Emerging Applications of Fluorescence Spectroscopy to Cellular Imaging: Lifetime Imaging, Metal-Ligand Probes, Multi-Photon Excitation and Light Quenching

Advances in time-resolved fluorescence spectroscopy can be applied to cellular imaging. Fluorescence lifetime imaging microscopy (FLIM) creates image contrast based on the decay time of sensing probes at each point in a two-dimensional image. FLIM allows imaging of Ca2+ and other ions without the need for wavelength-ratiometric probes. Ca2+ imaging can be performed by FLIM with visible wavelength excitation. Instrumentation for FLIM is potentially simple enough to be present in most research laboratories. Applications of fluorescence are often limited by the lack of suitable fluorophores. New, highly photostable probes allow off-gating of the prompt autofluorescence, and measurement of rotational motion of large macromolecules. These luminescent metal-ligand complexes will become widely utilized. Modem pulse lasers allow new experiments based on non-linear phenomena. With picosecond and femtosecond lasers fluorophores can be excited by simultaneous absorption of two or three photons. Hence, Ca2+ probes, membrane probes, and even intrinsic protein fluorescence can be excited with red or near infrared wavelengths, without ultraviolet lasers or optics. Finally, light itself can be used to control the excited state population. By using light pulses whose wavelength overlaps the emission spectrum of a fluorophore one can modify the excited state population and orientation. This use of non-absorbed light to modify emission can have wide reaching applications in cellular imaging

Increasing the sensitivity of DNA microarrays by metal-enhanced fluorescence using surface-bound silver nanoparticles

The effects of metal-enhanced fluorescence (MEF) have been measured for two dyes commonly used in DNA microarrays, Cy3 and Cy5. Silver island films (SIFs) grown on glass microscope slides were used as substrates for MEF DNA arrays. We examined MEF by spotting biotinylated, singly-labeled 23 bp DNAs onto avidin-coated SIF substrates. The fluorescence enhancement was found to be dependent on the DNA spotting concentration: below ∼12.5 μM, MEF increased linearly, and at higher concentrations MEF remained at a constant maximum of 28-fold for Cy5 and 4-fold for Cy3, compared to avidin-coated glass substrates. Hybridization of singly-labeled oligonucleotides to arrayed single-stranded probes showed lower maximal MEF factors of 10-fold for Cy5 and 2.5-fold for Cy3, because of the smaller amount of immobilized fluorophores as a result of reduced surface hybridization efficiencies. We discuss how MEF can be used to increase the sensitivity of DNA arrays, especially for far red emitting fluorophores like Cy5, without significantly altering current microarray protocols

DNA crunching by a viral packaging motor: Compression of a procapsid-portal stalled Y-DNA substrate

AbstractMany large double-stranded DNA viruses employ high force-generating ATP-driven molecular motors to package to high density their genomes into empty procapsids. Bacteriophage T4 DNA translocation is driven by a two-component motor consisting of the procapsid portal docked with a packaging terminase-ATPase. Fluorescence resonance energy transfer and fluorescence correlation spectroscopic (FRET-FCS) studies of a branched (Y-junction) DNA substrate with a procapsid-anchoring leader segment and a single dye molecule situated at the junction point reveal that the “Y-DNA” stalls in proximity to the procapsid portal fused to GFP. Comparable structure Y-DNA substrates containing energy transfer dye pairs in the Y-stem separated by 10 or 14 base pairs reveal that B-form DNA is locally compressed 22–24% by the linear force of the packaging motor. Torsional compression of duplex DNA is thus implicated in the mechanism of DNA translocation

