High Background Signal Research Articles

Use of Nuclear Medicine Technology for Clinical Molecular Imaging: a Message from the Associate Editor.

Molecular imaging is defined as the visual representation, characterization, and quantification of biological processes at the cellular and subcellular levels within intact living organisms; this can be achieved by various imaging technologies such as nuclear imaging, optical imaging, magnetic resonance imaging (MRI), ultrasound imaging, and computed tomography (CT) [1]. Although these modalities are actively used for sensing molecular and cellular processes in preclinical studies, the majority of imaging techniques remain unapplied in the clinical field due to various unresolved issues, such as safety, poor tissue penetration of the imaging signal, and limited sensitivity. So far, nuclear or fusion imaging modalities with the nuclear medicine component are the actively used molecular imaging modalities in the clinical field [1–3]. Nuclear imaging has many fundamental principles as molecular imaging. Nuclear medicine imaging has provided molecular information since the early years of imaging technology. In 1951, Dr. Benedict Cassen successfully imaged the thyroid gland with his rectilinear scanner; his imaging principle is still widely used in daily clinical practices to elucidate iodine avidity of benign and malignant thyroid follicular cells. F-18 FDG PET imaging is probably the most well-known clinical molecular imaging technique that can be used to visualize activity of glucose metabolism. The imaging technique is widely used in the oncologic arena as the majority of tumors utilize glucose more actively than normal tissues. High avidity of radioiodine in thyroid follicular cells functions as the tracer for thyroid diseases, while high glucose avidity of tumor functions as F-18 FDG for oncologic disorders. The elucidation of pathophysiology of certain diseases lead to the development of new diagnostic tracers, which in turn shed more light on the disease pathophysiology. This virtuous cycle would aid in uncovering diseases at an early phase and might increase the chance of complete cure by identifying appropriate molecular signatures of diseases. Nuclear molecular imaging was and still is the leading molecular imaging technology. Although nuclear molecular imaging has distinct advantages over other imaging modalities, it has its own limitations, such as low spatial/temporal resolution and high background signal. Multimodal fusion imaging might be a good strategy to overcome the disadvantages of molecular imaging modalities; PET/CT or PET/MRI is already widely installed and actively used in clinics to provide the best molecular imaging service [4]. In fact, clinical molecular imaging already plays indispensable roles in optimal patient care, and its diagnostic and theranostic potentials are increasingly recognized through better understanding of disease pathophysiology in combination with rapid advancements of electronic and biologic technologies. Constant contributions of nuclear medicine physicians and nuclear/biological scientists will retain nuclear molecular imaging as the leading clinical imaging technology to visualize hidden molecular events in human studies and link successful ideas from basic preclinical environment to clinical translation [5]. Nuclear Medicine and Molecular Imaging would be the platform for the advancement of nuclear molecular imaging and provide cutting edge * Byeong-Cheol Ahn abc2000@knu.ac.kr

Life Sciences Discovery and Technology Highlights

Life Sciences Discovery and Technology Highlights

Advances in Gated CW STED Microscopy: Toward a Versatile Implementation

Stimulated emission depletion (STED) microscopy based on time-gated detection is a straightforward implementation, which substantial reduces the STED beam (peak) intensity to obtain effective sub-diffraction imaging. Furthermore, when combined with STED beam operating in continuous-wave (CW) lowers the complexity and the cost with respect to the early STED implementations, the so called gated CW-STED implementation (gCW-STED). However, the time-gated detection reduces the fluorescence signal that forms the images. Hence, in a situation of high background and/or weak signal, the images degrade in terms of signal-to-noise/background ratio (SNR and SBR) and the benefits of time-gated detection could vanish.In this work we reported about synergetic combinations of hardware and software methods able to compensate for the SNR and SBR reduction. In particular we demonstrate the benefits of (i) low intensity-noise laser to further reduce the STED beam intensity [1]; (ii) dedicated time gating electronics to optimize the fluorescence collection efficiency [2]; (iii) synchronous detection methods to remove the anti-Stokes emission background caused by the direct excitation from the STED beam [3]; (iv) tailored image deconvolution algorithms to improve the contrast. The combination of the proposed methods with two-photon-excitation (TPE) gCW-STED microscopy are also demonstrated.[1]I. Coto Hernandez, M. D’Amora, a Diaspro, and G. Vicidomini, “Influence of laser intensity noise on gated CW-STED microscopy,” Laser Phys. Lett., vol. 11, no. 9, p. 095603, 2014.[2]I. C. Hernandez, M. Buttafava, G. Boso, A. Diaspro, A. Tosi, and G. Vicidomini, “Gated STED microscopy with time-gated single-photon avalanche diode,” Biomed. Opt. Express, vol. 6, no. 6, p. 2258, 2015.[3]I. Coto Hernandez, C. Peres, F. C. Zanacchi, M. Amora, S. Christodoulou, P. Bianchini, A. Diaspro, and G. Vicidomini, “A new filtering technique for removing anti-Stokes emission background in gated CW-STED microscopy,” J. Biophotonics, 1-5, 2014.

