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Enrico Gratton

Publications and source records attributed to Enrico Gratton.

At least 19 recordsLinked to original sources
PubMed2003

Detection of the fast neuronal signal on the motor cortex using functional frequency domain near infrared spectroscopy.

Using non-invasive near infrared spectroscopy fast changes in the range of ms in the optical properties of neurons during brain activity have been described. Since the signal is small, the system to detect it has to be highly noise optimized. We used a frequency-domain tissue oximeter, whose laser diodes were modulated at 110 MHz and the amplitude (AC), mean intensity (DC) and phase (phi) of the modulated optical signal was measured at 96 Hz sample rate. In two volunteers, 36 and 37 years old, the probe consisting of 4 crossed source detector pairs was placed above the motor cortex (C3 position), contralateral to the hand performing the tapping exercise. The tapping frequency was set at 2.5 times the heart rate of the subject to avoid the influence of harmonics on the signal. An electronic device recorded the tapping movement. Control-data were obtained from a solid medium of approximately the same optical properties as the human head. To reduce physiological noise the arterial pulsatility was removed using an adaptive filter, the data was detrended by a high pass filter and a cross correlation function between the optical data and the tapping signal was calculated. The instrumental noise of the control data was very low (AC mean 0.0015% +/- SD 0.00092%, DC 0.00037% +/- 0.00023% and phi 0.00083 degrees +/- 0.00042 degrees). On the head the noise level was AC 0.0042% +/- 0.0031%, DC 0.0021% +/- 0.0012% and phi 0.0020 degrees +/- 0.0017 degrees. In 14 DC, 5 AC and 0 phi out of 30 locations a fast signal was detected, which was higher (p<0.001) than the noise level. This signal disappeared during non-tapping periods. With the signal to noise ratio that we have achieved single subject measurements become feasible.

Animals↗
PubMed2003

Mapping of hemodynamics on the human calf with near infrared spectroscopy and the influence of the adipose tissue thickness.

We investigated the influence of the adipose tissue thickness (ATT) on near-infrared spectroscopy (NIRS) measurements of the absorption coefficient (mu a), the reduced scattering coefficient (mu s') and changes in concentrations of oxyhemoglobin ([O2Hb]) and deoxyhemoglobin ([HHb]). We used a frequency domain spectrometer and a special probe to generate maps of these parameters on the human calf during venous occlusion. For ATT below 6 mm mu a remained constant, whereas for ATT between 6 and 14 mm mu a decreased quickly and became almost constant again for ATT larger than 14 mm. Mu s' was not significantly altered by the ATT but the values showed a high variability between subjects. We found significantly different changes in both the [O2Hb] and the [HHb] between the proximal and distal locations of measurement. Although ATT influences the recovery of the optical properties of the underlying tissue, these differences depending on the location cannot be sufficiently explained by the ATT for the following reasons. The ATT varied little within one subject (mean difference 0.88 +/- 1.80 mm). The inter-subject variability was 5 times higher. For a given ATT within one subject we observed different values for changes in [O2Hb] and [HHb] depending on the measurement location. Moreover for a smaller ATT the difference between the values of delta[O2Hb] and delta[HHb] proximal versus distal were more distinct. The thinner the overlying tissue (ATT) the higher is the proportion of muscle tissue in the probed tissue volume. Therefore these differences are most likely coming from the muscle tissue rather than the ATT. This indicates that although the ATT has an evident influence on the measurement of optical parameters and hemodynamics and should therefore be recognized when performing NIRS measurements, other factors will have to be considered as well.

Adipose Tissue↗
PubMed2003

The roles of changes in deoxyhemoglobin concentration and regional cerebral blood volume in the fMRI BOLD signal.

To study the behavior of cerebral physiological parameters and to further the understanding of the functional magnetic resonance imaging (fMRI) blood-oxygen-level-dependent (BOLD) effect, multisource frequency-domain near-infrared and BOLD fMRI signals were recorded simultaneously during motor functional activation in humans. From the near-infrared data information was obtained on the changes in cerebral blood volume and oxygenation. To relate our observations to changes in cerebral blood flow the well-known "balloon" model was employed. Our data showed that the deoxyhemoglobin concentration is the major factor determining the time course of the BOLD signal. The increase in cerebral blood oxygenation during functional activation is due to an increase in the velocity of blood flow, and occurs without significant swelling of the blood vessels.

