956 research outputs found
LIVE-PAINT Supplementary Videos: Compressed versions
No full textCompressed versions of the files of Oi, Curran; Horrocks, Mathew; Regan, Lynne. (2020). LIVE-PAINT Supplementary Videos, [dataset]. University of Edinburgh. Quantitative Biology, Biochemistry and Biotechnology. https://doi.org/10.7488/ds/2801.
## File listing ##
* "SupplementaryMovie1_-_mKO_blinking_compressed.mp4" LIVE-PAINT blinking behavior shown in S. cerevisiae using the fluorescent protein mKO and the reversible interaction pair SYNZIP17-SYNZIP18. Scale bar is 1 micron.
* "SupplementaryMovie2_-_mOrange_blinking_compressed.mp4" LIVE-PAINT blinking behavior shown in S. cerevisiae using the fluorescent protein mOrange and the reversible interaction pair SYNZIP17-SYNZIP18. Scale bar is 1 micron.
* "SupplementaryMovie3_-_cofilin_tracking_compressed.mp4" Video tracking cofilin in S. cerevisiae, where cofilin is C-terminally tagged with SYNZIP18 and labeled with separately expressed SYNZIP17-mNeonGreen. Scale bar is 5 microns.
* "SupplementaryMovie4_-_cofilin_tracking_compressed.mp4" Tracking the diffusion of a single cofilin "spot" in S. cerevisiae, where cofilin is C-terminally tagged with SYNZIP18 and labeled with separately expressed SYNZIP17-mNeonGreen. Scale bar is 1 micron
Author, Geraldine Brooks at the National Library of Australia for the 2009 Ray Mathew Lecture, Canberra, 23 October 2009 [picture] /
No full textTitle from acquisitions documentation.; Part of the collection: Portraits of author, Geraldine Brooks during her visit to the National Library of Australia for the 2009 Ray Mathew Lecture, Canberra, 23 October 2009.; Acquired in digital format; access copy available online.; Mode of access: Internet via World Wide Web.; Photographed by a staff member of the National Library of Australia
LIVE-PAINT Supplementary Videos: Compressed versions
No full textCompressed versions of the files of Oi, Curran; Horrocks, Mathew; Regan, Lynne. (2020). LIVE-PAINT Supplementary Videos, [dataset]. University of Edinburgh. Quantitative Biology, Biochemistry and Biotechnology. https://doi.org/10.7488/ds/2801. ## File listing ## * "SupplementaryMovie1_-_mKO_blinking_compressed.mp4" LIVE-PAINT blinking behavior shown in S. cerevisiae using the fluorescent protein mKO and the reversible interaction pair SYNZIP17-SYNZIP18. Scale bar is 1 micron. * "SupplementaryMovie2_-_mOrange_blinking_compressed.mp4" LIVE-PAINT blinking behavior shown in S. cerevisiae using the fluorescent protein mOrange and the reversible interaction pair SYNZIP17-SYNZIP18. Scale bar is 1 micron. * "SupplementaryMovie3_-_cofilin_tracking_compressed.mp4" Video tracking cofilin in S. cerevisiae, where cofilin is C-terminally tagged with SYNZIP18 and labeled with separately expressed SYNZIP17-mNeonGreen. Scale bar is 5 microns. * "SupplementaryMovie4_-_cofilin_tracking_compressed.mp4" Tracking the diffusion of a single cofilin "spot" in S. cerevisiae, where cofilin is C-terminally tagged with SYNZIP18 and labeled with separately expressed SYNZIP17-mNeonGreen. Scale bar is 1 micron.Oi, Curran; Horrocks, Mathew; Regan, Lynne. (2020). LIVE-PAINT Supplementary Videos: Compressed versions, [moving image]. University of Edinburgh. Quantitative Biology, Biochemistry and Biotechnology. https://doi.org/10.7488/ds/2839
Super-resolution imaging of proteins in live cells using reversibly interacting peptide pairs
No full textSuper-resolution techniques have revolutionised our ability to observe cellular structures
with significantly higher resolution than traditional microscopy. Despite the
number of super-resolution microscopy techniques available, live cell super-resolution
imaging remains challenging. For example, while Photo-activated localisation microscopy
(PALM) can be used in vivo, it necessitates the direct fusion of a fluorophore
to the protein of interest. This approach can be problematic because a direct fusion
to a fluorescent protein can disrupt the normal function and localisation of the protein
being studied. Moreover, once the fluorescent protein is photobleached, no more data
can be collected from that molecule.
