Download (.pdf)The statistics of protein expression ratios for cellular fluorescence studies
Co-authored with Joachim D. Mueller
DOI: 10.1007/s00249-012-0792-x
Fluorescence studies of cellular protein-protein interactions commonly employ transient cotransfection to express two... more Fluorescence studies of cellular protein-protein interactions commonly employ transient cotransfection to express two proteins carrying distinct fluorescent labels. Because transiently transfected cells differ significantly in their expression level, the concentration ratio of the two expressed proteins varies, which in turn influences the measured fluorescence signal. Knowledge of the statistics of protein expression ratios is of considerable interest both from a fundamental point of view and for cellular fluorescence studies. Despite the perceived randomness of transient transfection, we were able to develop a quantitative model that describes the average and distribution of the protein expression ratio from a cell population. We show that the expression ratio is proportional to the molar plasmid ratio and relate the distribution to the finite number of active plasmids in the cell. The process of cationic lipid-mediated transfection is explored in more detail. Specifically, the influence of lipoplexes on the statistics of the expression ratio is examined. We further demonstrate that the transfection model provides a quantitative description of fluorescence fluctuation experiments, where only a fraction of the proteins are labeled.
Quantifying Adsorbed Protein on Surfaces using Confocal Fluorescence Microscopy.
by Alan Ryder
D.M. Togashi, A. G. Ryder, and G. Heiss, Colloids and Surfaces B: Biointerfaces. 72(2), 219-229, (2009).
The quantification and analysis of protein adsorption on solid surfaces are of significant importance in many areas of... more The quantification and analysis of protein adsorption on solid surfaces are of significant importance in many areas of biosensors, biomaterials, and biomedical devices research. The accurate, in-situ, measurement of multiple physicochemical properties from the thin protein layers which adsorb on surfaces is critical to understanding biocompatibility, surface chemistry factors, and the performance of implanted medical devices. To implement such studies, new tools and simple protocols based on instrumentation available in typical bioscience laboratories are desirable. In this work, we have developed an approach using confocal fluorescence microscopy to quantify the amount of Bovine Serum Albumin (BSA) adsorbed onto a flat hydrophilic glass surface, under different pH conditions. This approach which can be implemented using most confocal fluorescence microscopes is described in detail and its limitations are discussed. This quantitative method coupled with the Langmuir model allowed for the determination of adsorption parameters at pH 2.0, 4.0, 7.4, and 9.2. The adsorption parameters were validated by comparison with literature values obtained from different techniques for a similar protein-surface system. The Derjanguin-Landau-Verwey-Overbeek (DLVO) theory was then used for a detailed analysis of these parameters, to understand in general terms how pH affected the surface adsorption interactions.
Fluorescence Lifetime Imaging study of a thin protein layer on solid surfaces.
by Alan Ryder
D.M. Togashi and A.G. Ryder, Experimental & Molecular Pathology, 82(2). 135-141, (2007).
Understanding the fundamental interactions between proteins and solid surfaces is essential in the area of implantable... more Understanding the fundamental interactions between proteins and solid surfaces is essential in the area of implantable medical devices. Fluorescence methods offer the sensitivity required to study the formation of the initial thin protein layers that mediate biocompatibility of materials. Thin protein layers (bovine serum albumin labelled with 1-anilino-8-naphthalenesulfonate, BSA–ANS) deposited on several surfaces (glass, silicon, stainless steel, polystyrene, and silver island film) were studied using confocal frequency domain Fluorescence Lifetime Imaging Microscopy (FLIM) and single-point multifrequency lifetime analysis techniques. FLIM provides spatial information about both fluorophores located on the surface and physicochemical parameters of the surface microenvironment. The average fluorescence lifetimes (τav) of the adsorbed BSA–ANS generated by the contact between a protein solution and the material surface were measured by the multifrequency modulation and phase shift. Results indicate that τav values of the albumin complexes on the surfaces (∼12 ns) are, in general, shorter than τav found in the bulk solution (∼14 ns). For some surfaces, like polystyrene and silver island film the differences in τav of the adsorbed BSA–ANS were found to be much greater. The differences in fluorescence lifetimes may indicate structural changes in the BSA protein induced by contact with the surface.
A fluorescence analysis of ANS bound to bovine serum albumin: binding properties revisited by using energy transfer.
by Alan Ryder
D.M. Togashi and A.G. Ryder. Journal of Fluorescence, 18(2), 519-526, (2008).