Adolescent↗
PubMed2003

Neonatal development of the stratum corneum pH gradient: localization and mechanisms leading to emergence of optimal barrier function.

Although basal permeability barrier function is established at birth, the higher risk for infections, dermatitis, and percutaneous absorption of toxic agents may indicate incomplete permeability barrier maturation in the early neonatal period. Since stratum corneum (SC) acidification in adults is required for normal permeability barrier homeostasis, and lipid processing occurs via acidic pH dependent enzymes, we hypothesized that, in parallel with the less acidic surface pH, newborn SC would exhibit signs of incomplete barrier formation. Fluorescence lifetime imaging reveals that neonatal rat SC acidification first becomes evident by postnatal day 3, in extracellular "microdomains" at the SC- stratum granulosum (SG) interface, where pH-sensitive lipid processing is known to occur. This localized acidification correlated temporally with efficient processing of secreted lamellar body contents to mature extracellular lamellar bilayers. Since expression of the key acidifying mechanism NHE1 is maximal just prior to birth, and gradually declines over the first postnatal week, suboptimal SC acidification at birth cannot be attributed to insufficient NHE1 expression, but could instead reflect reduced NHE1 activity. Expression of the key lipid processing enzyme, beta-glucocerebrosidase (beta-GlcCer'ase), develops similar to NHE1, excluding a lack of beta-GlcCer'ase protein as rate limiting for efficient lipid processing. These results define a postnatal development consisting of initial acidification in the lower SC followed by outward progression, which is accompanied by formation of mature extracellular lamellar membranes. Thus, full barrier competence appears to require the extension of acidification in microdomains from the SC/SG interface outward toward the skin surface in the immediate postnatal period.

Acids↗
PubMed2003

Localized irregularities in hemoglobin flow and oxygenation in calf muscle in patients with peripheral vascular disease detected with near-infrared spectrophotometry.

PURPOSE: Near-infrared spectrophotometry is used to measure flow, concentration, and oxygenation of hemoglobin in arterioles, capillaries, and venules several centimeters deep in tissue. The purpose of this study was to investigate the distribution of flow, concentration, and oxygenation of hemoglobin in calf muscle in patients with documented peripheral arterial occlusive disease (PVD), patients with risk factors for PVD,and healthy younger subjects at rest. METHOD: With a frequency-domain near-infrared spectrophotometer and a specially designed probe, we generated maps at 22 locations simultaneously of hemoglobin flow, concentration, and oxygenation, with the venous occlusion method. Eight legs of 7 patients with diagnosed PVD (PVD group), 10 legs of 8 patients with normal ankle-brachial index but with risk factors for PVD (RF group), and 16 legs of 8 healthy subjects (H group) were studied. RESULTS: Global mean values were significantly (P <.05) different between the three groups for oxygen consumption (PVD group, 0.027 +/- 0.009 mL/100 g/min; RF group, 0.038 +/- 0.017 mL/100 g/min; H group, 0.022 +/- 0.020 mL/100 g/min), venous oxygen saturation (PVD, 59.7% +/- 15.4%; RF, 69.6% +/- 10.5%; H, 80.8% +/- 4.5%), and, at 60 s of venous occlusion, concentration changes in oxyhemoglobin (PVD, 4.48 +/- 3.25 micromol/L; RF, 8.44 +/- 2.33 micromol/L; H, 6.85 +/- 4.57 micromol/L), deoxyhemoglobin (PVD, 3.60 +/- 0.73 micromol/L; RF, 4.39 +/- 1.30 micromol/L; H, 2.36 +/- 1.79 micromol/L), and total hemoglobin (PVD, 8.07 +/- 3.83 micromol/L; RF, 12.83 +/- 2.75 micromol/L; H, 9.21 +/- 6.34 micromol/L). No significant difference was found between the three groups for hemoglobin flow (PVD, 0.92 +/- 0.69 micromol/100 mL/min; RF, 1.68 +/- 0.50 micromol/100 mL/min; H, 1.44 +/- 1.17 micromol/100 mL/min) and blood flow (PVD, 0.45 +/- 0.28 mL/100 g/min; RF, 0.77 +/- 0.21 mL/100 g/min; H, 0.62 +/- 0.50 mL/100 g/min). All parameters featured a distribution dependent on location. CONCLUSION: Mean value for venous oxygen saturation was higher in healthy subjects compared to patients with documented PVD. In patients with PVD, areas of lower oxygenation were clearly discernible. At distal locations of calf muscle, significant correlations between reduced hemoglobin flow, venous oxygen saturation, oxyhemoglobin, and total hemoglobin and reduced ankle-brachial index were found. Maps revealed localized irregularities in oxyhemoglobin, total hemoglobin, and venous oxygen saturation in patients with PVD. Near-infrared spectrophotometry is a noninvasive bedside technique that can enable determination of blood flow and oxygenation in tissue and may provide a method for evaluating patients with PVD.

