Multicolor Probes Research Articles

Screening results and mutation frequency analysis of G6PD deficiency in 1,291,274 newborns in Huizhou, China: a twenty-year experience.

This study aimed to investigate the incidence rate and spectrum of gene mutations of Glucose-6-phosphate dehydrogenase (G6PD) deficiency in the Huizhou city of southern China to provide a scientific basis for disease prevention and control in the area. From March 2003 to December 2022, newborn screening for G6PD enzyme activity was carried out in Huizhou city using the fluorescence quantitative method. Infants who tested positive during the initial screening were diagnosed using the nitroblue tetrazolium ratio method, while a subset of infants received further gene mutation analysis using the multicolor probe melting curve analysis method. A total of 1,291,274 newborns were screened and the screening rate has increased from 20.39% to almost 100%. In the 20-year period, 57,217 (4.43%) infants testing positive during the initial screening. Out of these infants, 49,779 (87%) were recalled for confirmatory testing. G6PD deficiency was confirmed in 39,261 of the recalled infants, indicating a positive predictive value of 78.87%. The estimated incidence rate of G6PD deficiency in the region was 3.49%, which was significantly higher than the average incidence rate of 2.1% in southern China. On the other hand, seven pathogenic G6PD variants were identified in the analysis of the 99 diagnosed infants with the most common being c.1388 G > A (48.5%), followed by c.95 A > G (19.2%), c.1376 G > T (15.2%), c.871 G > A (9.1%), c.1360 C > T (3.0%), c.392 G > T (3.0%), and c.487 G > A (1.0%). The incidence of G6PD deficiency in newborns in the Huizhou city was higher than the southern China average level, while the types and frequencies of gene mutations were found to vary slightly from other regions. Our findings suggested that free government screening and nearby diagnosis strategies could reduce the incidence of G6PD deficiency in the area.

Water-soluble iridium(III) complexes as multicolor probes for one-photon, two-photon and fluorescence lifetime imaging

Fluorescence microscopy imaging provides an indispensable way to visualize morphological details in tissue with subcellular resolution. Of the various imaging agents currently available, heavy-metal complex based probes offer a powerful tool for live cell imaging, primarily due to their excellent photophysical properties, including evident Stokes shifts, high photoluminescence efficiency and relatively long emission lifetime of phosphorescent signals. Herein, we have designed and synthesized seven water soluble cationic iridium(III) solvato complexes (1 − 7) for live cell imaging. The emission colors of these iridium(III) complexes can be tuned from green to red in PBS buffer (phosphate buffered saline). In particular, multicolor phosphorescent imaging for cytoplasm staining of living cells has been realized. More importantly, two-photon excited imaging and fluorescence lifetime imaging microscopy have also successfully been applied for complex 7.

First Comprehensive Characterization of Phayre's Leaf-Monkey (Trachypithecus phayrei) Karyotype.

The chromosomal homologies of human (Homo sapiens—HSA) and Trachypithecus phayrei (TPH—Phayre’s leaf-monkey, family Cercopithecidae) have previously been studied by using classical chromosome staining/banding and fluorescence in situ hybridization (FISH) from the 1970s to 1990s. In this study, we carried out molecular cytogenetics applying human multicolor banding (MCB), locus-specific, and human heterochromatin-specific probes to establish the first detailed chromosomal map of TPH, which was not available until now. Accordingly, it was possible to precisely determine evolutionary-conserved breakpoints (ECBs) and the orientation of evolutionary-conserved segments compared to HSA. It could be shown that five chromosomes remained completely unchanged between these two species, and 16 chromosomes underwent only intrachromosomal changes. In addition, 50 ECBs that failed to be resolved in previous reports were exactly identified and characterized in this study. It could also be shown that 43.5% of TPH centromere positions were conserved and 56.5% were altered compared to HSA. Interestingly, 82% ECBs in TPH corresponded to human fragile sites. Overall, this study is an essential contribution to future studies and reviews on chromosomal evolution in Cercopithecidae.