Determination of binding parameters such as the number of ligands and the respective binding constants require a... more Determination of binding parameters such as the number of ligands and the respective binding constants require a considerable number of experiments to be performed. These involve accurate determination of either free and/or bound ligand concentration irrespective of the measurement technique applied. Then, an appropriate theoretical model is used to fit the experimental data, and to extract the binding parameters. In this work, the interaction between bovine serum albumin (BSA) and 1-anilino-8-naphthalene sulphonate (ANS) is revisited. Using steady state fluorescence spectroscopy, the binding isotherm of BSA/ANS was obtained applying the Halfman-Nishida approach. The binding parameters, site number, and binding site association constants, were determined from the stoichiometric Adair model and Job’s plot. The binding parameters obtained were then correlated to the distance of the respective binding site to the tryptophan residues using the energy transfer technique. This approach, that uses both tryptophans independently from each other, is presented as a tool to help understand the binding mechanism of the albumin fluorescent complex. The results show that ANS molecules bind to BSA in up to five different binding sites. Energy transfer from the tryptophan residues to the BSA/ANS complex shows that the four highest affinity binding sites ( >10,000 M-1 ) are located at a reasonably close (18 Å to 27 Å) to at least one of two tryptophan residues, while the lowest affinity binding site (~10,000 M-1 ) is located over 34 Å away from the both tryptophans.
The application of structured-light illumination to hydrocarbon-bearing fluid inclusions.
by Alan Ryder
N.J.F. Blamey, A.G. Ryder, M. Feely, P. Dockery, and P. Owens, Geofluids, 8, 102-112, (2008).
Structured-light illumination (SLI) based microscopy offers geologists a new perspective for the first-pass... more Structured-light illumination (SLI) based microscopy offers geologists a new perspective for the first-pass examination of hydrocarbon-bearing (HCFI) and small aqueous fluid inclusion (AFI) assemblages. This optical sectioning technique provides rapid, confocal like imaging, using relatively simple and inexpensive instrumentation. The 3D fluorescent images of HCFI planes and large single HCFI, permits the visualisation of the relationships between HCFI assemblages, examination of HCFI morphology, and volume estimates of the fluorescent components within HCFI. By the use of normal white light illumination, SLI image capture, and varying acquisition time one can also image AFI because of the random movements of vapour bubbles within the inclusions. This provides the near simultaneous visualisation of HCFI and AFI which is of significant importance for the study of sedimentary basins and petroleum reservoirs. SLI is a unique and accessible 3D petrographic tool, with practical advantages over conventional epifluorescence and conventional confocal laser scanning microscopy.
Frequency Domain Fluorescence Lifetime Study of Crude Petroleum Oils.
by Alan Ryder
P. Owens, A.G. Ryder, and N.J.F. Blamey. Journal of Fluorescence, 18(5), 997-1006, (2008).
Frequency domain (FD) fluorescence lifetime data was collected for a series of 20 crude petroleum oils using a 405 nm... more
Frequency domain (FD) fluorescence lifetime data was collected for a series of 20 crude petroleum oils using a 405 nm excitation source and over a spectral range of ~426 to ~650 nm. Average fluorescence Lifetimes were calculated using three different models: discrete multi-exponential, Gaussian distribution, and Lorentzian distribution. Fitting the data to extract accurate average lifetimes using the various models proved easier and less time consuming for the FD data than with Time Correlated Single Photon Counting (TCSPC) methods however the analysis of confidence intervals to the computed average lifetimes proved cumbersome for both methods. For the lifetime distributions, the data generally fit to two to three decay term distributions and the uncertainty in the average lifetime was reduced in comparison to the discrete lifetime decay fits. Correlations between average fluorescence lifetimes and physical and chemical parameters of the crude oils were made with a view to developing a quantitative model for predicting the gross chemical composition of crude oils. No significant differences in correlation were observed for the discrete multi-exponential fits between TCSPC or FD data. Overall the Gaussian model (FD data) gave the best correlations with chemical composition. The lifetime trends enable a qualitative correlation to some bulk oil parameters.
