Laser safety aspects for refractive eye surgery with femtosecond laser pulses

We report on laser safety aspects for near infrared femtosecond laser refractive surgery. In particular, the transmittance of microjoule laser pulses at 1040 nm through the cornea during flap procedures based on femtosecond laser induced multiphoton ionization and photodisruption has been determined. When using focusing optics with a numerical aperture of 0.3, more than 20% of the incident NIR photons are propagating towards the retina. In addition, self-focusing, white light and second harmonic generation, and destructive photodisruptive side effects have to be considered when using such high energy laser pulses of amplified laser systems. Microjoule femtosecond laser pulses in combination with low NA objectives have the potential to induce destructive intraocular side effects. Further studies are required to evaluate the damage potential of the transmitted photons absorbed by the retinal pigment epithelium and other intraocular compartments. Because of the fact that flaps can be also generated with low nanojoule energy femtosecond laser pulses of non-amplified MHz lasers in combination with high NA objectives, a compromise between procedure time, pulse energy and numerical apertures has to be found for safe ocular femtosecond laser surgery.     

Two-photon microscopy using fiber-based nanosecond excitation

Two-photon excitation fluorescence (TPEF) microscopy is a powerful technique for sensitive tissue imaging at depths of up to 1000 micrometers. However, due to the shallow penetration, for in vivo imaging of internal organs in patients beam delivery by an endoscope is crucial. Until today, this is hindered by linear and non-linear pulse broadening of the femtosecond pulses in the optical fibers of the endoscopes. Here we present an endoscope-ready, fiber-based TPEF microscope, using nanosecond pulses at low repetition rates instead of femtosecond pulses. These nanosecond pulses lack most of the problems connected with femtosecond pulses but are equally suited for TPEF imaging. We derive and demonstrate that at given cw-power the TPEF signal only depends on the duty cycle of the laser source. Due to the higher pulse energy at the same peak power we can also demonstrate single shot two-photon fluorescence lifetime measurements.OCIS codes: (180.4315) Nonlinear microscopy, (180.2520) Fluorescence microscopy, (060.2350) Fiber optics imaging, (140.3510) Lasers, fiber, (060.4370) Nonlinear optics, fibers     

Multimodal fiber source for nonlinear microscopy based on a dissipative soliton laser

Recent developments in high energy femtosecond fiber lasers have enabled robust and lower-cost sources for multiphoton-fluorescence and harmonic-generation imaging. However, picosecond pulses are better suited for Raman scattering microscopy, so the ideal multimodal source for nonlinear microcopy needs to provide both durations. Here we present spectral compression of a high-power femtosecond fiber laser as a route to producing transform-limited picosecond pulses. These pulses pump a fiber optical parametric oscillator to yield a robust fiber source capable of providing the synchronized picosecond pulse trains needed for Raman scattering microscopy. Thus, this system can be used as a multimodal platform for nonlinear microscopy techniques.OCIS codes: (320.7140) Ultrafast processes in fibers, (180.5655) Raman microscopy, (190.4970) Parametric oscillators and amplifiers     

Lowered threshold energy for femtosecond laser induced optical breakdown in a water based eye model by aberration correction with adaptive optics

In femtosecond laser ophthalmic surgery tissue dissection is achieved by photodisruption based on laser induced optical breakdown. In order to minimize collateral damage to the eye laser surgery systems should be optimized towards the lowest possible energy threshold for photodisruption. However, optical aberrations of the eye and the laser system distort the irradiance distribution from an ideal profile which causes a rise in breakdown threshold energy even if great care is taken to minimize the aberrations of the system during design and alignment. In this study we used a water chamber with an achromatic focusing lens and a scattering sample as eye model and determined breakdown threshold in single pulse plasma transmission loss measurements. Due to aberrations, the precise lower limit for breakdown threshold irradiance in water is still unknown. Here we show that the threshold energy can be substantially reduced when using adaptive optics to improve the irradiance distribution by spatial beam shaping. We found that for initial aberrations with a root-mean-square wave front error of only one third of the wavelength the threshold energy can still be reduced by a factor of three if the aberrations are corrected to the diffraction limit by adaptive optics. The transmitted pulse energy is reduced by 17% at twice the threshold. Furthermore, the gas bubble motions after breakdown for pulse trains at 5 kilohertz repetition rate show a more transverse direction in the corrected case compared to the more spherical distribution without correction. Our results demonstrate how both applied and transmitted pulse energy could be reduced during ophthalmic surgery when correcting for aberrations. As a consequence, the risk of retinal damage by transmitted energy and the extent of collateral damage to the focal volume could be minimized accordingly when using adaptive optics in fs-laser surgery.OCIS codes: (110.1080) Active or adaptive optics, (140.3440) Laser-induced breakdown, (140.3300) Laser beam shaping, (330.4460) Ophthalmic optics and devices, (170.4470) Ophthalmology, (330.3350) Vision - laser damage     

