Nat Methods Research Articles

A transgenic approach to live imaging of heparan sulfate modification patterns.

Heparan sulfate (HS) glycosaminoglycan chains contain highly modified HS domains that are separated by sections of sparse or no modification. HS domains are central to the role of HS in protein binding and mediating protein-protein interactions in the extracellular matrix. Since HS domains are not genetically encoded, they are impossible to visualize and study with conventional methods in vivo. Here we describe a transgenic approach using previously described single chain variable fragment (scFv) antibodies that bind HS in vitro and on tissue sections with different specificities. By engineering a secretion signal and a fluorescent protein to the scFvs and transgenically expressing these fluorescently tagged antibodies in Caenorhabditis elegans, we are able to directly visualize specific HS domains in live animals (Attreed et al. Nat Methods 9(5):477-479, 2012). The approach allows concomitant colabeling of multiple epitopes, the study of HS dynamics and, could lend itself to a genetic analysis of HS domain biosynthesis or to visualize other nongenetically encoded or posttranslational modifications.

PONDEROSA-C/S: client-server based software package for automated protein 3D structure determination.

Peak-picking Of Noe Data Enabled by Restriction Of Shift Assignments-Client Server (PONDEROSA-C/S) builds on the original PONDEROSA software (Lee et al. in Bioinformatics 27:1727–1728. doi:10.1093/bioinformatics/btr200, 2011) and includes improved features for structure calculation and refinement. PONDEROSA-C/S consists of three programs: Ponderosa Server, Ponderosa Client, and Ponderosa Analyzer. PONDEROSA-C/S takes as input the protein sequence, a list of assigned chemical shifts, and nuclear Overhauser data sets (13C- and/or 15N-NOESY). The output is a set of assigned NOEs and 3D structural models for the protein. Ponderosa Analyzer supports the visualization, validation, and refinement of the results from Ponderosa Server. These tools enable semi-automated NMR-based structure determination of proteins in a rapid and robust fashion. We present examples showing the use of PONDEROSA-C/S in solving structures of four proteins: two that enable comparison with the original PONDEROSA package, and two from the Critical Assessment of automated Structure Determination by NMR (Rosato et al. in Nat Methods 6:625–626. doi:10.1038/nmeth0909-625, 2009) competition. The software package can be downloaded freely in binary format from http://pine.nmrfam.wisc.edu/download_packages.html. Registered users of the National Magnetic Resonance Facility at Madison can submit jobs to the PONDEROSA-C/S server at http://ponderosa.nmrfam.wisc.edu, where instructions, tutorials, and instructions can be found. Structures are normally returned within 1–2 days.Electronic supplementary materialThe online version of this article (doi:10.1007/s10858-014-9855-x) contains supplementary material, which is available to authorized users.

Abstract 531: Mutagenic Gene Trapping to Study Novel Genes in Zebrafish Cardiovascular Development

The zebrafish has emerged as an excellent model for cardiovascular research thanks to its ex-utero and rapid embryonic development, its embryonic transparent nature, and its capacity to survive in the absence of a functional cardiovascular system during the first week of development, which enables functional characterization of mutations that would otherwise induce lethality in traditional murine models. The aim of this project is to identify and characterize novel genes involved in zebrafish cardiovascular development using mutagenic gene trapping, a technique that generates random insertional mutations across the genome. We use the well-defined RP2 gene-breaking transposon system, which not only mutates, but also fluorescently tags the trapped gene product(s) (Clark, Nat Methods, 2011). RP2 also introduces loxP sites into the mutant locus, which can be used for Cre-mediated phenotype rescue by microinjection of Cre recombinase, or by crossing to tissue-specific Cre lines. From over 3000 RP2-injected embryos, 141 fish showed germline transmission. Among them, 51 expressed strong fluorescence in different tissues, including the heart, vessels, notochord, central nervous system (CNS), and eyes. Three cardiovascular lines were selected for phenotypic characterization and functional studies. RP2#C2 strain expressed fluorescence in the heart valves, CNS, eyes and pectoral fin buds. Inverse PCR in RP2#C2 demonstrated a trapped gene at meis4.1a, which encodes a homeobox transcription factor that has not been previously studied in zebrafish. RP2#121 strain expressed strong fluorescence in cardiac and skeletal muscles. RP2#91 strain showed expression in the vasculature and demonstrated a trapped gene at pdgfra. Homozygous RP2#91 mutants showed severe defects in the heart, blood flow, and other body parts including the head and musculature. We are currently creating a panel of tissue-specific Cre lines targeting tissues such as cardiomyocytes, endothelial cells and smooth muscle cells for spatiotemporal rescuing of the mutant phenotype. The generated zebrafish protein-trap lines are invaluable tools to annotate gene function, dissect the molecular mechanisms of cardiovascular development, and potentially serve as disease models.

