Carey K. Johnson

Two-photon microscopy with wavelength switchable fiber laser excitation

Two-photon scanning fluorescence microscopy has become a powerful tool for imaging living cells and tissues. Most applications of two-photon microscopy employ a Ti:sapphire laser excitation source, which is not readily portable or rapidly tunable. This work explores the use of two-photon fiber laser excitation (TP-FLEX) as an excitation source for scanning two-photon microscopy. We have further demonstrated the use of a photonic crystal fiber (PCF) for facile tuning of the excitation wavelength over the range from 810 nm to 1100 nm. We generated two-photon fluorescence images at excitation wavelengths from 850 nm to 1100 nm detected on a scanning-stage microscope. By PCF wavelength tuning the dye BODIPY fl was selectively excited at 1000 nm whereas MitoTracker red was excited preferentially at 1100 nm. We discuss the potential for fiber laser sources coupled with PCF wavelength tuning as an attractive tunable excitation source for two-photon scanning fluorescence microscopy.

Time-resolved anisotropic two-photon spectroscopy

Abstract Two-photon excitation (TPE) in a randomly oriented liquid sample generates an anisotropic distribution of excitations that can be probed by a secondary spectroscopic transition, e.g. fluorescence or transient absorption. The orientation dependence of the secondary transition dipole (and therefore of the fluorescence or transient-absorption signal) can be exploited to generate information on molecular symmetry and orientation that is not available in two-photon absorption alone. A theoretical formalism is developed here for the orientationally averaged two-photon absorptivity taking into account the orientation of the secondary transition dipole. Spherical tensor formalism is employed to distinguish isotropic and anisotropic components of the two-photon absorptivity, and several two-photon anisotropy parameters are defined. The theory describes time-resolved detection of TPE by transient-absorption or fluorescence decay measurements. Time-resolved measurements with time-resolution that is fast relative to the rotational correlation times are shown to provide new information, including the two-photon anisotropies. Applications to TPE fluorescence anisotropy measurements and TPE induced anisotropic transient absorption are described, and illustrated by experimental measurements on bacteriorhodopsin.

Fluorescence Properties of Fluorescein, Tetramethylrhodamine and Texas Red Linked to a DNA Aptamer¶

Abstract We report the picosecond time-scale fluorescence dynamics of a dye-labeled DNA oligonucleotide or “aptamer” designed to bind specifically to Immunoglobulin E. Comparison of the photophysics of Texas Red (TR), fluorescein and 5′-carboxytetramethylrhodamine (TAMRA)-labeled aptamers reveals surprising differences with significant implications for measurements of oligonucleotide structure and dynamics. The fluorescence decay of the TR-aptamer is a simple single exponential with a weak temperature dependence. The fluorescence decay of the fluorescein-aptamer (fl-aptamer) is pH dependent and displays a complex temperature dependence with significant changes on melting of the aptamer tertiary structure. Despite its similarities to TR, TAMRA is strongly quenched when conjugated to the aptamer and displays complex fluorescence kinetics best described by a distributed rate model. Using the maximum entropy method, we have discovered two highly temperature-dependent fluorescence lifetimes for the TAMRA-aptamer. One of these lifetimes is similar to that of free TAMRA and displays the same temperature dependence. The other lifetime is quenched and displays a temperature dependence characteristic of a charge transfer reaction. These data set TR apart as an attractive alternative to TAMRA and fluorescein for studies such as fluorescence polarization and fluorescence resonance energy transfer, where environmental sensitivity of the probe is not desired.

