Fast topographic optical imaging using encoded search focal scan.

A central quest in optics is to rapidly extract quantitative information from a sample. Existing topographical imaging tools allow non-contact and three-dimensional measurements at the micro and nanoscales and are essential in applications including precision engineering and optical quality control. However, these techniques involve acquiring a focal stack of images, a time-consuming process that prevents measurement of moving samples. Here, we propose a method for increasing the speed of topographic imaging by orders of magnitude. Our approach involves collecting a reduced set of images, each integrated during the full focal scan, whilst the illumination is synchronously modulated during exposure. By properly designing the modulation sequence for each image, unambiguous reconstruction of the object height map is achieved using far fewer images than conventional methods. We describe the theoretical foundations of our technique, characterise its performance, and demonstrate sub-micrometric topographic imaging over 100 µm range of static and dynamic systems at rates as high as 67 topographies per second, limited by the camera frame rate. The high speed of the technique and its ease of implementation could enable a paradigm shift in optical metrology, allowing the real-time characterisation of large or rapidly moving samples.

Scan-less microscopy based on acousto-optic encoded illumination.

Several optical microscopy methods are now available for characterizing scientific and industrial processes at sub-micron resolution. However, they are often ill-suited for imaging rapid events. Limited by the trade-off between camera frame-rate and sensitivity, or the need for mechanical scanning, current microscopes are optimized for imaging at hundreds of frames-per-second (fps), well-below what is needed in processes such as neuronal signaling or moving parts in manufacturing lines. Here, we present a scan-less technology that allows sub-micrometric imaging at thousands of fps. It is based on combining a single-pixel camera with parallelized encoded illumination. We use two acousto-optic deflectors (AODs) placed in a Mach-Zehnder interferometer and drive them simultaneously with multiple and unique acoustic frequencies. As a result, orthogonal light stripes are obtained that interfere with the sample plane, forming a two-dimensional array of flickering spots - each with its modulation frequency. The light from the sample is collected with a single photodiode that, after spectrum analysis, allows for image reconstruction at speeds only limited by the AOD's bandwidth and laser power. We describe the working principle of our approach, characterize its imaging performance as a function of the number of pixels - up to 400 × 400 - and characterize dynamic events at 5000 fps.

Rapid quantification of 3D ultrasound fields with wavefront sensing and Schlieren tomography.

The rapid and precise characterization of three-dimensional (3D) pressure fields inside water is paramount for ultrasound (US) applications in fields as relevant as biomedicine and acoustic trapping. The most conventional way is to scan point-by-point a needle hydrophone across the field of interest, which is an intrinsically invasive and slow process. With typical acquisition times of hours and even days, this method remains impractical in many realistic scenarios. Alternatively, optical techniques can be used to non-invasively and rapidly measure the changes in light intensity or phase induced by pressure differences. However, these techniques remain largely qualitative: extracting precise pressure values can require extensive calibration, and complex processing, or can be limited to low-pressure ranges. Here, we report how combining wavefront sensing and Schlieren tomography enables rapid and direct quantification of 3D pressure fields while obviating any calibration steps. By simultaneously capturing optical phase and intensity information of the US-perturbed fluid using a Wavefront Sensor and Schlieren projections, respectively, 3D pressure fields over several millimeters cubic can be reconstructed after a few seconds. We present a detailed description of the approach and prove its feasibility by characterizing the US field after an acoustic lens, which is in excellent agreement with calibrated hydrophone measurements and simulations. These results are a significant step forward toward the precise and real-time characterization of ultrasound patterns.

Enhanced light focusing inside scattering media with shaped ultrasound.

