Laser nanosurgery in cell biology

Since their first use in the early 60's, pulsed lasers have become increasingly popular for their ability to ablate biological tissue. Short laser pulses allow high precision surgery for biological and medical applications with minimal invasiveness. Performing highly targeted manipulation and ablation allows experiments impossible so far in development biology, cellular biology or even assisted reproductive technologies and laser surgery has been increasingly used over the last five years to answer key questions in Biology. Recently, picosecond UV and femtosecond IR laser pulses have been used to cleave microtubules and to severe actin stress fibers in vivo with a spatial precision in the submicrometer range to study their dynamics without affecting cell viability. We review recent findings on the underlying principles of pulsed laser nanosurgery mechanisms showing how the use of ultra short laser pulses increases precision and non-invasiveness of laser surgery. We show how the understanding of the surgical process allows one to distinguish between single cell ablation in living organisms or intracellular nanosurgery in living cells and we review recent applications to the study of forces and the quantification of cytoskeleton dynamics.     

Effect of photodynamic therapy on the skin using the ultrashort laser ablation

Photodynamic Therapy (PDT) with 5‐aminolevulinic acid (ALA) is known to be limited for applications in tumours of large volume mainly due to the limited penetration of topical photosensitization. The results show that micro‐holes created using a femtosecond laser before PDT significantly increased the depth of PDT effect in the healthy tissue. The combination of ultrashort laser ablation technique with PDT showed an important scientific breakthrough related to transportation and delivery of drugs into the deeper regions of the tissue. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Qualitative tissue differentiation by analysing the intensity ratios of atomic emission lines using laser induced breakdown spectroscopy (LIBS): prospects for a feedback mechanism for surgical laser systems

The research work presented in this paper focuses on qualitative tissue differentiation by monitoring the intensity ratios of atomic emissions using ‘Laser Induced Breakdown Spectroscopy’ (LIBS) on the plasma plume created during laser tissue ablation. The background of this study is to establish a real time feedback control mechanism for clinical laser surgery systems during the laser ablation process. Ex‐vivo domestic pig tissue samples (muscle, fat, nerve and skin) were used in this experiment. Atomic emission intensity ratios were analyzed to find a characteristic spectral line for each tissue. The results showed characteristic elemental emission intensity ratios for the respective tissues. The spectral lines and intensity ratios of these specific elements varied among the different tissue types. The main goal of this study is to qualitatively and precisely identify different tissue types for tissue specific laser surgery. (© 2013 WILEY‐VCH Verlag GmbH &Co. KGaA, Weinheim)     

In-vivo intratissue ablation by nanojoule near-infrared femtosecond laser pulses

Non-invasive intratissue ablation was performed in the cornea of living rabbits by using 80 MHz near-infrared intense nanojoule femtosecond laser pulses. The intratissue surgical effect was induced by multiphoton absorption at a wavelength of 800 nm and was ascertained by histological examination. Highly precise intratissue ablation was obtained with no detrimental effects to the overlying or underlying layers. Activated keratocytes in the laser-treated corneas were detected with two-photon imaging postoperatively. Intratissue femtosecond laser ablation thus has potential as a effective technique in refractive surgery for the treatment of visual disorders. This work was supported in part by the German Science Foundation.     

Influence of environmental conditions in bovine bone ablation by ultrafast laser

Ultrafast lasers are promising tools for surgical applications requiring precise tissue cutting. Shallow ablation depth and slow rate as well as collateral damage are common barriers limiting the use of laser in clinical applications. Localized cooling with water and/or air jet is known to reduce collateral thermal damage. We studied the influence of environmental conditions including air, compressed air flow, still water and water jet on ablation depth, ablation rate and surface morphology on bovine bone samples with an 800 nm femtosecond laser. At 15 J/cm², no thermal effect was observed by electron microscopy and Raman spectroscopy. The experimental results indicate that environmental conditions play a significant role in laser ablation. The deepest cavity and highest ablation rate were achieved under the compressed air flow condition, which is attributed to debris removal during the ablation process. The shallowest ablation depth and lowest ablation rates were associated with water flushing. For surface morphology, smooth surface and the absence of microcracks were observed under air flow conditions, while rougher surfaces and minor microcracks were observed under other conditions. These results suggest that ultrafast ablation of bone can be more efficient and with better surface qualities if assisted with blowing air jet.      

