Cell imaging and manipulation by nonlinear optical microscopy.

Advances in the technologies for labeling and imaging biological samples drive a constant progress in our capability of studying structures and their dynamics within cells and tissues. In the last decade, the development of numerous nonlinear optical microscopies has led to a new prospective both in basic research and in the potential development of very powerful noninvasive diagnostic tools. These techniques offer large advantages over conventional linear microscopy with regard to penetration depth, spatial resolution, three-dimensional optical sectioning, and lower photobleaching. Additionally, some of these techniques offer the opportunity for optically probing biological functions directly in living cells, as highlighted, for example, by the application of second harmonic generation to the optical measurement of electrical potential and activity in excitable cells. In parallel with imaging techniques, nonlinear microscopy has been developed into a new area for the selective disruption and manipulation of intracellular structures, providing an extremely useful tool of investigation in cell biology. In this review we present some basic features of nonlinear microscopy with regard both to imaging and manipulation, and show some examples to illustrate the advantages offered by these novel methodologies.

Global versus local linear beat-to-beat analysis of the relationship between arterial pressure and pulse transit time during dynamic exercise.

Global linear analysis has been traditionally performed to verify the relationship between pulse transit time (PTT) and systolic arterial pressure (SAP) at the level of their spontaneous beat-to-beat variabilities: PTT and SAP have been plotted in the plane (PTT,SAP) and a significant linear correlation has been found. However, this relationship is weak and in specific individuals cannot be found. This result prevents the utilization of the SAP-PTT relationship to derive arterial pressure changes from PTT measures on an individual basis. We propose a local linear approach to study the SAP-PTT relationship. This approach is based on the definition of short SAP-PTT sequences characterized by SAP increase (decrease) and PTT decrease (increase) and on their search in the SAP and PTT beat-to-beat series. This local approach was applied to PTT and SAP series derived from 13 healthy humans during incremental supine dynamic exercise (at 10, 20 and 30% of the nominal individual maximum effort) and compared to the global approach. While global approach failed in some subjects, local analysis allowed the extraction of the gain of the SAP-PTT relationship in all subjects both at rest and during exercise. When both local and global analyses were successful, the local SAP-PTT gain is more negative than the global one as a likely result of noise reduction.

The dependence of the conductance of the hemocyanin channel on applied potential and ionic concentration with mono- and divalent cations.

Incorporation of Megatura crenulata hemocyanin into phosphatidylcholine black lipid membranes results in the formation of ion channels. Channel properties depend on many factors, three of which are examined in this work: type and concentration of electrolyte and applied voltage. Eight cations at different concentrations have been used. Instantaneous conductance of the channel is a saturating function of both applied voltage and ionic strength of the bathing solution with monovalent cations, but only of ionic strength with divalent cations. Steady-state voltage-conductance relations are nonlinear for both signs but show slight saturation with ionic strength. Relaxation towards the steady state can be fitted by two exponentials with different time constants. All experimental data are fitted postulating the existence of a mechanism of voltage gating of the channel, and of discrete negative charge near its mouth. Specific and nonspecific binding of cations is required.

Ion transport through hemocyanin channels in oxidized cholesterol artificial bilayer membranes.

Incorporation of molluscan hemocyanins, obtained either from the blood of Megatura crenulata or Paludina vivipara, into oxidized-cholesterol black lipid films results in the formation of ionic channels. Channel conductance depends on the type of electrolyte present, ranging in our experiments from about 20 to 500 pS. It rises in a non-linear way as the salt concentration is increased, showing a saturation effect. An observed pH dependence of channel conductance suggests that there is a negative fixed charge associated with the pore. We discuss a model based on a simplified form of the Gouy-Chapman theory of the electrified double layer to explain the experimental results.

Different patterns of atrial activation in idiopathic atrial fibrillation: simultaneous multisite atrial mapping in patients with paroxysmal and chronic atrial fibrillation.