Light focusing is the primary enabler of various scientific and industrial processes including laser materials processing and microscopy. However, the scattering of light limits the depth at which current methods can operate inside heterogeneous media such as biological tissue, liquid emulsions, and composite materials. Several approaches have been developed to address this issue, but they typically come at the cost of losing spatial or temporal resolution, or increased invasiveness. Here, we show that ultrasound waves featuring a Bessel-like profile can locally modulate the optical properties of a turbid medium to facilitate light guiding. Supported by wave optics and Monte Carlo simulations, we demonstrate how ultrasound enhances light focusing a factor of 7 compared to conventional methods based on placing optical elements outside the complex medium. Combined with point-by-point scanning, images of samples immersed in turbid media with an optical density up to 15, similar to that of weakly scattering biological tissue, can be reconstructed. The quasi-instantaneous generation of the shaped-ultrasound waves, together with the possibility to use transmission and reflection architectures, can pave the way for the real-time control of light inside living tissue.

Preparation of anisotropic multiscale micro-hydrogels via two-photon continuous flow lithography.

HYPOTHESIS: Polymeric anisotropic soft microparticles show interesting behavior in biological environments and hold promise for drug delivery and biomedical applications. However, self-assembly and substrate-based lithographic techniques are limited by low resolution, batch operation or specific particle geometry and deformability. Two-photon polymerization in microfluidic channels may offer the required resolution to continuously fabricate anisotropic micro-hydrogels in sub-10 µm size-range. EXPERIMENTS: Here, a pulsed laser source is used to perform two-photon polymerization under microfluidic flow of a poly(ethylene glycol) diacrylate (PEGDA) solution with the objective of realizing anisotropic micro-hydrogels carrying payloads of various nature, including small molecules and nanoparticles. The fabrication process is described via a reactive-convective-diffusion system of equations, whose solution under proper auxiliary conditions is used to corroborate the experimental observations and sample the configuration space. FINDINGS: By tuning the flow velocity, exposure time and pre-polymer composition, anisotropic PEGDA micro-hydrogels are obtained in the 1-10 µm size-range and exhibit an aspect ratio varying from 1 to 5. Furthermore, 200 nm curcumin-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles and 100 nm ssRNA-encapsulating lipid nanoparticles were entrapped within square PEGDA micro-hydrogels. The proposed approach could support the fabrication of micro-hydrogels of well-defined morphology, stiffness, and surface properties for the sustained release of therapeutic agents.

Nanopatterning with Photonic Nanojets: Review and Perspectives in Biomedical Research.

Nanostructured surfaces and devices offer astounding possibilities for biomedical research, including cellular and molecular biology, diagnostics, and therapeutics. However, the wide implementation of these systems is currently limited by the lack of cost-effective and easy-to-use nanopatterning tools. A promising solution is to use optical methods based on photonic nanojets, namely, needle-like beams featuring a nanometric width. In this review, we survey the physics, engineering strategies, and recent implementations of photonic nanojets for high-throughput generation of arbitrary nanopatterns, along with applications in optics, electronics, mechanics, and biosensing. An outlook of the potential impact of nanopatterning technologies based on photonic nanojets in several relevant biomedical areas is also provided.

Volumetric Lissajous confocal microscopy with tunable spatiotemporal resolution.

Dynamic biological systems present challenges to existing three-dimensional (3D) optical microscopes because of their continuous temporal and spatial changes. Most techniques are rigid in adapting the acquisition parameters over time, as in confocal microscopy, where a laser beam is sequentially scanned at a predefined spatial sampling rate and pixel dwell time. Such lack of tunability forces a user to provide scan parameters, which may not be optimal, based on the best assumption before an acquisition starts. Here, we developed volumetric Lissajous confocal microscopy to achieve unsurpassed 3D scanning speed with a tunable sampling rate. The system combines an acoustic liquid lens for continuous axial focus translation with a resonant scanning mirror. Accordingly, the excitation beam follows a dynamic Lissajous trajectory enabling sub-millisecond acquisitions of image series containing 3D information at a sub-Nyquist sampling rate. By temporal accumulation and/or advanced interpolation algorithms, the volumetric imaging rate is selectable using a post-processing step at the desired spatiotemporal resolution for events of interest. We demonstrate multicolor and calcium imaging over volumes of tens of cubic microns with 3D acquisition speeds of 30 Hz and frame rates up to 5 kHz.

Zebrafish structural development in Mueller-matrix scanning microscopy.

