In-vivo tracking of harmonic nanoparticles: a study based on a TIGER widefield microscope [Invited]

Abstract

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.

NIR-to-NIR Imaging: Extended Excitation Up to 2.2 μm Using Harmonic Nanoparticles with a Tunable hIGh EneRgy (TIGER) Widefield Microscope

“…The dependence of the SHG intensity with respect to fundamental power in LiNbO 3 has previously been investigated in ref. [23] and was found in accordance with our investigations using the TIGER microscope for KNbO 3 [13], but also from other authors and harmonic nanoparticles (cf. BaTiO 3 [24] and BFO [25]) with similar nonlinear optical properties to LiNbO 3 [26].…”

“…Widefield nonlinear optical imaging is performed using the TIGER microscope introduced in Ref. [13]: a tunable, high-energy fs-pulse laser system (identical to the one used for nonlinear diffuse fs-pulse reflectometry) is collinearly coupled via a beam stabilization system, model type: Aligna 4D (TEM Messtechnik GmbH, Hannover, Germany), with the widefield illumination beam path of an inverted microscope (type: FV3000, Olympus Europa SE & Co. KG, Hamburg, Germany. Excitation is possible with wavelengths in the range from 630-2500 nm, thus covering all four biological optical windows NIR-I, NIR-II, NIR-III, and NIR-IV.…”

“…Shifting the interest towards the NIR-III and NIR-IV windows is challenging because not only biomarkers that can be excited here are needed, but also on the detection side, a suitable camera system is needed if the emission appears in this spectral range [12]. In this article, we address this task by studying nonlinear NIR-to-NIR imaging at the example of harmonic nanoparticles (HNPs) in a Tunable hIGh EneRgy (TIGER) widefield microscope [13] with a particular focus on the excitation within the NIR-III and NIR-IV biological windows, i.e., a marker-based approach in multiphoton microscopy. While marker-free (unlabeled) microscopy has emerged already as powerful tool in medical-technical applications, the development of nanoparticle-based approaches aims at molecular monitoring, optogenetics and drug release [14,15], as it has been recently successfully demonstrated with harmonic LiNbO 3 [16].…”

Thin Patterned Lithium Niobate Films by Parallel Additive Capillary Stamping of Aqueous Precursor Solutions

“…The LiNbO 3 rims appear dark and the holes, where the surface of the ITO substrate is exposed, appear bright. Microscopic scanning of the SHG [ 24 ] in the same area yielded the SHG map shown in Figure 3b. The SHG signal reproduces the structure of the holey LiNbO 3 film apparent from the CLSM image (Figure 3a) and is in line with the LiNbO 3 patterns imaged by scanning electron microscopy (SEM, Figure 1a,b) and AFM (Figure 1c).…”

“…Nonlinear Imaging: The setup consisted of a laser scanning microscope FV3000 (Olympus) optically combined with a regeneratively amplified femtosecond laser (Pharos-HE-20, Light Conversion Inc.) described in detail by Vittadello et al [24] The fs laser acts as a pump source for an optical parametric amplifier (OPA) (Orpheus-Twins F, Light Conversion). The fs pulses are adjusted collinearly to the optical path of the integrated continuous lasers in the microscope scanning unit.…”