Comparison of reflectance confocal microscopy and two-photon second harmonic generation microscopy in fungal keratitis rabbit model ex vivo.

Fungal keratitis is an infection of the cornea by fungal pathogens. Diagnosis methods based on optical microscopy could be beneficial over the conventional microbiology method by allowing rapid and non-invasive examination. Reflectance confocal microscopy (RCM) and two-photon second harmonic generation microscopy (TPSHGM) have been applied to pre-clinical or clinical studies of fungal keratitis. In this report, RCM and TPSHGM were characterized and compared in the imaging of a fungal keratitis rabbit model ex vivo. Fungal infection was induced by using two strains of fungi: aspergillus fumigatus and candida albicans. The infected corneas were imaged in fresh condition by both modalities sequentially and their images were analyzed. Both RCM and TPSHGM could detect both fungal strains within the cornea based on morphology: aspergillus fumigatus had distinctive filamentous structures, and candida albicans had round structures superficially and elongated structures in the corneal stroma. These imaging results were confirmed by histology. Comparison between RCM and TPSHGM showed several characteristics. Although RCM and TPSHGM images had good correlation each other, their images were slightly different due to difference in contrast mechanism. RCM had relatively low image contrast with the infected turbid corneas due to high background signal. TPSHGM visualized cells and collagen in the cornea clearly compared to RCM, but used higher laser power to compensate low autofluorescence. Since these two modalities provide complementary information, combination of RCM and TPSHGM would be useful for fungal keratitis detection by compensating their weaknesses each other.

Engineering Signaling Aptamers That Rely on Kinetic Rather Than Equilibrium Competition.

During the past decade, aptasensors have largely been designed on the basis of the notion that ligand-modulated equilibration between aptamer conformations could be exploited for sensing. One implementation of this strategy has been to denature the aptamer with an antisense oligonucleotide, wait for dissociation of the antisense oligonucleotide, and stabilize the folded, signaling conformer with a ligand. However, there is a large kinetic barrier associated with releasing the oligonucleotide from the aptamer to again obtain an active, binding conformation. If the length of the antisense oligonucleotide is decreased to make dissociation from the aptamer more favorable, higher background signals are observed. To improve the general methodology for developing aptasensors, we have developed a novel and robust strategy for aptasensor design in which an oligonucleotide kinetically competes with the ligand for binding rather than having to be released from a stable duplex. While the oligonucleotide can induce conformational change, it initially chooses between the aptamer and a molecular beacon (MB), a process that does not require a lengthy pre-equilibration. Using an anti-ricin aptamer as a starting point, we developed a "competitive" aptasensor with a measured limit of detection (LOD) of 30 nM with an optical readout and as low as 3 nM for ricin toxin A-chain (RTA) detection on an electrochemical platform.

