Spatial tomography of light resolved in time, spectrum, and polarisation.

Measuring polarisation, spectrum, temporal dynamics, and spatial complex amplitude of optical beams is essential to studying phenomena in laser dynamics, telecommunications and nonlinear optics. Current characterisation techniques apply in limited contexts. Non-interferometric methods struggle to distinguish spatial phase, while phase-sensitive approaches necessitate either an auxiliary reference source or a self-reference, neither of which is universally available. Deciphering complex wavefronts of multiple co-propagating incoherent fields remains particularly challenging. We harness principles of spatial state tomography to circumvent these limitations and measure a complete description of an unknown beam as a set of spectrally, temporally, and polarisation resolved spatial state density matrices. Each density matrix slice resolves the spatial complex amplitude of multiple mutually incoherent fields, which over several slices reveals the spectral or temporal evolution of these fields even when fields spectrally or temporally overlap. We demonstrate these features by characterising the spatiotemporal and spatiospectral output of a vertical-cavity surface-emitting laser.

Formation of nanochannels in sapphire with ultrashort Bessel pulses.

We explore, both by numerical simulations and experimentally, the flexibility in controlling Bessel beam parameters by re-imaging it into transparent material with a demagnifying collimator for the formation of high-aspect ratio nanochannels. Analysis of nanochannels produced by in-house precision-made axicon with 275 fs pulses in sapphire reveals the intensity threshold of ∼7.2 × 1013 W/cm2 required to create the cylindrical microexplosion. We estimate that the maximum applied pressure during the process was 1.5 TPa and that the resulting density of compressed sapphire in the nanochannel's shells are ∼1.19 ± 0.02 times higher than the pristine crystal, and higher than what was achieved before in spherical microexplosion with Gaussian pulses.

Controlling the non-linear emission of upconversion nanoparticles to enhance super-resolution imaging performance.

Upconversion nanoparticles (UCNPs) exhibit unique optical properties such as photo-emission stability, large anti-Stokes shift, and long excited-state lifetimes, allowing significant advances in a broad range of applications from biomedical sensing to super-resolution microscopy. In recent years, progress on nanoparticle synthesis led to the development of many strategies for enhancing their upconversion luminescence, focused in particular on heavy doping of lanthanide ions and core-shell structures. In this article, we investigate the non-linear emission properties of fully Yb-based core-shell UCNPs and their impact on the super-resolution performance of stimulated excitation-depletion (STED) microscopy and super-linear excitation-emission (uSEE) microscopy. Controlling the power-dependent emission curve enables us to relax constraints on the doping concentrations and to reduce the excitation power required for accessing sub-diffraction regimes. We take advantage of this feature to implement multiplexed super-resolution imaging of a two-sample mixture.

Simultaneous super-linear excitation-emission and emission depletion allows imaging of upconversion nanoparticles with higher sub-diffraction resolution.

Upconversion nanoparticles (UCNPs) are becoming increasingly popular as biological markers as they offer photo-stable imaging in the near-infrared (NIR) biological transparency window. Imaging at NIR wavelengths benefits from low auto-fluorescence background and minimal photo-damage. However, as the diffraction limit increases with the wavelength, the imaging resolution deteriorates. To address this limitation, recently two independent approaches have been proposed for imaging UCNPs with sub-diffraction resolution, namely stimulated emission-depletion (STED) microscopy and super linear excitation-emission (uSEE) microscopy. Both methods are very sensitive to the UCNP composition and the imaging conditions, i.e. to the excitation and depletion power. Here, we demonstrate that the imaging conditions can be chosen in a way that activates both super-resolution regimes simultaneously when imaging NaYF4:Yb,Tm UCNPs. The combined uSEE-STED mode benefits from the advantages of both techniques, allowing for imaging with lateral resolution about six times better than the diffraction limit due to STED and simultaneous improvement of the axial resolution about twice over the diffraction limit due to uSEE. Conveniently, at certain imaging conditions, the uSEE-STED modality can achieve better resolution at four times lower laser power compared to STED mode, making the method appealing for biological applications. We illustrate this by imaging UCNPs functionalized by colominic acid in fixed neuronal phenotype cells.