Zebrafish are powerful animal models for understanding biological processes and the molecular mechanisms involved in different human diseases. Advanced optical techniques based on fluorescence microscopy have become the main imaging method to characterize the development of these organisms at the microscopic level. However, the need for fluorescence probes and the consequent high light doses required to excite fluorophores can affect the biological process under observation including modification of metabolic function or phototoxicity. Here, without using any labels, we propose an implementation of a Mueller-matrix polarimeter into a commercial optical scanning microscope to characterize the polarimetric transformation of zebrafish preserved at different embryonic developmental stages. By combining the full polarimetric measurements with statistical analysis of the Lu and Chipman mathematical decomposition, we demonstrate that it is possible to quantify the structural changes of the biological organization of fixed zebrafish embryos and larvae at the cellular scale. This convenient implementation, with low light intensity requirement and cheap price, coupled with the quantitative nature of Mueller-matrix formalism, can pave the way for a better understanding of developmental biology, in which label-free techniques become a standard tool to study organisms.

Fast Acoustic Light Sculpting for On-Demand Maskless Lithography.

Light interference is the primary enabler of a number of optical maskless techniques for the large-scale processing of materials at the nanoscale. However, methods controlling interference phenomena can be limited in speed, ease of implementation, or the selection of pattern designs. Here, an optofluidic system that employs acoustic standing waves in a liquid to produce complex interference patterns at sub-microsecond temporal resolution, faster than the pulse-to-pulse period of many commercial laser systems, is presented. By controlling the frequency of the acoustic waves and the motion of a translation stage, additive and subtractive direct-writing of tailored patterns over cm2 areas with sub-wavelength uniformity in periodicity and scalable spatial resolution, down to the nanometric range, are demonstrated. Such on-the-fly dynamic control of light enhances throughput and design flexibility of optical maskless lithography, helping to expand its application portfolio to areas as important as plasmonics, electronics, or metamaterials.

A tissue chamber chip for assessing nanoparticle mobility in the extravascular space.

Although a plethora of nanoparticle configurations have been proposed over the past 10 years, the uniform and deep penetration of systemically injected nanomedicines into the diseased tissue stays as a major biological barrier. Here, a 'Tissue Chamber' chip is designed and fabricated to study the extravascular transport of small molecules and nanoparticles. The chamber comprises a collagen slab, deposited within a PDMS mold, and an 800 µm channel for the injection of the working solution. Through fluorescent microscopy, the dynamics of molecules and nanoparticles was estimated within the gel, under different operating conditions. Diffusion coefficients were derived from the analysis of the particle mean square displacements (MSD). For validating the experimental apparatus and the protocol for data analysis, the diffusion D of FITC-Dextran molecules of 4, 40 and 250 kDa was first quantified. As expected, D reduces with the molecular weight of the dextran molecules. The MSD-derived diffusion coefficients were in good agreement with values derived via fluorescence recovery after photobleaching (FRAP), an alternative technique that solely applies to small molecules. Then, the transport of six nanoparticles with similar hydrodynamic diameters (~ 200 nm) and different surface chemistries was quantified. Surface PEGylation was confirmed to favor the diffusion of nanoparticles within the collagen slab, whereas the surface decoration with hyaluronic acid (HA) chains reduced nanoparticle mobility in a way proportional to the HA molecular weight. To assess further the generality of the proposed approach, the diffusion of the six nanoparticles was also tested in freshly excised brain tissue slices. In these ex vivo experiments, the diffusion coefficients were 5-orders of magnitude smaller than for the Tissue Chamber chip. This was mostly ascribed to the lack of a cellular component in the chip. However, the trends documented for PEGylated and HA-coated nanoparticles in vitro were also confirmed ex vivo. This work demonstrates that the Tissue Chamber chip can be employed to effectively and efficiently test the extravascular transport of nanomedicines while minimizing the use of animals.

Enhanced volumetric imaging in 2-photon microscopy via acoustic lens beam shaping.