Molecular structure and thermodynamic predictions to create highly sensitive microRNA biosensors

Many studies have established microRNAs (miRNAs) as post-transcriptional regulators in a variety of intracellular molecular processes. Abnormal changes in miRNA have been associated with several diseases. However, these changes are sometimes subtle and occur at nanomolar levels or lower. Several biosensing hurdles for in situ cellular/tissue analysis of miRNA limit detection of small amounts of miRNA. Of these limitations the most challenging are selectivity and sensor degradation creating high background signals and false signals. Recently we developed a reporter+probe biosensor for let-7a that showed potential to mitigate false signal from sensor degradation. Here we designed reporter+probe biosensors for miR-26a-2-3p and miR-27a-5p to better understand the effect of thermodynamics and molecular structures of the biosensor constituents on the analytical performance. Signal changes from interactions between Cy3 and Cy5 on the reporters were used to understand structural aspects of the reporter designs. Theoretical thermodynamic values, single stranded conformations, hetero- and homodimerization structures, and equilibrium concentrations of the reporters and probes were used to interpret the experimental observations. Studies of the sensitivity and selectivity revealed 5–9 nM detection limits in the presence and absence of interfering off-analyte miRNAs. These studies will aid in determining how to rationally design reporter+probe biosensors to overcome hurdles associated with highly sensitive miRNA biosensing.

Bioluminescence activated by antibody-enzyme fragment complementation following target engagement

Event Abstract Back to Event Bioluminescence activated by antibody-enzyme fragment complementation following target engagement Andrew S. Dixon1, Brett K. Baumgartner1, Sylvia Krippner2 and Shawn Owen1 1 University of Utah, Pharmaceutics and Pharmaceutical Chemistry, United States 2 University of Marburg, Pharmacy, Germany Diagnostic and imaging applications using antibodies require conjugating or labeling the antibody in order to obtain a measurable readout[1],[2]. As such, the readout signal is active prior to antibody-antigen binding, requiring significant effort to prevent high background and false signals. The same problem plagues therapeutic antibody-enzyme constructs – the enzyme is constantly active. We created a superior theranostic method in which the diagnostic signal or therapeutic activity is completely dormant until antibody binds to the target antigen. Our approach, termed Target Engaged Complementation (TEC), utilizes enzymes split into inactive components. Each half of the split enzyme is fused to an individual antibody fragment (Fab). The binding of the Fabs places the enzyme fragments into proximity where they complement to reform the active enzyme. Thus, activity is completely dependent on the antibody binding to the target antigen (Figure 1). Materials and Methods: Potential non-overlapping antibody binding partners were identified in silico using reported crystal structures: trastuzumab (Herceptin ®) – 1N8Z; pertuzumab (Perjeta ®) – 1S78. The luciferase NanoLuc enzyme (Promega) was selected for diagnostic applications[3]. Fab-enzyme fusion proteins were produced recombinantly using the pF1k plasmid expressed in Shuffle T7 competent e. coli (NEB) and purified by affinity chromatography. Assays were performed by incubating live cells with Fab-enzyme fusions. Nano-Glo® (Promega) luciferase reagent was added and luminescence was measured on a M1000 Pro Tecan. Results and Discussion: We selected the cancer biomarker HER2 as a target for an initial validation of the TEC concept and selected several antibody pairs from in silico measurements. The criteria for potential pairs are: 1) do not bind to overlapping epitopes and 2) bind so that the distance between pairs allows structural complementation of the split enzyme. We created fusion proteins from anti-HER2 Fabs and fragments from the small luciferase NanoLuc® which has been previously split into fragments that reconstitute activated enzyme following complementation (Figure 2)[4]. Lumiescence assays reveal the ability of these constructs to detect HER2 on the ovarian cancer cell line SKOV3 (Figure 2) with a signal to noise ratio ~300:1. In addition, TEC was effective in detecting the shed extracellular domain of HER2 in solution (data not shown). Neither assay required any washing steps to remove unbound antibody. As such, the specific interaction of antibody with antigen is sufficient to force enzyme complementation to detect an important clinical cancer biomarker. Conclusions: In addition to diagnostics, TEC may be used for therapeutic applications. An original inception of targeted therapeutics involved an approach termed Antibody-Dependent Enzyme Prodrug Therapy (ADEPT), where a full enzyme was fused with an antibody and used to convert a prodrug[5]. The ability of the enzyme to continuously convert prodrug at the tumor site is extremely appealing, yet first compositions of ADEPT suffered from a constitutively active enzyme that activated the prodrug at off-target sites. TEC is an alternative approach that can potentially activate prodrugs specifically to the tumor site. Success has the field-shifting potential of creating a unique theranostic platform applicable to oncology and other clinically relevant areas.

