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Anatomic tracing is recognized as a critical source of knowledge on brain circuitry that can be used to assess the accuracy of diffusion MRI (dMRI) tractography. However, most prior studies that have performed such assessments have used dMRI and tracer data from different brains and/or have been limited in the scope of dMRI analysis methods allowed by the data. In this work, we perform a quantitative, voxel-wise comparison of dMRI tractography and anatomic tracing data in the same macaque brain. An ex vivo dMRI acquisition with high angular resolution and high maximum b-value allows us to compare a range of q-space sampling, orientation reconstruction, and tractography strategies. The availability of tracing in the same brain allows us to localize the sources of tractography errors and to identify axonal configurations that lead to such errors consistently, across dMRI acquisition and analysis strategies. We find that these common failure modes involve geometries such as branching or turning, which cannot be modeled well by crossing fibers. We also find that the default thresholds that are commonly used in tractography correspond to rather conservative, low-sensitivity operating points. While deterministic tractography tends to have higher sensitivity than probabilistic tractography in that very conservative threshold regime, the latter outperforms the former as the threshold is relaxed to avoid missing true anatomical connections. On the other hand, the q-space sampling scheme and maximum b-value have less of an impact on accuracy. Finally, using scans from a set of additional macaque brains, we show that there is enough inter-individual variability to warrant caution when dMRI and tracer data come from different animals, as is often the case in the tractography validation literature. Taken together, our results provide insights on the limitations of current tractography methods and on the critical role that anatomic tracing can play in identifying potential avenues for improvement., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Green fluorescent protein (GFP) and its counterparts are modern molecular biology research tools indispensable in many experimental systems. Within fungi, researchers studying Saccharomyces cerevisiae and other model ascomycetes have access to a wide variety of fluorescent proteins. Unfortunately, many of these tools have not crossed the phylum divide into the Basidiomycota, where only GFP S65T, Venus, Ds-Red, and mCherry are currently available. To address this, we searched the literature for potential candidates to be expressed in the human fungal pathogen Cryptococcus neoformans and identified a suite of eight more modern fluorescent proteins that span the visible spectrum. A single copy of each fluorophore was heterologously expressed in Safe Haven 1 and their fluorescence intensities compared in this encapsulated yeast. mTurquoise2, mTFP1, Clover, mNeonGreen, mRuby3, and Citrine were highly visible under the microscope, whereas Superfolder GFP and mMaroon1 were not. Expressed fluorophores did not impact growth or virulence as demonstrated by an in vitro spotting assay and murine inhalation model, respectively., (Crown Copyright © 2020. Published by Elsevier Inc. All rights reserved.)

Regulatory T cells (Tregs), expressing the transcription factor Foxp3, are defined as immunosuppressive cells able to modulate a variety of immune cells in order to avoid unwanted and excessive immune responses; however, in the tumor context, Treg function contribute to inhibit immune surveillance, thus promoting cancer progression. In tumor microenvironment, where the availability of metabolic resources is strongly limited, Tregs are expanded and may exploit different metabolic routes to achieve a metabolic advantage, prevailing over effector cells. In this context an important role of lipid metabolism has been described thanks to the possibility to evaluate the intracellular lipid content selectively in tumor-infiltrating Tregs (TUM-Tregs). Taking into account the heterogeneous and complex build of tumor mass, we set-up a combined protocol that optimizes tumor-infiltrating lymphocytes (TIL) extraction from the tissue through a Percoll density gradient, and assesses ex vivo the lipid load in whole TUM-Treg population, evaluating by flow cytometry the incorporation of an intensely fluorescent lipophilic fluorophore able to specifically stain neutral lipids. This method provides an important advantage compared to the traditional technique based on microscopy, whose lipid level evaluation is limited to a tissue section, and hence may not be representative of the entire population., (© 2020 Elsevier Inc. All rights reserved.)