Adult↗
PubMed2003

Fluorescence lifetime imaging for the two-photon microscope: time-domain and frequency-domain methods.

Fluorescence lifetime images are obtained with the laser scanning microscope using two methods: the time-correlated single-photon counting method and the frequency-domain method. In the same microscope system, we implement both methods. We perform a comparison of the performance of the two approaches in terms of signal-to-noise ratio (SNR) and the speed of data acquisition. While in our practical implementation the time-correlated single-photon counting technique provides a better SNR for low-intensity images, the frequency-domain method is faster and provides less distortion for bright samples.

Adenylate Kinase↗
PubMed2003

Camptothecin poly[n-(2-hydroxypropyl) methacrylamide] copolymers in antitopoisomerase-I tumor therapy: intratumor release and antitumor efficacy.

Soluble copolymers of camptothecin (CPT), based on poly[N-(2-hydroxypropyl) methacrylamide] (pHPMA), were obtained by conjugation through the degradable spacers -Gly-Phe-Leu-Gly- or -Gly-6-aminohexanoyl-Gly-. We investigated to what extent passive accumulation and retention of hydroxypropyl methacrylamide copolymer of CPT (pHPMA-CPT) in tumors and modulation of the drug release influence efficacy. Release of CPT in vivo was detected by time-resolved phase-shift fluorescence imaging on tumor specimens, based on the evidence that free and bound drug had different fluorescence lifetimes in solution. HT-29 murine specimens, obtained at several times after treatment with (3)H-labeled free CPT, pHPMA-Gly-Phe-Leu-Gly-CPT, or pHPMA-Gly-6-aminohexanoyl-Gly-CPT, were either imaged for time-resolved phase-shift fluorescence or subjected to autoradiography. Phase shifts of CPT conjugates were equal or longer than those of free CPT, indicating the presence of both free and polymer-bound drug in the tumor, in agreement with autoradiograms. pHPMA-Gly-Phe-Leu-Gly-CPT underwent relevant intratumor hydrolysis during the first 24 h, whereas the hydrolysis of pHPMA-Gly-6-aminohexanoyl-Gly-CPT was slow. The latter showed antitumor activity at doses from 10 to 22.5 mg/kg/day against s.c. HT-29, A2780, M14, and A549 s.c. xenografts. Moreover, inhibition of tumor growth lasted for up to 73-88 days, and cures were observed on mice with orthotopic implanted HT-29; pHPMA-Gly-Phe-Leu-Gly-CPT was 2-fold more potent than pHPMA-Gly-6-aminohexanoyl-Gly-CPT but less tolerated. Our data suggest that the efficacy of pHPMA-CPT copolymers is related to their intratumor accumulation, and in vivo properties of releasing CPT by esterolytic and proteolytic degradation.

Acrylamides↗
PubMed2002

Relationship between erythrocyte membrane phase properties and susceptibility to secretory phospholipase A2.

