A system has been developed that allows for optical and fluidic manipulation of gametes. The optical manipulation is performed by using a single-point gradient trap with a 40× oil immersion PH3 1.3 NA objective on a Zeiss inverted microscope. The fluidic manipulation is performed by using a custom microfluidic chamber designed to fit into the short working distance between the condenser and objective. The system is validated using purple sea urchin Strongylocentrotus purpuratus gametes and has the potential to be used for mammalian in vitro fertilization and animal husbandry.
The purpose of this study is to analyze human sperm motility and energetics in media with different viscosities. Multiple experiments were performed to collect motility parameters using customized computer tracking software that measures the curvilinear velocity (VCL) and the minimum laser power (Pesc) necessary to hold an individual sperm in an optical trap. The Pesc was measured by using a 1064 nm Nd:YVO4 continuous wave laser that optically traps motile sperm at a power of 450 mW in the focused trap spot. The VCL was measured frame by frame before trapping. In order to study sperm energetics under different viscous conditions sperm were labeled with the fluorescent dye DiOC6(3) to measure membrane potentials of mitochondria in the sperm midpiece. Fluorescence intensity was measured before and during trapping. The results demonstrate a decrease in VCL but an increase in Pesc with increasing viscosity. Fluorescent intensity is the same regardless of the viscosity level indicating no change in sperm energetics. The results suggest that, under the conditions tested, viscosity physically affects the mechanical properties of sperm motility rather than the chemical pathways associated with energetics.
This study combines microfluidics with optical microablation in a microscopy system that allows for high-throughput manipulation of oocytes, automated media exchange, and long-term oocyte observation. The microfluidic component of the system transports oocytes from an inlet port into multiple flow channels. Within each channel, oocytes are confined against a microfluidic barrier using a steady fluid flow provided by an external computer-controlled syringe pump. This allows for easy media replacement without disturbing the oocyte location. The microfluidic and optical-laser microbeam ablation capabilities of the system were validated using surf clam (Spisula solidissima) oocytes that were immobilized in order to permit ablation of the 5 μm diameter nucleolinus within the oocyte nucleolus. Oocytes were the followed and assayed for polar body ejection.
In two previous studies we have conducted combined laser subcellular microsurgery and optical trapping
on chromosomes in living cells1, 2. In the latter study we used two separate microscopes, one for the trap
and one for the laser scissors, thus requiring that we move the cell specimen between microscopes and
relocate the irradiated cells. In the former paper we combined the 1064 nm laser trap and the 532 nm laser
scissors into one microscope. However, in neither study did we have multiple traps allowing for more
flexibility in application of the trapping force. In the present paper we describe a combined laser scissors
and tweezers microscope that (1) has two trapping beams (both moveable via rapid scanning mirrors (FSM-
300, Newport Corp.), (2) uses a short pulsed tunable 200 fs 710-990 nm Ti:Sapphire laser for laser
microsurgery, and (3) also has the option to use a 337 nm 4 ns UV laser for subcellular surgery. The two
laser tweezers and either of the laser ablation beams can be used in a cell surgery experiment. The system
is integrated into the robotic-controlled RoboLase system3. Experiments on mitotic chromosomes of rat
kangaroo PTK2 cells are described.
Relative concentrations of Streptococcus mutans and Streptococcus sanguis are important parameters in the study of dental caries, but current methods of measuring these concentrations are time consuming and prone to inaccuracies. We investigate the use of Raman spectroscopy for measuring relative concentrations of these two bacterial species in solid mixtures. To our knowledge, this is the first time Raman spectroscopy has been used to analyze bacterial mixtures rather than to identify the species of a pure colony. Mixtures of the two streptococcal species in various ratios are measured for 200 s using a home-built Raman microscope. Spectral correlations with bacterial content were identified via partial least-squares analysis. The relative concentrations of S. mutans in subsequent samples are predicted with a root mean squared error below 5%. In clinical plaque samples, this sort of accuracy would enable discrimination between normal and dangerously elevated levels of S. mutans. Samples with and without salivary proteins are predicted with equal accuracy. This result shows the potential of Raman spectroscopy for analyzing mixed populations of bacteria, such as those that occur in oral plaques.
Raman spectroscopy has been employed to measure the varying concentrations of two oral bacteria in simple mixtures. Evaporated droplets of centrifuged mixtures of Streptococcus sanguis and Streptococcus mutans were analyzed via Raman microspectroscopy. The concentration of s. sanguis was determined based upon the measured Raman spectrum, using partial least squares cross-validation, with an r2 value of 0.98.