# Recent Advances in Intrinsically Fluorescent
## 3 Application
### Bioimaging
Compared with the commonly used flow cytometry, this method shows the advantages of simple operation and a low cost, making it a promising application prospect in the identification of tumor cells and tissues. It is worth noting that PDA nanodots that are oxidized by ROS display one-photon and two-photon fluorescence properties, providing a tool for directly observing animal organization structure and activities under free animal activities. In the above studies, PDA nanoparticles or copolymers all enter cells non-specifically through endocytosis, which may limit practical applications. Fortunately, the abundant functional groups on the surface of PDA allow it  
Figure 4: Under different pH levels: (**A**) CLSM images, (**B**) TEM of (a) pH = 3.0 (b) pH = 5.0 (c) pH = 7.0 and (**C**) PL spectra [87]. Reprinted/adapted with permission from Ref. [87]. 2018 Elsevier. to be connected to other recognition groups or biological cross-linking agents without the need for additional surface modification. PDA as a drug carrier has been widely reported and applied [90; 91; 92]. When conducting a drug test evaluation, it is also necessary to connect a fluorescent dye as a probe to ascertain whether the drug enters the cell and releases it. Fluorescent polydopamine can be used as a multifunctional experimental platform for carriers and fluorescent probes at the same time, which is conducive to simplifying the experimental process and combining with other functional groups. [PERSON] et al. prepared highly emissive and biocompatible FPDA with sensitive detection of Fe\({}^{3+}\) ions and targeted cell imaging of live cells [55]. The cRGD peptides and GPR120 primary antibodies were used for targeted cell imaging and membrane protein labeling, respectively, by the common method of EDC/NHS connection. Because the surface of PDA is rich in carboxyl groups, it can also be easily connected to other types of peptides or proteins through the EDC/NHS connection method. In the above studies, FPDA nanoparticles or copolymers can only enter the cell through endocytosis and imaging in the cytoplasm due to the blocking of nuclear pore complexes [93]. The nucleus plays a key role in the main physiological activities of the cell, including cell metabolism, growth, and differentiation [93; 94; 95], and real-time in situ observation of the nucleus in the live cell is indispensable for the study of cell behavior and events. [PERSON] et al. have reported polydopamine fluorescent nanoparticles with nucleus imaging in label-free, in situ, real-time, and long-term manners [96]. They incubated N-(3-(Dimethylamino)-propyl)-N-ethyl carbodiimide (EDC), DA, and cells in a cell culture medium for 3 hours and observing them under a confocal microscope by excitation at 405-nm wavelength (Figure 5). The results showed bright blue fluorescence in the nucleus but almost no fluorescence in the cytoplasm. However, the mechanism of the bright fluorescence of FPDA produced by the reaction of EDC and DA was unclear. As a nitrogen-containing biological cross-linking agent, it could be speculated that EDC weakens the \(\pi\)-\(\pi\) stacking by doping with N atoms. More ingeniously, the generated FPDA can only stay in the nucleus due to the existence of nuclear pores and shows a strong nuclear imaging specificity. As mentioned earlier, the method of doping with N atoms can improve the fluorescence intensity and photostability of PDA. Using nitrogen-containing biological cross-linkers or other low-cytotoxic chemicals to generate PDA in situ in cells will be an interesting research direction. From the above discussion, FPDA shows good application prospects in fluorescence imaging and overcomes the defects of many traditional fluorescent dyes, such as complex preparation operations, a fluorescent background, and high cytotoxicity. However, it was found that the current FPDA bioimaging is at the cell level and has not been applied to in vivo imaging, which is mainly attributed to the lack of relevant imaging equipment or harsh use conditions and environments.