Femtosecond pulsed laser ablation to enhance drug delivery across the skin

Laser poration of the skin locally removes its outermost, barrier layer, and thereby provides a route for the diffusion of topically applied drugs. Ideally, no thermal damage would surround the pores created in the skin, as tissue coagulation would be expected to limit drug diffusion. Here, a femtosecond pulsed fiber laser is used to porate mammalian skin ex vivo. This first application of a hollow core negative curvature fiber (HC‐NCF) to convey a femtosecond pulsed, visible laser beam results in reproducible skin poration. The effect of applying ink to the skin surface, prior to ultra‐short pulsed ablation, has been examined and Raman spectroscopy reveals that the least, collateral thermal damage occurs in inked skin. Pre‐application of ink reduces the laser power threshold for poration, an effect attributed to the initiation of plasma formation by thermionic electron emission from the dye in the ink. Poration under these conditions significantly increases the percutaneous permeation of caffeine in vitro. Dye‐enhanced, plasma‐mediated ablation of the skin is therefore a potentially advantageous approach to enhance topical/transdermal drug absorption. The combination of a fiber laser and a HC‐NCF, capable of emitting and delivering femtosecond pulsed, visible light, may permit a compact poration device to be developed.

Using a femtosecond pulsed, visible laser beam to create an array of micropores in dyed mammalian skin, with little collateral, thermal damage, leads to an enhancement in the percutaneous permeation of caffeine in vitro.     

Investigation of the differentiation of ex vivo nerve and fat tissues using laser‐induced breakdown spectroscopy (LIBS): Prospects for tissue‐specific laser surgery

In the present study, the elemental compositions of fat and nerve tissue during their plasma mediated laser ablation are studied in the context of tissue differentiation for laser surgery applications by using Laser‐Induced Breakdown Spectroscopy (LIBS). Tissue samples of porcine fat and nerve were prepared as ex vivo experimental objects. Plasma mediated laser ablation is performed using an Nd : YAG laser in open air and under normal stray light conditions. The performed measurements suggest that the two tissue types show a high similarity in terms of qualitative elemental composition while at the same time revealing a distinct difference in the concentration of the constituent elements. Different analysis approaches are evaluated and discussed to optimize the tissue‐differentiation performance of the LIBS approach.

Plasma mediated laser tissue ablation.     

Micropatterning and characterization of electrospun poly(ε‐caprolactone)/gelatin nanofiber tissue scaffolds by femtosecond laser ablation for tissue engineering applications

Experimental investigations aimed at assessing the effectiveness of femtosecond (FS) laser ablation for creating microscale features on electrospun poly(ε‐caprolactone) (PCL)/gelatin nanofiber tissue scaffold capable of controlling cell distribution are described. Statistical comparisons of the fiber diameter and surface porosity on laser‐machined and as‐spun surface were made and results showed that laser ablation did not change the fiber surface morphology. The minimum feature size that could be created on electrospun nanofiber surfaces by direct‐write ablation was measured over a range of laser pulse energies. The minimum feature size that could be created was limited only by the pore size of the scaffold surface. The chemical states of PCL/gelatin nanofiber surfaces were measured before and after FS laser machining by attenuated total reflectance Fourier transform infrared (ATR‐FTIR) spectroscopy and X‐ray photoelectron spectroscopy (XPS) and showed that laser machining produced no changes in the chemistry of the surface. In vitro, mouse embryonic stem cells (mES cells) were cultured on as‐spun surfaces and in laser‐machined microwells. Cell densities were found to be statistically indistinguishable after 1 and 2 days of growth. Additionally, confocal microscope imaging confirmed that spreading of mES cells cultured within laser‐machined microwells was constrained by the cavity walls, the expected and desired function of these cavities. The geometric constraint caused statistically significant smaller density of cells in microwells after 3 days of growth. It was concluded that FS laser ablation is an effective process for microscale structuring of these electrospun nanofiber tissue scaffold surfaces. Biotechnol. Bioeng. 2011; 108:116–126. © 2010 Wiley Periodicals, Inc.     

Evaluation of bone healing following Er:YAG laser ablation in rat calvaria compared with bur drilling

The Er:YAG laser is currently used for bone ablation. However, the effect of Er:YAG laser irradiation on bone healing remains unclear. The aim of this study was to investigate bone healing following ablation by laser irradiation as compared with bur drilling. Rat calvarial bone was ablated using Er:YAG laser or bur with water coolant. Er:YAG laser effectively ablated bone without major thermal changes. In vivo micro‐computed tomography analysis revealed that laser irradiation showed significantly higher bone repair ratios than bur drilling. Scanning electron microscope analysis showed more fibrin deposition on laser‐ablated bone surfaces. Microarray analysis followed by gene set enrichment analysis revealed that IL6/JAK/STAT3 signaling and inflammatory response gene sets were enriched in bur‐drilled bone at 6 hours, whereas the E2F targets gene set was enriched in laser‐irradiated bone. Additionally, Hspa1a and Dmp1 expressions were increased and Sost expression was decreased in laser‐irradiated bone compared with bur‐drilled bone. In granulation tissue formed after laser ablation, Alpl and Gblap expressions increased compared to bur‐drilled site. Immunohistochemistry showed that osteocalcin‐positive area was increased in the laser‐ablated site. These results suggest that Er:YAG laser might accelerate early new bone formation with advantageous surface changes and cellular responses for wound healing, compared with bur‐drilling.      