Herpesviruses encode their own microRNAs.

Featured Article: Pfeffer S, Sewer A, Lagos-Quintana M, Sheridan R, Sander C, Grasser FA, van Dyk LF, Ho CK, Shuman S, Chien M, Russo JJ, Ju J, Randall G, Lindenbach BD, Rice CM, Simon V, Ho DD, Zavolan M, Tuschl T. Identification of microRNAs of the herpesvirus family. Nat Methods 2005;2:269–76.2 In 2001, the term microRNA (miRNA)3 was proposed for the first time after 3 groups reported the identification of hundreds of such small (22 nucleotides) noncoding RNAs in the nematode worm, Drosophila, and humans. Today, with the advent of next generation sequencing technologies, the number of validated miRNAs is nearing 2000 for humans alone, and the number of publications containing the keyword “miRNA” is up to 6000 for the year 2013. What are miRNAs? We now know quite a lot about their biogenesis, their modes of action, and their roles in a wide variety of biological processes (1). They derive from large primary transcripts, which are …

Abstract W P361: Telomere G-tail Length Shortening is Associated With Endothelial Dysfunction in Patients With Chronic Cerebrovascular Disease and Comorbidity

Background and Purpose: Telomere length of leukocytes is associated with cardiovascular risk as well as ischemic stroke and poststroke mortality. In addition, leukocyte telomere length shortening is thought to be associated with increased carotid intimal medial thickness (IMT) and endothelial dysfunction evaluated by flow mediated dilation (FMD). Recently, telomeric 3’-overhang (G-tail) lengths has been essential for the biological effects of telomere dysfunction. The aim of this study was to investigate the associations between telomere G-tail lengths of leukocytes and the physiological studies. Methods: Patients with cerebrovascular diseases history and other neurological problems were enrolled in this study (n=63; 39 male, 69.8±10.0 years). Telomere G-tail lengths were measured by G-tail telomere hybridization protection assay (H Tahara et al. Nat Methods. 2005). The mean value of maximal IMT measured in the distal CCA far wall (CCA-IMT) and plaque score were measured by carotid ultrasonography. Endothelial dysfunction was defined as a FMD≤4.5%. Results: Telomere G-tail length (18947.0±4066.6 RLU/μg DNA) was positively correlated with total telomere length (230330.8±25830.9 RLU/μg DNA; R 2 =0.261, p<0.001). In addition, telomere G-tail length was negatively correlated with age (R 2 =0.217, p<0.001) and positively correlated with FMD (R 2 =0.136, p=0.003). Telomere G-tail length was not related to CCA-IMT and plaque score. The patients with endothelial dysfunction (n=39, 61.9%) were older (72.4±7.6 vs. 65.5±11.8, P=0.007), more frequently male (74.4% vs. 41.7%, P=0.016) and more frequently had hypertension (89.7% vs. 37.5%, P<0.001) than the patients without. After multivariate analysis with age, sex, presence of stroke and comorbidities, the lowest quartile of telomere G-tail length was independently associated with the endothelial dysfunction (OR 3.85, 95%CI 1.58-12.42, P=0.002), although there was no significant association between total telomere length and endothelial dysfunction on multivariate analysis. Conclusion: Telomere G-tail length might be more useful marker for the endothelial dysfunction which was evaluated by FMD than total telomere length.

