2.1.

Specimen

Semen samples collected from several domestic dogs were cryogenically frozen according to a standard protocol.^{16, 17} Studies on human sperm have shown that properly freezing, storing, and thawing sperm has no significant effect on escape force.¹⁸ Furthermore, we compared frozen-thawed and fresh dog sperm from the same semen sample and found that the swimming speed and escape laser power distributions were statistically the same (swimming speed: $P>0.06$ ; escape power: $P>0.9$ , data not shown). Therefore, frozen-thawed semen samples in this study are considered comparable to fresh samples.

For each experiment, a sperm sample is thawed in a water bath $(37°\mathrm{C})$ for approximately $1\min$ and its contents are transferred to an Eppendorf centrifuge tube. The sample is centrifuged at $2000\mathrm{rpm}$ for $10\min$ (the centrifuge tip radius is $8.23\mathrm{cm}$ ). The supernatant is removed and the remaining sperm pellet is suspended in $1\mathrm{mL}$ of pre-warmed media [ $1\mathrm{mg}$ of bovine serum albumin (BSA) per $1\mathrm{mL}$ of Biggers, Whittens, and Whittingham (BWW), osmolality of $270\text{to}300\mathrm{mmol}∕\mathrm{kg}$ water,¹⁹ pH of 7.2 to 7.4]. Note that this media is non-capacitating, as it has a low concentration of bicarbonate²⁰ $\left(4\mathrm{mM}\right)$ . Therefore, the sperm do not achieve hyperactivity.

To monitor the voltage potential across the inner membrane of the mitochondria, sperm are labeled with $\mathrm{Di}\mathrm{O}{\mathrm{C}}_2\left(3\right)$ ( $3,3^{\prime }$ -dithyloxacarbocyanine, $30\mathrm{nM}$ final dye concentration, Molecular Probes, Invitrogen Corp., Carlsbad, California). $\mathrm{Di}\mathrm{O}{\mathrm{C}}_2\left(3\right)$ is a cationic cyanine dye that primarily accumulates in the mitochondria of a cell in response to the electrochemical proton gradient, or MP. The probe emits both a red and green fluorescence. The ratiometric parameter (red/green intensity) is a size-independent measure of MP, as the green fluorescence varies with size and red fluorescence is dependent²¹ on both size and MP. After the dye is added, the cells are incubated for $20\min$ in a $37°\mathrm{C}$ water bath and then centrifuged for $10\min$ $\left(2000\mathrm{rpm}\right)$ . The pellet is suspended in the media by “flicking” the tube according to the protocol for the MitoProbe assay kit (Invitrogen Corp.) for flow cytometry. To test sensitivity of both the probe and the system to changes in MP (see Sec. 2.4), an aliquot of sperm are exposed to the proton ionophore CCCP (carbonyl cyanide 3-chlorophenylhydrazone, $50\mu \mathrm{M}$ final concentration, Molecular Probes, Invitrogen Corp., Carlsbad, California), which is known^{7, 22} to decrease the magnitude of the MP. CCCP and $\mathrm{Di}\mathrm{O}{\mathrm{C}}_2\left(3\right)$ are added to the sperm simultaneously.

Final dilutions of $\sim \mathrm{30,000}\text{sperm}∕\mathrm{mL}$ of media are used in the experiments. The sperm dilution is loaded into a $3\mathrm{mL}$ Rose tissue culture chamber and mounted into a microscope stage holder according to previously described methods.²³ The sample is kept at $37°\mathrm{C}$ using an air curtain incubator (NEVTEK, ASI 400 Air Stream Incubator, Burnsville, Virginia). A thermocouple is attached to the Rose chamber to ensure temperature stability.

2.2.

Hardware, Software, and Optical Design

The optical system, shown in Fig. 1a , is adapted after Nascimento, ¹⁰ A single point gradient trap is generated using an $\mathrm{Nd}:\mathrm{Y}\mathrm{V}{\mathrm{O}}_4$ continuous wave $1064\text{-}\mathrm{nm}$ wavelength laser (Spectra Physics, BL-106C, Mountain View, California), coupled into a Zeiss Axiovert S100 microscope equipped with a phase III, $40\times$ , 1.3 numerical aperture (NA), oil immersion objective (Zeiss, Thornwood, New York). The laser power in the specimen plane is attenuated by rotating the polarizer, which is mounted in a stepper-motor-controlled rotating mount (Newport Corporation, Model PR50PP, Irvine, California).