In vivo three- and four-photon fluorescence microscopy using a 1.8 µm femtosecond fiber laser system

Multiphoton microscopy has enabled us to image cellular dynamics in vivo. However, the excitation wavelength for imaging with commercially available lasers is mostly limited between 0.65–1.04 µm. Here we develop a femtosecond fiber laser system that produces ∼150 fs pulses at 1.8 µm. Our system starts from an erbium-doped silica fiber laser, and its wavelength is converted to 1.8 µm using a Raman shift fiber. The 1.8 µm pulses are amplified with a two-stage Tm:ZBLAN fiber amplifier. The final pulse energy is ∼1 µJ, sufficient for in vivo imaging. We successfully observe TurboFP635-expressing cortical neurons at a depth of 0.7 mm from the brain surface by three-photon excitation and Clover-expressing astrocytes at a depth of 0.15 mm by four-photon excitation.     

Femtosecond laser disruption of mitochondria in living cells

Studying intact organelles and their function in a living cell is essential to understand cell dynamics. Femtosecond laser pulses in the near-infrared region have potential applications in nanosurgery in cell biology. We investigate the disruption of subcellular organelles in living cells by focusing femtosecond laser pulses inside the cells. When intense femtosecond laser pulses are tightly focused at the targeted organelles, the intensity around the focal volume can become high enough to result in a permanently damaged region in the cell with sub-micron size. Femtosecond laser disruption offers precise control of material removal or modifications of organelles in the cell. The subcellular disruption of mitochondria in living cells was demonstrated by restaining experiments. Femtosecond laser-based nanosurgery has the possibility to provide information on the function and dynamics of organelles in living cells.     

Characterization of multiphoton microscopy in the bone marrow following intravital laser osteotomy

The bone marrow is an important site where all blood cells are formed from hematopoietic stem cells and where hematologic malignancies such as leukemia emerge. It is also a frequent site for metastasis of solid tumors such as breast cancer and prostate cancer. Intravital microscopy is a powerful tool for studying the bone marrow with single cell and sub-cellular resolution. To improve optical access to this rich biological environment, plasma-mediated laser ablation with sub-microjoule femtosecond pulses was used to thin cortical bone. By locally removing a superficial layer of bone (local laser osteotomy), significant improvements in multiphoton imaging were observed in individual bone marrow compartments in vivo. This work demonstrates the utility of scanning laser ablation of hard tissue with sub-microjoule pulses as a preparatory step to imaging.OCIS codes: (120.0120) Instrumentation, measurement, and metrology; (170.1020) Ablation of tissue; (180.5810) Scanning microscopy     

Femtosecond diode-based time lens laser for multiphoton microscopy

We demonstrate a near-infrared, femtosecond, diode laser-based source with kW peak power for two-photon microscopy. At a wavelength of 976 nm, the system produces sub-ps pulses operating at a repetition rate of 10 MHz with kilowatt class peak powers suitable for deep tissue two-photon microscopy. The system, integrated with a laser-scanning microscope, images to a depth of 900 µm in a fixed sample of PLP-eGFP labeled mouse brain tissue. This represents a significant development that will lead to more efficient, compact, and accessible laser sources for biomedical imaging.     

