Biomedical subjects

J R Lakowicz

Publications and source records attributed to J R Lakowicz.

At least 91 records · Page 5Linked to original sources

PubMed1996

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.

Absorptiometry, Photon↗

PubMed1996

Synthesis and evaluation of Ru-complexes as anisotropy probes for protein hydrodynamics and immunoassays of high-molecular-weight antigens.

We investigated three unsymmetrical Ru-complexes, namely [Ru(bpy)2 (phen-ITC)]2+, [Ru(bpy)2(dcbpy)] and [Ru(bpy)2(mcbpy)]+ for use as probes for rotational diffusion and in immunoassays of high-molecular-weight antigens. For this purpose we synthesized reactive forms of these metal-ligand complexes and conjugated them to human serum albumin (HSA). The maximal anisotropies (r0) for the HSA-bound forms in frozen solution are 0.23, 0.17 and 0.14 for the (dcbpy), (mcbpy) and (phen-ITC) derivatives, respectively. The activated Ru metal-ligand complexes have either one or two NHS-esters or an isothiocyanate group as the reactive moiety. The usefulness of these complexes in immunoassays was determined by titration of the labeled HSA with polyclonal anti-HSA. The highest steady state anisotropy (r) values (0.190) were observed for the [Ru(bpy)2(dcbpy)]-labeled HSA on titration with polyclonal antibody. However, a relative increase in the steady state anisotropy (r/r0) on titration with polyclonal antibody was found for the phen-ITC probe (96%), as compared to the dcbpy (83%) or mcbpy (79%) derivatives. These findings were confirmed by time-resolved frequency-domain measurements. In particular the higher mean correlation times calculated for the phen-ITC derivative suggests reduced local probe motion for this probe when bound to HSA as compared to the (mcbpy) and (dcbpy) conjugates.

Anisotropy↗

PubMed1996

Fluorescence detection of the anticancer drug topotecan in plasma and whole blood by two-photon excitation.

The anticancer drug topotecan was detected in human plasma and whole blood using two-photon excitation at 730 or 820 nm. These wavelengths are longer than the main absorption bands of hemoglobin. Two-photon excitation of topotecan was demonstrated by a quadratic dependence of the emission intensity on the incident power, compared to a linear dependence for one-photon excitation at 410 nm. The observed emission centered at 525 nm was shown to be topotecan from the similarity of the emission spectrum and decay times observed for one-photon and two-photon excitation. Topotecan was detected at concentrations as low as 0.05 and 1 microM in plasma and whole blood, respectively. Since skin blood and tissues are translucent at long wavelengths, these results suggest the possibility of homogeneous or noninvasive clinical sensing with two-photon excitation.

Antineoplastic Agents↗

PubMed1996

Fluorescence polarization immunoassay of a high-molecular-weight antigen using a long wavelength-absorbing and laser diode-excitable metal-ligand complex.

We describe a fluorescence polarization immunoassay (FPI) based on an osmium metal-ligand complex, Os(bpy)2(dcbpy). The Os complex was selected for its long wavelength absorption, which allows excitation with wavelengths up to 720 nm. At these wavelengths tissue absorbance and autofluorescence are minimal, and excitation can be accomplished with LEDs or laser diodes. The Os complex displays a lifetime of 19 ns and a high initial anisotropy at two excitation wavelengths, 505 and 690 nm. A reactive NHS-ester of Os- (bpy)2(dcbpy) was synthesized containing a NHS ester, and used to label the human serum albumin (HSA) or anti-HSA. The FPIs of HSA were performed with monoclonal and polyclonal antibodies using excitation at 505 or 685 nm. The results showed the potential of the Os-ligand complex to be used as a long excitation wavelength FPI probe for potential use in homogeneous immunoassays with simple excitation sources.

2,2'-Dipyridyl↗

PubMed1996

Distance distributions from the tyrosyl to disulfide residues in the oxytocin and [Arg8]-vasopressin measured using frequency-domain fluorescence resonance energy transfer.

