Efficient optical clearance is fundamental for whole brain imaging. In particular, clearance of the brain without membrane damage is required for the imaging of lipophilic tracer-labeled neural tracts. Relying on an ascending gradient of fructose solutions, SeeDB can achieve sufficient transparency of the mouse brain while ensuring that the plasma membrane remains intact. However, it is challenging to extend this method to larger mammalian brains due to the extremely high viscosity of the saturated fructose solution. Here we report a SeeDB-derived optical clearing method, termed FRUIT, which utilizes a cocktail of fructose and urea. As demonstrated in the adult mouse brain, combination of these two highly water-soluble clearing agents exerts a synergistic effect on clearance. More importantly, the final FRUIT solution has low viscosity so as to produce transparency of the whole adult rabbit brain via arterial perfusion, which is impossible to achieve with a saturated fructose solution. In addition to good compatibility with enhanced yellow fluorescent protein, the cocktail also preserves the fluorescence of the lipophilic tracer DiI. This work provides a volume-independent optical clearing method which retains the advantages of SeeDB, particularly compatibility with lipophilic tracers.
For 1,319 nm laser radiation, the ocular axial length has great, while the retinal pigmentation has only slight influence on retinal damage threshold. The relative lower direct transmittance of human eye media makes the retina more resistant to laser lesion, which should be considered when deriving human maximum permissible exposure (MPE).
The retinal damage effects induced by transitional near-infrared (NIR) lasers have been investigated for years. However, the damage threshold dependence on exposure duration has not been revealed. In this paper, the in-vivo retinal damage ED50 thresholds were determined in chinchilla grey rabbits for 1319 nm laser radiation for exposure durations from 0.1 s to 10 s. The incident corneal irradiance diameter was fixed at 5 mm. The ED50 thresholds given in terms of the total intraocular energy (TIE) for exposure durations of 0.1, 1 and 10 s were 1.36, 6.33 and 28.6 J respectively. The ED50 thresholds were correlated by a power law equation, ED50 = 6.31t0.66 [J] where t is time [s], with correlation coefficient R = 0.9999. There exists a sufficient safety margin (factor of 28~60) between the human ED50 thresholds derived from the rabbit and the maximum permissible exposure (MPE) values in the current laser safety standards.
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