A biomarker (ONOO-)-activated multicolor fluorescent probe for early detection and assessment of arthritis

To understand the entangled relationship between reactive oxygen species (ROS) and arthritis, it is of great interest to urgently need an effective bioanalytical method to detect biomarker peroxynitrite (ONOO-) of arthritis in vivo. In contrast to the previously reported ONOO- fluorescent probes, multicolor fluorescent probe could be applied to complex environments for multi-component analysis and provide multichannel imaging results. However, multicolor fluorescent probes for ONOO- detecting have not been reported so far. Herein, an ONOO--activated multicolor fluorescent probe (MULTI-ONOO) based on FRET was developed for quantitative detecting and visualization of ONOO- levels in arthritis. This probe mainly contained an acceptor (near-infrared xanthane fluorophore) and a donor (naphthalimide fluorophore). In the presence of ONOO-, the oxonium ion undergoes a strong nucleophilic response between ONOO- and xanthane to generate coumarin (CM) and naphthimide (NP) fluorophores with multicolor signal output. Impressively, MULTI-ONOO has a rapid response time (~20 s) and outstanding detection limits as low as 11.6 nM. Finally, this probe was successfully adopted to detect endogenous ONOO- in living cells, arthritis tissues and rat model. Thus, this ONOO--activated multicolor probe could provide a robust tool for investigating the role of ONOO- between in different inflamed diseases.

Analyte-restrained silver coating of gold nanostructures: an efficient strategy to advance multicolorimetric probes

Visual detection based on gold nanorods (AuNRs) has gained tremendous attention in sensing applications owing to the potential for simple, inexpensive, instrument-free, and on-site detection. The proper selection of the mechanism involved in the interaction between the analyte and the nanostructure plays a significant role in designing a selective and multicolorimetric probe for visual purposes. A winning mechanism to develop multicolorimetric probes is the silver metalization of AuNRs. Herein, an unprecedented idea is presented to expand the variety of multicolorimetric sensors relying on the mechanism of silver deposition. We introduce the anti-silver deposition mechanism in which the analyte directly or indirectly restrains the silver coating of AuNRs. To ascertain the anti-silver deposition mechanism, we have exploited the proposed idea for the direct detection of nitrate. The presence of nitrate (as restrainer agent), which was firstly treated with ascorbic acid (as reducing agent), induced a decrease in the spectral blueshift of AuNRs along with diverse sharp color transitions from reddish-orange (blank) to maroon, wine, berry/purple, dark blue, teal, green, seafoam, and mint. The difference in the spectrum area of the probe in the absent (S o) and presence (S) of nitrate were linearly proportional to nitrate concentration in the range of 0.5–5.5 mmol l−1 and the limit of detection was calculated to be 465 μmol l−1. Furthermore, the practicability of the multicolor probe was assessed by the determination of nitrate in complex environmental samples.

Neuron Activity Dependent Redox Compartmentation Revealed with a Second Generation Red-Shifted Ratiometric Sensor.

Oxidative stress is a hallmark of several aging and trauma related neurological disorders, but the precise details of how altered neuronal activity elicits subcellular redox changes have remained difficult to resolve. Current redox sensitive dyes and fluorescent proteins can quantify spatially distinct changes in reactive oxygen species levels, but multicolor probes are needed to accurately analyze compartment-specific redox dynamics in single cells that can be masked by population averaging. We previously engineered genetically encoded red-shifted redox-sensitive fluorescent protein sensors using a Förster resonance energy transfer relay strategy. Here, we developed a second-generation excitation ratiometric sensor called rogRFP2 with improved red emission for quantitative live-cell imaging. Using this sensor to measure activity-dependent redox changes in individual cultured neurons, we observed an anticorrelation in which mitochondrial oxidation was accompanied by a concurrent reduction in the cytosol. This behavior was dependent on the activity of Complex I of the mitochondrial electron transport chain and could be modulated by the presence of cocultured astrocytes. We also demonstrated that the red fluorescent rogRFP2 facilitates ratiometric one- and two-photon redox imaging in rat brain slices and Drosophila retinas. Overall, the proof-of-concept studies reported here demonstrate that this new rogRFP2 redox sensor can be a powerful tool for understanding redox biology both in vitro and in vivo across model organisms.

