Application of 30-MHz acoustic scattering to the study of human red blood cells

A technique for simultaneously measuring the scattering amplitude of individual particles at two angles is applied to human red blood cells. Using a Rayleigh scattering model, the density and compressibility of the cells may be determined given a priori knowledge of their volume. A calibration method relying on measurements of the bulk properties of particle suspensions is described. Red cell properties in hypotonic and hypertonic hosts are compared with a homogeneous mixture model, and a linear relation between hemoglobin content and scattering amplitude at a 90 deg scattering angle is established.     

Effects of intra- and extracellular space properties on diffusion and T(2) relaxation in a tissue model

The purpose of this study was to investigate the effects of biophysical factors on the diffusion and the relaxation time T(2) independently. Certain properties of the extracellular and the intracellular space may change radically in pathological conditions resulting in water diffusion changes. A tissue model consisting of red blood cells was studied. The extra- and intracellular spaces were modified osmotically and by suspending medium concentration. Diffusion measurements were evaluated with regard to the effective medium theory. Neither the nature of the protein in the extracellular space nor an increased level of intracellular hydration caused a significant net water diffusion change in the cell suspension. The relaxation time T(2) exhibited very little dependence on the extracellular volume fraction or the concentration or the nature of the protein in the extracellular space. An increased level of intracellular hydration resulted in systematically larger T(2) values. It seems probable that increases in extracellular protein concentrations or in the extent of intracellular hydration do not play a significant role in the diffusion changes detected in pathological conditions. T(2) appears to depend on the level of hydration or the total water content but is seemingly less dependent of the concentration and the nature of the extracellular protein in our model solutions.     

Vocal stability in functional dysphonic versus healthy voices at different times of voice loading

Functional (nonorganic) dysphonia is often characterized by vocal instability. The purpose of the prospective study was to examine whether there is a difference in vocal instability of functional dysphonic voices compared with healthy ones, this means whether electroglottographic perturbation values differ (1) between healthy and dysphonic voices and (2) between two subgroups of the dysphponic voices (hpertonic and hypotonic dysphonic voices). Twenty-three patients with hypertonic functional dysphonia, 9 with hypotonic functional dysphonia and 31 healthy nonsmokers, were each examined electroglottographically before (Ex 1), immediately after (Ex 2), and 1 hour after (Ex 3) voice loading. Perturbations of frequency, amplitude, quasi-open-quotient, and contact-index were calculated from the EGG signal. At all three times of examination, hypertonic dysphonic voices showed higher perturbations than healthy voices, and they had higher perturbations than hypotonic dysphonic voices before and 1 hour after voice loading. Hypotonic dysphonic voices showed higher perturbations than healthy voices only 1 hour after voice loading. Voice loading induced different reactions in dysphonic voices: Some voices showed increased perturbations, and others exhibited normal or even decreased perturbation immediately after voice loading. Examination of electroglottographic-derived perturbations immediately after voice loading seems not to be useful. Differentiation of hypertonic and hypotonic dysphonic voices was possible with an estimated sensitivity of 88.9% and a specificity of 87.0% by using the sum of the amplitude-perturbation and the quasi-open-quotient-perturbation measured before voice loading.     

Gd-DTPA adrenal gland enhancement at 1.5 T

The change in relative signal intensity of normal adrenal glands in 31 patients was evaluated following bolus administration of 0.1 mmol/kg of gadolinium diethylenetriamine pentacetic acid (Gd-DTPA). A marked increase in relative intensity of greater than 300% was observed within 2.5 min following contrast administration upon comparison of pre- and postcontrast T1-weighted gradient-echo images (TR = 47 msec, TE = 13 msec, pulse angle 80 degrees). Significantly elevated relative intensities of 55% and 44% persisted on postcontrast T1-weighted spin-echo images obtained at further delay times averaging 8 and 20 min, respectively, when compared to the identical precontrast sequence.     

Induced renal artery stenosis in rabbits: magnetic resonance imaging, angiography, and radionuclide determination of blood volume and blood flow

To investigate the ability of MRI to detect alterations due to renal ischemia, a rabbit renal artery stenosis (RAS) model was developed. Seven rabbits had RAS induced by surgically encircling the artery with a polyethylene band which had a lumen of 1 mm, 1 to 2 weeks prior to imaging. The stenosis was confirmed by angiography, and the rabbits were then imaged in a 1.4 T research MRI unit. T1 was calculated using four inversion recovery sequences with different inversion times. Renal blood flow, using 113Sn-microspheres, and regional water content by drying were then measured. The average T1 of the inner medulla was shorter for the ischemia (1574 msec) than for the contralateral kidney (1849 msec), while no change ws noted in the cortex. Ischemic kidneys had less distinct outer medullary zones on IR images with TI = 600 msec than did contralateral or control kidneys. Blood flow to both the cortex and medulla were markedly reduced in ischemic kidneys compared with contralateral kidneys (119.5 vs. 391 ml/min/100 gm for cortex and 19.8 vs. 50.8 ml/min/100 gm for medulla). Renal water and blood content were less affected. Our rabbit model of renal artery stenosis with MRI, radionuclide, and angiographic correlation has the potential to increase our understanding of MR imaging of the rabbit kidney.     

