This book highlights the key concepts underlying design and application of optical biosensors in diverse contexts. It begins with an historical perspective on the field, moving on to foundations of engineering biosensors and development of fluorescent imaging approaches. The core chapters focus on the design and application of specific optical biosensors in vivo, either for tracking certain biomolecules or visualizing certain cellular events in various living systems. The final section addresses several additional applications, including use in high throughput drug screening and in integrated methods that involve modeling or optogenetics.