Near-infrared (NIR) marker-based imaging is of growing importance for deep tissue imaging and is based on a considerable reduction of optical losses at large wavelengths. We aim to extend the range of NIR excitation wavelengths particularly to values beyond 1.6 μm in order to profit from the low loss biological windows NIR-III and NIR-IV. We address this task by studying NIR-excitation to NIR-emission conversion and imaging in the range of 1200 up to 2400 nm at the example of harmonic Mg-doped lithium niobate nanoparticles (i) using a nonlinear diffuse femtosecond-pulse reflectometer and (ii) a Tunable hIGh EneRgy (TIGER) widefield microscope. We successfully demonstrate the existence of appropriate excitation/emission configurations in this spectral region taking harmonic generation into account. Moreover, NIR-imaging using the most striking configurations NIR-III to NIR-I, based on second harmonic generation (SHG), and NIR-IV to NIR-I, based on third harmonic generation (THG), is demonstrated with excitation wavelengths from 1.6–1.8 μm and from 2.1–2.2 μm, respectively. The advantages of the approach and the potential to additionally extend the emission range up to 2400 nm, making use of sum frequency generation (SFG) and difference frequency generation (DFG), are discussed.
In vivo tracking of harmonic nanoparticles (HNPs) in living animals is a technique not yet exploited, despite the great potential offered by these markers, due to a lack of an appropriate tool. The main drawback is the necessity to excite nonlinear effects in the millimeter area in a widefield mode with a sufficient signal to noise ratio. Our approach to this problem consists in a redesign of the laser space parameters in a region of high energy per pulse and low repetition rate in the kHz regime, in counter-trend with the actual microscope research technology. We realise this by means of a regeneratively amplified fs-laser system, creating an easy alignable and reproducible Tunable hIGh EneRgy (TIGER) widefield microscope. This one is successfully applied for HNPs tracking in the blood flow of the heart system of a Drosophila larvae, a powerful platform to study socially relevant diseases, such as congenital heart defects in human beings. It is possible to follow nonlinear emitting marker in a remarkable field-of-view of up to 1.5 × 1.5 mm2 at 70 frame per seconds. The impact of the energy per pulse, the pulse repetition rate as well as of the photon energy on the SNR is determined and the optimum setup conditions are deduced. At the same time, wavelengths of fundamental and harmonic pulses are carefully considered and tailored to match the transmission fingerprint of the Drosophila larvae. Our findings clearly demonstrate the large impact of precise pulse parameter management in the view of the optical features of the sample, the optical setup and the photosensitivity of the detector. A step-by-step instruction for more general use of the technique is described, opening the path for addressing biological research questions that require far-field imaging at high frame rates with exceedingly high spatial and temporal precision.
Time‐resolved nonlinear optical sum‐frequency generation (SFG) of two differently colored, infrared (sub‐)picosecond laser pulses is studied by means of nonlinear diffuse femtosecond‐pulse reflectometry using lithium niobate nanoparticle pellets. The visible SFG emission exhibits an asymmetric pulse shape in the time domain which is explained within the framework of light propagation in random media. The analysis of the spectro‐temporal data set indicates that LiNbO3 nanoparticle pellets can be used for an alternative type of an optical correlator, e.g., for the determination of a pulse's chirp parameter. This finding is generalized by means of numerical simulations. As a result, ultrashort pulse shapes can now be predicted inside nanoscaled, densely packed media with a nonlinear optical response on a comprehensive basis, giving rise to a number of prospective fields of applications.
A lack of suitable markers hampered the exploitation of the third and fourth biological windows, an appealing approach for safe in-vivo imaging. Harmonic nanoparticles and nonlinear microscopy offer a valuable solution to bridge this gap.
A widefield nonlinear optical microscope based on pulses having an energy in the µJ and a repetition rate in the kHz regime is conceived to perform harmonic nanoparticles in-vivo tracking in a Drosophila larva.
In-vivo tracking based on harmonic nanoparticles is so far not exploited because of a lack on an appropriate tool. Here we present a new approach based on the re-design of the laser space parameters.
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