Biology Reference
In-Depth Information
D. Laser Surgery of Cells and Processes
Genetically targeted cell disruption and laser-ablation have been used to deter-
mine the function of neurons. To genetically eliminate an identified neuron, one
can express apoptotic genes such as ced-3 and ced-4, ''degenerin family'' genes
such as mec-4, mec-10, deg-1,andunc-8, or toxins such as ricin A and diphtheria
toxin-A under neuronal promoters ( Harbinder et al., 1997; Shaham and Horvitz,
1996 ). The caveats of these genetic methods are lack specific promoters to
express cell-toxic proteins, disrupting neuronal development and affecting sur-
rounding cells. In these cases, physically killing cells by laser has its advantages.
Laser-ablation was traditionally done by first identifying the cell under Nomarski
microscope, and then followed by laser operation. Transgenic labeling has greatly
simplified the cell identification process. More recently, laser has been success-
fully used to sever axons or dendrites. We summarize the two types of laser
ablation below.
(1) Cell ablation: To make a traditional laser-ablation system, one needs a laser
source, a microscope, and optics to direct the beam of the laser into the
microscope objective. The fluorescent compound microscope used to find
targeting neurons should have an objective with a numerical aperture of at
least 1.25 to focus the laser beam. The laser pulse energy should be bigger
than 5 mJ in order to kill cells. Optics is used to shape the beam to enter the
specimen from a full range of angles to a focus at a point on the image plane.
The traditional cell-ablation system has been widely used in the C. elegans
field even before the discovery of GFP; the detailed information of this
system can be found in Bargmann and Avery ' s review paper ''Laser killing
of cells in Caenorhabditis elegans'' ( Bargmann and Avery, 1995 ).
(2) Axon severing: A new application of laser ablation is to severing axons or nerve
processes ( Wu et al., 2007; Yanik et al., 2004 ). Two types of lasers can be used to
perform axotomy.
(a) Femtosecond lasers: A KMLabs MTS Ti-Sapphire oscillator (Kapteyn-
Murnane Laboratories, Boulder, CO) pumped by a Verdi V5 (Coherent,
Santa Clara, CA) or a KMLabs Cascade laser that can be operated in mode-
locked continuous wave (80 MHz) or cavity-dumped (1-100 kHz) modes
can be used to generate laser. The pulse energy can be attenuated using
neutral density filters and controlled pulse delivery with an electromechan-
ical shutter (Uniblitz VS14 with VMM-T1 controller; Vincent Associates,
Rochester, NY).
(b) Conventional laser: Axotomy can also be performed using a Photonics
Micropoint VSL pulsed UV laser (Photonics Instruments, St. Charles,
IL). The laser beam is delivered to an imaging microscope equipped for
simultaneous laser and GFP illumination via a Photonics Instruments adap-
tor, and surgery can be performed by using a Plan Neofluar 100/N.A. 1.3
objectives.
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