Protein correlation profiling-SILAC to study protein-protein interactions.

An interactome describes the global organization of protein interactions within a cell and is typically generated using affinity purification-mass spectrometry (AP-MS), yeast two-hybrid screening, or protein-fragment complementation assays (Gavin et al. Nature 440: 631-636, 2006; Krogan et al. Nature 440: 637-643, 2006; Uetz et al. Nature 403: 623-627, 2000; Tarassov et al. Science 320: 1465-1470, 2008). These techniques have been widely used to depict the interactome as we know it today but current models of interactomes do not contain stoichiometric or temporal information. In this chapter we describe size-exclusion chromatography (SEC) combined with protein correlation profiling-stable isotope labeling by amino acids in cell culture (PCP-SILAC) to generate dynamic chromatographs for thousands of proteins (Kristensen et al. Nat Methods 9: 907-909, 2012). Using the precise co-elution of two proteins as evidence that they interact, it is possible to identify similar numbers of protein interactions without overexpression or creating fusion proteins as other high-throughput techniques require. In addition, triplex SILAC allows us to quantify protein stoichiometry and temporal changes to the interactome following perturbation. Finally, SEC-PCP-SILAC is very time efficient since it generates two orders of magnitude fewer samples for LC-MS analysis and avoids the tedious tagging and purification steps, making it possible for everyone with a single mass spectrometer to study the interactome.

Advanced Spim Microscopy Towards the Study of Nanoparticle Uptake Consequence on Small Organisms

In the last years, particular attention has been addressed to the study of possible effect of silver nanoparticles (AgNPs) on the development of Danio Rerio embryos [1][2]. In this work we focus our attention on the possible biological consequence of small-sized silver nanoparticles (8-10nm) on Danio Rerio development by assessing different end-points and exposure periods through a multi-level investigation. This study outlines classical analysis of malformations and molecular characterization using advanced fluorescence imaging techniques.In particular, selective plane illumination microscopy (SPIM) has been proven to be a powerful tool within the developmental biology scenario, thanks to its imaging capabilities with high cellular and subcellular resolution [3][4] in thick samples.Furthermore, an inverted SPIM configuration [5] has been proven to open the application of light sheet microscopy to a wider range of samples, thus becoming a better alternative to confocal imaging techniques for 3D imaging of Danio Rerio embryos.In our studies the use of confocal and light sheet based microscopy techniques allows for the analysis of nanoparticles possible effects on the cytoskeletal architecture. We investigate the possible perturbation induced by AgNPs on the structural integrity of filamentous actin (F-actin) in the embryos in a dose dependent manner.Our work was partially supported by PRIN 2010JFYFY2_002.(1) Asharani P.V. et al., Nanotechnology; 19(25):255102 (2008).(2) Myrzakhanova M. et al., Biomed Res Int.; 2013:205183 (2013).(3) Cella Zanacchi F. etal., Nat Methods, 8, no. 12, pp. 1047-1049 (2011).(4) Cella Zanacchi F. et al., PLoS One, 8(7):e67667 (2013).(5) Wu Y. et al., PNAS,108 (43)(2011).

Development of a Red Genetically-Encoded Voltage Indicator and its use with Channelrhodopsin for All-Optical Electrophysiology

