Henryk Malak

A 10‐GHz frequency‐domain fluorometer

We developed a frequency‐domain fluorometer which operates from 4 to 2000 MHz. The modulated excitation is provided by the harmonic content of a laser pulse train (3.76 MHz, 5 ps) from a synchronously pumped and cavity dumped dye laser. The phase angle and modulation of the emission are measured with a microchannel‐plate photomultiplier (PMT). Cross‐correlation detection is performed outside the PMT. The high‐frequency signals for cross correlation were obtained by multiplication of the output from a 500‐MHz frequency synthesizer. The performance was verified in several ways, including measurement of known time delays and examination of standard fluorophores. The detector displayed no detectable color effect, with the 300–600‐nm difference being less than 5 ps. The precision of the measurements is adequate to detect differences of 20 ps for decay times of 500 ps. A correlation time of 53 ps was found for indole in water at 20 °C. The shortest correlation time we measured was 15 ps for indole in methanol/w...

Metal-ligand complexes as a new class of long-lived fluorophores for protein hydrodynamics.

We describe the use of asymmetric Ru-ligand complexes as a new class of luminescent probes that can be used to measure rotational motions of proteins. These complexes are known to display luminescent lifetimes ranging from 10 to 4000 ns. In this report, we show that the asymmetric complex Ru(bpy)2(dcbpy) (PF6)2 displays a high anisotropy value when excited in the long wavelength absorption band. For covalent linkage to proteins, we synthesized the N-hydroxy succinimide ester of this metal-ligand complex. To illustrate the usefulness of these probes, we describe the intensity and anisotropy decays of [Ru(bpy)2(dcbpy)] when covalently linked to human serum albumin, concanavalin A (ConA), human immunoglobulin G (IgG), and Ferritin, and measured in solutions of increased viscosity. These data demonstrate that the probes can be used to measure rotational motions on the 10 ns to 1.5 microseconds timescale, which so far has been inaccessible using luminescence methods. The present probe [Ru(bpy)2(dcbpy)] can be regarded as the first of a class of metal-ligand complexes, each with different chemical reactivity and spectral properties, for studies of macromolecular dynamics.

Three-photon excitation in fluorescence microscopy

We show experiments proving the feasibility of scanning fluorescence microscopy by three-photon excitation. Three-photon excitation fluorescence axial images are shown of polystyrene beads stained with the fluorophore 2,5-bis(4-biphenyl)oxazole (BBO). Three-photon excitation is performed at an excitation wavelength of 900 nm and with pulses of 130 fs duration provided by a mode-locked titanium sapphire laser. Fluorescence is collected between 350 and 450 nm. The fluorescence image signal features a third-order dependence on the excitation power, also providing intrinsic 3-D imaging. The resolution of a three-photon excitation microscope is increased over that of a comparable two-photon excitation microscope.

Optical assay for glucose based on the luminescnence decay time of the long wavelength dye Cy5

An optical assay for glucose is described based on the luminescence decay time of a long wavelength dye (Cy5) which can be excited with currently available red laser diodes. Concanavalin A was covalently labeled with Cy5 which served as the donor in an assay based on fluorescence resonance energy transfer (FRET). The acceptor was Malachite Green which was covalently linked to insulin which served as a carrier protein. To provide binding affinity for ConA Malachite Green insulin was also covalently labeled with maltose (MIMG). Binding of Cy5ConA to MIMG resulted in a decreased intensity and decay time of Cy5 as observed by time-correlated single photon counting. Glucose was detected by competitive displacement of MIMG from Cy5ConA, resulting in increased intensity and decay time. This glucose assay has several features which can result in practical real world assays for glucose. The long absorption wavelength of Cy5 allows excitation with red laser diodes, which can be readily pulsed or amplitude-modulated for time-domain or frequency-domain decay time measurements. Additionally, decay times can be measured through skin using long wavelength excitation and emission, suggesting the possibility of an implanted glucose sensor. And finally, the assay affinity and reversibility can in principle be adjusted by controlling the extent and type of sugar labeling of the carrier protein.

Fluorescence of reduced nicotinamides using one- and two-photon excitation

We examined the steady-state and time-resolved emission of NADH and NAMH resulting from one-photon and two-photon excitation. Similar emission spectra were observed for both modes of excitation. The fundamental anisotropy of NADH is near 0.54 for two-photon excitation from 690 to 740 nm, which is 46% higher than the value of 0.37 observed for one-photon excitation. This observation of a higher anisotropy with two-photon excitation was consistent with INDO/SDCI calculations of the one- and two-photon transitions. Minor differences in the multi-exponential decays of NADH were observed for one- and two-photon excitation, but presently available resolution does not allow us to conclude the decays are distinct. NADH-LADH-IBA complex formation led to an order of magnitude larger of the average lifetimes of NADH fluorescence resulting from one- and two-photon excitation. Fluorescence intensity and fluorescence anisotropy decays of NADH was double-exponential for both modes of excitation and show that the observed heterogeneity of the fluorescence decay kinetics of reduced nicotinamides arises from the inherent photoprocess of the dihydronicotinamide chromophore and not due to any intramolecular interactions with adenine part of NADH. Such interactions are responsible for the depolarization of NADH fluorescence observed for excitation wavelength below 300 nm for OPE and 600 nm for TPE, respectively. NADH displays a low cross-section for two-photon excitation which suggests that fluorescence from NADH will be moderately difficult to observe with two-photon fluorescence microscopy, and may not interfere with observations of TPIF of other extrinsic probes used to label cells.

