Intracellular nanosurgery and cell enucleation using a picosecond laser

Abstract

SummaryLiving cells are highly organized in space and time, which makes spatially and temporally confined manipulations an indispensable tool in cell biology. Laser-based nanosurgery is an elegant method that allows precise ablation of intracellular structures. Here, we show cutting of fluorescently labelled microtubules and mitotic spindles in fission yeast, performed with a picosecond laser coupled to a confocal microscope. Diverse effects from photo-bleaching to partial and complete breakage are obtained by varying the exposure time, while simultaneously imaging the structures of interest. Using this system we developed an efficient technique to generate enucleated cells without perturbing the distribution of other organelles. This enucleation method can be used to study the cytoskeleton in a nucleus-free environment, as well as the role of the nucleus in cell growth and a variety of cellular functions.

At the cutting edge: applications and perspectives of laser nanosurgery in cell biology

“…Cutting spindle MTs close to one of the poles leads to asymmetric division where one daughter cell inherits the entire genetic material and the other one has no nucleus. This effect is due to the forces generated by the aster MTs that pull the poles apart (Raabe et al , 2009 ).…”

“…Pulsed lasers suitable for nanosurgery range from diode lasers (Raabe et al , 2009 ) over frequency-doubled (532 nm) or tripled (355 nm) Nd:YAG lasers (Colombelli et al , 2004 ;Caussinus et al, 2008;Solon et al , 2009 ) to mode-locked Ti:Sa lasers, which are also used for two-photon microscopy ( Table 1 ; Konig et al , 1999 ;Heisterkamp et al , 2005 ;Kohli and Elezzabi , 2009 ).…”

“…This enables researchers to visualize and ablate objects that are even smaller that the diffraction limit if the laser is targeted to the fl uorescent signal. The fl uorescent label can decrease the plasma formation threshold if it absorbs the ablation laser light effi ciently (Botvinick et al , 2004 ;Raabe et al, 2009 ). Therefore, even more precise nanosurgery results are achievable labeling the structures of interest with the right fl uorophore (Botvinick et al , 2004 ).…”

“…This is optimally solved if the laser nanosurgery scanner can be controlled independently during the imaging mode. Examples of commercially available solutions are confocal microscopes Olympus FV1000 SIM (Raabe et al , 2009 ;Solon et al , 2009 ) and Zeiss LSM Duo. In these systems a second scanner is available for photoperturbations (nanosurgery with appropriate laser sources) while the fi rst scanner is continuously imaging.…”

“…They usually guarantee a high repetition rate with pulse durations in the ps-range but relatively low pulse energy. To achieve nanosurgery effects a high number of pulses usually has to be used with this laser option (Raabe et al , 2009 ;Tinevez et al , 2009 ).…”