Biomedical subjects

Stanley W Botchway

Publications and source records attributed to Stanley W Botchway.

6 recordsLinked to original sources

PubMed2005

Single- and multi-photon excited fluorescence from serotonin complexed with beta-cyclodextrin.

The fluorescence of serotonin on binding with beta-cyclodextrin has been studied using both steady state and time-resolved methods. Steady state fluorescence intensity of serotonin at 340 nm showed approximately 30% increase in intensity on binding with K(A) approximately 60 dm(3) mol(-1) and the fluorescence lifetimes showed a corresponding increase. In contrast, the characteristic green fluorescence ('hyperluminescence') of serotonin observed upon multiphoton near-infrared excitation with sub-picosecond pulses was resolved into two lifetime components assigned to free and bound serotonin. The results are of interest in relation to selective imaging and detection of serotonin using the unusual hyperluminescence emission and in respect to recent determinations of serotonin by capillary electrophoresis in the presence of cyclodextrin. The results also suggest that hyperluminescence occurs from multiphoton excitation of a single isolated serotonin molecule.

Electrophoresis, Capillary↗

PubMed2005

A novel method for observing proteins in vivo using a small fluorescent label and multiphoton imaging.

A novel method for the fluorescence detection of proteins in cells is described in the present study. Proteins are labelled by the selective biosynthetic incorporation of 5-hydroxytryptophan and the label is detected via selective two-photon excitation of the hydroxyindole and detection of its fluorescence emission at 340 nm. The method is demonstrated in this paper with images of a labelled protein in yeast cells.

5-Hydroxytryptophan↗

PubMed2005

The use of time-resolved fluorescence imaging in the study of protein kinase C localisation in cells.

BACKGROUND: Two-photon-excitation fluorescence lifetime imaging (2P-FLIM) was used to investigate the association of protein kinase C alpha (PKCalpha) with caveolin in CHO cells. PKCalpha is found widely in the cytoplasm and nucleus in most cells. Upon activation, as a result of increased intracellular Ca2+ and production of DAG, through G-protein coupled-phospholipase C signalling, PKC translocates to a variety of regions in the cell where it phosphorylates and interacts with many signalling pathways. Due to its wide distribution, discerning a particular interaction from others within the cell is extremely difficult. RESULTS: Fluorescence energy transfer (FRET), between GFP-PKCalpha and DsRed-caveolin, was used to investigate the interaction between caveolin and PKC, an aspect of signalling that is poorly understood. Using 2P-FLIM measurements, the lifetime of GFP was found to decrease (quench) in certain regions of the cell from approximately 2.2 ns to approximately 1.5 ns when the GFP and DsRed were sufficiently close for FRET to occur. This only occurred when intracellular Ca2+ increased or in the presence of phorbol ester, and was an indication of PKC and caveolin co-localisation under these conditions. In the case of phorbol ester stimulated PKC translocation, as commonly used to model PKC activation, three PKC areas could be delineated. These included PKCalpha that was not associated with caveolin in the nucleus and cytoplasm, PKCalpha associated with caveolin in the cytoplasm/perinuclear regions and probably in endosomes, and PKC in the peripheral regions of the cell, possibly indirectly interacting with caveolin. CONCLUSION: Based on the extent of lifetime quenching observed, the results are consistent with a direct interaction between PKCalpha and caveolin in the endosomes, and possibly an indirect interaction in the peripheral regions of the cell. The results show that 2P-FLIM-FRET imaging offers an approach that can provide information not only confirming the occurrence of specific protein-protein interactions but where they occur within the cell.

Animals↗

PubMed2003

Nanoscale spatial induction of ultraviolet photoproducts in cellular DNA by three-photon near-infrared absorption.

The high-resolution spatial induction of ultraviolet (UV) photoproducts in mammalian cellular DNA is a goal of many scientists who study UV damage and repair. Here we describe how UV photoproducts can be induced in cellular DNA within nanometre dimensions by near-diffraction-limited 750 nm infrared laser radiation. The use of multiphoton excitation to induce highly localized DNA damage in an individual cell nucleus or mitochondrion will provide much greater resolution for studies of DNA repair dynamics and intracellular localization as well as intracellular signalling processes and cell-cell communication. The technique offers an advantage over the masking method for localized irradiation of cells, as the laser radiation can specifically target a single cell and subnuclear structures such as nucleoli, nuclear membranes or any structure that can be labelled and visualized by a fluorescent tag. It also increases the time resolution with which migration of DNA repair proteins to damage sites can be monitored. We define the characteristics of localized DNA damage induction by near-infrared radiation and suggest how it may be used for new biological investigations.

Animals↗

PubMed2003

Isotope effects on the picosecond time-resolved emission spectroscopy of tris(2,2'-bipyridine)ruthenium (II).

The excited-state properties of the transition metal complexes tris(2,2'-bipyridine) ruthenium(II) and tris(4,7-diphenyl-1,10-phenanthroline) ruthenium(II) are examined using picosecond time-resolved luminescence spectroscopy. For both complexes, direct observation of a short-lived high-energy emission with a lifetime of less than 4 ps is reported. Upon deuteriation of the complexes the lifetime of the high-energy emission shows a marked increase with a biexponential decay (20 and approximately 300 ps components). Examination by time-resolved excited-state resonance Raman shows that for the perprotio complexes features attributable to the 3MLCT excited state are formed within 4 ps, while for the perdeuterio a rise time of approximately 20 ps is observed in the 3MLCT features. The results indicate that the emission in both cases may be 1MLCT in origin and are discussed with respect to heterogeneous electron transfer.

Journal Article↗

PubMed2003

Investigation of multiphoton-induced fluorescence from solutions of 5-hydroxytryptophan.

It is reported (J. B. S. Shear, C. Xu and W. W. Webb, Photochem. Photobiol. 1997, 65, 931) that multiphoton near infrared excitation of 5-hydroxytryptophan results in a transient product with green fluorescence. Visible fluorescence from multiphoton excitation enables detection of 5-hydroxytryptophan with extremely high sensitivity and also has potential applications in imaging of biological systems and investigation of protein dynamics. The characteristic fluorescence at 500 nm has now also been observed in a two laser experiment whereby 308 nm photolysis of the solution is followed by an excitation step at 430 nm. Fluorescence was observed in aerated and deaerated solutions and in the presence of ascorbate. Enhancement of fluorescence was observed on addition of ethanol. Transient absorption experiments with 308 nm photolysis showed the formation of three transient species. In the presence of ascorbate the radical formed by photoionisation was quenched, revealing a long-lived species (tau > 1 ms) with a similar absorption spectrum, which is ascribed to the fluorescing species. Fluorescence induced by multiphoton excitation had a lifetime of 910 +/- 10 ps and was also unaffected by ascorbate. In the presence of organic solvents there was an increase in fluorescence lifetime, but a decrease in overall fluorescence intensity. The fluorescence intensity and fluorescence lifetime both decreased in acidic solution (pH < 3). The results indicate that the fluorescence does not originate from the 5-indoxyl radical as previously suggested but from one or more other transient products which require further characterisation.

5-Hydroxytryptophan↗