Auto-fluorescence of a silk fibroin-based scaffold and its interference with fluorophores in labeled cells

Abstract

Silk fibroin is increasingly emerging as an important biomaterial for tissue engineering applications. The ability to fluorescently image silk matrices under a microscope would be helpful in differentiating embedded labeled cells from background signal, critical for the study of silk-based engineered tissues. In this study, we fabricated a scaffold using freeze drying and confirmed its structure by X-ray diffraction and Fourier transform infrared spectroscopy. We then examined the fluorescence of the silk fibroin scaffold using confocal microscopy, both before and after cell seeding and fluorescent labeling. We subsequently investigated the fluorescent signature of the silk fibroin scaffold chemically. Fluorophore-labeled cells seeded into the scaffold showed the same fluorescent color as the scaffold itself when excited by the same wavelength of light. UV-Vis and fluorescence spectroscopy of a silk fibroin solution indicated absorption and emission maxima at 277 and 345 nm, respectively, which is a typical protein-derived signal. HPLC and GC-MS were used to detect quercetin and quercetin derivatives, without success. We therefore conclude that unlike silk cocoons, the fluorescent behavior of silk fibroin scaffolds does not derive from quercetin and its derivatives but from the intrinsic fluorescence of fibroin protein. We also find that the fluorescent signals deriving from a scaffold and from labeled cells embedded in it can be distinguished when the different optical channels are merged.

Tumor-Stroma Interactions Alter the Sensitivity of Drug in Breast Cancer

“…This is observed in our present findings (Figure 3A). Silk fibroin being autofluorescent in nature also appear green in DOX free samples (Amirikia et al, 2018). The incorporation of DOX increase the noise, leading to blur fluorescent images.…”

Recent Advances in Fluorescent Silk Fibroin

“…Since the SF scaffold releases fluorescence at wavelengths identical in color to the fluorescent dyes themselves, the intrinsic fluorescence of SF can interfere with the signal from such fluorescent dyes (Georgakoudi et al, 2007). Amirikia et al (2018) fabricated an SF scaffold using the freeze-drying method and examined the fluorescence of the SF scaffold both prior and after cell seeding and fluorescent labeling using confocal microscopy. They subsequently explored the fluorescent index of the SF scaffold chemically.…”

Silk/Fibroin Microcarriers for Mesenchymal Stem Cell Delivery: Optimization of Cell Seeding by the Design of Experiment

“…Evaluation of cell adhesion was performed with Calcein staining (Life Technologies): each sample was treated with 2 µM of Calcein-AM in saline solution for 10 min at 37 °C and 5% CO 2 . The auto-fluorescence of silk fibroin after exposure to green light was used to better discriminate the surface of adhesion [46]. Then, micrographs were obtained by observing cells with a fluorescence microscope (Olympus IX71).…”