Influence of temperature and viscosity on anthracene rotational diffusion in organic solvents: Molecular dynamics simulations and fluorescence anisotropy study

Molecular dynamics simulations and fluorescence anisotropy decay measurements are used to investigate the rotational diffusion of anthracene in two organic solvents—cyclohexane and 2-propanol—at several temperatures. Molecular dynamics simulations of 1 ns length were performed for anthracene in cyclohexane (at 280, 296, and 310 K) and in 2-propanol (at 296 K). The calculated time constants for reorientation of the short in-plane axis were 7–9 and 11–16 ps at 296 K in cyclohexane and 2-propanol, respectively, in excellent agreement with corresponding fluorescence depolarization measurements of 8 and 14 ps. The measured rotational reorientation times and the calculated average rotational diffusion coefficients varied in accord with Debye–Stokes–Einstein theory. Their magnitudes were close to values predicted for an ellipsoid of shape and size equivalent to an anthracene molecule, and exhibited predictable variation with external conditions—increasing with temperature and decreasing with solvent viscosity. H...

Fluorescence and Rotational Dynamics of Dityrosine

We have examined the lifetimes and rotational correlation times of dityrosine emission by time-correlated single-photon counting. We first noticed dityrosine fluorescence in samples of tyrosine and tyrosine dipeptides by its characteristic red-shifted emission at 400 to 430 nm. The longer rotational correlation time relative to tyrosine proved that this fluorescence emanated from a distinct species. Comparison with the fluorescence properties of synthesized dityrosine established the identity of the emitting species. Fluorescence intensity decays of dityrosine are generally characterized by two decay components, one with a lifetime in the range of 150 to 800 ps and another between 2.5 and 4.5 ns. We found no evidence for an excited-state reaction, since a rising phase (negative-amplitude component) was not observed. In the pH range from 4 to 10, two ground-state species exist in equilibrium with pKa ≈ 7. Both species exhibit two fluorescence decays. The average fluorescence lifetime increases gradually with pH over the pH range from 4 to 10 and decreases at pH 2. Anisotropy decays were measured for dityrosine and the alanine–dityrosine–alanine and leucine–dityrosine–leucine dipeptides. The rotational correlation times of dityrosine and dityrosine dipeptides increase linearly with van der Waals volumes. The slope indicates a stronger solute–solvent interaction than predicted with “stick” boundary conditions. It is suggested that these interactions result from the presence of two zwitterionic pairs.

Nonresonant hyper‐Raman and hyper‐Rayleigh scattering in benzene and pyridine

Nonresonant hyper‐Raman and hyper‐Rayleigh spectra excited at 1064 nm are reported for neat benzene and pyridine. The theory of Herzberg–Teller vibronic coupling in nonresonant and preresonant hyper‐Raman scattering is developed. Nonresonant hyper‐Raman scattering is shown to be vibronically induced by modes that efficiently couple strongly allowed one‐photon and two‐photon transitions. A weak and broad (55 cm−1) hyper‐Rayleigh band was observed in benzene and attributed to collective scattering, while in pyridine, a much more intense and much narrower hyper‐Rayleigh band was observed. Only the a2u vibration (ν11) was observed in the hyper‐Raman spectrum of benzene, while several strong bands were observed in pyridine. Possible vibronic‐coupling pathways are discussed for these modes. In addition, the observed hyper‐Raman spectrum of pyridine is compared to a recent calculation.

Detecting intramolecular dynamics and multiple Förster resonance energy transfer states by fluorescence correlation spectroscopy.

Fluorescence correlation spectroscopy (FCS) is a robust method for the detection of intramolecular dynamics in proteins but is also susceptible to interference from other dynamic processes such as triplet kinetics and photobleaching. We describe an approach for the detection of intramolecular dynamics in proteins labeled with a FRET dye pair based on global fitting to the two autocorrelation functions (green-green and red-red) and the two cross-correlation functions (green-red and red-green). We applied the method to detect intramolecular dynamics in the Ca(2+) signaling protein calmodulin. Dynamics were detected on the 100 mus time scale in Ca(2+)-activated calmodulin, whereas in apocalmodulin dynamics were not detected on this time scale. Control measurements on a polyproline FRET construct (Gly-Pro(15)-Cys) demonstrate the reliability of the method for isolating intramolecular dynamics from other dynamic processes on the microsecond time scale and confirm the absence of intramolecular dynamics of polyproline. We further show the sensitivity of the initial amplitudes of the FCS auto- and cross-correlation functions to the presence of multiple FRET states, static or dynamic. The FCS measurements also show that the diffusion of Ca(2+)-calmodulin is slower than that of apocalmodulin, indicating either a larger average hydrodynamic radius or shape effects resulting in a slower translational diffusion.

