Electron Energy Loss and One- and Two-Photon Excited SERS Probing of “Hot” Plasmonic Silver Nanoaggregates

We report electron energy loss spectroscopy (EELS) and one- and two-photon excited surface-enhanced Raman scattering (SERS) and hyper Raman studies on plasmonic silver nanoaggregates. By comparison with computations, EELS imaging reveals an inverse relationship between local field intensity in an optical experiment and electron energy loss intensity at energies corresponding to excitation wavelengths used for optical probing. This inverse relation exists independent on specific nanoaggregate geometries and is mainly controlled by the gap size between the particles forming the aggregate. The ratio between two- and one-photon excited SERS measured at different excitation wavelengths provides information about local fields in the hottest spots and their dependence on the photon energy. Our data verify experimentally the predicted increase of local optical fields in the hot spots with increasing wave lengths. The reported findings show new experimental ways to characterize local fields of plasmonic nanostructures. This is of particular importance for complex structures which are not easily approachable by computations.     

Resonance Raman scattering of cytochrome P450 BM3 and effect of imidazole inhibitors

Resonance Raman scattering from cytochrome P450 BM3 is obtained with a Raman microprobe using 406-nm excitation with an accumulation time of a few seconds. The small sample size and rapid measurement time make the routine characterization of P450 systems by resonance Raman spectroscopy easier. Addition of imidazole and imidazole derivatives as inhibitors causes the appearance of additional peaks due to vinyl modes, increases the relative intensity of symmetric modes that would be A(1g) in D(4h) symmetry, and causes a large drop in the intensity of nu(11). This information indicates that the ligation of imidazoles to the heme iron causes the alignment of the vinyl modes with the plane of the heme ring and reduces the out of plane distortion of the ring. The effect of both inhibitors is similar but there is a subtle difference in the extent of the reduction in the intensity of nu(11), which suggests that steric effects within the pocket are having some effect.     

Surface Plasmon Resonances of Metallic Nanostars/Nanoflowers for Surface-Enhanced Raman Scattering

We investigate theoretically the optical properties associated to plasmon resonances of metal nanowires with cross section given by low-order Chebyshev nanoparticles (like rounded-tip nanostars or nanoflowers). The impact of the nanoflower shape is analyzed for varying symmetry and deformation parameter through the spectral dependence of resonances and their corresponding near field distributions. Large field intensity enhancements are obtained at the gaps between petals, apart from the tips themselves, which make these nanostars/nanoflowers specially suitable to host molecules for surface-enhanced Raman scattering sensing applications.     

Photobleaching in two-photon excitation microscopy

The intensity-squared dependence of two-photon excitation in laser scanning microscopy restricts excitation to the focal plane and leads to decreased photobleaching in thick samples. However, the high photon flux used in these experiments can potentially lead to higher-order photon interactions within the focal volume. The excitation power dependence of the fluorescence intensity and the photobleaching rate of thin fluorescence samples ( approximately 1 microm) were examined under one- and two-photon excitation. As expected, log-log plots of excitation power versus the fluorescence intensity and photobleaching rate for one-photon excitation of fluorescein increased with a slope of approximately 1. A similar plot of the fluorescence intensity versus two-photon excitation power increased with a slope of approximately 2. However, the two-photon photobleaching rate increased with a slope > or =3, indicating the presence of higher-order photon interactions. Similar experiments on Indo-1, NADH, and aminocoumarin produced similar results and suggest that this higher-order photobleaching is common in two-photon excitation microscopy. As a consequence, the use of multi-photon excitation microscopy to study thin samples may be limited by increased photobleaching.     

Surface-enhanced resonance Raman spectroscopy of porphyrin and metalloporphyrin species in systems with Ag nanoparticles and their assemblies

The advantages of systems with Ag nanoparticles and their assemblies for surface-enhanced resonance Raman scattering (SERRS) spectral investigation, detection and determination of porphyrin species are demonstrated. SERRS spectral detection limits of the testing porphyrin species (including porphyrin aggregates) in these systems are shown to be, on average, 10(2)-10(3) lower than detection limits by resonance Raman scattering (RRS). Systems with Ag nanoparticles modified by anionic organosulfur spacers enable us to obtain SERRS spectra of unperturbed cationic porphyrin species. In the case of thiopheneacetate-modified Ag particles prepared by laser ablation, no negative effect of the spacer on the spectral detection limit of the porphyrin was observed. Systems with isolated Ag nanoparticles allow for obtaining SERRS spectra of porphyrin species upon excitation into the Soret electronic absorption band which leads to at least a 10-fold decrease in the detection limit.     