Normally, cell membranes resist hydrolysis by secretory phospholipase A(2). However, upon elevation of intracellular calcium, the cells become susceptible. Previous investigations demonstrated a possible relationship between changes in lipid order caused by increased calcium and susceptibility to phospholipase A(2). To further explore this relationship, we used temperature as an experimental means of manipulating membrane physical properties. We then compared the response of human erythrocytes to calcium ionophore at various temperatures in the range of 20-50 degrees C using fluorescence spectroscopy and two-photon fluorescence microscopy. The steady state fluorescence emission of the environment-sensitive probe, laurdan, revealed that erythrocyte membrane order decreases systematically with temperature throughout this range, especially between 28 and 45 degrees C. Furthermore, the ability of calcium ionophore to induce increased membrane order and susceptibility to phospholipase A(2) depended similarly on temperature. Both responses to calcium influx were enhanced as membrane fluidity increased. Analysis of the spatial distribution of laurdan fluorescence at several temperatures indicated that the ordering effect of intracellular calcium on fluid membranes generates an increase in the number of fluid-solid boundaries. Hydrolysis of the membrane appeared to progress outward from these boundaries. We conclude that phospholipase A(2) prefers to hydrolyze lipids in fluid regions of human erythrocyte membranes, but primarily when those regions coexist with domains of ordered lipids.

Erythrocyte Membrane↗
PubMed2002

NHE1 regulates the stratum corneum permeability barrier homeostasis. Microenvironment acidification assessed with fluorescence lifetime imaging.

The outermost epidermal layer, the stratum corneum (SC), exhibits an acidic surface pH, whereas the pH at its base approaches neutrality. NHE1 is the only Na(+)/H(+) antiporter isoform in keratinocytes and epidermis, and has been shown to regulate intracellular pH. We now demonstrate a novel function for NHE1, as we find that it also controls acidification of extracellular "microdomains" in the SC that are essential for activation of pH-sensitive enzymes and the formation of the epidermal permeability barrier. NHE1 expression in epidermis is most pronounced in granular cell layers, and although the surface pH of NHE1 knockout mice is only slightly more alkaline than normal using conventional pH measurements, a more sensitive method, fluorescence lifetime imaging, demonstrates that the acidic intercellular domains at the surface and of the lower SC disappear in NHE1 -/- animals. Fluorescence lifetime imaging studies also reveal that SC acidification does not occur through a uniform gradient, but through the progressive accumulation of acidic microdomains. These findings not only visualize the spatial distribution of the SC pH gradient, but also demonstrate a role for NHE1 in the generation of acidic extracellular domains of the lower SC, thus providing the acidification of deep SC interstices necessary for lipid processing and barrier homeostasis.

Animals↗
PubMed2002

Unfolding of pyridoxal 5'-phosphate-dependent O-acetylserine sulfhydrylase probed by time-resolved tryptophan fluorescence.

Proteins utilizing pyridoxal 5'-phosphate as a coenzyme constitute a large superfamily and are currently classified into three functional groups and five structural fold types. Despite the variability of sequences and catalyzed reactions, they share relevant structural, dynamic and functional properties. Therefore, they constitute an optimal system to investigate the relative influence of primary sequence and coenzyme interactions on folding pathways, structural stability and enzymatic function. O-Acetylserine sulfhydrylase is a dimeric pyridoxal 5'-phosphate dependent enzyme that catalyzes the synthesis of L-cysteine from O-acetylserine and sulfide. The time-resolved fluorescence study of O-acetylserine sulfhydrylase unfolding, here reported, indicates that the coenzyme stabilizes the protein structure. The dependence on denaturant concentration of tryptophan lifetimes in the holo- and apo-enzyme demonstrates that the interactions with the coenzyme stabilize the C-terminal domain to a higher extent with respect to the N-terminal domain. This result is discussed in terms of a linkage between the differential stabilization brought about by the coenzyme and the different degrees of conformational flexibility required by the specialized functional role of distinct protein regions.

Circular Dichroism↗
PubMed2002

Ligand binding to somatostatin receptors induces receptor-specific oligomer formation in live cells.

Heptahelical receptors (HHRs) are generally thought to function as monomeric entities. Several HHRs such as somatostatin receptors (SSTRs), however, form homo- and heterooligomers when activated by ligand binding. By using dual fluorescent ligands simultaneously applied to live cells monotransfected with SSTR5 (R5) or SSTR1 (R1), or cotransfected with R5 and R1, we have analyzed the ligand receptor stoichiometry and aggregation states for the three receptor systems by fluorescence resonance energy transfer and fluorescence correlation spectroscopy. Both homo- and heterooligomeric receptors are occupied by two ligand molecules. We find that monomeric, homooligomeric, and heterooligomeric receptor species occur in the same cell cotransfected with two SSTRs, and that oligomerization of SSTRs is regulated by ligand binding by a selective process that is restricted to some (R5) but not other (R1) SSTR subtypes. We propose that induction by ligand of different oligomeric states of SSTRs represents a unique mechanism for generating signaling specificity not only within the SSTR family but more generally in the HHR family.