Time-resolved two-photon induced anisotropy decay: The rotational diffusion regime

Two‐photon excitation (TPE) of randomly oriented chromophores in solution generates an anisotropic distribution. In a previous paper [Chem. Phys. 179, 513 (1994)], the polarization dependence of the TPE signal probed by a secondary spectroscopic transition (fluorescence or transient absorption) was determined. In this paper, the time dependence of anisotropic two‐photon induced fluorescence or transient absorption signals due to rotational diffusion is treated in spherical tensor formalism. The two‐photon signal in general contains isotropic (orientation independent) and anisotropic (orientation dependent) contributions. The latter decay with up to five exponential components. Four time‐dependent anisotropy parameters can be defined and measured, allowing additional information, not available in conventional one‐photon fluorescence depolarization measurements, to be determined. The special case of one‐color TPE is discussed in particular. It is shown that by measurement of the linear and circular anisotro...

Cometary airbursts and atmospheric chemistry: Tunguska and a candidate Younger Dryas event

We find agreement between models of atmospheric chemistry changes from ionization for the A.D. 1908 Tunguska (Siberia region, Russia) airburst event and nitrate enhancement in Greenland Ice Sheet Project 2 (GISP2H and GISP2) ice cores, plus an unexplained ammonium spike. We then consider a candidate cometary impact at the Younger Dryas onset (YD). The large estimated NO x production and O 3 depletion are beyond accurate extrapolation, but the ice core peak is much lower, possibly because of insufficient sampling resolution. Ammonium and nitrate spikes in both Greenland Ice Core Project (GRIP) and GISP2 ice cores have been attributed to biomass burning at the onset of the YD. A similar result is well resolved in Tunguska ice core data, but that forest fire was far too small to account for this. Direct input of ammonia from a comet into the atmosphere is adequate for YD ice core data, but not for the Tunguska data. An analog of the Haber process with hydrogen contributed by cometary or surface water, atmospheric nitrogen, high pressures, and possibly catalytic iron from a comet could in principle produce ammonia, accounting for the peaks in both data sets.

FRET-FCS detection of intralobe dynamics in calmodulin.

Fluorescence correlation spectroscopy (FCS) can be coupled with Förster resonance energy transfer (FRET) to detect intramolecular dynamics of proteins on the microsecond time scale. Here we describe application of FRET-FCS to detect fluctuations within the N-terminal and C-terminal domains of the Ca(2+)-signaling protein calmodulin. Intramolecular fluctuations were resolved by global fitting of the two fluorescence autocorrelation functions (green-green and red-red) together with the two cross-correlation functions (green-red and red-green). To match the Förster radius for FRET to the dimensions of the N-terminal and C-terminal domains, a near-infrared acceptor fluorophore (Atto 740) was coupled with a green-emitting donor (Alexa Fluor 488). Fluctuations were detected in both domains on the time scale of 30 to 40 μs. In the N-terminal domain, the amplitude of the fluctuations was dependent on occupancy of Ca(2+) binding sites. A high amplitude of dynamics in apo-calmodulin (in the absence of Ca(2+)) was nearly abolished at a high Ca(2+) concentration. For the C-terminal domain, the dynamic amplitude changed little with Ca(2+) concentration. The Ca(2+) dependence of dynamics for the N-terminal domain suggests that the fluctuations detected by FCS in the N-terminal domain are coupled to the opening and closing of the EF-hand Ca(2+)-binding loops.