Fs‐Laser Scissors for Photobleaching, Ablation in Fixed Samples and Living Cells, and Studies of Cell Mechanics

Abstract

The use of ultrashort laser pulses for microscopy has steadily increased over the past years. In this so-called multiphoton microscopy, laser pulses with pulse duration around 100 femtoseconds (fs) are used to excite fluorescence within the samples. Due to the high peak powers of fs lasers, the absorption mechanism of the laser light is based on nonlinear absorption. Therefore, the fluorescence signal is highly localized within the bulk of biological materials, similar to a confocal microscope. However, this nonlinear absorption mechanism can not only be used for imaging but for selective alteration of the material at the laser focus: The absorption can on one hand lead to the excitation of fluorescent molecules of fluorescently tagged cells by the simultaneous absorption of two or three photons or on the other hand, in case of higher order processes, to the creation of free-electron plasmas and, consequently, plasma-mediated ablation. Typical imaging powers are in the range of tens of milliwatts using 100-fs pulses at a repetition rate of 80-90 MHz, while pulse energies needed for ablation powers are as low as a few nanojoules when using high numerical aperture microscope objectives for focusing the laser radiation into the sample. Since the first demonstration of this technique, numerous applications of fs lasers have emerged within the field of cellular biology and microscopy. As the typical wavelengths of ultrashort laser systems lie in the near infrared between 800 and 1000 nm, high penetration depth can be achieved and can provide the possibility of imaging and manipulating the biological samples with one single laser system.