Multifaceted applications of pre-mature chromosome condensation in radiation biodosimetry

Background Biodosimetry with persistent cytogenetic indicators in peripheral blood lymphocytes (PBLs) plays crucial role in regulatory/medical management of individuals overexposed to radiation. Conventional methods require ∼48 h culture and have limited dose range (0.1–5Gy) applications due to checkpoint arrest/poor stimulation. G0-Phase Premature chromosome condensation (G0-PCC) allows chromosome aberration analysis within hours after blood collection. Due to high skill demand, applications of G0-PCC were not very well explored and being re-visited worldwide. Among all aberrations, analysis of excess chromosomal fragments is quickest. Radiation dose response curve for the fragments has been reported. Purpose In present study, excess fragment analysis has been addressed in detail, in addition to validation of radiation dose response curve, gender variation in the response, dose dependent repair kinetics, minimum detection limit (MDL), duration and accuracy of final dose estimation with 5blindfolded, ex vivo irradiated samples have been studied. In extension, feasibility of multiparametric dosimetry with Fluorescent in situ hybridization (FISH) based endpoints were qualitatively explored. Material and methods PBLs were exposed to Gamma-Radiation and G0-PCC was performed at different time points. Decay kinetics and dose response curve were established. Gender Variation of the frequency of the fragments was assessed at 0, 2 and 4 Gy. FISH was performed with G0-PCC applying centromere probe, whole chromosome paints, multi-color FISH and multi-color banding probes. Results Radiation response curve for fragments was found to be linear (Slope 1.09 ± 0.031 Gy-1). Background frequency as well as dose response did not show significant gender bias. Based on variation in background frequency of fragments MDL was calculated to be ∼0.3 Gy. Kinetics of fragment tested at 0, 4, 8, 16 and 24 h showed exponential decay pattern from 0 to 8 h and without further decay. Final dose estimation of five samples was completed within 13 man-hours. Dicentric chromosomes, translocations, insertions and breaks were identifiable in combination with centromere FISH and WCP. Advanced methods employing multicolor FISH and multi-color banding were also demonstrated with PCC spreads. Conclusion G0-PCC, can be useful tool for high dose biodosimetry with quick assessment of fragment frequency. Further, it holds potential for multi-parametric dosimetry in combination with FISH.

Qualitative Real-Time PCR Method for Poisonous Entoloma rhodopolium-Related Species in Japan: Real-Time PCR Method for Entoloma Mushrooms

Qualitative real-time PCR method for three poisonous Entoloma rhodopolium-related species in Japan was established using specific primers and FAM, VIC, Texas Red, Cy5-labeled probes. The use of multicolor probes can extend the method to simultaneous detection of different targets. Standard plasmids were constructed as reference materials. Designed primers and probes in the method detect only a target species, and the detection limit was 12.5 copies or below. This indicates it is highly specific and sensitive enough to detect the poisonous mushrooms in food residues. Next, we applied the method to four food residue samples obtained from food poisoning cases. The real-time PCR method did identify all of four samples as E. subrhodopolium and E. pseudorhodopolium, whereas PCR-RFLP did not. The method established here revealed Entoloma rhodopolium-related species in Hokkaido were different species such as E. eminens and unknown species.

