FISHji: new ImageJ macros for the quantification of fluorescence in epifluorescence images

Fluorescence in situ hybridization (FISH) is based on the use of fluorescent staining dyes, however, the signal intensity of the images obtained by microscopy is seldom quantified with accuracy by the researcher. The development of innovative digital image processing programs and tools has been trying to overcome this problem, however, the determination of fluorescent intensity in microscopy images still has issues due to the lack of precision in the results and the complexity of existing software. This work presents FISHji, a set of new ImageJ methods for automated quantification of fluorescence in images obtained by epifluorescence microscopy. To validate the methods, results obtained by FISHji were compared with results obtained by flow cytometry. The mean correlation between FISHji and flow cytometry was high and significant, showing that the imaging methods are able to accurately assess the signal intensity of fluorescence images. FISHji are available for non-commercial use at http://paginas.fe.up.pt/nazevedo/.This work was financially supported by: Project UID/EQU/00511/2013-LEPABE, by the FCT/MEC with national funds and when applicable co-funded by FEDER in the scope of the P2020 Partnership Agreement; Project NORTE-07-0124-FEDER-000025—RL2_Environment&Health, by FEDER funds through Programa Operacional Factores de Competitividade—COMPETE, by the Programa Operacional do Norte (ON2) program and by national funds through FCT—Fundação para a Ciência e a Tecnologia; DNA mimics Research Project PIC/IC/82815/2007, PhD Fellowship SFRH/BD/72999/2010 and Post-Doctoral fellowship SFRH/BPD/78846/2011

Detection of Dehalococcoides spp. by peptide nucleic acid fluorescent in situ hybridization

Chlorinated solvents including tetrachloroethene (perchloroethene and trichloroethene), are widely used industrial solvents. Improper use and disposal of these chemicals has led to a widespread contamination. Anaerobic treatment technologies that utilize Dehalococcoides spp. can be an effective tool to remediate these contaminated sites. Therefore, the aim of this study was to develop, optimize and validate peptide nucleic acid (PNA) probes for the detection of Dehalococcoides spp. in both pure and mixed cultures. PNA probes were designed by adapting previously published DNA probes targeting the region of the point mutations described for discriminating between the Dehalococcoides spp. strain CBDB1 and strain 195 lineages. Different fixation, hybridization and washing procedures were tested. The results indicated that the PNA probes hybridized specifically and with a high sensitivity to their corresponding lineages, and that the PNA probes developed during this work can be used in a duplex assay to distinguish between strain CBDB1 and strain 195 lineages, even in complex mixed cultures. This work demonstrates the effectiveness of using PNA fluorescence in situ hybridization to distinguish between two metabolically and genetically distinct Dehalococcoides strains, and they can have strong implications in the monitoring and differentiation of Dehalococcoides populations in laboratory cultures and at contaminated sites.The authors gratefully acknowledge the University of Porto-Mobile Program and the Portuguese Science and Technology Foundation (FCT) for the research project (DNA mimics - reference PIC/IC/82815/2007), a PhD grant (SFRH/BD/44876/2011) and two research positions (Ciencia 2008 Program). The authors wish to thank Lorenz Adrian and Ivonne Nijenhuis (Helmholtz Center for Environmental Research) and Steven Zinder (Cornell University) for providing Dehalococcoides strains 195 and CBDB1. In addition, the assistance of Julian Renpenning (Helmholtz Center for Environmental Research) and Heather Fullerton (Cornell University) is greatly appreciated

Supplementary Material for: Detection of <b><i>Dehalococcoides</i></b> spp. by Peptide Nucleic Acid Fluorescent in situ Hybridization

