1.

Introduction

Over the past several decades, laser scissors have proven an invaluable tool for microdissection in cell and developmental biology.¹ Through their continual refinement, a high level of precision in single-cell manipulation has been achieved. Noteworthy cellular applications of optical scissors include the ablation of axons,² microtubules,^{3, 4} and chromosomes,^{5, 6, 7} as well as plasma membrane poration.⁸ Recently, optical scissors have seen extensive use in DNA damage repair research by exposing a linear path across the nucleus of interphase cells followed by the monitoring of DNA damage recognition and repair in the altered region.^{9, 10, 11, 12, 13, 14, 15}

Current approaches to laser-induced linear DNA damage consist primarily of moving the sample back and forth under a stationary focused laser beam via the microscope stage (motorized or manual), or moving the beam via a fast-scanning galvomirror (FSM) placed in the beam path (Fig. 1 ). In both cases of scanning, linear DNA damage is possible, but certain practical limitations exist when an area of damage that extends beyond the diffraction limited spot is desired. In cases where the microscope stage is moved, damage occurs to a greater degree on the edges of the linear path due to the lag time associated with changing the stage direction (when repeated paths over the same cell region are necessary), as well as when initiation and termination of stage movement are required. When an FSM is used to steer the beam, curvature at the edges of long linear cuts arises due to the steep angle to which the beam is reflected off the mirror surface.

Fig. 1

Schematic of line scissors and tweezers setup. POL: polarizer; BE: beam expander; M: folding mirror; ND: neutral density filter; DM1: dichroic mirror (reflects IR and transmits UV); RCL: rotatable cylindrical lens; SM: XY-scanning mirror; VAP: variable circular aperture; L1 and L2: 1:1 telescope; HBO: mercury lamp; EX: excitation filter; DM2: dichroic mirror (reflects UV and IR and transmits blue); DM3: dichroic mirror (reflects UV–blue and IR, but transmits green–red); MO: microscope objective; SC: sample chamber; CON: condenser; HAL: halogen lamp; EM: emission filter.

Additionally, uniformity of damage production across a linear target zone may be difficult to achieve by both the stage moving and the FSM approaches since both methods utilize trains of pulses to damage the target material. Since the focal plane of the diffraction limited spot is circular, regions along a linearly exposed path may be exposed to overlapping pulses [Fig. 2a ] or no pulses, all dependent on either the rate of the stage movement or the rate of the FSM movement. At though each can be minimized by tuning the distance between delivered pulses, minimization of one will result in exaggeration of the other. These variations make it difficult to compare damage in the exposed path from one experiment to the next.

Fig. 2

Control of line scissors dimension and orientation. (a) Illustration of the inherent variability in irradiance delivered to a linear ROI when using a diffraction-limited spot profile. The dark blue areas received greater than average total irradiance compared to the majority of the ROI, whereas the white areas bordered by light blue regions and dark lines represent areas that received little if any irradiance as compared to the majority of the ROI. (b) Intensity profile (along length) of the elliptically focused spot monitored by fluorescence; (c) line cuts on RBC in different radial orientations of the cylindrical lens; and (d) point and (e) controlled line cuts generated by varied restriction of the beam by the aperture. (Color online only.)

In an attempt to improve the quality of focused laser-induced damage in living cells, we have developed an optical system that incorporates a cylindrical lens in the beam path coupled to an automated electronically controlled rotating mount and an automated iris. This optical system stretches the beam into a linear pattern and provides precise control over the length and radial orientation of the exposure pattern. By shaping the laser beam into a uniform linear pattern, it is possible to achieve a higher throughput of exposed cells, with an increased dosimetric uniformity and accuracy.

2.

Materials and Method

3.

Results and Discussion

In order to determine the damage capabilities of the optical line scissors, the induction of DNA DSBs was assayed. Immediately following laser exposure, cells were fixed and immunostained for the phosphorylated H2AX $\left(\gamma \mathrm{H}2\mathrm{AX}\right)$ (Fig. 3 ). $\gamma \mathrm{H}2\mathrm{AX}$ was selected because it is a well-accepted marker for DNA DSBs in mammalian cells^{18, 19, 20} and produces prominent foci even where low levels of damage have occurred. Laser scanning confocal microscopic analysis of HeLa cells whose nuclei were exposed to the line-shaped scissors showed clear $\gamma \mathrm{H}2\mathrm{AX}$ focus formation confined to the beam profile at dosimetry levels similar to other methods of laser-induced DNA damage (Fig. 3). From the Z-stack fluorescence reconstruction, $\gamma \mathrm{H}2\mathrm{AX}$ fluorescence staining was observed over a cylinder with radius of $3\mu \mathrm{m}$ .

Fig. 3

DNA damage induced by line scissors. (a) Laser scanning confocal image of HeLa cells stained for $\gamma \mathrm{H}2\mathrm{AX}$ after 25, 50, and 100 pulse exposures. (b) XY, XZ, and YZ cross-sectional view of the DNA damage induced by the line scissors in a HeLa cell. (c) Composite image showing overlay of the fluorescence and phase images.

