scholarly journals Live-Cell Imaging and Measurement of Intracellular pH in Filamentous Fungi Using a Genetically Encoded Ratiometric Probe

2009 ◽  
Vol 8 (5) ◽  
pp. 703-712 ◽  
Author(s):  
Tanja Bagar ◽  
Kirsten Altenbach ◽  
Nick D. Read ◽  
Mojca Benčina
Keyword(s):  
Intracellular Ph ◽  
Chimeric Protein ◽  
Weak Acid ◽  
Cytoplasmic Ph ◽  
Ph Gradients ◽  
Ph Values ◽  
Ph Imaging ◽  
Carbonyl Cyanide ◽  
Ratio Imaging ◽  
Ratiometric Probe

ABSTRACT A novel, genetically encoded, ratiometric pH probe (RaVC) was constructed to image and measure intracellular pH in living hyphae of Aspergillus niger. RaVC is a chimeric protein based on the pH-sensitive probe pHluorin, which was partially codon optimized for expression in Aspergillus. Intracellular pH imaging and measurement was performed by simultaneous, dual-excitation confocal ratio imaging. The mean cytoplasmic pH measured was 7.4 to 7.7 based on calibrating RaVC in situ within nigericin-treated hyphae. Pronounced, longitudinal cytoplasmic pH gradients were not observed in the apical 20 μm of actively growing hyphae at the periphery of 18-h-old colonies. The cytoplasmic pH remained unchanged after prolonged growth in buffered medium with pH values between 2.5 or 9.5. Sudden changes in external pH significantly changed cytoplasmic pH by <1.3 pH units, but it returned to its original value within 20 min following treatment. The weak acid and antifungal food preservative sorbic acid caused prolonged, concentration-dependent intracellular acidification. The inhibition of ATPases with N-ethylmaleimide, dicychlohexylcarbodimide, or sodium azide caused the cytoplasmic pH to decrease by <1 pH unit. Treatment with the protonophore carbonyl cyanide m-chlorophenylhydrazone or cyanide p-(trifluoromethoxy) phenylhydrazone reduced the cytoplasmic pH by <1 pH unit. In older hyphae from 32-h-old cultures, RaVC became sequestered within large vacuoles, which were shown to have pH values between 6.2 and 6.5. Overall, our study demonstrates that RaVC is an excellent probe for visualizing and quantifying intracellular pH in living fungal hyphae.

Design and validation of a new ratiometric intracellular pH imaging probe using lanthanide-doped upconverting nanoparticles

2017 ◽  
Vol 46 (40) ◽  
pp. 13957-13965 ◽  
Author(s):  
Shuoren Du ◽  
Javier Hernández-Gil ◽  
Hao Dong ◽  
Xiaoyu Zheng ◽  
Guangming Lyu ◽  
...  
Keyword(s):  
Intracellular Ph ◽  
Quantitative Imaging ◽  
Living Cells ◽  
Ph Sensitive ◽  
Upconversion Nanoparticles ◽  
Imaging Probe ◽  
Ph Imaging ◽  
Ratiometric Probe ◽  
Subcellular Level

A ratiometric probe based on upconversion nanoparticles modified with a pH sensitive moiety for the quantitative imaging of pH at the subcellular level in living cells.

Intracellular pH of isolated perfused turtle heart

1960 ◽  
Vol 199 (6) ◽  
pp. 1112-1114 ◽  
Author(s):  
William J. Waddell ◽  
Harold F. Hardman
Keyword(s):  
Intracellular Ph ◽  
Salt Solution ◽  
Weak Acid ◽  
Extracellular Ph ◽  
Chrysemys Picta ◽  
Ph Values ◽  
Painted Turtles ◽  
Turtle Heart

Hearts from 15 painted turtles ( Chrysemys picta) were perfused for 30 minutes at 23°–26°C with a buffered salt solution. The pH of the perfusing solution was varied between 6.5 and 9.5. The intracellular pH values of the ventricles were calculated from the distribution of the weak acid, 5.5-dimethyl-2,4-oxazolidinedione (DMO). It was found that the extracellular pH (pH e) and the intracellular pH (pH i) under these conditions could be related by the equation: pH i = 0.617 pH e + 2.98.

