SignificancePrecise laser ablation at the single-cell level is demonstrated on onion epidermal and human neuroblastoma cells for the first time using a 1.95 μm thulium-doped fiber laser (TDFL). The high-precision laser ablation demonstrated paves the way for micro-dissection and surgery in scientific and clinical applications.AimWe aimed to ablate individual target cells with pulsed laser radiation while minimizing damage to neighboring cells and to study the effects of pulse variation on laser ablation outcome.ApproachAn advanced 1.95 μm nanosecond-pulsed TDFL is developed offering a high degree of pulse control and coupled to a microscope to enable simultaneous ablating and monitoring. A reflective microscope objective is used to focus the light onto the sample without chromatic aberrations. Systematic studies of ablation outcomes using different pulse parameters are then performed.ResultsSingle-cell ablation is achieved, with a precision of 31.3±0.1 μm for onion epidermal cells and 20 μm for fixed human neuroblastoma cells, in which the latter demonstrates the ability to target fixed individual cells in a sample of up to 250,000 cells. Careful control of the pulse parameters produced ablation without carbonization and cavitation bubbles.ConclusionsSingle-cell level ablation harnessing a TDFL is clearly demonstrated on onion epidermal cells and human neuroblastoma cells. The TDFL, with an easily accessible range of wavelengths, provides significant opportunities in the field of biology and medicine for stimulation, dissection, and surgical applications.
Osteoarthritis (OA), a degenerative joint disease presenting as loss of cartilage, is a leading cause of disability worldwide, increasingly with aging populations. Early detection is crucial for effective treatment since there is no definitive cure, yet, current assessment techniques fall short and rely on ionising radiation or invasive procedures. We report an application of multimodal “spectromics”, low-level abstraction data fusion of non-destructive NIR Raman and NIR-SWIR absorption spectroscopy, providing an enhanced, interpretable “fingerprint” for diagnosis of OA in human cartilage. Under multivariate statistical analyses and supervised machine learning, cartilage was classified with high precision and disease state predicted accurately. Discriminatory features within the spectromics fingerprint elucidated clinically relevant tissue components (OA biomarkers). Further, we have developed an automated goniometric 3D hyperspectral mapping setup, and characterised OA cartilage on whole human femoral heads post hip arthroplasty for spatially-resolved spectromics. These results lay foundation for minimally invasive, deeply penetrating, label-free, chemometric diagnosis of the hip.
We demonstrate precise cellular laser ablation on SH-SY5Y and onion cells by using an advanced 1.95μm nanosecond-pulsed thulium-doped fibre laser (TDFL). The TDFL offers a high degree of control on pulse parameters, which enables good thermal and mechanical confinement during the laser ablation and results in a high precision of 30μm, with minimal carbonisation or collateral damage to surrounding cells. The realisation of precise cellular ablation from a TDFL will open up new applications in microsurgery for disease treatment, benefitting patients and researchers worldwide.
Multiphoton microscopies are an invaluable tool in biomedical imaging given their inherent capabilities for label free imaging, optical sectioning, chemical and structural specificity. They comprise various types of Coherent Raman microscopies (CR), such as Coherent Anti-Stokes Raman Scattering (CARS), Stimulated Raman Loss (SRL) or Stimulated Raman Gain, different kinds of Harmonic Generation imaging (HG) such as Second and Third Harmonic Generation (SHG and THG respectively), and Multiphoton Autofluorescence imaging (MA) such as Two and Three Photon Excited Autofluorescence (TPEAF and ThPEAF respectively). Despite their significant advantages, multiphoton microscopies, comparably to all other types of optical microscopies, exhibit limited penetration depth in tissue due to absorption and scattering. In this work we explore the advantages of multiphoton microscopies in hard and soft deep tissue imaging when using excitation wavelengths in the range of Short-Wavelength Infrared (SWIR) windows which occur between 1000 nm and 2500 nm. These spectral windows have notable merits including longer attenuation lengths and none or very low signal absorption observed for almost all kinds of multiphoton microscopy. We show results of using excitations in the SWIR windows, generated by standard as well as novel sources, such as a thulium fibre laser, in different types of multiphoton microscopy on a variety of hard and soft tissue samples (bone, cartilage and other tissue types) and demonstrate the advantages of using excitations in this wavelength range, including longer penetration depth and high resolution for deep tissue imaging.
Digital micromirror devices (DMDs) have found many scientific research applications. We present adaptive optics techniques exploiting the point spread function (PSF) of a DMD pixel to enhance the fidelity of image-projection-based laser machining. Femtosecond laser pulses with intensity profiles spatially shaped by a DMD were demagnified to a sample via a microscope objective, with ~10 DMD mirrors, each of width ~10µm, approximately projecting to the optical setup diffraction limit of ~1µm. A single DMD mirror then scales geometrically to dimensions well below the diffraction limit, permitting various techniques to enhance machining. By digitally shifting an intensity mask on the DMD between pulses while the sample remains static, machined features with resolutions below the single-exposure diffraction limit are produced (similar to pitch splitting multiple exposure techniques), with a reduction of <2.5x achieved in nickel. By combining digital image shifts with real-time sample image recognition algorithms, point-to-point positional accuracy is camera-resolution-limited (~500nm) rather than translation stage-limited. Furthermore, the PSF allows near-continuous intensity distributions rather than binary on/off intensity patterns, and have been used to produce variable-depth surface texturing (up to 40nm depth changes with 2µm period demonstrated in metals) features via single shots. Algorithms have been used to automate optical proximity corrections for arbitrary intensity masks in order to reduce machining errors due to optical filtering. These techniques are being combined to produce <1cm2 size, highly complex substrates for the production of biologically-friendly cell growth assays, with the viability of human bone stem cells on flexible substrates demonstrated.
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