OBJECTIVES: We aimed to evaluate: 1) the behavior of electrical activity simultaneously in different atrial regions during atrial fibrillation (AF); 2) the difference of atrial activation between paroxysmal and chronic AF; 3) the atrial refractoriness dispersion; and 4) the correlation between the effective refractory periods (ERPs) and the FF intervals. BACKGROUND: Little data exist on the electrophysiologic characteristics of the different atrial regions in patients with AF. A more detailed knowledge of the electrical activity during AF may provide further insights to improve treatment of AF. METHODS: Right and left atria were extensively mapped in 30 patients with idiopathic AF (18 paroxysmal and 12 chronic). In different atrial locations, we analyzed 1) the FF interval duration; and 2) the grade of organization and, in case of organized electrical activity, the direction of atrial activation. Furthermore, in patients with paroxysmal AF, we determined the atrial ERP, evaluated the ERP dispersion and assessed the presence of a correlation between the ERPs and the FF intervals. RESULTS: In patients with chronic AF, we observed a shortening of the FF intervals and a greater prevalence of disorganized activity in all the atrial sites examined. In patients with paroxysmal AF, a significant dispersion of refractoriness was observed. The right lateral wall showed longer FF intervals and more organized atrial activity and, unexpectedly, the shortest mean ERPs. In contrast, the septal area showed shorter FF intervals, greater disorganization and the longest mean ERPs. CONCLUSIONS: Electrical activity during AF showed a significant spatial inhomogeneity, which was more evident in patients with paroxysmal AF. The mean FF intervals did not correlate with the mean ERPs.

Second-harmonic generation sensitivity to transmembrane potential in normal and tumor cells.

Second-harmonic generation (SHG) is emerging as a powerful tool for the optical measurement of transmembrane potential in live cells with high sensitivity and temporal resolution. Using a patch clamp, we characterize the sensitivity of the SHG signal to transmembrane potential for the RH 237 dye in various normal and tumor cell types. SHG sensitivity shows a significant dependence on the type of cell, ranging from 10 to 17% per 100 mV. Furthermore, in the samples studied, tumor cell lines display a higher sensitivity compared to normal cells. In particular, the SHG sensitivity increases in the cell line Balb/c3T3 by the transformation induced with SV40 infection of the cells. We also demonstrate that fluorescent labeling of the membrane with RH 237 at the concentration used for SHG measurements does not induce any measurable alteration in the electrophysiological properties of the cells investigated. Therefore, SHG is suitable for the investigation of outstanding questions in electrophysiology and neurobiology.

Absorption rate density (ARD) computation in microwave hyperthermia by the finite-difference time-domain method.

A mathematical model has been developed, which is able to predict power distributions in biological tissues during microwave hyperthermia delivered by waveguide applicators. The numerical solutions of Maxwell's equations was obtained by the finite-difference time-domain (FDTD) technique. Two improvements with respect to the standard implementation of FDTD were introduced: a separation between the source and load calculations (based on the Schelkunoff equivalence principle) and a simple routine that automatically recognises the steady state. Two commercially available applicators, a dual-ridged and a side-loaded waveguide, were modelled using their theoretical aperture fields. The absorption rate density (ARD) distributions delivered by these applicators were measured through phantom thermal dosimetry and compared with the patterns estimated by the simulation.

Absorbed power distributions from single or multiple waveguide applicators during microwave hyperthermia.

A theoretical model has been developed to calculate the power distributions in biological-like tissues in direct contact with waveguide applicators. A numerical solution of the coupled integral equations is performed by means of their transformation into matrix equations by using the method of moments, where the biological tissue and the applicator are described by two independent matrices. The model permits an accurate evaluation of the electric field at the aperture. The application of this method for the evaluation of arrays is also discussed. Power contour plots produced by applicators routinely used in clinics have been computed and compared with experimental results obtained with tissue-equivalent phantoms for three typical bodies: homogeneous, stratified and inhomogeneous.

An automated method for mapping human tissue permittivities by MRI in hyperthermia treatment planning.

This paper presents an automatic method to obtain tissue complex permittivity values to be used as input data in the computer modelling for hyperthermia treatment planning. Magnetic resonance (MR) images were acquired and the tissue water content was calculated from the signal intensity of the image pixels. The tissue water content was converted into complex permittivity values by monotonic functions based on mixture theory. To obtain a water content map by MR imaging a gradient-echo pulse sequence was used and an experimental procedure was set up to correct for relaxation and radiofrequency field inhomogeneity effects on signal intensity. Two approaches were followed to assign the permittivity values to fat-rich tissues: (i) fat-rich tissue localization by a segmentation procedure followed by assignment of tabulated permittivity values; (ii) water content evaluation by chemical shift imaging followed by permittivity calculation. Tests were performed on phantoms of known water content to establish the reliability of the proposed method. MRI data were acquired and processed pixel-by-pixel according to the outlined procedure. The signal intensity in the phantom images correlated well with water content. Experiments were performed on volunteers' healthy tissue. In particular two anatomical structures were chosen to calculate permittivity maps: the head and the thigh. The water content and electric permittivity values were obtained from the MRI data and compared to others in the literature. A good agreement was found for muscle, cerebrospinal fluid (CSF) and white and grey matter. The advantages of the reported method are discussed in the light of possible application in hyperthermia treatment planning.

