RESUMO
Multiphoton microscopes employ femtosecond lasers as light sources because the high peak power of the ultrashort pulse allows for multiphoton excitation of fluorescence in the examined sample. However, such short pulses are susceptible to broadening in a microscope's highly dispersive optical elements and require careful dispersion management, otherwise decreasing excitation efficiency. Here, we have developed a 10 nJ Yb:fiber picosecond laser with an integrated pulse picker unit and evaluated its performance in multiphoton microscopy. Our results show that performance comparable to femtosecond pulses can be obtained with picosecond pulses only by reducing the pulse repetition rate and that such pulses are significantly less prone to the effect of chromatic dispersion. These findings proved that the temporal pulse compression is not always efficient, and it can be omitted by using a smaller and easier-to-use all-fiber setup.
RESUMO
We characterized and analyzed the effect of intracavity spectral filtering in the Er:fiber laser mode-locked with a semiconductor saturable absorber mirror (SESAM). We studied the dispersive properties of bandpass filters and their influence on the characteristics of generated soliton pulses. Our analysis showed that various sideband structures were induced by the filter dispersion profiles and shaped through the interaction of the soliton with the dispersive wave. In addition, intracavity filtering improved the intensity and phase noise of the laser significantly, and we showed optimal filtering conditions for both types of noise. By adding a 10â nm bandpass filter to the laser resonator, the intensity and phase noise were improved 2- and 2.6 times, respectively.
RESUMO
High-resolution imaging techniques capable of detecting identifiable endogenous fluorophores in the eye along with genetic testing will dramatically improve diagnostic capabilities in the ophthalmology clinic and accelerate the development of new treatments for blinding diseases. Two-photon excitation (TPE)-based imaging overcomes the filtering of ultraviolet light by the lens of the human eye and thus can be utilized to discover defects in vitamin A metabolism during the regeneration of the visual pigments required for the detection of light. Combining TPE with fluorescence lifetime imaging (FLIM) and spectral analyses offers the potential of detecting diseases of the retina at earlier stages before irreversible structural damage has occurred. The main barriers to realizing the benefits of TPE for imaging the human retina arise from concerns about the high light exposure typically needed for informative TPE imaging and the requirement to correlate the ensuing data with different states of health and disease. To overcome these hurdles, we improved TPE efficiency by controlling temporal properties of the excitation light and employed phasor analyses to FLIM and spectral data in mouse models of retinal diseases. Modeling of retinal photodamage revealed that plasma-mediated effects do not play a role and that melanin-related thermal effects are mitigated by reducing pulse repetition frequency. By using noninvasive TPE imaging we identified molecular components of individual granules in the retinal pigment epithelium and present their analytical characteristics.
Assuntos
Biópsia/métodos , Imagem Óptica/métodos , Retina/diagnóstico por imagem , Animais , Modelos Animais de Doenças , Corantes Fluorescentes , Camundongos , Camundongos Endogâmicos C57BL , Retina/química , Doenças Retinianas/diagnóstico por imagem , Epitélio Pigmentado da Retina/química , Epitélio Pigmentado da Retina/diagnóstico por imagemRESUMO
Droplet microfluidics disrupted analytical biology with the introduction of digital polymerase chain reaction and single-cell sequencing. The same technology may also bring important innovation in the analysis of bacteria, including antibiotic susceptibility testing at the single-cell level. Still, despite promising demonstrations, the lack of a high-throughput label-free method of detecting bacteria in nanoliter droplets prohibits analysis of the most interesting strains and widespread use of droplet technologies in analytical microbiology. We use a sensitive and fast measurement of scattered light from nanoliter droplets to demonstrate reliable detection of the proliferation of encapsulated bacteria. We verify the sensitivity of the method by simultaneous readout of fluorescent signals from bacteria expressing fluorescent proteins and demonstrate label-free readout on unlabeled Gram-negative and Gram-positive species. Our approach requires neither genetic modification of the cells nor the addition of chemical markers of metabolism. It is compatible with a wide range of bacterial species of clinical, research, and industrial interest, opening the microfluidic droplet technologies for adaptation in these fields.
Assuntos
Bactérias Gram-Negativas/isolamento & purificação , Bactérias Gram-Positivas/isolamento & purificação , Ensaios de Triagem em Larga Escala , Técnicas Analíticas Microfluídicas , Nanopartículas/química , Análise de Célula Única , Bactérias Gram-Negativas/citologia , Bactérias Gram-Positivas/citologia , Tamanho da Partícula , Propriedades de SuperfícieRESUMO
In this Letter, we demonstrate an all-polarization-maintaining, stretched-pulse Tm-doped fiber laser generating â¼200 fs pulses centered at 1945 nm. As a saturable absorber, a graphene/poly(methyl methacrylate) composite was used. To the best of our knowledge, this is the first demonstration of stretched-pulse operation of a graphene-based fiber laser at 2 µm.
