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.