Congenital long QT syndrome inducing 2:1 atrioventricular block: early detection in fetal life.

A case is reported of congenital long QT interval associated with fixed 2:1 atrioventricular block. The bradycardia was detected at 16 weeks of gestational age. The atrioventricular block was due to an extremely delayed ventricular repolarization. Early detection of bradycardia in fetal life and the demonstration of a normal positive correlation between QT duration and ventricular rate suggest that, in this case, the syndrome may be due to an anomaly of the myocardial cells rather than to imbalance of the sympathetic nervous system.

Continuous monitoring of myocardial acid-base status during intermittent warm blood cardioplegia.

OBJECTIVE: Intermittent warm blood cardioplegia (IWBC) is a well-established technique for myocardial protection during cardiac operations. According to standardized protocols, IWBC administration is currently performed every 15-20 min regardless of any individual variable and in the absence of any instrumental monitoring. We devised a new system for continuous measurement of the acid-base status of coronary sinus blood for on-line evaluation of myocardial oxygenation during IWBC. METHODS: In 19 patients undergoing cardiac surgery for coronary artery bypass graft and/or valve surgery and receiving IWBC (34-37 degrees C) by antegrade induction (3 min) and retrograde or antegrade maintenance (2 min) every 15 min, continuous monitoring of myocardial oxygenation and acid/base status was performed by means of a multiparameter PO(2), PCO(2), pH, and temperature sensor (Paratrend7 (R), Philips Medical System) inserted into the coronary sinus. RESULTS: Mean cross-clamping time was 76+/-26 min; ischemic time was 13+/-0.2 min. pH decline was not linear, showing an initial fast decline, a point of flexus, and a progressive slow decline. After every ischemic period, the pH adaptation curve showed a complex pattern reaching step-by-step lower minimum levels (7.28+/-0.14 during the first ischemic period, to 7.16+/-0.19 during the third ischemic period - P=0.003). PO(2) decreased rapidly at 90% in 5.0+/-1.2 min after every reperfusion. During ischemia, PCO(2) increased steadily at 1.6+/-0.1 mmHg per minute, with progressively incomplete removal after successive reperfusion, and progressive increase of maximal level (42+/-12 mmHg during the first ischemic period, to 53+/-23 mmHg during the third ischemic period - P=0.05). CONCLUSIONS: Myocardial oxygen, carbon dioxide, and pH show marked changes after repeated IWBC. Myocardial ischemia is not completely reversed by standardized reperfusions, as reflected by steady deterioration of PCO(2) and pH after each reperfusion. Progressive increase of reperfusion durations or direct monitoring of myocardial oxygenation could be advisable in cases of prolonged cross-clamping time.

Quantitative colorimetric analysis of liquid crystal films (LCF) for phantom dosimetry in microwave hyperthermia.

A fully quantitative analysis of liquid crystal film (LCF) color patterns, in phantom thermal dosimetry for microwave hyperthermia, is presented. An accurate determination of absorption rate density (ARD) is achieved by color image computer processing. This work is proven to be an improvement upon the semi-quantitative or qualitative descriptions of LCF colors performed essentially by visual analysis of photographs. Temperature-induced chromatic distributions are acquired as R, G, B (red, green, blue) signals by a CCD camera connected to a PC frame grabber board. These data, stored into three 512 x 512 memory buffers, are then converted to H, S, I (hue, saturation, intensity) colorimetric system. Provided a suitable calibration of the LCF, the H quantity can be transformed to temperature using a monotonic relationship. In this way, a temperature accuracy lower than 0.2 degrees C and a spatial resolution less than 1 mm are obtained. A sequence of thermal maps can be acquired and stored on disk at a maximum rate of 1 image/2 s, and then the ARD is calculated at each pixel of the map using the least squares method.

Experimental approach for testing the uncoupling between cardiovascular variability series.

