Combined intracellular three-dimensional imaging and selective nanosurgery by a nonlinear microscope

Year: 2005

Authors: Sacconi L., Tolić-Nørrelykke I.M., Antolini R., Pavone FS.

Autors Affiliation: University of Trento, via Sommarive 14, 38050 Povo (TN), Italy; European Laboratory for Non-linear Spectroscopy, via Nello Carrara 1, 50019 Sesto Fiorentino (Fl), Italy; Rugjer Boškoviæ Institute, Bijenièka 54, 10000 Zagreb, Croatia University of Trento, via Sommarive 14, 38050 Trento, Italy; European Laboratory for Non-linear Spectroscopy, via Nello Carrara 1, 50019 Florence, Italy; University of Florence and INFM Sezione di Firenze, Department of Physics, via Sansone 1, 50019 Florence, Italy

Abstract: We use near-IR femtosecond laser pulses for a combination of microscopy and nanosurgery on fluorescently labeled structures within living cells. Three-dimensional reconstructions of microtubule structures tagged with green fluorescent protein (GFP) are made during different phases of the cell cycle. Further, the microtubules are dissected using the same laser beam but with a higher laser power than for microscopy. We establish the viability of this technique for the cells of a fission yeast, which is a common model to study the mechanics of cell division. We show that nanosurgery can be performed with submicrometer precision and without visible collateral damage to the cell. The energy is primarily absorbed by the GFP molecules, and not by other native structures in the cell. GFP is particularly suitable for multiphoton excitation, as its excitation wavelength near 900 nm is benign for most cellular structures. The ability to use GFP to label structures for destruction by multiphoton excitation may be a valuable tool in cell biology. (c) 2005 Society of Photo-Optical instrumentation Engineers.


Volume: 10 (1)      Pages from: 014002-1  to: 014002-5

KeyWords: two-photon excitation; laser nanosurgery; green fluorescent protein; microtubules;
DOI: 10.1117/1.1854675

Citations: 72
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