Recent developments of novel targeted therapies are contributing to the increased long-term survival of cancer patients; however, drug-induced cardiotoxicity induced by cancer drugs remains a serious problem in clinical settings. Nevertheless, there are few in vitro cell-based assays available to predict this toxicity, especially from the aspect of morphology. Here, we developed a simple two-dimensional (2D) morphological assessment system, 2DMA, to predict drug-induced cardiotoxicity in cancer patients using human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) with image-based high-content analysis in a high-throughput manner. To assess the effects of drugs on cardiomyocytes, we treated iPSC-CMs with 28 marketed pharmaceuticals and measured two key parameters: number of cell nuclei and sarcomere morphology. Drugs that significantly perturbed these two parameters at concentrations ≤30 times the human C max value were regarded as positive in the test. Based on these criteria, the sensitivity and specificity of the 2DMA system were 81% and 100%, respectively. Moreover, the translational predictability of 2DMA was comparable with that of a three-dimensional cardiotoxicity assay. RNA sequencing further revealed that the expression levels of several genes related to sarcomere components decreased following treatment with sunitinib, suggesting that inhibition of the synthesis of proteins that comprise the sarcomere contributes to drug-induced sarcomere disruption. Based on these features, the 2DMA system provides mechanistic insight with high predictability of cancer drug-induced cardiotoxicity in humans, and could thus contribute to the reduction of drug attrition rates at early stages of drug development., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Biosensors are biological molecules able to detect and report the presence of a target molecule by the emission of a signal. Nucleic acids are particularly appealing for the design of such molecule since their great structural plasticity makes them able to specifically interact with a wide range of ligands and their structure can rearrange upon recognition to trigger a reporting event. A biosensor is typically made of three main domains: a sensing domain that is connected to a reporting domain via a communication module in charge of transmitting the sensing event through the molecule. The communication module is therefore an instrumental element of the sensor. This module is usually empirically developed through a trial-and-error strategy with the testing of only a few combinations judged relevant by the experimenter. In this work, we introduce a novel method combining the use of droplet-based microfluidics and next generation sequencing. This method allows to functionally characterize up to a million of different sequences in a single set of experiments and, by doing so, to exhaustively test every possible sequence permutations of the communication module. Here, we demonstrate the efficiency of the approach by isolating a set of optimized RNA biosensors able to sense theophylline and to convert this recognition into fluorescence emission., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Emerging carbon dots (CDs) are widely used as fluorescent probes in biological and environmental fields, nevertheless, the control of CDs based on different detection mechanisms is rarely reported. In this paper, green luminescent CDs (G-CDs) were prepared by a facile hydrothermal treatment of benzoxazine monomers (BZM). The obtained G-CDs showed pH dependent photoluminescence, which could be designed as fluorescence turn-on and turn-off sensors. The G-CDs exhibited weak photoluminescence at pH = 7.0 and could be turned on by Zn(II) selectively with the limitation of 0.32 μM in the concentration range from 1 to 100 μM. When pH = 10.0, Cr(VI) could quench the strong fluorescence of G-CDs efficiently, and the limit of detection was 0.99 μM with a linear range of 1-50 μM. Furthermore, the fluorescence turn-on and turn-off performance of G-CDs was attributed to the intramolecular charge transfer (ICT) of Zn(II) and the inner filter effect (IFE) of Cr(VI), respectively. The excellent probes were successfully applied for the detection of Zn(II) in biological system and Cr(VI) in environment., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Type I, II, and V CRISPR-Cas systems are RNA-guided dsDNA targeting defense mechanisms found in bacteria and archaea. During CRISPR interference, Cas effectors use CRISPR-derived RNAs (crRNAs) as guides to bind complementary sequences in foreign dsDNA, leading to the cleavage and destruction of the DNA target. Mutations within the target or in the protospacer adjacent motif can reduce the level of CRISPR interference, although the level of defect is dependent on the type and position of the mutation, as well as the guide sequence of the crRNA. Given the importance of Cas effectors in host defense and for biotechnology tools, there has been considerable interest in developing sensitive methods for detecting Cas effector activity through CRISPR interference. In this chapter, we describe an in vivo fluorescence-based method for monitoring plasmid interference in Escherichia coli. This approach uses a green fluorescent protein reporter to monitor varying plasmid levels within bacterial colonies, or to measure the rate of plasmid-loss in bacterial populations over time. We demonstrate the use of this simple plasmid-loss assay for both chromosomally integrated and plasmid-borne CRISPR-Cas systems., (© 2019 Elsevier Inc. All rights reserved.)

Fluorescent labeling of proteins is a critical requirement for single-molecule imaging studies. Many protein labeling strategies require harsh conditions or large epitopes that can inactivate the target protein, either by decreasing the protein's enzymatic activity or by blocking protein-protein interactions. Here, we provide a detailed protocol to efficiently label CRISPR-Cas complexes with a small fluorescent peptide via sortase-mediated transpeptidation. The sortase tag consists of just a few amino acids that are specifically recognized at either the N- or the C-terminus, making this strategy advantageous when the protein is part of a larger complex. Sortase is active at high ionic strength, 4°C, and with a broad range of organic fluorophores. We discuss the design, optimization, and single-molecule fluorescent imaging of CRISPR-Cas complexes on DNA curtains. Sortase-mediated transpeptidation is a versatile addition to the protein labeling toolkit., (© 2019 Elsevier Inc. All rights reserved.)

Dysregulated cellular metabolism is an emerging hallmark of cancer. Improved methods to profile aberrant metabolic activity thus have substantial applications as tools for diagnosis and understanding the biology of malignant tumors. Here we describe the utilization of a bioorthogonal ligation to fluorescently detect the TCA cycle oncometabolite fumarate. This method enables the facile measurement of fumarate hydratase activity in cell and tissue samples, and can be used to detect disruptions in metabolism that underlie the genetic cancer syndrome hereditary leiomyomatosis and renal cell cancer (HLRCC). The current method has substantial utility for sensitive fumarate hydratase activity profiling, and also provides a foundation for future applications in diagnostic detection and imaging of cancer metabolism., (© 2019 Elsevier Inc. All rights reserved.)

Astrocytes are the most abundant cell type in the brain and are a crucial part of solving its mysteries. Originally assumed to be passive supporting cells, astrocyte's functions are now recognized to include active modulation and information processing at the neural synapse. The full extent of the astrocyte contribution to neural processing remains unknown. This is, in part, due to the lack of methods available for astrocyte identification and analysis. Existing strategies employ genetic tools like the astrocyte specific promoters glial fibrillary acidic protein (GFAP) or Aldh1L1 to create transgenic animals with fluorescently labeled astrocytes. Recently, small molecule targeting moieties have enabled the delivery of bright fluorescent dyes to astrocytes. Here, we review methods for targeting astrocytes, with a focus on a recently developed methylpyridinium targeting moiety's development, chemical synthesis, and elaboration to provide new features like light-based spatiotemporal control of cell labeling., (© 2019 Elsevier Inc. All rights reserved.)

