Ez c1 gold version 3

Measurements of fluorescence intensities were performed in MII oocytes having either heterogeneous (perinuclear and/or pericortical) or homogeneous (small aggregates) mt distribution pattern. Oocytes showing abnormal mt distribution pattern were excluded from this analysis. In each individual oocyte, MitoTracker and DCF fluorescence intensities were measured at the equatorial plane, as in previous studies from our unit performed in human [55 (link)] and animal oocytes [20 (link), 40 (link), 47 , 50 (link)], with the aid of the EZ-C1 Gold Version 3.70 image analysis software platform for Nikon C1 (Nikon Instruments) confocal microscope. A circle of an area = 100 in diameter (arbitrary value) was drawn to measure only the cytoplasmic area (512 × 512 pixels). The fluorescence intensity encountered within the programmed scan area was recorded and plotted against the conventional pixel unit scale (0–255). Fluorescence intensity was expressed as arbitrary densitometric units (ADU). Parameters related to fluorescence intensity were maintained at constant values for all measurements. In detail, images were taken under fixed scanning conditions with respect to laser energy, signal detection (gain), and pinhole size.

In each individual oocyte, MitoTracker and DCF fluorescence intensities and the Manders’ overlap coefficient [29 (link)], indicating the extent of mitochondria/ROS colocalization, were measured at the equatorial plane using the EZ-C1 Gold Version 3.70 image analysis software platform for a Nikon C1 confocal microscope. A circular area was drawn to measure only the region including cell cytoplasm. The fluorescence intensity within the scanned area (512 × 512 pixels) was recorded and 16-bit images were obtained. Mitochondrial membrane potential (ΔΨ) and intracellular ROS concentrations were recorded as the fluorescence intensity emitted by each probe and expressed as arbitrary densitometric units (ADUs). Sample signals were expressed as percentages of the signal of the sample used as a control (EH treatment). Variables related to fluorescence intensity, such as laser energy, signal detection (gain), and pinhole size, were maintained at constant values for all measurements. In mitochondria/ROS colocalization analysis, threshold levels were kept constant, for all measurements, at 10% of the maximum pixel intensity.

In each individual oocyte, MitoTracker and DCF fluorescence intensities were measured at the equatorial plane and at the excitation/emission, as described above by use of EZ-C1 Gold Version 3.70 image analysis software platform for Nikon C1 confocal microscope. A circular area was drawn in order to measure only the region including cell cytoplasm. The fluorescence intensity within the programmed scan area was recorded and plotted against the conventional pixel unit scale (0–255). Mitochondrial activity and intracellular ROS levels were recorded as MitoTracker Orange CMTM Ros and DFC fluorescence intensity in arbitrary densitometric units (ADU). Parameters related to fluorescence intensity, such as laser energy, signal detection (gain), and pinhole size, were maintained at constant values for all measurements. The degree of mitochondria-ROS colocalization, reported as a biomarker of healthy oocytes [62 (link),63 (link)] was quantified by the overlap coefficient between MitoTraker Orange CMTM Ros and DCF fluorescence intensity signals.

In each individual MII oocyte, MitoTracker and DCF fluorescence intensities were measured at the equatorial plane, at the excitation/emission as described above by use of EZ-C1 Gold Version 3.70 image analysis software platform for the Nikon C1 confocal microscope. A circular area was drawn in order to measure only the region including cell cytoplasm. For the blastocysts, a circle was traced on all the 25 acquired focal planes. The fluorescence intensity within the programmed scan area was recorded and plotted against the conventional pixel unit scale (0–2.16255). Mitochondrial activity and intracellular ROS levels were recorded as MitoTracker Orange CMTMRos and DFC fluorescence intensity in arbitrary densitometric units (ADU). Parameters related to fluorescence intensity, such as laser energy, signal detection (gain), and pinhole size, were maintained at constant values for all measurements.

In each individual oocyte, MitoTracker and DCF fluorescence intensities were measured at the equatorial plane and at the excitation/emission, as described above, using the EZ-C1 Gold Version 3.70 image analysis software platform for Nikon C1 confocal microscope. A circular area was drawn in order to measure only the region including cell cytoplasm. The fluorescence intensity within the programmed scan area was recorded and plotted against the conventional pixel unit scale (0–255). Mitochondrial activity and intracellular ROS levels were recorded as MitoTracker Orange CMTM Ros and DFC fluorescence intensity in arbitrary densitometric units (ADUs). Parameters related to fluorescence intensity, such as laser energy, signal detection (gain), and pinhole size, were maintained at constant values for all measurements. The degree of mitochondria/ROS colocalization, reported as a biomarker of healthy oocytes [61 (link),62 (link)], was quantified by the overlap coefficient between MitoTraker Orange CMTM Ros and the DCF fluorescence intensity signals.

In each individual oocyte, MitoTracker Orange CMTM Ros and DCF fluorescence intensities were measured at the equatorial plane, as in previous studies in human [34 (link)] and animal oocytes [25 (link),26 (link),29 (link),32 (link)], with the aid of the EZ-C1 Gold Version 3.70 image analysis software platform for Nikon C1 (Nikon Instruments) confocal microscope. A circle of area = 100 in diameter (arbitrary value) was drawn to measure only the cytoplasmic area (512 by 512 pixels). The fluorescence intensity encountered within the programmed scan area was recorded and plotted against the conventional pixel unit scale (0–255). Fluorescence intensity was expressed as Arbitrary Densitometric Units (ADU). Parameters related to fluorescence intensity were maintained at constant values for all measurements. In detail, images were taken under fixed scanning conditions with respect to laser energy, signal detection (gain) and pinhole size.

In each individual MII oocyte, MitoTracker Orange CMTM Ros and DCF fluorescence intensities were measured at equatorial plane, with the EZ-C1 Gold Version 3.70 image analysis software for Nikon C1 confocal microscope. A circle area was drawn to measure the area including cell cytoplasm and fluorescence intensity encountered within the scan area was recorded and plotted against a conventional pixel unit scale (0–255). Sample signals were expressed as percentage of the control sample. [54 (link), 62 (link)].