Simultaneous, Single-Cell Measurement of Messenger RNA, Cell Surface Proteins, and Intracellular Proteins.

Nucleic acid content can be quantified by flow cytometry through the use of intercalating compounds; however, measuring the presence of specific sequences has hitherto been difficult to achieve by this methodology. The primary obstacle to detecting discrete nucleic acid sequences by flow cytometry is their low quantity and the presence of high background signals, rendering the detection of hybridized fluorescent probes challenging. Amplification of nucleic acid sequences by molecular techniques such as in situ PCR have been applied to single-cell suspensions, but these approaches have not been easily adapted to conventional flow cytometry. An alternative strategy implements a Branched DNA technique, comprising target-specific probes and sequentially hybridized amplification reagents, resulting in a theoretical 8,000- to 16,000-fold increase in fluorescence signal amplification. The Branched DNA technique allows for the quantification of native and unmanipulated mRNA content with increased signal detection and reduced background. This procedure utilizes gentle fixation steps with low hybridization temperatures, leaving the assayed cells intact to permit their concomitant immunophenotyping. This technology has the potential to advance scientific discovery by correlating potentially small quantities of mRNA with many biological measurements at the single-cell level.

Light-up and FRET aptamer reporters; evaluating their applications for imaging transcription in eukaryotic cells

The regulation of RNA transcription is central to cellular function. Changes in gene expression drive differentiation and cellular responses to events such as injury. RNA trafficking can also have a large impact on protein expression and its localization. Thus, the ability to image RNA transcription and trafficking in real time and in living cells is a worthwhile goal that has been difficult to achieve. The availability of “light-up” aptamers that cause an increase in fluorescence of their ligands when bound by the aptamer have shown promise for reporting on RNA production and localization in vivo. Here we have investigated two light-up aptamers (the malachite green aptamer and the Spinach aptamers) for their suitabilities as reporters of RNA expression in vivo using two eukaryotic cell types, yeast and mammalian. Our analysis focused on the aptamer ligands, their contributions to background noise, and the impact of tandem aptamer strings on signal strength and ligand affinity. Whereas the background fluorescence is very low in vitro, this is not always true for cell imaging. Our results suggest the need for caution in using light-up aptamers as reporters for imaging RNA. In particular, images should be collected and analyzed by operators blinded to the sample identities. The appropriate control condition of ligand with the cells in the absence of aptamer expression must be included in each experiment. This control condition establishes that the specific interaction of ligand with aptamer, rather than nonspecific interactions with unknown cell elements, is responsible for the observed fluorescent signals. High background signals due to nonspecific interactions of aptamer ligands with cell components can be minimized by using IMAGEtags (Intracellular Multiaptamer GEnetic tags), which signal by FRET and are promising RNA reporters for imaging transcription.

Ultra sensitive firefly luciferase‐based protein‐protein interaction assay (FlimPIA) attained by hinge region engineering and optimized reaction conditions

Detecting and assaying protein-protein interactions are significant research procedures in biology and biotechnology. We recently reported a novel assay to detect protein-protein interaction, i.e. firefly luminescent intermediate-based protein-protein interaction assay (FlimPIA) using two mutant firefly luciferases (Flucs), which complement each other's deficient half reaction. This assay detects neighboring of two mutant Flucs, namely, a "Donor" that catalyzes the adenylation of firefly luciferin to produce a luciferyl-adenylate intermediate, and an "Acceptor" that catalyzes the subsequent light emitting reaction. However, its rather high background signal, derived from the remaining adenylation activity of the Acceptor, has limited its usefulness. To reduce this background signal, we introduced a mutation (R437K) into the hinge region of the Acceptor, while maintaining the oxidative activity. Interestingly, the signal/background (S/B) ratio of the assay was markedly improved by the addition of coenzyme A and reduction of the ATP concentration, probably due to reduced inhibition by dehydroluciferyl-adenylate formed during the catalysis and an increased ATP-based Km value of the Acceptor, respectively. As a result, a significantly improved maximal S/B ratio from 2.5 to ∼40 was attained, which promises wider use of the assay in in vitro diagnostics, drug discovery, and expanding our knowledge of various biological phenomena.

