Determination of Electrophoretic Mobility Distributions through the Analysis of Individual Mitochondrial Events by Capillary Electrophoresis with Laser-Induced Fluorescence Detection

Duffy, Ciarán F.; Fuller, Kathryn M.; Malvey, Megan W; O’Kennedy, Richard; Arriaga, Edgar A.

doi:10.1021/ac010939i

Cited by 76 publications

(124 citation statements)

References 32 publications

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“…Previous reports on the CE-LIF analysis of mitochondrial samples, showed that for samples containing ∼50 events, the electrophoretic mobility histograms are flat and broadly distributed [8], while for samples containing ∼2000, the corresponding histograms appear more Gaussian in nature [21]. This suggests that sample size is important when analyzing individual particles and statistical descriptions are needed to help identify how the number of events affects a distribution.…”

Section: Introductionmentioning

confidence: 98%

“…The capability of CE systems, equipped with laser-induced fluorescence (LIF) detectors, to separate individual organelles has also been demonstrated [8]. Although flow cytometry was historically used first to detect individual organelles [9,10], CE has the additional analytical advantage of being able to separate organelles based on their electrophoretic mobilities [11][12][13][14].…”

Section: Introductionmentioning

confidence: 99%

“…Although flow cytometry was historically used first to detect individual organelles [9,10], CE has the additional analytical advantage of being able to separate organelles based on their electrophoretic mobilities [11][12][13][14]. In general, the electrophoretic mobility of a particle stems from its size [12,14,19] and surface characteristics [13,19], which makes CE useful for comparing the size or surface characteristics of individually detected biological species, such as proteins [15], mitochondria [8,16], nuclei [17], and acidic organelles [18]. The size and surface characteristics of these particles may be highly heterogeneous, making it extremely important to analyze samples with a sufficient number of individual species so that an accurate statistical description is observed.…”

Section: Introductionmentioning

confidence: 99%

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Evaluation of individual particle capillary electrophoresis experiments via quantile analysis

Whiting

Arriaga

2007

Journal of Chromatography A

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The number of particles in a sample heavily influences the shape of the distribution describing the corresponding individual particle measurements. Selecting an adequate number of particles that prevents biases due to sample size is particularly difficult for complex biological systems in which statistical distributions are not normal. Quantile analysis is a powerful statistical technique that can rapidly compare differences between multiple distributions of individual particles. This report utilizes quantile analysis to show that the number of events detected affects the mobility distributions for rat liver and mouse liver mitochondria, sample individual particles, when analyzed via capillary electrophoresis with laser-induced fluorescence. When the mitochondrial sample is small (e.g. <78), there are not enough events to obtain statistically relevant mobility data. Adsorption to the capillary surface also significantly affects the mobility distribution at a small number of events in uncoated and dynamically coated capillaries. These adsorption effects can be overcome when the mitochondrial load on the capillary is sufficiently large (i.e. >609 and >1426 events for mouse liver on uncoated capillaries and rat liver on dynamically coated capillaries, respectively). It is anticipated that quantile analysis can be used to study other distributions of individual particles, such as nanoparticles, organelles, and biomolecules, and that distributions of these particles will also be dependant on sample size.

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Section: Introductionmentioning

confidence: 98%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Evaluation of individual particle capillary electrophoresis experiments via quantile analysis

Whiting

Arriaga

2007

Journal of Chromatography A

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“…Fig. 2), which is due to the size heterogeneity of mitochondrial particles and the presence of some mitochondrial fragments and aggregates in the sample [32]. Fortunately, particles that are similar in size to mitochondria have been shown to have electrophoretic mobilities independent of size [33].…”