3D sub-diffraction imaging in a conventional confocal configuration by exploiting super-linear emitters.

Sub-diffraction microscopy enables bio-imaging with unprecedented clarity. However, most super-resolution methods require complex, costly purpose-built systems, involve image post-processing and struggle with sub-diffraction imaging in 3D. Here, we realize a conceptually different super-resolution approach which circumvents these limitations and enables 3D sub-diffraction imaging on conventional confocal microscopes. We refer to it as super-linear excitation-emission (SEE) microscopy, as it relies on markers with super-linear dependence of the emission on the excitation power. Super-linear markers proposed here are upconversion nanoparticles of NaYF4, doped with 20% Yb and unconventionally high 8% Tm, which are conveniently excited in the near-infrared biological window. We develop a computational framework calculating the 3D resolution for any viable scanning beam shape and excitation-emission probe profile. Imaging of colominic acid-coated upconversion nanoparticles endocytosed by neuronal cells, at resolutions twice better than the diffraction limit both in lateral and axial directions, illustrates the applicability of SEE microscopy for sub-cellular biology.

Subcellular spatial resolution achieved for deep-brain imaging in vivo using a minimally invasive multimode fiber.

Achieving intravital optical imaging with diffraction-limited spatial resolution of deep-brain structures represents an important step toward the goal of understanding the mammalian central nervous system1-4. Advances in wavefront-shaping methods and computational power have recently allowed for a novel approach to high-resolution imaging, utilizing deterministic light propagation through optically complex media and, of particular importance for this work, multimode optical fibers (MMFs)5-7. We report a compact and highly optimized approach for minimally invasive in vivo brain imaging applications. The volume of tissue lesion was reduced by more than 100-fold, while preserving diffraction-limited imaging performance utilizing wavefront control of light propagation through a single 50-µm-core MMF. Here, we demonstrated high-resolution fluorescence imaging of subcellular neuronal structures, dendrites and synaptic specializations, in deep-brain regions of living mice, as well as monitored stimulus-driven functional Ca2+ responses. These results represent a major breakthrough in the compromise between high-resolution imaging and tissue damage, heralding new possibilities for deep-brain imaging in vivo.

Extended depth of field imaging through multicore optical fibers.

Compact microendoscopes use multicore optical fibers (MOFs) to visualize hard-to-reach regions of the body. These devices typically have a large numerical aperture (NA) and are fixed-focus, leading to blurry images from a shallow depth of field with little focus control. In this work, we demonstrate a method to digitally adjust the collection aperture and therefore extend the depth of field of lensless MOF imaging probes. We show that the depth of field can be more than doubled for certain spatial frequencies, and observe a resolution enhancement of up to 78% at a distance of 50µm from the MOF facet. Our technique enables imaging of complex 3D objects at a comparable working distance to lensed MOFs, but without the requirement of lenses, scan units or transmission matrix calibration. Our approach is implemented in post processing and may be used to improve contrast in any microendoscopic probe utilizing a MOF and incoherent light.

Statistically strong label-free quantitative identification of native fluorophores in a biological sample.

Bioimaging using endogenous cell fluorescence, without any external biomarkers makes it possible to explore cells and tissues in their original native state, also in vivo. In order to be informative, this label-free method requires careful multispectral or hyperspectral recording of autofluorescence images followed by unsupervised extraction (unmixing) of biochemical signatures. The unmixing is difficult due to the scarcity of biochemically pure regions in cells and also because autofluorescence is weak compared with signals from labelled cells, typically leading to low signal to noise ratio. Here, we solve the problem of unsupervised hyperspectral unmixing of cellular autofluorescence by introducing the Robust Dependent Component Analysis (RoDECA). This approach provides sophisticated and statistically robust quantitative biochemical analysis of cellular autofluorescence images. We validate our method on artificial images, where the addition of varying known level of noise has allowed us to quantify the accuracy of our RoDECA analysis in a way that can be applied to real biological datasets. The same unsupervised statistical minimisation is then applied to imaging of mouse retinal photoreceptor cells where we establish the identity of key endogenous fluorophores (free NADH, FAD and lipofuscin) and derive the corresponding molecular abundance maps. The pre-processing methodology of image datasets is also presented, which is essential for the spectral unmixing analysis, but mostly overlooked in the previous studies.

