Fluorescence enhancement of fluorophores tethered to different sized silver colloids deposited on glass substrate

We studied fluorescence enhancements of fluorescein tethered to silver colloids of different size. Thiolated 23-mer oligonucleotide (ss DNA-SH) was bound selectively to silver colloids deposited on 3-aminopropyltriethoxysilane (APS)-treated quartz slides. Fluorescein-labeled complementary oligonucleotide (ss Fl-DNA) was added in an amount significantly lower than the amount of unlabeled DNA tethered to the colloids. The hybridization kinetics, observed as an increase in fluorescence emission, on small (30-40 nm) and large (> 120 nm) colloids were similar. However, the final fluorescence intensity of the sample with large colloids was about 50% higher than that observed for the sample with small colloids. The reference sample without ss DNA-SH was used to estimate the fluorescence enhancements of fluorescein tethered to the small colloids (E = 2.7) and to the large colloids (E = 4.1) due to its steady fluorescence signal. The proposed method, based on controlled hybridization with minimal amount of fluorophore labeled ss DNA, can be used to reliably estimate the fluorescence enhancements on any silver nanostructures.     

Molecular heterogeneity of O-acetylserine sulfhydrylase by two-photon excited fluorescence fluctuation spectroscopy

O-acetylserine sulfhydrylase, a homo-dimeric enzyme from Salmonella typhimurium, covalently binds one pyridoxal 5'-phosphate molecule per subunit as a fluorescent coenzyme. Different tautomers of the Schiff base between the coenzyme and lysine 41 generate structured absorption and fluorescence spectra upon one-photon excitation. We investigated the protein population heterogeneity by fluorescence correlation spectroscopy and lifetime techniques upon two-photon excitation. We sampled the fluorescence intensity from a small number of molecules (approximately 10) and analyzed the distribution of photon counts to separately determine the number and the fluorescence brightness of excited protein molecules. The changes in the average number of molecules and in the fluorescence brightness with the excitation wavelength indicate the presence of at least two fluorescent species, with two-photon excitation maxima at 660 and 800 nm. These species have been identified as the enolimine and ketoenamine tautomers of the protein-coenzyme internal aldimine. Their relative abundance is estimated to be 4:1, whereas the ratio of their two-photon cross sections is reversed with respect to the single-photon excitation case. Consistent results are obtained from the measurement of the lifetime decays, which are sensitive to the excited-state heterogeneity. At least two components were detected, with lifetimes of approximately 2.5 and 0.5 ns. The lifetimes are very close to the values measured in bulk solutions upon one-photon excitation and attributed to the ketoenamine tautomer and to a dipolar species formed upon proton dissociation in the excited state.     

Dimethyl-pepep: a DNA probe in two-photon excitation cellular imaging

Dimethyl-pepep (D-pepep), a newly developed and very efficient two-photon absorber, has been tested here for two-photon excitation (TPE) cellular imaging. The spectral characteristics of the dye following one-photon excitation (OPE) and TPE (excitation and emission spectra, fluorescence lifetime, molecular brightness, saturation intensity) are reported. In vitro interaction studies with biomolecules show that dimethyl-pepep has a large affinity for DNA. A comparison with a widely used DNA stainer, 4-6-diamidino-2-phenylindole (DAPI) bound to DNA shows that the D-pepep brightness is one order of magnitude higher than that of DAPI, making this dye suitable for microscopy and imaging applications. TPE images taken from double-stained yeast Saccharomyces cerevisiae cells have revealed that D-pepep localizes mainly in the nucleus, similarly to DAPI, and in mitochondria, although to a minor extent. Preliminary tests have shown that the dye cellular toxicity is negligible.     

Two-photon induced fluorescence of Cy5-DNA in buffer solution and on silver island films

We report the observation of a strong two-photon induced fluorescence emission of Cy5-DNA within the tunable range of a Ti:Sapphire laser. The estimated two-photon cross-section for Cy5-DNA of 400GM is about 3.5-fold higher than it was reported for rhodamine B. The fundamental anisotropies of Cy5-DNA are close to the theoretical limits of 2/5 and 4/7 for one- and two-photon excitation, respectively. We also observed an enhanced two-photon induced fluorescence (TPIF) of Cy5-DNA deposited on silver island films (SIFs). In the presence of SIFs, the TPIF is about 100-fold brighter. The brightness increase of Cy5-DNA TPIF near SIFs is mostly due to enhanced local field.     