Generation of intracellular pH gradients in single cardiac myocytes with a microperfusion system

2000 ◽  
Vol 278 (4) ◽  
pp. H1371-H1382 ◽  
Author(s):  
Kenneth W. Spitzer ◽  
Philip R. Ershler ◽  
Rita L. Skolnick ◽  
Richard D. Vaughan-Jones
Keyword(s):  
Intracellular Ph ◽  
Ventricular Myocytes ◽  
Weak Acid ◽  
Confocal Imaging ◽  
Ion Diffusion ◽  
External Application ◽  
Ph Gradients ◽  
Regional Myocardial Ischemia ◽  
Single Ventricular Myocytes ◽  
Myocyte Function

This study describes the use of a microperfusion system to create rapid, large regional changes in intracellular pH (pHi) within single ventricular myocytes. The spatial distribution of pHi in single myocytes was measured with seminaphthorhodafluor-1 fluorescence using confocal imaging. Changes in pHi were induced by local external application of NH4Cl, CO2, or sodium propionate. Local application was achieved by simultaneously directing two parallel square microstreams, each 275 μm wide, over a single myocyte oriented perpendicular to the direction of flow. One stream contained the control solution, and the other contained a weak acid or base. End-to-end, stable pHi gradients as large as 1 pH unit were readily created with this technique. This result indicates that pH within a single cardiac cell may not always be spatially uniform, particularly when weak acid or base gradients are present, which can occur, for example, in regional myocardial ischemia. The microperfusion method should be useful for studying the effects of localized acidosis on myocyte function, estimating intracellular ion diffusion rates, and, possibly, inducing regional changes in other important intracellular ions.

A profluorescent ratiometric probe for intracellular pH imaging

Talanta ◽  
2015 ◽  
Vol 131 ◽  
pp. 666-671 ◽  
Author(s):  
Qingqing Zhang ◽  
Ming Zhou
Keyword(s):  
Intracellular Ph ◽  
Ph Imaging ◽  
Ratiometric Probe

Estimation of cellular pH gradients with 31P-NMR in intact rabbit renal tubular cells

1984 ◽  
Vol 247 (3) ◽  
pp. C188-C196 ◽  
Author(s):  
S. Adler ◽  
E. Shoubridge ◽  
G. K. Radda
Keyword(s):  
Ph Gradient ◽  
Cytoplasmic Ph ◽  
Buffer Solution ◽  
Ph Gradients ◽  
Tubular Cells ◽  
Renal Tubular Cells ◽  
Ph Values ◽  
Intact Rabbit ◽  
Renal Tubular ◽  
External Ph

To determine intracellular pH gradients rabbit renal cortical tubular cells were prepared by collagenase separation, suspended in a Krebs-Ringer buffer solution, and gassed with 95% O2-5% CO2 in a special nuclear magnetic resonance (NMR) probe. Renal tubular cellular pH was determined simultaneously from the distribution of 14C-dimethadione (DMO) (pHDMO) or the chemical shift of inorganic phosphate (pHNMR). Experiments were performed at different external pH values (pHe) ranging between 6.52 and 7.20. pHNMR, a measure of cytoplasmic pH, changed by an amount equal to the change in pHe. pHDMO, however, a measure of cytoplasmic plus mitochondrial pH, changed less than pHe as the latter increased. pHDMO, higher than pHNMR at low pHe, became equal to pHNMR at higher pHe values. By use of assumed mitochondrial volumes of 30-40% mitochondrial pH was calculated from pHDMO and pHNMR. Mitochondrial pH remained relatively constant over the entire pHe range studied. Since cytoplasmic pH fell as pHe was lowered, the transmitochondrial pH gradient increased at low pHE values. These findings suggest that the transmitochondrial pH gradient may be important in regulating metabolism.

scholarly journals Intracellular pH in single motile cells.

