The relationship between the size of mitochondria and the intensity of light that they scatter in different energetic states

Knight, Vincent A.; Wiggins, Philippa M.; Harvey, J.D.; O’Brien, Judith A.

doi:10.1016/0005-2728(81)90220-6

Cited by 22 publications

(30 citation statements)

References 14 publications

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“…In agreement with previous observations, 26,30,31,39,40 we found superior prediction of size using FSC compared to SSC (see Experimental Section). In contrast, SSC, which we associated with the concept of “granularity”, 26,30 has also been associated with mitochondrial bioenergetic state (e.g., polarization of the inner membrane, organelle swelling, or the state of ATP synthase in the mitochondrial membrane). 26,30 …”

Section: Resultssupporting

confidence: 93%

“…In contrast, SSC, which we associated with the concept of “granularity”, 26,30 has also been associated with mitochondrial bioenergetic state (e.g., polarization of the inner membrane, organelle swelling, or the state of ATP synthase in the mitochondrial membrane). 26,30 …”

Section: Resultsmentioning

confidence: 97%

“…It has been reported previously that isolated mitochondria can polarize and respire normally in the absence of an ETC substrate (e.g., succinate) as long as a “permeant ion” such as K + is present in the buffer. 24–26 Furthermore, the presence of this ion facilitates valinomycin (a K + ionophore) mediated depolarization of mitochondria. Of note, we found JC-9 dye to be incompatible with CCCP (or FCCP) because treatment with this depolarizing agent appeared to dramatically affect the amount of JC-1 in solution, which ultimately could alter the red/green ratio of mitochondria.…”

Section: Methodsmentioning

confidence: 99%

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Identification and Characterization of Mitochondrial Subtypes in Caenorhabditis elegans via Analysis of Individual Mitochondria by Flow Cytometry

et al. 2016

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Mitochondrial bioenergetics has been implicated in a number of vital cellular and physiological phenomena, including aging, metabolism, and stress resistance. Heterogeneity of the mitochondrial membrane potential (Δψ), which is central to organismal bioenergetics, has been successfully measured via flow cytometry in whole cells but rarely in isolated mitochondria from large animal models. Similar studies in small animal models, such as Caenorhabditis elegans (C. elegans), are critical to our understanding of human health and disease but lack analytical methodologies. Here we report on new methodological developments that make it possible to investigate the heterogeneity of Δψ in C. elegans during development and in tissue-specific studies. The flow cytometry methodology described here required an improved collagenase-3-based mitochondrial isolation procedure and labeling of mitochondria with the ratiometric fluorescent probe JC-9. To demonstrate feasibility of tissue-specific studies, we used C. elegans strains expressing blue-fluorescent muscle-specific proteins, which enabled identification of muscle mitochondria among mitochondria from other tissues. This methodology made it possible to observe, for the first time, critical changes in Δψ during C. elegans larval development and provided direct evidence of the elevated bioenergetic status of muscle mitochondria relative to their counterparts in the rest of the organism. Further application of these methodologies can help tease apart bioenergetics and other biological complexities in C. elegans and other small animal models used to investigate human disease and aging.

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Section: Resultssupporting

confidence: 93%

Section: Resultsmentioning

confidence: 97%

Section: Methodsmentioning

confidence: 99%

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Identification and Characterization of Mitochondrial Subtypes in Caenorhabditis elegans via Analysis of Individual Mitochondria by Flow Cytometry

et al. 2016

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confidence: 99%

“…The volume changes are commonly followed by observing either the intensity of light scattered at 900 to the incident beam, or the absorbance of a suspension, the assumption being that as mitochondria swell, their relative refractive index decreases and so does the intensity of light they scatter. Measurements of the light scatter (7,10) demonstrate that changes in the intensity of scattered light are not reliable indices of changes ofvolume of mitochondria, and that changes in conformation with changes in metabolic state dominate changes in light scatter. At present, we do not know if the flow cytometric analysis that considered uniquely the objects passing through the laser beam has provided similar information.…”

mentioning

confidence: 99%

Flow Cytometric Analysis of Rhodamine 123 Fluorescence during Modulation of the Membrane Potential in Plant Mitochondria

Petit

1992

Plant Physiol.

