Wavelength-dependent conformational changes in collagen after mid-infrared laser ablation of cornea

We ablated porcine corneas with a free electron laser tuned to either 2.77 or 6.45 μm, two matched wavelengths that predominantly target water and protein, respectively. The ejected nonvolatile debris and the crater left behind were examined by circular dichroism, Raman spectroscopy, and scanning electron microscopy to characterize the postablation conformation of collagen proteins. We found near-complete unfolding of collagen secondary and tertiary structure at either ablating wavelength. On the other hand, we found excess fibril swelling and evidence for excess cis-hydroxyproline in the 6.45-μm debris. These results support the hypothesis that the favorable ablative properties of protein-targeting wavelengths rest on selective heating of tissue proteins.     

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.      

Liquid sample delivery techniques for serial femtosecond crystallography

X-ray free-electron lasers overcome the problem of radiation damage in protein crystallography and allow structure determination from micro- and nanocrystals at room temperature. To ensure that consecutive X-ray pulses do not probe previously exposed crystals, the sample needs to be replaced with the X-ray repetition rate, which ranges from 120 Hz at warm linac-based free-electron lasers to 1 MHz at superconducting linacs. Liquid injectors are therefore an essential part of a serial femtosecond crystallography experiment at an X-ray free-electron laser. Here, we compare different techniques of injecting microcrystals in solution into the pulsed X-ray beam in vacuum. Sample waste due to mismatch of the liquid flow rate to the X-ray repetition rate can be addressed through various techniques.     

Theoretical and physical aspects of excimer laser trabeculotomy (ELT) ab interno with the AIDA laser with a wave length of 308 mm]

Ablation of tissue structures containing and surrounded by water differs from tissue ablation at a surface, not only theoretically, but also in terms of outcome. In contrast to the situation often observed with surface ablation, it is shown that the trabecular meshwork in the anterior chamber of the eye can be ablated cleanly and accurately with the 308 nm Excimer laser without causing collateral thermal damage. The reason for this is that in the trabecular meshwork, the ratio of radiation-absorbing tissue to water--which absorbs very little energy--is very small. A marked cooling effect thus results, which permits the development of only a very small amount of collateral thermal damage at the boundaries of the ablation zone.     

Ultrafast Mid-IR Laser Scalpel: Protein Signals of the Fundamental Limits to Minimally Invasive Surgery

Lasers have in principle the capability to cut at the level of a single cell, the fundamental limit to minimally invasive procedures and restructuring biological tissues. To date, this limit has not been achieved due to collateral damage on the macroscale that arises from thermal and shock wave induced collateral damage of surrounding tissue. Here, we report on a novel concept using a specifically designed Picosecond IR Laser (PIRL) that selectively energizes water molecules in the tissue to drive ablation or cutting process faster than thermal exchange of energy and shock wave propagation, without plasma formation or ionizing radiation effects. The targeted laser process imparts the least amount of energy in the remaining tissue without any of the deleterious photochemical or photothermal effects that accompanies other laser wavelengths and pulse parameters. Full thickness incisional and excisional wounds were generated in CD1 mice using the Picosecond IR Laser, a conventional surgical laser (DELight Er:YAG) or mechanical surgical tools. Transmission and scanning electron microscopy showed that the PIRL laser produced minimal tissue ablation with less damage of surrounding tissues than wounds formed using the other modalities. The width of scars formed by wounds made by the PIRL laser were half that of the scars produced using either a conventional surgical laser or a scalpel. Aniline blue staining showed higher levels of collagen in the early stage of the wounds produced using the PIRL laser, suggesting that these wounds mature faster. There were more viable cells extracted from skin using the PIRL laser, suggesting less cellular damage. β-catenin and TGF-β signalling, which are activated during the proliferative phase of wound healing, and whose level of activation correlates with the size of wounds was lower in wounds generated by the PIRL system. Wounds created with the PIRL systsem also showed a lower rate of cell proliferation. Direct comparison of wound healing responses to a conventional surgical laser, and standard mechanical instruments shows far less damage and near absence of scar formation by using PIRL laser. This new laser source appears to have achieved the long held promise of lasers in minimally invasive surgery.     