Spore-derived color-tunable multi-doped carbon nanodots as sensitive nanosensors and intracellular imaging agents

Fluorescent carbon dots hold great promise in biomedical real-time tracking due to their outstanding optical properties and biocompatibility. Here, we report a one-pot synthetic strategy to produce multi-doped carbon dots (MCDs) with bright and color-tunable emission. The resultant MCDs are doped by abundant biogenic elements (O, N, P), so that they contain a variety of surface functional groups. Such hetero-doping and functionalization make MCDs display strong fluorescent emission and excitation-wavelength-dependent characteristic, good aqueous solubility as well as high photo-stability and ion-stability. Moreover, the as-prepared MCDs response to the change of pH values and exhibit high selective and sensitive detection of Fe3+ ions under blue light illumination with the limit of detection (LOD) of 15.9 nM. Such a Fe3+ sensing ability is further demonstrated in real water samples and extended to intracellular multi-color monitoring of Fe3+. More interestingly, the MCDs can also sense the gastrointestinal bacteria Escherichia coli, exhibiting linear sensing dependent behavior. With low cytotoxicity and tunable emission, such MCDs hold great potential as efficient multi-color probes and imaging agents for clinical diagnosis.

Full color emitting fluorescent carbon material as reversible pH sensor with multicolor live cell imaging

Carbon-based nano materials are developed as a cytocompatible alternative to semiconducting quantum dots for bioimaging and fluorescence-based sensing. The green alternatives for the synthesis of carbon materials are imminent. The present study demonstrates microwave based one step quick synthesis of fluorescent carbon material (FCM) having three variants: (i) un-doped fluorescent carbon material (UFCM) (ii) nitrogen doped FCM (N@FCM), and (iii) nitrogen & phosphorus co-doped FCM (N-P@FCM) using sugarcane extract as a carbon source. The N doping was performed using ethylenediamine and phosphoric acid was used for P doping. The heteroatom doped FCM were synthesized due to insolubility of UFCM in water. Unlike, UFCM, the N@FCM and N-P@FCM were found to be highly soluble in water. The N-P@FCM shows highest quantum yield among the three. The N-P@FCM was explored for alkaline pH sensing and it shows a quenching of fluorescence in the pH range 09–14. The sensing behaviour shows reversibility and high selectivity. Further, the sensor was also investigated for their biocompatibility and hence employed as a promising multicolour probe for cancer cell imaging. The generality in cell imaging was investigated by flow cytometry. The hetero-atom doped green carbon-dots may open new avenues for sensing and selective cellular targeting.

Abstract 756: Molecular characterization of triploid and tetraploid urine FISH samples

Abstract Urothelial carcinomas (UC) are characterized, among other aberrations, by chromosome 3, 7, 17 gains and 9p21 (p16) loss. Since UC cells readily exfoliate into the urine, molecular cytogenetics techniques have been used to detect cells consistent with UC diagnosis and follow-up: a multitarget Fluorescence In Situ Hybridization (FISH) test, composed of 3, 7 and 17 centromeres and 9p21 locus multicolor probes, has been proved useful for atypical cells identification in both void urine and lower and upper urinary tract samples before morphological changes are apparent through cytological and histological examination. To date, there are no uniform criteria for urine FISH scoring though the test is commonly considered positive following the UroVysion Bladder Cancer Kit criteria (Abbott Molecular): 4 or more cells with a gain of 2 or more chromosomes (polysomy), and/or 12 or more cells with 9p21 homozygous deletion. This definition of polysomy includes trisomy and tetrasomy classes, but, as tetraploidy may represent cells at S/G2 phase, this criterion may lead to false-positives; moreover it has been described that also inflammation induces tetraploidy, and that small percentages of normal urine cells show trisomy/tetrasomy. On the other hand, triploidy and tetraploidy may have a direct role in tumorigenesis, as an intermediate state in the development of cancer aneuploidy. At the moment little is reported about the clinical correlations of urine FISH cases showing only trisomy/tetrasomy and more data are needed to clarify the diagnostic value of triploid and tetraploid urothelial cells. Aiming to better classify patients with a trisomy/tetrasomy urine FISH result, we extracted genomic DNA from 50 fixed urine cell suspensions, previously tested by urine FISH, and investigated them for the presence of fibroblast growth factor receptor 3 (FGFR3) and telomerase reverse transcriptase (TERT) promoter mutations, which constitute the most recurrent somatic alterations in BC. In particular frequent mutations in exons 7, 10 and 15 of FGFR3 characterize non-muscle invasive UC while mutations in the core promoter region of TERT gene, mainly at -124 and -146 positions from the ATG start site, have emerged as the most frequent somatic genetic alteration in both non-muscle invasive and muscle invasive UC. All specimens were subjected to PCR amplification and Sanger sequencing for FGFR3 exon 7 and 10, and for the C228T and C250T TERT promoter mutations. A preliminary analysis of 20 urine FISH cases showed 4 samples with trisomy/tetrasomy, and 1 of these 4 had a mutation of the TERT promotor. Complete results of the FGFR3 and TERT promoter molecular analysis will be presented and correlated to both FISH results and patients clinical data, in order to evaluate if the presence of FGFR3 and/or TERT promoter mutations in urine samples with FISH detected trisomy/tetrasomy may help to identify patients with a higher risk of recurrence and progression. Citation Format: Lorenza Pecciarini, Valeria De Pascali, Ilaria Francaviglia, Anna Talarico, Chiara Iacona, Massimo Freschi, Maria Giulia Cangi, Claudio Doglioni. Molecular characterization of triploid and tetraploid urine FISH samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 756. doi:10.1158/1538-7445.AM2017-756