Study of water adsorption in Poly(N-isopropylacrylamide) thin films using fluorescence emission of 3-hydroxyflavone probes.
by Alan Ryder
C. Morris, B. Szczupak, A.S. Klymchenko, and A.G. Ryder. Macromolecules. 43(22), 9488-9494, (2010).
The non-contact, measurement of water uptake in micro-scale (1-100 microns), thermoresponsive... more The non-contact, measurement of water uptake in micro-scale (1-100 microns), thermoresponsive Poly(N-isopropylacrylamide) thin films is challenging. We assessed the efficacy of three different 3-Hydroxyflavone (3-HF) based fluorophores; to monitor water uptake in pNIPAM thin films close to the Lower Critical Solution Temperature (LCST) at 25 and 37 ºC. These 3-HF fluorophores undergo excited state intramolecular proton transfer, yielding emission from normal (N*) and tautomeric (T*) excited-state forms. The emission intensity ratio, log(IN*/IT*) and N* band position are environmentally sensitive. Water adsorption in pNIPAM thin films follows a non-linear, two phase process: at low relative humidity, the adsorbed water disrupts polymer-fluorophore hydrogen bonding, yielding small changes in log(IN*/IT*) ratios, and overall intensity, at higher relative humidity, these intensity parameters (but not fluorescence lifetime) change dramatically, indicating a larger change in local polarity. These probes are thus sensitive indicators of water uptake in pNIPAM.
Low temperature Fluorescence Study of Crude Petroleum Oils.
by Alan Ryder
P. Owens and A.G. Ryder, Energy & Fuels, 25(11), 5022-5032, (2011).
We have studied the low-temperature (133−298 K) fluorescence emission of crude petroleum oils using a combination of... more We have studied the low-temperature (133−298 K) fluorescence emission of crude petroleum oils using a combination of steady-state and time-resolved measurements. This was done to first see if we could generate linear correlations between oil composition information and the fluorescence measurements, and second to better understand how static and dynamic quenching affect fluorescence emission. It was observed that the fluorescence intensity and lifetime of the crude oils increased rapidly with decreasing temperature down to the freezing point and then either remained constant or surprisingly began to decrease slightly. These changes could not be correlated accurately with the compositional data available. However, despite the very large variations in sample composition, it was found that these lifetime-temperature changes followed simple Arrhenius and Eyring behavior. For the cold liquid phase, an Arrhenius model enabled the calculation of an intrinsic lifetime, the magnitude of which was inversely related to the degree of static quenching. The low values of the calculated activation energies (4.6 to 19.2 kJmol−1) implied that in the liquid phase, non-radiative decay was primarily diffusion based quenching. At the lowest temperatures, when all samples have solidified, the lifetime data followed Eyring like behavior, giving typical enthalpy and entropy values of −1 kJmol−1 and from −67 to −93 JK−1mol−1 respectively. The Eyring model was used to describe the non-radiative decay mechanism arising from vibrational coupling from the fluorophores to the surrounding matrix. This modeling of the temperature dependence of fluorescence lifetime has provided a clearer, quantitative picture of the fluorescence quenching processes in crude petroleum oils.
Investigating trypthopan quenching of fluorescein fluorescence under protolytic equilibrium.
by Alan Ryder
D.M. Togashi, B. Szczupak, A.G. Ryder, A. Calvet, and M. O'Loughlin, Journal of Physical Chemistry A, 113(12), 2757-2767, (2009).
Fluorescein is one of most used fluorescent labels for characterizing biological systems, such as proteins, and is... more
Fluorescein is one of most used fluorescent labels for characterizing biological systems, such as proteins, and is used in fluorescence microscopy. However, if fluorescein is to be used for quantitative measurements involving proteins then one must account for the fact that the fluorescence of fluorescein-labeled protein can be affected by the presence of intrinsic amino acids residues, such as tryptophan (Trp). There is a lack of quantitative information to explain in detail the specific processes that are involved, and this makes it difficult to evaluate quantitatively the photophysics of fluorescein-labeled proteins. To address this, we have explored the fluorescence of fluorescein in buffered solutions, in different acidic and basic conditions, and at varied concentrations of tryptophan derivatives, using steady-state absorption and fluorescence spectroscopy, combined with fluorescence lifetime measurements. Stern-Volmer analyses show the presence of static and dynamic quenching processes between fluorescein and tryptophan derivatives. Nonfluorescent complexes with low association constants (5.0-24.1 M-1) are observed at all pH values studied. At low pH values, however, an additional static quenching contribution by a sphere-of-action (SOA) mechanism was found. The possibility of a proton transfer mechanism being involved in the SOA static quenching, at low pH, is discussed based on the presence of the different fluorescein prototropic species. For the dynamic quenching process, the bimolecular rate constants obtained (2.5-5.3 × 109 M-1s-1) were close to the Debye-Smoluchowski diffusion rate constants. In the encounter controlled reaction mechanism, a photoinduced electron transfer process was applied using the reduction potentials and charges of the fluorophore and quencher, in addition to the ionic strength of the environment. The electron transfer rate constants (2.3-6.7 × 109 s-1) and the electronic coupling values (5.7-25.1 cm-1) for fluorescein fluorescence quenching by tryptophan derivatives in the encounter
complex were then obtained and analyzed.