Multiscale photoacoustic tomography using reversibly switchable bacterial phytochrome as a near-infrared photochromic probe.

Photoacoustic tomography (PAT) of genetically encoded probes allows imaging of targeted biological processes with high spatial resolution at depths. Here, we combined multi-scale photoacoustic imaging with, for the first time, a reversibly switchable non-fluorescent bacterial phytochrome BphP1. With a heme-derived biliverdin chromophore, BphP1 has the most red-shifted absorption among reported genetically encoded probes, and is reversibly photoconvertible between its red and near-infrared light absorption states. We combined single-wavelength PAT with efficient BphP1 photoswitching, enabling differential imaging that substantially removed background signals, enhanced detection sensitivity, increased penetration depth, and improved spatial resolution. In doing so, we monitored tumor growth and metastasis with a ~100 µm resolution at depths approaching 10 mm using photoacoustic computed tomography, and imaged individual cancer cells with a sub-optical-diffraction resolution of ~140 nm using photoacoustic microscopy. This technology is promising for biomedical studies at different length scales.

Optimization of strand displacement amplification-sensitized G-quadruplex DNAzyme-based sensing system and its application in activity detection of uracil-DNA glycosylase

As an isothermal nucleic acid amplification technique, strand displacement amplification (SDA) reaction has been introduced in G-quadruplex DNAzyme-based sensing system to improve the sensing performance. To further provide useful information for the design of SDA-amplified G-quadruplex DNAzyme-based sensors, the effects of nicking site number in SDA template DNA were investigated. With the increase of the nicking site number from 1 to 2, enrichment of G-quadruplex DNAzyme by SDA is changed from a linear amplification to an exponential amplification, thus greatly increasing the amplification efficiency and subsequently improving the sensing performance of corresponding sensing system. The nicking site number cannot be further increased because more nicking sites might result in high background signals. However, we demonstrated that G-quadruplex DNAzyme enrichment efficiency could be further improved by introducing a cross-triggered SDA strategy, in which two templates each has two nicking sites are used. To validate the proposed cross-triggered SDA strategy, we used it to develop a sensing platform for the detection of uracil-DNA glycosylase (UDG) activity. The sensor enables sensitive detection of UDG activity in the range of 1×10−4–1U/mL with a detection limit of 1×10−4U/mL.

Portable kit for identification and detection of drugs in human urine using surface-enhanced Raman spectroscopy.

A portable kit was demonstrated for rapid and reliable surface-enhanced Raman scattering (SERS) detection of drugs in human urine. This kit contains two sealed reagent tubes, a packet of standardized SERS substrates, and a mini Raman device. A 3 min pretreatment for separating amphetamines from human urine was developed with an extraction rate of >80% examined by ultraperformance liquid chromatography (UPLC). Simultaneously, highly reproducible two-dimensional (2D) gold nanorod (GNR) arrays were assembled by the use of methoxymercaptopoly(ethylene glycol) (mPEG-SH) capping. Thirty batches of GNR arrays produced the 1001 cm(-1) intensity of methamphetamine (MA) molecules with a relative standard deviation (RSD) of 7.9%, and a 21 × 21 μm(2) area mapping on a 2D GNR array produced a statistical RSD of <10%, implying an excellent reproducibility and uniformity. The detection limit of amphetamines in human urine was at least 0.1 ppm. Moreover, the portable kit was successfully used for detecting MA, 3,4-methylenedioxymethamphetamine (MDMA), and methcathinone (MC) in 30 volunteers' urine samples with various clinical natures, and the dual-analyte detection of MA and MDMA implied a good capability of multiplex analysis. UPLC examination and the SERS recovery test clearly indicated that our pretreatment procedure was sufficient to lower the high background signals caused by complex components in urine and demonstrated the practicability and the resistance to false positives, which is a vital problem for law enforcement applications. The excellent performance of our portable kit promises a great prospective toward a rapid, reliable, and on-spot analyzer, especially for public safety and healthcare.

Two-Photon Sensing and Imaging of Endogenous Biological Cyanide in Plant Tissues Using Graphene Quantum Dot/Gold Nanoparticle Conjugate.

