Global analysis of fluorescence fluctuation data

Over the last decade the number of applications of fluorescence correlation spectroscopy (FCS) has grown rapidly. Here we describe the development and application of a software package, FCS Data Processor, to analyse the acquired correlation curves. The algorithms combine strong analytical power with flexibility in use. It is possible to generate initial guesses, link and constrain fit parameters to improve the accuracy and speed of analysis. A global analysis approach, which is most effective in analysing autocorrelation curves determined from fluorescence fluctuations of complex biophysical systems, can also be implemented. The software contains a library of frequently used models that can be easily extended to include user-defined models. The use of the software is illustrated by analysis of different experimental fluorescence fluctuation data sets obtained with Rhodamine Green in aqueous solution and enhanced green fluorescent protein in vitro and in vivo.An erratum to this article can be found at Victor V. Skakun, Mark A. Hink and Anatoli V. Digris contributed equally to this work.     

A method to quantify spontaneous and in vivo induced thioguanine-resistant mouse lymphocytes

A clonogenic assay to quantify thioguanine (TG)-resistant (TGr) spleen lymphocytes in the mouse has been developed to support studies of in vivo mutation affecting the hypoxanthine phosphoribosyltransferase (hprt) locus. Lymphocytes are cultured in 96-well microtiter plates for 9 days with proliferation initiated by the mitogen concanavalin A and supported thereafter by conditioned medium containing interleukin-2. Lymphocytes are plated at high densities (4-8 X 10(5)/well) with TG and irradiated L5178Y lymphoma cells (10(4)/well) to detect the presence of TGr cells. To determine the cloning efficiency without TG lymphocytes are plated at a low density (10/well) with irradiated L5178Y cells and irradiated lymphocytes (4-8 X 10(5)/well). Proliferation of cells is detected by [3H]thymidine incorporation and scintillation spectrometry. Spontaneous frequencies of TGr clones are independent of TG dose from 0.2 to 10 micrograms/ml and independent of cell density over the range cited. The TGr clones tested have less than 10% hypoxanthine incorporation in vivo relative to unselected clones and have stable phenotypes in the absence of selection. The spontaneous frequency of TGr cells ranged from 1 to 3 X 10(-6). In vivo treatment of mice intraperitoneally with ethylnitrosourea 15 days prior to in vitro culture resulted in a linear dose-related increase of TGr cells, with 70.2 mg/kg inducing a frequency of TGr cells of 2 X 10(-5).     

A spectrophotometric method to quantify linear DNA

A spectrophotometric method for quantification of linear DNA is described. The assay measures ADP produced following digestion of linear DNA by an ATP-dependent deoxyribonuclease. Cleavage of the phosphodiester bond of the DNA substrate is proportional to ADP formed in the reaction which follows typical Michaelis-Menten kinetics (K(m) of 0.6 microM, and a V(max) of 30 nmol/min/mg). The enzyme requires Mg(2+)-ATP and Mg(2+)-DNA as substrates, although the results suggest a requirement for yet another metal ion which may be enzyme bound. Both single-stranded and double-stranded linear DNA are substrates, as demonstrated by comparable initial velocity measurements. However, covalently closed circular (CCC) and nicked open circular DNA are not substrates for the enzyme. The rate of hydrolysis of ATP is not inhibited by 1 microg RNA or covalently closed circular DNA. The product (ADP) formed in the reaction is coupled to NADH oxidation using pyruvate kinase and lactate dehydrogenase. NAD formed in the reaction is monitored spectrophotometrically as a loss in absorbance at 340 nm. This assay directly measures the amount of linear DNA present in preparations of supercoiled (CCC) plasmid DNA, and has direct utility for monitoring the quality of plasmid preparations for gene therapy.     