Loop mediated isothermal amplification (LAMP) assay is sensitive, prompt, high throughput and field deployable technique for nucleic acid amplification under isothermal conditions. In this study, we have developed and optimized four different visualization methods of loop-mediated isothermal amplification (LAMP) assay to detect Pfcrt K76T mutants of P. falciparum and compared their important features for one-pot in-field applications. Even though all the four tested LAMP methods could successfully detect K76T mutants of P. falciparum, however considering the time, safety, sensitivity, cost and simplicity, the malachite green and HNB based methods were found more efficient. Among four different visual dyes uses to detect LAMP products accurately, hydroxynaphthol blue and malachite green could produce long stable color change and brightness in a close tube-based approach to prevent cross-contamination risk. Our results indicated that the LAMP offers an interesting novel and convenient best method for the rapid, sensitive, cost-effective, and fairly user friendly tool for detection of K76T mutants of P. falciparum and therefore presents an alternative to PCR-based assays. Based on our comparative analysis, better field based LAMP visualization method can be chosen easily for the monitoring of other important drug targets (Kelch13 propeller region)., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Nucleotide hydrolases play integral yet poorly understood roles in several metallocluster biosynthetic pathways. For example, the cytosolic iron-sulfur cluster assembly (CIA) is initiated by the CIA scaffold, an ATPase which builds new iron-sulfur clusters for proteins localized to the cytosol and the nucleus in eukaryotic organisms. While in vivo studies have demonstrated the scaffold's nucleotide hydrolase domain is vital for its function, in vitro approaches have not revealed tight allosteric coupling between the cluster scaffolding site and the ATPase site. Thus, the role of ATP hydrolysis has been hard to pinpoint. Herein, we describe methods to probe the nucleotide affinity and hydrolysis activity of the CIA scaffold from yeast, which is comprised of two homologous polypeptides called Nbp35 and Cfd1. In particular, we report two different equilibrium binding assays that make use of commercially available fluorescent nucleotide analogs. Importantly, these assays can be applied to probe nucleotide affinity of both the apo- and holo-forms of the CIA scaffold. Generally, these fluorescent nucleotide analogs have been underutilized to probe metal trafficking NTPase because one of the most commonly used probes, mantATP, which is labeled with the methylanthraniloyl probe via the 2' or 3' sugar hydroxyls, has an absorption which overlaps with the UV-Vis features of many metal-binding proteins. However, by exploiting analogs like BODIPY-FL and trinitrophenyl-labeled nucleotides which have better photophysical properties for metalloprotein applications, these approaches have the potential to reveal the mechanistic underpinnings of NTPases required for metallocluster biosynthesis., (© 2018 Elsevier Inc. All rights reserved.)

The unique features of Mesoporous Silica Nanoparticles (MSNs) provide a suitable platform to carry fluorescence dyes for various bioimaging applications. Several strategies have been developed to conjugate a variety of dyes either in the pores or on the surfaces of MSNs to form the fluorescence MSNs (FMSNs). In this chapter, we will discuss recent research progress and future development of FMSNs for living system imaging. We will first describe different strategies for the fabrications of FMSNs. Then, we will discuss the recent developments of cellular and intracellular imaging including self-probe for the interactions of FMSNs with the cells, receptor and organelle labeling, sensing and tracking of biological system, and monitoring the drug delivery and release processes. Moreover, we will include the applications of FMSNs as contrast agents for in vivo imaging. Finally, we will conclude and highlight the challenges and opportunities for MSNs in medical applications., (© 2018 Elsevier Inc. All rights reserved.)

The encapsulation of fluorescent dyes inside silica nanoparticles is advantageous to improve their quality as probes. Inside the particle, the fluorophore is protected from the external conditions and its main emission parameters remains unchanged even in the presence of quenchers. On the other hand, the amine-functionalized nanoparticle surface enables a wide range of applications, as amino groups could be easily linked with different biomolecules for targeting purposes. This kind of nanoparticle is regularly synthesized by methods that employ templates, additional nanoparticle formation or multiple pathway process. However, a one-step synthesis will be an efficient approach in this sort of bifunctional hybrid nanoparticles. A co-condensation sol-gel synthesis of hybrid fluorescent silica nanoparticle where developed. The chemical and morphological characterization of the particles where investigated by DRIFTS, XPS, SEM and SAXS. The nanoparticle fluorescent properties were also assessed by excitation-emission matrices and time resolved experiments. We have developed a one-pot synthesis method that enables the simultaneous incorporation of functionalities, the fluorescent molecule and the amino group, by controlling co-condensation process. An exhaustive characterization allows the definition of the spatial distribution of the fluorescent probe, fluorescein isothiocyanate, inside the particle and reactive amino groups on the surface of the nanoparticle with diameter about 100nm., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Protein-based fluorescent biosensors are powerful tools for quantitative detection of biomolecules or drugs with high sensitivity under physiological conditions. However, conventional methods for construction of biosensors require structural data with high resolution or amino acid sequence information in most cases, which hampers applicability of this method to structurally complicated receptor proteins. To sidestep such limitations, we recently developed a new method that employs ligand-directed chemistry coupled with a bimolecular fluorescence quenching and recovery system, which enabled the conversion of various kinds of membrane-bound receptors to "turn-on" type fluorescent sensors. Here, we describe a protocol for construction of "turn-on" type fluorescent biosensors based on the GABA A receptor which permits quantitative analysis of the ligand affinity., (© 2017 Elsevier Inc. All rights reserved.)

Genetically encoded fluorescent sensors are essential tools in modern biological research, and recent advances in fluorescent proteins (FPs) have expanded the scope of sensor design and implementation. In this review we compare different sensor platforms, including Förster resonance energy transfer (FRET) sensors, fluorescence-modulated single FP-based sensors, translocation sensors, complementation sensors, and dimerization-based sensors. We discuss elements of sensor design and engineering for each platform, including the incorporation of new types of FPs and sensor screening techniques. Finally, we summarize the wide range of sensors in the literature, exploring creative new sensor architectures suitable for different applications., (© 2017 Elsevier Inc. All rights reserved.)

Fluorescent proteins of different colors are useful probes to study protein structure and function, and to investigate cellular events and conditions. Large efforts have focused on engineering new properties into fluorescent proteins via rational design and directed evolution. In addition to applications in imaging of protein expression level and subcellular localization, fluorescent proteins have been increasingly engineered to act as biosensors to track concentrations of small-molecule metabolites, enzyme activities, and protein conformational changes in living cells. Unlike small-molecule fluorescence biosensors, fluorescent proteins are genetically encodable, and thus can be expressed inside living cells. Attachment of organelle-specific signals to the proteins allows their localization to be specified. Recently, a new class of fluorescent protein biosensors has been developed to include unnatural amino acids as the sensing element. The unique chemical and physical properties of the unnatural amino acids enable sensor designs that cannot be realized by using the standard genetic code with the 20 canonical amino acids. In this chapter, we detail the general procedure for the genetic incorporation of unnatural amino acids. We further present two protocols for the in vitro and in vivo detection of hydrogen peroxide (H 2 O 2 ) using a fluorescent protein biosensor that contains an unnatural amino acid, p-boronophenylalanine., (© 2017 Elsevier Inc. All rights reserved.)