Chlorinated solvents including tetrachloroethene (perchloroethene and trichloroethene), are widely used industrial solvents. Improper use and disposal of these chemicals has led to a widespread contamination. Anaerobic treatment technologies that utilize <i>Dehalococcoides</i> spp. can be an effective tool to remediate these contaminated sites. Therefore, the aim of this study was to develop, optimize and validate peptide nucleic acid (PNA) probes for the detection of <i>Dehalococcoides</i> spp. in both pure and mixed cultures. PNA probes were designed by adapting previously published DNA probes targeting the region of the point mutations described for discriminating between the <i>Dehalococcoides</i> spp. strain CBDB1 and strain 195 lineages. Different fixation, hybridization and washing procedures were tested. The results indicated that the PNA probes hybridized specifically and with a high sensitivity to their corresponding lineages, and that the PNA probes developed during this work can be used in a duplex assay to distinguish between strain CBDB1 and strain 195 lineages, even in complex mixed cultures. This work demonstrates the effectiveness of using PNA fluorescence in situ hybridization to distinguish between two metabolically and genetically distinct <i>Dehalococcoides</i> strains, and they can have strong implications in the monitoring and differentiation of <i>Dehalococcoides</i> populations in laboratory cultures and at contaminated sites

Detection and discrimination of biofilm populations using locked nucleic acid/2'-O-methyl-RNA fluorescence in situ hybridization (LNA/2'OMe-FISH)

Multispecies biofilms are the dominant form of biofilms found in Nature. The application of fluorescence in situ hybridization (FISH)-based techniques to the discrimination of biofilm populations might contribute to the understanding of microorganism interactions in these structures, and might allow the development of efficient strategies to prevent or minimize biofilm-associated diseases. This work presents the first study that develops, optimizes and validates a multiplex FISH procedure using locked nucleic acid (LNA) and 2-O-methyl RNA (2OMe) oligonucleotides probes for the in vitro discrimination within mixed populations. As a case study, Escherichia coli, the major cause of urinary tract infections (UTIs), and three other atypical colonizers of urinary catheters (Delftia tsuruhatensis, Achromobacter xylosoxidans and Burkholderia fungorum) with unproven pathogenic potential, were selected. Specific probes for these species were designed and optimized for specific hybridization in multiplex experiments. Results showed that the LNA/2OMe-FISH method performed well in multiplex experiments and presented a good correlation with total and cultivability counts, regardless of the cells physiological state. In fact, the method was also able to report variations of viable but non-cultivable populations. Further analysis of mixed biofilm structures by confocal laser scanning microscopy provided a clear discrimination in three dimensions between the location of the different populations.This work was funded by FEDER funds through the Operational Programme for Competitiveness Factors - COMPETE, ON.2 - O Novo Norte -North Portugal Regional Operational Programme and National Funds through FCT - Foundation for Science and Technology under the projects: PEst-C/EQB/UI0511, NORTE-07-0124-FEDER-000025 - RL2_ Environment & Health and Project "DNA mimics" PIC/IC/82815/2007; PhD Fellowship [SFRH/BD/82663/2011]; and Postdoctoral Fellowship [SFRH/BPD/74480/2010]. The authors would like to thank to M. Fenice and A. Steinbuchel for kindly providing the D. tsuruhatensis BM90 and A. xylosoxidans B3 strains, respectively

Controversies in using urine samples for prostate cancer detection: PSA and PCA3 expression analysis

PURPOSE: Prostate cancer (PCa) is one of the most commonly diagnosed malignancies in the world. Although PSA utilization as a serum marker has improved prostate cancer detection it still presents some limitations, mainly regarding its specificity. The expression of this marker, along with the detection of PCA3 mRNA in urine samples, has been suggested as a new approach for PCa detection. The goal of this work was to evaluate the efficacy of the urinary detection of PCA3 mRNA and PSA mRNA without performing the somewhat embarrassing prostate massage. It was also intended to optimize and implement a methodological protocol for this kind of sampling. MATERIALS AND METHODS: Urine samples from 57 patients with suspected prostate disease were collected, without undergoing prostate massage. Increased serum PSA levels were confirmed by medical records review. RNA was extracted by different methods and a preamplification step was included in order to improve gene detection by Real-Time PCR. RESULTS: An increase in RNA concentration with the use of TriPure Isolation Reagent. Despite this optimization, only 15.8% of the cases showed expression of PSA mRNA and only 3.8% of prostate cancer patients presented detectable levels of PCA3 mRNA. The use of a preamplification step revealed no improvement in the results obtained. CONCLUSION: This work confirms that prostate massage is important before urine collection for gene expression analysis. Since PSA and PCA3 are prostate specific, it is necessary to promote the passage of cells from prostate to urinary tract, in order to detect these genetic markers in urine samples