In this thesis, I describe the development and use of LIVE-PAINT, a novel live-cell
super-resolution microscopy technique. In LIVE-PAINT, a peptide-protein or peptidepeptide
pair, one fused to the protein of interest and the other to a fluorescent protein,
reversibly interact. When the peptide pair bind, a blink is observed, and the precise
location can be determined. In a few minutes, enough binding events occur to generate
an image of the protein of interest with a resolution of around 20 nanometres.
Initially, this work optimises and applies LIVE-PAINT for diffraction-limited and
super-resolution imaging of proteins within live budding yeast cells. I then demonstrate
that the small peptide tag used to label the protein of interest makes LIVE-PAINT a
valuable tool for imaging proteins that are sensitive to direct fusions to fluorescent
proteins. In addition, I validate that LIVE-PAINT enables replenishment of signal
throughout imaging. This is because the imaging peptide, the peptide-labelled fluorescent
protein, is expressed separately from the target protein, creating a pool of imaging
peptides within the cell that can replenish those that are photobleached during imaging.
I utilise this property of LIVE-PAINT to track moving proteins over long periods of
time.
Subsequently, I describe how I adapted the LIVE-PAINT system to apply this
technique to the more complex environment of live mammalian cells. I show that
LIVE-PAINT successfully yields diffraction-limited and super-resolution images of
proteins located in various organelles. This is the first time that interacting peptide pairs
have been used to facilitate point accumulation for imaging in nanoscale topography
(PAINT) based super-resolution imaging in live mammalian cells. These results are
obtained through both transient transfections of labelled proteins and stably integrated
versions. Through this work I generate several new cell lines which can be shared
with other researchers allowing them to use this technique to gain new insights into the
proteins they study.
Furthermore, this thesis explores improvements to the LIVE-PAINT method. I
demonstrate that peptides as small as 5 residues can be used for LIVE-PAINT imaging.
This will broaden the applicability of LIVE-PAINT to a wider range of proteins
that cannot tolerate modifications. To harness the increased brightness of synthetic
fluorescent dyes compared to fluorescent proteins, I developed mammalian cell lines
expressing a HaloTag fused to a LIVE-PAINT peptide. I show that the exogenous
addition of the binding partner to HaloTag, HaloLigand, labelled with a synthetic dye,
to these cells, enables LIVE-PAINT imaging with synthetic dyes. Lastly, I validate that
LIVE-PAINT can be multiplexed by using orthogonal peptide-protein pairs to image
two proteins concurrently in live cells.
In summary, this thesis presents the development and optimisation of LIVE-PAINT,
an innovative peptide-based super-resolution imaging technique tailored for live cell
imaging. While this work explores select applications of LIVE-PAINT, it is anticipated
that this novel technique will have a broad spectrum of applications
The design and synthesis of peptides for fluorescence imaging applications
No full textTesting new function - does this work?? Fluorescence microscopy is a tool
routinely utilised to address biological questions. Direct visualisation of features of interest, confirmation of protein identity or detecting the presence of a posttranslational modification enable researchers to study differences between healthy and pathological phenotypes. As the questions get more complex, more advanced imaging techniques are required to address the limit of resolution, probe specificity to target and fluorescent background. This thesis explores two advancements to the field of fluorescence microscopy: the development of a novel imaging platform
and the introduction of a new imaging modality.