The need for effective genetically-encoded voltage indicators is widely recognized. Several prototypes have been made to date by fusing one or more fluorescent proteins to a voltage sensitive membrane protein (1). However, one factor that has limited the utility of these indicators is poor voltage sensitivity, making it a challenge to express the indicators in neurons and still observe detectable responses (2). In addition, none of the genetically-encoded voltage indicators reported can be combined with channelrhodopsin for optical readout of electrical activity in an all-optical electrophysiology setup. To address these limitations, we set up a directed evolution strategy to screen for both voltage indicator brightness and voltage sensitivity. First, bacterial expression enables us to screen thousands of clones in colonies of E. coli to improve the brightness of the indicator. Second, a medium throughput mammalian electric field stimulation system allows us to test for function of the voltage indicators. This screening has resulted in a family of red voltage indicators (VSDRs). They consist of the voltage-sensing domain of Ciona intestinalis voltage-sensitive phosphatase linked to red fluorescent protein mApple. The fluorescence intensity of the VSDRs increases in response to depolarization. We show that the combination of signal size and response speed of VSDRs allows the reliable detection of spontaneous action potentials in cultured mammalian neurons in single trials with widefield fluorescent light microscopy. Critically, we also demonstrate that VSDRs faithfully reports signals from all-optical electrophysiology experiments using channelrhodopsin to depolarize mammalian cells.1. B. J. Baker et al., J. Neurosci. Methods. 2007, 161, 32-38.2. W. Akemann et al., Nat Methods. 7, 2010, 643-649.

High-Speed Hopping Probe Scanning Ion Conductance Microscopy

Vertically protruding stereocilia on the apical surface of the inner ear hair cells represent the ultimate challenge for scanning probe imaging. We have previously imaged these structures, which are largely unresolvable by optical microscopy, using hopping probe scanning ion conductance microscopy (HPSICM) in glutaraldehyde-fixed mammalian auditory hair cells (Novak et al. Nat Methods, 2009). However, it required a considerable amount of time (∼44 min per bundle of ∼8x8 μm). To study live cells, we needed to significantly increase the speed of imaging.For Z-movement we have now used a faster piezo assembly with a resonant frequency of ∼18 kHz, similar to the ones we used for hair bundle deflection. Despite having a less sensitive strain gauge sensor, the vertical resolution of the system remained the same (∼5nm). After adjusting the proportional-integral-derivative controller of the Z-scanner (∼50μs delay) and increasing the speed of approach, we were able to obtain high-resolution images of live hair cell bundles at a frame rate of 12 min/bundle or less.We tested the performance of the improved HPSICM system in live rat inner hair cells (IHC) and showed, for the first time in live cells, the presence of characteristic stereocilia features at an X-Y resolution of ∼11nm. We also imaged IHC bundles from the Shaker2 and Whirler mice due to their short stereocilia with abundant stereocilia links (typically ∼5nm in diameter and ∼100-300nm in length). We confirmed the reproducibility of links in continuous time-lapse scanning and also their absence after chemical disruption with BAPTA-buffered Ca2+-free medium.Our results demonstrate that the improved HPSICM technique successfully visualizes the extremely convoluted surface of stereocilia in live auditory hair cells at a high resolution and a faster speed.Supported by NIDCD/NIH (R01DC008861).

Roles for text mining in protein function prediction.

The Human Genome Project has provided science with a hugely valuable resource: the blueprints for life; the specification of all of the genes that make up a human. While the genes have all been identified and deciphered, it is proteins that are the workhorses of the human body: they are essential to virtually all cell functions and are the primary mechanism through which biological function is carried out. Hence in order to fully understand what happens at a molecular level in biological organisms, and eventually to enable development of treatments for diseases where some aspect of a biological system goes awry, we must understand the functions of proteins. However, experimental characterization of protein function cannot scale to the vast amount of DNA sequence data now available. Computational protein function prediction has therefore emerged as a problem at the forefront of modern biology (Radivojac et al., Nat Methods 10(13):221-227, 2013).Within the varied approaches to computational protein function prediction that have been explored, there are several that make use of biomedical literature mining. These methods take advantage of information in the published literature to associate specific proteins with specific protein functions. In this chapter, we introduce two main strategies for doing this: association of function terms, represented as Gene Ontology terms (Ashburner et al., Nat Genet 25(1):25-29, 2000), to proteins based on information in published articles, and a paradigm called LEAP-FS (Literature-Enhanced Automated Prediction of Functional Sites) in which literature mining is used to validate the predictions of an orthogonal computational protein function prediction method.

