High-throughput capillary-electrophoresis analysis of the contents of a single mitochondria

Abstract

We present a technique for labeling the contents of acidic organelles and rapidly releasing, separating, and detecting their labeled contents with laser-induced fluorescence. We have performed solution-phase separation of the contents of single mitochondria and single 100 nm vesicles, which represents a demonstration of an analyzed volume of approximately 1 aL. Our strategy to label the acidic contents of the mitochondrion relies on the use of the membrane-permeable dye, Oregon Green diacetate succinimidyl ester, and a membrane-permeable base to raise intramitochondrial pH. In order to measure the contents, we utilized a glass microfluidic chip and high voltage gradient for millisecond capillary electrophoresis separation after single-mitochondrion photolysis. We observed heterogeneity among a population of mitochondria with respect to a constituent chemical component.

Figure 1

(A) A schematic diagram of the instrument, including the connections among the modules and essential optics. The USB-DAQ triggers the opening of a mechanical shutter for confocal illumination then activates the HV power supply. Once the power supply has reached full voltage, the USB-DAQ board triggers the MCS card (which is located in the PC). The MCS card records the photon counts from the single photon avalanche diode (SPAD); at 50% time of the full data collection run, the MCS card sends a TTL pulse to the nanosecond pulsed UV laser to initiate photolysis. The 488 nm laser is shuttered such that while aiming, it is configured for epi-fluorescence (to show the location of all mitochondria within the field of view), and during data acquisition it is configured for line confocal detection with the SPAD. Epi. indicates epifluorescence optics, Con. indicates confocal optics, Obj. indicates objective lens. (B) Schematic and images showing the essential elements of the microfluidic chip design including the 750 μm wide, 120 μm deep inlet and outlet channels and the 50 μm wide, ~1 μm deep separation channels. Light micrographs of the two respective side profiles are shown at bottom.

Figure 2

(A-D) Schematics showing the sequence of events in a capillary-electrophoresis experiment, starting with (A) a target (vesicle or mitochondrion) aligned in the UV laser focus, which caused the target to be lysed with a single nanosecond UV laser pulse (B); the insets show the target vesicle (arrow) before and after lysis. The released components (C) are separated as they travel toward and across the probe volume (blue elliptical focus) (D). (E) Simulation of 1-D free diffusion (black) overlaid on data collected (red) with the line confocal probe volume parked at 2 μm from the release point, and 2-D free diffusion (black) overlaid on data collected (red) at 7 μm from the release point.

Figure 3

Electropherogram of a single 100 nm vesicle (1 attoliter in volume) containing Oregon Green (OG) and OG labeled glutamate (OG-Glu) after single-pulse photolysis. The data shows resolved glutamate and free dye peaks. The inset above shows a bulk electropherogram of the glutamate labeling products prior to encapsulation; the images to the right shows the vesicle prior to (top image) and after (bottom image) photolysis; the targeted vesicle is indicated with an arrow.

Figure 4

(A) Schematic showing the labeling scheme for encapsulated contents. Solutions 1 and 2 show the Control Samples containing Oregon Green Diacetate Succinimidyl Ester (OGDA-SE) without (1) and with (2) Glutamate (Glu). The flow chart shows acidic, glutamate-containing vesicles (red circles) are suspended in a basic buffer (3). To this solution (3), OGDA-SE was added without (4) and with (5) benzylethanolamine (BEA), which raised the intra-vesicular pH to basic. The red vesicle in (4) represents acidic intra-vesicular pH, while the clear vesicle in (5) represents basic intra-vesicular pH (same pH as the extra-vesicular solution). Both solutions (4) and (5) were purified with a size-exclusion column to remove extra-vesicular OGDA-SE, Glu, and OG-Glu (solutions 6 and 7). (B) Electropherograms of the corresponding numbered solutions in the labeling scheme. The vesicles were lysed with Triton X-100 and analyzed with CE. A small amount of OG-Glu was seen in (4) and (5) because of the presence of a small amount of Glu in the extra-vesicular solution that reacted with OGDA-SE, because the size-exclusion column might not be 100% efficient in removing all extra-vesicular Glu and because a small amount of lysis of the vesicles might have released some Glu into the extra-vesicular solution.

Figure 5

(A) Single-mitochondrion electropherograms obtained without the use of benzylethanolamine (BEA) (top trace) and with BEA (bottom trace). Inset shows before and after photolysis of a mitochondrion prior to capillary-electrophoresis separation. (B) Bulk electropherograms of mitochondrial lysate without (top) and with (bottom) BEA in the labeling step. (C) Histogram of integrated photon counts from 1.1 to 1.3 msec, which shows a multi-modal distribution among those mitochondria displaying a peak at this retention time.