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The fluorescent dye rhodamine 123, which selectively accumulates in mitochondria based on the membrane potential, was used with flow cytometry to evaluate variations in activity of mitochondria isolated from plant tissues. In the presence of succinate and ATP, potato (Solanum tuberosum L.) tuber mitochondrial activity was affected by metabolic inhibitors and compounds that modify the membrane potential. The more uniform the mitochondrial population, the higher the observed membrane potential. The reactive population corresponds to the proportion of intact mitochondria (94-97%) defined by classic methods. Changes in the light-scattering properties are more related to internal modifications affecting the inner membrane-matrix system of the mitochondria during metabolic modulation than to specific volume change or outer membrane surface modifications. We tested our approach using an Arum maculatum preparation that contains three different types of mitochondria and demonstrated the validity of the light-scatter measurements to distinguish the a, ,B, and y mitochondria and to measure their ability to built up a membrane potential in the presence of succinate. These results demonstrate clearly that flow cytometric techniques using rhodamine 123 can be employed to study the activity in isolated plant mitochondria.Fluorescent probes have been applied as optical indicators ofthe membrane potential differences in several types ofcells, isolated organelles, and lipid vesicles (1,4,20,31). The technique relies on membrane potential-dependent partitioning of charged lipophilic dye molecules across the membrane. Changes in membrane potential result in changes in the intensity of dye fluorescence, termed "redistribution signals" (4). Lipophilic cationic dyes have been used successfully to measure changes in the membrane potential of in situ or isolated mitochondria of yeast cells (9,20), several kinds of animal cells (27), and, more recently, plant cells and protoplasts (13,15,26). In plant mitochondria, the dyes most widely used for mitochondria are either derivatives of rhodamine or cyanine dyes developed by Waggoner (30, 31). The laser dye, Rh 123,' has been extensively employed as a fluorescent stain of mitochondria in living cells (27). Because Rh 123 is an aromatic cation, it has been assumed to distribute itself electrophoretically into the mitochondrial matrix in response to AI.At high concentrations, Rh 123 has toxic side effects, but Emaus et al. (6) have recently demonstrated that, at concentrations that do not inhibit mitochondrial function, Rh 123 is indeed a sensitive and specific probe of AI in isolated mitochondria. Agents known to depolarize or deenergize mitochondria, such as uncouplers (valinomycin) and respiratory inhibitors (KCN, SHAM), decreased Rh123 fluorescence of mitochondria in cultured cells, whereas nigericin, which collapses ApH and raises AI, increases fluorescence. For Rh 123, the energy-linked changes were accompanied by dye uptake into the matrix space and the concentration ratio in-to...

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Mitochondrial Subtype Identification and Characterization

2018

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Healthy, functional mitochondria are central to many cellular and physiological phenomena, including aging, metabolism, and stress resistance. A key feature of healthy mitochondria is a high membrane potential (Δψ) or charge differential (i.e., proton gradient) between the matrix and inner mitochondrial membrane. Mitochondrial Δψ has been extensively characterized via flow cytometry of intact cells, which measures the average membrane potential within a cell. However, the characteristics of individual mitochondria differ dramatically even within a single cell, and thus interrogation of mitochondrial features at the organelle level is necessary to better understand and accurately measure heterogeneity. Here we describe a new flow cytometric methodology that enables the quantification and classification of mitochondrial subtypes (via their Δψ, size, and substructure) using the small animal model C. elegans. Future application of this methodology should allow research to discern the bioenergetic and mitochondrial component in a number of human disease and aging models, including, C. elegans, cultured cells, small animal models, and human biopsy samples. © 2018 by John Wiley & Sons, Inc.

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The relationship between the size of mitochondria and the intensity of light that they scatter in different energetic states

Cited by 22 publications

References 14 publications

Identification and Characterization of Mitochondrial Subtypes in Caenorhabditis elegans via Analysis of Individual Mitochondria by Flow Cytometry

Identification and Characterization of Mitochondrial Subtypes in Caenorhabditis elegans via Analysis of Individual Mitochondria by Flow Cytometry

Flow Cytometric Analysis of Rhodamine 123 Fluorescence during Modulation of the Membrane Potential in Plant Mitochondria

Mitochondrial Subtype Identification and Characterization

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