The two-photon laser-induced fluorescence of the tumor-localizing photosensitizer hematoporphyrin derivative. Resonance-enhanced 750 nm two-photon excitation into the near-UV Soret band

The tumor-localizing photosensitizer hematoporphyrin derivative (HPD) is shown to undergo a simultaneous two-photon excitation into the near-ultraviolet Soret band system upon intense laser irradiation at 750 nm, a spectral region where there is no significant HPD one-photon absorbance in aqueous solution. Subsequent to this excitation, internal conversion and vibrational relaxation occur, resulting in the population of the vibrationless level of the first electronically excited singlet state. This state relaxes by two channels, the emission of fluorescence in the spectral region 600-700 nm and intersystem crossing into the triplet manifold, followed by near-resonant electronic energy transfer with surrounding oxygen to result in the generation of highly reactive singlet molecular oxygen (1 delta g). Evidence for the two-photon excitation consists in the observation both of the HPD fluorescence spectrum in the region of 615 nm as a result of 750 nm excitation and the quadratic dependence of this fluorescence emission intensity upon the excitation laser intensity. Since, in general, the penetration depth of ultraviolet and visible light into tissue varies directly with wavelength (red penetrating more deeply than blue), these studies suggest the possibility that two-photon-induced localization of tumor-bound HPD might facilitate the detection of deeper lying tumors than allowed by the current one-photon photolocalization method.     

Tryptophan fluorescence intensity and anisotropy decays of human serum albumin resulting from one-photon and two-photon excitation.

We measured the emission spectra, intensity decays and anisotropy decays of the single tryptophan residue of human serum albumin (HSA) resulting from one-photon (295-298 nm) and two-photon (590-596) excitation. The emission spectra and intensity decays were independent of the mode of excitation. The anisotropy decays were superficially similar for one- and two-photon excitation. However, upon consideration of the different orientation photoselection for one- and two-photon excitation, the anisotropy data reveal different angles between the absorption and emission oscillators for one-photon and two-photon excitation. This result suggests different relative one-photon and two-photon cross-sections for the 1La and 1Lb transitions of the indole residue. This first report of the time-resolved anisotropy decay of a protein resulting from two-photon excitation suggests that such measurement will yield insights into the complex photophysical properties of tryptophan residues in proteins.     

Three-photon induced fluorescence of the calcium probe Indo-1.

We report the calcium-dependent emission spectral properties of the calcium probe Indo-1 for three-photon excitation. We found that Indo-1 could be readily excited with the femtosecond pulses from a mode-locked Ti:sapphire laser at 885 nm. This wavelength is too long for two-photon excitation, which is expected to occur for wavelengths no longer than twice the longest single-photon absorption wavelength of 400 nm. For excitation at 885 nm the emission intensity was found to depend on the cube of the laser power, as expected for simultaneous interaction with three photons. At wavelengths below 840 nm the emission intensity depends on the square of the laser power, indicating two-photon excitation at shorter wavelengths. The intensity decays of Indo-1 were found to be dependent on Ca2+ and essentially identical for one- and three-photon excitation. The emission anisotropy of Indo-1 was found to be considerably higher for three-photon excitation than for one-photon excitation, consistent with cos6 theta photoselection, as compared with cos2 theta photoselection for one-photon excitation. The high values of the anisotropy are in agreement with those expected for a three-photon process. Calcium-dependent emission spectra were observed for Indo-1 with three-photon excitation, demonstrating that three-photon excitation of Indo-1 can be used for calcium imaging by emission intensity ratio measurements. The calcium-dependent emission spectra indicate a higher three-photon cross-section for the calcium-free form of Indo-1 than for the calcium-bound form. The possible advantages of three-photon excitation include the availability of the appropriate wavelengths with solid-state lasers, enhanced spatial resolution due to a reduced size of the excited volume, absence of light quenching, and possibly high selectivity of the three-photon excitation process.     

Three-photon excitation of N-acetyl-L-tyrosinamide.