RESUMO
We report a study on a hybrid mode-locked fiber laser with two saturable absorbers: slow and fast, integrated in a single device. Amorphous antimony telluride (Sb(2)Te(3)) layer was deposited on side-polished fiber to form the slow saturable absorber due to the third order nonlinear susceptibility of Sb(2)Te(3). Additionally, an unsymmetrical design of the device causes polarization-dependent losses and together with polarization controller allows to use a nonlinear polarization evolution to form the artificial fast saturable absorber. Sub-200 fs soliton pulses with 0.27 nJ of pulse energy were generated in the hybrid mode-locked Er-doped fiber laser. Differences in the dynamics of mode-locked laser are further investigated with the use of slow and fast saturable absorbers solely, and compared with the hybrid device. Joint operation of two saturable absorbers enhances the laser performance and stability. The conducted experiments allowed to define roles of each mechanism on the pulse shaping in the laser cavity.
RESUMO
We report on the generation of dissipative solitons in an Er-doped fiber laser mode-locked by antimony telluride (Sb 2 Te 3) topological insulator in the near-zero dispersion regime. Layers of Sb 2 Te 3 were deposited on a side-polished (D-shaped) fiber using a pulsed magnetron sputtering technique. Sub-170-fs pulses with 34-nm full width at half-maximum (FWHM) and 0.21 nJ of pulse energy were obtained from an all-fiber, ring-shaped laser cavity after the compression in a single-mode fiber.
RESUMO
Noninvasive imaging of endogenous retinal fluorophores, including vitamin A derivatives, is vital to developing new treatments for retinal diseases. Here, we present a protocol for obtaining in vivo two-photon excited fluorescence images of the fundus in the human eye. We describe steps for laser characterization, system alignment, positioning human subjects, and data registration. We detail data processing and demonstrate analysis with example datasets. This technique allays safety concerns by allowing for the acquisition of informative images at low laser exposure. For complete details on the use and execution of this protocol, please refer to Boguslawski et al. (2022).1.
RESUMO
In this work we demonstrate comprehensive studies on graphene oxide (GO) and reduced graphene oxide (rGO) based saturable absorbers (SA) for mode-locking of Er-doped fiber lasers. The paper describes the fabrication process of both saturable absorbers and detailed comparison of their parameters. Our results show, that there is no significant difference in the laser performance between the investigated SA. Both provided stable, mode-locked operation with sub-400 fs soliton pulses and more than 9 nm optical bandwidth at 1560 nm center wavelength. It has been shown that GO might be successfully used as an efficient SA without the need of its reduction to rGO. Taking into account simpler manufacturing technology and the possibility of mass production, GO seems to be a good candidate as a cost-effective material for saturable absorbers for Er-doped fiber lasers.
Assuntos
Tecnologia de Fibra Óptica/instrumentação , Grafite/química , Lasers de Estado Sólido , Absorção , Desenho de Equipamento , Análise de Falha de Equipamento , Luz , Oxirredução , Óxidos/químicaRESUMO
Severe non-healing infections are often caused by multiple pathogens or by genetic variants of the same pathogen exhibiting different levels of antibiotic resistance. For example, polymicrobial diabetic foot infections double the risk of amputation compared to monomicrobial infections. Although these infections lead to increased morbidity and mortality, standard antimicrobial susceptibility methods are designed for homogenous samples and are impaired in quantifying heteroresistance. Here, we propose a droplet-based label-free method for quantifying the antibiotic response of the entire population at the single-cell level. We used Pseudomonas aeruginosa and Staphylococcus aureus samples to confirm that the shape of the profile informs about the coexistence of diverse bacterial subpopulations, their sizes, and antibiotic heteroresistance. These profiles could therefore indicate the outcome of antibiotic treatment in terms of the size of remaining subpopulations. Moreover, we studied phenotypic variants of a S. aureus strain to confirm that the profile can be used to identify tolerant subpopulations, such as small colony variants, associated with increased risks for the development of persisting infections. Therefore, the profile is a versatile instrument for quantifying the size of each bacterial subpopulation within a specimen as well as their individual and joined heteroresistance.
Assuntos
Infecções Estafilocócicas , Staphylococcus aureus , Humanos , Staphylococcus aureus/genética , Pseudomonas aeruginosa , Antibacterianos/farmacologia , Bactérias , Testes de Sensibilidade MicrobianaRESUMO
We report the development of a widely-tunable femtosecond fiber laser system and its application for two-photon vision studies. The source is based on an Er-doped fiber laser with spectral shift up to 2150 nm, followed by a second harmonic generation module to generate a frequency-doubled beam tunable from 872 to 1075 nm. The source delivers sub-230 fs pulses with nearly-constant duration over the entire tuning range, with output powers between 0.68-1.24 mW, which corresponds to a pulse energy of 13.2-24.1 pJ. Such pulse energy is sufficient for employing a system for measurements of two-photon scotopic spectral sensitivity of two-photon vision in humans. The laser parameters allow for very efficient and safe two-photon stimulation of the human visual system, as proved by a good separation between one- and two-photon thresholds for wavelengths below 950 nm, which we have confirmed for 3 healthy subjects.