Kinetics of a collagen-like polypeptide fragmentation after mid-IR free-electron laser ablation

Tissue ablation with mid-infrared irradiation tuned to collagen vibrational modes results in minimal collateral damage. The hypothesis for this effect includes selective scission of protein molecules and excitation of surrounding water molecules, with the scission process currently favored. In this article, we describe the postablation infrared spectral decay kinetics in a model collagen-like peptide (Pro-Pro-Gly)₁₀. We find that the decay is exponential with different decay times for other, simpler dipeptides. Furthermore, we find that collagen-like polypeptides, such as (Pro-Pro-Gly)₁₀, show multiple decay times, indicating multiple scission locations and cross-linking to form longer chain molecules. In combination with data from high-resolution mass spectrometry, we interpret these products to result from the generation of reactive intermediates, such as free radicals, cyanate ions, and isocyanic acid, which can form cross-links and protein adducts. Our results lead to a more complete explanation of the reduced collateral damage resulting from infrared laser irradiation through a mechanism involving cross-linking in which collagen-like molecules form a network of cross-linked fibers.     

生物组织激光消融阈值的光谱特性

在一个宽光谱范围内研究不同激光作用下生物组织的消融,对理解激光与组织间相互作用及开发激光在外科的新应用有着极其重要的意义。其中消融阈值及其与激光波长的函数依赖关系是激光外科研究的重点。阐述了消融阈值的物理描述,并对消融阈值的波长依赖关系进行了初步探讨。     

Histopathologic and morphometric evaluation of the skin abnormalities induced by erbium:YAG and carbon dioxide lasers in 10 patients

In the 1960s, carbon dioxide (CO2) laser therapy started to be applied to eliminate wrinkles, actinic scars, and acne because of its capacity of induce intracellular water vaporization. However, recent studies have shown the efficacy of the erbium laser in removing delicate and moderate scars. Furthermore, the postoperative lesions induced by the erbium laser seem to resolve faster and with less erythematous pattern compared with lesions induced by the CO2 laser. The purpose of this study was to determine the immediate pathologic alterations caused by single applications of CO2 and erbium lasers and their association in human skin shreds.Ten white female patients aged 30 to 63 years underwent rhytidectomy, and their respective shreds, which were prepared for excision, were tattooed with the CO2 laser, the erbium laser, or a combination of both in random order and number of applications, before final removal. This project was approved by the local ethical committee. After surgical removal, these tattooed shreds were fixed in 10% buffered formalin and submitted to histopathologic analysis. Morphometric studies demonstrated the normal skin thickness and thickness of the laser-treated area, and their subtraction resulted in the ablation damage values. Residual thermal damage corresponded to the thickness of the affected skin from the most superficial layer of tissue in the laser-treated area down to the deepest dermal area with basophilic degeneration of collagen fibers.Our results showed that two CO2 applications resulted in greater ablation and residual thermal damage when compared with only one CO2 application. The same was true in comparisons of one and two applications of the erbium laser. Both results were statistically significant (p < 0.05). When one isolated erbium and one isolated CO2 application were compared, ablation damage was greater in the former group, although with no statistical significance. One CO2 plus one erbium application compared with one isolated CO2 application showed similar ablation damage but greater residual thermal damage in the latter group (p < 0.05). These observations might contribute to our understanding of the lesions caused in the human skin by erbium and CO2 lasers and eventually help determine the ideal laser combination for the appropriate surgical treatment.     

