Abstract
Silk fibroin is a well-known biopolymer that is used in several applications in which interactions with biological tissue are required. Fibroin is extremely versatile and can be shaped to form several constructs that are useful in tissue engineering applications. Confocal imaging is usually performed to test cell behavior on a construct, and, in this context, the fibroin intrinsic fluorescence is regarded as a problem. In addition, the intrinsic fluorescence is not intense enough to provide useful morphological images. In fact, to study the construct’s morphology, other techniques are used (i.e., SEM and Micro-CT). In this work, we propose a method based on fluorescence energy transfer (FRET) to suppress the fibroin intrinsic fluorescence and move it to a higher wavelength that is accessible to confocal microscopy for direct imaging. This was done by creating two FRET couples by dispersing two fluorophores (2,5-diphenyloxazole (PPO) and Lumogen F Violet 570 (LV)) into the fibroin matrix and optimizing their percentages to suppress the fibroin intrinsic fluorescence. With the optimized composition, we produced an electrospun mat, and the dimensions of the fibers were accurately determined by confocal microscopy.
Highlights
Silk fibroin is the internal protein of the silk fiber and is the main protein responsible for its mechanical strength
We demonstrated the possibility of using fluorescence energy transfer (FRET) to shift the silk fibroin intrinsic fluorescence emission toward a longer wavelength and by confocal microscopy to study the morphology on a silk construct
Two amino acids structures responsible for the intrinsic fluorescence of silk fibroin are shown in Figure 1A and Figure 1B (Tyrosine), while the structure of the fluorophore used are reported in Figure 1C (PPO) and Figure 1D (LV)
Summary
Silk fibroin is the internal protein of the silk fiber and is the main protein responsible for its mechanical strength. Fibroin possesses several interesting properties, including an incredibly high mechanical strength, an excellent biocompatibility, and a high transparency when used as a film. Fibroin found its main applications in Tissue Engineering [7,8] as a source for the development of scaffolds [9,10,11], and, in recent years, in frontier applications encountering other disciplines as in the case of bio-electronics [12] and in bio-optics [13,14]. The morphological analysis of silk fibroin constructs is usually obtained using imaging techniques, such as secondary electron microscopy (SEM), micro-computer tomography (micro-CT), and optical microscopy (OM). Fluorescence and confocal microscopy have been used by dissolving specific fluorophores and observing their emissions when excited at their specific excitation wavelengths [15]
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