Image credits Shutterstock/cybrain
Recently, a new generation of materials has arisen, having the ability to absorb near infrared radiations with lower energy and emitting higher energy visible radiation via a nonlinear optical process. This relatively new branch of fluorescent materials has a huge number of applications in fields such as imaging, biosensing, in vitro and in vivo analytic detection.
These materials show unique properties, such as large Stokes shift, minimal photobleaching, high penetration depth for imaging and sharp emission bands, making them extremely attractive as an alternative to currently used imaging probes.
How do these UCNPs Work?
To understand the mechanism of upconversion we should first understand the process of relatively simple downconversion in conventional fluorophores. In conventional fluorophores, fluorescence is generated by the luminescence phenomenon, in which a fluorophore absorbs photons of a certain wavelength, and the electrons are excited to a higher energy state from their stable ground state. When these electrons return to their ground state, photons are emitted with energy equivalent to the difference between the energies of the excited state and the ground state of the fluorophore.
Most of these conventional fluorophores, such as fluorescein and rhodamine, follow the principle of Stokes law; according to which, the wavelength of the emitted radiation is longer than the radiation that was absorbed. Upconverting nanoparticles, on the other hand, follows the principle of the anti-Stokes law which means they absorb near-infrared (NIR) radiation and emit visible range photons.
They are called upconverting on the premises that emission energies higher than that of the absorbed radiation, violate Stokes law. Upconversion was first demonstrated by François Auzel in 1966.
Image credits Ghulam Jalani, Rafik Naccache, Derek H. Rosenzweig, Lisbet Haglund, Fiorenzo Vetrone, and Marta Cerruti (J. Am. Chem. Soc., 2016, 138 (3), pp 1078–1083)
Advantages of UCNPs Over Downconversion Fluorophores
These upconverting nanoparticles have various advantages over downconverting fluorophores. One of the major advantages is low autofluorescence, which is an inherent problem in fluorescence microscopy of tissues.
Autofluorescence is emission by natural biomolecules in the imaging region that are not marked by the biological marker. This happens because these naturally occurring biomolecules such as NADPH, melanin, Flavin, and collagen etc., show fluorescence in UV range. It interferes with the emission signals of interest, and thus the image produced is considerably ambiguous. But since upconversion happens in NIR light, the possibility of autofluorescence is significantly reduced resulting in higher signal-to-noise ratio.
Another extremely important advantage is deep tissue penetration. NIR light has low scattering effect in biological samples and thus can penetrate deeper than lower wavelengths.
UCNPs are extremely resistant to external environmental factors such as temperatures and pH, enabling them to handle higher emissions in host matrices. Image multiplexing is also possible by employing these UCNPs, since their emission wavelengths can be varied by changing the concentration of the dopants in the main matrix.
Targeted upconversion probes
Huang et. al. at Fudan University, has contributed significantly to small-animal imaging with targeted upconversion material probes. The surface of hydrothermally synthesized NaYF4:Yb, Er was functionalized by folic acid (FA). They performed measurements for folate receptor expression of FR (+) cervical carcinoma (HeLa) cells with FR (-) human breast cancer cells (MCF-7) as a control, by exciting the sample with 980 nm light and receiving 500-560 nm (red light) and 495-570 nm (green light).
Next, they injected UCNPs-FA and UCNPs-NH2 in vivo into the tail of a mouse that had a HeLa tumor on the right flank. After 24 hours, the signal from UCNPs-FA showed its corresponding signal in contrast to the untargeted probe, which did not show any signal in the vicinity of the tumor. This work demonstrates the applicability of these nanoparticles in targeted imaging. Similar works have been reported for other functionalization and different orientations such as in core-shell configurations and bi-functional materials.
Great progress has been achieved in the past few years in the development of UCNPs, but, in spite of to the advantages mentioned in this article, there are a few challenges to the commercialization of these upconverting nanoparticles.
For example, their application is severely limited due to its low dispersibility in water. Their bio-compatibility and hydrophilicity is a direct reflection of the functionalization and surface modification carried out on these UCNPs. A few studies have shown that there is no obvious toxicity in the in vitro or in vivo assessment, but their effects on small animals over a longer time period are still unknown.
Some promising research has been reported in the application of UCNPs for theranostics, which is a combination of therapy and diagnostics by the single formulation. It will enable the physician to monitor the therapeutic action in real time, which will be of tremendous value in terms of cost cutting and time-saving.