Femtosecond Laser Axotomy in Caenorhabditis elegans and Collateral Damage Assessment Using a Combination of Linear and Nonlinear Imaging Techniques

In this work highly localized femtosecond laser ablation is used to dissect single axons within a living Caenorhabditis elegans (C. elegans). We present a multimodal imaging methodology for the assessment of the collateral damage induced by the laser. This relies on the observation of the tissues surrounding the targeted region using a combination of different high resolution microscopy modalities. We present the use of Second Harmonic Generation (SHG) and Polarization Sensitive SHG (PSHG) to determine damage in the neighbor muscle cells. All the above is done using a single instrument: multimodal microscopy setup that allows simultaneous imaging in the linear and non-linear regimes and femtosecond-laser ablation.     

Plasmonic Enhancement During Femtosecond Laser Drilling of Sub-wavelength Holes in Metals

Precise ablation of metals using tightly focused femtosecond laser pulses with intensities close to the damage threshold can yield sub-wavelength, nanometer-sized holes or craters. These structures in metals can exhibit plasmonic effects, thereby affecting the interactions involved. We numerically simulate light propagation inside such holes and model the ablation process. We show that surface plasmon resonances can be excited at near-infrared and visible wavelengths. At resonance wavelengths, significant enhancement of aspect ratio is possible. Our results show that plasmonic effects are essential for the understanding of precision laser processing of metals, and they can be exploited to significantly enhance the performance of laser micro- and nano-machining.     

Imaging of the DNA damage‐induced dynamics of nuclear proteins via nonlinear photoperturbation

Understanding the cellular response to DNA strand breaks is crucial to decipher the mechanisms maintaining the integrity of our genome. We present a novel method to visualize how the mobility of nuclear proteins changes in response to localized DNA damage. DNA strand breaks are induced via nonlinear excitation with femtosecond laser pulses at λ = 1050 nm in a 3D‐confined subnuclear volume. After a time delay of choice, protein mobility within this volume is analysed by two‐photon photoactivation of PA‐GFP fusion proteins at λ = 775 nm. By changing the position of the photoactivation spot with respect to the zone of lesion the influence of chromatin structure and of the distance from damage are investigated. As first applications we demonstrate a locally confined, time‐dependent mobility increase of histone H1.2, and a progressive retardation of the DNA repair factor XRCC1 at damaged sites. This assay can be used to map the response of nuclear proteins to DNA damage in time and space. (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

用二次谐波成像技术研究经飞秒激光切削后角膜变化

本文用二次谐波成像技术（second harmonic generation SHG）来研究飞秒激光切削后角膜结构的变化.在生物学研究,材料科学等方面都有很广泛应用的SHG成像技术能在不破坏的角膜情况下获得高对比度的角膜层析图像,分辨率为500 nm,实验装置是利用现有的双光子显微镜.本文还根据成像结果评价了飞秒激光在角膜切削中的质量,为飞秒激光微米级的精确切削和临床应用提供了实验支持.     

飞秒激光与生物细胞作用机理及应用

飞秒激光是自1960年第一台激光器诞生以来,过去20年间由激光科学发展起来的最强有力的新工具之一。飞秒激光由于脉冲持续时间短、瞬时功率大、聚焦尺寸小的特点,使得其在超快、超强和超精细领域有着广阔的应用前景。其中最重要的一个方向是飞秒激光在生物细胞方面的应用。细胞是生命活动的基本单位。所有的病源微观上都体现在细胞中细胞器的工作,所以用飞秒激光作用在病体的细胞器上达到治疗的目的,是一个很有前景的领域。由于生物大分子和水几乎不吸收近红外光,故应用近红外飞秒激光对细胞进行手术,同时可在不损伤细胞活性的前提下对细胞进行实验。这种激光手术技术已被用于对细胞内结构进行切割和蚀除。介绍了该技术在细胞领域中的一些应用,如纳米手术、基因转染和染色体切割等;还介绍了飞秒激光技术与生物细胞中主要细胞器的祛除的原理、飞秒激光细胞操作与手术系统和实验中荧光成像、多光子成像显微镜等手段。     

Enhanced operation of femtosecond lasers and applications in cell transfection

In this work we present a review and discussion on the enhancement of femtosecond (fs) lasers for use within biophotonics with a particular focus on their use in optical transfection techniques. We describe the broad range of source options now available for the generation of femtosecond pulses before briefly reviewing the application of fs laser in optical transfection studies. We show that major performance enhancements may be obtained by optimising the spatial and temporal performance of the laser source before considering possible future directions in this field. In relation to optical transfection we describe how such laser sources initiate a multiphoton process to permeate the cell membrane in a transient fashion. We look at aspects of this technique including the ability to combine transfection with optical trapping. For future implementation of such transfection we explore the role of new sources and “nondiffracting” light fields. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

