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Pulse energy dependence of subcellular dissection by femtosecond laser pulses

A. Heisterkamp, I. Z. Maxwell, E. Mazur, J. M. Underwood, J. A. Nickerson, S. Kumar, and D. E. Ingber

Open Access Open Access

Abstract

Precise dissection of cells with ultrashort laser pulses requires a clear understanding of how the onset and extent of ablation (i.e., the removal of material) depends on pulse energy. We carried out a systematic study of the energy dependence of the plasma-mediated ablation of fluorescently-labeled subcellular structures in the cytoskeleton and nuclei of fixed endothelial cells using femtosecond, near-infrared laser pulses focused through a high-numerical aperture objective lens (1.4 NA). We find that the energy threshold for photobleaching lies between 0.9 and 1.7 nJ. By comparing the changes in fluorescence with the actual material loss determined by electron microscopy, we find that the threshold for true material ablation is about 20% higher than the photobleaching threshold. This information makes it possible to use the fluorescence to determine the onset of true material ablation without resorting to electron microscopy. We confirm the precision of this technique by severing a single microtubule without disrupting the neighboring microtubules, less than 1 µm away.

©2005 Optical Society of America

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Figures (4)
Fig. 1.
Fig. 1. (a) Cuts through fluorescently-labeled actin fibers in a fixed endothelial cell obtained by irradiation with femtosecond laser pulses of energies between 1.8 nJ and 4.4 nJ. (b) Fluorescence intensity profile along the actin bundle outlined in the image

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Fig. 2.
Fig. 2. Cuts in the nucleus of a fixed endothelial cell at various laser energies, imaged by (a) fluorescence microscopy and (b) electron microscopy.

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Fig. 3.
Fig. 3. Pulse energy dependence of the ablation width of cuts in the nucleus of endothelial cells measured by fluorescence microscopy (filled circles) and TEM (open circles) in three different cells (a)–(c).

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Fig. 4.
Fig. 4. (a) Fluorescence microscope image of GFP-labeled microtubule network in an endothelial cell. (b) time-lapse sequence showing rapid retraction of microtubule due to depolymerization. The cross hair shows the position targeted by the laser; the triangles show the retracting ends of the microtubule.

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