We observed emission from the tyrosine derivative N-acetyl-L-tyrosinamide (NATyrA) when excited with the fundamental output of a femtosecond Ti:Sapphire laser from 780 to 855 nm. The dependence on incident laser power indicates a three-photon process. The emission spectra and intensity decay in glycerol-water (30:70) at 5 degrees C were found to be identical for one- and three-photon excitation. Also the excitation spectrum of three-photon-induced fluorescence of NATyrA corresponds to the one-photon excitation spectrum. The time-zero or fundamental anisotropy spectrum was reconstructed from the frequency-domain anisotropy decays. The three-photon anisotropies are similar or larger than the one-photon anisotropies. These three-photon anisotropies are surprising given the near zero values known for tyrosine with two-photon excitation. The observations indicate that one- and three-photon excitation directly populates the same singlet excited states(s). However, the origin of the anisotropies with multi-photon excitation of tyrosine remain unclear and unpredictable.     

Two-photon fluorescence excitation of macroscopic areas on planar waveguides

In this paper, we report the first successful demonstration, to our knowledge, of two-photon fluorescence excitation (TPFE) using planar thin-film waveguide structures of macroscopic excitation dimensions (square millimeters to square centimeters in size). The high intensity of excitation light required for TPFE is available not only at a single focus point but along the whole trace of the beam guided in the waveguide structure. Line profiles of the fluorescence excited by TPFE show excellent correlation with the geometry of the launched laser beams. A clear second-order dependence of the fluorescence intensity on the excitation intensity confirms the two-photon character of fluorescence generation. Spectra of the emission generated by one-photon excitation and by two-photon excitation show only minor differences.     

The two-photon induced fluorescence of the tumor localizing photosensitizer hematoporphyrin derivative via 1064 nm photons from a 20 ns Q-switched Nd-YAG laser

We demonstrate the direct 1064 nm two-photon excitation of hematoporphyrin derivative (HPD), a complex mixture of photosensitizing porphyrins which is selectively retained in tumor tissue and used in cancer photochemotherapy. Although 1064 nm is outside of the one-photon HPD absorption spectrum, two-photon induced fluorescence from HPD was observed following excitation by the 20 ns output of an amplified, Q-switched Nd-YAG laser at peak power levels of 0.1 to 3 GW/cm2. Evidence for the successful two-photon excitation to vibrational levels of the S1 state consists of the observation of the known HPD fluorescence spectrum exhibiting peaks at approximately 615 and 675 nm, with the observed two-photon induced fluorescence intensity exhibiting a quadratic dependence on the excitation laser intensity as required for a direct two-photon process. More generally, these results suggest the possibility for the achievement of photosensitized oxidations utilizing photons of lower energy than that required for single photon excitation, offering the potential for both greater selectivity and a reduction in competing photochemical processes.     

Two-photon excitation fluorescence spectrum of the light-harvesting complex LH2 from Chromatium minutissimum within 650-745 nm range is determined by two-photon absorption of bacteriochlorophyll rather than of carotenoids

Two-photon fluorescence excitation spectra of the peripheral light-harvesting complex LH2 from the purple photosynthetic bacterium Chromatium minutissimum were examined within the expected spectral range of the optically forbidden S1 singlet state of carotenoids. LH2 preparations isolated from wild-type and carotenoid-depleted cells were used. 100-fs laser pulses in the range of 1300-1490 nm with an energy of 7-9 mW (corresponding to one-photon absorption between 650 and 745 nm) were used for two-photon fluorescence excitation. It was shown that two-photon fluorescence excitation spectra of LH2 complex from wild and carotenoid-depleted cells are very similar to each other and to the two-photon fluorescence excitation spectrum of bacteriochlorophyll a in acetone. It was concluded that direct two-photon excitation of bacteriochlorophyll a determines the fluorescence of both samples within the 650-745 nm spectral range.     

