A topographic analysis of metabolic pathways in single living cells by multisite microfluorometry

Kohen, Elli; Kohen, Cahide; Thorell, B.; Bartick, Philip

doi:10.1016/0014-4827(79)90331-8

Cited by 14 publications

(1 citation statement)

References 15 publications

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“…A general dictum has been that quantification was at best difficult or, at worst, impossible (8,9). A number of quantitative studies have been undertaken to measure various intraceUular metabolic states (10)(11)(12)(13), oxygen (14,15), pH (16,17), and various kinetic processes such as diffusion coefficients of fluorescent molecules (18)(19)(20)(21). In general, these measurements have been restricted to and integrated over a small area of the microscope field, with little effort to retain spatial information.…”

mentioning

confidence: 99%

Digital imaging fluorescence microscopy: spatial heterogeneity of photobleaching rate constants in individual cells.

Benson¹,

Bryan²,

Plant³

et al. 1985

The Journal of Cell Biology

160

114

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Photobleaching and related photochemical processes are recognized experimental barriers to quantification of fluorescence by microscopy. We have measured the kinetics of photobleaching of fluorophores in living and fixed cells and in microemulsions, and have demonstrated the spatial variability of these processes within individual cells. An inverted fluorescence microscope and a high-sensitivity camera, together with high-speed data acquisition by a computer-controlled image processor, have been used to control precisely exposure time to excitation light and to record images. To improve the signal-to-noise ratio, 32 digital images were integrated. After correction for spatial variations in camera sensitivity and background fluorescence, the images of the relative fluorescence intensities for 0.065 ~m 2 areas in the object plane were obtained.To evaluate photobleaching objectively, an algorithm was developed to fit a three-parameter exponential equation to 20 images recorded from the same microscope field as a function of illumination time. The results of this analysis demonstrated that the photobleaching process followed first-order reaction kinetics with rate constants that were spatially heterogeneous and varied, within the same cell, between 2-and 65-fold, depending on the fluorophore. The photobleaching rate constants increased proportionally with increasing excitation intensity and, for benzo(a)pyrene, were independent of probe concentration over three orders of magnitude (1.25 /~M to 1.25 mM). The propensity to photobleach was different with each fluorophore. Under the cellular conditions used in these studies, the average rates of photobleaching decreased in this order: N-(7-nitrobenz-2-oxa-1,3-diazole)-23,24-dinor-5-cholen-22-amine-3fl-ol > acridine orange > rhodamine-123 > benzo(a)pyrene > fluorescein > tetramethylrhodamine > 1,1'dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine. The photobleaching appears to be an oxidation reaction, in that the addition of saturated solutions of Na2S2Os to mineral oil microemulsions eliminated photobleaching of N-(7-nitrobenz-2-oxa-1,3-diazole)-23, 24-dinor-5-cholen-22-amine-3fl-ol or benzo(a)pyrene. We identified experimental conditions to observe, without detectable photobleaching, fluorophores in living cells, which can not be studied anaerobically. Useful images were obtained when excitation light was reduced to eliminate photobleaching, as determined from zero-time images calculated from the exponential fit routine. The results show that, with conventional illumination and photographic methods, the fluorescence of regions with high rate constants for photobleaching are significantly underrepresented, and can be as much as 20-fold.Fluorescence microscopy is an attractive method to study the dynamics of various cellular processes and to elucidate the internal architecture of individual cells that uses appropriately labeled fluorescent antibodies and proteins. Work by a large

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mentioning

confidence: 99%

Digital imaging fluorescence microscopy: spatial heterogeneity of photobleaching rate constants in individual cells.

Benson¹,

Bryan²,

Plant³

et al. 1985

The Journal of Cell Biology

160

114

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Metabolic control and compartmentation in single living cells

Kohen¹,

Kohen²,

Hirschberg³

et al. 1983

Cell Biochemistry & Function

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Microspectrofluorometry of cell coenzymes (NAD(P)H, flavins) in conjunction with sequential microinjections into the same cell of metabolites and modifiers, reveals aspects of the regulatory mechanisms of transient redox changes of mitochondrial and extramitochondrial nicotinamide adenine dinucleotides. The injection of ADP in the course of an NAD(P)H transient produced by glycolytic (e.g. glucose 6-phosphate, G6P) or mitochondrial (e.g. malate) substrate leads to sharp reoxidation (state III, Chance and Williams, 1955), followed by a spontaneous state III to IV transition, and an ultimate return to original redox steady state. The response to ADP alone is biphasic, i.e. a small oxidation-reduction transient followed by a larger reverse transient. Similarities between responses to injected ATP and ADP suggest possible intracellular interconversions. Sequential injections of glycolytic and Krebs cycle substrates into the same cell, produce a two-step NAD(P) response, possibly revealing the intracellular compartmentation of this coenzyme. A two-step NAD(P)H response to sequentially injected fructose 1,6-diphosphate and G6P indicates the dynamic or even structural compartmentation of glycolytic phosphate esters in separate intracellular pools. The intracellular regulation and compartmentation of bioenergetic pathways and cell-to-cell metabolic inhomogeneities provide the basis on which the quantitative biochemistry of the intact living cell may be reconciled with these in situ findings.

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