Three-dimensional imaging at high-spatiotemporal resolutions and over large penetration depths is key for unmasking the dynamics and structural organization of complex biological systems. However, the need to axially shift the focus, with consequent limitations in imaging speed, and signal degradation at large depths due to scattering effects, makes this task challenging. Here, we present a novel approach in 2-photon excitation microscopy that allows fast volumetric imaging and enhanced signal-to-background (S/B) in thick tissue. Our technique is based on ultrafast beam shaping at each pixel by means of an acoustic optofluidic lens. Shaping the excitation beam with different phase profiles enables both high-speed axial focus shifting, for continuous volumetric imaging, and controlled aberrated imaging, advantageous for out-of-focus background removal. We provide a theoretical description of our approach, and demonstrate volumetric imaging of neuronal cells from a mouse brain slice with enhancements in S/B up to a factor of 10 over a depth of 600 µm.

Combination of scanning probe technology with photonic nanojets.

Light focusing through a microbead leads to the formation of a photonic nanojet functional for enhancing the spatial resolution of traditional optical systems. Despite numerous works that prove this phenomenon, a method to appropriately translate the nanojet on top of a region of interest is still missing. Here, by using advanced 3D fabrication techniques we integrated a microbead on an AFM cantilever thus realizing a system to efficiently position nanojets. This fabrication approach is robust and can be exploited in a myriad of applications, ranging from microscopy to Raman spectroscopy. We demonstrate the potential of portable nanojets by imaging different sub-wavelength structures. Thanks to the achieved portability, we were able to perform a detailed optical characterization of the resolution enhancement induced by the microbead, which sheds light into the many contradictory resolution claims present in literature. Our conclusions are strongly supported by rigorous data analysis and by numerical simulations, all in perfect agreement with experimental results.

Selectable light-sheet uniformity using tuned axial scanning.

Light-sheet fluorescence microscopy (LSFM) is an optical sectioning technique capable of rapid three-dimensional (3D) imaging of a wide range of specimens with reduced phototoxicity and superior background rejection. However, traditional light-sheet generation approaches based on elliptical or circular Gaussian beams suffer an inherent trade-off between light-sheet thickness and area over which this thickness is preserved. Recently, an increase in light-sheet uniformity was demonstrated using rapid biaxial Gaussian beam scanning along the lateral and beam propagation directions. Here we apply a similar scanning concept to an elliptical beam generated by a cylindrical lens. In this case, only z-scanning of the elliptical beam is required and hence experimental implementation of the setup can be simplified. We introduce a simple dimensionless uniformity statistic to better characterize scanned light-sheets and experimentally demonstrate custom tailored uniformities up to a factor of 5 higher than those of unscanned elliptical beams. This technique offers a straightforward way to generate and characterize a custom illumination profile that provides enhanced utilization of the detector dynamic range and field of view, opening the door to faster and more efficient 2D and 3D imaging.

Direct Laser Printing of Tailored Polymeric Microlenses.

We report a laser-based approach for the fast fabrication of high-optical-quality polymeric microlenses and microlens arrays with controllable geometry and size. Our strategy consists of the direct laser printing of microdroplets of a highly viscous UV prepolymer at targeted positions, followed by photocuring. We study the morphological characteristics and imaging performance of the microlenses as a function of the substrate and laser parameters and investigate optimal printing conditions and printing mechanisms. We show that the microlens size and focusing properties can be easily tuned by the laser pulse energy, with minimum volumes below 20 fL and focal lengths ranging from 7 to 50 µm.

Three-dimensional particle tracking via tunable color-encoded multiplexing.

We present a novel 3D tracking approach capable of locating single particles with nanometric precision over wide axial ranges. Our method uses a fast acousto-optic liquid lens implemented in a bright field microscope to multiplex light based on color into different and selectable focal planes. By separating the red, green, and blue channels from an image captured with a color camera, information from up to three focal planes can be retrieved. Multiplane information from the particle diffraction rings enables precisely locating and tracking individual objects up to an axial range about 5 times larger than conventional single-plane approaches. We apply our method to the 3D visualization of the well-known coffee-stain phenomenon in evaporating water droplets.

