A novel fluorescence imaging technique combining deconvolution microscopy and spectral analysis for quantitative detection of opportunistic pathogens

Puil, M. Le; Biggerstaff, John; Weidow, Brandy; Price, Jeffery R.; Naser, Saleh A.; White, David C.; Alberte, Randall S.

doi:10.1016/j.mimet.2006.05.019

Cited by 11 publications

(10 citation statements)

References 25 publications

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“…Major problems we encountered during quantitation from the small glass slide chip included variability in fluorescence signals, interference from nearby neighbors, and out-of-focus blur. 16,18,21 To overcome them, we applied 2-D restorative deconvolution (i.e., Lucy-Richardson algorithm 22 ) during postimage processing, which could reassign out-of-focus blur mathematically to its point of origin and therefore permit counting of the absolute number of cells on the same ROI.…”

Section: Discussionmentioning

confidence: 99%

High-Throughput Screening of Small Molecule Ligands Targeted to Live Bacteria Surface

et al. 2013

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The discovery of small molecule ligands targeted to the surface of live pathogenic bacteria would enable an entirely new class of antibiotics. We report the development and validation of a microarray-based high-throughput screening platform for bacteria that exploits 300 μm diameter chemical spots in a 1” × 3” nano-layered glass slide format. Using 24 model compounds and 4 different bacterial strains we optimized the screening technology, including fluorophore-based optical deconvolution for automated scoring of affinity and cyan-magenta-yellow-black (CMYK) color-coding for scoring of both affinity and specificity. The latter provides a lossless, one-dimensional view of multidimensional data. By linking in silico analysis with cell binding affinity and specificity, we could also begin to identify the physicochemical factors that affect ligand performance. The technology we describe could form the foundation for developing new classes of antibiotics.

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Section: Discussionmentioning

confidence: 99%

High-Throughput Screening of Small Molecule Ligands Targeted to Live Bacteria Surface

et al. 2013

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“…Although the problems associated with autofluorescence were less severe in our seawater sample, compared to those in coral tissues, the removal of spectra originating from sources other than fluorescent probes (e.g., fluorescent eukaryotic and bacterial cells) significantly facilitated the identification of target organisms. Second, the technology not only enabled the separation of probe-conferred and other interfering spectra, but also the separation of multiple different probe-conferred spectra assigned to target organisms Le Puil et al 2006).…”

Section: Improving Accuracy Of Target Detection In Fish Using Spectramentioning

confidence: 99%

Imaging Marine Bacteria with Unique 16S rRNA V6 Sequences by Fluorescence in situ Hybridization and Spectral Analysis

Hasegawa

Welch

Valm

et al. 2010

Geomicrobiology Journal

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Fluorescence in situ hybridization (FISH) combined with spectral analysis was performed to image specific bacteria from seawater using probes targeting the V6 hypervariable region of small subunit ribosomal RNA (SSU rRNA), corresponding to positions 984 to 1047 of E. coli 16S rRNA gene. For each target, we designed two probes, each with a distinct fluorescent reporter and a unique hybridization site. Spectral imaging analysis of bacteria incubated with a pair of specific probes and a general bacterial probe enabled the detection of three probe-conferred spectra in each target cell, increasing the reliability of target identification in a diverse bacterial community.

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“…The imaging platform allowed acquisition of specific spectral signatures for each pixel of the image and, therefore, allowed separation of the microsphere fluorescent signal from the inherent noise signal of the sample material. Based on previous works (Chang et al, 2003;Biggerstaff et al, 2006;Le Puil et al, 2006), a quantitation protocol was derived by way of AutoDeblur ® (AutoQuant Imaging Inc., Watervliet, NY). Fluorescence areas were quantified for each image to obtain the density of microspheres in each field of view.…”

Section: Microsphere Tracersmentioning

confidence: 99%

Challenges for Coring Deep Permafrost on Earth and Mars

et al. 2008

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A scientific drilling expedition to the High Lake region of Nunavut, Canada, was recently completed with the goals of collecting samples and delineating gradients in salinity, gas composition, pH, pe, and microbial abundance in a 400 m thick permafrost zone and accessing the underlying pristine subpermafrost brine. With a triple-barrel wireline tool and the use of stringent quality assurance and quality control (QA/QC) protocols, 200 m of frozen, Archean, mafic volcanic rock was collected from the lower boundary that separates the permafrost layer and subpermafrost saline water. Hot water was used to remove cuttings and prevent the drill rods from freezing in place. No cryopegs were detected during penetration through the permafrost. Coring stopped at the 535 m depth, and the drill water was bailed from the hole while saline water replaced it. Within 24 hours, the borehole iced closed at 125 m depth due to vapor condensation from atmospheric moisture and, initially, warm water leaking through the casing, which blocked further access. Preliminary data suggest that the recovered cores contain viable anaerobic microorganisms that are not contaminants even though isotopic analyses of the saline borehole water suggests that it is a residue of the drilling brine used to remove the ice from the upper, older portion of the borehole. Any proposed coring mission to Mars that seeks to access subpermafrost brine will not only require borehole stability but also a means by which to generate substantial heating along the borehole string to prevent closure of the borehole from condensation of water vapor generated by drilling.

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A novel fluorescence imaging technique combining deconvolution microscopy and spectral analysis for quantitative detection of opportunistic pathogens

Cited by 11 publications

References 25 publications

High-Throughput Screening of Small Molecule Ligands Targeted to Live Bacteria Surface

High-Throughput Screening of Small Molecule Ligands Targeted to Live Bacteria Surface

Imaging Marine Bacteria with Unique 16S rRNA V6 Sequences by Fluorescence in situ Hybridization and Spectral Analysis

Challenges for Coring Deep Permafrost on Earth and Mars

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