Three-color femtosecond source for simultaneous excitation of three fluorescent proteins in two-photon fluorescence microscopy

We demonstrate a fiber-based, three-color femtosecond source for simultaneous imaging of three fluorescent proteins (FPs) using two-photon fluorescence microscopy (2PM). The three excitation wavelengths at 775 nm, 864 nm and 950 nm, are obtained through second harmonic generation (SHG) of the 1550-nm pump laser and the 1728-nm and 1900-nm solitons generated through soliton self-frequency shift (SSFS) in a large-mode-area (LMA) fiber. These energetic pulses are well matched to the two-photon excitation peaks of red, cyan and yellow fluorescent proteins (TagRFPs, TagCFPs, and TagYFPs) for efficient excitation. We demonstrate simultaneous 2PM of human melanoma cells expressing a “rainbow” combination of these three fluorescent proteins.OCIS codes: (060.4370) Nonlinear optics, fibers; (180.2520) Fluorescence microscopy; (180.4315) Nonlinear microscopy; (190.2620) Harmonic generation and mixing     

Phototoxicity induced in living HeLa cells by focused femtosecond laser pulses: a data-driven approach

Nonlinear optical microscopy is a powerful label-free imaging technology, providing biochemical and structural information in living cells and tissues. A possible drawback is photodamage induced by high-power ultrashort laser pulses. Here we present an experimental study on thousands of HeLa cells, to characterize the damage induced by focused femtosecond near-infrared laser pulses as a function of laser power, scanning speed and exposure time, in both wide-field and point-scanning illumination configurations. Our data-driven approach offers an interpretation of the underlying damage mechanisms and provides a predictive model that estimates its probability and extension and a safety limit for the working conditions in nonlinear optical microscopy. In particular, we demonstrate that cells can withstand high temperatures for a short amount of time, while they die if exposed for longer times to mild temperatures. It is thus better to illuminate the samples with high irradiances: thanks to the nonlinear imaging mechanism, much stronger signals will be generated, enabling fast imaging and thus avoiding sample photodamage.     

Nanoprocessing of subcellular targets using femtosecond laser pulses

In this paper we review the work done in our laboratory on femtosecond laser dissection within single cells and living organisms. Precise dissection of biological material with ultrashort laser pulses requires a clear understanding of the pulse-energy dependence of the onset and extent of plasma-mediated ablation (i.e., the removal of material). We carried out a systematic study of the energy dependence of the plasma-mediated ablation of fluorescently-labeled subcellular structures in the cytoskeleton and in nuclei of fixed endothelial cells using femtosecond, near-infrared laser pulses focused through a high-numerical aperture objective lens (1.4 NA). We performed laser nanosurgery in live cells, where we ablated a single mitochondria and severed cytoskeletal filaments without compromising the cell membrane or the cell's viability. We also cut dendrites in living C. elegans without affecting the neighboring neurons. This nanoprocessing technique enables non-invasive manipulation of the structural machinery of cells and tissues down to several hundred nanometer resolution.     

Mesoporous carbon nanospheres deposited onto D-shaped fibers for femtosecond pulse generation

We demonstrate nonlinear optical modulation by combining mesoporous carbon nanospheres (MCNs) and D-shaped fibers (DFs). The MCNs are prepared by the silica-assisted strategy and the DFs are fabricated through a precision wheel polishing method. When the MCNs are deposited onto the DF as the saturable absorbers (SAs), the SAs possess broadband linear absorption and nonlinear saturable absorption properties. As the DF-MCNs SA is integrated into the laser cavities, ultrafast lasers at 1.56 and 2 μm were realized with minimal pulse duration down to a few hundreds of femtoseconds. Compared with the film and microfiber-based MCNs-SAs, the DF-MCNs SAs exhibit greater robustness and stronger evanescent field, and are more effective at generating femtosecond pulses. Our results verify that DF-MCNs as a kind of cost-effective and easily-prepared SA would be of great importance for stable and high-power femtosecond fiber lasers.

We demonstrate nonlinear optical modulation by combining mesoporous carbon nanospheres (MCNs) and D-shaped fibers (DFs).     