Selective fluorescence functionalization of dye-doped polymerized structures fabricated by direct laser writing (DLW) lithography.

The continuous development of the vast arsenal of fabrication techniques is a pivotal factor in the breakthrough of nanotechnology. Although the broad interest is generally focused on the reduction of the dimensions of the fabricated structures, localized functionalization of the nanomaterials emerges as a key factor closely linked to their potential applications. In particular, fabrication of spatially selective fluorescence nanostructures is highly demanded in nanophotonics, as for example in three-dimensional (3D) optical data storage (ODS), where massive storage capacity and fast writing-reading processes are promised. We have developed an innovative method to control the location and intensity of the fluorescence signal in dye-doped photopolymerized structures fabricated with Direct Laser Writing (DLW) lithography. Well-defined fluorescent pixels (area = 0.24 µm(2)) were written inside a polymer matrix with the help of a femtosecond pulsed laser (multiphoton absorption) via a thermally-induced di-aggregation of a fluorescent dye. Moreover, we have accomplished a fine control of the fluorescence intensity which can increase the storage capacity of ODS systems fabricated with this approach.

Sub-wavelength Laser Nanopatterning using Droplet Lenses.

When a drop of liquid falls onto a screen, e.g. a cell phone, the pixels lying underneath appear magnified. This lensing effect is a combination of the curvature and refractive index of the liquid droplet. Here, the spontaneous formation of such lenses is exploited to overcome the diffraction limit of a conventional laser direct-writing system. In particular, micro-droplets are first laser-printed at user-defined locations on a surface and they are later used as lenses to focus the same laser beam. Under conditions described herein, nanopatterns can be obtained with a reduction in spot size primarily limited by the refractive index of the liquid. This all-optics approach is demonstrated by writing arbitrary patterns with a feature size around 280 nm, about one fourth of the processing wavelength.

λ/20 axial control in 2.5D polymerized structures fabricated with DLW lithography.

An astonishing λ/20 height control is accomplished in 2.5D photopolymerized structures by taking advantage of the induced expansion of the resin. Our nanofabrication method is a one-pot approach with two processing steps: (i) regular 2.5D photopolymerization of the resin monomer by using multiphoton direct laser writing (DLW) lithography and (ii) spatially-selective irradiation of the photopolymerized features before development resulting in a nanometer-controlled height increase of the structure. The UV-visible-NIR sub-wavelength axial feature size (~40 nm) of this method allows fabricating devices with applications in multiple technological fields such as nanoelectronics and photonics.

Simultaneous multiplane confocal microscopy using acoustic tunable lenses.

Maximizing the amount of spatiotemporal information retrieved in confocal laser scanning microscopy is crucial to understand fundamental three-dimensional (3D) dynamic processes in life sciences. However, current 3D confocal microscopy is based on an inherently slow stepwise process that consists of acquiring multiple 2D sections at different focal planes by mechanical or optical z-focus translation. Here, we show that by using an acoustically-driven optofluidic lens integrated in a commercial confocal system we can capture an entire 3D image in a single step. Our method is based on continuous axial scanning at speeds as high as 140 kHz combined with fast readout. In this way, one or more focus sweeps are produced on a pixel by pixel basis and the detected photons can be assigned to their corresponding focal plane enabling simultaneous multiplane imaging. We exemplify this method by imaging calibration and biological fluorescence samples. These results open the door to exploring new fundamental processes in science with an unprecedented time resolution.

Simultaneous imaging of multiple focal planes for three-dimensional microscopy using ultra-high-speed adaptive optics.

Traditional white-light and fluorescent imaging techniques provide powerful methods to extract high-resolution information from two-dimensional (2-D) sections, but to retrieve information from a three-dimensional (3-D) volume they require relatively slow scanning methods that result in increased acquisition time. Using an ultra-high speed liquid lens, we circumvent this problem by simultaneously acquiring images from multiple focal planes. We demonstrate this method by imaging microparticles and cells flowing in 3-D microfluidic channels.