Applications of Tightly Focused Ultrafast Laser in the Fabrication of Micro Total Analysis Systems and Biological Research.

Abstract

Tight focusing of femtosecond pulses accentuates the non-linear effect of optical breakdown to achieve damage regions smaller than the light resolution limit. When pulse energy is near the breakdown threshold, optical breakdown using tightly focused ultrafast laser is extraordinary precise and induces minimal collateral damage. These characteristics enable the surgical manipulation of sub-cellular structures and its importance becomes apparent when dealing with intrinsically mechanical cellular processes such as mitosis. We examine the role of polar ejection forces (PEFs) in guiding directional instability of vertebrate mitotic chromosomes. Taking advantage of extremely precise character of femtosecond pulsed laser microsurgery, we abruptly alter PEFs by severing chromosome arms. Reduction of PEFs increases the amplitude of directional instability without altering other characteristics, or the speed of chromosome movement. We find that PEFs limit the range of chromosome oscillation by increasing the probability that motors at a leading kinetochore abruptly fail or disengage, leading to a direction reversal. From the relation between the change in oscillation amplitude and the length of the chromosome arm shortened, we are able to map the distribution of PEFs across spindle, which is surprisingly different from distributions previously assumed. These results allow us to differentiate between models of directional instability, and reveal relations between forces within the spindle and chromosome movements fundamental to the intrinsically mechanical mitotic process. In the course of developing this surgical technique, we fabricated devices such as nanochannels and resistive-pulsed sensors in glass in order to study the capabilities of laser-induced optical breakdown. We found microbubbles created with this technique highly damped and contribute little collateral damage compared to shockwave-forming cavitation bubbles produced by longer pulsed lasers. In their own right, these studies contribute to the field of medical diagnosis and biodefense by establishing methods capable of speeding up chemical separation and detection of viruses. Transitioning from glass to cells, we studied the effect of microsurgical removal of proteinaceous ASC aggregates, which play an important role in inflammatory diseases.