A colorimetric method to quantify endo-polygalacturonase activity

We report a new colorimetric assay to quantify endo-polygalacturonase activity, which hydrolyzes polygalacturonic acid to produce smaller chains of galacturonate. Some of the reported polygalacturonase assays measure the activity by detecting the appearance of reducing ends such as the Somogyi-Nelson method. As a result of being general towards reducing groups, the Somogyi-Nelson method is not appropriate when studying polygalacturonase and polygalacturonase inhibitors in plant crude extracts, which often have a strong reducing power. Ruthenium Red is an inorganic dye that binds polygalacturonic acid and causes its precipitation. In the presence of polygalacturonase, polygalacturonic acid is hydrolyzed bringing about a corresponding gain in soluble Ruthenium Red. The described assay utilizes Ruthenium Red as the detection reagent which has been used previously in plate-based assays but not in liquid medium reactions. The new method measures the disappearance of the substrate polygalacturonic acid and is compared to the Somogyi-Nelson assay. The experimental results using lemon peel, a fern fronds and castor leaf crude extracts demonstrate that the new method provides a way to the quickly screening of polygalacturonase activity and polygalacturonase inhibitors in plant crude extracts containing high amounts of reducing power. On the other hand, the Ruthenium Red assay is not able to determine the activity of an exo-polygalacturonase as initial velocity and thus would allow the differentiation between endo- and exo-polygalacturonase activities.     

A general method to quantify quasi-equivalence in icosahedral viruses

A quantitative, atom-based, method is described for comparing protein subunit interfaces in icosahedral virus capsids with quasi-equivalent surface lattices. An integrated, normalized value (between 0 and 1) based on equivalent residue contacts (Q-score) is computed for every pair of subunit interactions and scores that are significantly above zero readily identify interfaces that are quasi-equivalent to each other. The method was applied to all quasi-equivalent capsid structures (T=3, 4, 7 and 13) in the Protein Data Bank and the Q-scores were interpreted in terms of their structural underpinnings. The analysis allowed classification of T=3 structures into three groups with architectures that resemble different polyhedra with icosahedral symmetry. The preference of subunits to form dimers in the T=4 human Hepatitis B virus capsid (HBV) was clearly reflected in high Q-scores of quasi-equivalent dimers. Interesting differences between the classical T=7 capsid and polyoma-like capsids were also identified. Application of the method to the outer-shell of the T=13 Blue tongue virus core (BTVC) highlighted the modest distortion between the interfaces of the general trimers and the strict trimers of VP7 subunits. Furthermore, the method identified the quasi 2-fold symmetry in the inner capsids of the BTV and reovirus cores. The results show that the Q-scores of various quasi-symmetries represent a "fingerprint" for a particular virus capsid architecture allowing particle classification into groups based on their underlying structural and geometric features.     

A cost-effective method to quantify biological surface sediment reworking

We propose a simple and inexpensive method to determine the rate and pattern of surface sediment reworking by benthic organisms. Unlike many existing methods commonly used in bioturbation studies, which usually require sediment sampling, our approach is fully non-destructive and is well suited for investigating non-cohesive fine sediments in streams and rivers. Optical tracer (e.g. luminophores or coloured sand) disappearance or appearance is assessed through time based on optical quantification of surfaces occupied by tracers. Data are used to calculate surface sediment reworking (SSR) coefficients depicting bioturbation intensities. Using this method, we evaluated reworking activity of stream organisms (three benthic invertebrates and a fish) in laboratory microcosms mimicking pool habitats or directly in the field within arenas set in depositional zones. Our method was sensitive enough to measure SSR as low as 0.2 cm² day^?1, such as triggered by intermediate density (774 m^?2) of Gammarus fossarum (Amphipoda) in microcosms. In contrast, complex invertebrate community in the field and a fish (Barbatula barabatula) in laboratory microcosms were found to yield to excessively high SSR (>60 cm² day^?1). Lastly, we suggest that images acquired during experiments can be used for qualitative evaluation of species-specific effects on sediment distribution.     

