*2.7. Fluorescence Spectroscopy*

Fluorescence spectroscopy utilizes the ability of the targeted molecules to emit light at a di fferent wavelength than the optical excited source (Figure 8) [115]. The information from the emitted photon can then be constructed to produce 2D images with high temporal and spatial resolution [116]. In biology, fluorescence can happen with most biological molecules with the appropriate excitation. However, for specific applications, like cell-based therapy and TE, the emitted signal from the fluorophore molecules can be enhanced through direct or indirect labeling. In the case of indirect labeling, the targeted cells can be engineered, like gene transfection, to express fluorescence proteins like GFP [117]. In indirect labeling, the targeted molecules are attached to certain functional fluorescent molecules which can be activated through an optical excited source [118]. In both cases, the excited wavelength needs to be considered carefully since it can a ffect the specific photophysical properties of the fluorophores, such as photostability, quantum yield, Stokes shift, and fluorescence lifetime [118].

Nanomaterials have emerged as an e ffective candidate for direct labeling in fluorescent spectroscopy. Di fferent materials such as quantum dots (QDs) [119], polymers [115,120,121], organic dyes [122], upconversion nanoparticles (UCNPs) [123], and gold nanoparticles (AuNPs) [115] have been considered depending on the specific applications. For cellular tracking, the functional nanomaterials can be attached ex vivo to the targeted protein on the cell surface or they can be infused inside through the process of di ffusion or active transport [118]. When excited optically, these nanomaterials can be used to distinguish di fferent performance and functionality of the matrix-embedded cells. In addition to cellular studies, nanomaterials, such as QDs [116] and UCNPs [123], have also been used to label hydrogel sca ffold structures to study their degradation. These materials require di fferent excitation wavelengths such as ultraviolet (UV) or visible light for QDs and dyes [115], while UCNPs are more sensitive to NIR wavelengths [123]. However, UV and visible wavelengths have di fferent shortcomings, such as limited penetration depth and potential disintegration of the biological molecules and sca ffolds [118]. In addition, even though QDs have excellent optical properties, their suboptimal biocompatibility and biodegradability represent major challenges that hinder their applications in TE [115]. NIR fluorophores can resolve those disadvantages as well as minimize the autofluorescence from cells and tissues, which allows them to be used in a wide range of in vivo tracking of hydrogel degradation (Figure 9) [115].

**Figure 9.** Fluorescence contrast of tumor growth when the mouse is injected with upconversion nanoparticles. Reproduced with permission from Ref. [115].

In addition to fluorescent properties, certain metallic nanoparticles like gold nanoparticles can have multi-functional properties, contributing to tissue mechanical properties, electrical conductivity and (cell) di fferentiation, and photothermal e ffect, when they are integrated into the biopolymer sca ffold. Such particles, therefore, are good candidates for multifunctional applications such as cancer detection assays and optically-controlled on–o ff microfluidic devices [124].