The line shapes of the water proton resonances of red blood cells containing carbonyl hemoglobin, deoxyhemoglobin, and methemoglobin: implications for the interpretation of proton MRI at fields of 1.5 T and below

The 300 MHz (7 T) water proton resonances of suspensions of red blood cells containing paramagnetic deoxyhemoglobin or methemoglobin can be resolved into two broad lines assignable to intra- and extracellular water which undergoes rapid T2 relaxation by diffusion in magnetic field gradients induced by the intracellular paramagnets. The width of the resolved lines allowed an estimate of the maximum contribution that diffusion makes to T2 relaxation at 7 T. The dependence of the diffusion contribution on the square of the strength of the static magnetic field suggest that diffusion makes a small contribution to water proton T2 relaxation at 1.5 T compared to 7 T, and a negligible one at 0.5 T in early and intermediate hematomas containing deoxyhemoglobin or methemoglobin in intact red blood cells. At the lower field strengths, water proton T2 relaxation is apparently dominated by the rapid chemical exchange (mean lifetime tau = 10 msec) between the intra- and extracellular environments.     

Diffusion generated T1 and T2 contrast

In MR images of porous organic samples (such as roots or wood) in water media, the sample is often surrounded by a bright ring, with a corresponding decreased T1 value in T1 maps. When the medium is removed, or contrast agents are added, the ring disappears, indicating that the signal does not originate in the outer layers of the sample, but from the medium itself. It can be shown that this "bright ring effect" is only observed when the medium experiences a reduction in T1 when permeating the sample. In order to investigate this effect, a computer model was used to simulate the diffusion of magnetisation between regions that exhibit different relaxation constants. Using this model, the origin of the signal increase was found to be an inflow effect, as diffusion transports relaxed magnetisation from the boundary regions of the sample into the surrounding medium. In the case of the "bright ring" around the plants described above, a mixing of short T1 values from within the sample and long T1 values within the medium occurs, yielding a "transition region" between the two values. There, a signal increase can be observed at T1 weighted images, compared to the signal from the medium beyond this transition region. The width of the transition region is on the order of magnitude of the diffusion displacement that is calculated from the T1 value as diffusion time. In addition to causing the bright ring around the plant samples, this diffusion effect also limits the resolution of the relaxation time maps. This effect is not limited to T1 relaxation but also applies to T2 relaxation. However, at high B0 field strengths such as those used in this study (11.7 T), a T2 effect is not usually observed due to the considerably shorter T2 times in plants (about 50 ms, compared to T1 times of higher than 1 s). Because the diffusion length during this T2 relaxation is short with respect to the resolution of the imaging experiments, no T2 ring effect is seen.     

Measurement of Cell Volume Changes by Fluorescence Self-Quenching

At high concentrations, certain fluorophores undergo self-quenching, i.e., fluorescence intensity decreases with increasing fluorophore concentration. Accordingly, the self-quenching properties can be used for measuring water volume changes in lipid vesicles. In cells, quantitative determination of water transport using fluorescence self-quenching has been complicated by the requirement of relatively high (mM) and often toxic loading concentrations. Here we report a simple method that uses low (M) loading concentrations of calcein-acetoxymethyl ester (calcein-AM) to obtain intracellular concentrations of the fluorophore calcein suitable for measurement of changes in cell water volume by self-quenching. The relationship between calcein fluorescence intensity, when excited at 490 nm (its excitation maximum), and calcein concentration was investigated in vitro and in various cultured cell types. The relationship was bell-shaped, with the negative slope in the concentration range where the fluorophore undergoes fluorescence self-quenching. In cultured retinal pigment epithelial cells, calcein fluorescence and extracellular osmolarity were linearly related. A 25-mOsm hypertonic challenge corresponded to a decrease in calcein fluorescence with high signal-to-noise ratio (>15). Similar results were obtained with the fluorophore BCECF when excited at its isosbestic wavelength (436 nm). The present results demonstrate the usefulness of fluorescence self-quenching to measure rapid changes in cell water volume.     

