Histochemistry of Single Molecules

Anna C. Croce, Giovanni Bottiroli

Carlo Pellicciari, Marco Biggiogera

Excitation of biological substrates with light at a suitable wavelength can give rise to a light emission in the ultraviolet (UV)-visible, near-infrared (IR) spectral range, called autofluorescence (AF). This is a widespread phenomenon, ascribable to the general presence of biomolecules acting as endogenous fluorophores (EFs) in the organisms of the whole life kingdom. In cytochemistry and histochemistry, AF is often an unwanted signal enhancing the background and affecting in particular the detection of low signals or rare positive labeling spots of exogenous markers. Conversely, AF is increasingly considered as a powerful diagnostic tool because of its role as an intrinsic biomarker directly dependent on the nature, amount, and microenvironment of the EFs, in a strict relationship with metabolic processes and structural organization of cells and tissues. As a consequence, AF carries multiple information that can be decrypted by a proper analysis of the overall emission signal, allowing the characterization and monitoring of cell metabolism in situ, in real time and in the absence of perturbation from exogenous markers. In the animal kingdom, AF studies at the cellular level take advantage of the essential presence of NAD(P)H and flavins, primarily acting as coenzymes at multiple steps of common metabolic pathways for energy production, reductive biosynthesis and antioxidant defense. Additional EFs such as vitamin A, porphyrins, lipofuscins, proteins, and neuromediators can be detected in different kinds of cells and bulk tissues, and can be exploited as photophysical biomarkers of specific normal or altered morphofunctional properties, from the retinoid storage in the liver to aging processes, metabolic disorders or cell transformation processes. The AF phenomenon involves all living system, and literature reports numerous investigations and diagnostic applications of AF, taking advantage of continuously developing self-assembled or commercial instrumentation and measuring procedures, making almost impossible to provide their comprehensive description. Therefore a brief summary of the history of AF observations and of the development of measuring systems is provided, along with a description of the most common EFs and their metabolic significance. From our direct experience, examples of AF imaging and microspectrofluorometric procedures performed under a single excitation in the near-UV range for cell and tissue metabolism studies are then reported.