Selective excitation and analysis of NaF:Cu+electronic transitions using two‐color, two‐photon spectroscopy

1989 ◽  
Vol 90 (8) ◽  
pp. 3915-3930 ◽  
Author(s):  
John M. Berg ◽  
Donald S. McClure
2002 ◽  
Vol 68 (2) ◽  
pp. 901-909 ◽  
Author(s):  
Thomas R. Neu ◽  
Ute Kuhlicke ◽  
John R. Lawrence

ABSTRACT A major limitation for the use of two-proton laser scanning microscopy (2P-LSM) in biofilm and other studies is the lack of a thorough understanding of the excitation-emission responses of potential fluorochromes. In order to use 2P-LSM, the utility of various fluorochromes and probes specific for a range of biofilm constituents must be evaluated. The fluorochromes tested in this study included classical nucleic acid-specific stains, such as acridine orange (AO) and 4",6"-diamidino-2-phenylindole (DAPI), as well as recently developed stains. In addition, stains specific for biofilm extracellular polymeric substances (EPS matrix components) were tested. Two-photon excitation with a Ti/Sapphire laser was carried out at wavelengths from 760 to 900 nm in 10-nm steps. It was found that autofluorescence of phototrophic organisms (cyanobacteria and green algae) resulted in strong signals for the entire excitation range. In addition, the coenzyme F420-related autofluorescence of methanogenic bacteria could be used to obtain images of dense aggregates (excitation wavelength, 780 nm). The intensities of the emission signals for the nucleic acid-specific fluorochromes varied. For example, the intensities were similar for excitation wavelengths ranging from 780 to 900 nm for AO but were higher for a narrower range, 780 to 810 nm, for DAPI. In selective excitation, fading, multiple staining, and combined single-photon-two-photon studies, the recently developed nucleic acid-specific fluorochromes proved to be more suitable regardless of whether they are intended for living or fixed samples. Probes specific for proteins and glycoconjugates allowed two-photon imaging of polymeric biofilm constituents. Selective excitation-emission was observed for Calcofluor White M2R (780 to 800 nm) and SyproOrange (880 to 900 nm). In addition, fluor-conjugated concanavalin A lectins were examined and provided acceptable two-photon emission signals at wavelengths ranging from 780 to 800 nm. Finally, CellTracker, a fluorochrome suitable for long-term labeling of microbial eucaryote cells, was found to give strong emission at wavelengths ranging from 770 to 810 nm. If fluorochromes have the same two-photon excitation cross section, they are suitable for multiple staining and multichannel recording. Generally, if an appropriate excitation wavelength and fluorochrome were used, it was possible to obtain more highly resolved images for thick biofilm samples with two-photon laser microscopy than with conventional single-photon laser microscopy. Due to its potential for higher resolution in light-scattering tissue-like material, such as biofilms, and extremely localized excitation, 2P-LSM is a valuable addition to conventional confocal laser scanning microscopy with single-photon excitation. However, further development of the method and basic research are necessary to take full advantage of nonlinear excitation in studies of interfacial microbial ecology.


2010 ◽  
Vol 102 (2) ◽  
pp. 435-439 ◽  
Author(s):  
Daniel S. Correa ◽  
Vinicius Tribuzi ◽  
Marcos R. Cardoso ◽  
Lino Misoguti ◽  
Cleber R. Mendonca

2010 ◽  
Vol 14 (04) ◽  
pp. 314-323 ◽  
Author(s):  
Joseph M. Beames ◽  
Timothy D. Vaden ◽  
Andrew J. Hudson

We will present resonant two-photon ionization spectra for meso-tetraphenylporphyrin, H 2 TPP , measured under isolated conditions. The polycrystalline compound was vaporized, in vacuo, using both thermal and laser desorption, and seeded into a supersonic expansion of an inert-carrier gas. The molecules remain largely intact in the gaseous phase. However, the two techniques for vaporizing H 2 TPP give different internal temperatures for the isolated substrate, with greater vibrational cooling achieved using laser desorption. A comparison of the peak positions and intensities in the resonant two-photon ionization spectra of thermal- and laser-desorbed molecules provides an insight into the vibrational structure of the Q band. In particular, the greater contribution made by electronic transitions originating from higher vibrational levels in the ground state of H 2 TPP is emphasized. We conclude that vibronic coupling in the ground electronic state plays an important role in a quantum-mechanical interpretation of the Q band.


1999 ◽  
Vol 5 (S2) ◽  
pp. 500-501
Author(s):  
Henryk Malak

A dye, Phloxine B, a common food coloring and one of the active components of a photoreactive insecticide was recently approved by Food and Drug Administration as D&C Red #28 for use in drugs and cosmetics. Phloxine B is also one of the most widely use stain in fluorescence microscopy. However, in spite of the widespread interest in multi-photon spectroscopy and imaging, no information is available on the electronic transitions properties of Phloxine B with red edge fluorescence excitation and with multi-photon excitation.In the present report we described the steady state and time-resolved fluorescent properties of Phloxine B with up-convert photon excitation and with two-photon excitation. We examined the electronic transitions properties of Phloxine B when was excited by femtosecond pulses from a mode-locked titanium sapphire laser or a mode-locked optical parametric oscillator laser. Phloxine B under excitation wavelengths above 775 nm was found to display two-photon excitation fluorescence with a spectrum maximum at 580 nm, which emission is consistent with one-photon excitation fluorescence spectrum. At the red edge excitation of Phloxine B, from 590 nm to 650 nm, we observed one-photon excitation fluorescence indicating that Phloxine B behaves like upconverting dye with one-photon excitation process.


2010 ◽  
Vol 498 (1-3) ◽  
pp. 18-21 ◽  
Author(s):  
Ditte Mogensen ◽  
Kurt V. Mikkelsen ◽  
Henrik G. Kjaergaard

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