Fast In Vivo Imaging of SHG Nanoprobes with Multiphoton Light-Sheet Microscopy.

Two-photon light-sheet microscopy (2P-SPIM) provides a unique combination of advantages for fast and deep fluorescence imaging in live tissues. Detecting coherent signals such as second-harmonic generation (SHG) in 2P-SPIM in addition to fluorescence would open further imaging opportunities. However, light-sheet microscopy involves an orthogonal configuration of illumination and detection that questions the ability to detect coherent signals. Indeed, coherent scattering from micron-sized structures occurs predominantly along the illumination beam. By contrast, point-like sources such as SHG nanocrystals can efficiently scatter light in multiple directions and be detected using the orthogonal geometry of a light-sheet microscope. This study investigates the suitability of SHG light-sheet microscopy (SHG-SPIM) for fast imaging of SHG nanoprobes. Parameters that govern the detection efficiency of KTiOPO4 and BaTiO3 nanocrystals using SHG-SPIM are investigated theoretically and experimentally. The effects of incident polarization, detection numerical aperture, nanocrystal rotational motion, and second-order susceptibility tensor symmetries on the detectability of SHG nanoprobes in this specific geometry are clarified. Guidelines for optimizing SHG-SPIM imaging are established, enabling fast in vivo light-sheet imaging combining SHG and two-photon excited fluorescence. Finally, microangiography was achieved in live zebrafish embryos by SHG imaging at up to 180 frames per second and single-particle tracking of SHG nanoprobes in the blood flow.

Polarized Luminescence of Anisotropic LaPO4:Eu Nanocrystal Polymorphs.

Lanthanide elements exhibit highly appealing spectroscopic properties that are extensively used for phosphor applications. Their luminescence contains precise information on the internal structure of the host materials. Especially, the polarization behavior of the transition sublevel peaks is a fingerprint of the crystal phase, symmetry, and defects. However, this unique feature is poorly explored in current research on lanthanide nanophosphors. We here report on a detailed investigation of the evolution of Eu3+ luminescence during the thermally induced phase transition of LaPO4 nanocrystal hosts. By means of c-axis-aligned nanocrystal assemblies, we demonstrate a dramatic change of the emission polarization feature corresponding to the distinct Eu3+ site symmetries in different LaPO4 polymorphs. We also show that changes of the nanocrystal structure can be identified by this spectroscopic method, with a much higher sensitivity than the X-ray diffraction analysis. This new insight into the nanostructure-luminescence relationship, associated with the unprecedented polarization characterizations, provides a new methodology to investigate phase transitions in nanomaterials. It also suggests a novel function of lanthanide emitters as orientation-sensing nanoprobes for innovative applications such as in bioimaging or microfluidics.

Corrigendum: Monitoring the orientation of rare-earth-doped nanorods for flow shear tomography.

This corrects the article DOI: 10.1038/nnano.2017.111.

Metrology of Multiphoton Microscopes Using Second Harmonic Generation Nanoprobes.

In multiphoton microscopy, the ongoing trend toward the use of excitation wavelengths spanning the entire near-infrared range calls for new standards in order to quantify and compare the performances of microscopes. This article describes a new method for characterizing the imaging properties of multiphoton microscopes over a broad range of excitation wavelengths in a straightforward and efficient manner. It demonstrates how second harmonic generation (SHG) nanoprobes can be used to map the spatial resolution, field curvature, and chromatic aberrations across the microscope field of view with a precision below the diffraction limit and with unique advantages over methods based on fluorescence. KTiOPO4 nanocrystals are used as SHG nanoprobes to measure and compare the performances over the 850-1100 nm wavelength range of several microscope objectives designed for multiphoton microscopy. Finally, this approach is extended to the post-acquisition correction of chromatic aberrations in multicolor multiphoton imaging. Overall, the use of SHG nanoprobes appears as a uniquely suited method to standardize the metrology of multiphoton microscopes.

Monitoring the orientation of rare-earth-doped nanorods for flow shear tomography.

Rare-earth phosphors exhibit unique luminescence polarization features originating from the anisotropic symmetry of the emitter ion's chemical environment. However, to take advantage of this peculiar property, it is necessary to control and measure the ensemble orientation of the host particles with a high degree of precision. Here, we show a methodology to obtain the photoluminescence polarization of Eu-doped LaPO4 nanorods assembled in an electrically modulated liquid-crystalline phase. We measure Eu3+ emission spectra for the three main optical configurations (σ, π and α, depending on the direction of observation and the polarization axes) and use them as a reference for the nanorod orientation analysis. Based on the fact that flowing nanorods tend to orient along the shear strain profile, we use this orientation analysis to measure the local shear rate in a flowing liquid. The potential of this approach is then demonstrated through tomographic imaging of the shear rate distribution in a microfluidic system.

A portable extensional rheometer for measuring the viscoelasticity of pitcher plant and other sticky liquids in the field.

BACKGROUND: Biological fluids often have interesting and unusual physical properties to adapt them for their specific purpose. Laboratory-based rheometers can be used to characterise the viscoelastic properties of such fluids. This, however, can be challenging as samples often do not retain their natural properties in storage while conventional rheometers are fragile and expensive devices ill-suited for field measurements. We present a portable, low-cost extensional rheometer designed specifically to enable in situ studies of biological fluids in the field. The design of the device (named Seymour) is based on a conventional capillary break-up extensional rheometer (the Cambridge Trimaster). It works by rapidly stretching a small fluid sample between two metal pistons. A battery-operated solenoid switch triggers the pistons to move apart rapidly and a compact, robust and inexpensive, USB 3 high speed camera is used to record the thinning and break-up of the fluid filament that forms between the pistons. The complete setup runs independently of mains electricity supply and weighs approximately 1 kg. Post-processing and analysis of the recorded images to extract rheological parameters is performed using open source software. RESULTS: The device was tested both in the laboratory and in the field, in Brunei Darussalam, using calibration fluids (silicone oil and carboxymethyl cellulose solutions) as well as Nepenthes pitcher plant trapping fluids as an example of a viscoelastic biological fluid. The fluid relaxation times ranged from 1 ms to over 1 s. The device gave comparable performance to the Cambridge Trimaster. Differences in fluid viscoelasticity between three species were quantified, as well as the change in viscoelasticity with storage time. This, together with marked differences between N. rafflesiana fluids taken from greenhouse and wild plants, confirms the need for a portable device. CONCLUSIONS: Proof of concept of the portable rheometer was demonstrated. Quantitative measurements of pitcher plant fluid viscoelasticity were made in the natural habitat for the first time. The device opens up opportunities for studying a wide range of plant fluids and secretions, under varying experimental conditions, or with changing temperatures and weather conditions.