Highly fluorescent N,S-co-doped carbon dots: synthesis and multiple applications

N and S co-doped carbon dots (N,S-CDs) synthesized from biomass can be used as multicolor probes for cell imaging. The N,S-CDs can selectively and sensitively detect Cr(vi) in real lake water, human serum and living cells.

Photoswitchable non-fluorescent thermochromic dye-nanoparticle hybrid probes.

Photoswitchable fluorescent proteins with controllable light–dark states and spectral shifts in emission in response to light have led to breakthroughs in the study of cell biology. Nevertheless, conventional photoswitching is not applicable for weakly fluorescent proteins and requires UV light with low depth penetration in bio-tissue. Here we introduce a novel concept of photoswitchable hybrid probes consisting of thermochromic dye and absorbing nanoparticles, in which temperature-sensitive light–dark states and spectral shifts in absorption can be switched through controllable photothermal heating of doped nanoparticles. The proof-of-concept is demonstrated through the use of two different types of temperature-sensitive dyes doped with magnetic nanoparticles and reversibly photoswitched by a near-infrared laser. Photoacoustic imaging revealed the high contrast of these probes, which is sufficient for their visualization in cells and deep tissue. Our results suggest that these new photoswitchable multicolour probes can be used for multimodal cellular diagnostics and potentially for magnetic and photothermal therapy.

The application of multicolor probe melting curve analysis for the prenatal diagnosis of β thalassemia

Objective To establish a method of multicolor melting curve analysis for the prenatal diagnosis of β thalassemia. Methods Methodology establishment. A total of 95 cases, including 9 fetal villi samples(10-13 weeks)and 86 amniotic fluid samples(18-24 weeks)were collected by Center for Prenatal Diagnosis of Shenzhen Maternity and Child Healthcare Hospital between January 2014 and December 2014. A double-blind test was done to detect the mutations of beta globin gene by means of reverse dot (RDB) blot and multicolor melting curve analysis(MMCA). The consistency of the two methods is compared. Results The results of 93 cases detected by MMCA and RDB are completely consistent. The results of the 2 cases detected by MMCA after correction are the same as the results detected by RDB. Finally, the coincidence rate of the result was 100%. Conclusion MMCA can be applied to the prenatal diagnosis of β thalassemia as an effective supplement to RDB.(Chin J Lab Med, 2016, 39: 192-196) Key words: beta-Thalassemia; Prenatal diagnosis; Reverse transcriptase polymerase chain reaction; Nucleic Acid Hybridization

Nanocluster Beacons for Detection of a Single N6-Methyladenine Epigenetic Modification