Section: Identifying Sampling Effects On Mitochondrial Distributionsmentioning

confidence: 96%

Determining under‐ and oversampling of individual particle distributions in microfluidic electrophoresis with orthogonal laser‐induced fluorescence detection

et al. 2008

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This report investigates the effects of sample size on the separation and analysis of individual biological particles using microfluidic devices equipped with an orthogonal LIF detector. A detection limit of 17 6 1 molecules of fluorophore is obtained using this orthogonal LIF detector under a constant flow of fluorescein, which is a significant improvement over epifluorescence, the most common LIF detection scheme used with microfluidic devices. Mitochondria from rat liver tissue and cultured 143B osteosarcoma cells are used as model biological particles. Quantile-quantile (q-q) plots were used to investigate changes in the distributions. When the number of detected mitochondrial events became too large (.72 for rat liver and .98 for 143B mitochondria), oversampling occurs. Statistical overlap theory is used to suggest that the cause of oversampling is that separation power of the microfluidic device presented is not enough to adequately separate large numbers of individual mitochondrial events. Fortunately, q-q plots make it possible to identify and exclude these distributions from data analysis. Additionally, when the number of detected events became too small (,55 for rat liver and ,81 for 143B mitochondria) there were not enough events to obtain a statistically relevant mobility distribution, but these distributions can be combined to obtain a statistically relevant electrophoretic mobility distribution.

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“…The design and setup of the electrophoresis system with sheath flow cuvette postcolumn LIF detection used in this study have been previously described [32,33]. A single-line argon-ion laser (488 nm, 12 mW) was used for fluorescence excitation (Melles Griot, Irvine, CA).…”

Section: Cementioning

confidence: 99%

CE analysis of the acidic organelles of a single cell

2007

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CE analysis of the acidic organelles of a single cellThe properties of organelles within a cell have been shown to be highly heterogeneous. Until now, it has been unclear just how much of this heterogeneity is endemic to the organelle subpopulations themselves and how much is actually due to stochastic cellular noise. An attractive approach for investigating the origins of heterogeneity among the organelles of a single cell is CE with LIF detection (CE-LIF). As a proof of principle, in this report we optimize and use a single cell CE-LIF method to investigate the properties of endocytic (acidic) organelles. Our results show that the properties of individual acidic organelles containing Alexa Fluor ® 488 Dextran suggest that there are two groups of CCRF-CEM cells: a group with a high dextran content per cell, and a group with a low dextran content per cell. Furthermore, the individual organelle measurements of the single cells allow us to compare in each group the distributions of doxorubicin content per acidic organelle and electrophoretic mobilities of these organelles. Keywords IntroductionHeterogeneity among cells of the same type is known to result partly from intracellular processes, such as pinocytosis [1], the expression of proteins participating in DNA replication [2], and cellular responses to drug treatments [3], and partly from the microenvironment in which the cells reside [4]. Within the cell, heterogeneity at the subcellular level, which refers to variations in the properties of organelles of a given type, has remained practically uncharacterized up to this point. There is a great need to develop and apply single cell approaches capable of measuring the different facets of cellular heterogeneity at the subcellular level. Among the existing techniques for investigating subcellular heterogeneity, chemical imaging has been used to probe the heterogeneity of subcellular properties such as organellar pH, ion concentration, and protein interactions [5,6]. Flow cytometry [7] and CE with LIF detection (CE-LIF) [7,8] have both been used to analyze individual organelles in samples prepared from thousands to millions of cells. With all of these techniques, it has been impossible to track down which organelles originate from the same cell, information that is needed if we are to determine cell-to-cell variations.In order to determine cell-to-cell variations, single cells have been analyzed by CE [9], chromatography [10], MS [11], enzymatic radiolabeling [12], sensitive voltammetric detection [13], and flow cytometry [14]. CE has been used to describe the contents and functions of single cells (e.g., protein expression [15], release of neurotransmitters, and cell secretion [16]). In regard to the subcellular analysis of single cells, CE has been used to sample small subcellular regions, and to measure properties of the whole nucleus of a single cell [17,18]. The ability to detect individual mitochondrial particles by CE-LIF after their release from single cells has been recently described [19]. This report sho...

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Determination of Electrophoretic Mobility Distributions through the Analysis of Individual Mitochondrial Events by Capillary Electrophoresis with Laser-Induced Fluorescence Detection

Cited by 76 publications

References 32 publications

Evaluation of individual particle capillary electrophoresis experiments via quantile analysis

Evaluation of individual particle capillary electrophoresis experiments via quantile analysis

Determining under‐ and oversampling of individual particle distributions in microfluidic electrophoresis with orthogonal laser‐induced fluorescence detection

CE analysis of the acidic organelles of a single cell

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