Sensitivity of individual‐donation and minipool nucleic acid amplification test options in detecting window period and occult hepatitis B virus infections

Several comparison studies showed that the Ultrio assay (Novartis Diagnostics) used in individual-donation nucleic acid amplification testing (ID-NAT) format was as sensitive as the TaqScreen assay (Roche) on minipools of six donations (MP6), but the sensitivity of HBV DNA detection has been improved in the new Ultrio Plus version of the assay. A head-to-head comparison study was designed to compare the clinical sensitivity of the Ultrio and Ultrio Plus assay in ID, MP4, and MP8 formats using TaqScreen MP6 as a reference assay. Plasma samples of 107 hepatitis B surface antigen (HBsAg)-negative, HBV ID-NAT (Ultrio) positive-yield samples and 29 HBV DNA-negative, HBsAg-positive samples were used for comparison of NAT options in replicate testing of dilutions. Viral loads and relative sensitivities were determined by probit analysis against the Eurohep standard. Ultrio Plus detected a significantly (p < 0.00001) higher proportion of replicate assays on HBV NAT yields (77%) than Ultrio ID (62%) and TaqScreen MP6 (47%), whereas Ultrio Plus MP4 and MP8 detected 53 and 41%, respectively. On HBsAg-yield samples missed by Ultrio screening, the reactivity rate increased significantly (p < 0.0001) from 23% in Ultrio to 65% in Ultrio Plus and further to 72% (p = 0.10) in the TaqScreen assay. The overall improvement factor of the analytical sensitivity offered by the target enhancer reagent in the Ultrio Plus assay was 2.5 (2.0-3.1)-fold on the Ultrio yield samples, but 43 (11-350)-fold on the HBsAg yields. In ID-NAT format the analytical sensitivity of TaqScreen relative to Ultrio Plus was 2.0 (1.0-4.2), 0.9 (0.7-1.3), and 1.6 (0.9-3.0) on the Eurohep standard, HBV NAT-, and HBsAg-yield samples respectively. The clinical sensitivity of the currently available commercial NAT methods is mainly driven by the pool size.

Abstract 221: MitoROMK Expression Mitigates the Cellular Response to Oxidative Stress

We have recently reported that mitoROMK overexpression protects against, while knockdown of native ROMK exacerbates, oxidative stress-induced cell death and have proposed mitoROMK as the molecular correlate of the cardioprotective mitoKATP channel (Foster, Ho, et al Circ Res 2012). In order to better understand the mechanisms of protection, here we examined how alterations of ROMK expression affect glutathione redox responses during acute oxidative stress, utilizing cytoplasmic or mitochondrially targeted, genetically-encoded GSSG sensors. The tandem protein construct Grx1-roGFP2, a fusion of human glutaredoxin and redox sensitive GFP (Gutscher et al, Nat Methods 2008) was incorporated into adenoviral gene transfer vectors for expression in cardiac-derived H9C2 cells. Cells were subjected to treatment with the Complex III inhibitor Antimycin A (50µM) to induce endogenous mitochondrial reactive oxygen species (ROS) production, and changes in the glutathione redox potential were determined ratiometrically. MitoROMK overexpressing cells showed a significantly lower rate of mitochondrial matrix GSH oxidation secondary to H2O2 scavenging compared to both control and ROMK knockdown cell lines, indicating enhanced ability to maintain ROS balance. Increases in cytoplasmic GSSG were also blunted by mitoROMK overexpression. To determine if ROMK expression affected ROS-induced mitochondrial calcium overload, we also employed the mitochondrially-targeted ratiometric Ca2+ probe GEM-GECO (Zhao et al Science 2011). The same Antimycin A challenge led to large mitochondrial Ca2+ increase in control and knockdown cell lines, but this response was significantly suppressed in mitoROMK overexpressing cells. This could not be explained by differences in baseline mitochondrial membrane potential (ΔΨm; determined using the potentiometric probe tetramethylrhodamine methyl ester), because both control cells and ROMK overexpressors showed similar ΔΨm, while ROMK knockdown cells were more depolarized. The results suggest that enhanced cell survival in mitoROMK overexpressing cells is mediated by improved mitochondrial ROS balance and calcium handling, providing a key molecular link between mitochondrial K+ channels and cardioprotection.