One main source of cyanide (CN(-)) exposure for mammals is through the plant consumption, and thus, sensitive and selective CN(-) detection in plants tissue is a significant and urgent work. Although various fluorescence probes have been reported for CN(-) in water and mammalian cells, the detection of endogenous biological CN(-) in plant tissue remains to be explored due to the high background signal and large thickness of plant tissue that hamper the effective application of traditional one-photo excitation. To address these issues, we developed a new two-photo excitation (TPE) nanosensor using graphene quantum dots (GQDs)/gold nanoparticle (AuNPs) conjugate for sensing and imaging endogenous biological CN(-). With the benefit of the high quenching efficiency of AuNPs and excellent two-photon properties of GQDs, our sensing system can achieve a low detection limit of 0.52 μM and deeper penetration depth (about 400 μm) without interference from background signals of a complex biological environment, thus realizing sensing and imaging of CN(-) in different types of plant tissues and even monitoring CN(-) removal in food processing. To the best of our knowledge, this is the first time for fluorescent sensing and imaging of CN(-) in plant tissues. Moreover, our design also provides a new model scheme for the development of two-photon fluorescent nanomaterial, which is expected to hold great potential for food processing and safety testing.

Real-time analysis of dual-display phage immobilization and autoantibody screening using quartz crystal microbalance with dissipation monitoring.

Over the last three decades, phage display technology has been used for the display of target-specific biomarkers, peptides, antibodies, etc. Phage display-based assays are mostly limited to the phage ELISA, which is notorious for its high background signal and laborious methodology. These problems have been recently overcome by designing a dual-display phage with two different end functionalities, namely, streptavidin (STV)-binding protein at one end and a rheumatoid arthritis-specific autoantigenic target at the other end. Using this dual-display phage, a much higher sensitivity in screening specificities of autoantibodies in complex serum sample has been detected compared to single-display phage system on phage ELISA. Herein, we aimed to develop a novel, rapid, and sensitive dual-display phage to detect autoantibodies presence in serum samples using quartz crystal microbalance with dissipation monitoring as a sensing platform. The vertical functionalization of the phage over the STV-modified surfaces resulted in clear frequency and dissipation shifts revealing a well-defined viscoelastic signature. Screening for autoantibodies using antihuman IgG-modified surfaces and the dual-display phage with STV magnetic bead complexes allowed to isolate the target entities from complex mixtures and to achieve a large response as compared to negative control samples. This novel dual-display strategy can be a potential alternative to the time consuming phage ELISA protocols for the qualitative analysis of serum autoantibodies and can be taken as a departure point to ultimately achieve a point of care diagnostic system.

Immuno-based detection of extracellular vesicles in urine as diagnostic marker for prostate cancer.

Extracellular vesicles (including the subclass exosomes) secreted by cells contain specific proteins and RNA that could be of interest in determining new markers. Isolation/characterization of PCa-derived exosomes from bodily fluids enables us to discover new markers for this disease. Unfortunately, isolation with current techniques (ultracentrifugation) is labor intensive and other techniques are still under development. The goal of our study was to develop a highly sensitive time-resolved fluorescence immunoassay (TR-FIA) for capture/detection of PCa-derived exosomes. In our assay, biotinylated capture antibodies against human CD9 or CD63 were incubated on streptavidin-coated wells. After application of exosomes, Europium-labeled detection antibodies (CD9 or CD63) were added. Cell medium from 37 cell lines was taken to validate this TR-FIA. Urine was collected (after digital rectal exam) from patients with PCa (n = 67), men without PCa (n = 76). As a control, urine was collected from men after radical prostatectomy (n = 13), women (n = 16) and patients with prostate cancer without digital rectal exam (n = 16). Signal intensities were corrected for urinary PSA and creatinine. This TR-FIA can measure purified exosomes with high sensitivity and minimal background signals. Exosomes can be measured in medium from 37 cell lines and in urine. DRE resulted in a pronounced increase in CD63 signals. After DRE and correction for urinary PSA, CD9 and CD63 were significantly higher in men with PCa. This TR-FIA enabled us to measure exosomes with high sensitivity directly from urine and cell medium. This TR-FIA forms the basis for testing different antibodies directed against exosome membrane markers to generate disease-specific detection assays.