An automated method to quantify microglia morphology and application to monitor activation state longitudinally in vivo

Microglia are specialized immune cells of the brain. Upon insult, microglia initiate a cascade of cellular responses including a characteristic change in cell morphology. To study the dynamics of microglia immune response in situ, we developed an automated image analysis method that enables the quantitative assessment of microglia activation state within tissue based solely on cell morphology. Per cell morphometric analysis of fluorescently labeled microglia is achieved through local iterative threshold segmentation, which reduces errors caused by signal-to-noise variation across large volumes. We demonstrate, utilizing systemic application of lipopolysaccharide as a model of immune challenge, that several morphological parameters, including cell perimeter length, cell roundness and soma size, quantitatively distinguish resting versus activated populations of microglia within tissue comparable to traditional immunohistochemistry methods. Furthermore, we provide proof-of-concept data that monitoring soma size enables the longitudinal assessment of microglia activation in the mouse neocortex imaged via 2-photon in vivo microscopy. The ability to quantify microglia activation automatically by shape alone allows unbiased and rapid analysis of both fixed and in vivo central nervous system tissue.     

Time-integrated fluorescence cumulant analysis in fluorescence fluctuation spectroscopy

We introduce a new analysis technique for fluorescence fluctuation data. Time-integrated fluorescence cumulant analysis (TIFCA) extracts information from the cumulants of the integrated fluorescence intensity. TIFCA builds on our earlier FCA theory, but in contrast to FCA or photon counting histogram (PCH) analysis is valid for arbitrary sampling times. The motivation for long sampling times lies in the improvement of the signal/noise ratio of the data. Because FCA and PCH theory are not valid in this regime, we first derive a theoretical model of cumulant functions for arbitrary sampling times. TIFCA is the first exact theory that describes the effects of sampling time on fluorescence fluctuation experiments. We calculate factorial cumulants of the photon counts for various sampling times by rebinning of the original data. Fits of the data to models determine the brightness, the occupation number, and the diffusion time of each species. To provide the tools for a rigorous error analysis of TIFCA, expressions for the variance of cumulants are developed and tested. We demonstrate that over a limited range rebinning reduces the relative error of higher order cumulants, and therefore improves the signal/noise ratio. The first four cumulant functions are explicitly calculated and are applied to simple dye systems to test the validity of TIFCA and demonstrate its ability to resolve species.     

A simple method to quantify maturation of wound collagen

While investigating the effect of NSAIDs and Zn(II) (NSAIDs)2 complexes on healing of dead space wounds we found that both NSAIDs and Zn(II) (NSAIDs)2 reduced collagen content to a comparable extent yet only NSAIDs reduced breaking strength (BS) of wounds. Since collagen content and maturation are the two important determinants of BS it was obvious that only NSAIDs reduced maturation. A simple method to assess the extent of maturation in available collagen is being reported in the present study. While all NSAIDs significantly reduced the maturation of collagen, Zn(II) (NSAIDs)2 complexes per se did not affect the maturation of collagen of 10-day old. Though, chronologically, granulation tissues obtained from both the treated groups were 10-day old, maturation-wise the age of NSAIDs and Zn(II) (NSAIDs)2 treated granulation was 6 and 12 days respectively.     