We have examined the fluorescence intensity decays of oxytocin and [Arg8]-vasopressin resulting from the single tyrosyl residue in each peptide, and the intensity decay of the Asu1,6-analogues in which the disulfide bridge is substituted by a CH2-CH2 bridge. Viscosity-dependent steady state and intensity decay measurements indicated that fluorescence resonance energy transfer (FRET) from tyrosyl phenol to the disulfide bridge is responsible for the decrease in fluorescence relative to the Asu-analogues. The frequency-domain phase and modulation data for the tyrosyl donor were interpreted in terms of fluorescence resonance energy transfer (FRET) to the weakly absorbing disulfide bridge and a distribution of donor-to-acceptor distances. Energy transfer efficiencies were determined from both time-resolved and steady-state measurements. Fitting the frequency-domain phase and modulation data to a Gaussian distance distribution indicated that the average inter-chromophoric distance (Rav) is similar in both compounds, Rav = 7.94 A for oxytocin and Rav = 8.00 A for vasopressin. However, the width of the distance distribution is narrower for vasopression (hw = 2.80 A) than for oxytocin (hw = 3.58 A), which is consistent with restriction of the tyrosine phenol motion due to its stacking wih the Phe3 side chain of vasopressin. Finally, the recovered distance distribution functions are compared with histograms describing the distance between the chromophores during the course of long, in vacuo, molecular dynamics runs using the computer program CHARMm and the QUANTA 3.0 parameters.

Amino Acid Sequence↗

PubMed1996

Two-photon induced fluorescence of linear alkanes; a possible intrinsic lipid probe.

We measured the fluorescence emission spectra and intensity decays of the linear alkane tetradecane when excited at 300 nm by two-photon excitation. The unquenched lifetime of tetradecane in neat solution is near 4.4 ns. The emission of tetradecane centered at 210 nm is collisionally quenched by oxygen, n-propanol and water. These results suggest that aliphatic groups in non-polar environments can display good fluorescence, and that the aliphatic side chains of detergents and lipids may serve as an intrinsic fluorescent probe of micelles and bilayers.

1-Propanol↗

PubMed1996

Fluorescence spectral properties of troponin C mutant F22W with one-, two-, and three-photon excitation.

We report the first measurements of protein fluorescence with three-photon excitation, using a mutant of troponin C (TnC) that contains a single tryptophan residue F22W. From the emission intensity dependence on laser power we determine that TnC F22W displays one-, two-, and three-photon excitation at 285, 570, and 855 nm, respectively. The emission spectra and intensity decays are identical for one-, two-, or three-photon excitation. The steady-state and time 0 anisotropies are distinct for each mode of excitation, but the correlation times were the same, suggesting that three-photon excitation of proteins can be accomplished without significant effects of the locally intense illumination. The excitation anisotropy spectrum from 830 to 900 nm displays only negative values, suggesting dominant excitation via the 1Lb state of tryptophan from 830 to 900 nm.

Animals↗

PubMed1996

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.

Biophysical Phenomena↗

PubMed1996

Two-color two-photon excitation of fluorescence.

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-terphenyl 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.

Fluorescence Polarization↗

PubMed1996

On the possibility of evanescent wave excitation distal from a solid-liquid interface using light quenching.

Evanescent wave illumination with total internal reflection is often used to provide excitation near a quartz-water interface. We now show that evanescent illumination at one wavelength and incident angle, coupled with light quenching at a second wavelength and incident angle, can be used for selective excitation of fluorophores located up to 5000 A into the aqueous phase. The displacement of the fluorophore population from the solid-liquid interface depends on the angles of incidence of the excitation and quenching beams and the optical power of the quenching beam. Light quenching with an evanescent wave was demonstrated to be experimentally possible using Pyridine2 and a light-quenching wavelength of 736 nm. The use of combined evanescent wave excitation and evanescent wave quenching could provide selective excitation of fluorophores in the cytoplasmic region of cells and may provide improved response times for optical sensors based on evanescent excitation.

Fluorescence↗

PubMed1996

Emerging applications of fluorescence spectroscopy to cellular imaging: lifetime imaging, metal-ligand probes, multi-photon excitation and light quenching.

Advances in time-resolved fluorescence spectroscopy can be applied to cellular imaging. Fluorescence lifetime imaging microscopy (FLIM) creates image contrast based on the decay time of sensing probes at each point in a two-dimensional image. FLIM allows imaging of Ca2+ and other ions without the need for wavelength-ratiometric probes. Ca2+ imaging can be performed by FLIM with visible wavelength excitation. Instrumentation for FLIM is potentially simple enough to be present in most research laboratories. Applications of fluorescence are often limited by the lack of suitable fluorophores. New, highly photostable probes allow off-gating of the prompt autofluorescence, and measurement of rotational motion of large macromolecules. These luminescent metal-ligand complexes will become widely utilized. Modern pulse lasers allow new experiments based on non-linear phenomena. With picosecond and femtosecond lasers fluorophores can be excited by simultaneous absorption of two or three photons. Hence, Ca2+ probes, membrane probes, and even intrinsic protein fluorescence can be excited with red or near infrared wavelengths, without ultraviolet lasers or optics. Finally, light itself can be used to control the excited state population. By using light pulses whose wavelength overlaps the emission spectrum of a fluorophore one can modify the excited state population and orientation. This use of non-absorbed light to modify emission can have wide reaching applications in cellular imaging.