Gated Luminescence Imaging of Silicon Nanoparticles.

The luminescence lifetime of nanocrystalline silicon is typically on the order of microseconds, significantly longer than the nanosecond lifetimes exhibited by fluorescent molecules naturally present in cells and tissues. Time-gated imaging, where the image is acquired at a time after termination of an excitation pulse, allows discrimination of a silicon nanoparticle probe from these endogenous signals. Because of the microsecond time scale for silicon emission, time-gated imaging is relatively simple to implement for this biocompatible and nontoxic probe. Here a time-gated system with ∼10 ns resolution is described, using an intensified CCD camera and pulsed LED or laser excitation sources. The method is demonstrated by tracking the fate of mesoporous silicon nanoparticles containing the tumor-targeting peptide iRGD, administered by retro-orbital injection into live mice. Imaging of such systemically administered nanoparticles in vivo is particularly challenging because of the low concentration of probe in the targeted tissues and relatively high background signals from tissue autofluorescence. Contrast improvements of >100-fold (relative to steady-state imaging) is demonstrated in the targeted tissues.

Förster Resonance Energy Transfer between Quantum Dot Donors and Quantum Dot Acceptors.

Förster (or fluorescence) resonance energy transfer amongst semiconductor quantum dots (QDs) is reviewed, with particular interest in biosensing applications. The unique optical properties of QDs provide certain advantages and also specific challenges with regards to sensor design, compared to other FRET systems. The brightness and photostability of QDs make them attractive for highly sensitive sensing and long-term, repetitive imaging applications, respectively, but the overlapping donor and acceptor excitation signals that arise when QDs serve as both the donor and acceptor lead to high background signals from direct excitation of the acceptor. The fundamentals of FRET within a nominally homogeneous QD population as well as energy transfer between two distinct colors of QDs are discussed. Examples of successful sensors are highlighted, as is cascading FRET, which can be used for solar harvesting.

Efficient cellular solid-state NMR of membrane proteins by targeted protein labeling.

Solid-state NMR spectroscopy (ssNMR) has made significant progress towards the study of membrane proteins in their native cellular membranes. However, reduced spectroscopic sensitivity and high background signal levels can complicate these experiments. Here, we describe a method for ssNMR to specifically label a single protein by repressing endogenous protein expression with rifampicin. Our results demonstrate that treatment of E. coli with rifampicin during induction of recombinant membrane protein expression reduces background signals for different expression levels and improves sensitivity in cellular membrane samples. Further, the method reduces the amount of time and resources needed to produce membrane protein samples, enabling new strategies for studying challenging membrane proteins by ssNMR.Electronic supplementary materialThe online version of this article (doi:10.1007/s10858-015-9936-5) contains supplementary material, which is available to authorized users.

Identifying differential transcription factor binding in ChIP-seq.

ChIP seq is a widely used assay to measure genome-wide protein binding. The decrease in costs associated with sequencing has led to a rise in the number of studies that investigate protein binding across treatment conditions or cell lines. In addition to the identification of binding sites, new studies evaluate the variation in protein binding between conditions. A number of approaches to study differential transcription factor binding have recently been developed. Several of these methods build upon established methods from RNA-seq to quantify differences in read counts. We compare how these new approaches perform on different data sets from the ENCODE project to illustrate the impact of data processing pipelines under different study designs. The performance of normalization methods for differential ChIP-seq depends strongly on the variation in total amount of protein bound between conditions, with total read count outperforming effective library size, or variants thereof, when a large variation in binding was studied. Use of input subtraction to correct for non-specific binding showed a relatively modest impact on the number of differential peaks found and the fold change accuracy to biological validation, however a larger impact might be expected for samples with more extreme copy number variations between them. Still, it did identify a small subset of novel differential regions while excluding some differential peaks in regions with high background signal. These results highlight proper scaling for between-sample data normalization as critical for differential transcription factor binding analysis and suggest bioinformaticians need to know about the variation in level of total protein binding between conditions to select the best analysis method. At the same time, validation using fold-change estimates from qRT-PCR suggests there is still room for further method improvement.