Cryopreservation of carrot (Daucus carota l.) cell suspensions and protoplasts by vitrification

Carrot cell suspensions and protoplasts were successfully cryopreserved by vitrification. Cells were precultured in liquid Murashige and Skoog medium containing 0.175 M sucrose for 3 d and then in liquid MS medium containing 0.4 M sorbitol for 1 d. After loading of the precultured carrot cells in 25 % PVS2 at room temperature for 5 min and treatment with 100 % PVS2 at 0 degrees C for 7.5 min, they were quenched in liquid nitrogen. Optimal survival was 83.3 % (based on the triphenyl tetrazolium chloride reduction assay) following warming and unloading. Recovered cells retained the ability to regenerate plantlets in vitro. In the case of vitrification of protoplasts isolated from carrot cell suspensions, the optimal loading and dehydration durations were 5 min in 25% PVS2 and 3 min 100 % PVS2 respectively. Survival of 47 % of the untreated control (based on the FDA-PI (fluorescein diacetate-propidium iodide) staining) was achieved after cryopreservation.     

Methodology for the measurement and analysis of relaxation times in proton imaging

Measurements of proton T1 and T2 were performed on GdCl3 solutions (20 less than T2 less than 500 msec, 90 less than T1 less than 1000 msec) on large-bore NMR imaging systems operating at 1.0T and 1.5T. CPMG multi-echo (ME), multiple saturation recovery (MSR) and modified fast inversion recovery (MFIR) pulse sequences as well as a sequence that combines and interleaves T1 and T2 weighted data acquisition (which we call "multiple saturation-recovery multiple-echo" (MSRME) were used. The relaxation data are compared to those obtained on a small bore NMR spectrometer operated at 1.5T. T1 and T2 values for the solutions were found to be the same within 10% for the two fields. Reproducibility of measurements of T1, T2 and the unnormalized spin density of the solutions was better than 5%. Systematic errors, amenable to correction through calibration, are noted in the imager T1 and T2 values. T1 and T2 values for some typical neural tissues at 1.5T and body tissue at 1.0T for human volunteers were obtained and are tabulated.     

Photodynamic damage study of HeLa cell line using ALA

The present study evaluates the photodynamic damage with 5-aminolevulinic acid (5-ALA) using HeLa as experimental model. HeLa cell line was irradiated with red light (He-Ne laser, λ = 632.8 CW nm). The influence of different incubation times and concentrations of 5-ALA, different irradiation doses and various combinations of photosensitizer and light doses on the cellular viability of HeLa cells were studied. The optimal uptake of photosensitizer ALA in HeLa cells was investigated by means of PpIX fluorescence intensity by exciting the HeLa cell suspension at 450 nm and a detection wavelength set at 690 nm. Cells viability was determined by means of trypan blue solution. The spectrometric measurements showed that the maximal cellular uptake of 5-ALA occurred after 4 h in vitro incubation. We found that the combination with 5-ALA and laser irradiation leads to time/concentration-dependent increase of cells death and also energy doses-dependent enlarge the cells death. The fluorescence intensity after PDD of carcinoma cells reduce when compared with the control group. The fluorescence emission spectral profiles after PDD of carcinoma cells showed a dip around 425–525 nm when compared with the control group. This may be due to the damage of mitochondria component of cells. The percentage of HeLa cells after PDD shows that the percentage of cells survival rate as function of laser dose (power). Hence it is clear that at 200 μg/ml ALA and 20 mW laser irradiation, more than 70% of HeLa cells were dead after 15 min.     

In vivo evaluation of the reproducibility of T1 and T2 measured in the brain of patients with multiple sclerosis.

The precision (reproducibility) of relaxation times derived from magnetic resonance images of patients with multiple sclerosis (MS) were investigated. Measurements of 10 MS patients were performed at 1.5 T on two occasions within 1 wk. T1 and T2 was measured using a partial saturation inversion recovery sequence (6 points) and a Carr-Purcell-Meiboom-Gill phase alternating-phase shift multiple spin-echo sequence with 32 echoes. Regions of interest (ROI) were placed both in apparently normal white matter and plaques. The precision (+/- 1.96 SD) and the confidence intervals for T1 and T2 for white matter and plaques were calculated. The precision of T1 for white matter and plaques was respectively +/- 94 msec and +/- 208 msec. The precision of T2 for white matter and plaques was respectively +/- 18 msec and +/- 26 msec. For all measurements the coefficient of variation was about 9%. Judging from our own study and others as well, a precision better than 10% for T1 and T2 would seem unrealistic at present.     