FRI0056 Antibody-based proximity ligation assay combined with flow cytometry is a powerful tool to detect active IL-7 receptors in arthritis

BackgroundIL-7 is a leading growth factor for memory T cells and is believed to play a central role in the sustained inflammation observed in many chronic inflammatory diseases, including rheumatoid...

FRI0051 Application of a multiplex gene polymorphism assay for variants associated with rheumatoid arthritis susceptibility - results of 168 snps tested in the optima study

BackgroundFactors related to increased susceptibility to rheumatoid arthritis (RA) have been described, with numerous genes and alleles having been identified that may be associated with the onset of the disease...

Abstract 252: Molecular characterization of cancer stem cells isolated from primary colon cancer cells.

Abstract Colon cancer is the 3rd most common type of cancer and the 4th leading cause of cancer-related deaths worldwide. OncoTrack is an international consortium that has launched one of Europe's largest collaborative academic-industry research projects to develop and assess novel approaches for identification of new markers for colon cancer (1). To this end, colorectal tumors and metastases, as well as isolated circulating tumor cells and cancer stem cells are subjected to high-throughput sequencing of DNA and RNA. The laboratory data are combined with clinical data and used for in silico modeling of the patients' tumors and prediction of biomarkers as well as a tailored therapy. These predictions are validated using patient-specific in vitro (primary cells) and in vivo (xenografts) models. Recent data show that a small subpopulation of cells within different tumors, including colon cancer, is responsible for tumor growth and maintenance. Due to their functional similarities to adult tissue stem cells and their likely origin, these cells have been termed cancer stem cells. We aim at characterizing patient-derived primary cell cultures and cancer stem cells isolated from these. Primary tumors and metastases from colon cancer patients are cultivated in vitro using a matrigel-based, serum-free culture system. This system allows for long-term expansion of primary colon cancer cells in vitro (2). Using this methodology, we were able to establish over 35 novel, patient-specific colon cancer cell lines, both from primary tumors and metastases. Using previously established markers of colon cancer stem cells (CD44+, CD133+, CD166+, EpCAM-high, Wnt-high), cells are sorted by flow cytometry and subsequently subjected to genome-wide analyses using next-generation sequencing. These experiments allow for a detailed characterization of colon cancer stem cells at the level of transcriptome and methylome. It has been shown that tumors are heterogeneous with regard to gene mutations and expression of cancer stem cell markers (3). Using molecular tools such as padlock probes (4), we are analyzing the distribution of gene mutations identified by next-generation sequencing within primary cells as well as primary tumors. Moreover, we are addressing the expression pattern of cancer stem cell markers in these cell types. These data will help in a better understanding of individual tumors and assist in design of a personalized therapy. OncoTrack is a project funded by the Innovative Medicines Initiative Joint Undertaking (IMI JU). 1 Elsner M, Nature Biotechnology 29, 378 (2011); www.oncotrack.eu 2 Sato T el al., Gastroenterology. 2011 Nov;141(5):1762-72. 3 Gerlinger M et al., N Engl J Med. 2012 Mar 8;366(10):883-92. 4 Larsson C, et al., Nat Methods. 2010 May;7(5):395-7. Citation Format: Martin Lange, Dirk Schumacher, Thomas Hauling, Christian Regenbrecht, Oliver Politz, Mats Nilsson, Jens Hoffmann, Reinhold Schaefer, David Henderson. Molecular characterization of cancer stem cells isolated from primary colon cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 252. doi:10.1158/1538-7445.AM2013-252

Abstract 3403: A stochastically arising subpopulation of B-RafV600E-expressing melanoma continues division in the presence of vemurafenib.

