Dynamic range and background filtering in raster image correlation spectroscopy.

Raster-scan image correlation spectroscopy (RICS) enables researchers to measure molecular translational diffusion constants and concentrations from standard confocal laser scanning microscope images and is suitable for measuring a wide range of mobility, especially fast-diffusing molecules. However, as RICS analysis is based on the spatial autocorrelation function of fluorescence images, it is sensitive to the presence of fluorescent structures within the image. In this study, we investigate methods to filter out immobile or slow moving background structures and their impact on RICS results. Both the conventional moving-average subtraction-based method and cross-correlation subtraction-based method are rationalized and quantified. Simulated data and experimental measurements in living cells stress the importance of optimizing the temporal resolution of background filtering for reliable RICS measurements. Finally, the capacity of RICS analysis to separate two species is studied.

Optical sensing of mechanical pressure based on diffusion measurement in polyacrylamide cell-like barometers.

Diffusion and transport of small molecules within hydrogel networks are of high interest for biomedical and pharmaceutical research. Herein, using fluorescence correlation spectroscopy (FCS), we experimentally showed that the diffusion time in the hydrogel was directly related to the mechanical state (compression or swelling) and thus to the volume fraction of the gel. Following this observation, we developed cell-like barometers in the form of PAA microbeads, which when incorporated between cells and combined with a diffusion-based optical readout could serve as the first biosensors to measure the local pressure inside the growing biological tissues. To illustrate the potential of the present method, we used multicellular spheroids (MCS) as a tissue model, and it was observed that the growth-associated tissue stress was lower than 1 kPa, but significantly increased when an external compressive stress was applied.

Cell-like pressure sensors reveal increase of mechanical stress towards the core of multicellular spheroids under compression.

The surrounding microenvironment limits tumour expansion, imposing a compressive stress on the tumour, but little is known how pressure propagates inside the tumour. Here we present non-destructive cell-like microsensors to locally quantify mechanical stress distribution in three-dimensional tissue. Our sensors are polyacrylamide microbeads of well-defined elasticity, size and surface coating to enable internalization within the cellular environment. By isotropically compressing multicellular spheroids (MCS), which are spherical aggregates of cells mimicking a tumour, we show that the pressure is transmitted in a non-trivial manner inside the MCS, with a pressure rise towards the core. This observed pressure profile is explained by the anisotropic arrangement of cells and our results suggest that such anisotropy alone is sufficient to explain the pressure rise inside MCS composed of a single cell type. Furthermore, such pressure distribution suggests a direct link between increased mechanical stress and previously observed lack of proliferation within the spheroids core.

Wavelet transform analysis of chromatin texture changes during heat shock.

Texture analysis can be a useful tool to investigate the organization of chromatin. Approaches based on multiscale analysis and in particular the 'à trou' wavelet analysis has already been used for microscopy (Olivo Marin). In order to analyse texture changes, the statistical properties of the wavelet coefficient images were summarized by the first four statistical orders: mean, standard deviation, skewness and kurtosis of the coefficient image histogram. The 'à trou' transform provided a representation of the wavelet coefficients and texture parameters with the same statistical robustness throughout the scale spaces. It was applied for quantifying chromatin texture and heat-induced chromatin changes in living cells. We investigated the changes by both laser scanning and spinning disk confocal microscopies and compared the texture parameters before and after increasing duration of heat shock exposure (15 min, 30 min and 1 h). Furthermore, as activation of the heat shock response also correlates with a rapid localization of HSF1 within a few nuclear structures termed nuclear stress bodies (nSBs), we compared the dynamics of nSBs formation with that of textural changes during 1 h of continuous heat shock. Next, we studied the recovery phase following a 1-h heat shock. Significant differences were observed, particularly affecting the perinucleolar region, even for the shortest heat shock time affecting mostly the skewness and standard deviation. Furthermore, progressive changes could be observed according to the duration of heat shock, mostly affecting fine details (pixel-wise changes) as revealed by the parameters, obtained from the first- and second-order wavelet coefficients. 'A trou' wavelet texture analysis provided a sensitive and efficient tool to investigate minute changes of chromatin.

