Laser-based techniques offer powerful tools for studying the complex cellular mechanisms underlying neurodegeneration and neuroprotection. Laser scissors enable the precise induction of axonal injury, allowing investigation into axonal degenerative processes and evaluation of potential neuroprotective treatments. Laser scissors can also be utilized to kill a single cell and monitor the response in nearby astrocytes. For example, using this method, we found that a single-cell death can be disruptive to spontaneous calcium activity in surrounding cells. Additionally, focused lasers can be utilized to induce cavitation bubbles that produce shockwaves and subject cells to conditions like those experienced during a blast-induced traumatic brain injury. This allows us to have a window into the immediate cellular responses to traumatic brain injury and investigate potential mechanisms that may be targeted to mitigate damage. Laser-induced shockwaves have allowed us to study calcium signaling in response to nearby injury and determine that the immediate calcium response differs between cells that die and stay alive.
Neuronal responses to injury are of interest to the development of methods to mitigate damage and stimulate repair. We utilized a single pulse from a 1030nm laser to create a laser induced shockwave (LIS) to subject neuronal cells to injury and compare the effects of injury on neurons from an Alzheimer’s Disease (AD) mouse model and wild type (WT) mice. We found differences in the calcium response to LIS in AD versus WT neurons. Additionally, we found that LIS induced cell death led to a calcium elevation which differed from that in cells that stayed alive. Therefore, the calcium response can be utilized to separate dead cells from live cells.
Axonal degeneration is a key component of neurodegenerative diseases such as Huntington’s disease, Alzheimer’s disease, and amyotrophic lateral sclerosis (ALS). (NAm), an NAD+ precursor, has long since been implicated in axonal protection and reduction of degeneration. On the other hand, hydrogen peroxide (H2O2) has been implicated in oxidative stress and axonal degeneration. The effects of laser-induced axonal damage in wild-type (WT) and Huntington’s disease(HD) mouse dorsal root ganglion neurons (DRGs) treated with NAm or H2O2 were investigated and the cell body width, axon width, axonal strength, and axon shrinkage post laser-induced injury were measured. We found that HD mouse DRGs have increased strength against laser damage compared to wild-type DRGs. We additionally found that treatment with NAm reduces the neuronal strength against laser damage in both WT and HD DRGs. Interestingly, when comparing HD DRGs treated with H2O2 and WT DRGs treated with H2O2, we found that treatment with H2O2 reduced the time required for the RoboLase laser system to cut through HD DRGs. We additionally found that both NAm and H2O2 treatments resulted in morphological changes in both WT and HD DRG cell bodies, respectively. We did not find any difference in shrinkage across the models. Ultimately, our results suggest that H2O2 at the same concentration may have less damaging effects on WT neurons than previously expected. Our results additionally indicate that higher concentrations of NAm, previously deemed to be safe, may have a neurotoxic effect rather than an axonal protective effect on HD and WT DRGs.
Single-cell green algae (C. Reinhardtii) is a key model organism to study ciliogenesis. Cilia have important roles in sensory signaling pathways and in clearing the airways of mucus and dirt in multiple systems of the human body. As cilia are found on most eukaryotic cells, defects in ciliogenesis result in many symptoms and disorders. We are testing the hypothesis that when a flagellum is removed, the long flagellum shrinks because it is competing with the shorter regrowing flagellum. We used a 780-nm 200-fs laser to perform laser ablation to amputate one of two flagella on wildtype and mutant algae. Fla3 and Fla10 mutants were altered to inhibit the KAP kinesin motor that drives the intraflagellar transport (IFT) pathway. Impaired IFT pathways would demonstrate a lag in response to flagellar length equalization and a reduced disassembly rate. Quantified images following the long flagellum for 20 min post-ablation demonstrate a delayed disassembly rate in the Fla3 mutant compared to wildtype; Fla10 was inconclusive. Therefore, it was concluded that the proper function of KAP motor protein serves a significant role in length control of cilia. In the future, we will compare the assembly rates of flagellar regrowth for the wildtype and mutants.
Astrocytes in the brain migrate to sites of injury where they can take up damaging molecules extruded from injured cells to protect neurons. The astrocytic response to cell death is critical to our understanding of ways to mitigate secondary injury from a traumatic brain injury(TBI). We previously showed that a laser could be used to induce a single cell death (photolysis) in order to monitor the surrounding astrocytic response. We found that photolysis leads to a calcium transient in surrounding astrocytes. Here we show that cells treated with the internal calcium chelator BAPTA-AM do not exhibit a transient. Similarly, cells whose endoplasmic reticulum (ER) has been depleted through blocking of the SERCA pump do not show a calcium increase. Cells treated with EGTA to chelate external calcium showed no statistical significance when compared to cells in regular medium with calcium. Therefore, it is concluded that the ER stores are largely responsible for the cytosolic calcium transient.