Oblique scanning 2‐photon light‐sheet fluorescence microscopy for rapid volumetric imaging

Light‐sheet fluorescence microscopy (LSFM) is a powerful tool for biological studies because it allows for optical sectioning of dynamic samples with superior temporal resolution. However, LSFM using 2 orthogonally co‐aligned objectives requires a special sample geometry, and volumetric imaging speed is limited due to physical sample translation. This paper describes an oblique scanning 2‐photon LSFM (OS‐2P‐LSFM) that eliminates these limitations by using a single objective near the sample and a refractive scanning‐descanning system. This system also provides improved light‐sheet confinement against scattering by using a 2‐photon Bessel beam. The OS‐2P‐LSFM hold promise for studying structural, functional and dynamic aspects of living tissues and organisms because it allows for high‐speed, translation‐free and scattering‐robust 3D imaging of large biological specimens.      

The photon counting histogram in fluorescence fluctuation spectroscopy.

Fluorescence correlation spectroscopy (FCS) is generally used to obtain information about the number of fluorescent particles in a small volume and the diffusion coefficient from the autocorrelation function of the fluorescence signal. Here we demonstrate that photon counting histogram (PCH) analysis constitutes a novel tool for extracting quantities from fluorescence fluctuation data, i.e., the measured photon counts per molecule and the average number of molecules within the observation volume. The photon counting histogram of fluorescence fluctuation experiments, in which few molecules are present in the excitation volume, exhibits a super-Poissonian behavior. The additional broadening of the PCH compared to a Poisson distribution is due to fluorescence intensity fluctuations. For diffusing particles these intensity fluctuations are caused by an inhomogeneous excitation profile and the fluctuations in the number of particles in the observation volume. The quantitative relationship between the detected photon counts and the fluorescence intensity reaching the detector is given by Mandel's formula. Based on this equation and considering the fluorescence intensity distribution in the two-photon excitation volume, a theoretical expression for the PCH as a function of the number of molecules in the excitation volume is derived. For a single molecular species two parameters are sufficient to characterize the histogram completely, namely the average number of molecules within the observation volume and the detected photon counts per molecule per sampling time epsilon. The PCH for multiple molecular species, on the other hand, is generated by successively convoluting the photon counting distribution of each species with the others. The influence of the excitation profile upon the photon counting statistics for two relevant point spread functions (PSFs), the three-dimensional Gaussian PSF conventionally employed in confocal detection and the square of the Gaussian-Lorentzian PSF for two photon excitation, is explicitly treated. Measured photon counting distributions obtained with a two-photon excitation source agree, within experimental error with the theoretical PCHs calculated for the square of a Gaussian-Lorentzian beam profile. We demonstrate and discuss the influence of the average number of particles within the observation volume and the detected photon counts per molecule per sampling interval upon the super-Poissonian character of the photon counting distribution.     

Laser irradiation and Raman spectroscopy of single living cells and chromosomes: Sample degradation occurs with 514.5 nm but not with 660 nm laser light

In Raman spectroscopic measurements of single cells (human lymphocytes) and chromosomes, using a newly developed confocal Raman microspectrometer and a laser excitation wavelength of 514.5 nm, degradation of the biological objects was observed. In the experiments high power microscope objectives were used, focusing the laser beam into a spot approximately 0.5 micron in diameter. At the position of the laser focus a paling of the samples became visible even when the laser power on the sample was reduced to less than 1 mW. This was accompanied by a gradual decrease in the intensity of the Raman signal. With 5 mW of laser power the events became noticeable after a period of time in the order of minutes. It is shown that a number of potential mechanisms, such as excessive sample heating due to absorption of laser light, multiple photon absorption, and substrate heating are unlikely to play a role. In experiments with DNA solutions and histone protein solutions no evidence of photo damage was found using laser powers up to 25 mW. No degradation of cells and chromosomes occurs when laser light of 660 nm is used. The most plausible explanation therefore seems to be that the sample degradation is the result of photochemical reactions initiated by laser excitation at 514.5 nm of as yet unidentified sensitizer molecules or complexes present in chromosomes and cells but not in purified DNA and histone protein samples.     