In eukaryotes, physiological cell functions rely on the preservation of the size and activity of the mitochondrial network. Mitophagy provides a key contribution in this setting by ensuring the removal of permeabilized or supernumerary mitochondria. Throughout the past decade, mitophagy has attracted considerable attention from both fundamental researchers and translational investigators, both of whom have called for the development of techniques that allow the precise quantification of mitophagy (as opposed to general autophagy). In this chapter, we present morphological, biochemical, and fluorescence-based approaches to measure autophagy in mammalian cells, and discuss recent progress in mouse models for the assessment of mitophagy in vivo., (© 2017 Elsevier Inc. All rights reserved.)

It is widely acknowledged that mitochondria are highly active structures that rapidly respond to cellular and environmental perturbations by changing their shape, number, and distribution. Mitochondrial remodeling is a key component of diverse biological processes, ranging from cell cycle progression to autophagy. In this chapter, we describe different methodologies for the morphological study of the mitochondrial network. Instructions are given for the preparation of samples for fluorescent microscopy, based on genetically encoded strategies or the employment of synthetic fluorescent dyes. We also propose detailed protocols to analyze mitochondrial morphometric parameters from both three-dimensional and bidimensional datasets. Finally, we describe a protocol for the visualization and quantification of mitochondrial structures through electron microscopy., (© 2017 Elsevier Inc. All rights reserved.)

Histone chaperones-key actors in the dynamic organization of chromatin-interact with the various histone variants to ensure their transfer in and out of chromatin. In vitro chromatin assembly assays and isolation of protein complexes using tagged histone variants provided first clues concerning their binding specificities and mode of action. Here, we describe an in vivo method using SNAP-tag-based imaging to assess the de novo deposition of histones and the role of histone chaperones. This method exploits cells expressing SNAP-tagged histones combined with individual cell imaging to visualize directly de novo histone deposition in vivo. We show how, by combining this method with siRNA-based depletion, we could assess the function of two distinct histone chaperones. For this, we provide the details of the method as applied in two examples to characterize the function of the histone chaperones CAF-1 and HIRA. In both cases, we document the impact of their depletion on the de novo deposition of the histone variants H3.1 and H3.3, first in a normal context and second in response to DNA damage. We discuss how this cellular assay offers means to define in a systematic manner the function of any chosen chaperone with respect to the deposition of a given histone variant., (© 2016 Elsevier Inc. All rights reserved.)

Chromatin accessibility is modulated by structural transitions that provide timely access to the genetic and epigenetic information during many essential nuclear processes. These transitions are orchestrated by regulatory proteins that coordinate intricate structural modifications and signaling pathways. In vitro reconstituted chromatin samples from defined components are instrumental in defining the mechanistic details of such processes. The bottleneck to appropriate in vitro analysis is the production of high quality, and quality-controlled, chromatin substrates. In this chapter, we describe methods for in vitro chromatin reconstitution and quality control. We highlight the strengths and weaknesses of various approaches and emphasize quality control steps that ensure reconstitution of a bona fide homogenous chromatin preparation. This is essential for optimal reproducibility and reliability of ensuing experiments using chromatin substrates., (© 2016 Elsevier Inc. All rights reserved.)

Recent improvements in high-throughput sequencing technologies underscore the pervasiveness of circular RNA (circRNA) expression in animal cells. CircRNAs are distinct from their linear counterparts because they lack the 5' caps and 3' tails that typically help determine the cellular fate of a transcript. However, due to the lack of free ends, circRNAs are impervious to exonucleases and thus can evade normal RNA turnover mechanisms. Most circRNAs are derived from protein-coding pre-mRNAs, via a mechanism called "back-splicing." Existing methods of circRNA expression thus typically involve genes that have been engineered to contain sequence elements that promote back-splicing. We recently uncovered an anciently conserved mechanism of RNA circularization in metazoans that involves splicing of tRNA introns. This splicing mechanism is completely independent from that of pre-mRNAs. In this chapter, we detail an orthogonal method that involves splicing of intron-containing tRNAs in order to produce circRNAs in vivo. We utilize fluorescence-based RNA reporters to characterize the expression, localization, and stability of these so-called tRNA intronic circular RNAs. Because tRNA biogenesis is essential for all cellular life, this method provides a means to express ultrastable, high-copy, circRNA effectors in a wide variety of metazoan cell types., (© 2016 Elsevier Inc. All rights reserved.)

Cell communications are essential to the organization, development, and maintenance of multicellular organisms. Much of this communication involves changes in RNA transcription and is dynamic. Most methods for studying transcription require interrupting the continuity of cellular function by sacrificing the communicating cells and capturing gene expression information by periodic sampling of individual cells or the population. The IMAGEtag technology to quantify RNA levels in living cells, demonstrated here in yeast, allows individual cells to be tracked over time as they respond to different environmental cues. IMAGEtags are short RNAs consisting of strings of a variable number of tandem aptamers that bind small-molecule ligands. The aptamer strings can vary in length and in configuration of aptamer constituents, such as to contain multiples of the same aptamer or two or more different aptamers that alternate in their occurrence. A minimum effective length is about five aptamers. The maximum length is undefined. The small-molecule ligands are enabled for imaging as fluorophore conjugates. For each IMAGEtag, two fluorophore conjugates are provided, which are FRET pairs. When a cell expresses an RNA containing an IMAGEtag sequence, the aptamers bind their ligands and bring the fluorophores into sufficiently close proximity to allow FRET. The background fluorescence of both fluorophores is minimal in the FRET channel. These features endow IMAGEtags with the sensitivity to report on mRNA expression levels in living cells., (© 2016 Elsevier Inc. All rights reserved.)