In cardiovascular variability analysis, the significance of the coupling between two series is commonly assessed by defining a zero level on the magnitude-squared coherence (MSC). Although the use of the conventional value of 0.5 does not consider the dependence of MSC estimates on the analysis parameters, a theoretical threshold Tt is available only for the weighted covariance (WC) estimator. In this study, an experimental threshold for zero coherence Te was derived by a statistical test from the sampling distribution of MSC estimated on completely uncoupled time series. MSC was estimated by the WC method (Parzen window, spectral bandwidth B = 0.015, 0.02, 0.025, 0.03 Hz) and by the parametric autoregressive (AR) method (model order M= 4, 8, 12, 16), on time series with length L = 180, 300, 420, 540 s. Te decreased with increasing B and L and with decreasing M (range: 0.11-0.54 for WC estimator, 0.06-0.46 for AR estimator). Values for the typical parameter settings of WC and AR estimation (B = 0.025 Hz; M = 8; L = 300 s) were, respectively, 0.24 and 0.17. Moreover, Tt was always higher (range: 0.12-0.65) and the results were less dependable than those for Te in defining the zero level of MSC. Thus, with the proposed method, the hypothesis of uncoupling is rejected by accounting for the parameters that affect the confidence of spectral and cross-spectral estimates. The broad applicability of this approach should favour its introduction for assessing the significance of the coupling between cardiovascular variability series.

Beat-to-beat measurement and analysis of the R-T interval in 24 h ECG Holter recordings.

This study assesses the feasibility of beat-to-beat measurement of the R-T interval in Holter ECG recordings. The low sampling rate of the Holter system was increased by a specific interpolating filter, and the precision and accuracy of two T-wave fiducial point (T-wave maximum: Tm, T-wave end: Te) detection algorithms were compared. The results of the validation tests show better performance of the Tm measurement procedure in the presence of high noise levels. The overall process for the beat-to-beat R-T interval measurement was then tested on ECG Holter recordings collected during free and controlled respiration. Finally, the R-Tm and the corresponding R-R intervals were measured on 24 h ECG recordings of healthy subjects and the spectral analysis was applied to the constructed series. Both R-R and R-Tm spectra show two main frequency components (low-frequency approximately 0.1 Hz, high-frequency approximately 0.25 Hz) changing in their power ratios continuously throughout the 24 h period. The method described seems to provide a dynamic index of the sympatho-vagal balance at the ventricle that can be useful for a deeper understanding of ventricular repolarisation duration variability.

Principal component analysis and cluster analysis for measuring the local organisation of human atrial fibrillation.

The distribution of atrial electrogram types has been proposed to characterise human atrial fibrillation. The aim of this study was to provide computer procedures for evaluating the local organisation of intracardiac recordings during AF as an alternative to off-line manual classification. Principal component analysis (PCA) reduced the data set to a few representative activations, and cluster analysis (CA) measured the average dissimilarity between consecutive activations of an intracardiac signal. The data set consisted of 106 bipolar signals recorded on 11 patients during electrophysiological studies for catheter ablation. Performances of PCA and CA in distinguishing between organised (type I) and disorganised (type II/III, Wells criteria) were assessed, in comparison with manual reading, by evaluating the predictive parameters of the classification analysis. Both methods gave high accuracy (92% for PCA and 89% for CA), confirming the feasibility of on-line characterisation of AF. Sensitivity was lower than specificity (81% against 98% for PCA, and 77% against 97% for CA), with seven out of eight misclassifications of PCA in common with CA. Differences between manual and computer analysis may be related to the higher resolution of PCA and CA in the measurement of the organisation of atrial activations. These procedures are suitable for providing automatic (by CA) or semi-automatic (by PCA) measures of the extent of local organisation of AF in the pre-ablation treatment phase.

Performance assessment of standard algorithms for dynamic R-T interval measurement: comparison between R-Tapex and R-T(end) approach.

Three automatic approaches to ventricular repolarisation duration measurement (R-Tapex, R-T(end threshold) and R-T(end fitting) methods) are compared on computer-generated and real ECG signals, in relation to their reliability in the presence of the most common electrocardiographic artefacts (i.e. additive broadband noise and additive and multiplicative periodical disturbances). Simulations permit the evaluation of the amount of R-T beat-to-beat variability induced by the artefacts. The R-T(end threshold) method performs better than the R-T(end fitting) one, and, hence, the latter should be used with caution when R-T(end) variability is addressed. Whereas the R-Tapex method is more robust with regard to broadband noise than the R-T(end threshold) one, the reverse situation is observed in the presence of periodical amplitude modulations. A high level of broadband noise dose not prevent the detection of the central frequency of underlying R-T periodical changes. Comparison between the power spectra of the beat-to-beat R-T variability series obtained from three orthogonal ECG leads (X,Y,Z) is used to assess the amount of real and artefactual variability in 13 normal subjects at rest. The R-Tapex series displays rhythms at high frequency (HF) with a percentage power on the Z lead (57.1 +/- 4.9) greater than that on the X and Y leads (41.9 +/- 4.6 and 46.1 +/- 4.9, respectively), probably because of respiratory-related artefacts affecting the Z lead more remarkably. More uniform HF power distributions over X,Y,Z leads are observed in the R-T(end threshold) series (31.8 +/- 3.8, 39.2 +/- 4.1 and 35.1 +/- 4.2, respectively), thus suggesting minor sensitivity of the R-T(end threshold) measure to respiratory-related artefacts.