Analysis of antifungal and anticancer effects of the extract from Pelargonium zonale

The extract from Pelargonium zonale stalks exhibits activity against Candida albicans and exerts an effect on the HeLa cell line. The action against C. albicans cells was analysed using light, CLSM, SEM, and TEM microscopes. The observations indicate that the extract influenced fungal cell morphology and cell metabolic activity. The morphological changes include cell wall damage, deformations of cell surfaces, and abnormalities in fungal cell shape and size. Cells of C. albicans treated with the extract exhibited disturbances in the budding pattern and a tendency to form agglomerates and multicellular chains. The P. zonale extract caused a significant decrease in the metabolic activity of C. albicans cells. Cells died via both apoptosis and necrosis. The antitumor activity of the extract was analysed using the MTT assay. The P. zonale extract exhibited minor cytotoxicity against the HeLa cell line but a dose-dependent cytopathic effect was noticed. The P. zonale extract is a promising source for the isolation of antifungal and anticancer compounds.     

The intensity of the 1602 cm−1 band in human cells is related to mitochondrial activity

We report a Raman band at 1602 cm⁻¹ in the spectra of human cells, which previously had only been observed in mitochondria of yeast cells. This band, which has not yet been assigned to a particular molecular species, was found to occur in HeLa cells, peripheral blood lymphocytes, human mesenchymal stem cells and bovine chondrocytes. The band is proposed as an indicator of the activity of mitochondria in cells. Cells were cultured with and without serum or temporarily deprived of serum. The band can be observed for all these variations in cell culture methodology. The band intensity decreases under the influence of an increase of the calcium ion concentration in the surrounding medium. Copyright © 2009 John Wiley & Sons, Ltd.     

低能离子注入宫颈癌细胞的红外光谱分析

利用离子注入机所产生的低能N+模仿宇宙中低能离子作用于人宫颈癌细胞(HeLa cell),探索其对人类细胞的影响及作用机制。因实验中的低能离子产生和加速要在真空中进行,细胞在离子注入同时将受到真空的影响,为此研究人员利用石蜡油保护细胞以防止注入时的水份蒸发。注入处理完毕后收集细胞,采用傅里叶变换红外光谱法(FTIR)分析真空和低能N+束注入后细胞中大分子的相对含量、构型及其构象变化等方面的信息。结果表明：(1)不同处理后的样品在3 300 cm-1附近吸收谱带存在明显差异。对照样品的特征峰位为3 300 cm-1,而其他样品中除了注入5×1014 N+·cm-2外,红外吸收峰均向长波数方向移动,真空2×1015 N+·cm-2样品的频移尤为明显至3 420 cm-1处。(2)与对照样品相比较,各处理样品的1 378 cm-1处吸收峰峰位均向长波数方向频移。(3)处理样品相对于对照样品而言,2 360 cm-1处吸收峰均向长波数方向移动。该结果说明低能离子注入处理可以引起细胞中核酸、蛋白的含量和构象变化。     

Water self-diffusion in Chlorella sp. studied by pulse field gradient NMR

The water self-diffusion behavior in chlorella water suspension was investigated by pulsed field gradient NMR technique. Three types of water was determined, which differs according to the self-diffusion coefficients; bulk water, extracellular and intracellular water. Intracellular and extracellular water self-diffusion were restricted, and the sizes of restriction regions were 3.4 microm and 17 microm, respectively. The water molecular exchange process between these three diffusion regions was investigated. The residence time and exchange rate constant for chlorella cells were obtained. The cell wall permeability determined from the rate constant as 3 x 10(-6) m/s agreed with the permeability 10(-6) m/s obtained from time dependence of intracellular water self-diffusion coefficient. The structural cluster model of chlorella cell is estimated to describe the extracellular water self-diffusion in chlorella water suspension.     

Comparison of pharmacokinetics, intracellular localizations and sonodynamic efficacy of endogenous and exogenous protoporphyrin IX in sarcoma 180 cells

Purpose

The aim of the present study was to investigate the differences in pharmacokinetics, sub-cellular localizations and sonodynamic efficacy between endogenous and exogenous protoporphyrin IX (endo-PpIX and exo-PpIX) in sarcoma 180 (S180) cells.

Materials and methods

The 5-aminolevulinic acid (ALA)-derived endo-PpIX and exo-PpIX pharmacokinetic profiles were determined by the fluorescence intensity of cell extracts with a spectrophotometer based on a standard curve. The changes in their sub-cellular localization patterns over a prolonged incubation time were evaluated by laser scanning confocal microscopy. The cytotoxic effects of 5-ALA-mediated sonodynamic therapy (ALA-SDT) and exogenous PpIX-mediated sonodynamic therapy (PpIX-SDT) were also evaluated by the MTT assay.