One caveat of $\gamma \mathrm{H}2\mathrm{AX}$ staining as a method of DSB detection is that $\gamma \mathrm{H}2\mathrm{AX}$ is known to spread from the actual site of damage to flanking regions of the genome as part of the repair pathway.²¹ Therefore, although $\gamma \mathrm{H}2\mathrm{AX}$ is useful as a probe to detect if the line scissors can induce DSBs, it lends itself to overestimation of the actual volume of damage induction. To more precisely resolve the zone of DNA damage, parallel experiments were conducted using antibodies against the DSB recognition factor Nbs1. Nbs1 is one of the first recognition factors recruited to DSB damage in mammalian cells and remains largely confined to the actual damage area as compared to $\gamma \mathrm{H}2\mathrm{AX}$ .^{21, 22} Confocal microscopy analysis of HeLa cells exposed to the line scissors and immunostained for Nbs1 showed a tight linear accumulation (with a cylinder with radius of $2\mu \mathrm{m}$ ) of antibody staining corresponding to the region of the linear beam profile exposure [Fig. 4a, 4b, 4c ]. It may further be noted that the spread of staining also depends on the intensity (or number of pulses) of the laser scissors beam.

Fig. 4

DNA repair factor (NbS1) accumulation in areas of damage induced by line scissors. (a) Laser scanning confocal image of NbS1-Alexa staining in HeLa cells exposed to 150 pulses. (b) XY, XZ, and YZ cross-sectional view of the accumulation of repair factor in areas of DNA damage induced by the line scissors in a CFPAC cell. The green line represents the position at which the XZ plane has been acquired. The red line represents the position at which the YZ plane has been acquired. The blue line represents the XY plane position relative to the XZ and YZ planes. (c) Composite image showing overlay of the fluorescence and phase images. Note that all cells in this image were exposed to 150 pulses to illustrate that the threshold for NbS1 recruitment is below 150 pulses. (Color online only.)

In addition, a laser-dose-dependent staining of $\gamma \mathrm{H}2\mathrm{AX}$ and Nbs1 was observed (Fig. 5 ). Increased localization of $\gamma \mathrm{H}2\mathrm{AX}$ and Nbs1 accumulation is observed with increase in line scissors dose in HeLa cells. Figure 6 shows the comparison of the three methods of applying a laser beam to induce linear patterns of DNA damage in HeLa cells: fast scanning mirror, scanning stage, and line scissors. Fluorescence images of $\gamma \mathrm{H}2\mathrm{AX}$ -Alexa staining in cells exposed to different numbers of laser pulses (same energy/pulse) shows that the line scissors is a comparatively simple tool for inducing DNA damage. Since the line scissors requires no dynamic movement of components (such as a scanning stage or an FSM) for laser energy deposition, the linear pattern of damage is more uniform. Additionally, this feature of the line scissors decreases the time necessary to complete damage induction, since deposition of the laser energy is governed only by the repetition rate of the laser (in our case, $<1\mathrm{s}$ for $50\mathrm{Hz}$ ), whereas methods employing repositioning of the stage requires $2\text{to}4\mathrm{s}$ per nucleus, and methods requiring repositioning of the scanning mirror required $\sim 2\mathrm{s}$ per nucleus. It should be noted that the application of this approach using solid-state lasers would further increase throughput to subsecond exposure times per nucleus.

Fig. 5

Line scissors dose-dependent $\gamma \mathrm{H}2\mathrm{AX}$ localization and NbS1 accumulation. Laser scanning confocal images of $\gamma \mathrm{H}2\mathrm{AX}$ and NbS1-Alexa staining in CFPAC cells exposed to (a) 50, (b) 100, and (c) 150 pulses. All images are at the same magnification. Scale bar: $10\mu \mathrm{m}$ . Note that nonlabeled cells in this image were exposed to 100 pulses to further illustrate that the threshold for NbS1 recruitment is between 100 and 150 pulses.

Fig. 6

Comparison of the scanning mirror, scanning stage, and line scissors induced DNA damage in HeLa cells. Fluorescence images of $\gamma \mathrm{H}2\mathrm{AX}$ -Alexa staining in cells exposed to (a) 50, (b) 100, and (c) 150 pulses. Scale bar: $10\mu \mathrm{m}$ .

4.

Conclusion

The linear laser-induced production of damage by the line scissors described in this report utilizes the unique incorporation of a rotatable cylindrical lens in the beam path in combination with a rotatable polarizer for beam attenuation. The level of control over exposed target length and radial orientation of the linearized beam achieved with this optical configuration is a major improvement over existing techniques. This method also has the added benefit of a smooth distribution of energy over the irradiated region. Furthermore, since the full dimension of the exposed region is filled by the beam itself, less time is required to produce damage in each cell, thus greatly increasing the amount of cellular throughput. The line scissors approach should be applicable to other applications such as high-throughput dissection of cells as well as subcellular structures. The incorporation of line tweezers on this system adds microstructure rotation and alignment capabilities²³ that we aim to take advantage of in the future to produce a high-throughput method for the dissection of cells and particles in suspension.