Three-photon induced fluorescence of 2,5-diphenyloxazole with a femtosecond Ti:sapphire laser

Abstract We report emission spectra and time-resolved intensity and anisotropy decays resulting from three-photon excitation of fluorescence. The emission intensity of 2,5-diphenyloxazole (PPO) was found to depend on the third power of the excitation intensity at 870 nm. This wavelength is too long to excite PPO by a two-photon process. The emission spectrum, intensity decay, and rotational correlation times were found to be identical for one- (290 nm) and three-photon (870 nm) excitation. However, the time-zero anisotropy ( r 0 = 0.61) observed from the time-resolved anisotropy decay, and observed in frozen solution, was larger than for one- or two-photon excitation. The possibility of three-photon excitation with a Ti:sapphire laser suggests wide ranging applications in biophysics and fluorescence microscopy.

Two-Color Two-Photon Excitation of Fluorescence

Abstract— We report the observation of two‐photon excitation of an organic fluorophore with two different wavelengths, a phenomenon we refer to as two‐color two‐photon (2C2P) excitation. Ultraviolet emission of p‐Merphenyl at 340 nm was observed when the sample was illuminated with both 375 and 750 nm pulses from a picosecond dye laser. The emission of p‐terphenyl was about 100‐fold and more than 1000‐fold less for illumination at only 375 or 750 nm, respectively. Observation of the 2C2P signal required temporal and spatial overlap of the 375 and 750 nm pulses. The amplitude of the signal depended on the polarization of each beam. 2C2P excitation can have applications in fluorescence microscopy and elsewhere when spatially localized excitation is desirable.

Intramolecular dynamics in the environment of the single tryptophan residue in staphylococcal nuclease.

The dipole relaxational dynamics in the environment of a single tryptophan residue Trp-140 in staphylococcal nuclease was studied by time-resolved (multi-frequency phase-modulation) spectroscopy and selective red-edge excitation. The long-wavelength position of the fluorescence spectrum (at 343 nm) and the absence of red-edge excitation effects at 0 and 20 degrees C indicate that this residue is surrounded by very mobile protein groups which relax on the subnanosecond time scale. For these temperatures (0-20 degrees C) the steady-state emission spectra did not show the excitation-wavelength dependent shifts (red-edge effects) for excitation wavelengths from 295 to 308 nm; however, the anisotropy decay rate is slow (tens of nanoseconds). This suggests that the spectral relaxation is due to mobility of the surrounding groups rather than the motion of the tryptophan itself. The motions of the tryptophan surrounding are substantially retarded at reduced temperatures in viscous solvent (60% glycerol). The temperature dependence of the difference in position of fluorescence spectra at excitation wavelengths 295 and 305 nm demonstrate the existence of red-edge effect at sub-zero temperatures, reaching a maximum value at -50 degrees C, where the steady-state emission spectrum is shifted to 332 nm. The excitation and emission wavelength dependence of multi-frequency phase-modulation data at the half-transition point (-40 degrees C) demonstrates the existence of the nanosecond dipolar relaxations. At -40 degrees C the time-dependent spectral shift is close to monoexponential with the relaxation time of 1.4 ns.

Long-lifetime metal-ligand complexes as luminescent probes for DNA

We examined the intensity and anisotropy decays of DNA labeled with two ruthenium metalligand complexes, [Ru(bpy)2(dppz)]2+ and [Ru(phe)2(dppz)]2+. Both complexes display high emission anisotropies in the absence of rotational diffusion, making them suitable probes for rotational motions. When bound to DNA, these complexes display decay times as long as 294 ns, providing long-lived probes of DNA dynamics. The decay times of both complexes were rather insensitive to dissolved oxygen. We examined anisotropy decays of these complexes bound to B-form DNA. The anisotropy decays revealed correlation times near 10, 50, and several hundred nanoseconds, suggesting that these probes are sensitive to a wide range of DNA motions. The use of metalligand complexes should allow resolution of both the torsional and bending motions of DNA, the latter of which has been mostly inaccessible using shorter-lived fluorescent probes bound to DNA.

Fluorescence anisotropy of tyrosine using one-and two-photon excitation

We examined the emission spectra and steady-state anisotropy of tyrosyl fluorescence with two-photon excitation from 565 to 578 nm. The emission spectra of phenol and N-acetyl-L-tyrosinamide (NATyrA) were all the same for one-photon excitation (OPE) and two-photon excitation (TPE), and the tyrosine emission from ribonuclease A showed 10-nm shift to longer wavelengths with TPE. Surprisingly, the anisotropy of tyrosine, NATyrA and Leu5-enkephalin in frozen solution were near zero for TPE as compared to near 0.3 for OPE. Low values of the anisotropy near 0.05 were also found for phenol and ribonuclease A. A low anisotropy appears to be a basic characteristic of tyrosine or tyrosyl residues with two-photon excitation.