Q开关Nd:YAG 1064nm和532nm激光对犬心肌切割作用的研究

目的 :研究 10 64nm和 53 2nm波长激光在激光能量为 14 0mJ/pulse(脉冲 )时对犬心肌切割效率。方法 :用Q开关Nd :YAG 10 64和 53 2nm波长脉冲激光分别照射犬离体和在体心肌组织 ,光学显微镜和偏振光学显微镜行组织学分析 ,观察不同条件下激光切割组织的深度和光热对组织的损伤。结果 :离体和在体实验 ,10 64nm波长激光的切割效率高于 53 2nm(p <0 .0 1)。在体和离体实验显示 10 64nm激光能量和重复率相同时 ,所致的切割效率无明显差异 (p >0 .0 5) ,血液对 10 64nm激光的切割效率影响较小。相反 ,在 53 2nm时血液对其影响较大 ,相同的激光能量和重复率 ,离体实验切割效率高于在体 (p <0 .0 1)。 10 64nm激光所致的光热和机械损伤均轻于 53 2nm激光。结论 :在切割效率方面 ,10 64nm激光比 53 2nm更适用于TMLR。 10 64nmQ开关Nd :YAG激光可通过光导纤维传输 ,是TMLR的一个有潜力的激光源     

Coherent convergent-beam time-resolved X-ray diffraction

The use of coherent X-ray lasers for structural biology allows the use of nanometre diameter X-ray beams with large beam divergence. Their application to the structure analysis of protein nanocrystals and single particles raises new challenges and opportunities. We discuss the form of these coherent convergent-beam (CCB) hard X-ray diffraction patterns and their potential use for time-resolved crystallography, normally achieved by Laue (polychromatic) diffraction, for which the monochromatic laser radiation of a free-electron X-ray laser is unsuitable. We discuss the possibility of obtaining single-shot, angle-integrated rocking curves from CCB patterns, and the dependence of the resulting patterns on the focused beam coordinate when the beam diameter is larger or smaller than a nanocrystal, or smaller than one unit cell. We show how structure factor phase information is provided at overlapping interfering orders and how a common phase origin between different shots may be obtained. Their use in refinement of the phase-sensitive intensity between overlapping orders is suggested.     

Incoherent manipulation of the photoactive yellow protein photocycle with dispersed pump-dump-probe spectroscopy

Photoactive yellow protein is the protein responsible for initiating the "blue-light vision" of Halorhodospira halophila. The dynamical processes responsible for triggering the photoactive yellow protein photocycle have been disentangled with the use of a novel application of dispersed ultrafast pump-dump-probe spectroscopy, where the photocycle can be started and interrupted with appropriately tuned and timed laser pulses. This "incoherent" manipulation of the photocycle allows for the detailed spectroscopic investigation of the underlying photocycle dynamics and the construction of a fully self-consistent dynamical model. This model requires three kinetically distinct excited-state intermediates, two (ground-state) photocycle intermediates, I(0) and pR, and a ground-state intermediate through which the protein, after unsuccessful attempts at initiating the photocycle, returns to the equilibrium ground state. Also observed is a previously unknown two-photon ionization channel that generates a radical and an ejected electron into the protein environment. This second excitation pathway evolves simultaneously with the pathway containing the one-photon photocycle intermediates.     

Critical evaluation of indications for the holmium:YAG laser and the neodymium:YAG laser in orthopedic surgery based on an in vitro study]

This is an in vitro study of the biophysical effects of holmium:YAG and neodymium-YAG lasers that was prompted by the poor clinical results obtained with lumbar percutaneous laser discus decompression (PLDD). In the absence of adequate cooling, ablation of tissue with the holmium:YAG laser causes thermal damage to the surrounding tissues. Utilizing the immediate colour-independent laser coupling effect, the holmium:YAG laser removes soft and hard tissue immediately. The low tissue penetrating power (max. 0.32 mm), together with the use of irrigation, avoids thermal problems, and this laser type with its high pulse energy and frequency is to be recommended for arthroscopic surgery. In contrast, the effects of the neodymium:YAG laser are highly dependent on tissue colour. Using this laser on light-coloured tissue only diffuse warming but no ablation of soft tissue was often seen. The depth of tissue penetration seen in our study was 0.58 mm, but is greatly dependent on the duration of application, and is much larger with long application times. In conclusion, we believe that the neodymium:YAG laser is more suitable for percutaneous intradiscal procedures than the holmium:YAG laser. For arthroscopic surgery, the holmium:YAG laser will be the better choice. The effect of each type of laser depends not only on its physical properties, but also on tissue properties (light or dark-coloured, thermal conductivity) and duration of application.     