Application of fluorescence lifetime measurements on single petroleum-bearing fluid inclusions to demonstrate multicharge history in petroleum reservoirs.
by Alan Ryder
Blamey NJF, Conliffe JF, Parnell J, Ryder AG, and Feely M, Geofluids, 9, 330-337, (2009).
A petrographic and microthermometric study of fluid inclusions in Jurassic and Cretaceous sandstones from the... more A petrographic and microthermometric study of fluid inclusions in Jurassic and Cretaceous sandstones from the Porcupine Basin, offshore Ireland was integrated with innovative fluorescence lifetime measurements of hydrocarbon bearing fluid inclusions to determine the compositions of the fluids associated with diagenesis and post-diagenetic fluid migration and the extent of hydrocarbon and aqueous fluid migration pathways. Petrographic analyses indicate that Jurassic strata were the main fluid migration pathways for hydrocarbon fluids and that hydrocarbon migration occurred relatively late in the diagenetic history of these sandstones. UV fluorescence and fluorescence lifetime measurements have recognized at least two chemically distinct hydrocarbon groups (Types 1a and 1b) with dissimilar lifetime-wavelength (tau-lambda) profiles, consistent with at least two petroleum charges derived from different sources. Primary aqueous inclusions in authigenic cements show that cementation in Cretaceous sandstones occurred at relatively shallow levels at low temperatures (<50 C), while inclusions in authigenic cements in Jurassic sandstones were trapped at higher temperatures (70–120 C) and deeper levels. Aqueous fluid inclusions in intergranular trails indicate that post-cementation fluid migration occurred at high temperatures (up to 175 C). These high temperature fluid migrations are interpreted to be associated with plume-related activity during the opening of the North Atlantic.
Hydrocarbon migration in Jurassic sandstones from the Porcupine Basin, offshore Ireland: evidence from fluid inclusion studies.
by Alan Ryder
J. Conliffe, N.J.F. Blamey, M. Feely, J. Parnell, and A.G. Ryder, Petroleum Geoscience, 16(1), 67-76, (2010).
Fluid inclusions in Jurassic and Cretaceous sandstones from the Porcupine Basin, offshore Ireland were examined to... more Fluid inclusions in Jurassic and Cretaceous sandstones from the Porcupine Basin, offshore Ireland were examined to determine the extent of hydrocarbon and aqueous fluid migration pathways. Fluid inclusion petrographic and microthermometric observations were collected from hydrocarbon-bearing and aqueous fluid inclusions. This data was combined with innovative fluorescence lifetime measurements of hydrocarbon-bearing fluid inclusions in order to investigate fossil petroleum systems. Inter- and intragranular trails of hydrocarbon-bearing fluid inclusions are common in Jurassic sandstones, but have only been recorded in a single Cretaceous sandstone. This indicates that Jurassic strata were the main fluid migration pathways for hydrocarbon fluids and that hydrocarbon migration occurred relatively late in the diagenetic history of these sandstones. The absence of hydrocarbon-bearing fluid inclusions in Cretaceous sandstones indicates that the majority of hydrocarbon migration in the Porcupine Basin was along lateral migration pathways with only limited vertical migration of hydrocarbons into overlying strata. Fluid inclusion microthermometry, UV fluorescence and fluorescence lifetime measurements have recognised the migration of at least two generations of hydrocarbons, reflecting variations in the maturity and sources of hydrocarbons across the Porcupine Basin. Microthermometric data from primary aqueous inclusions in authigenic cements show that cementation in Cretaceous sandstones occurred at low temperatures (< 50°C) at relatively shallow levels. In contrast inclusions in authigenic cements in Jurassic sandstones were trapped at much higher temperatures (70 to 120°C), consistent with cementation at deeper levels. Aqueous fluid inclusions in intergranular trails indicate that post-cementation fluid migration occurred at temperatures of up to 175°C, higher that predicted from geothermal gradients. These high temperature fluid migrations are interpreted to be associated with plumerelated activity during the opening of the North Atlantic.
Polarity assessment of thermoresponsive poly(NIPAM-co-NtBA) copolymer films using fluorescence methods.
by Alan Ryder
B. Szczupak, A.G. Ryder, D.M. Togashi, A.S. Klymchenko, Y.A. Rochev, A. Gorelov, and T.J. Glynn, Journal of Fluorescence, 20(3), 719-731, (2010).