飞秒激光在白内障手术中的应用新进展

飞秒激光辅助的白内障手术是目前最热门的白内障手术之一。即在计算机系统引导下利用飞秒激光进行透明角膜切口的制作,晶状体核裂解和晶状体前囊膜的切开,明显降低了传统超声乳化手术中的并发症,具有十分广阔的临床应用前景。然而飞秒激光辅助的白内障手术目前仍然处于临床应用初级阶段,其昂贵的价格是影响其研究和应用的主要原因,手术安全性及远期屈光效果仍然需要长期的随访观察。本文根据文献资料,就飞秒激光在白内障手术中的优势及临床应用的局限性进行综述。     

Real‐time monitoring of incision profile during laser surgery using shock wave detection

Lack of sensory feedback during laser surgery prevents surgeons from discerning the exact location of the incision, which increases duration and complexity of the treatment. In this study we demonstrate a new method for monitoring of laser ablation procedures. Real‐time tracking of the exact three dimensional (3D) lesion profile is accomplished by detection of shock waves emanating from the ablation spot and subsequent reconstruction of the incision location using time‐of‐flight data obtained from multiple acoustic detectors. Here, incisions of up to 9 mm in depth, created by pulsed laser ablation of fresh bovine tissue samples, were successfully monitored in real time. It was further observed that, by utilizing as little as 12 detection elements, the incision profile can be characterized with accuracy below 0.5 mm in all three dimensions and in good agreement with histological examinations. The proposed method holds therefore promise for delivering high precision real‐time feedback during laser surgeries. (© 2013 WILEY‐VCH Verlag GmbH &Co. KGaA, Weinheim)     

Femtosecond Laser‐Etched MXene Microsupercapacitors with Double‐Side Configuration via Arbitrary On‐ and Through‐Substrate Connections

The capacitance of microsupercapacitors (MSCs) can double if both sides of substrates are used to construct MSCs. Nevertheless, achieving electric connections of MSCs through substrates is a challenge due to the difficulty in precisely positioning each MSC couple that has two of the same MSCs units on two sides. In this work, taking advantage of the synchronous etching on both sides of transparent polyethylene terephthalate substrates by femtosecond laser pulses, a double‐sided configuration is attained with high precision in the alignment of back‐to‐back MSC couples and versatile double‐side MSCs are realized via arbitrary on‐ and through‐substrate connections of MXene MSC units. The MXene double‐side MSC fabricated by the series connection of 12 spiral pattern MXene MSC units with interdigital electrodes of 10 μm width interspace can output a large working voltage of 7.2 V. Additionally, femtosecond laser etching brings the transformation of MXene into titania near‐etched edges with a lateral distance less than 1 µm. Such a small laser‐affected area has little influence on the capacitive performance, which is one of advantages for femtosecond laser over conventional lasers. This research is valuable for one‐step manufacturing of highly integrated MSCs in the field of miniaturized energy storage systems.     

Force communication in multicellular tissues addressed by laser nanosurgery

Cell contractility is a prominent mechanism driving multicellular tissue development and remodeling. Forces originated by the actomyosin cytoskeleton not only act within the cell body but can also propagate many layers away from the contraction source and grant tissues the ability to organize collectively and to achieve robust remodeling through development. Tissue tension is being thoroughly investigated in model organisms and increasing evidence is revealing the major role played by the communication, dynamics and propagation of cell-to-cell physical forces in multicellular remodeling. Recently, pulsed-laser-based surgery has fostered in vivo experimental studies to investigate intracellular and supracellular forces in action. The technique offers a unique method to perturb mechanical equilibrium in a subpopulation of cells or in a single cell, while the overall tissue remains intact. In particular, improved ablation precision with short laser pulses and the combination of this technique with biophysical models now allow an in-depth understanding of the role of cellular mechanics in tissue morphogenesis. We first characterize laser ablation modes available to perform intracellular, cellular, or multi-cellular ablation via the example of the model monolayer tissue of the amnioserosa of Drosophila by relating subnanosecond laser pulse energy to ablation efficiency and the probability of cavitation bubble formation. We then review recent laser nanosurgery experiments that have been performed in cultured cells and that tackle actomyosin mechanics and provide molecular insights into force-sensing mechanisms. We finally review studies showing the central role of laser ablation in revealing the nature and orientation of forces involved in intracellular contractility and force mechanosensing in tissue development, e.g., axis elongation, branching morphogenesis, or tissue invagination. We discuss the perspectives offered by the technique in force-based cell-cell communication and mechanosensing pathways.