Gold nanoparticles (Au NPs) are one of the most studied nanosystems [1], [2], [3], [4], [5], [6]. They are especially suitable for application in bionanotechnology and nanomedicine due to their chemical stability and biocompatibility [3], [7]. Au NP exhibit surface plasmon resonant absorption in the visible part of the electromagnetic spectrum, while the position of the resonance is sensitive to the environmental changes [8]. This enables monitoring of the nanoparticle interactions with biological molecules using optical spectroscopy. The adsorption of various biomolecules onto the surface of Au NPs is also a good path for the fabrication of complex nanosystems with desired properties [9], [10]. Functionalized Au NPs have been applied in different fields of biotechnology and medicine, including sensing [6], [11], [12] and microscopy [13], [14], as well as photothermal [15], radio- [16], and photodynamic [17], [18] therapies of cancer.

Information on spatial distribution of Au NP within a biological system is of importance for understanding of their biological activity. So far, structural techniques such as transmission electron microscopy [19], as well as noninvasive optical methods that include surface-enhanced Raman scattering, Rayleigh light scattering and confocal Raman microscopy [1], were used to investigate the nanoparticle-cell interactions and the migration of nanoparticles in cells. Our recent results demonstrated that synchrotron radiation deep-ultraviolet (DUV) fluorescence microscopy is a convenient method for determination of the accumulation of antibiotics [20], [21], [22], [23], [24], and nanoparticles [25], [26], [27], [28], [29] within an individual cell with a spatial resolution of ∼ 150 nm. For example, DUV fluorescent imaging studies of bacteria incubated with tryptophan functionalized silver [27] and gold [29] nanoparticles and showed that it was possible to distinguish the fluorescent signals of the nanostructures from the autofluorescence of the cells, due to a relatively simple morphology and small size ( ∼ 3 μm) of these prokaryotic cells.When it comes to more complex cells, such as human tissue cells, the signal differentiation is far from a trivial since the biomaterial is highly heterogeneous. In an attempt to localize the fluorescent signal that comes from the nanoparticles, we used a novel approach and followed the photobleaching of the fluorescent centers in human hepatocellular carcinoma Huh7.5.1 cells.

This study is focused on Au NP functionalized by both tryptophan and riboflavin, a resonance energy transfer (RET) pair of molecules, and the investigation of the RET efficiency in these hybrid nanosystems. RET is a dipole-dipole interaction process that occurs between a molecule in an excited state (donor) and a molecule in the ground state (acceptor), if the fluorescence band of the donor overlaps the excitation band(s) of the acceptor [30], [31], [32], [33]. It was shown that RET efficiency for a pair of molecules can be affected by the metal surface [34], [35], [36], [37], [38], [39], [40], [41], [42], which has been extensively studied both theoretically and experimentally. However, there is no general consensus on the role of metal on RET process, mainly due to different basic systems used in theoretical and experimental studies [43]. Theoretical investigations usually assume bare metal nanoparticles [37], [38], [39], [40], [44], [45] and a single RET pair of the fluorophores placed at some distance from the metallic surface, while in most of experimental studies [36], [46], [47], there are intermediate linkers between particles surfaces and fluorophores. In the present study, we used the simple chemical procedure for the fabrication of bare Au NP that were directly functionalized by tryptophan and riboflavin, i. e. without the use of intermediate ligands. In this way it was possible to study the effects of the metal surface on RET process directly.

An important consequence of RET is that it changes photobleaching dynamics of the donor molecule by providing additional channel for the deexcitation. This effect is the basis for photobleaching RET imaging [48]. It will be shown that the Au NPs change the efficiency of RET between tryptophan and riboflavin and, consequently, affect the photobleaching dynamics further of the donor molecules. Our aim is to exploit this effect in order to detect the functionalized nanoparticles in human cancer cells. It is important to emphasize that we use biomolecules, which are common in biological systems. Moreover, tryptophan is often chosen as a donor molecule in RET studies related to microorganisms or tissues [30], while riboflavin is a vitamin critical in oxidation and reduction processes in cells. They both contribute to the autofluorescence of the cells and are responsible for photochemical sensitivity of the biological material. Therefore, this study also shows that it is possible to distinguish between these biomolecules that are attached to Au NP and those that are already present in cells by using standard optical techniques. This approach is in the line with current trends presented in a recent review by Sahl, Hell and Jakobs [49] on fluorescence nanoscopy. They suggested the use of certain physical processes as the auxiliary tools in attempts to break the diffraction barrier, i. e. to increase our ability to differentiate between nanoobjects and particular molecules in cells.