Quantitative measurements of both the copy number and spatial distribution of large fractions of the transcriptome in single cells could revolutionize our understanding of a variety of cellular and tissue behaviors in both healthy and diseased states. Single-molecule fluorescence in situ hybridization (smFISH)-an approach where individual RNAs are labeled with fluorescent probes and imaged in their native cellular and tissue context-provides both the copy number and spatial context of RNAs but has been limited in the number of RNA species that can be measured simultaneously. Here, we describe multiplexed error-robust fluorescence in situ hybridization (MERFISH), a massively parallelized form of smFISH that can image and identify hundreds to thousands of different RNA species simultaneously with high accuracy in individual cells in their native spatial context. We provide detailed protocols on all aspects of MERFISH, including probe design, data collection, and data analysis to allow interested laboratories to perform MERFISH measurements themselves., (© 2016 Elsevier Inc. All rights reserved.)

Hypoxia induces G1 arrest in many cancer cell types. Tumor cells are often exposed to hypoxia/reoxygenation, especially under acute hypoxic conditions in vivo. In this study, we investigated cell-cycle kinetics and clonogenic survival after hypoxia/reoxygenation in HeLa cells expressing fluorescent ubiquitination-based cell cycle indicator (Fucci). Hypoxic treatment halted cell-cycle progression during mid-S to G2 phase, as determined by the cell cycle-regulated E3 ligase activities of SCF(Skp2) and APC/C(Cdh1), which are regulators of the Fucci probes; however, the DNA content of the arrested cells was equivalent to that in G1 phase. After reoxygenation, time-lapse imaging and DNA content analysis revealed that all cells reached G2 phase, and that Fucci fluorescence was distinctly separated into two fractions 24h after reoxygenation: red cells that released from G2 arrest after repairing DNA double-strand breaks (DSBs) exhibited higher clonogenic survival, whereas most cells that stayed green contained many DSBs and exhibited lower survival. We conclude that hypoxia disrupts coordination of DNA synthesis and E3 ligase activities associated with cell-cycle progression, and that DSB repair could greatly influence cell-cycle kinetics and clonogenic survival after hypoxia/reoxygenation., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Endoplasmic reticulum resident protein 44 (ERp44) is a member of the protein disulfide isomerase family and functions in oxidative protein folding in the endoplasmic reticulum. A structurally flexible C-terminal tail (C-tail) of ERp44 plays critical roles in dynamically regulating ERp44's function in protein folding quality control. The structure-function dynamics of ERp44's C-tail may be studied further using fluorescence and other techniques, if methods are found to label the C-tail site-specifically with a fluorescent group or segmentally with other desired labels. Here we have developed such methods, employing split inteins capable of protein trans-splicing, and identifying atypical S1 split inteins able to function efficiently at a suitable split site in the ERp44 sequence. One method demonstrated segmental expression of ERp44 for segmental labeling of the C-tail, another method efficiently added a commercially available fluorescent group to the C-terminus of ERp44, and both methods may also be generally useful for studying other proteins., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Skeletal muscle excitation-contraction coupling is triggered by the concerted action of two enormous Ca(2+) channel complexes, the dihydropyridine receptor and the type 1 ryanodine receptor. Recent advances in our understanding of the structure of these large Ca(2+) channels have been driven by fluorescence resonance energy transfer (FRET)-based analysis. A methodological challenge in conducting these FRET measurements is the ability to site-specifically label these huge ion channels with donor and acceptor fluorophores capable of undergoing energy transfer. In this chapter, we detail specific protocols for tagging large membrane proteins with these fluorescent probes using three orthogonal labeling methods: fluorescent protein fusions, biarsenical reagents directed to engineered tetracysteine tags, and Cy3/5 nitrilotriacetic acid conjugates that bind to poly-histidine tags., (© 2015 Elsevier Inc. All rights reserved.)

Caspases, which constitute a family of cysteine proteases, are highly conserved in multicellular organisms and function as a central player in apoptosis. The detection of apoptosis is intrinsically difficult because dying cells are rapidly removed from tissues by phagocytosis. Thus, the development of a method for detecting caspase activation is critical for the in vivo study of apoptosis. In this chapter, we describe a genetically encoded fluorescent probe for live imaging of caspase activation., (© 2014 Elsevier Inc. All rights reserved.)

It is essential for a cell to maintain a proper mitochondrial quality and quantity for normal cellular functions. Damaged or unwanted mitochondria can be selectively removed through mitophagy. Mitophagy research has attracted great attention from life sciences and biomedical fields, it is thus important for the community to properly measure mitophagy. Here, we will focus on the current techniques that have been used to monitor mitophagy in mammalian cells, including morphological and biochemical parameters for monitoring the occurrence of mitophagy.

The ability of white and brown adipose tissue to efficiently take up long-chain fatty acids is key to their physiological functions in energy storage and thermogenesis, respectively. Several approaches have been taken to determine uptake rates by cultured cells and primary adipocytes including radio- and fluorescently labeled fatty acids. In addition, the recent description of activatable bioluminescent fatty acids has opened the possibility for expanding these in vitro approaches to real-time monitoring of fatty acid uptake kinetics by adipose depots in vivo. Here, we will describe some of the most useful experimental paradigms to quantitatively determine long-chain fatty acid uptake by adipocytes in vitro and provide the reader with detailed instruction on how bioluminescent probes for in vivo imaging can be synthesized and used in living mice., (© 2014 Elsevier Inc. All rights reserved.)