Excitation spectra and brightness optimization of two-photon excited probes

Two-photon probe excitation data are commonly presented as absorption cross section or molecular brightness (the detected fluorescence rate per molecule). We report two-photon molecular brightness spectra for a diverse set of organic and genetically encoded probes with an automated spectroscopic system based on fluorescence correlation spectroscopy. The two-photon action cross section can be extracted from molecular brightness measurements at low excitation intensities, while peak molecular brightness (the maximum molecular brightness with increasing excitation intensity) is measured at higher intensities at which probe photophysical effects become significant. The spectral shape of these two parameters was similar across all dye families tested. Peak molecular brightness spectra, which can be obtained rapidly and with reduced experimental complexity, can thus serve as a first-order approximation to cross-section spectra in determining optimal wavelengths for two-photon excitation, while providing additional information pertaining to probe photostability. The data shown should assist in probe choice and experimental design for multiphoton microscopy studies. Further, we show that, by the addition of a passive pulse splitter, nonlinear bleaching can be reduced--resulting in an enhancement of the fluorescence signal in fluorescence correlation spectroscopy by a factor of two. This increase in fluorescence signal, together with the observed resemblance of action cross section and peak brightness spectra, suggests higher-order photobleaching pathways for two-photon excitation.     

Red fluorescent protein from Discosoma as a fusion tag and a partner for fluorescence resonance energy transfer

The biochemical and biophysical properties of a red fluorescent protein from a Discosoma species (DsRed) were investigated. The recombinant DsRed expressed in E. coli showed a complex absorption spectrum that peaked at 277, 335, 487, 530, and 558 nm. Excitation at each of the absorption peaks produced a main emission peak at 583 nm, whereas a subsidiary emission peak at 500 nm appeared with excitation only at 277 or 487 nm. Incubation of E. coli or the protein at 37 degrees C facilitated the maturation of DsRed, resulting in the loss of the 500-nm peak and the enhancement of the 583-nm peak. In contrast, the 500-nm peak predominated in a mutant DsRed containing two amino acid substitutions (Y120H/K168R). Light-scattering analysis revealed that DsRed proteins expressed in E. coli and HeLa cells form a stable tetramer complex. DsRed in HeLa cells grown at 37 degrees C emitted predominantly at 583 nm. The red fluorescence was imaged using a two-photon laser (Nd:YLF, 1047 nm) as well as a one-photon laser (He:Ne, 543.5 nm). When fused to calmodulin, the red fluorescence produced an aggregation pattern only in the cytosol, which does not reflect the distribution of calmodulin. Despite the above spectral and structural complexity, fluorescence resonance energy transfer (FRET) between Aequorea green fluorescent protein (GFP) variants and DsRed was achieved. Dynamic changes in cytosolic free Ca2+ concentrations were observed with red cameleons containing yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), or Sapphire as the donor and RFP as the acceptor, using conventional microscopy and one- or two-photon excitation laser scanning microscopy. Particularly, the use of the Sapphire-DsRed pair rendered the red cameleon tolerant of acidosis occurring in hippocampal neurons, because both Sapphire and DsRed are extremely pH-resistant.     

Fluorescence properties of labeled proteins near silver colloid surfaces

The fluorescence properties of a monolayer of labeled avidin molecules were studied near silver island films. We first adsorbed a monolayer of biotinylated-BSA as a base that was used to capture labeled avidin molecules. For labeled avidin on silver island films, we observed an increase of the fluorescence intensity of between 18 and 80 with one-photon excitation and up to several hundredfold or larger with two-photon excitation. The probes were moderately more photostable in the presence of silver islands. There was also a dramatic decrease in the lifetimes with the amplitude-weighted values decreasing from 7- to 35-fold. The data suggest that these spectral changes are due to both increased rates of excitation near the metallic particles and increases in the rates of radiative decay. Because these silver island surfaces are very heterogeneous, we are hopeful that larger increases in intensity and photostability can be obtained for probes situated at an optimal distance from the ideal island surfaces.     

Picosecond multiphoton scanning near-field optical microscopy.

We have implemented simultaneous picosecond pulsed two- and three-photon excitation of near-UV and visible absorbing fluorophores in a scanning near-field optical microscope (SNOM). The 1064-nm emission from a pulsed Nd:YVO4 laser was used to excite the visible mitochondrial specific dye MitoTracker Orange CM-H2TMRos or a Cy3-labeled antibody by two-photon excitation, and the UV absorbing DNA dyes DAPI and the bisbenzimidazole BBI-342 by three-photon excitation, in a shared aperture SNOM using uncoated fiber tips. Both organelles in human breast adenocarcinoma cells (MCF 7) and specific protein bands on polytene chromosomes of Drosophila melanogaster doubly labeled with a UV and visible dye were readily imaged without photodamage to the specimens. The fluorescence intensities showed the expected nonlinear dependence on the excitation power over the range of 5-40 mW. An analysis of the dependence of fluorescence intensity on the tip-sample displacement normal to the sample surface revealed a higher-order function for the two-photon excitation compared to the one-photon mode. In addition, the sample photobleaching patterns corresponding to one- and two-photon modes revealed a greater lateral confinement of the excitation in the two-photon case. Thus, as in optical microscopy, two-photon excitation in SNOM is confined to a smaller volume.     

