Dissection of DNA damage and repair pathways in live cells by femtosecond laser microirradiation and free-electron modeling.

Understanding and predicting the outcome of the interaction of light with DNA has a significant impact on the study of DNA repair and radiotherapy. We report on a combination of femtosecond pulsed laser microirradiation at different wavelengths, quantitative imaging, and numerical modeling that yields a comprehensive picture of photon-mediated and free-electron-mediated DNA damage pathways in live cells. Laser irradiation was performed under highly standardized conditions at four wavelengths between 515 nm and 1,030 nm, enabling to study two-photon photochemical and free-electron-mediated DNA damage in situ. We quantitatively assessed cyclobutane pyrimidine dimer (CPD) and γH2AX-specific immunofluorescence signals to calibrate the damage threshold dose at these wavelengths and performed a comparative analysis of the recruitment of DNA repair factors xeroderma pigmentosum complementation group C (XPC) and Nijmegen breakage syndrome 1 (Nbs1). Our results show that two-photon-induced photochemical CPD generation dominates at 515 nm, while electron-mediated damage dominates at wavelengths ≥620 nm. The recruitment analysis revealed a cross talk between nucleotide excision and homologous recombination DNA repair pathways at 515 nm. Numerical simulations predicted electron densities and electron energy spectra, which govern the yield functions of a variety of direct electron-mediated DNA damage pathways and of indirect damage by •OH radicals resulting from laser and electron interactions with water. Combining these data with information on free electron-DNA interactions gained in artificial systems, we provide a conceptual framework for the interpretation of the wavelength dependence of laser-induced DNA damage that may guide the selection of irradiation parameters in studies and applications that require the selective induction of DNA lesions.

Mechanisms of corneal intrastromal laser dissection for refractive surgery: ultra-high-speed photographic investigation at up to 50 million frames per second.

Every year, more than a million refractive eye surgeries using femtosecond lasers are performed but the intrastromal cutting process remains an area of development. We investigated the mechanisms of laser dissection in cornea by ultra-high-speed photography. We found that the intrastromal bubble forms multiple lobes along the elongated laser plasma and the overlying lobes expand along the corneal lamellae. Videography demonstrated that the cutting process relies on crack propagation in the stroma along the bubble lobes with the crack originating from the pre-existing bubble layer. These insights are important for further improvement of the cutting mechanisms in refractive surgery.

Optical Vortex Beam for Gentle and Ultraprecise Intrastromal Corneal Dissection in Refractive Surgery.

Purpose: We introduce a novel focus shaping concept for intrastromal corneal dissection that facilitates cleavage along corneal lamellae, and we analyze laser-tissue interactions governing cutting effectiveness and mechanical side effects. Methods: Focus shaping was achieved by a spiral phase plate that converts an incident Gaussian beam into a Laguerre-Gaussian beam with a helical phase. Such vortex beams have zero intensity at their center, are propagation invariant, and possess a ring focus equal in length to the Gaussian focus but with a larger diameter. Cutting precision and the required absorbed energy for flap dissection were compared for Gaussian and vortex beams on ex vivo porcine corneal specimens at pulse durations between 480 fs and 9 ps. Cutting quality and bubble formation were characterized by scanning electron microscopy and macro photography. Results: With the vortex beam, the cuts were much smoother. Bubble formation was markedly reduced because cutting can be performed close to the bubble threshold, whereas with the Gaussian beam energies well above threshold are needed. Although the incident energy at the flap dissection threshold was slightly larger for the vortex beam, the absorbed energy was much smaller and contributed more effectively to cutting. This reduced plasma-induced pressure more than sevenfold. Conclusions: The vortex beam approach for corneal dissection is a simple, versatile, and cost-effective way of improving the precision of refractive surgery while reducing bubble formation and pressure-related mechanical side effects. Translational Relevance: Phase plates for propagation invariant vortex beams are easily implemented in the beam path of next-generation clinical devices.

Correction of hyperopia by intrastromal cutting and liquid filler injection.

Correction of hyperopia requires an increase of the refractive power by steepening of the corneal surface. Present refractive surgical techniques based on corneal ablation (LASIK) or intrastromal lenticule extraction (SMILE) are problematic due to epithelial regrowth. Recently, it was shown that correction of low hyperopia can be achieved by implanting intracorneal inlays or allogeneic lenticules. We demonstrate a steepening of the anterior corneal surface after injection of a transparent, liquid filler material into a laser-dissected intrastromal pocket. We performed the study on ex-vivo porcine eyes. The increase of the refractive power was evaluated by optical coherence tomography (OCT). For a circular pocket, injection of 1 µl filler material increased the refractive power by +4.5 diopters. An astigmatism correction is possible when ellipsoidal intrastromal pockets are created. Injection of 2 µl filler material into an ellipsoidal pocket increased the refractive power by +10.9 dpt on the short and +5.1 dpt on the long axis. OCT will enable to monitor the refractive change during filler injection and is thus a promising technique for real-time dosimetry.

Probing the immune and healing response of murine intestinal mucosa by time-lapse 2-photon microscopy of laser-induced lesions with real-time dosimetry.

Gut mucosa is an important interface between body and environment. Immune response and healing processes of murine small intestinal mucosa were investigated by intravital time-lapse two-photon excited autofluorescence microscopy of the response to localized laser-induced damage. Epithelial lesions were created by 355-nm, 500-ps pulses from a microchip laser that produced minute cavitation bubbles. Size and dynamics of these bubbles were monitored using a novel interferometric backscattering technique with 80 nm resolution. Small bubbles (< 2.5 µm maximum radius) merely resulted in autofluorescence loss of the target cell. Larger bubbles (7-25 µm) affected several cells and provoked immigration of immune cells (polymorphonuclear leucocytes). Damaged cells were expelled into the lumen, and the epithelium healed within 2 hours by stretching and migration of adjacent epithelial cells.