Abstract Patients with melanomas expressing B-RafV600E benefit from treatment with B-Raf-targeted therapeutics, such as vemurafenib. Initial responses to these agents are occasionally profound, but the duration of response is variable from patient to patient, with some tumors recurring within a few weeks and others requiring months before tumors progress. The source of this variability of response is under intense investigation. A number of acquired resistance mechanisms have been described, but it is not known whether they pre-exist or develop after treatment initiation. To address this question, we tracked the cellular response to PLX4720 (the vemurafenib research compound) over 96 hours in the A375 and SK-MEL-5 cell line models of B-RafV600E-expressing melanoma. Both cell lines exhibited a significant early reduction in cell number compared to control, but after 60 hours of treatment cells numbers began to increase even in the presence of 8 μM drug, albeit at a slow rate. This “rebound” proliferation effect was not due to drug decay since re-administration of the drug did not prevent it. We further examined the rebound effect at the single cell level using our newly described Fractional Proliferation assay (Tyson et al., Nat Methods, 2012, 9(9):923-928, doi:10.1038/nmeth.2138). The rebound corresponded to a dividing cell subpopulation whose size and rate of division depend on the concentration of drug. This subpopulation does not represent rare non-responding variants within the cell lines, but rather it arises stochastically as sibling cells do not always exhibit the same phenotypic response. Investigation of the molecular correlates of this dividing subpopulation, which may act as the reservoir from which the resistant cells arise, are ongoing. Citation Format: Darren R. Tyson, Chengwei Peng, Keisha N. Hardeman, Bishal Paudel, Vito Quaranta. A stochastically arising subpopulation of B-RafV600E-expressing melanoma continues division in the presence of vemurafenib. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3403. doi:10.1158/1538-7445.AM2013-3403

Super Resolution Imaging of Thick Samples by Means of Selective Plane Illumination Microscopy

Recently, super-resolution techniques based on single molecule localization became a popular tool to understand biological processes and image structures below the diffraction limit. In the last few years a fast development in the field allowed multicolor and 3D super-resolution imaging of biological samples. However, super-resolution techniques are currently well established at the cellular level but their application to more complicate samples, such as tissues and embryos, still remain a challenging task. within this context, recently, the coupling of localization based techniques and selective plane illumination microscopy [1], allowed to extend the application range to thicker tissues ( 50 μm), by coupling far-field individual molecule localization (IML) and selective plane illumination microscopy (SPIM) both in the linear and non linear regime. Particular attention is addressed to the advantages provided by two photon photo-activation in scattering environments.IML-SPIM allows to image cellular spheroids with sub-diffraction resolution in depth and can be applied to image cellular substructures in Zebrafish early developmental stages as well.(1) Cella Zanacchi F. et al., “Live-cell 3D super-resolution imaging in thick biological samples.,” Nat Methods, 8, no. 12, pp. 1047-1049, (2011)(2) Truong T. V. et al. “Deep and fast live imaging with two-photon scanned light-sheet microscopy.,” Nat Methods, 8, no. 9, pp. 757-760, (2011). Huisken, J., et al. Science 305, 1007-1009 (2004).

Two-Photon Excitation and Selective Plane Illumination Microscopy: A Combination to Minimize Scattering Effects While Imaging Thick Samples