Caspases are a family of integral proteases playing a role in apoptosis. The importance of apoptosis in disease has made these proteases not only an attractive drug target but also a focal point for measuring apoptosis in vivo. The critical role caspases play in determining cell death has led to the development of a wide array of technologies to measure caspase activity in vivo, ranging from small molecule PET imaging reagents to fluorescent and luminescent protein-based reporters used in whole animal and cell-based applications. This chapter reviews this wide range of technologies available as well as the most appropriate applications for each reagent and the mechanism of how it measures caspase activity in vivo., (© 2014 Elsevier Inc. All rights reserved.)

The actin cytoskeleton is very dynamic and highly regulated by multiple associated proteins in vivo. Understanding how this system of proteins functions in the processes of actin network assembly and disassembly requires methods to dissect the mechanisms of activity of individual factors and of multiple factors acting in concert. The advent of single-filament and single-molecule fluorescence imaging methods has provided a powerful new approach to discovering actin-regulatory activities and obtaining direct, quantitative insights into the pathways of molecular interactions that regulate actin network architecture and dynamics. Here we describe techniques for acquisition and analysis of single-molecule data, applied to the novel challenges of studying the filament assembly and disassembly activities of actin-associated proteins in vitro. We discuss the advantages of single-molecule analysis in directly visualizing the order of molecular events, measuring the kinetic rates of filament binding and dissociation, and studying the coordination among multiple factors. The methods described here complement traditional biochemical approaches in elucidating actin-regulatory mechanisms in reconstituted filamentous networks., (© 2014 Elsevier Inc. All rights reserved.)

Microtubules (MTs) polymerize from soluble αβ-tubulin and undergo rapid dynamic transitions to depolymerization at their ends. Microtubule-associated regulator proteins modulate polymerization dynamics in vivo by altering microtubule plus end conformations or influencing αβ-tubulin incorporation rates. Biochemical reconstitution of dynamic MT polymerization can be visualized with total internal reflection fluorescence (TIRF) microscopy using purified MT regulators. This approach has provided extensive details on the regulation of microtubule dynamics. Here, I describe a general approach to reconstitute MT dynamic polymerization with TOG domain microtubule regulators from the XMAP215/Dis1 and CLASP families using TIRF microscopy. TIRF imaging strategies require nucleation of microtubule polymerization from surface-attached, stabilized MTs. The approaches described here can be used to study the mechanism of a wide variety of microtubule regulatory proteins., (© 2014 Elsevier Inc. All rights reserved.)

Formin homology proteins (formins) are responsible for the formation of actin structures such as actin stress fibers, actin cables, and cytokinetic contractile rings. Formins are the major actin filament (F-actin) nucleators in the cell. Because formins remain bound to the barbed end after nucleating an actin filament, it was expected that formins might rotate along the double-helical structure of F-actin during processive actin elongation (helical rotation). Here, we describe a method to detect the rotational movement of F-actin elongating from immobilized formins using single-molecule fluorescence polarization (FLP). Tetramethylrhodamine (TMR) attached to Cys-374 of actin emits polarized fluorescence at ≈45° with respect to the filament axis. When the TMR-labeled F-actin laying at 45° in the visual field rotates, the vertical- and horizontal-polarized fluorescence (FLV and FLH, respectively) of TMR alternately become bright. This technique allowed us to demonstrate the helical rotation of mDia1, a mammalian formin. Adenosine triphosphate (ATP) hydrolysis in actin subunits is not required for helical rotation; however, ATP appears to contribute to accelerating actin elongation by mDia1. When helical rotation is limited by trapping both mDia1 and the pointed-end side, the processive filament elongation is blocked. Thus, mDia1 faithfully rotates along the long-pitch helix of F-actin. In this chapter, we introduce the theoretical concept of single-molecule FLP, the optical setup, the preparation of adenosine diphosphate-bound actin, and the procedure to observe the rotational movement of F-actin elongating from immobilized formins., (© 2014 Elsevier Inc. All rights reserved.)

Composed of both RNA and protein components, the ribosome is one of the largest macromolecular machines in life responsible for the production of all protein. Interestingly, the major catalytic center of the ribosome (the peptidyl transferase center) and much of the binding regions for both mRNA and tRNA are composed of RNA making the ribosome one of the most complex and widely studied ribozymes. Further, large-scale conformational rearrangements throughout the ribosome are required for proper function making the ribosome a riboswitch as well. Recent advances in single-molecule biophysics have significantly augmented our understanding of ribosome function as both a ribozyme and riboswitch. Here, we discuss single-molecule Förster resonance energy transfer and its application to the study of the ribosome. Also, we describe how these experiments are designed from sample preparation to data acquisition and analysis. The general approach and methods described here can be generally applied to many other biological systems.

The generation in vitro of cultured red blood cells (cRBC) could become an alternative to classical transfusion products. However, even when derived from healthy donors, the cRBC generated in vitro from hematopoietic stem cells may display alterations resulting from a poor controlled production process. In this context, we attempted to monitor the quality of the transfusion products arising from new biotechnologies. For that purpose, we developed an in vitro erythrophagocytosis (EP) test with the murine fibroblast cell line MS-5 and human macrophages (reference method). We evaluated 38 batches of cRBC, at the stage of reticulocyte, generated from CD34(+) cells isolated from placental blood or by leukapheresis. We showed that (i) the EP test performed with the MS-5 cell line was sensitive and can replace human macrophages for the evaluation of cultured cells. (ii) The EP tests revealed disparities among the batches of cRBC. (iii) The viability of the cells (determined by calcein-AM test), the expression of CD47 (antiphagocytosis receptor) and the externalization of phosphatidylserine (PS, marker of phagocytosis) were not critical parameters for the validation of the cRBC. (iv) Conversely, the cell deformability determined by ektacytometry was inversely correlated with the intensity of the phagocytic index. Assuming that the culture conditions directly influence the quality of the cell products generated, optimization of the production mode could benefit from the erythrophagocytosis test., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Polyacrylamide gel electrophoresis (PAGE) is a powerful tool for analyzing RNA samples. Denaturing PAGE provides information on the sample composition and structural integrity of the individual RNA species. Nondenaturing gel electrophoresis allows separation of the conformers and alternatively folded RNA species. It also can be used to resolve RNA protein complexes and to detect RNA complex formation by analyzing changes in the electrophoretic mobility of the RNA. RNA can be visualized within gels by different methods depending on the nature of the detection reagent. RNA molecules can be stained with various dyes, including toluidine blue, SYBR green, and ethidium bromide. Radioactively labeled RNA molecules are visualized by autoradiography, and fluorescently labeled RNA molecules can be observed with a fluorescence scanner. Generally, gels between 0.4 and 1.5mm thick are used for analytical PAGE. Gels thinner than 1mm are fragile and thus usually are not stained but rather are used for radiolabeled RNA. The gels are dried and the radiolabeled RNA is visualized by autoradiography., (© 2013 Elsevier Inc. All rights reserved.)

