The Spectral-Polarimetric Hypersensor (SPH) is a new compact, solid state, video rate imaging system with no moving parts capable of capturing up to 16 bands of both spectral and polarimetric content simultaneously. For these imagers, which are an adaptation of a plenoptic system, all elements for a given super pixel sample the same point in the scene, thus providing the ability to accurately measure point sources. Various scenes are imaged using two systems that span the VNIR (0.4 – 0.95nm) and SWIR (0.9-1.7m) wavebands. For each imager, twelve spectral components and four polarization states are captured simultaneously. The VNIR system captures full data cubes of 512x512 (spatial pixels) x 12 (spectral bands) x 4 (polarization states) at frame rates up to 30 Hz while the SWIR system captures full data cubes of 320x256 (spatial pixels) x 12 (spectral) x 4 (polarization states) at frame rates up to 30 Hz. From the acquired images, we compute the Stokes vector components, calculate DoLP, polarization angle, and obtain a spectral signature of objects in the scene. This data is then used to carry out target identification and clutter suppression in real-time, i.e. video frame rates.
We combine laser tweezers with custom computer tracking software and robotics to analyze the motility [swimming speed, VCL (curvilinear velocity), and swimming force in terms of escape laser power (Pesc)] and energetics [mitochondrial membrane potential (MP)] of individual sperm. Domestic dog sperm are labeled with a cationic fluorescent probe, DiOC2(3), that reports the MP across the inner membrane of the mitochondria located in the sperm's midpiece. Individual sperm are tracked to calculate VCL. Pesc is measured by reducing the laser power after the sperm is trapped using laser tweezers until the sperm is capable of escaping the trap. The MP is measured every second over a 5-s interval during the tracking phase (sperm is swimming freely) and continuously during the trapping phase. The effect of the fluorescent probe on sperm motility is addressed. The sensitivity of the probe is measured by assessing the effects of a mitochondrial uncoupling agent (CCCP) on MP of free swimming sperm. The effects of prolonged exposed to the laser tweezers on VCL and MP are analyzed. The system's capabilities are demonstrated by measuring VCL, Pesc, and MP simultaneously for individual sperm. This combination of imaging tools is useful to quantitatively assess sperm quality and viability.
This paper describes a robust single sperm tracking algorithm (SSTA) that can be used in laser optical trapping and sperm motility studies. The algorithm creates a region of interest (ROI) centered about a sperm selected by the user. SSTA contrast enhances the ROI image and implements a modified four-class thresholding method to extract the tracked sperm as it transitions in and out of focus. The nearest neighbor method is complemented with a speed-check feature to aid tracking in the presence of additional sperm or other particles. SSTA has a collision-detection feature for real or perceived collision or near-miss cases between two sperm. Subsequent postcollision analysis employs three criteria to distinguish the tracked sperm in the image. The efficacy of SSTA is validated through examples and comparisons to commercially available computer-aided sperm tracking systems.
Sperm cells from a domestic dog were treated with oxacarbocyanine DiOC2(3), a ratiometrically-encoded membrane potential fluorescent probe in order to monitor the mitochondria stored in an individual sperm's midpiece. This dye normally emits a red fluorescence near 610 nm as well as a green fluorescence near 515 nm. The ratio of red to green fluorescence provides a substantially accurate and precise measurement of sperm midpiece membrane potential. A two-level computer system has been developed to quantify the motility and energetics of sperm using video rate tracking, automated laser trapping (done by the upper-level system) and fluorescent imaging (done by the lower-level system). The communication between these two systems is achieved by a networked gigabit TCP/IP cat5e crossover connection. This allows for the curvilinear velocity (VCL) and ratio of the red to green fluorescent images of individual sperm to be written to the hard drive at video rates. This two-level automatic system has increased experimental throughput over our previous single-level system (Mei et al., 2005) by an order of magnitude.