Two-photon excitation of rhenium metal-ligand complexes

We describe the emission spectral properties of two rhenium metal-ligand complexes with one and two-photon excitation, Re(bpy)2(CO)3Cl and [Re(bpy)(CO)3CH3CN]+, where bpy is 2,2’-bipyridyl and CH3CN is acetonitrile. Similar emission spectra and intensity decay times characteristic of the metal-to-ligand charge transfer state were observed for one- and two-photon excitation. The lifetime and quantum yield of the acetonitrile complex are approximately 14-fold higher than that of the chloride complex. Both complexes display high anisotropies near 0.33 in frozen solution with one-photon excitation. Two-photon excitation results in anisotropies about 40% larger, consistent with the increased photoselection expected for two-photon absorption. These complexes display single rotational correlation times in glycerol, but the correlation time of the charged acetonitrile complex is 3 to 4-fold larger. These results show that rhenium complexes can be used as hydrodynamic probes with one- or two-photon excitation

Extending the Propagation Distance of a Silver Nanowire Plasmonic Waveguide with a Dielectric Multilayer Substrate

Chemical synthesized silver nanowires have been proved to be the efficient architecture for Plasmonic waveguides, but the high propagation loss prevents their widely applications. Here, we demonstrate that the propagation distance of the plasmons along the Ag NW can be extended if the Ag NW was placed on a dielectric multilayer substrate containing a photonic band gap, but not placed on a commonly used glass substrate. The propagation distance at 630 nm wavelength can reach 16 um even that the Ag NW is as thin as 90 nm in diameter. Experimental and simulation results further show that the polarization of this propagating plasmon mode was nearly parallel to the surface of the dielectric multilayer, so it was excited by a transverse-electric polarized Bloch surface wave propagating along a polymer nanowire with diameter at only about 170 nm on the same dielectric multilayer. Numerical simulations were also carried out and consistent with the experiment results. Our work provides a platform to extend the propagation distance of plasmonic waveguide and also for the integration between photonic and plasmonic waveguides on the nanometre scale.Comment: 5 pages, 4 figure

Boronic acid fluorescent sensors for monosaccharide signaling based on the 6-methoxyquinolinium heterocyclic nucleus: progress toward noninvasive and continuous glucose monitoring

Abstract-The synthesis, characterization, and spectral properties of strategically designed boronic acid containing fluorescent sensors, o-, m-, p-BMOQBA, for the potential detection of tear glucose concentrations when immobilized in plastic disposable contact lenses is described. The new probes, BMOQBAs, consist of the 6-methoxyquinolinium nucleus as a fluorescent indicator, and the boronic acid moiety as a glucose chelating group. A control compound BMOQ, which has no boronic acid group and therefore does not bind monosaccharides has also been prepared. In this paper, we show that structural design considerations of the new probes have afforded for their compatibility within the lenses, with reduced probe sugar-bound pK a favorable with the mildly acidic lens environment. In addition, the new probes are readily water soluble, have high quantum yields, and can be prepared by a simple one-step synthetic procedure

Wavelength-ratiometric nearphysiological ph sensors based on 6-aminoquinolinium boronic acid probes

Abstract We describe the pH response of a set of isomeric water-soluble fluorescent probes based on both the 6-aminoquinolinium and boronic acid moieties. These probes show spectral shifts and intensity changes with pH, in a wavelength-ratiometric and colorimetric manner. Subsequently, changes in pH can readily be determined around the physiological level. Although boronic acid containing probes are known to exhibit pH sensitivity along with an ability for saccharide binding/chelating, the new probes reported here are considered to be unique and show an unperturbed pH response, even in the presence of high concentrations of background saccharide, such as with glucose and fructose, allowing for the predominant pH sensitivity. The response of the probes is based on the ability of the boronic acid group to interact with strong bases like OH − , changing from the neutral form of the boronic acid group, R-B(OH) 2 , to the anionic ester, R-B − (OH) 3 , form, which is an electron donating group. The presence of an electron deficient quaternary heterocyclic nitrogen center and a strong electron donating amino group in the 6-position of the quinolinium backbone, provides for the spectral changes observed upon OH − complexation. In addition, by comparing the results obtained with systems separately incorporating 6-methoxy or 6-methyl substituents, the suppressed response towards monosaccharides, such as with glucose and fructose, can clearly be observed for these systems. Finally we compare our results to those of a control compound, BAQ, which does not contain the boronic acid group, allowing a rationale of the spectral changes to be made