Calcium↗

PubMed1995

A lifetime-based fluorescence resonance energy transfer sensor for ammonia.

A lifetime-based optical NH3 sensor based on the principle of fluorescence resonance energy transfer was developed. The sensor consisted of sulforhodamine 101 as the donor, bromocresol green as the acceptor, ethyl cellulose as the polymer support, and tributyl phosphate as the plasticizer. When the concentration of NH3 changed, it caused a change in the decay time of the SR101, which was measured by phase-modulation fluorometry. At 100 MHz, increasing the concentration of NH3 from 0 to 175 ppm resulted in a decrease in phase angle of about 31 degrees and an increase in modulation of about 18%. Oxygen and carbon dioxide did not interfere with the sensor. However, a 30% relative humidity could cause a downward shift of the response by 5 degrees, while additional increase in the relative humidity to 100% showed little further effect. For a film thickness of 40 microns, the typical response and recovery times for 90% of total signal change were 1 and 2.5 min, respectively. The phase angle measurements for the same sample were reproducible for 5 days, with no special care of the film sample.

Ammonia↗

PubMed1995

Fluorescence energy transfer immunoassay based on a long-lifetime luminescent metal-ligand complex.

We describe an immunoassay based on fluorescence resonance energy transfer (FRET). The antigen was human serum albumin (HSA), which was labeled with a ruthenium-ligand complex, [Ru(bpy)2(phen-ITC)]2+. The antibody (IgG) to HSA was labeled with a nonfluorescent absorber, Reactive Blue 4. Association of the Ru-labeled HSA with the antibody was detected by three spectral parameters, a decreased quantum yield of Ru-HSA, a decrease in its fluorescence lifetime, and an increase in its fluorescence anisotropy. The steady-state anisotropy of Ru-HSA increased approximately eightfold upon binding to the antibody. These spectral effects were observed both in the direct association of the Ru-HSA with Reactive Blue 4-labeled antibody, and in a competitive assay format wherein unlabeled HSA competed with Ru-HSA for the binding sites on the antibody. Some nonspecific interactions of HSA may have occurred with Reactive Blue 4-labeled AHA, a difficulty which can be avoided with a different acceptor. The use of FRET provides a reliable means to alter the spectral properties upon antigen-antibody binding. The advantages of a ruthenium-ligand fluorophore include its long-wavelength absorption and emission, long fluorescence lifetime, and high photo-stability. Long wavelengths minimize problems of autofluorescence from biological samples, and long life-times allow off-gating of the prompt autofluorescence.

Antigen-Antibody Complex↗

PubMed1995

A lifetime-based optical CO2 gas sensor with blue or red excitation and stokes or anti-stokes detection.

We describe the fabrication and characterization of an optical CO2 sensor based on the change in fluorescence lifetimes due to fluorescence resonance energy transfer from a pH-insensitive donor, sulforhodamine 101, to a pH-sensitive acceptor, either m-cresol purple or thymol blue, entrapped in an ethyl cellulose film. A phase transfer agent allows incorporation of the dyes and water into the film, while providing an initially basic environment for the acceptor. Diffusion of CO2 into the water entrapped in the film produced carbonic acid, causing a pH-dependent decrease in the spectral overlap of the acceptor absorbance with the donor emission, and decreased energy transfer, resulting in increased SR101 donor lifetimes. The lifetime changes were detected as a change in the phase of the emission, relative to the modulated excitation, and were insensitive to excitation intensities and emission signal levels. In addition to an externally modulated 442-nm light source, we excited the sensor with a directly modulated 635-nm laser diode and detected the anti-Stokes emission. The CO2 sensor is not fragile and can provide stable readings for weeks. The use of fluorescence resonance energy transfer, along with the simple entrainment procedure, allows facile change of the CO2 response range through change of the acceptor dye and the use of laser diode excitation sources.

Chromogenic Compounds↗

PubMed1995

Fluorescence polarization immunoassay of a high-molecular-weight antigen based on a long-lifetime Ru-ligand complex.

