Optical microscopy is developing into nanoscopy and multimodal microscopy the better to decipher the functioning mechanisms in living systems, and investigating biological specimens at molecular level using fluorescence as a mechanism of contrast. Results have demonstrated the potential to provide information at the Angstrom level. Other optical methods now offer more in terms of spatial and temporal resolution, making it possible to study the delicate and complex relationship between structure and function in cells. Modern optical microscopes also use the decisive advantage provided by artificial intelligence algorithms. All in all, a rapidly changing field with an increasing number of questions to be answered. This book contains the lectures presented as Course 210 of the prestigious International School of Physics Enrico Fermi, Multimodal and Nanoscale Optical Microscopy, held in Varenna, Italy from 11 to 16 July 2021. Topics covered include fluorescence; linear and nonlinear microscopy; label-free with Mueller matrix and Brillouin microscopy; F-methods such as FRAP, FLIM, FRET and FCS; super-resolution, phototoxicity and photodamage; optical and magnetic trapping; image formation; and bioimage analysis, among others. The book also includes selected contributions of a number of young researchers. Packed with novel ideas, new instruments, challenging theoretical approaches and amazing applications, the book will be of interest to all those working in the field of multimodal and nanoscale optical microscopy.