Fig. 1

(a) Optical schematic showing the optical components used to generate and control the laser tweezers and (b) imaging setup showing the illumination sources, filters, and cameras used to image the sperm in both phase contrast and fluorescence.

The imaging setup, shown in Fig. 1b, was adapted after Mei ¹² Two dual video adapters are used to incorporate the laser into the microscope and simultaneously image the sperm in phase contrast and fluorescence. The laser beam enters the side port of the first dual video adapter and is transmitted to the microscope. A filter (Chroma Technology Corp., Model E700SP-2P, Rockingham, Vermont) is used to prevent back reflections of IR laser light from exiting the top port of the adapter but allow reflected visible light coming from the specimen to pass to the second video adapter. The specimen is viewed in phase contrast using red light filtered from the halogen lamp (Chroma Technology Corp., Model D680/60 X) and in fluorescence using the arc lamp (Zeiss FluoArc). The fluorescence filter cube contains an HQ $500∕20\text{-}\mathrm{nm}$ excitation filter and a dichroic beamsplitter with a $505\text{-}\mathrm{nm}$ cut-on wavelength. The second dual video adapter attached to the top port of the first video adapter uses a filter cube to separate the phase information (reflects $>670\mathrm{nm}$ ) from the fluorescence (transmits $500\text{to}670\mathrm{nm}$ ). The phase contrast images are filtered through a filter (Chroma Technology Corp., Model HQ 675/50M) and acquired by a CCD camera (Cohu, Model 7800, San Diego, California, operating at $40\text{frames}∕\mathrm{s}$ ) coupled to a variable zoom lens system (0.33 to $1.6\times$ magnification) to increase the field of view. For the fluorescent images, a Dual-View system (Optical-Insights, Tucson, Arizona) splits the red and green fluorescent light emitted by the specimen to produce a copy of the image for each color. Fluorescent emission filters are placed in this emission-splitting system (green fluorescence emitter: HQ $535∕40\text{-}\mathrm{nm}$ M filter; red fluorescence emitter: HQ $605∕50\text{-}\mathrm{nm}$ M filter, Chroma Technology Corp.). The Dual-View system is coupled to a digital camera (Quantix 57, Roper Scientific Inc., Tucson, Arizona) that captures the fluorescent images.

The hardware and software to perform the experiments in this paper are described in greater detail elsewhere.¹⁵ Briefly, two computers are networked together. An upper-level computer that acquires and displays the images from the Cohu CCD is responsible for tracking and trapping the sperm of interest. The lower-level computer is prompted by the upper-level computer to acquire the fluorescent images of the sperm’s mitochondria from the Quantix CCD. From the image, the lower level computer calculates the ratio (red/green) value.

Once the user selects the sperm of interest, the upper level computer tracks the sperm and calculates the VCL (in micrometers per second) in real time. Note that other motility parameters including lateral head movement, straight line velocity, and smoothed-path velocity that are typical of a CASA system are also calculated.^{10, 13, 14} Nascimento found that several of these parameters had near equal influence on the variability in the data set.¹⁰ Therefore, for the purpose of this paper, only VCL is used to describe sperm swimming speed, as it is a more comprehensive parameter, accounting for both forward progression and lateral head movement.¹¹ During this tracking phase, the microscope stage moves the sperm to the center of the field of view if it nears the edge. In addition, the sperm is relocated to a defined $(x,y)$ coordinate every second and a command is sent to the lower-level computer to acquire a fluorescent image. For track-and-trap experiments, the sperm is relocated to the laser trap coordinates after being tracked and fluorescently imaged for $5\mathrm{s}$ . Once the sperm is successfully trapped by the laser tweezers, the lower-level computer continuously acquires fluorescent images. Laser power is either kept constant (see Sec. 2.5) or is attenuated (see Sec. 2.6). If the power is attenuated, the power at which the sperm is capable of escaping the trap (Pesc, in milliwatts) is recorded by the upper-level computer. The sperm is tracked and fluorescently imaged for an additional $5\mathrm{s}$ once it is released from or escapes the optical trap. Data for each tracked sperm, including $(x,y)$ trajectory coordinates in the field of view, stage movement, instantaneous VCL, and average VCL, for each time point is saved to a file on the upper-level computer. The fluorescent data—including the average, maximum, and minimum ratio values—for each image are saved to a file on the lower-level computer.

