Femtosecond near‐infrared laser pulses as a versatile non‐invasive tool for intra‐tissue nanoprocessing in plants without compromising viability

Abstract

SummaryIn this report, we describe a highly reproducible femtosecond near-infrared (NIR) laser-based nanoprocessing technique that can be used both for non-invasive intra-tissue nanodissection of plant cell walls as well as selective destruction of a single plastid or part thereof without compromising the viability of the cells. The ultra-precise intra-tissue nanoprocessing is achieved by the generation of high light intensity (10 12 W cm ±2 ) by diffraction-limited focusing of the radiation of an NIR (l = 740 and 800 nm) femtosecond titanium-sapphire laser to a sub-femtolitre volume and subsequent highly localized instantaneous plasma formation. Following nanosurgery, electron microscopical analysis of the corresponding cellular target areas revealed clean non-staggering lesions across the cell wall with a cut width measuring less than 400 nm. To our knowledge, this is the smallest cut made non-invasively within a plant tissue. Further evidence, including two-photon imaging of chlorophyll¯uorescence, revealed that a single target chloroplast or part thereof can be completely knocked out using intense ultra-fast NIR pulses without any visible deleterious effect on the adjacent plastids. The vitality of the cells after nanoprocessing has been ascertained by exclusion of propidium iodide from the cells as well as by the presence of cytoplasmic streaming. The potential applications of this technical advance include developmental biology applications, particularly studies addressing spatio-temporal control of ontogenetic events and cell±cell interactions, and gravitational biology applications.

Surgical applications of femtosecond lasers

“…Recent work includes using metal nanoparticles to preferentially absorb light and damage particular locations on the chromosome [84] and a demonstration of an integrated 1-GHz repetition rate system for multiphoton tomography and surgery with staining [85]. Femtosecond laser ablation has also selectively disrupted individual plastids in plant tissue [86] and mitochondria in HeLa [87] and capillary endothelial [36] cells. In summary, subcellular femtosecond laser ablation of chromosomes and organelles results in experimentally-straightforward dissections of unprecedented precision but have yet to move beyond the proof-of-principle phase to become common tools in cell biology [88][89][90][91].…”

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

“…This combines effi cient delivery of membrane impermeable substances and high cell viability (Tirlapur and Konig , 2002b ;Kohli et al , 2005a,b ;Stracke et al , 2005 ;Barrett et al , 2006 ;Baumgart et al , 2008 ;Uchugonova et al , 2008 ). Furthermore, when applied to plant cells, laser nanosurgery gives the possibility to dissect the cell wall in living tissues (Tirlapur and Konig , 2002a ) individual chloroplasts without compromising the cell viability. This fi nding paved the way for the ablation of other membrane bound organelles such as mitochondria in mammalian cultured cells (Figure 2 B; Khodjakov et al , 2004b ;Watanabe et al , 2004 ;Shen et al , 2005 ;Shimada et al , 2005 ).…”

“…Furthermore, the size and positioning of membrane bound organelles could be studied after partial laser-mediated removal. As discussed before, different studies showed the possibility to ablate chloroplasts and mitochondria (Tirlapur and Konig , 2002a ;Watanabe et al , 2004 ;Shen et al , 2005 ;Shimada et al , 2005 ) but their subsequent biogenesis and spatial re-organization has not yet been addressed. Given the fact that organelle localization is emerging as a crucial determinant for signaling events and activation of different pathways (Korolchuk et al , 2011 ), research on organelle shape and spatial organization as well as their biogenesis could take advantage of laser nanosurgery and micropatterning to gain insights into topics that are otherwise diffi cult to address.…”

Sub-100 nm material processing and imaging with a sub-15 femtosecond laser scanning microscope

“…4 Intracellular nanosurgery within living cells has been performed based on multiphoton ionization in a subfemtoliter excitation volume where four or more photons are simultaneously absorbed which can lead to the formation of plasma. [4][5][6] Furthermore, intratissue nanoprocessing has been realized in plants 7,8 and in ocular tissue 9,10 by the same group using nanojoule femtosecond NIR laser microscopes. Nanodissection of fixed chromosomes has been reported in 2001.…”

“…12 One major application of these novel nanotools in life sciences is targeted transfection [13][14][15][16] and the optical injection of substances into living cells by the generation of transient nanopores in the cellular membrane. 17 This pioneering work of using femtosecond laser microscopes as nanoprocessing tools [4][5][6][7][8][9][10][11][12][13][14][15][16][17] was done with 80 MHz nanojoule (about 100 mW mean power) laser pulses at a large pulse width of 200-300 fs at the target. When applying extremely ultrashort laser pulses such as 10 fs to the nanoprocessing microscope, pulse broadening occurs due to group velocity dispersion effects that can result in in situ pulse widths at the target of much more than 300 fs.…”