Animals↗
PubMed2002

Different time evolution of oxyhemoglobin and deoxyhemoglobin concentration changes in the visual and motor cortices during functional stimulation: a near-infrared spectroscopy study.

Neurovascular coupling is the generic term for changes in cerebral metabolic rate of oxygen (CMRO(2)), cerebral blood flow, and cerebral blood volume related to brain activity. The goal of this paper is to better understand the effects of neurovascular coupling in the visual and motor cortices using frequency-domain near-infrared spectroscopy. Maps of concentration changes in oxyhemoglobin [O(2)Hb], deoxyhemoglobin [HHb], and total hemoglobin of the visual and motor cortices were generated during stimulation using a reversing checkerboard screen and palm-squeezing, respectively. Seven healthy volunteers of 18-37 years of age were included. In the visual cortex the patterns of [O(2)Hb] and [HHb] were strongly linearly correlated (r(2) > 0.8 in 13 of a total of 24 locations). In 20 locations the change in [O(2)Hb] was larger than 0.25 microM. The mean slope of the linear regression between [O(2)Hb] and [HHb] was -3.93 +/- 0.31 (SE). The patterns of the [O(2)Hb] and [HHb] traces over the motor cortex looked different. The [O(2)Hb] reached its maximum change a few seconds before the [HHb] reached its minimum. This was confirmed by the linear regression analysis (r(2) > 0.8 in none of 40 locations). In 20 locations the change in [O(2)Hb] was larger than 0.25 microM. The mean slope of the regression line was -1.76 +/- 0.20, which is significantly higher than that in the motor cortex (P < 0.0000001). Patterns of [O(2)Hb] and [HHb] differ among cortex areas. This implies that the regulation of perfusion in the visual cortex is different from that in the motor cortex. There is evidence that the CMRO(2) increases substantially in the visual cortex, while this is not the case for the motor cortex.

Blood-Brain Barrier↗
PubMed2002

Functional frequency-domain near-infrared spectroscopy detects fast neuronal signal in the motor cortex.

Millisecond changes in the optical properties of the human brain during stimulation were detected in five volunteers using noninvasive frequency-domain near-infrared spectroscopy. During a motor stimulation task we found highly significant signals, which were directly related to neuronal activity and exhibited much more localized patterns than the slow hemodynamic signals that are also detected by the near-infrared method. We considerably reduced the noise in the instrumental system and improved data analysis algorithms. With the achieved signal-to-noise ratio, single subject measurements were feasible without the requirement of particularly strong stimuli and within a reasonable period of measurement of 5 min at a mean signal-to-noise ratio of 3.6. The advantage of this noninvasive technique with respect to electrical recording is that it is able to detect neuronal activity with the relatively high spatial resolution of 8 mm.

Dominance, Cerebral↗
PubMed2002

Two-photon fluorescence lifetime imaging of the skin stratum corneum pH gradient.

Two-photon fluorescence lifetime imaging is used to identify microdomains (1-25 microm) of two distinct pH values within the uppermost layer of the epidermis (stratum corneum). The fluorophore used is 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF), whose lifetime tau (pH 4.5, tau = 2.75 ns; pH 8.5, tau = 3.90 ns) is pH dependent over the pH range of the stratum corneum (pH 4.5 to pH 7.2). Hairless mice (SKH1-hrBR) are used as a model for human skin. Images (< or =50 microm x 50 microm) are acquired every 1.7 microm from the stratum corneum surface to the first viable layer (stratum granulosum). Acidic microdomains (average pH 6.0) of variable size (~1 microm in diameter with variable length) are detected within the extracellular matrix of the stratum corneum, whereas the intracellular space of the corneocytes in mid-stratum corneum (25 microm diameter) approaches neutrality (average pH 7.0). The surface is acidic. The average pH of the stratum corneum increases with depth because of a decrease in the ratio of acidic to neutral regions within the stratum corneum. The data definitively show that the stratum corneum acid mantle results from the presence of aqueous acidic pockets within the lipid-rich extracellular matrix.