Improved two-photon imaging of living neurons in brain tissue through temporal gating

We optimize two-photon imaging of living neurons in brain tissue by temporally gating an incident laser to reduce the photon flux while optimizing the maximum fluorescence signal from the acquired images. Temporal gating produces a bunch of ~10 femtosecond pulses and the fluorescence signal is improved by increasing the bunch-pulse energy. Gating is achieved using an acousto-optic modulator with a variable gating frequency determined as integral multiples of the imaging sampling frequency. We hypothesize that reducing the photon flux minimizes the photo-damage to the cells. Our results, however, show that despite producing a high fluorescence signal, cell viability is compromised when the gating and sampling frequencies are equal (or effectively one bunch-pulse per pixel). We found an optimum gating frequency range that maintains the viability of the cells while preserving a pre-set fluorescence signal of the acquired two-photon images. The neurons are imaged while under whole-cell patch, and the cell viability is monitored as a change in the membrane’s input resistance.OCIS codes: (170.3880) Medical and biological imaging, (170.3660) Light propagation in tissues, (140.0140) Lasers and laser optics, (170.2520) Fluorescence microscopy, (170.6930) Tissue, (110.0110) Imaging systems     

Widely-tunable synchronisation-free picosecond laser source for multimodal CARS,SHG, and two-photon microscopy

We demonstrate a continuous wave (CW) seeded synchronization-free optical parametric amplifier (OPA) pumped by a picosecond, 1 µm laser and show its performance when used as a simple yet powerful source for label-free coherent anti-Stokes Raman scattering (CARS), concurrent second harmonic generation (SHG), and two-photon fluorescence microscopy in an epi-detection geometry. The average power level of above 175 mW, spectral resolution of 8 cm⁻¹, and 2 ps pulse duration are well optimized for CARS microscopy in bio-science and bio-medical imaging systems. Our OPA is a much simpler setup than either the “gold-standard” laser and optical parametric oscillator (OPO) combination traditionally used for CARS imaging, or the more recently developed OPA systems pumped with femtosecond pulses [1]. Rapid and accurate tuning between resonances was achieved by changing the poled channels and temperature of the periodically-poled lithium niobate (PPLN) OPA crystal together with the OPA seed wavelength. The Pump-Stokes frequency detuning range fully covered the C-H stretching band used for the imaging of lipids. By enabling three multiphoton techniques using a compact, synchronization free laser source, our work paves the way for the translation of label-free multi-photon microscopy imaging from biomedical research to an imaging based diagnostic tool for use in the healthcare arena.     

Suppression of the non-linear background in a multimode fibre CARS endoscope

Multimode fibres show great potential for use as miniature endoscopes for imaging deep in tissue with minimal damage. When used for coherent anti-Stokes Raman scattering (CARS) microscopy with femtosecond excitation sources, a high band-width probe is required to efficiently focus the broadband laser pulses at the sample plane. Although graded-index (GRIN) fibres have a large bandwidth, it is accompanied by a strong background signal from four-wave mixing and other non-linear processes occurring inside the fibre. We demonstrate that using a composite probe consisting of a GRIN fibre with a spliced on step-index fibre reduces the intensity of the non-linear background by more than one order of magnitude without significantly decreasing the focusing performance of the probe. Using this composite probe we acquire CARS images of biologically relevant tissue such as myelinated axons in the brain with good contrast.     

Dual-wavelength multimodal multiphoton microscope with SMA-based depth scanning

We report on a multimodal multiphoton microscopy (MPM) system with depth scanning. The multimodal capability is realized by an Er-doped femtosecond fiber laser with dual output wavelengths of 1580 nm and 790 nm that are responsible for three-photon and two-photon excitation, respectively. A shape-memory-alloy (SMA) actuated miniaturized objective enables the depth scanning capability. Image stacks combined with two-photon excitation fluorescence (TPEF), second harmonic generation (SHG), and third harmonic generation (THG) signals have been acquired from animal, fungus, and plant tissue samples with a maximum depth range over 200 µm.     

Possible retina damage potential of the femtosecond laser in situ keratomileusis (fs-LASIK) refractive surgery