Preparative polyacrylamide gel electrophoresis (PAGE) is a powerful tool for purifying RNA samples. Denaturing PAGE allows separation of nucleic acids that differ by a single nucleotide in length. It is commonly used to separate and purify RNA species after in vitro transcription, to purify naturally occurring RNA variants such as tRNAs, to remove degradation products, and to purify labeled RNA species. To preserve RNA integrity following purification, RNA is usually visualized by UV shadowing or stained with ethidium bromide or SYBR green dyes., (© 2013 Elsevier Inc. All rights reserved.)

In combination with synthetic fluorescent probes, fluorescence microscopy has emerged as a powerful technique to investigate the production, localization, trafficking, and function of biomolecules in living systems in a noninvasive manner. Prompted by our interest in providing a molecular tool to disentangle the complicated interrelationship between H2O2 and NO in the signal transduction and oxidative pathways, our laboratory has developed a single fluorescent probe, FP-H2O2-NO, that can report H2O2, NO, and H2O2/NO with three different sets of fluorescence signal patterns. In this chapter, we provide essential information about the probe FP-H2O2-NO in order to assist researchers interested to apply our probe to investigate H2O2 and NO biology. We describe the use of FP-H2O2-NO with the representative examples of imaging both exogenous and endogenous H2O2 and NO in live Hela and RAW 264.7 macrophage cells by fluorescence microscopy., (Copyright © 2013 Elsevier Inc. All rights reserved.)

GABA(B) receptors are G-protein-coupled receptors (GPCRs) that are activated by GABA, the principal inhibitory neurotransmitter in the central nervous system. Cell surface mobility of GABA(B) receptors is a key determinant of the efficacy of slow and prolonged synaptic inhibition initiated by GABA. Therefore, experimentally monitoring receptor mobility and how this can be regulated is of primary importance for understanding the roles of GABA(B) receptors in the brain, and how they may be therapeutically exploited. Unusually for a GPCR, heterodimerization between the R1 and R2 subunits is required for the cell surface expression and signaling by prototypical GABA(B) receptors. Here, we describe a minimal epitope-tagging method, based on the incorporation of an α-bungarotoxin binding site (BBS) into the GABA(B) receptor, to study receptor internalization in live cells using a range of imaging approaches. We demonstrate how this technique can be adapted by modifying the BBS to monitor the simultaneous movement of both R1 and R2 subunits, revealing that GABA(B) receptors are internalized as heteromers., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Most current studies of reactive oxygen species (ROS) production report globally averaged measurements within the cell; however, ROS can be produced in distinct subcellular locations and have local effects in their immediate vicinity. A microfluidic platform for high-throughput single-cell imaging allows mitochondrial ROS production to be monitored as varying in both space and time. Using this systems biology approach, single-cell variability can be viewed within a population. We discuss single-cell monitoring of contributors to mitochondrial redox state-mitochondrial hydrogen peroxide or superoxide-through the use of a small molecule probe or targeted fluorescent reporter protein. Jurkat T lymphoma cells were stimulated with antimycin A and imaged in an arrayed microfluidic device over time. Differences in single-cell responses were observed as a function of both inhibitor concentration and type of ROS measurement used., (Copyright © 2013 Elsevier Inc. All rights reserved.)

HyPer, a ratiometric genetically encoded fluorescent sensor, is a popular tool for intracellular hydrogen peroxide detection. When expressed in cultured cells, the freely diffusing version of the sensor (HyPer-cyto) detects temporal patterns of H2O2 generation. However, rapid diffusion of the probe within the nucleocytoplasmic compartment averages the H2O2 signal even in cases of local oxidant production. Consequently, we immobilized the sensor within specific subcellular compartments allowing it to monitor local increases in H2O2. Here, we provide a protocol of ratiometric imaging and ImageJ-based quantification of H2O2 microdomains produced by cells upon physiological stimulation., (Copyright © 2013 Elsevier Inc. All rights reserved.)