NanoCluster Beacons (NCBs) are a new type of activatable molecular probes that are low cost, easy to prepare and have high fluorescence enhancement ratios. NCBs employ DNA-templated, few-atom silver nanoclusters (DNA/Ag NCs, with about 2∼20 silver atoms per cluster) as reporters which can significantly “light up” through interactions with a nearby DNA sequence (called an enhancer). Taking advantage of this fluorescence tunability by altering the surrounding ligands, a property that is not commonly seen among existing reporters, NCB soon evolved to a multicolor probe, termed chameleon NanoCluster Beacon (cNCB), for single-nucleotide polymorphism (SNP) detection. Here we bring the NCB detection to the next level by designing a new NCB specifically for N6-methyladenine (hereafter denoted as m6A) detection. m6A is a methylation modification abundant in prokaryotic genomes, and also found in lower eukaryotes and higher plants. So far detection of m6A relies on methods such as TLC, HPLC, MS, and enzymatic reactions, which are often laborious, expensive, with low specificity and varying reactivity. Whereas high-resolution melting analysis is able to detect a single m6A modification within a target DNA via the destabilizing effect of m6A, HRM cannot pinpoint the location of m6A in the sequence. A simple and cost-effective way to identify single m6A at any specific sites is therefore highly desired. Here, we developed a robust, simple, enzyme-free and hybridization-based method for m6A detection with “pinpoint specificity”, using a new type of silver cluster-based DNA probe which we term methyladenine-specific NanoCluster Beacon (maNCB). To date, there is no hybridization technique that has the potential to reach these remarkable results. Y.-A. Chen, J.M. Obliosca, Y.-L. Liu, C. Liu, M.L. Gwozdz, and H.-C. Yeh, “NanoCluster Beacons enable detection of a single N6-methyladenine,” JACS, http://dx.doi.org/10.1021/jacs.5b06038, 2015.

Karyotyping human and mouse cells using probes from single-sorted chromosomes and open source software.

Multispectral karyotyping analyzes all chromosomes in a single cell by labeling them with chromosome-specific probes conjugated to unique combinations of fluorophores. Currently available multispectral karyotyping systems require the purchase of specialized equipment and reagents. However, conventional laser scanning confocal microscopes that are capable of separating multiple overlapping emission spectra through spectral imaging and linear unmixing can be utilized for classifying chromosomes painted with multicolor probes. Here, we generated multicolor chromosome paints from single-sorted human and mouse chromosomes and developed the Karyotype Identification via Spectral Separation (KISS) analysis package, a set of freely available open source ImageJ tools for spectral unmixing and karyotyping. Chromosome spreads painted with our multispectral probe sets can be imaged on widely available spectral laser scanning confocal microscopes and analyzed using our ImageJ tools. Together, our probes and software enable academic labs with access to a laser-scanning spectral microscope to perform multicolor karyotyping in a cost-effective manner.

Abstract 4833: Single-cell genetic analysis reveals insights into clonal development of cervical cancer and confirms TERC as an early and dominant aberration

