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

Sacconi, Leonardo; Tolic-Norrelykke, Iva M.; Antolini, R.; Pavone, Francesco S.

doi:10.1117/1.1854675

Cited by 98 publications

(53 citation statements)

References 16 publications

(16 reference statements)

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“…Femtosecond laser ablation can sever single fibers without disrupting the rest of the cell, allowing in situ perturbation of the cytoskeleton. Three proof-of-principle experiments in 2005 determined the optimal laser parameters and submicrometer surgical precision of femtosecond laser ablation of fluorescently-labelled cytoskeletal fibers [33,36,93]. Post-surgery dynamics demonstrate that actin is under tension and microtubules are under spring-like compression [33].…”

Section: Chromosomes and Organellesmentioning

confidence: 99%

Surgical applications of femtosecond lasers

Chung¹,

Mazur

2009

Journal of Biophotonics

229

136

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Femtosecond laser ablation permits non‐invasive surgeries in the bulk of a sample with submicrometer resolution. We briefly review the history of optical surgery techniques and the experimental background of femtosecond laser ablation. Next, we present several clinical applications, including dental surgery and eye surgery. We then summarize research applications, encompassing cell and tissue studies, research on C. elegans, and studies in zebrafish. We conclude by discussing future trends of femtosecond laser systems and some possible application directions. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

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Section: Chromosomes and Organellesmentioning

confidence: 99%

Surgical applications of femtosecond lasers

Chung¹,

Mazur

2009

Journal of Biophotonics

229

136

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“…1 In parallel to its application in imaging, multiphoton absorption has also been used as a tool for the selective disruption of intracellular structures. 2,3 A similar approach has also been applied in vivo, where two-photon imaging and laser-induced lesions have been combined. 4,5 Other groups have taken advantage of multiphoton absorption to ablate or dissect individual neurons.…”

mentioning

confidence: 99%

“…These experimental parameters were extrapolated from our previous investigations in cultured cells. 3 In a first series of experiments, this methodology was applied to irradiate a single dendrite in the somatosensory cortex. Briefly, we found that the morphological consequences of irradiating a single dendrite can be grouped into two categories of biological response: 1. transient swelling with recovery, and 2. complete dendritic dissection.…”

mentioning

confidence: 99%

In vivo multiphoton nanosurgery on cortical neurons

et al. 2007

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Abstract. Two-photon microscopy has been used to perform high spatial resolution imaging of spine plasticity in the intact neocortex of living mice. Multiphoton absorption has also been used as a tool for the selective disruption of cellular structures in living cells and simple organisms. In this work, we exploit the spatial localization of multiphoton excitation to perform selective lesions on the neuronal processes of cortical neurons in living mice expressing fluorescent proteins. Neurons are irradiated with a focused, controlled dose of femtosecond laser energy delivered through cranial optical windows. The morphological consequences are then characterized with time lapse 3-D two-photon imaging over a period of minutes to days after the procedure. This methodology is applied to dissect single dendrites with submicrometric precision without causing any visible collateral damage to the surrounding neuronal structures. The spatial precision of this method is demonstrated by ablating individual dendritic spines, while sparing the adjacent spines and the structural integrity of the dendrite. The combination of multiphoton nanosurgery and in vivo imaging in mammals represents a promising tool for neurobiology and neuropharmacology research. The nonlinear nature of two-photon fluorescence provides an absorption volume spatially confined to the focal region. The localization of the excitation is maintained even in strongly scattering tissues, allowing deep high-resolution microscopy. In combination with fluorescent protein expression techniques, two-photon microscopy has become an indispensable tool to image cortical plasticity in living mice.1 In parallel to its application in imaging, multiphoton absorption has also been used as a tool for the selective disruption of intracellular structures.2,3 A similar approach has also been applied in vivo, where two-photon imaging and laser-induced lesions have been combined. 4,5 Other groups have taken advantage of multiphoton absorption to ablate or dissect individual neurons. Multiphoton nanosurgery has been performed in worms to study axon regeneration 6 and dissect the role of specific neurons in behavior.7 Nevertheless, the potential of this technique has not been fully explored in the mammalian central nervous system. We demonstrate a method for performing multiphoton nanosurgery in the central nervous system of mice, and discuss the widespread applications in neurobiology research.We exploited the spatial localization and deep penetration of multiphoton excitation to perform selective lesions on the neuronal processes of cortical neurons in mice expressing fluorescent proteins ͑thy1-YFP-H and thy1-GFP-M transgenic lines 8 ͒. Neurons were irradiated with a focused, controlled dose of femtosecond laser energy delivered through a permanent cranial optical window. Transgenic mice aged 3 to 5 months were used in all experiments unless otherwise stated. Mice were deeply anaesthetized with an intraperitoneal injection of ketamine ͑0.13 mg per g body weight͒ and xylazine ͑0....

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“…It has been shown that applying a pulsed laser on interphase MTs induces catastrophe in a reproducible manner and generates a new plus-and minus-end. These new tips behave as in normal MTs regarding their polymerization and depolymerization dynamics (Figure 1 A; Colombelli et al , 2005 ;Sacconi et al , 2005 ;Wakida et al , 2007 ). This observation qualifi es laser nanosurgery as an important technique for the study of the physiological behavior of MTs.…”

Section: Microtubulesmentioning

confidence: 56%

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

Ronchi

Terjung

Pepperkok³

2012

BCHM

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Laser-mediated nanosurgery has become popular in the last decade because of the previously unexplored possibility of ablating biological material inside living cells with sub-micrometer precision. A number of publications have shown the potential applications of this technique, ranging from the dissection of sub-cellular structures to surgical ablations of whole cells or tissues in model systems such as Drosophila melanogaster or Danio rerio . In parallel, the recent development of micropatterning techniques has given cell biologists the possibility to shape cells and reproducibly organize the intracellular space. The integration of these two techniques has only recently started yet their combination has proven to be very interesting. The aim of this review is to present recent applications of laser nanosurgery in cell biology and to discuss the possible developments of this approach, particularly in combination with micropattern-mediated endomembrane organization.

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

Cited by 98 publications

References 16 publications

Surgical applications of femtosecond lasers

Surgical applications of femtosecond lasers

In vivo multiphoton nanosurgery on cortical neurons

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

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