Note: multi-confocal fluorescence correlation spectroscopy in living cells using a complementary metal oxide semiconductor-single photon avalanche diode array.

Living cells are heterogeneous and rapidly changing biological samples. It is thus desirable to measure molecular concentration and dynamics in many locations at the same time. In this note, we present a multi-confocal setup capable of performing simultaneous fluorescence correlation spectroscopy measurements, by focusing the spots with a spatial light modulator and acquiring data with a monolithic 32 × 32 single-photon avalanche photodiode array. A post-processing method is proposed to correct cross-talk effects between neighboring spots. We demonstrate the applicability of our system by simultaneously measuring the diffusion of free enhanced Green Fluorescent Protein (eGFP) molecules at nine different points in living cells.

Measurement of surface concentration of fluorophores by fluorescence fluctuation spectroscopy.

Fluorescence fluctuation spectroscopy is applied to study molecules passing through a small observation volume, usually subjected to diffusive or convective motion in a liquid phase. We suggest that such a technique could be used to measure the areal absolute concentration of fluorophores deposited on a substrate or embedded in a thin film, with a resolution of a few micrometers. The principle is to translate the solid substrate in front of a confocal fluorescence microscope objective and to record the subsequent fluctuations of the fluorescence intensity. The validity of this concept is investigated on model substrates (fluorescent microspheres) and DNA biochips.

Continuous photobleaching in vesicles and living cells: a measure of diffusion and compartmentation.

We present a comprehensive and analytical treatment of continuous photobleaching in a compartment, under single photon excitation. In the very short time regime (t<0.1 ms), the diffusion does not play any role. After a transition (or short time regime), one enters in the long time regime (t>0.1-5 s), for which the diffusion and the photobleaching balance each other. In this long time regime, the diffusion is either fast (i.e., the photobleaching probability of a molecule diffusing through the laser beam is low) so that the photobleaching rate is independent of the diffusion constant and dependent only of the laser power, or the diffusion is slow (i.e., the photobleaching probability is high) and the photobleaching rate is mainly dependent on the diffusion constant. We illustrate our theory by using giant unilamellar vesicles ranging from approximately 10 to 100 microm in diameter, loaded with molecules of various diffusion constants (from 20 to 300 microm2/s) and various photobleaching cross sections, illuminated under laser powers between 3 and 100 microW. We also demonstrated that information about compartmentation can be obtained by this method in living cells expressing enhanced green fluorescent proteins or that were loaded with small FITC-dextrans. Our quantitative approach shows that molecules freely diffusing in a cellular compartment do experience a continuous photobleaching. We provide a generic theoretical framework that should be taken into account when studying, under confocal microscopy, molecular interactions, permeability, etc.

Photobleaching, mobility, and compartmentalisation: inferences in fluorescence correlation spectroscopy.

In living cells the transport and diffusion of molecules is constrained by compartments of various sizes. This paper is an attempt to show that the size of these compartments can in principle be estimated experimentally from Fluorescence Correlation Spectroscopy (FCS) combined with the measurement of the photobleaching rate. In this work, confocal fluorescence microscopy experiments have been carried out on giant unilamellar vesicles, a system that mimics cellular compartmentalisation. We have developed numerical and analytical models to describe the fluorescence decay due to photobleaching in this geometry, which has enabled us to point out two regimes depending on the value of the parameter P(B) = sigma(B)P/D (where sigma(B) is the photobleaching cross section of the dye, D its diffusion constant, and P the laser power (in photon/s)). In particular, when P(B) << 1 (i.e. in the fast diffusion regime), the photobleaching rate is independent of the diffusion constant and scales like sigma(B)P/R2, in agreement with the experimental results. On the other hand, the standard diffusion models used to analyse the FCS data do not take into account the effects of the fluorescence decay on the autocorrelation curve. We show here how to correct the raw data for these drawbacks.

In vitro and in vivo investigations on fluoroquinolones; effects of the P-glycoprotein efflux transporter on brain distribution of sparfloxacin.