This study examines the use of optical trapping as a quantitative measure of sperm motility. The effects of laser trap duration and laser trapping power on sperm motility are described between sperm swimming force, swimmimg speed, and speed of progression (SOP) score. Sperm (SOP scores of 2–4) were trapped by a continuous-wave 1064 nm single-point gradient laser trap. Trap duration effects were quantified for 15, 10, and 5 seconds at 420 mW laser power. Laser power effects were quantified at powers of 420 mW, 350 mW, 300 mW, and 250 mW for five seconds. Swimming force, swimming speed, and SOP score relationships were examined at a trap duration and trapping power shown to minimally affect sperm motility. Swimming forces were measured by trapping sperm and subsequently decreasing laser power until the sperm escaped the trap. Swimming trajectories were calculated by custom-built software, and SOP scores were assigned by three qualified sperm scoring experts. A ubiquitous class of sperm were identified that swim with relatively high forces that are uncorrelated to swimming speed. It is concluded that sperm swimming forces measured by optical trapping provide new and valuable quantitative information to assess sperm motility.
The purpose of this study is to seek a correlation between the swimming forces of sperm, their swimming speed and the speed of progression score (SOP), which is given to them by the generally applied subjective 1 - 5 system. This study also examines the effects of length of exposure to the laser trap and laser power on sperm motility. Sperm with SOPs of 2-4 were trapped by a continuous wave 1064 nm single point gradient laser trap. To study trap duration effects, sperm were trapped for fifteen, ten and five seconds at 420mW laser power. To study laser power effects within the trap, powers of 420mW, 350mW, 300mW, and 250mW were applied for a constant duration (5 seconds). The correlations between sperm swimming force, swimming speed and speed of progression were examined at a trap duration and trapping power that were statistically shown to not significantly affect sperm motility. Swimming forces were measured by trapping sperm and subsequently decreasing laser power until the sperm were capable of escaping the trap (escape laser power is directly proportional to swimming force). Swimming speeds were calculated by custom built software, and speed of progression scores were assigned in a blind study by fertility experts examining offline video of the experiments. Sperm swimming force measurements by optical trapping may be a method to quantitatively measure sperm vitality that augments currently used methods.
As a powerful and noninvasive tool, laser trapping has been widely applied for the confinement and physiological study of biological cells and organelles. Researchers have used the single spot laser trap to hold individual sperm and quantitatively evaluated the motile force generated by a sperm. Early studies revealed the relationship between sperm motility and swimming behavior and helped the investigations in medical aspects of sperm activity. As sperm chemotaxis draws more and more interest in fertilization research, the studies on sperm-egg communication may help to explain male or female infertility and provide exciting new approaches to contraception. However, single spot laser trapping can only be used to investigate an individual target, which has limits in efficiency and throughput. To study the chemotactic response of sperm to eggs and to characterize sperm motility, an annular laser trap with a diameter of several hundred microns is designed, simulated with ray tracing tool, and implemented. An axicon transforms the wavefront such that the laser beam is incident on the microscope objective from all directions while filling the back aperture completely for high efficiency trapping. A trapping experiment with microspheres is carried out to evaluate the system performance. The power requirement for annular sperm trapping is determined experimentally and compared with theoretical calculations. With a chemo-attractant located in the center and sperm approaching from all directions, the annular laser trapping could serve as a speed bump for sperm so that motility characterization and fertility sorting can be performed efficiently.
The purpose of this study is to determine the effects of the working distance on the accuracy of confocal scanning laser tomography using the Heidelberg Retina Tomograph II. Twenty eyes of normal patients were imaged and the topographies of the retinal surfaces were recorded. Each eye was imaged first at the optimum working distance,
establishing the baseline exam, and then re-imaged at four different working distances (one at a shorter distance than optimum, three more at longer distances than optimum, variation done in 2 mm increments). The recorded data at various working distances was compared to the baseline data. The deviation from the baseline was compared to the
normal standard deviation for the instrument reported in the literature. Data is within the normal standard deviation when staying between -2 mm and +4 mm of optimum working distance. Some stereometric parameters vary greater than the normal standard deviation if working distance is more than +4 mm from optimum. To minimize error in recorded data, the operator of the Heidelberg Retinal Tomograph II should image the patient’s eye between -2 mm and
+4 mm of optimum working distance. Staying in this range should provide results that vary within the normal standard deviation.