Protein motions visualized by femtosecond time-resolved crystallography: The case of photosensory vs photosynthetic proteins

Proteins are dynamic objects and undergo conformational changes when functioning. These changes range from interconversion between states in equilibrium to ultrafast and coherent structural motions within one perturbed state. Time-resolved serial femtosecond crystallography at free-electron X-ray lasers can unravel structural changes with atomic resolution and down to femtosecond time scales. In this review, we summarize recent advances on detecting structural changes for phytochrome photosensor proteins and a bacterial photosynthetic reaction center. In the phytochrome structural changes are extensive and involve major rearrangements of many amino acids and water molecules, accompanying the regulation of its biochemical activity, whereas in the photosynthetic reaction center protein the structural changes are smaller, more localized, and are optimized to facilitate electron transfer along the chromophores. The detected structural motions underpin the proteins’ function, providing a showcase for the importance of detecting ultrafast protein structural dynamics.     

Dose‐dependent study of effects of 532‐nm continuous wave laser on rat skin: A mechanistic insight

Visible lasers emitting in the green spectral region are being routinely employed in various medical and defense fields namely treatment of pigmented lesions, tattoo inks, port wine stains, dazzling the target or mob dispersal. Despite their increasing applications, lasers also tend to pose occupational hazards to operators, ancillary personnel, individuals undergoing laser therapies. This study was aimed at investigating the effects of different doses of 532‐nm continuous wave laser on rat skin. The present study demonstrated that higher fluences of 532‐nm continuous wave (CW) laser induces significant tissue damage through induction of tumor necrosis factor‐α, cyclooxygenase‐2, tumor protein (p53), PARP 1, caspase3 which in turn leads to tissue damage and cell death. Furthermore, level of heat shock proteins, pAkt were found up‐regulated as a cope up response to laser‐induced stress. On the basis of the findings, irradiation with 532‐nm CW laser up to 2.5 J/cm² was found within the safe exposure limits. Thus, it is probably the first attempt to demonstrate the tissue damage induced by 532‐nm CW laser on skin, which may help in choosing safe laser dose for certain skin‐based applications and evolving methods to ameliorate laser‐inflicted injuries.     

7 ? resolution in protein two-dimensional-crystal X-ray diffraction at Linac Coherent Light Source

Membrane proteins arranged as two-dimensional crystals in the lipid environment provide close-to-physiological structural information, which is essential for understanding the molecular mechanisms of protein function. Previously, X-ray diffraction from individual two-dimensional crystals did not represent a suitable investigational tool because of radiation damage. The recent availability of ultrashort pulses from X-ray free-electron lasers (XFELs) has now provided a means to outrun the damage. Here, we report on measurements performed at the Linac Coherent Light Source XFEL on bacteriorhodopsin two-dimensional crystals mounted on a solid support and kept at room temperature. By merging data from about a dozen single crystal diffraction images, we unambiguously identified the diffraction peaks to a resolution of 7 Å, thus improving the observable resolution with respect to that achievable from a single pattern alone. This indicates that a larger dataset will allow for reliable quantification of peak intensities, and in turn a corresponding increase in the resolution. The presented results pave the way for further XFEL studies on two-dimensional crystals, which may include pump–probe experiments at subpicosecond time resolution.     