Gold@Silver@Gold Core Double-Shell Nanoparticles: Synthesis and Aggregation-Enhanced Two-Photon Photoluminescence Evaluation

A facile, straightforward, and low-cost method is proposed to synthesize gold@silver@gold core double-shell nanoparticles. The technique is a seed-mediated growth protocol that contains four steps of (1) gold seed synthesis, (2) gold seed growth, (3) silver layer coating through silver salt reduction, and (4) gold layer deposition via gold precursor reduction. The prepared nanoparticles had a narrow size distribution and the average particle size of 28 ± 1 nm. Cysteine was introduced to the nanoparticles solution as a coupling agent to assemble nanoparticles. Aggregation-induced two-photon photoluminescence enhancement of three types assembled nanoparticles, i.e., gold@silver@gold, gold@silver, and gold nanoparticles, was studied. It was observed that the assembled core double-shell nanoparticles presented huge enhancement in two-photon photoluminescence signal in comparison with two other nanoparticles. Moreover, the gold@silver@gold nanoparticle is a stable and biocompatible plasmonic nanosystem. This paper provides a novel candidate for two-photon photoluminescence excitation sensing and imaging for biomedical applications.

     

Dual-photon excitation of albumin

Fluorescence emission after two-photon excitation at 580 nm is observed in albumin by means of Nd:YAG laser at room temperature. The two-photon excitation spectral range 550-590 nm was obtained. The experimental results show that albumin fluorescence originates from tryptophan residues.     

Multiphoton excitation fluorescence microscopy and spectroscopy of in vivo human skin

Multiphoton excitation microscopy at 730 nm and 960 nm was used to image in vivo human skin autofluorescence from the surface to a depth of approximately 200 microm. The emission spectra and fluorescence lifetime images were obtained at selected locations near the surface (0-50 microm) and at deeper depths (100-150 microm) for both excitation wavelengths. Cell borders and cell nuclei were the prominent structures observed. The spectroscopic data suggest that reduced pyridine nucleotides, NAD(P)H, are the primary source of the skin autofluorescence at 730 nm excitation. With 960 nm excitation, a two-photon fluorescence emission at 520 nm indicates the presence of a variable, position-dependent intensity component of flavoprotein. A second fluorescence emission component, which starts at 425 nm, is observed with 960-nm excitation. Such fluorescence emission at wavelengths less than half the excitation wavelength suggests an excitation process involving three or more photons. This conjecture is further confirmed by the observation of the super-quadratic dependence of the fluorescence intensity on the excitation power. Further work is required to spectroscopically identify these emitting species. This study demonstrates the use of multiphoton excitation microscopy for functional imaging of the metabolic states of in vivo human skin cells.     

Amplified protein sensing using deep purple fluorophores on homogeneous Au substrates

We report here the first observation of appreciable enhancement of fluorescence induced by large Au colloids. Au monolayers with Au colloids of 40 nm, 59 nm, and 81 nm in radii, were formed on silane modified glass surfaces, respectively. The nanoparticle densities were varied by varying the deposition times and documented by scanning electron microscopy. Two types of samples were prepared, with large inter-particle distance and hence little or no inter-particle coupling and small inter-particle distance with inter-particle coupling. The fluorescence enhancement was examined by using a self assembled monolayer of the fluorophore-protein conjugate with Deep Purple as a fluorophore and Bovine Serum Albumin as protein (DP-BSA). The data show the over 15 fold enhancement under optimized conditions and reveal strong variations with both inter-particle distance and particle size. Nanostructures of appropriate size with optimized inter-particle distance thus prepared can produce promising substrates for decent fluorescence enhancement. We demonstrated that the Au colloid monolayers on glass surfaces are promising substrates for fluorescence enhancement with outstanding macroscopic homogeneity. This important feature will pave the way for the application of our substrates in biotechnology and life sciences such as imaging and sensing of biomolecules in proteomics.