Laser induced shockwave (LIS) can be utilized to subject neuronal cells to conditions similar to those occurring during a blast induced traumatic brain injury. We utilized a 532nm Coherent Flare laser to induce a shockwave near cells which had been transfected with a FRET calcium biosensor (D3CPV) so that we could monitor the immediate cellular responses. Our shockwave system was characterized with a high-speed camera to monitor cavitation bubble dynamics and calculate the shear forces cells were subjected to. We found that we could induce forces which have been previously shown to induce injury. Using both phase and fluorescence microscopy we monitored the effects of shear on our cells. We found that at distances up to 120 microns from the laser focal point cells experienced shears greater than 10kPa. At those distances cell fragmentation was observed. Cells that survived and expressed the FRET biosensor demonstrated an immediate calcium elevation irrespective of extracellular or cytoplasmic calcium concentration. Cells recovered to pre-shockwave calcium levels within ~30s. In conclusion, LIS can be utilized to simultaneously monitor the neuronal response to shear stress and nearby cell death or injury.
Traumatic Brain Injury (TBI) occurs when an external force injures the brain. While clinical outcomes of TBI can vary widely in severity, few mechanisms of neurodegeneration following TBI have been identified for treatment. Understanding mechanotransduction in cells is key to understanding cellular response to injury. This has been previously studied using a variety of optical techniques such as laser tweezers, laser ablation, and others. We propose a model utilizing photodisruption for studying the early pathogenesis of TBI in primary neuron cultures by generating laser-induced shockwaves (LISs). Photodisruption allows for the generation of spatiotemporally defined shear stress against cells. The shear stress exerted by the shockwave is between 0 - 50 kPa depending on the distance from the shockwave epicenter. Cells typically situated at a distance from the epicenter of 50 m undergo necrosis while viability is preserved for those located at a distance of 100 m. An optical system was developed that allows single cells to be selectively studied in response to LISs. Approximate timescales of each of the effects culminating in shockwave generation span several orders of magnitude from nanoseconds to milliseconds. Thus, our system utilizes Pockels cells — a high-speed, electro-optical shutter — to capture shockwave dynamics. The force measurement system is characterized by imaging stages over the period of cavitation then, violent expansion and collapse of microbubbles responsible for shockwave generation. Here, we visualize LISs and observe subsequent, morphological responses elicited by cells under a range of forces generated from optical breakdown.
Traumatic Brain Injury (TBI) occurs when an external force injures the brain. While clinical outcomes of TBI can vary widely in severity, few mechanisms of neurodegeneration following TBI have been identified for treatment. We propose a model for studying TBI using laser-induced shockwaves (LISs). An optical system was developed that allows single cells to be studied in response to LISs. Our system utilizes an optically-coupled force measurement component that allows for the visualization of shockwave dynamics. Here, the force measurement system is characterized by imaging stages over the period of violent expansion and collapse of microbubbles responsible for shockwave generation.
Region specific DNA breaks can be created in single cells using laser light that damages DNA but does not directly generate reactive oxygen species (ROS). We have examined the cellular response to directly generated DNA breaks in single cells. Using a combination of ROS specific dyes and oxidase inhibitors we have found that the oxidase and chromatin remodeling protein Lysine demethylase I (LSD1) generates detectable ROS as a byproduct of its chromatin remodeling activity during the initial DNA damage response. ROS is produced at detectable amounts primarily within the first 3 minutes post irradiation. LSD1 activity has been previously associated with transcriptional regulation therefore these findings have implications for regulation of gene expression following DNA damage particularly in cells with altered redox states.
Traumatic brain injury (TBI) represents a major treatment challenge in both civilian and military medicine; on the cellular level, its mechanisms are poorly understood. As a method to study the dysfunctional repair mechanisms following injury, laser induced shock waves (LIS) are a useful way to create highly precise, well characterized mechanical forces. We present a simple model for TBI using laser induced shock waves as a model for damage. Our objective is to develop an understanding of the processes responsible for neuronal death, the ways in which we can manipulate these processes to improve cell survival and repair, and the importance of these processes at different levels of biological organization. The physics of shock wave creation has been modeled and can be used to calculate forces acting on individual neurons. By ensuring that the impulse is in the same regime as that occurring in practical TBI, the LIS model can ensure that in vitro conditions and damage are similar to those experienced in TBI. This model will allow for the study of the biochemical response of neurons to mechanical stresses, and can be combined with microfluidic systems for cell growth in order to better isolate areas of damage.