Use of dynamic light scattering to determine second virial coefficient in a semidilute concentration regime

The present work discusses an alternative procedure to obtain static light scattering (SLS) parameters in a dilute and semidilute concentration regime from a dynamic light scattering (DLS) instrument that uses an avalanche photodiode (APD) for recording the scattered intensity signal. An APD enables one to perform both SLS and DLS measurements by photon counting and photon correlation, respectively. However, due to the associated recovery time, the APDs are susceptible to saturation (above 1000 kcps), which may limit the measurements in systems that scatter too much light. We propose an alternative way of obtaining the SLS parameters with instruments that use APD for recording signal intensities.     

High-order photobleaching of green fluorescent protein inside live cells in two-photon excitation microscopy

Combination of green fluorescent protein (GFP) and two-photon excitation fluorescence microscopy (TPE) has been used increasingly to study dynamic biochemical events within living cells, sometimes even in vivo. However, the high photon flux required in TPE may lead to higher-order photobleaching within the focal volume, which would introduce misinterpretation about the fine biochemical events. Here we first studied the high-order photobleaching rate of GFP inside live cells by measuring the dependence of the photobleaching rate on the excitation power. The photobleaching rate under one- and two-photon excitation increased with 1-power and 4-power of the incident intensity, respectively, implying the excitation photons might interact with excited fluorophore molecules and increase the probability of photobleaching. These results suggest that in applications where two-photon imaging of GFP is used to study dynamic molecular process, photobleaching may ruin the imaging results and attention should be paid in interpreting the imaging results.     

Photosynthetic performance of phototrophic biofilms in extreme acidic environments

Photosynthesis versus irradiance curves and their associated photosynthetic parameters from different phototrophic biofilms isolated from an extreme acidic environment (Río Tinto, SW, Spain) were studied in order to relate them to their species composition and the physicochemical characteristics of their respective sampling locations. The results indicated that the biofilms are low light acclimated showing a photoinhibition model; only floating communities of filamentous algae showed a light saturation model. Thus, all the biofilms analysed showed photoinhibition over 60 μmol photon m(-2) s(-1) except in the case of Zygnemopsis sp. sample, which showed a light-saturated photosynthesis model under irradiations higher that 200 μmol photon m(-2) s(-1). The highest values of compensation light intensity (I(c)) were showed also by Zygnemosis sp. biofilm (c. 40 μmol photon m(-2) s(-1)), followed by Euglena mutabilis and Chlorella sp. samples (c. 20 μmol photon m(-2) s(-1)). The diatom sample showed the lowest I(c) values (c. 5 μmol photon m(-2) s(-1)). As far as we know this is the first attempt to determine the photosynthetic activity of low pH and heavy metal tolerant phototrophic biofilms, which may give light in the understanding of the ecological importance of these biofilms for the maintenance of the primary production of these extreme and unique ecosystems.     

Size Dependence of Nanoparticle-SERS Enhancement from Silver Film over Nanosphere (AgFON) Substrate

The dependence of nanoparticle size on surface-enhanced Raman scattering (SERS) from silver film over nanospheres substrate is studied. For a range of nanosphere sizes from 430 to 1,500 nm, optimum SERS signal is obtained with a nanosphere size of 1,000 nm at an excitation wavelength of 532 nm. We have clarified the physical origin of this optimization in an unambiguious way as due to resonant plasmonic excitations from 3D finite-difference time-domain simulations, as well as with the assistance of UV-visible reflectance spectrum.     

Conformational dependence of the Raman scattering intensities from polynucleotides

The intensity of Raman scattering from the various Raman active vibrations of poly-(riboadenylic acid), poly(ribocytidylic acid), poly(ribouridylic acid), and poly(riboinosinic acid) in moderately dilute solutions were examined as the temperature was changed to alter their conformation. It was found that certain highly intense, highly polarized Raman bands from the totally symmetric, i.e., in-plane, ring vibrations of the nucleic acid bases become less intense as the chains become more ordered in solution. Since these vibrations occur at frequencies which are markedly different for each type of base, Raman spectroscopy appears to provide a new method for the characterizing of the average conformation of each of the bases in solution. A theory for the resonant Raman effect is given in which it is shown that, a decrease in resonant Raman intensity is to be expected if one obtains a decrease in the intensity of the corresponding ultraviolet absorption band with which the incident light is resonant. If it is assumed that certain Raman bands derive their intensity predominantly from the first few ultraviolet absorption intensities, then a qualitative explanation of our observed conformational dependence of the ordinary Raman intensities can be obtained.     