We describe a new class of fluorescence polarization immunoassays based on the luminescence from an asymmetrical Ru-ligand complex. We found that such a complex displays larger polarization values than those of comparable symmetrical complexes and appear to be highly photostable in aqueous solution. We synthesized a conjugatable Ru-ligand complex, which was used to label human serum albumin (HSA) as the antigen. The Ru-ligand complex displays a long decay time near 400 ns when covalently linked to proteins. We found that the steady-state polarization of labeled HSA was sensitive to binding of anti-HSA, resulting in a 200% increase in polarization. The labeled HSA was also used in a competitive format using unlabeled HSA as the antigen. The time-resolved anisotropy decays demonstrate increased correlation times for labeled HSA in the presence of anti-HSA, an effect which was partially reversed in the presence of unlabeled HSA. These results demonstrate the potential of the metal-ligand complexes to be used in the fluorescence polarization immunoassay of high-molecular-weight analytes. The use of such metal-ligand complexes enable fluorescence polarization immunoassays which bypass the usual limitation to low-molecular-weight antigens, which is a consequence of the 2-5 ns decay time of the previously used fluorophores.

2,2'-Dipyridyl↗

PubMed1995

Synthesis and interaction of fluorescent thapsigargin derivatives with the sarcoplasmic reticulum ATPase membrane-bound region.

Fluorescent derivatives of thapsigargin (TG) were synthesized by replacing the C8-butanoyl chain with a dansyl (DTG) or eosin (ETG) moiety. DTG and ETG retain the inhibitory effect of TG on the sarcoplasmic reticulum (SR) ATPase, displaying a 2 and 10 microM Ki, respectively. Steady state and lifetime fluorescence measurements are consistent with energy transfer between tryptophanyl residues assigned to the ATPase membrane-bound region and DTG. This phenomenon exhibits saturation behavior, occurs in the presence of DTG concentrations producing ATPase inhibition, and is partially prevented by inhibitory concentrations of TG. Although long range conformational effects of TG binding affect the fluorescence properties of endogenous tryptophans as well as of a fluorescein 5'-isothiocyanate (FITC) label of the ATPase extramembranous region, no significant energy transfer was detected between DTG and the FITC label. It is concluded that the inhibitors partition within the membrane and the binding domain resides within or near the membrane-bound region of the ATPase.

Animals↗

PubMed1995

Fluorescence energy transfer in one dimension: frequency-domain fluorescence study of DNA-fluorophore complexes.

Fluorescence resonance energy transfer among linear DNA bound fluorophores was carried out to study the process in one dimension. The donor fluorescence intensity decays in the case of energy transfer in one dimension are stretched exponential and show exp[-(t/tau)1/6] time dependence, which results in an initial more rapid decay and subsequent slower decay at long times when compared to those in higher dimensions. DNA-bound 4',6'-diamidino-2-phenyl indole (DAPI), acridine orange (AO), and ethidium bromide (EB) were used as donors. The acceptors were in the case of DAPI AO and EB; in the case of AO nile blue (NB), methylene blue (MB), and crystal violet (CV); and NB, MB, and oxazine 750 in the case of EB. As expected, the donor intensity decays became highly heterogeneous upon energy transfer and were characterized by the simultaneous presence of both highly and marginally quenched donors. The intensity decays for all three donors in the presence of various acceptors are satisfactorily described by the Förster model of energy transfer in one dimension. The intensity decays also allow for clear rejection of a two- or three-dimensional model. The experimentally recovered critical Förster distances (R0) ranged between 37 A in the case of DAPI and EB to 70 A in the case of AO and CV donor-acceptor pairs. These recovered R0 values compare reasonably with those calculated from spectral properties if we use values of 1.25 for k2, and 1.5 for the refractive index of DNA. The k2 value will be even higher, between 1.5 and 2.0, if the consensus DNA refractive index of 1.75 is used. These k2 values strongly suggest that the dipoles of the acceptor chromophores when bound to DNA are not randomly oriented but are aligned preferentially in plane.

Acridine Orange↗

PubMed1995

Possibility of simultaneously measuring low and high calcium concentrations using Fura-2 and lifetime-based sensing.

We characterized the fluorescence probe Fura-2 for calcium measurements using frequency-domain phase-modulation fluorometry. By the use of different excitation wavelengths from 345 to 380 nm, the apparent calcium dissociation constants can be altered from 41 nM to 1.92 microM Ca2+. This change in apparent Kd results from changes in the relative extent of excitation of the calcium-bound and calcium-free forms, and the excitation wavelength-dependent contribution of each form to the intensity decay. These results indicate that lifetime-based measurements with Fura-2 can be used for imaging of calcium over a wide range of concentrations. An additional favorable feature of Fura-2 is that the calcium-free form can be almost exclusively excited at wavelength of 390 nm or longer, and can thus be used as a reference providing the lifetime in the absence of calcium, without removing the calcium. Additionally, exposure of Fura-2 to intense illumination shifts but does not distort the frequency response. For cellular imaging, these favorable properties of Fura-2 may allow calibration of the calcium concentrations without the use of ionophores.

Calcium↗