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Biophysics is a new way of looking at living matter. It uses quantitative experimental, theoretical, and computational methods, thereby opening a new window for studying and understanding life processes. This textbook provides a brief introduction to the basics of the field, followed by in-depth discussions of more advanced biophysics subjects, going all the way to state-of-the-art experiments and their theoretical interpretations. The second edition presents some of the newest developments in the field (e.g., biomolecular condensates, loop extrusion), a new chapter on computational methods, and many computer exercises specially designed for this textbook.