RESUMO
BackgroundNoninvasive assessment of metabolic processes that sustain regeneration of human retinal visual pigments (visual cycle) is essential to improve ophthalmic diagnostics and to accelerate development of new treatments to counter retinal diseases. Fluorescent vitamin A derivatives, which are the chemical intermediates of these processes, are highly sensitive to UV light; thus, safe analyses of these processes in humans are currently beyond the reach of even the most modern ocular imaging modalities.MethodsWe present a compact, 2-photon-excited fluorescence scanning laser ophthalmoscope and spectrally resolved images of the human retina based on 2-photon excitation (TPE) with near-infrared light. A custom Er:fiber laser with integrated pulse selection, along with intelligent postprocessing of data, enables excitation with low laser power and precise measurement of weak signals.ResultsWe demonstrate spectrally resolved TPE fundus images of human subjects. Comparison of TPE data between human and mouse models of retinal diseases revealed similarity with mouse models that rapidly accumulate bisretinoid condensation products. Thus, visual cycle intermediates and toxic byproducts of this metabolic pathway can be measured and quantified by TPE imaging.ConclusionOur work establishes a TPE instrument and measurement method for noninvasive metabolic assessment of the human retina. This approach opens the possibility for monitoring eye diseases in the earliest stages before structural damage to the retina occurs.FundingNIH, Research to Prevent Blindness, Foundation for Polish Science, European Regional Development Fund, Polish National Agency for Academic Exchange, and Polish Ministry of Science and Higher Education.
Assuntos
Oftalmoscópios , Imagem Óptica , Retina , Doenças Retinianas , Animais , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Retina/diagnóstico por imagem , Retina/metabolismo , Doenças Retinianas/diagnóstico por imagem , Doenças Retinianas/genética , Doenças Retinianas/metabolismoRESUMO
Two-photon vision is a phenomenon associated with the perception of short pulses of near-infrared radiation (900-1200 nm) as a visible light. It is caused by the nonlinear process of two-photon absorption by visual pigments. Here we present results showing the influence of pulse duration and repetition rate of short pulsed lasers on the visual threshold. We compared two-photon sensitivity maps of the retina obtained for subjects with normal vision using a cost-effective fiber laser (λc = 1028.4 nm, τp = 12.2 ps, Frep = 19.17 MHz) and a solid-state laser (λc = 1043.3 nm, τp = 0.253 ps, Frep = 62.65 MHz). We have shown that in accordance with the description of two-photon absorption, the average optical power required for two-photon vision for a fiber laser is 4 times greater than that for a solid-state laser. Mean sensitivity measured for the first one is 5.9 ± 2.8 dB lower than for the second but still 17 dB away from the safety limit, confirming that picosecond light sources can be successfully applied in microperimetry. This development would dramatically reduce the cost and complexity of future clinical devices.
RESUMO
A femtosecond frequency-doubled erbium-doped fiber laser with an adjustable pulse repetition rate is developed and applied in two-photon excited fluorescence microscopy. The all-fiber laser system provides the fundamental pulse at 1560â nm wavelength with 22 fs duration for the second harmonic generation, resulting in 1.35 nJ, 60 fs pulses at 780â nm. The repetition rate is adjusted by a pulse picker unit built-in within the amplifier chain, directly providing transform-limited pulses for any chosen repetition rate between 1 and 12â MHz. We employed the laser source to drive a scanning two-photon excited fluorescence microscope for ex vivo rat skin and other samples' imaging at various pulse repetition rates. Due to compactness, ease of operation, and suitable pulse characteristics, the laser source can be considered as an attractive alternative for Ti:Sapphire laser in biomedical imaging.
RESUMO
Due to their broadband nonlinear optical properties, low-dimensional materials are widely used for pulse generation in fiber and solid-state lasers. Here we demonstrate novel materials, Bi2Te2Se (BTS) and Sn-doped Bi2Te2Se (BSTS), which can be used as a universal saturable absorbers for distinct spectral regimes. The material was mechanically exfoliated from a bulk single-crystal and deposited onto a side-polished fiber. We have performed characterization of the fabricated devices and employed them in polarization-maintaining ytterbium- and erbium-doped fiber lasers. This enabled us to obtain self-starting passively Q-switched regime at 1 µm and 1.56 µm. The oscillators emitted stable, linearly polarized radiation with the highest single pulse energy approaching 692 nJ. Both lasers are characterized by the best performance observed in all-polarization maintaining Q-switched fiber lasers with recently investigated new saturable absorbers, which was enabled by a very high damage threshold of the devices. This demonstrates the great potential of the investigated materials for the ultrafast photonics community.