Modal beam splitter: determination of the transversal components of an electromagnetic light field.

The transversal profile of beams can always be defined as a superposition of orthogonal fields, such as optical eigenmodes. Here, we describe a generic method to separate the individual components in a laser beam and map each mode onto its designated detector with low crosstalk. We demonstrate this with the decomposition into Laguerre-Gaussian beams and introduce a distribution over the integer numbers corresponding to the discrete orbital and radial momentum components of the light field. The method is based on determining an eigenmask filter transforming the incident optical eigenmodes to position eigenmodes enabling the detection of the state of the light field using single detectors while minimizing cross talk with respect to the set of filter masks considered.

Multimode fibre: Light-sheet microscopy at the tip of a needle.

Light-sheet fluorescence microscopy has emerged as a powerful platform for 3-D volumetric imaging in the life sciences. Here, we introduce an important step towards its use deep inside biological tissue. Our new technique, based on digital holography, enables delivery of the light-sheet through a multimode optical fibre--an optical element with extremely small footprint, yet permitting complex control of light transport processes within. We show that this approach supports some of the most advanced methods in light-sheet microscopy: by taking advantage of the cylindrical symmetry of the fibre, we facilitate the wavefront engineering methods for generation of both Bessel and structured Bessel beam plane illumination. Finally, we assess the quality of imaging on a sample of fluorescent beads fixed in agarose gel and we conclude with a proof-of-principle imaging of a biological sample, namely the regenerating operculum prongs of Spirobranchus lamarcki.

Compact multimode fiber beam-shaping system based on GPU accelerated digital holography.

Real-time, on-demand, beam shaping at the end of the multimode fiber has recently been made possible by exploiting the computational power of rapidly evolving graphics processing unit (GPU) technology [Opt. Express 22, 2933 (2014)]. However, the current state-of-the-art system requires the presence of an acousto-optic deflector (AOD) to produce images at the end of the fiber without interference effects between neighboring output points. Here, we present a system free from the AOD complexity where we achieve the removal of the undesired interference effects computationally using GPU implemented Gerchberg-Saxton and Yang-Gu algorithms. The GPU implementation is two orders of magnitude faster than the CPU implementation which allows video-rate image control at the distal end of the fiber virtually free of interference effects.

Laser-induced breakdown of an optically trapped gold nanoparticle for single cell transfection.

The cell selective introduction of therapeutic agents remains a challenging problem. Here we demonstrate spatially controlled cavitation instigated by laser-induced breakdown of an optically trapped single gold nanoparticle of diameter 100 nm. The energy breakdown threshold of the gold nanoparticle with a single nanosecond laser pulse at 532 nm is three orders of magnitude lower than water, which leads to nanocavitation allowing single cell transfection. We quantify the shear stress to cells from the expanding bubble and optimize the pressure to be in the range of 1-10 kPa for transfection. The method shows transfection of plasmid DNA into individual mammalian cells with an efficiency of 75%.

Transient transfection of mammalian cells using a violet diode laser.

We demonstrate the first use of the violet diode laser for transient mammalian cell transfection. In contrast to previous studies, which showed the generation of stable cell lines over a few weeks, we develop a methodology to transiently transfect cells with an efficiency of up to approximately 40%. Chinese hamster ovary (CHO-K1) and human embryonic kidney (HEK293) cells are exposed to a tightly focused 405-nm laser in the presence of plasmid DNA encoding for a mitochondrial targeted red fluorescent protein. We report transfection efficiencies as a function of laser power and exposure time for our system. We also show, for the first time, that a continuous wave laser source can be successfully applied to selective gene silencing experiments using small interfering RNA. This work is a major step towards an inexpensive and portable phototransfection system.