Microspectrofluorometry by digital image processing: Measurement of cytoplasmic pH

L. Tanasugarn, P. McNeil, G. T. Reynolds, D. L. Taylor

Research output: Contribution to journalArticle

84 Citations (Scopus)

Abstract

An interface of our microspectrofluorometer with an image processing system performs microspectrofluorometric measurements in living cells by digital image processing. Fluorescence spectroscopic parameters can be measured by digital image processing directly from microscopic images of cells, and are automatically normalized for pathlength and accessible volume. Thus, an accurate cytoplasmic 'map' of various spectroscopic parameters can be produced. The resting cytoplasmic pH of fibroblasts (3T3 cells) has been determined by measuring the ratio of fluorescein fluorescence exited by two successive wavelengths (489 and 452 nm). Fluorescein-labeled dextran microinjected into the cells is used as a pH indicator, since it is trapped in the cytoplasm but is excluded from the nucleus and other organelles. The average cytoplasmic pH is 6.83 (± 0.38). However, cytoplasmic pH exhibits a non-unimodal distribution, the lower mean pH being 6.74 (± 0.23). When 3T3 cells pinocytose medium containing fluorescein dextran, pinosomes peripheral to the nucleus exhibit a lower pH than those closer to the ruffling edge of the cell. The present image processing system is analyzed for linearity of detection, light scattering artifacts, signal to noise ratio, standard curves, and spatial resolution. The results obtained from digital image analysis are shown to be comparable to the results from standard microspectrofluorometry. We also discuss several other applications of this ratio imaging technique in cell biology.

Original languageEnglish (US)
Pages (from-to)717-724
Number of pages8
JournalJournal of Cell Biology
Volume98
Issue number2
DOIs
StatePublished - Jan 1 1984
Externally publishedYes

Fingerprint

3T3 Cells
Fluorescence
Pinocytosis
Signal-To-Noise Ratio
Fluorescein
Organelles
Artifacts
Cell Biology
Cytoplasm
Fibroblasts
Light
fluorescein-dextran

ASJC Scopus subject areas

  • Cell Biology

Cite this

Microspectrofluorometry by digital image processing : Measurement of cytoplasmic pH. / Tanasugarn, L.; McNeil, P.; Reynolds, G. T.; Taylor, D. L.

In: Journal of Cell Biology, Vol. 98, No. 2, 01.01.1984, p. 717-724.

Research output: Contribution to journalArticle

Tanasugarn, L. ; McNeil, P. ; Reynolds, G. T. ; Taylor, D. L. / Microspectrofluorometry by digital image processing : Measurement of cytoplasmic pH. In: Journal of Cell Biology. 1984 ; Vol. 98, No. 2. pp. 717-724.
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