The in-situ, non-contact, and non-destructive measurement of the physicochemical properties such as the polarity of... more The in-situ, non-contact, and non-destructive measurement of the physicochemical properties such as the polarity of thin (nm to microns), hydrophilic polymer films is desirable in many areas of polymer science. Polarity is a complex factor and encompasses a range of non-covalent interactions including dipolarity/polarizability and hydrogen bonding. A polarity measurement method based on fluorescence would be ideal, but the key challenge is to identify suitable probes which can accurately measure specific polarity related parameters. In this manuscript we assess a variety of fluorophores for measuring the polarity of a series of relatively hydrophilic, thermoresponsive N-isopropylacrylamide/N-tert-butylacrylamide (NIPAM/NtBA) copolymers. The emission properties of both pyrene and 3-Hydroxyflavone (3-HF) based fluorophores were measured in dry polymer films. In the case of pyrene, a relatively weak, linear relationship between polymer composition and the ratio of the first to the third vibronic band of the emission spectrum (I1/I3) is observed, but pyrene emission is very sensitive to temperature and thus not suitable for robust polarity measurements. The 3-HF fluorophores which can undergo an excited-state intramolecular proton transfer (ESIPT) reaction have a dual band fluorescence emission that exhibits strong solvatochromism. Here we used 4’-diethylamino-3-hydroxyflavone (FE), 5,6-benzo-4’-diethylamino-3-hydroxyflavone (BFE), and 4´-diethylamino-3-hydroxy-7-methoxyflavone (MFE)). The log ratio of the dual band fluorescence emission (log (IN*/IT*)) of 3-HF doped, dry, NIPAM-NtBA copolymer films were found to depend linearly on copolymer composition, with increasing hydrophobicity (greater NtBA fraction) leading to a decrease in the value of log (IN*/IT*). However, the ESIPT process in the polymer matrix was found to be irreversible, non-equilibrated and occurs over a much longer timescale in comparison to the results previously reported for liquid solvents.
Monitoring local unfolding of Bovine Serum Albumin in the denaturation process using steady-state and time-resolved fluorescence spectroscopy.
by Alan Ryder
D.M. Togashi, A.G. Ryder, and D. O’Shaughnessy, Journal of Fluorescence, 20(2), 441-452, (2010).
In a previous report (J. Fluoresc. 16, 153, 2006) we studied the chaotropiclly induced denaturation of Bovine Serum... more In a previous report (J. Fluoresc. 16, 153, 2006) we studied the chaotropiclly induced denaturation of Bovine Serum Albumin (BSA) using the fluorescence decay kinetics at different stages in the denaturation of BSA by guanidinium hydrochloride (GuHCl). In this work, we gain a more detailed insight into the BSA denaturation process by investigating the thermodynamics of the process. Structural changes were monitored spectrophotometrically via the intrinsic protein fluorescence from tryptophan residues, and the extrinsic fluorescence from 1,8-anilinonaphthalene sulphonate (ANS). ANS tends to locate in a variety of binding sites in BSA which are located in different domains, and these can be selectively populated using different, 1:1 and 1:10 molar ratios of BSA to ANS. The data from steady-state and time-resolved fluorescence spectroscopy were analyzed using thermodynamic two-state and three-state models and the lifetime data clearly indicated the formation of an intermediate state during denaturation. A global analysis using non-linear regression gave ΔGº(H2O,D)= 6.7 kcal.mol-1 for the complete unfolding of the BSA-ANS complexes, and ΔGº(H2O,I)=0.9 kcal.mol-1 for the first step to the intermediate. Therefore, the unfolding energy of the intermediate, which appears mostly at intermediate GuHCl concentrations (1.0 to 1.5 M), to the denatured state, is 5.8 kcal.mol-1. The lifetime analysis of the BSA-ANS complexes also shows clearly that there are differences in stability of the BSA domains, with domain III unfolding first at low GuHCl concentrations (<1.5M).
Quenching mechanism of Zn(salicylaldimine) by nitroaromatics
M.E. Germain, T.R. Vargo, B.A. McClure, J.J. Rack, P.G.Van Patten, M. Odoi, M.J. Knapp; Inorganic Chemistry, 47, 2008, 6203-6211
Excited State Distortion in Photochromic Ruthenium Sulfoxide Complexes
B. A. McClure, E. R. Abrams, J. J. Rack. J. Am. Chem. Soc. 2010, 132 (15), 5428-5436