Axonal injury and stress have long been thought to play a pathogenic role in a variety of neurodegenerative diseases. However, a model for studying single-cell axonal injury in mammalian cells and the processes of repair has not been established. The purpose of this study was to examine the response of neuronal growth cones to laser-induced axonal damage in cultures of embryonic rat hippocampal neurons and induced pluripotent stem cell (iPSC) derived human neurons. A 532-nm pulsed Nd:YVO4 picosecond laser was focused to a diffraction limited spot at a precise location on an axon using a laser energy/power that did not rupture the cell membrane (subaxotomy). Subsequent time series images were taken to follow axonal recovery and growth cone dynamics. After laser subaxotomy, axons thinned at the damage site and initiated a dynamic cytoskeletal remodeling process to restore axonal thickness. The growth cone was observed to play a role in the repair process in both hippocampal and iPSC-derived neurons. Immunofluorescence staining confirmed structural tubulin damage and revealed initial phases of actin-based cytoskeletal remodeling at the damage site. The results of this study indicate that there is a repeatable and cross-species repair response of axons and growth cones after laser-induced damage.
In this study, we investigated the effects of size and surrounding media viscosity on trapping of microspheres. A continuous wave ytterbium fiber laser with a 1064 nm wavelength was used to create an optical tweezers system for optical manipulation experiments. Briefly, the system consisted of an inverted microscope, and a 100X 1.4 NA oil immersion objective through which the laser beam converged to form the optical trap. The laser beam was collimated, steered, and coupled to the microscope through the epifluorescence microscope port. The laser power at the trap focal spot was determined by measuring the input power at the back aperture of the objective multiplied by the objective transmission factor at 1064 nm measured by a modified dual objective method. Polystyrene microspheres varying in diameter from 5 to 15 microns were suspended in liquid media in glass bottom petri dishes prior to trapping experiments. The microspheres were trapped at different trapping powers, and fluidic viscous drag forces where applied to the optically trapped microspheres by driving a computer controlled 2D motorized microscope stage at known velocities. The drag forces were calculated at the point that the microspheres fell out of the trap, based on the Stokes equation for flow around spheres. The data show a linear relationship between trapping force and trap power within the range of the microsphere diameters and media viscosity values used. The work includes calculation of the dimensionless trap efficiency coefficient (Q) at 1064 nm wavelength and the corresponding effects of media viscosity and microsphere size on (Q).
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.
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.
In this study, 532-nm picosecond and 800-nm femtosecond lasers are used in combination with fluorescently labeled tubulin to further elucidate microtubule depolymerization and the effect lasers may have on the resulting depolymerization. Depolymerization rates of targeted single microtubules are dependent on location with respect to the nucleus. Microtubules located near the nucleus exhibit a significantly faster depolymerization rate when compared to microtubule depolymerization rates near the periphery of the cell. Microtubules cut with the femtosecond laser depolymerize at a slower rate than unirradiated controls (p=0.002), whereas those cut with the picosecond laser depolymerize at the same rate as unirradiated controls (p=0.704). Our results demonstrate the ability of both the picosecond and femtosecond lasers to cut individual microtubules. The differences between the two ablation results are discussed.
In previous studies we have shown that the second harmonic 532 nm, from a picosecond frequency doubled Nd:YAG laser, can cleanly and selectively disrupt spindle fiber microtubules in live cells (Botvinick et al 2004, Biophys. J. 87:4303-4212). In the present study we have ablated different locations and amounts of the metaphase mitotic spindle, and followed the cells in order to observe the fate of the irradiated spindle and the ability of the cell to continue through mitosis. Cells of the rat kangaroo line (PTK2) were stably transfected by ECFP-tubulin and, using fluorescent microscopy and the automated RoboLase microscope, (Botvinick and Berns, 2005, Micros. Res. Tech. 68:65-74) brightly fluorescent individual cells in metaphase were irradiated with 0.2447 nJ/micropulse corresponding to an irradiance of 1.4496*10^7 J/(ps*cm^2) . Upon irradiation the exposed part of the mitotic spindle immediately lost fluorescence and the following events were observed in the cells over time: (1) immediate contraction of the spindle pole towards the cut, (2) recovery of connection between pole and cut microtubule, (3) completion of mitosis. This system should be very useful in studying internal cellular dynamics of the mitotic spindle.
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.
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