Eliminating Unwanted Far-Field Excitation in Objective-Type TIRF. Part I. Identifying Sources of Nonevanescent Excitation Light

Total internal reflection fluorescence microscopy (TIRFM) achieves subdiffraction axial sectioning by confining fluorophore excitation to a thin layer close to the cell/substrate boundary. However, it is often unknown how thin this light sheet actually is. Particularly in objective-type TIRFM, large deviations from the exponential intensity decay expected for pure evanescence have been reported. Nonevanescent excitation light diminishes the optical sectioning effect, reduces contrast, and renders TIRFM-image quantification uncertain. To identify the sources of this unwanted fluorescence excitation in deeper sample layers, we here combine azimuthal and polar beam scanning (spinning TIRF), atomic force microscopy, and wavefront analysis of beams passing through the objective periphery. Using a variety of intracellular fluorescent labels as well as negative staining experiments to measure cell-induced scattering, we find that azimuthal beam spinning produces TIRFM images that more accurately portray the real fluorophore distribution, but these images are still hampered by far-field excitation. Furthermore, although clearly measureable, cell-induced scattering is not the dominant source of far-field excitation light in objective-type TIRF, at least for most types of weakly scattering cells. It is the microscope illumination optical path that produces a large cell- and beam-angle invariant stray excitation that is insensitive to beam scanning. This instrument-induced glare is produced far from the sample plane, inside the microscope illumination optical path. We identify stray reflections and high-numerical aperture aberrations of the TIRF objective as one important source. This work is accompanied by a companion paper (Pt.2/2).     

Eliminating Unwanted Far-Field Excitation in Objective-Type TIRF. Part I. Identifying Sources of Nonevanescent Excitation Light

Total internal reflection fluorescence microscopy (TIRFM) achieves subdiffraction axial sectioning by confining fluorophore excitation to a thin layer close to the cell/substrate boundary. However, it is often unknown how thin this light sheet actually is. Particularly in objective-type TIRFM, large deviations from the exponential intensity decay expected for pure evanescence have been reported. Nonevanescent excitation light diminishes the optical sectioning effect, reduces contrast, and renders TIRFM-image quantification uncertain. To identify the sources of this unwanted fluorescence excitation in deeper sample layers, we here combine azimuthal and polar beam scanning (spinning TIRF), atomic force microscopy, and wavefront analysis of beams passing through the objective periphery. Using a variety of intracellular fluorescent labels as well as negative staining experiments to measure cell-induced scattering, we find that azimuthal beam spinning produces TIRFM images that more accurately portray the real fluorophore distribution, but these images are still hampered by far-field excitation. Furthermore, although clearly measureable, cell-induced scattering is not the dominant source of far-field excitation light in objective-type TIRF, at least for most types of weakly scattering cells. It is the microscope illumination optical path that produces a large cell- and beam-angle invariant stray excitation that is insensitive to beam scanning. This instrument-induced glare is produced far from the sample plane, inside the microscope illumination optical path. We identify stray reflections and high-numerical aperture aberrations of the TIRF objective as one important source. This work is accompanied by a companion paper (Pt.2/2).     

Quantitation of individual proteins by a two-stage electrophoresis procedure

Increasing use of X-ray absorption spectroscopy (edge and EXAFS) to determine the local structure of active sites of biological molecules has caused greater interest in and attention to the nature of X-ray damage and the integrity of the sample. In a complementary way, efficient X-ray photon collection from the sample is required in order to maximize data acquisition and minimize sample damage. This report describes systems currently in use at Stanford Synchrotron Radiation Laboratory and Cornell High Energy Synchrotron Source, comprising two types of low-temperature techniques for sample protection and fast X-ray photon detection of large solid-angle collection.     

Statistical mechanical analysis of Raman spectroscopic order parameter changes in pressure-induced lipid bilayer phase transitions.