A new alternative method to quantify residual structure in 'unfolded' proteins

Pig (pCSD1) and human (hCSD1) calpastatin domain 1 proteins were studied to characterize common features of the denatured state of proteins. These proteins were chosen for the present investigation, because pCSD1 was suggested previously to be unstructured in water even at 25 degrees C (1) [T. Konno et al., Biochim. Biophys. Acta 1342 (1997) 73-82]. hCSD1 could be expected to exhibit similar features on the basis of preliminary spectroscopic studies. In the present study, the experimental grounds for the estimate of residual structure in the unfolded state were differential scanning calorimetry heat capacity and circular dichroism (CD) measurements over the temperature range 10-80 degrees C. At selected temperatures, we studied also the effect of guanidinium hydrochloride (GdnHCl) which is known to promote further unfolding of the polypeptide chain. All other measurements were performed at pH 6 in pure water. The present results support the conclusion that the comparison of the experimentally obtained heat capacity data with theoretical heat capacity values calculated on the basis of a newly established increment system gives insight into the degree of hydration of the unfolded polypeptide chain. The percentage by which the experimental heat capacity of the unfolded polypeptide chain differs from the calculated heat capacity permits a quantitative estimate of the residual structure. This estimate is in good agreement with that based on CD absorption. The heat capacity approach has the advantage of comparing fully hydrated and partially hydrated residues in the same aqueous environment, whereas for example spectroscopic measurements, such as CD, are generally referred to the fully unfolded chain in concentrated urea or GdnHCl solutions. As the unfolded chains of pCSD1 and hCSD1 exhibit a smaller heat capacity than that calculated on the new peptide-based increment system [M. H?ckel et al., J. Mol. Biol. 291 (1999) 197-213], we conclude that the residues in the unfolded polypeptide chain are less hydrated than the same residues in oligopeptides. This suboptimal hydration is the result of residual structure in the chain as observed in both CD and heat capacity measurements.     

A method to quantify glucose utilization in vivo in skeletal muscle and white adipose tissue of the anaesthetized rat.

A quantitative method allowing determination of glucose metabolism in vivo in muscles and white adipose tissue of the anaesthetized rat is presented. A tracer dose of 2-deoxy[3H]glucose was injected intravenously in an anaesthetized rat and the concentration of 2-deoxy[3H]glucose was monitored in arterial blood. After 30-80 min, three muscles, the soleus, the extensor digitorum longus and the epitrochlearis, periovarian white adipose tissue and brain were sampled and analysed for their content of 2-deoxy[3H]glucose 6-phosphate. This content could be related to glucose utilization during the same time period, since (1) the integral of the decrease of 2-deoxy[3H]glucose in arterial blood was known and (2) correction factors for the analogue effect of 2-deoxyglucose compared with glucose in the transport and phosphorylation steps were determined from experiments in vitro. Glucose utilization was then measured by this technique in the tissues of post-absorptive rats in the basal state (0.1 munit of insulin/ml of plasma) or during euglycaemic-hyperinsulinaemic glucose clamp (8 munits of insulin/ml of plasma) and of 48 h-starved rats. Results corresponded qualitatively and quantitatively to the known physiological characteristics of the tissues studied.     

Cumulant analysis in fluorescence fluctuation spectroscopy

A novel technique for the analysis of fluorescence fluctuation experiments is introduced. Fluorescence cumulant analysis (FCA) exploits the factorial cumulants of the photon counts and resolves heterogeneous samples based on differences in brightness. A simple analytical model connects the cumulants of the photon counts with the brightness epsilon and the number of molecules N in the optical observation volume for each fluorescent species. To provide the tools for a rigorous error analysis of FCA, expressions for the variance of factorial cumulants are developed and tested. We compare theory with experiment by analyzing dye mixtures and simple fluorophore solutions with FCA. A comparison of FCA with photon-counting histogram (PCH) analysis, a related technique, shows that both methods give identical results within experimental uncertainty. Both FCA and PCH are restricted to data sampling times that are short compared to the diffusion time of molecules through the observation volume of the instrument. But FCA theory, in contrast to PCH, can be extended to treat arbitrary sampling times. Here, we derive analytical expressions for the second factorial cumulant as a function of the sampling time and demonstrate that the theory successfully models fluorescence fluctuation data.     

