Construction of a femtosecond laser microsurgery system

Abstract

Femtosecond laser microsurgery is a powerful method for studying cellular function, neural circuits, neuronal injury and neuronal regeneration because of its capability to selectively ablate sub-micron targets in vitro and in vivo with minimal damage to the surrounding tissue. Here, we present a step-by-step protocol for constructing a femtosecond laser microsurgery setup for use with a widely available compound fluorescence microscope. The protocol begins with the assembly and alignment of beam-conditioning optics at the output of a femtosecond laser. Then a dichroic mount is assembled and installed to direct the laser beam into the objective lens of a standard inverted microscope. Finally, the laser is focused on the image plane of the microscope to allow simultaneous surgery and fluorescence imaging. We illustrate the use of this setup by presenting axotomy in Caenorhabditis elegans as an example. This protocol can be completed in 2 d.

Figure 1

Optical system layout. See Table 1 for component list.

Figure 2

An exploded view of the dichroic mounting adapter. (a-b) (a) The dichroic mounting adapter is composed of a metal filter cube, which contains an infrared (IR) dichroic mirror (b). (c) The filter cube is attached to a BA1 standard base using glue. (d) The opposite face of the BA1 standard base attaches to a two-axis compact kinematic mount also using glue. (e) The compact kinematic mount is attached to a Ø1″ (1″) pedestal pillar post with a #8–32 × 1/4″ set screw. (f) The assembly comprising components a–e mounts to a Nikon adapter plate from a 70 mm stage-up kit so that the dichroic mirror sits in the beam path. (g-h) (g) This is accomplished by drilling a hole and using a #8–32 × 1/4″ screw (h) to position the dichroic mirror over the opening in the adapter plate. (i) The entire assembly is affixed to the microscope using screws included in the 70 mm stage-up kit. Figure 1 shows the location of the dichroic mounting adapter on the microscope.

Figure 3

Optical path for simultaneous epifluorescence imaging and laser axotomy. The femtosecond laser, indicated by the red line, passes through beam conditioning optics before being directed up by the near infrared (NIR) dichroic mirror into the back aperture of the objective lens. The epifluorescence excitation, indicated by the blue line, is simultaneously directed into the back aperture of the objective lens by the filter cube. The fluorescence emission, indicated by the green line, passes through multiple filters and is captured by the camera.

Figure 4

An exploded view of the beam expander. Lenses L1 and L2 (a and b, respectively) sit in their mounts that are attached to Ø1/2″ posts. Two Ø1″ irises (c) are also attached to Ø1/2″ posts. All four posts sit securely in Ø1/2″ post holders (d), one of which is attached to a single-axis stage (e) with rotatable micrometer and Ø1.5″ post clamp adapter plate (f), whereas the remaining three are attached to rail carriers (g). These four assemblies firmly attach to the 12″optical rail (h) which is mounted to the two Ø1.5″ posts by two Ø1.5″ post mounting clamps (i). The entire assembly is mounted using BA2 standard bases.

Figure 5

Use of the infrared (IR) alignment tool. The IR alignment tool is composed of an RMS IR-aligning disk and an RMS 45 to CFI 60 objective adapter. (a) Without lenses L1 and L2, the transmitted beam is directed to the center of the dichroic mirror, thus resulting in a glowing spot on the field of the disk. (b) Adjusting the dichroic mirror causes the transmitted beam to pass through the center hole and an additional spot caused by the reflection of the beam from the cover glass appears on the field of the disk. The reflected spot is caused by the non-normal incidence of the transmitted beam on the cover glass. Moving the transmitted spot half-way towards the initial location of the reflected spot by adjusting the upper periscope mirror and then moving the transmitted spot back to the center hole by adjusting the angle of the dichroic mirror, achieves normal incidence of the beam on the cover glass. (c) Normal incidence is indicated by both reflected and transmitted beams passing through the center. (d) Inserting both lenses L1 and L2 into the beam path (Steps 68–70), results in a large, symmetric circular illumination on the IR alignment tool.

Figure 6

Ablated patterns in permanent marker on cover glass under different alignment conditions. (a,b) When the image plane is focused on the boundary of the marked and unmarked glass surfaces, and when the system is properly aligned, the resulting cutting pattern is narrow and symmetric (a), whereas the firing pattern is relatively small and also symmetric (b). If the beam is clipped and/or lenses L1 and L2 are misaligned, the cutting pattern is wider in one direction than the orthogonal direction (c). In addition, the firing pattern becomes asymmetric. If the laser is out of focus, the cutting pattern is blurred and the firing pattern is larger (d). Scale bar is 50 µm.

Figure 7

Point spread function of the laser at the focal plane. The system described in this protocol generates a circular laser spot at the sample with a full width at half-maximum of 1.7 µm.

Figure 8

Femtosecond laser microsurgery. (a) A highly localized region (arrow) of a Caenorhabditis elegans mechanosensory neuron is ablated using the system described in this protocol. (b) Following surgery at point 1, the ablated process first retracts to point 2 and then regenerates to point 3. Scale bar represents 10 µm.