The postgenomic era of fluorescence microscopy: molecules, cells and tissues
Oncological research has been deeply involved in this change process, recognising the risks of a sterile approach based solely on in vitro models. The need for therapies targeting specific molecules realises a trait d’union starting from analysis of single molecule biochemical structures and extending to their role in the cellular environment both at intra and intercellular level. The analysed scenario subsequently evolves from clonal tumour populations to the intricate network of interactions established between the tumour and the host first examined in tissue biopsies and then in living animal models.
Modern optical microscopy has always represented an indispensable tool for biomedical research and has developed to adapt to the continuously changing requests dictated by the great heterogeneity of samples targeted by experimental approaches. Besides a someway obvious need to progressively increase resolution, optical microscopy must now extend its non invasive analytical ability from single cells to entire organism minimising the disturbance to life during the observation step.
The revolution of the multiphoton excitation microscope represented a big step toward the observation of complex living systems . Infrared high frequency sources provided an incomparable penetration depth. The ability to easily induce fluorescence from UV excitable molecules has been successfully employed to monitor concentration and functionality of metabolic and structural markers in vivo, such as NADH and collagen respectively. The developed assays supported the pioneer involvement of twophoton microscopes in clinical trials to realise a first approach to “optical biopsies” in the diagnosis of skin neoplasia .
One of the most relevant and successful transformations of the modern microscope is the ability to parallel an instrumental modification with the development of new assays for the determination of functional parameters. Besides the emerging field of nanoscopies, characterised by the birth of 4Pi and STED microscopes allowing the reduction of the gap in spatial resolution between optical and electron microscopy, our insights into the properties of single molecular species gained relevant advantages from the birth of new functional microscopy assays and techniques. This process culminated in the rediscovery of the "F Techniques", namely Fluorescence Recovery After Photobleaching (FRAP), Fluorescence Resonance Energy Transfer (FRET) and Fluorescence Correlation Spectroscopy (