The statistical mechanical cluster theory of Fisher as applied by Kanehisa and Tsong to phospholipid bilayers is modified to describe the effects of hydrostatic pressure on the state of an aqueous dispersion of the phospholipid dipalmitoyl phosphatidylcholine. A high pressure Raman scattering cell has been built to obtain the Raman spectra of aqueous dispersions of phospholipids as a function of the applied hydrostatic pressure from 0 to 100 atmospheres. Predicted thermal and pressure-induced phase transitions are compared with an experimentally obtained Raman order parameter derived from the ratio of two bands in the C-H stretching region of the Raman spectrum of the sample. The parameters of the theory are adjusted to obtain a satisfactory fit of the Raman order parameter versus temperature. The theory is then found to give an excellent prediction of the observed pressure dependence of the Raman order parameter with no changes in the adjustable parameters. The implications of the success of the theoretical fit is discussed. Particularly of interest is the rather high value of the critical temperature, Tc, for lipid bilayers which is predicted by the model.     

Preparation of macromolecular complexes for cryo-electron microscopy

This protocol describes the preparation of frozen-hydrated single-particle specimens of macromolecular complexes. First, it describes how to create a grid surface coated with holey carbon by first inducing holes in a Formvar film to act as a template for the holey carbon that is stable under cryo-electron microscopy (cryo-EM) conditions and is sample-friendly. The protocol then describes the steps required to deposit the homogeneous sample on the grid and to plunge-freeze the grid into liquid ethane at the temperature of liquid nitrogen, so that it is suitable for cryo-EM visualization. It takes 4-5 h to make several hundred holey carbon grids and about 1 h to make the frozen-hydrated grids. The time required for sample purification varies from hours to days, depending on the sample and the specific procedure required. A companion protocol details how to collect cryo-EM data using an FEI Tecnai transmission electron microscope that can subsequently be processed to obtain a three-dimensional reconstruction of the macromolecular complex.     

Design and performance of an ultraviolet resonance Raman spectrometer for proteins and nucleic acids.

We describe an ultraviolet resonance Raman (UVRR) spectrometer appropriate for structural studies of biological macromolecules and their assemblies. Instrument design includes the following features: a continuous wave, intracavity doubled, ultraviolet laser source for excitation of the Raman spectrum; a rotating cell (or jet source) for presentation of the sample to the laser beam; a Cassegrain optic with f/1.0 aperture for collection of the Raman scattering; a quartz prism dispersing element for rejection of stray light and Rayleigh scattering; a 0.75-m single grating monochromator for dispersion of the Raman scattering; and a liquid-nitrogen-cooled, charge-coupled device for detection of the Raman photons. The performance of this instrument, assessed on the basis of the observed signal-to-noise ratios, the apparent resolution of closely spaced spectral bands, and the wide spectrometer bandpass of 2200 cm-1, is believed superior to previously described UVRR spectrometers of similar design. Performance characteristics of the instrument are demonstrated in UVRR spectra obtained from standard solvents, p-ethylphenol, which serves as a model for the tyrosine side chain, the DNA nucleotide deoxyguanosine-5'-monophosphate, and the human tumor necrosis factor binding protein, which is considered representative of soluble globular proteins.     

The resonance Raman spectrum of carotenoids as an intrinsic probe for membrane potential. Oscillatory changes in the spectrum of neurosporene in the chromatophores of Rhodopseudomonas sphaeroides.

The resonance Raman spectrum of the carotenoid neurosporene is shown to be a sensitive monitor of absorption shifts, and thus changes in membrane potential, in chromatophores of the GlC mutant of Rhodopseudomonas sphaeroides. For a Raman excitation wavelength at 472.7 nm, the intensities of the two most prominent resonance Raman features (v1 and v2) respond very differently to small shifts in the absorption maxima. Thus, the ratio intensity v1/intensity v2 is a sensitive probe for absorption shifts. Changes in this ratio of approximately 20% were observed during a valinomycin induced diffusion potential. At 5 degrees C changes in the average intensity ratio of +6, -4 and -14% were brought about by oligomycin, FCCP and sodium deoxycholate, respectively. The changes in intensity ratio were temperature dependent and, in addition, effects due to the laser beam acting as an actinic light could be detected. Oscillatory changes were observed in absolute Raman and Rayleigh scattering intensities for chromatophores at 5 degrees C and for intact cells under growing conditions.