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Updated: Oct 17, 2026
Simultaneous pH Measurement in Endocytic and Cytosolic Compartments in Living Cells using Confocal Microscopy
Published on: April 28, 2014
Measurement of intracellular (compartmental) pH by 31P NMR in Aspergillus niger
S J Hesse1, G J Ruijter, C Dijkema
1Department of Molecular Physics, Wageningen Agricultural University, The Netherlands. stephan.hesse@algemeen.mgim.wau.nl
Abstract:
31P nuclear magnetic resonance (31P NMR) was used to monitor cytoplasmic and vacuolar pH values in the filamentous fungus Aspergillus niger. To obtain a homogeneous cell sample and to be able to perform long term in vivo NMR measurements A. niger mycelium was kept in a setup that allows perfusion of the cell plug within the NMR tube. Mycelial samples, however, became rapidly clogged during perfusion leading to (partial) anaerobiosis of the plug with subsequent acidification of the cytoplasm. As a result, only short-term NMR measurements (5-10 min) were possible using free mycelium. To increase and to prolong perfusion, A. niger was immobilized in Ca(2+)-alginate beads. Deteriorated spectra recorded under hypoxia could be completely restored in the presence of oxygen. With this system perfusion in the presence of citrate could be maintained for at least 18 h at much higher rates (15 ml min-1 compared with 4 ml min-1 for free mycelium). During this period 31P NMR spectra were highly invariable, indicating approximate steady-state intracellular conditions during long term measurements. Perfusion in the presence of glucose resulted in complete depletion of the vacuolar inorganic phosphate pool within 45 min and yielded a higher pH gradient over the tonoplast than when citrate was used (delta pH = 1.6 and 1.4, respectively).
Insights
Immobilizing Aspergillus niger in alginate beads enabled long-term in vivo monitoring of cellular pH using 31P nuclear magnetic resonance (NMR). This method overcame limitations of free mycelium, allowing stable measurements for extended periods.
Area of Science:
- Biochemistry
- Cell Biology
- Mycology
Background:
- Accurate measurement of intracellular pH is crucial for understanding cellular processes in fungi.
- Previous in vivo studies using 31P NMR in Aspergillus niger were limited by short measurement times due to mycelial clogging during perfusion.
Purpose of the Study:
- To develop a method for prolonged in vivo monitoring of cytoplasmic and vacuolar pH in Aspergillus niger using 31P NMR.
- To overcome the limitations of free mycelium perfusion for long-term NMR measurements.
Main Methods:
- 31P nuclear magnetic resonance (NMR) spectroscopy was employed to measure pH.
- Aspergillus niger was immobilized in calcium alginate beads to facilitate continuous perfusion.
- Perfusion rates and spectral quality were assessed under different conditions (hypoxia, oxygen, citrate, glucose).
Main Results:
- Immobilization in alginate beads significantly improved perfusion rates and allowed for stable, long-term (≥18 h) in vivo NMR measurements.
- Restoration of oxygen improved spectral quality under hypoxic conditions.
- Perfusion with glucose led to rapid depletion of vacuolar inorganic phosphate and a larger pH gradient across the tonoplast compared to citrate.
Conclusions:
- Alginate immobilization is an effective strategy for long-term in vivo NMR studies of filamentous fungi like Aspergillus niger.
- This improved methodology allows for stable monitoring of intracellular pH homeostasis and nutrient metabolism under various conditions.