Abstract In human cervical carcinogenesis, the gain of the long arm of chromosome 3 is the decisive genetic event that determines progression of dysplasia to invasive disease. Other recurrent genomic imbalances, such as gains on 1q, 5p, 8q, and 20q, and losses on 2p, 3p, and 11q are also observed. To better understand the patterns of clonal evolution during disease progression we developed a multiplex FISH assay with three multicolor probe panels for sequential hybridizations of cervical intraepithelial neoplasia (CIN3) and invasive cervical carcinoma (ICC), including synchronous CIN3/ICC lesions. Our FISH assay targets oncogenes COX2 (1q), TERC (3q), TERT (5p), MYC (8q), ZNF217 (20q), and tumor suppressor genes ING5 (2p), FHIT (3p), CHEK1 (11q), and centromere 7 as a ploidy control. Our results confirmed the dominant role of the gain of 3q: nineteen out of twenty lesions showed a major clonal population with TERC amplification. Nine out of the 20 lesions showed a concomitant loss of FHIT (3p) suggesting an isochromosome 3q formation, which makes the loss of FHIT the second most frequent clonal event in this group of samples. Other imbalances were present, but, as expected, less common. The copy number increases of 3q can occur either in diploid cells or after tetraploidization of the genome. Interestingly, in our samples only three CIN3 lesions showed major clones with signal patterns consistent with near-tetraploidy, while all other lesions, including all ICC lesions and the two synchronous CIN3/ICC lesions showed major signal patterns consistent with near-diploid cell populations. Although tetraploidization can occur already in premalignant lesions, and there is independent evidence that such chromosome doublings increase the risk of chromosome segregation errors, and therefore promote the subsequent acquisition of genomic imbalances, our data indicate that a majority of cervical precancerous and cancerous lesions acquire aberrations in a diploid genome. Independent of whether the 3q gain and other aberrations are acquired in a diploid or tetraploid background, it selects cells with a proliferative advantage, which is supported by our observations that (i) cells with identical patterns of 3q gain are adjacent to each other, hence indicating a clonal proliferation, and (ii) that the only commonality between the major clones in the synchronous CIN3 and ICC lesions was a TERC gain. We further examined overall cell-to-cell heterogeneity in CIN3 versus ICC samples. While overall levels of clonal heterogeneity were indistinguishable between CIN3 and ICC, phylogenetic models of single tumors suggested greater average numbers of mutations per cell in ICC versus CIN3. These findings improve our understanding of clonal development during cervical carcinogenesis and with that our understanding of genome dynamics at early stages of carcinogenesis. Citation Format: Leanora S. Hernandez, Amanda Bradley, Timo Gaiser, Sonia Andersson, E. Michael Gertz, Salim A. Chowdhury, Russel Schwartz, Alejandro Schäffer, Kerstin Heselmeyer-Haddad, Thomas Ried. Single-cell genetic analysis reveals insights into clonal development of cervical cancer and confirms TERC as an early and dominant aberration. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4833. doi:10.1158/1538-7445.AM2015-4833

Multicolour correlative imaging using phosphor probes

Correlative light and electron microscopy exploits the advantages of optical methods, such as multicolour probes and their use in hydrated live biological samples, to locate functional units, which are then correlated with structural details that can be revealed by the superior resolution of electron microscopes. One difficulty is locating the area imaged by the electron beam in the much larger optical field of view. Multifunctional probes that can be imaged in both modalities and thus register the two images are required. Phosphor materials give cathodoluminescence (CL) optical emissions under electron excitation. Lanthanum phosphate containing thulium or terbium or europium emits narrow bands in the blue, green and red regions of the CL spectrum; they may be synthesised with very uniform-sized crystals in the 10- to 50-nm range. Such crystals can be imaged by CL in the electron microscope, at resolutions limited by the particle size, and with colour discrimination to identify different probes. These materials also give emissions in the optical microscope, by multiphoton excitation. They have been deposited on the surface of glioblastoma cells and imaged by CL. Gadolinium oxysulphide doped with terbium emits green photons by either ultraviolet or electron excitation. Sixty-nanometre crystals of this phosphor have been imaged in the atmospheric scanning electron microscope (JEOL ClairScope). This probe and microscope combination allow correlative imaging in hydrated samples. Phosphor probes should prove to be very useful in correlative light and electron microscopy, as fiducial markers to assist in image registration, and in high/super-resolution imaging studies.

Mo1368 A Targeted Multicolor FISH Probe Set Increases the Diagnostic Sensitivity for Detecting Malignancy in Pancreatobiliary Brushings

Mo1368 A Targeted Multicolor FISH Probe Set Increases the Diagnostic Sensitivity for Detecting Malignancy in Pancreatobiliary Brushings

A multi-functional fluorescent scaffold as a multi-colour probe: design and application in targeted cell imaging

A new scaffold material based on a novel targeting strategy has been developed, benefiting from recent progress in the development of tailor-made fluorescent bioprobes for cellular imaging and opening a new viewpoint for further improvements in in vitro and in vivo imaging.