Real-time calcium measurements of live optically trapped microorganisms.

A system has been developed that allows for the real-time measurement of calcium dynamics in swimming sperm. Specifically, the ratiometric dye Indo-I is used as a fluorescent indicator of intracellular calcium dynamics. The dual emissions are collected by a high-sensitivity back-illuminated CCD camera coupled to a Dual-View imaging system. From the CCD, the images are sent to a custom developed algorithm which processes the images and outputs the calcium measurements in real-time. Additionally, sperm velocity and position data are processed and outputted in real-time. The velocity and position data are obtained using a separate coupled red light (>670 nm) phase contrast imaging setup that does not optically interfere with the fluorescent imaging. Using this system the effects of optical trapping on calcium dynamics was determined. Optical trapping of sperm with a decaying focused laser power of 510 mW to 3 mW over 8 seconds causes a statistically insignificant change in calcium dynamics between in-trap and out-of-trap conditions. Progesterone, a calcium activator, was added and sperm were trapped under the 8 second power decay conditions. Progesterone treated sperm has a statistically higher average calcium level than untreated sperm, but shows no statistical difference between progesterone treated in-trap and out-of-trap conditions. Trapping at 16 seconds at 510 mW without decay, which have been shown to decrease sperm motility, shows a statistical difference between baseline pre-trap and in-trap intracellular calcium levels.