Results

The exo-PpIX showed dose-dependent pharmacokinetics in which a plateau of intra- and extracellular content was observed 45 min after administration. However, the amount of ALA-derived endogenous intracellular PpIX, as well as extracellular PpIX in the same samples, showed linear accumulation with incubation time, which was independent of ALA concentration. Fluorescent imaging revealed that the exo-PpIX mainly accumulated at the plasma membrane in the early stage, whereas the ALA-derived PpIX initially localized in the mitochondria. Cells displayed sonodynamic damage by the synthesized endo-PpIX after addition of 1 mM ALA for 12 h, but the cytotoxicity induced by the equivalent amount of exo-PpIX was much more significant with increasing ultrasound intensities.

Conclusions

Our findings suggest that endo- and exo-PpIX in S180 cells differ not only in pharmacokinetics but also in sub-cellular localizations, which may affect their sonodynamic efficacy and mechanisms of inducing cell death.     

Nondestructive isolation of single cultured animal cells by femtosecond laser-induced shockwave

A shockwave induced by focusing an intense femtosecond laser onto a culture medium under a microscope was used to manipulate single cultured animal cells. Mouse NIH3T3 fibroblasts were cultured on a collagen matrix and placed on a microscope installation that included an intense femtosecond laser. Cells were detached individually from the matrix by the shockwave, after their filopodia were cut by focusing the laser directly on them. The appearance and locomotion of cells after detachment was monitored with a CCD camera. Filopodia of the detached cells were regenerated, and the cell adhered again to the matrix within 4 hrs after detachment. When the shockwave was induced at a pulse energy of 0.72 J/pulse, 80% of cells were successfully detached from the culture plate in a non-destructive manner. The force required to detach a cell was estimated to be a few N/m², which is larger than the photon force resulting from conventional laser trapping. PACS 42.62.Be; 47.40.Nm; 87.80.Rb     

Photophysical Study of Local Anesthetics in Reverse Micelles and Water-Ethanol Mixtures

Fluorescence emission from three tertiary amine local anesthetics, dibucaine (D), tetracaine (T), and procaine (P) was investigated in the membrane-mimetic environment of AOT/isooctane reverse micelles and water-ethanol mixtures. At room temperature all three show broad UV absorption bands and large Stokes' shifts in emission from aqueous medium. In reverse micelles the emission bands are blue-shifted, with the magnitude of this shift decreasing continuously as the waterto-surfactant molar ratio (w₀) of the micelles is increased. The emission intensity of D is much stronger in water and in the interior of micelles than in ethanol, whereas for T and P the emission in water is much weaker than in ethanol and in micelles. Whereas the behavior in micelles is the result of the increased hydrophobicity with respect to bulk water in their interior, that in waterethanol mixtures may be explained on the basis of state reversal of the excited electronic levels of D with increasing hydrogen bonding capability of the solvent mixture. The steady-state anisotropy for both T and P is unusually high in bulk water (0.27) and increases with increasing w₀ in micelles. However, the rotational correlation time of the anesthetics, as calculated using Perrin's equation, decreases with increasing w₀, as expected from physical considerations relating the size of the water pool to the ease of rotational relaxation of probe molecules inside micelles. Together, these results point to a marked difference in emission properties between D on one hand and T and P on the other, arising from differences in electronic characters of their primary emitting chromophores—quinoline for D, para amino benzoate for T and P.     

真空、低能离子注入后HeLa细胞的吸收光谱研究

在选用了石蜡油作为细胞耐受真空的保护剂基础上,分别采集了人宫颈癌细胞(HeLa细胞)经真空和不同剂量低能离子注入后的紫外吸收光谱.实验结果显示:HeLa细胞在202及260 nm附近均有特征性的吸收峰.数据的进一步分析发现:(1)HeLa细胞经真空处理后的紫外吸光值随着真空时间的延长而增加,且真空对细胞紫外吸收光谱的影响大于单纯的石蜡油浸泡影响;(2)真空对于HeLa细胞紫外吸光值的影响远低于低能离子注入的影响;(3)HeLa细胞在注入不同剂量的低能N+后,紫外吸光值会随着注入剂量的增大而增加.在上述分析的基础上,本文又探讨了低能N+束注入对肿瘤细胞的结构、各成分的组成比例及分子排列的影响,该研究为深入探索低能离子注入生物样品的作用机理奠定了基础.