We present a new and simple approach to fabricate wafer-scale, thin encapsulated, two-dimensional nanochannels by using conventional surface-micromachining technology and thin-film evaporation. The key steps to the realization of two-dimensional nanochannels are a fine etching of a sacrificial layer to create underetching spaces at the nanometer regime, and an accurate thin-film evaporation for encapsulation. Well-defined cross-sectional, encapsulated nanochannel arrays with dimensions as small as 20 nm in both width and height have been realized at the wafer-scale. The fabricated nanochannels with a channel length of 10mm have been used as a suitable fluidic platform for confining a solution containing nanomolar concentrations of Alexa fluorescent molecules. Initial results toward visualization of single Alexa molecules in the confined solution are reported., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Life cell imaging is a tool for cell biology that has provided invaluable insights into many dynamic processes such as cell division, morphogenesis, or endo- and exocytosis. While observing cells by time-lapse imaging is a standard procedure in many systems, this technique was until recently not available for blood stages of Plasmodium falciparum, the causative agent of the most severe form of human malaria. Here, we provide a detailed description of the procedure for time-lapse-based four-dimensional microscopy in blood stages of this important pathogen. With the widespread use of P. falciparum transfection to fluorescently tag proteins of interest, this technique provides a new tool to study the biology of malaria blood stages that is hoped to lead to a better appreciation of the dynamic processes in this life cycle phase., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Cellulose is generally found in the context of complex plant cell wall materials and mostly in association with other glycans. Cellulose-directed carbohydrate-binding modules (CBMs) can be readily adapted to a range of methods for the in situ imaging of cellulose structures within plant cell walls or other cellulose-based materials. Protocols for the preparation and selection of plant materials, their fixation and processing for preparation of sections for CBM labeling, and fluorescence imaging procedures are described. Approaches to direct methods in which CBMs are directly coupled to fluorophores and indirect methods in which staged incubations with secondary reagents are used for the fluorescence imaging of CBM binding to materials are discussed and presented., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The pathogenic Escherichia coli strain E. coli K1 is a primary causative agent of neonatal meningitis. Understanding how these bacteria cross the blood-brain barrier is vital to develop therapeutics. Here, we describe the use of live-cell imaging techniques to study E. coli K1 interactions with cellular markers following infection of human brain microvascular endothelial cells, a model system of the blood-brain barrier. We also discuss optimization of endothelial cell transfection conditions using nonviral transfection technique, bacterial labeling techniques, and in vitro assays to screen for fluorescent bacteria that retain their ability to invade host cells., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Viruses have evolved to mimic cellular ligands in order to gain access to their host cells for replication. Since viruses are simple in structure, they rely on host cells for all their transportation needs. Following single virus particles during the initial phase of infection, that is, virus entry into target cells, can reveal crucial information on the mechanism of pathogen infections and likewise cellular transport and membrane dynamics. Here, we give an overview on how to fluorescently label virus particles for live cell microscopy, and on how virus entry can be analyzed by single particle tracking experiments. Highlighted are strategies, on how to chemically introduce fluorophores into virions, and on how to extract quantitative information from live cell data., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The advent and wide use of image-based, high-content screening assay formats demands reliable solutions for cellular compartment segmentation to track critical events-for example, those reported by GFP fusions within cell cycle control pathways, signaling pathways, protein translocations, and those associated with drug-induced toxicity such as mitochondrial membrane depolarization, plasma membrane permeabilization, and reactive oxygen species. To meet this need, a series of nuclear/cytoplasmic discriminating probes has been developed: the supravital dyes DRAQ5™ and CyTRAK Orange™ and most recently the viability dye DRAQ7™. These are all spectrally compatible with GFP reporters offering new solutions in imaging and cytometry. As red/far-red emitting dyes, they provide convenient fluorescent emission signatures which are spectrally separated from the majority of commonly used reporter proteins (e.g., eGFP, YFP, mRFP), and a wide range of fluorescent tags such as Alexafluor 488, fluorescein, and Cy2 and fluorescent functional probes used to report cell health status or demark organellar structures. In addition, they are not excited by UV wavelengths thus avoiding complications of the frequently seen pharmacophore UV-autofluorescence in drug discovery. Conversely, their preferential red excitation reduces interference by biological sample autofluorescence. High water solubility and high-affinity DNA-binding properties provide a convenient means of stoichiometrically labeling cell nuclei in live cells without the aid of DMSO and can equally be used for fixed cells. Powerfully, they permit the simultaneous and differential labeling of both nuclear and cytoplasmic compartments in live and fixed cells to clearly render the precise location of cell boundaries which may be beneficial for quantitative expression measurements, cell-cell interactions, and most recently compound in vitro toxicology testing. In one case, DRAQ7™, the core structure has been chemically derivatized to render it intact-cell-membrane impermeant. This far-red viability dye can be more widely combined with other fluorescent reporters to reveal temporally separated events and shows negligible cytotoxicity as determined by sensitive bioassays., (Copyright © 2012 Elsevier Inc. All rights reserved.)

With the advent of fast genome analysis, many genes encoding novel putative cellulolytic enzymes are being identified in diverse bacterial and fungal genomes. The discovery of these genes calls for quick, robust, and reliable methods for qualitative and quantitative characterization of the enzymatic activities of the encoded proteins. Here, we describe the use of the polysaccharide analysis by carbohydrate gel electrophoresis (PACE) method, which was previously used, among other applications, to characterize various hemicellulose degrading enzymes; for structural elucidation of these carbohydrates; and for analysis of products resulting from enzymatic cleavage of cellulose. PACE relies on fluorescent labeling of mono-, oligo-, and polysaccharides at their reducing end and separation of the labeled carbohydrates by polyacrylamide gel electrophoresis. Labeling can be carried out before or after enzymatic digestion. PACE is very sensitive and allows analysis of both substrate specificities and kinetic properties of cellulolytic enzymes., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Proteases play causal roles in many aspects of the aggressive phenotype of tumors, yet many of the implicated proteases originate from tumor-associated cells or from responses of tumor cells to interactions with other cells. Therefore, to obtain a comprehensive view of tumor proteases, we need to be able to assess proteolysis in tumors that are interacting with their microenvironment. As this is difficult to do in vivo, we have developed functional live-cell optical imaging assays and 3D and 4D (i.e., 3D over time) coculture models. We present here a description of the probes used to measure proteolysis and protease activities, the methods used for imaging and analysis of proteolysis and the 3D and 4D models used in our laboratory. Of course, all assays have limitations; however, we suggest that the techniques discussed here will, with attention to their limitations, be useful as a screen for drugs to target the invasive phenotype of tumors., (Copyright © 2012 Elsevier Inc. All rights reserved.)