Improved late potential analysis in frequency domain.

This work presents a technique to improve the identification of late potentials (LP) in patients affected by greater arrhythmogenic right ventricular disease (GARVD). Several authors have documented the correlation between GARVD and LP by means of time domain analysis. Moreover, the high incidence of bundle branch block in patients affected by GARVD suggests LP analysis in the frequency domain be performed. The method of spectral mapping of the ECG with Fourier transform was adopted. This consists in dividing the ST segment into 25 subsegments and estimating their frequency components by means of the fast Fourier transform. Recently, it was documented that this technique suffers from poor reproducibility of results. Low reproducibility is the consequence of an improper localization of the analysed QRS segments. An algorithm to increase the QRS end point identification reproducibility is proposed. An optimal QRS filter was adopted as well as a technique based on the Hilbert transform. This technique allowed the reliability of the normality factor estimates to be improved. The computed normality factors on the XYZ leads and on the vector magnitude were used to classify patients and healthy subjects; 28 patients affected by greater arrhythmogenic right ventricular disease and 35 healthy subjects were analysed in the study. High sensitivity was obtained with respect to GARVD and clinical sustained ventricular tachycardia by means of a cluster analysis technique. By applying the technique proposed in this paper the identification of LP in GARVD was increased from 47% to 88%, when clinical sustained ventricular tachycardia was documented, whereas in patients affected by GARVD but not prone to sustained ventricular tachycardia LP identification increases from 18% to 64%.

[Stable ventricular tachycardia in arrhythmogenic dysplasia of the right ventricle in sportsmen]. / Stabil'naia zheludochkovaia takhikardiia pri aritmogennoi displazii pravogo zheludochka u sportsmenov.

Right ventricular arrhythmogenic dysplasia (RVAD) is a typical asymptomatic arrhythmogenic cardiopathy in athletes, which is occasionally concurrent with normal ventricular function and life-threatening arrhythmias. A total of 32 athletes (28 males and 4 females, mean age, 23 years, mean follow-up, 6.7 years) were examined for severe cardiac arrhythmias with left bundle branch block. The conclusive diagnosis of RVAD was established from clinical, echocardio-, and angiographic evidence. The protocol of the examination involved Holter monitoring, loading tests, electrophysiological study, two-dimensional echocardiography, cardiac angiography of the right and left ventricles, coronary angiography. The most severe arrhythmias were observed in athletes whose mean age was 23.4 years, 20 patients had sustained ventricular tachycardia (it occurred only in 19 who were indulging in sports), 6 presented with transient ventricular tachycardia, and 1 had ventricular fibrillation. They all had been considered fit for sports. The disease proceeded severely in 16 of 32 athletes (in 13 of 16 while indulging in sports), the conditions close to syncope were seen in 9 patients (8 had sustained ventricular tachycardias and 1 had transient ventricular tachycardias), syncopes were observed in 5 patients (sustained ventricular tachycardias).

Size controlled hydroxyapatite and calcium carbonate particles: synthesis and their application as templates for SERS platform.

An elegant route for hydroxyapatite (HA) particle synthesis via ionic exchange reaction is reported. Calcium carbonate particles (CaCO3) were recrystallized into HA beads in water solution with phosphate ions. The size of initial CaCO3 particles was controlled upon the synthesis by varying the amount of ethylene glycol (EG) in aqueous solution. The average size of HA beads ranged from 0.6±0.1 to 4.3±1.1µm. Silver nanoparticles were deposited on the surface of HA and CaCO3 particles via silver mirror reaction. Surface enhanced Raman scattering of silver functionalized beads was demonstrated by detecting Rhodamine B. CaCO3 and HA particles have a great potential for design of carrier which can provide diagnostic and therapeutic functions.