Animals↗
PubMed2002

Cellular characterization of adenylate kinase and its isoform: two-photon excitation fluorescence imaging and fluorescence correlation spectroscopy.

Adenylate kinase (AK) is a ubiquitous enzyme that regulates the homeostasis of adenine nucleotides in the cell. AK1beta (long form) from murine cells shares the same protein sequence as AK1 (short form) except for the addition of 18 amino acid residues at its N-terminus. It is hypothesized that these residues serve as a signal for protein lipid modification and targeting of the protein to the plasma membrane. To better understand the cellular function of these AK isoforms, we have used several modern fluorescence techniques to characterize these two isoforms of AK enzyme. We fused cytosolic adenylate kinase (AK1) and its isoform (AK1beta) with enhanced green fluorescence protein (EGFP) and expressed the chimera proteins in HeLa cells. Using two-photon excitation scanning fluorescence imaging, we were able to directly visualize the localization of AK1-EGFP and AK1beta-EGFP in live cells. AK1beta-EGFP mainly localized on the plasma membrane, whereas AK1-EGFP distributed throughout the cell except for trace amounts in the nuclear membrane and some vesicles. We performed fluorescence correlation spectroscopy measurements and photon-counting histogram analysis in specific domains of live cells. For AK1-EGFP, we observed only one diffusion component in the cytoplasm. For AK1beta-EGFP, we observed two distinct diffusion components on the plasma membrane. One corresponded to the free diffusing protein, whereas the other represented the membrane-bound AK1beta-EGFP. The diffusion rate of AK1-EGFP was slowed by a factor of 1.8 with respect to that of EGFP, which was 50% more than what we would expect for a free diffusing AK1-EGFP. To rule out the possibility of oligomer formation, we performed photon-counting histogram analysis to direct analyze the brightness difference between AK1-EGFP and EGFP. From our analysis, we concluded that cytoplasmic AK1-EGFP is monomeric. fluorescence correlation spectroscopy proved to be a powerful technique for quantitatively studying the mobility of the target protein in live cells. This technology offers advantages in studying protein interactions and function in the cell.

Adenylate Kinase↗
PubMed2002

Molecular brightness characterization of EGFP in vivo by fluorescence fluctuation spectroscopy.

We characterize the molecular properties of autofluorescence and transiently expressed EGFP in the nucleus and in the cytoplasm of HeLa cells by fluorescence correlation spectroscopy (FCS) and by photon counting histogram (PCH) analysis. PCH has been characterized and applied in vitro, but its potential for in vivo studies needs to be explored. Thus, this study mainly focuses on the characterization of PCH analysis in vivo. The strength of PCH lies in its ability to distinguish biomolecules by their molecular brightness value. Because the concept of molecular brightness is crucial for PCH analysis, we study the molecular brightness of EGFP and determine the statistical accuracy of its measurement under in vivo conditions. We started by characterizing the influence of autofluorescence on EGFP measurements. We found a molecular brightness of EGFP that is a factor of 10 higher than the brightness of the autofluorescence. Moment analysis demonstrates that the contribution of autofluorescence to fluorescence fluctuation experiments is negligible at EGFP concentrations of one protein per excitation volume. The molecular brightness of EGFP measured in the nucleus, the cytoplasm, and in vitro are identical and our study demonstrates that molecular brightness is a very stable and predictable quantity for cellular measurements. In addition to PCH, we also analyzed the autocorrelation function of EGFP. The diffusion coefficient of EGFP is a factor of 3 lower in vivo than compared to in vitro, and a simple diffusion process describes the autocorrelation function. We found that in the nucleus the fluorescence intensity is stable as a function of time, while measurements in the cytoplasm display fluorescence intensity drifts that complicate the data analysis. We introduce and discuss an analysis method that minimizes the influence of the intensity drifts on PCH analysis. This method allows us to recover the correct molecular brightness of EGFP even in the presence of drifts of the fluorescence intensity signal. We found the molecular brightness of EGFP to be a very robust parameter, and anticipate the use of PCH analysis for the study of oligomerization processes in vivo.

Cell Nucleus↗