1980 ◽  
Vol 86 (3) ◽  
pp. 885-890 ◽  
Author(s):  
J M Heiple ◽  
D L Taylor
Keyword(s):  
Intracellular Ph ◽  
Fluorescence Intensity ◽  
Divalent Cations ◽  
Weak Acid ◽  
Fluorescence Intensity Ratio ◽  
Cytoplasmic Ph ◽  
Standard Curve ◽  
Motile Cells ◽  
Significant Difference

Cytoplasmic pH in single living specimens of Chaos carolinensis is determined microfluorometrically by measuring the ratio of fluorescence intensity of microinjected fluorescein-thiocarbamyl (FTC)-ovalbumin at two different excitation wavelengths. The probe is evenly distributed throughout, and confined to, the cytoplasm, and the fluorescence intensity ratio depends only upon pH. It is independent of pathlength, concentration of probe, divalent cations, and ionic strength. Ratios are calibrated with a standard curve generated in situ by adjusting internal pH of FTC-ovalbumin-containing amebae with weak acid and weak base or by injection of strong buffers. With this technique, the average cytoplasmic pH of freely moving ameba is found to be 6.75 (SD +/- 0.3). The pH of a given spot relative to the morphology of a moving ameba remains fairly constant (+/- 0.05 U), whereas the pH of two different spots in the same cell may differ by as much as 0.4 U, and average pH in different amebae ranges from 6.3 to 7.4, with a suggestion of clustering about pH 6.5 and 6.8. During wound healing, there is a local, transient drop in pH (as great as 0.35 U) at the wound site upon puncture, proportional in extent to the degree of damage. Comparison of tails and advancing pseudopod tips reveals no significant difference in cytoplasmic pH at this level of spatial (50 microns diameter spot) and temporal (1.3 s) resolution. Fluctuations in intracellular pH and/or intracellular free Ca++ may be involved in regulation of cytoplasmic structure and contractility.

pH gradients in the developing teeth of young mice from autoradiography of [14C]DMO

1977 ◽  
Vol 232 (4) ◽  
pp. F364-F367 ◽  
Author(s):  
G. E. Lyman ◽  
W. J. Waddell
Keyword(s):  
Water Content ◽  
Companion Paper ◽  
Nucleation And Growth ◽  
Weak Acid ◽  
Ph Gradients ◽  
Ph Values ◽  
Autoradiographic Technique ◽  
Young Mice

The distribution of the weak acid [14C]DMO was studied in mice, 10-19 days old, by an autoradiographic technique that does not translocate or remove the compound. Calculations of pH values in areas of the developing teeth of these animals were made by comparing the photometric density of the distribution of [14C]DMO, as an indicator of pH, with that of 14C]NAAP, as an indicator of water content, which was reported in the companion paper. The cellular layers, i.e., odontoblasts, ameloblasts, etc., had pH values of 7.1 +/- 0.1; the pH values in enamel, predentine, and dentine ranged from 7.3 to 8.5. It is suggested that an alkaline matrix promotes nucleation and growth of hydroxyapatite.

Construction of Polysiloxane-Based Fluorescent Probe for Visualizing The pH Down-regulation

2021 ◽  
Author(s):  
Xiaoni Wang ◽  
Zhiming Gou ◽  
Minggang Tian ◽  
Yujing Zuo
Keyword(s):  
Fluorescent Probe ◽  
Intracellular Ph ◽  
Cell Apoptosis ◽  
Pathological Process ◽  
Down Regulation ◽  
Ph Values

Intracellular pH values change is a significant physiological and pathological process and plays vital roles in autophagy, self-repairing, and programmed cell apoptosis. A unique fluorescent probe (PN-1) based on polysiloxanes...

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