Description:

We have successfully used AttofluorTM RatioVisionTM digital imaging system to measure dynamics in the mitochondrial membrane potential (m), intracellular [Ca²⁺], and production of reactive oxygen species in fertilized mouse zygotes. More than one hundred embryos have been imaged. The m was measured by quantification of the change in fluorescence of cells stained with the probe tetra-methylrhodamine ethyl ester (TMRE) (Molecular Probes, Eugene, OR). Fluorescence was acquired at the excitation wavelength of 530 nm and emission of 580 nm and reduced with a neutral density filter. We showed that the mitochondrial uncoupler FCCP (750 nM) induced instant dissipation of m. Radiometric fluorescent dye Fura-2AM was used to measure intracellular [Ca²⁺]. Calcium imaging was conducted at excitation wavelength of 334/380nm and measured at emission wavelength of 520nm.

Progress:

We showed that FCCP induced an increase in intracellular [Ca²⁺]. The intracellular production of reactive oxygen species (ROS) was determined using the fluorescent probe CM-H2DCFDA (Molecular Probes), and CM-DCF fluorescence was measured at an excitation wavelength of 488 nm and an emission of 520 nm. ROS was significantly increased 20 min after FCCP challenge and continued to increase as exposure time was prolonged. The ROS production occurred gradually after disruption in m and the increase in intracellular [Ca²⁺].