An automatic system to study sperm motility and energetics

Abstract

An integrated robotic laser and microscope system has been developed to automatically analyze individual sperm motility and energetics. The custom-designed optical system directs near-infrared laser light into an inverted microscope to create a single-point 3-D gradient laser trap at the focal spot of the microscope objective. A two-level computer structure is described that quantifies the sperm motility (in terms of swimming speed and swimming force) and energetics (measuring mid-piece membrane potential) using real-time tracking (done by the upper-level system) and fluorescent ratio imaging (done by the lower-level system). The communication between these two systems is achieved by a gigabit network. The custom-built image processing algorithm identifies the sperm swimming trajectory in real-time using phase contrast images, and then subsequently traps the sperm by automatically moving the microscope stage to relocate the sperm to the laser trap focal plane. Once the sperm is stably trapped (determined by the algorithm), the algorithm can also gradually reduce the laser power by rotating the polarizer in the laser path to measure the trapping power at which the sperm is capable of escaping the trap. To monitor the membrane potential of the mitochondria located in a sperm’s mid-piece, the sperm is treated with a ratiometrically-encoded fluorescent probe. The proposed algorithm can relocate the sperm to the center of the ratio imaging camera and the average ratio value can be measured in real-time. The three parameters, sperm escape power, sperm swimming speed and ratio values of the mid-piece membrane potential of individual sperm can be compared with respect to time. This two-level automatic system to study individual sperm motility and energetics has not only increased experimental throughput by an order of magnitude but also has allowed us to monitor sperm energetics prior to and after exposure to the laser trap. This system should have application in both the human fertility clinic and in animal husbandry.

Chemosensory Ca2+ Dynamics Correlate with Diverse Behavioral Phenotypes in Human Sperm

“…The laser tweezers optical design, hardware, sperm tracking software, experimental procedure, and analysis of sperm have been described in detail elsewhere (52,53). Briefly, a single point gradient trap was generated using an Nd:YVO 4 continuous wave 1064-nm wavelength laser (BL-106C, Spectra Physics, Mountain View, CA), coupled to a Zeiss Axiovert S100 microscope equipped with a phase III, oil immersion objective (ϫ40 magnification; numerical aperture ϭ 1.3).…”

Optical manipulation for single‐cell studies

“…The system expands the availability of complex and expensive research technologies and research groups can collaborate in real time even over the continents. A similar automated system has been used to study sperm motility and energetics [157]. Here, a complex two level computer structure was used to quantify sperm motility and energetics using a real-time tracking and fluorescent ratio imaging.…”

Optomechatronic System For Automated Intra Cytoplasmic Sperm Injection

“…Different computer-assisted methods for sperm analyses based on multiobject tracking, motility, and energy estimation have been developed [19,20]. Here, we use a common technique, for sperm speed measurement, known as Particle Image Velocimetry (PIV).…”