2.3.

Effect of Probe on Sperm Motility

The effects of $\mathrm{Di}\mathrm{O}{\mathrm{C}}_2\left(3\right)$ on sperm motility are assessed. The swimming speed (VCL in micrometers per second) distribution of sperm exposed to $\mathrm{Di}\mathrm{O}{\mathrm{C}}_2\left(3\right)$ is compared to that of sperm not exposed to the probe (control). Sperm from each group are analyzed during the same time intervals. During the first time interval, both groups are viewed in phase contrast microscopy only. During the second time interval, again, both groups are viewed in phase contrast microscopy. However, the test group exposed to $\mathrm{Di}\mathrm{O}{\mathrm{C}}_2\left(3\right)$ are also illuminated with excitation light $\left(500\mathrm{nm}\right)$ from the arc lamp.

2.4.

Sensitivity to Changes in MP

The ability to measure changes in MP as well as the ability to detect and report changes in MP is tested. The test sperm group are exposed to both CCCP $\left(50\mu \mathrm{M}\right)$ and $\mathrm{Di}\mathrm{O}{\mathrm{C}}_2\left(3\right)$ $\left(30\mathrm{nM}\right)$ , whereas the control sperm group are labeled only with $\mathrm{Di}\mathrm{O}{\mathrm{C}}_2\left(3\right)$ $\left(30\mathrm{nM}\right)$ . Sperm are loaded onto the microscope and tracked for $10\mathrm{s}$ . A fluorescent image is acquired every second. The ratio value distributions of the test and control groups are compared.

2.5.

Track, Trap (Constant Power and Constant Duration), and Fluorescently Image

Sperm labeled with $\mathrm{Di}\mathrm{O}{\mathrm{C}}_2\left(3\right)$ are tracked and trapped under constant power $\left(460\mathrm{mW}\right)$ for a constant duration $\left(90\mathrm{s}\right)$ . For each sperm analyzed, fluorescent images are acquired approximately once every second during the $5\mathrm{s}$ prior to and after trapping and acquired continuously once the sperm is in the trap. Effects of prolonged exposure to the laser tweezers on MP and swimming speed (VCL in micrometers per second) are assessed.

2.6.

Track, Trap (Decaying Laser Power), and Fluorescently Image

Sperm labeled with $\mathrm{Di}\mathrm{O}{\mathrm{C}}_2\left(3\right)$ are tracked and trapped under decaying power. Again, for each sperm, fluorescent images are acquired once every second during the $5\mathrm{s}$ prior to and post trapping and acquired continuously once the sperm is in the trap. Examples of the various sperm responses to the optical trap are described.

3.1.

Effect of Probe on Sperm Motility

The swimming speed (VCL in micrometers per second) distribution of sperm cells exposed to $\mathrm{Di}\mathrm{O}{\mathrm{C}}_2\left(3\right)$ is compared to that of the control sperm using the Wilcoxon paired-sample test (the distributions are found not to be Gaussian, thus requiring the non-parametric test). The VCL distributions are found to be statistically equal, even when the probe is activated by the arc lamp (without arc lamp illumination; $P>0.3$ , $N_{\text{control}}=24$ , $N_{\mathrm{Di}\mathrm{O}{\mathrm{C}}_2\left(3\right)}=37$ ; with arc lamp illumination; $P>0.2$ , $N_{\text{control}}=23$ , $N_{\mathrm{Di}\mathrm{O}{\mathrm{C}}_2\left(3\right)}=19$ ). Thus, $\mathrm{Di}\mathrm{O}{\mathrm{C}}_2\left(3\right)$ does not adversely affect sperm motility.

3.2.