The role of mdr1a-encoded P-glycoprotein on transport of several fluoroquinolones across the blood-brain barrier was investigated. In vitro, P-glycoprotein substrates were selected by using a confluent monolayer of MDR1-LLC-PK1 cells. The inhibition of fluoroquinolones (100 microM) on transport of rhodamine-123 (1 microM) was compared with P-glycoprotein inhibitors verapamil (20 microM) and SDZ PSC 833 (2 microM). Subsequently, transport polarity of fluoroquinolones was studied. Sparfloxacin showed the strongest inhibition (26%) and a large polarity in transport, by P-glycoprotein activity. In vivo, using mdr1a (-/-) and wild-type mice, brain distribution of pefloxacin, norfloxacin, ciprofloxacin, fleroxacin and sparfloxacin was determined at 2, 4, and 6 h following intra-arterial infusion (50 nmol/min). Brain distribution of sparfloxacin was clearly higher in mdr1a (-/-) mice compared with wild-type mice. Sparfloxacin was infused (50 nmol/min) for 1, 2, 3 and 4 h in which intracerebral microdialysis was performed. At 4 h, in vivo recovery (dynamic-no-net-flux method) was 6.5+/-2.2 and 1.5+/-0.5%; brain(ECF) concentrations were 5.1+/-0.2 and 26+/-21 microM; and total brain concentrations were 7.2+/-0.3 and 23+/-0.3 microM in wild-type and mdr1a (-/-) mice, respectively. Plasma concentrations were similar (18.4+/-0.7 and 17.9+/-0.5 microM, respectively). In conclusion, sparfloxacin enters the brain poorly mainly because of P-glycoprotein activity at the blood-brain barrier.

Low-temperature polarized helium-3 for MRI applications.

The first 3He nuclear magnetic resonance (NMR) experiments using low-temperature prepolarization are presented. 3He cells were polarized at 4.2 K and 4.7 T, transported to another magnet, heated to room temperature, and used for NMR experiments at 2.35 T. Cells with and without a rubidium coating were tested. In both cases, the NMR signal was greater than 100 times the thermal equilibrium signal. No evidence of a rubidium coating effect on the longitudinal relaxation time T1 of 3He (500 mbar) at 4.2 K could be demonstrated. NMR gradient-echo images of the cells were acquired.

NMR imaging of thermally polarized helium-3 gas.

It is shown that thermally polarized 3He gas can be used to measure important physical parameters and to design, test, and tune imaging sequences. The bulk values of T1, T2, and the diffusion coefficient were measured in a glass cell containing a mixture of helium-3 (0.8 bar) and oxygen (0.2 bar). They were found to be T1 = 7 s, T2 = 2.4 s, and D = 1.6 cm2 s(-1). The relaxation times T2* and T1 and the apparent diffusion coefficient of thermally polarized helium-3 gas were measured in the rat lung, and these parameters were used to design a helium-3 optimized multi-spin-echo sequence which was shown to increase the signal-to-noise ratio sufficiently to obtain the first NMR-images of thermally polarized helium-3 in the rat lung.

Pharmacokinetic-pharmacodynamic contributions to the convulsant activity of fluoroquinolones in rats.

The in vivo convulsant activities in rats of five representative fluoroquinolones (FQs), norfloxacin, enoxacin, sparfloxacin, fleroxacin, and pefloxacin, were compared. The experimental approach allowed distinction between the drugs' ability to reach the pharmacological receptors at the level of the central nervous system (pharmacokinetic contribution) and their ability to interact with these receptors (pharmacodynamic contribution). The presence of a methyl group on the piperazine moiety decreased the pharmacodynamic contribution to the convulsant activity by severalfold, and the ratios of concentrations of the FQs in cerebrospinal fluid (CSF) to concentrations of unbound FQs in plasma varied from about 5 to 75% as a function of lipophilicity. Interestingly, FQs with the highest intrinsic convulsant activities had the lowest levels of diffusion in CSF and vice versa. This in vivo approach provides information complementary to that of in vitro experiments and should be recommended for early preclinical assessment of a new FQ's epileptogenic risk.

