Detection of heteroplasmy in individual mitochondrial particles.

Mitochondrial DNA (mtDNA) mutations have been associated with disease and aging. Since each cell has thousands of mtDNA copies, clustered into nucleoids of five to ten mtDNA molecules each, determining the effects of a given mtDNA mutation and their connection with disease phenotype is not straightforward. It has been postulated that heteroplasmy (coexistence of mutated and wild-type DNA) follows simple probability rules dictated by the random distribution of mtDNA molecules at the nucleoid level.

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

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 +/- 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.

Analysis of mitochondria isolated from single cells.

Bulk studies are not suitable to describe and study cell-to-cell variation, which is of high importance in biological processes such as embryogenesis, tissue differentiation, and disease. Previously, capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) was used to measure the properties of organelles isolated from millions of cells. As such, these bulk measurements reported average properties for the organelles of cell populations.

Fast electrophoretic analysis of individual mitochondria using microchip capillary electrophoresis with laser induced fluorescence detection.

The analysis of mitochondria by capillary electrophoresis usually takes longer than 20 min per replicate which may compromise the quality of the mitochondria due to degradation. In addition, low sample consumption may be beneficial in the analysis of rare or difficult samples. In this report, we demonstrate the ability to analyze individual mitochondrial events in picoliter-volume samples (approximately 80 pL) taken from a bovine liver preparation using microchip capillary electrophoresis with laser-induced fluorescence detection (micro-chip CE-LIF).

Determination of individual microsphere properties by capillary electrophoresis with laser-induced fluorescence detection.

Capillary electrophoresis with postcolumn laser-induced fluorescence detection was used to individually detect 6.0, 1.0, 0.

Determination of the cardiolipin content of individual mitochondria by capillary electrophoresis with laser-induced fluorescence detection.

We report the application of capillary electrophoresis (CE) with postcolumn laser-induced fluorescence (LIF) detection to measure the cardiolipin content of individual mitochondria from cultured NS1 cells. Mitochondria were isolated by differential centrifugation and stained with the fluorescent dye 10-N-nonyl acridine orange which stoichiometrically binds to cardiolipin in a 1:1 or 2:1 ratio depending on the dye concentration. The green fluorescence resulting from the 1:1 complex was chosen for analysis because it is substantially more intense than the red fluorescence resulting from the 2:1 complex.

Determination of electrophoretic mobility distributions through the analysis of individual mitochondrial events by capillary electrophoresis with laser-induced fluorescence detection.

Here we report on the analysis of mitochondrial preparations by capillary electrophoresis with postcolumn laser-induced fluorescence detection. Individual mitochondria are detected by fluorescent labeling with the mitochondrion-selective probe, 10-nonyl acridine orange. Interactions between the organelles and the capillary walls are controlled by derivatization of the capillaries with poly(acryloylaminopropanol).