Sensitivity to Changes in MP

Since the probe used in this study is typically applied in flow cytometry experiments, we wanted to verify that our custom system and method of analysis is sensitive to changes in MP. We also wanted to verify that this probe reports changes in MP. Figure 2 shows the ratio value over a $10\text{-}\mathrm{s}$ interval for sperm from the test group (with CCCP) and control group (without CCCP). The figure demonstrates that CCCP does indeed cause a decrease in MP and that both the probe and the system are capable of reporting such a decrease. The average ratio value of sperm exposed to CCCP ( $3.74\pm 0.75$ , $N_{\mathrm{Di}\mathrm{O}{\mathrm{C}}_2\left(3\right)}=33$ ) versus that of the control sperm ( $5.82\pm 0.41$ , $N_{\text{control}}=36$ ) is found to be statistically significantly different $(P⪡0.001)$ using the Student’s $T$ test (distributions are found to be Gaussian). The velocity of each sperm was also measured. The average VCL value of sperm exposed to CCCP $(67.15\pm 20.77)$ versus that of the control sperm $(70.39\pm 24.70)$ is found to be statistically the same $(P>0.56)$ using the Student’s $T$ test (distributions are found to be Gaussian).

Fig. 2

Effects of CCCP on mitochondrial membrane potential. The ratio value (red/green fluorescence) is plotted against time (in seconds). Ratio values are measured over a 10-s interval for test sperm group (with CCCP, in magenta) and control sperm group (without CCCP, in black). Each track represents an individual sperm.

3.3.

Track, Trap (Constant Power and Constant Duration), and Fluorescently Image

Figure 3 shows the ratio value prior to trapping, during trap, and after trapping plotted over time for two different sperm. For the sperm in Fig. 3a, there is an overall decline in ratio value over time as the sperm is held in the trap. Once released from the optical trap, the sperm’s ratio value does increase, however, it does not fully recover within $5\mathrm{s}$ to the original value it was prior to being trapped. Similarly, the sperm’s swimming speed, VCL, does not recover to its pre-trapping value. For the sperm in Fig. 3b, there is a slight decrease in ratio value while the sperm is in the trap. Again, neither swimming speed nor ratio value fully recover post trap to the pre-trapping values. A previous study had shown that trapping sperm for $15\mathrm{s}$ at a constant power of $420\mathrm{mW}$ in the focal volume had a negative effect on sperm motility.¹⁰ The results reported here are consistent with those findings.

Fig. 3

Track, trap (constant power, constant duration) and fluorescently image. The ratio value (red/green) is plotted against time (in seconds) for the three different phases: prior to trapping, during trap, and after trapping. Both the average ratio value (AveRat) and VCL prior to (pre) and after trapping (post) are inset in the figure for each sperm (a) and (b). A trap duration of $90\mathrm{s}$ has a negative effect on sperm motility and energetics. (Color online only.)

3.4.

Track, Trap (Decaying Laser Power), and Fluorescently Image

Pesc was plotted against VCL (data not shown) and showed the same positive correlation between the two parameters as found in previous studies¹⁰ (regressions applied to data sets found to be statistically equal, $P>0.2$ ). Figure 4 plots the ratio value over time for four sperm for the three different phases: prior to trapping, during trap, and after trapping. These four examples demonstrate the various responses the sperm have to the optical trap. The sperm in Fig. 4a did not escape the trap. After $10\mathrm{s}$ , the trapping power reaches the minimum $3.8\mathrm{mW}$ , at which point the trap turns off. This sperm’s VCL slightly increased after being trapped, but the average ratio value decreased. The sperm in Fig. 4b escaped the trap at $55\mathrm{mW}$ and had an approximate 18% increase in VCL after trapping. However, the average ratio value was nearly the same after trapping as it was prior to trapping. The sperm in Fig. 4c, although it escaped at a relatively high power, had a significant decrease in VCL, yet the average ratio value increased slightly. The sperm in Fig. 4d, which escaped the trap at $26\mathrm{mW}$ , also had a decrease in VCL and an increase in average ratio value.

Fig. 4

Track, trap (decaying laser power) and fluorescently image. The ratio value (red/green) is plotted against time (in seconds) for the three different phases: prior to trapping, during trap, and after trapping. Various escape powers and swimming speeds are represented by the four sperm. The average ratio value (AveRat) and VCL prior to (pre) and after trapping (post), as well as Pesc, are inset in the figure for each sperm (a) to (d). (Color online only.)