Statistical accuracy in fluorescence fluctuation experiments

The mathematical expression of the signal to noise ratio in fluorescence fluctuation experiments is derived for arbitrary sample profiles and for any mechanism of translational motion, and experimentally verified. The signal to noise ratio depends on the mean count rate per particle per dwell time, the mean number of particles per sample volume, time characteristics of the correlation function, sample profile characteristics, and the data collection time. Statistical accuracy of the third order moment of fluorescence intensity fluctuations is also studied. The optimum concentration for the third order moment analysis is about one particle per sample volume. Received: 13 February 1996 / Accepted: 20 September 1996     

A novel colorimetric method to quantify tannase activity of viable bacteria

A novel colorimetric method to quantify tannase activity of viable tannase-producing bacterial strains was developed through application of a visual reading method that was to detect the activity qualitatively. The novel method was sensitive enough to quantify the marginal tannase activity of strains that could not be otherwise measured by conventional spectrophotometric or colorimetric methods.     

An ELISA-based method to quantify osteocalcin carboxylation in mice

Osteocalcin was recently identified as an osteoblast-secreted hormone regulating insulin secretion and sensitivity. In mice and humans, osteocalcin can be present in the serum in carboxylated or undercarboxylated forms and it has been shown that it is the undercarboxylated form of osteocalcin which acts as a hormone. The study of osteocalcin different circulating forms in mouse serum, however, has been hampered by the absence of quantitative methodology. Here we described a triple enzyme-linked immunosorbent assay (ELISA) system for quantification of mouse total, carboxylated and uncarboxylated osteocalcin. That carboxylation of osteocalcin was decreased in mouse osteoblasts cultures treated with warfarin, an inhibitor of carboxylation validated this assay. This ELISA could also detect elevated levels of undercarboxylated osteocalcin in the serum of mice treated with warfarin and in the serum of Esp −/− mice, a mouse model known to have more undercarboxylated, i.e., active osteocalcin. These results show that this new ELISA system is a reliable method to assess carboxylation status of osteocalcin in cell culture supernatants as well as in mouse serum. Its use should facilitate the analysis of culture system or mouse model in which the hormonal activity of osteocalcin needs to be evaluated.     

A rapid method to quantify pro-oxidant activity in cultures of wood-decaying white-rot fungi

A new, rapid method for evaluation of lipid peroxidation promoting (pro-oxidant) activity in cultures of wood-decaying fungi was developed. The method is based on measurement of the rate of oxygen consumption in the reaction of linoleic acid peroxidation initiated by fungal culture filtrates. The liquid cultures of the white-rot fungi Bjerkandera adusta and Phanerochaete chrysosporium grown on wheat straw-containing glucose-peptone-corn steep liquor medium possessed significant levels of the pro-oxidant activity. Other white-rot fungi producing manganese peroxidase (MnP) were also found to show the activity. MnP demonstrated a crucial role as the major pro-oxidant agent in the fungal cultures. The total pro-oxidant activity may be considered as net result of the peroxidation by MnP and the inhibition by antioxidant compounds present in the fungal culture fluids.     

Chlorophyll a fluorescence transients: a fast data acquisition system to facilitate in vivo measurements

Characteristics of primary phases in chlorophyll a fluorescence transients based on room temperature in vivo measurement with a Plant Productivity Fluorometer (Brancker Model SF-10) can be greatly facilitated by coupling the instrument to a fast data acquisition system. The SF-10 was linked to a Multitech Industrial Corporation Microprofessor Microcomputer and further modified to ensure simultaneous onset of light activation and signal capture. Circuit diagrams and program listings are given in detail. This microprocessor system is capable of capturing signal changes over a minimum period of 200 milliseconds to a maximum of 6 seconds. Accuracy of recorded data is dependent on rate of change of the input signal and the recording time period. Acquisition and storage of 5000 points from zero to 300 milliseconds ensured clear resolution of F_o, I and D when played back over 120 seconds on a chart recorder. For routine use, the primary transient can be captured over 0–2 seconds and then replayed as an accompaniment to standard slower presentation of primary plus secondary transients. Coincidence of signal amplitude for F_p on both systems can then be ascertained while retaining adequate resolution of F_o and I.