checkpoint was obtained from different assay methods including flow cytometry, immunoblotting, and microscopy. Cancer research would benefit if these cellular properties could instead be measured by a single, quantitative, automated assay method, such as high content analysis (HCA). Thus, nocodazole-treated cells were fluorescently labeled for different cell cycle-associated properties, including DNA content, retinoblastoma (Rb) and histone H3 phosphorylation, p53 and p21(WAF1) expression, nuclear and cell sizes, and cell morphology, and automatically imaged, analyzed, and correlated using HCA. HCA verified that nocodazole-induced mitosis block resulted in tetraploid cells. Rb and histone H3 were maximally hyperphosphorylated by 24 h of nocodazole treatment, accompanied by cell and nuclear size decreases and cellular rounding. Cells remained tetraploid and mononucleated with longer treatments, but other targets reverted to G1 levels, including Rb and histone H3 dephosphorylation accompanied by cellular respreading. This was accompanied by increased p53 and p21(WAF1) expression levels. The range of effects accompanying nocodazole-induced block of mitosis and the resulting tetraploid cells' reversal to a pseudo-G1 state can be quantitatively measured by HCA in an automated manner, recommending this assay method for the large-scale biology challenges of modern cancer drug discovery.

Quantitative cell-based high-content screening for vasopressin receptor agonists using transfluor technology.

The authors demonstrate the use of a simple, universal G-protein-coupled receptor (GPCR) assay to screen for agonists for a specific GPCR. Cells stably expressing a green fluorescent protein (GFP)-labeled beta-arrestin fusion protein and the vasopressin V2 receptor (V2R) were used in a high-content screening (HCS) assay to screen a small peptide library for V2R agonists. Cells were treated with the peptides at a final concentration of 500 nM for 30 min. Agonist stimulation causes V2R internalization into endosomes. GFP-beta-arrestin remains associated with the V2R in endosomes, resulting in a fluorescent pattern of intracellular spots. Assay plates were automatically imaged and quantitatively analyzed using an HCS imaging platform and a fast turnkey image analysis application optimized for detection of receptor activation and intracellular spots. Hits were further evaluated to determine their potency. The combination of unique biology, automated high-content analysis, and a powerful means of validating hits results in better leads.

A quantitative cell-based high-content screening assay for the epidermal growth factor receptor-specific activation of mitogen-activated protein kinase.

The complexity of mitogen-activated protein kinase (MAPK) signaling pathways and their activation by different stimuli makes assaying the activation of particular MAPKs by specific receptors a challenging problem. The multiplexing capability of quantitative high-content screening (HCS) assays enables the simultaneous monitoring and correlation, in the same cell, of an MAPK's specific activation with a particular receptor's post-signaling behavior, such as its internalization. We demonstrate a cell-based HCS assay to quantify the epidermal growth factor (EGF) receptor-specific activation of the MAPK ERK. Activation was quantified by measuring immunofluorescently labeled phosphorylated extracellular signal-regulated protein kinases (ERK) in the nucleus. Specificity of ERK activation by the EGF receptor was simultaneously confirmed in the same cell by quantitatively monitoring fluorescent EGF's internalization and subsequent intracellular degradation. Quantitative analysis of the temporal behavior of these two activities showed that phosphorylated ERK's accumulation in the nucleus peaked at 5 min before falling to basal levels by 30 min. Cellular accumulation of fluorescent EGF was slower, peaking around 30 min, before being degraded. This assay strategy can serve as a paradigm to study other signaling pathways and their activation by specific receptors. The flexibility and multiplexing capability of HCS assays allow the use of additional targets to further qualify the specificity of response by including other MAPKs or receptors, to rule out cross-talk from competing signaling pathways, or to simultaneously monitor toxicity effects of compounds. This automated, non-subjective, easy-to-use assay procedure provides information rich, quantitative results, and demonstrates the potential of the HCS assay approach in deconvolving intracellular signaling pathways.