Combining on-line spectroscopy with synchrotron and X-ray free electron laser crystallography

With the recent advances in serial crystallography methods at both synchrotron and X-ray free electron laser sources, more details of intermediate or transient states of the catalytic reactions are being revealed structurally. These structural studies of reaction dynamics drive the need for on-line in crystallo spectroscopy methods to complement the crystallography experiment. The recent applications of combined spectroscopy and crystallography methods enable on-line determination of in crystallo reaction kinetics and structures of catalytic intermediates, sample integrity, and radiation-induced sample modifications, if any, as well as heterogeneity of crystals from different preparations or sample batches. This review describes different modes of spectroscopy that are combined with the crystallography experiment at both synchrotron and X-ray free-electron laser facilities, and the complementary information that each method can provide to facilitate the structural study of enzyme catalysis and protein dynamics.     

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.     

Preclinical evaluation of porcine colon resection using hollow core negative curvature fibre delivered ultrafast laser pulses

Ultrashort pulse lasers offer great promise for tissue resection with exceptional precision and minimal thermal damage. Surgery in the bowel requires high precision and minimal necrotic tissue to avoid severe complications such as perforation. The deployment of ultrashort lasers in minimally invasive or endoscopic procedures has been hindered by the lack of suitable optical fibres for high peak powers. However, recent developments of hollow core microstructured fibres provide potential for delivery of such pulses throughout the body. In this study, analysis of laser ablation via a scanning galvanometer on a porcine colon tissue model is presented. A thermally damaged region (<85 μm) and fine depth control of ablation using the pulse energies 46 and 33 μJ are demonstrated. It is further demonstrated that such pulses suitable for precision porcine colon resection can be flexibly delivered via a hollow core negative curvature fibre (HC‐NCF) and again ablation depth can be controlled with a thermally damaged region <85 μm. Ablation volumes are comparable to that of early stage lesions in the inner lining of the colon. This study concludes that the combination of ultrashort pulses and flexible fibre delivery via HC‐NCF present a viable route to new minimally invasive surgical procedures.     

Evaluation of the Efficacy of Excimer Laser Ablation of Cross-Linked Porcine Cornea

Background

Combination of riboflavin/UVA cross-linking (CXL) and excimer laser ablation is a promising therapy for treating corneal ectasia. The cornea is strengthened by cross-linking, while the irregular astigmatism is reduced by laser ablation. This study aims to compare the efficacy of excimer laser ablation on porcine corneas with and without cross-linking.

Methods and Findings

The porcine cornea was de-epithelialized and treated with 0.1% riboflavin solution for 30 minutes. A half of the cornea was exposed to UVA-radiation for another 30 minutes while the controlled half of the cornea was protected from the UVA using a metal shield. Photo therapeutic keratectomy (PTK) was then performed on the central cornea. Corneal thickness of 5 paired locations on the horizontal line, ±0.5, ±1.0, ±1.5, ±2.0, and ±2.5 mm from the central spot, were measured using optical coherence tomography prior to and after PTK. The ablation depth was then determined by the corneal thickness. There was a 9% difference (P<0.001) in the overall ablation depth between the CXL-half corneas (158±22 µm) and the control-half corneas (174±26 µm). The ablation depths of all 5 correspondent locations on the CXL-half were significantly smaller (P<0.001).

Conclusion

The efficacy of the laser ablation seems to be lower in cross-linked cornea. Current ablation algorithms may need to be modified for cross-linked corneas.     

IR-MALDI-LDI combined with ion mobility orthogonal time-of-flight mass spectrometry

Most MALDI instrumentation uses UV lasers. We have designed a MALDI-IM-oTOF-MS which employs both a Nd:YAG laser pumped optical parametric oscillator (OPOTEK, lambda = 2.8-3.2 microm at 20 Hz) to perform IR-LDI or IR-MALDI and a Nd:YLF laser (Crystalaser, lambda = 249 nm at 200 Hz) for the UV. Ion mobility (IM) gives a fast separation and analysis of biomolecules from complex mixtures in which ions of similar chemical type fall along well-defined "trend lines". Our data shows that ion mobility allows multiply charged monomers and multimers to be resolved; thus, yielding pure spectra of the singly charged protein ion which are virtually devoid of chemical noise. In addition, we have demonstrated that IR-LDI produced similar results as IR-MALDI for the direct tissue analysis of phospholipids from rat brain.