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Re(I) complexes have exposed highly suitable properties for cellular imaging (especially for fluorescent microscopy) such as low cytotoxicity, good cellular uptake, and differential staining. These features can be modulated or tuned by modifying the ligands surrounding the metal core. However, most of Re(I)-based complexes have been tested for non-walled cells, such as epithelial cells. In this context, it has been proposed that Re(I) complexes are inefficient to stain walled cells (i.e., cells protected by a rigid cell wall, such as bacteria and fungi), presumably due to this physical barrier hampering cellular uptake. More recently, a series of studies have been published showing that a suitable combination of ligands is useful for obtaining Re(I)-based complexes able to stain walled cells. This review summarizes the main characteristics of different fluorophores used in bioimage, remarking the advantages of d6-based complexes, and focusing on Re(I) complexes. In addition, we explored different structural features of these complexes that allow for obtaining fluorophores especially designed for walled cells (bacteria and fungi), with especial emphasis on the ligand choice. Since many pathogens correspond to bacteria and fungi (yeasts and molds), and considering that these organisms have been increasingly used in several biotechnological applications, development of new tools for their study, such as the design of new fluorophores, is fundamental and attractive.

In this study, we explored new properties of the bioinspired pyridine benzimidazole compound B2 (2,4-di-tert-butyl-6-(3H-imidazo[4,5-c]pyridine-2-yl)phenol) regarding its potential use as a differential biomarker. For that, we performed 1D 1HNMR (TOCSY), UV-Vis absorption spectra in different organic solvents, voltammetry profile (including a scan-rate study), and TD-DFT calculations that including NBO analyses, to provide valuable information about B2 structure and luminescence. In our study, we found that the B2 structure is highly stable, where the presence of an intramolecular hydrogen bond (IHB) seems to have a crucial role in the stability of luminescence, and its emission can be assigned as fluorescence. In fact, we found that the relatively large Stokes Shift observed for B2 (around 175 nm) may be attributed to the stability of the B2 geometry and the strength of its IHB. On the other hand, we determined that B2 is biocompatible by cytotoxicity experiments in HeLa cells, an epithelial cell line. Furthermore, in cellular assays we found that B2 could be internalized by passive diffusion in absence of artificial permeabilization at short incubation times (15 min to 30 min). Fluorescence microscopy studies confirmed that B2 accumulates in the endoplasmic reticulum (ER) and Golgi apparatus, two organelles involved in the secretory pathway. Finally, we determined that B2 exhibited no noticeable blinking or bleaching after 1 h of continuous exposure. Thus, B2 provides a biocompatible, rapid, simple, and efficient way to fluorescently label particular organelles, producing similar results to that obtained with other well-established but more complex methods.