A new approach for early assessment of the epileptogenic potential of quinolones.

The epileptogenic potential of pefloxacin and norfloxacin, two quinolone antibiotics, was investigated in vivo in three different animal species by measuring drug concentrations in cerebrospinal fluid (CSF), which is part of the biophase, at the onset of convulsions. Interestingly, the pefloxacin-to-norfloxacin concentration ratios in CSF were virtually constant across the species (7.0, 6.6, and 6.0 in mice, rats, and rabbits, respectively), suggesting that this approach could be used to predict the relative epileptogenic potential of quinolones in humans.

Development of a new quantitative approach for the isobolographic assessment of the convulsant interaction between pefloxacin and theophylline in rats.

PURPOSE: A new mathematical approach was developed to quantify convulsant interaction between pefloxacin and theophylline in rats. METHODS: Animals received each compound separately or in different combination ratios. Infusion was stopped at the onset of maximal seizures. Cerebrospinal fluid (CSF) and plasma samples were collected for HPLC drug determination. The nature and intensity of the pharmacodynamic (PD) interaction between drugs was assessed with a new modeling approach which includes (a) data transformation to create an essentially error-free X-variable and (b) estimation of an interaction parameter a by fitting a nonlinear hyperbolic model to the combination data with unweighted nonlinear regression. RESULTS: Drug disposition to the biophase was linear within the range of administered doses. The estimates of a suggested a Loewe antagonistic interaction between pefloxacin and theophylline at the induction of maximal seizures in rats. Similar intensity of PD interaction was observed at the dose and biophase level (alpha was -0.415 +/- 0.069 and -0.567 +/- 0.079, respectively). CONCLUSIONS: The suitability of the proposed model was assessed by Monte Carlo simulation. This new mathematical approach enabled the characterization of the Loewe antagonistic nature of the PD (convulsant) interaction between pefloxacin and theophylline, whereas previously used methodologies failed to do so.

Pharmacokinetic-pharmacodynamic contributions to the convulsant activity of pefloxacin and norfloxacin in rats.

The purpose of this investigation was to compare the convulsant activity of two quinolones differing, respectively, by the presence (pefloxacin) or absence (norfloxacin) of a methyl group on the piperazine moiety at the position 7 of their parent nuclei and consequently by their lipophilicity. An in vivo model was used, which can distinguish between ease in reaching pharmacological receptors at the central nervous system level, and ability to interact with these receptors. Male Sprague-Dawley rats (approximately 230g-300g) received an i.v. infusion of pefloxacin or norfloxacin at one of four different rates: 480, 960, 1440 and 1920 micromol/hr, until the onset of maximal seizures. This occurred after an average of 12.7 to 69.4 min. We found enough evidence to suggest that in these conditions the contribution of pefloxacin metabolites, including norfloxacin, to its convulsant activity was negligible. Doses of pefloxacin and concentrations in cerebrospinal fluid and plasma (total and unbound) at the pharmacodynamic endpoint were all independent of infusion rate, whereas only cerebrospinal fluid concentrations of norfloxacin were independent of infusion rate. The overall cerebrospinal fluid concentration of norfloxacin (47.3 +/- 9.9 micromol/liter) was about 8-fold lower than that of pefloxacin (380 +/- 27 micromol/liter), indicating that on average the "intrinsic convulsant activity" of norfloxacin is 8-fold greater than that of pefloxacin. However, total doses of pefloxacin and norfloxacin at the onset of maximal seizures were in the same order of magnitude (1500-2000 micromol/kg), suggesting that the higher ability of the more lipophilic pefloxacin to reach central nervous system compensates for its lower intrinsic convulsant activity.

Rifampin-induced nonresponsiveness of giant cell arteritis to prednisone treatment.

Rifampin is an enzymatic inducer known to increase steroid metabolism. We studied two patients with giant cell arteritis in whom rifampin caused nonresponsiveness to prednisone treatment. A prednisone pharmacokinetics study was done. When rifampin-prednisone treatment must be used in giant cell arteritis, we propose increasing the prednisone dosage to 2 mg/kg per day.