A new nanosecond UV laser at 355 nm: early results of corneal flap cutting in a rabbit model.

PURPOSE: A new 355 nm UV laser was used for corneal flap cutting in an animal model and tested for clinical and morphologic alterations. METHODS: Corneal flaps were created (Chinchilla Bastards; n = 25) with an UV nanosecond laser at 355 nm (150 kHz, pulse duration 850 ps, spot-size 1 µm, spot spacing 6 × 6 µm, side cut Δz 1 µm; cutting depth 130 µm) and pulse energies of 2.2 or 2.5 µJ, respectively. Following slit-lamp examination, animals were killed at 6, 12, and 24 hours after treatment. Corneas were prepared for histology (hematoxylin and eosin [HE], TUNEL-assay) and evaluated statistically, followed by ultrastructural investigations. RESULTS: Laser treatment was tolerated well, flap lift was easier at 2.5 µJ compared with 2.2 µJ. Standard HE at 24 hours revealed intact epithelium in the horizontal cut, with similar increase in corneal thickness at both energies. Irrespective of energy levels, TUNEL assay revealed comparable numbers of apoptotic cells in the horizontal and vertical cut at 6, 12, and 24 hours, becoming detectable in the horizontal cut as an acellular stromal band at 24 hours. Ultrastructural analysis revealed regular morphology in the epi- and endothelium, while in the stroma, disorganized collagen lamellae were detectable representing the horizontal cut, again irrespective of energy levels applied. CONCLUSIONS: This new UV laser revealed no epi- nor endothelial damage at energies feasible for corneal flap cutting. Observed corneal swelling was lower compared with existing UV laser studies, albeit total energy applied here was much higher. Observed loss of stromal keratinocytes is comparable with available laser systems. Therefore, this new laser is suitable for refractive surgery, awaiting its test in a chronic environment.

Femtosecond-laser-induced nanocavitation in water: implications for optical breakdown threshold and cell surgery.

We determined the bubble radius R_(max) for femtosecond optical breakdown in water at 347, 520, and 1040 nm with an unprecedented accuracy (+/-10 nm). At threshold, R_(max) was smaller than the diffraction-limited focus radius and ranged from 190 nm to 320 nm. The increase of R_(max) with laser energy E_(L) is slowest at 347 nm, providing optimum control of cell surgery. Experimental results agree with a model of bubble formation in heated and thermoelastically stretched liquids. Theory predicts a threshold temperature T_(th) approximately equal to 168 degrees C. For T>300 degrees C, a phase explosion sets in, and R_(max) increases rapidly with E_(L).

Principles of laser-induced separation and transport of living cells.

Separation and transport of defined populations of living cells grown on a thin membrane can be achieved by laser microdissection (LMD) of the sample of interest, followed by a laser-induced forward transport process [laser pressure "catapulting" (LPC)] of the dissected cell cluster. We investigate the dynamics of LMD and LPC with focused and defocused UV-A laser pulses by means of time-resolved photography. Catapulting is driven by plasma formation when tightly focused pulses are used, and by confined thermal ablation at the bottom of the sample for defocused catapulting. With both modalities, the initial specimen velocity amounts to about 50 to 60 ms. Time-resolved photography of live cell catapulting reveals that in defocused catapulting, strong shear forces arise when the sample is accelerated out of the culture medium covering the cells. By contrast, pulses focused at the periphery of the specimen cause a fast rotational movement that minimizes the flow of culture medium parallel to the sample surface, and thus the resulting shear stresses. Therefore, the recultivation rate of catapulted cells is much higher when focused pulses are used. Compared to collateral damage by mechanical forces, side effects by heat and UV exposure of the cells play only a minor role.

Principles of laser microdissection and catapulting of histologic specimens and live cells.

Rapid contact- and contamination-free procurement of specific samples of histologic material for proteomic and genomic analysis as well as separation and transport of living cells can be achieved by laser microdissection (LMD) of the sample of interest followed by a laser-induced forward transport process [laser pressure "catapulting," (LPC)] of the dissected material. We investigated the dynamics of LMD and LPC with focused and defocused laser pulses by means of time-resolved photography. The working mechanism of microdissection was found to be plasma-mediated ablation. Catapulting is driven by plasma formation, when tightly focused pulses are used, and by ablation at the bottom of the sample for moderate and strong defocusing. Driving pressures of several hundred megapascals accelerate the specimen to initial velocities of 100-300 m/s before it is rapidly slowed down by air friction. With strong defocusing, driving pressure and initial flight velocity decrease considerably. On the basis of a characterization of the thermal and optical properties of the histologic specimens and supporting materials used, we calculated the temporal evolution of the heat distribution in the sample. After laser microdissection and laser pressure catapulting (LMPC), the samples were inspected by scanning electron microscopy. Catapulting with tightly focused or strongly defocused pulses results in very little collateral damage, while slight defocusing involves significant heat and UV exposure of up to about 10% of the specimen volume, especially if samples are catapulted directly from a glass slide. Time-resolved photography of live-cell catapulting revealed that in defocused catapulting strong shear forces originate from the flow of the thin layer of culture medium covering the cells. By contrast, pulses focused at the periphery of the specimen cause a fast rotational movement that makes the specimen wind its way out of the culture medium, thereby undergoing much less shear stresses. Therefore, the recultivation rate of catapulted cells was much higher when focused pulses were used.