The study covers the design and construction of the Full Spectrum Fluorescence Lifetime Imaging Microscope (FS-FLIM): a new imaging platform. The FS-FLIM maximises the information collected in a fluorescence imaging experiment. It is capable of 3-in-1 imaging, collecting fluorescence intensity and lifetime data at 512 wavelengths simultaneously. The instrument is compatible with a wide range of samples. The spectral region observed can be matched to the emitters present in the sample. Moreover, a wide range of lifetimes (from sub-nanosecond to tens of nanoseconds) can be recorded using the FS-FLIM. The performance of the
constructed instrument is validated through solution lifetime measurements of several fluorescent dyes, and its application in environment sensing is described using a model system.
Next, the thesis focuses on developing fluorescent peptides for imaging applications. Although peptides can be labelled with a range of fluorophores, this study mostly uses a selenium-derivatised nitrobenzoxadiazole dye - SeNBD, a recent exciting addition to the imaging toolbox. The fluorogenic properties of the new dye are characterised in this study, and the incorporation of the dye on-column using standard solid state peptide synthesis methods is described. The switch-on character, as well as the small size of the dye, are leveraged throughout the work, with short peptide sequences and internally-labelled peptide sequences developed as a new generation of peptide probes.
The specificity of peptides enables their application as therapeutic agents
for otherwise undruggable proteins. One such example is α-synuclein, a protein associated with Parkinson’s disease (PD). It is commonly thought that aggregation of α-synuclein and formation of cytotoxic inclusions is involved in PD progression. An α-helical bacteria-derived peptide, phenol soluble modulin α3 (PSMα), was previously shown to bind to small oligomeric α-synuclein species. Here, the peptide sequence was derivatised with several labels to explore its applications in imaging. Pilot experiments to detect α-synuclein species in vitro were conducted to confirm the binding to the protein-of-interest. A biotin-labelled peptide analogue was used to perform immunohistochemistry staining on patient tissue for in situ detection. Lastly, first attempts at fluorescence lifetime imaging of α-synuclein on the FS-FLIM instrument were made.
To further illustrate the capabilities of SeNBD dye specifically, and its potential
for super-resolution imaging, a range of sequences targeting the PDZ domain
(a common structural motif of anchoring and signalling proteins) were then
considered. The chosen sequences were only a few amino acids long, however they
demonstrated retained transient binding to the protein-of-interest upon labelling
with SeNBD. Moreover, an internally-labelled sequence was also synthesised and successfully used to target PDZ domains. The transient nature of the peptideprotein binding was utilised in a Point Accumulation in Nanoscale Topography (PAINT) experiment, where the on-off binding enabled precise localisation of the dye molecules and super-resolution image reconstruction. The PDZ contained in post-synaptic density proteins of a brain-derived sample was successfully imaged, and nanoclusters of the protein super-resolved, corroborating literature findings. Furthermore, an extension to the approach using a coiled-coil interacting peptide pair: 101A and 101B, was investigated as an alternative approach for proteiniv peptide interaction pairs for super-resolution microscopy. Whilst the delivery of
the synthesised targeting sequence (101B) proved challenging, expressing 101B attached to the target protein in cellulo, and subsequent staining with fluorescent 101A sequence produced promising results and enabled direct visualisation and super-resolution imaging of TOM-20 and LAMP-1 in fixed HEK cells.