Imaging thick samples in optical fluorescence microscopy still represents an open challenge because sample-induced aberrations can lead to a worsening of the imaging quality and a wrong interpretation of the data collected. Two-photon excitation microscopy is a suitable tool to enhance the penetration depth capabilities of a microscopy system but may still be affected by scattering effects, generating an out-of-focus fluorescence, which can result in a shift of the intensity excitation distribution (1). Selective Plane Illumination Microscopy represents an optimal tool to perform imaging of large samples, and recently it has been combined with two photon excitation (2) in order to improve the performances of such a system while imaging deep into a scattering sample. The aim of this work is to characterize how relevant scattering effects are in distorting the shape of a light sheet. A comparison between single and two-photon excitation light sheet has been performed measuring the excitation distribution profiles in fluorescent immobile phantom samples mimicking the optical properties of some biological tissues. Results show that two photon excitation is able to preserve the shape of the light sheet even in strong scattering samples, while single photon light sheet shows a strong shift in the intensity excitation distribution as penetration depth and scattering strength increase. To show the performances of the imaging of the system, a 3D reconstruction of a tumor spheroid, a suitable model for a thick scattering sample, is reported.1. Theer, P. Denk, W. “On the fundamental imaging-depth limit in two-photon microscopy”. J Opt Soc Am A, 23 (12), (2006).2. Truong, T. et al. “Deep and fast live imaging with two-photon scanned light-sheet microscopy.” Nat Methods, 8 (9) (2011)

STED Microscopy with Time-Gated Detection:Benefits and Limitations

In a stimulated emission depletion (STED) microscope the region in which fluorescence markers can emit spontaneously shrinks with continued STED beam action after a singular excitation event. This fact has been recently used [1] to substantially improve the effective spatial resolution in STED nanoscopy using pulsed excitation, continuous wave (CW) STED beams and by sorting photons depending by them arrival-times (time-gated detection). We present theoretical/experimental data that characterize the time evolution of the effective detection volume of a STED microscope and illustrate the physical basis, the benefits, and the limitations of this new STED implementation, namely gated CW-STED (gCW-STED). Among all the STED implementations, gCW-STED provides the highest effective resolution at low light intensity and is in essence limited (only) by the reduction of the signal that is associated with gating. Time-gated detection also strongly reduces the influence of local variations of the fluorescence lifetime on STED microscopy resolution. Finally, combination of gCW-STED with fluorescent correlation spectroscopy (FCS) is discussed: gCW-STED offers the unique property of tuning the effective detection volume by sorting photons in time. [1] Vicidomini G, Moneron G, Han KY, Westphal V, Ta H, et al. (2011) Sharper low-power STED nanoscopy by time gating. Nat Methods 8: 571-573.

Developing Photoactivatable Fluorescent Proteins for Diffraction-Limited and Superresolution Imaging

Photoactivatable fluorescent proteins (PAFPs) are molecules that switch to a new fluorescent state in response to specific light activation, and play vital roles in super-resolution imaging. There are three classes of PAFPs: dark-to-bright photoactivators (PAFPs), irreversible photoconverters (PCFPs), and reversible highlighters (RSFP). However, compared to traditional fluorescent proteins (such as GFP or RFP), only limited PAFPs are available for super-resolution microscopy.Previously, we developed several novel PAFPs, mGeos, with various switching rates, photon numbers and brightness1. And based on the crystal structure of green state mEos2, we evolved two truly monomeric and bright RSFPs, mEos3.1 and mEos3.2, with the good photochemical properties including rapid maturation rate, high photon budget and extremely high labeling density2. Here we present new generation of mGeos2 which are true monomeric PAFPs. These novel fluorescent proteins are suitable for both single color and dual color PALM superresolution imaging, and have a broad brand of applications in traditional fluorescence microscopy such as dynamic tracking and pulse chase labeling of proteins.1. Chang H, Zhang M, Ji W, Chen J, Zhang Y, Liu B, Lu J, Zhang J, Xu P, Xu T, A new series of reversibly switchable fluorescent proteins with beneficial properties for various applications, Proc Natl Acad Sci U S A. 2012 Mar 20;109(12):4455-60.2. Zhang M, Chang H, Zhang Y, Yu J, Wu L, Ji W, Chen J, Liu B, Lu J, Liu Y, Zhang J, Xu P, Xu T. Rational design of true monomeric and bright photoactivatable fluorescent proteins. Nat Methods. 2012 May 13;9(7):727-9.