Since the early 1990s, the laser in situ keratomileusis (LASIK) procedure has been successfully implemented in refractive eye surgery to correct ametropia. Rapid development in new laser technology enables the application of ultrashort laser pulses in the femtosecond (fs) regime that marks advancement in conventional standard procedures of refractive surgery by eliminating the use of mechanical knives. With the fs-LASIK procedure, ultrashort laser pulses focus in the near-infrared spectral range and create a laser-induced breakdown (LIB) that disrupts the corneal tissue. During the cutting process not all of the pulse energy is deposited in the cornea. Approximately half the remaining energy is propagated through the eye and reaches the retina, as well as the strongly absorbing layers behind.In the current standard refractive surgery procedures, such as photorefractive keratomileusis (PRK) and LASIK, retina damage is reduced because of the complete absorption of ultraviolet irradiation by corneal tissue during ablation.The aim of this project was to investigate possible retina damage under fs-LASIK conditions and to optimize external parameters during surgery, such as pulse energy and numerical aperture, to reduce risk potential. The process was simulated with a conventional fs-laser system integrated into an experimental set-up. To investigate the retina damage thresholds, in vivo investigations were carried out systematically on enucleated porcine eyes. Damage thresholds were determined in terms of macroscopic and histopathological observations. The results of these studies indicate possible retina damage.     

Self-synchronized reflection-mode acousto-optic imaging system utilizing nanosecond laser pulses

We present an acousto-optic imaging system operating in reflection-mode and utilizing a pair of compact, triggerable lasers with 532 and 1064 nm wavelength and nanosecond pulse duration. The system maps the fluence rate distribution of light transmitted through optically scattering samples. The imaging is performed using an acousto-optic probe comprising an ultrasound linear array with attached optical fiber on one side and a camera on the other. The described hardware configuration images samples with access restricted to one side only and ensures mobility of the entire setup. The major challenge of the introduced approach is mitigating the effects of laser parameter instabilities and precise synchronization of ultrasound and laser pulses. We solved this issue by developing an electronic feedback circuit and a microcontroller-based synchronization and control system triggering the ultrasound scanner. Schematics and details regarding control algorithms are introduced. The imaging performance of the system is demonstrated on examples of results obtained for solid, acoustically-homogeneous and optically scattering phantoms with and without light absorbing inclusions present. Adjusting the size and location of the region of interest within the camera sensor matrix and the number of laser pulses illuminating every frame allows for significant improvements in terms of the achievable peak signal to noise ratio. We demonstrate that the developed synchronization algorithm and system play a crucial role in ensuring imaging quality and accuracy.     

Preliminary fsLIBS study on bone tumors

The aim of this study is to evaluate the capability of femtosecond Laser Induced Breakdown Spectroscopy (fsLIBS) to discriminate between normal and cancerous bone, with implications to femtosecond laser surgery procedures. The main advantage of using femtosecond lasers for surgery is that the same laser that is being used to ablate can also be used for a feedback system to prevent ablation of certain tissues. For bone tumor removal, this technique has the potential to reduce the number of repeat surgeries that currently must be performed due to incomplete removal of the tumor mass. In this paper, we performed fsLIBS on primary bone tumor, secondary tumor in bone, and normal bone. These tissues were excised from consenting patients and processed through the UC Davis Cancer Center Biorepository. For comparison, each tumor sample had a matched normal bone sample. fsLIBS was performed to characterize the spectral signatures of each tissue type. A minimum of 20 spectra were acquired for each sample. We did not detect significant differences between the fsLIBS spectra of secondary bone tumors and their matched normal bone samples, likely due to the heterogeneous nature of secondary bone tumors, with normal and cancerous tissue intermingling. However, we did observe an increase in the fsLIBS magnesium peak intensity relative to the calcium peak intensity for the primary bone tumor samples compared to the normal bone samples. These results show the potential of using femtosecond lasers for both ablation and a real-time feedback control system for treatment of primary bone tumors.OCIS codes: (300.0300) Spectroscopy, (300.6365) Spectroscopy, laser induced breakdown, (170.6510) Spectroscopy, tissue diagnostics, (170.4730) Optical pathology, (170.1020) Ablation of tissue     

Synchronously pumped Raman laser for simultaneous degenerate and nondegenerate two-photon microscopy

Two-photon fluorescence microscopy is a nonlinear imaging modality frequently used in deep-tissue imaging applications. A tunable-wavelength multicolor short-pulse source is usually required to excite fluorophores with a wide range of excitation wavelengths. This need is most typically met by solid-state lasers, which are bulky, expensive, and complicated systems. Here, we demonstrate a compact, robust fiber system that generates naturally synchronized femtosecond pulses at 1050 nm and 1200 nm by using a combination of gain-managed and Raman amplification. We image the brain of a mouse and view the blood vessels, neurons, and other cell-like structures using simultaneous degenerate and nondegenerate excitation.