Overall, the work presented herein showcased a range of fluorescent peptides that could be used across versatile fluorescence imaging platforms. The small novel dye, SeNBD, enabled super-resolution imaging using peptide sequences containing less than 10 amino acids. The small size of the peptides decreased the linkage error and ensured the fluorescent signal was localised at target of interest. The environment sensing properties of the dye were explored in preliminary experiments to observe α-synuclein on a purpose-built novel microscope, the FS-FLIM. It is hoped the techniques developed here could further progress research in the field
of neurodegeneration and beyond
Ventriloquism Days: In Conversation with David Mathew
No full textDavid Mathew is the author of three novels – O My Days, Creature Feature, and most recently Ventriloquists – and a volume of short stories entitled Paranoid Landscapes. His wide areas of interest include psychoanalysis, linguistics, distance learning, prisons and online anxiety. With approximately 600 published pieces to his name, including a novel based on his time working in the education department of a maximum security prison (O My Days), he has published widely in academic, journalistic and fiction outlets. In addition to his writing, he co-edits The Journal of Pedagogic Development (at the University of Bedfordshire, UK), teaches academic writing, and he particularly enjoys lecturing in foreign countries and learning about wine. He is a member of the Tavistock Society of Psychotherapists and Allied Professionals, Evidence Informed Policy and Practice in Education in Europe (EIPPEE), and the European Association for the Teaching of Academic Writing. He was also a member of The Health Technology Assessment programme (www.hta.ac.uk), as part of the NIHR Evaluation, Trials and Studies Coordinating Centre at the University of Southampton (2009-2013). We met at his home in the south-east of England in November 2014 to discuss his approaches to writing and his new novel, Ventriloquists
Fifty Forensic Fables
No full textThis book does for the legal profession in England what George Ade's fables do more broadly. These are enjoyable tales with pleasing caricatures. All the actors are humans. A funny appendix follows The Story of an Ancient Line through twelve generations. The book shows what fable meant earlier in this century.This is a hardbound book (hard cover)This book has a dust jacket (book cover)O (Theo Mathew
LIVE-PAINT Supporting Datasets
No full textWe present LIVE-PAINT, a new approach to super-resolution fluorescent imaging inside live cells. In LIVE-PAINT only a short peptide sequence is fused to the protein being studied, unlike conventional super-resolution methods, which rely on directly fusing the biomolecule of interest to a large fluorescent protein, organic fluorophore, or oligonucleotide. LIVE-PAINT works by observing the blinking of localized fluorescence as this peptide is reversibly bound by a protein that is fused to a fluorescent protein. We have demonstrated the effectiveness of LIVE-PAINT by imaging a number of different proteins inside live S. cerevisiae. Not only is LIVE-PAINT widely applicable, easily implemented, and the modifications minimally perturbing, but we also anticipate it will extend data acquisition times compared to those previously possible with methods that involve direct fusion to a fluorescent protein.Oi, Curran; Horrocks, Mathew; Gidden, Zoe; Regan, Lynne. (2020). LIVE-PAINT Supporting Datasets, [dataset]. University of Edinburgh. Quantitative Biology, Biochemistry and Biotechnology. https://doi.org/10.7488/ds/2859
LIVE-PAINT Supplementary Videos
No full textWe present LIVE-PAINT, a new approach to super-resolution fluorescent imaging inside live cells. In LIVE-PAINT only a short peptide sequence is fused to the protein being studied, unlike conventional super-resolution methods, which rely on directly fusing the biomolecule of interest to a large fluorescent protein, organic fluorophore, or oligonucleotide. LIVE-PAINT works by observing the blinking of localized fluorescence as this peptide is reversibly bound by a protein that is fused to a fluorescent protein. We have demonstrated the effectiveness of LIVE-PAINT by imaging a number of different proteins inside live S. cerevisiae. Not only is LIVE-PAINT widely applicable, easily implemented, and the modifications minimally perturbing, but we also anticipate it will extended data acquisition times compared to those previously possible with methods that involve direct fusion to a fluorescent protein.Oi, Curran; Horrocks, Mathew; Regan, Lynne. (2020). LIVE-PAINT Supplementary Videos, [dataset]. University of Edinburgh. Quantitative Biology, Biochemistry and Biotechnology. https://doi.org/10.7488/ds/2801
The Psalter in the Description of Jesus’ Passion from the Gospel of St. Mathew
No full textThe author focuses on the quotations from the psalms that we find in the description of Jesus’ Passion in the Gospel of St. Mathew. It turns out that almost all the quotations from the psalms (with the exception of 26, 64: Ps 109, 1 LXX) stress the human nature of Jesus, i.e. they are anthropologically oriented. The author discusses each of the seven quotations in the context of the psalm, and then in the context of Jesus’ Passion. Following partly the Gos¬pel of St. Mark, St. Mathew enhances in the reader a belief that Jesus in His Passion is the Suffering Just and the suffering poor Jehovah
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