As next generation lunar missions and interplanetary human spaceflight grow closer, the ability to assess habitat surface microbial content quickly and accurately has become increasingly significant. Current state of the art technology relies on astronaut swabbing of surfaces and subsequently performing molecular analysis on the samples to determine the microbial burden. To alleviate this burden, Nanohmics Inc., proposes to continue advanced development of an autonomous, fluorescence imaging detector (AFID) for microbial mapping demonstrated during the Phase I program. The key components of the unmanned, aerial, 3D-sensing AFID system are a custom fluorescence detector with excitation sources controlled by embedded image acquisition and processing that uses spectral fingerprints and machine learning to differentiate between bacteria, fungi, and other organic material. The goal of the Phase II program will be design, optimization, and performance demonstration of the AFID system ability to generate a microorganism map of the total bioburden on simulated habitat surfaces relevant to future human spaceflight. The final AFID prototype will be advanced to TRL 5-6 over the course of the Phase II program with the ability to distinguish bacteria (detection threshold > 500 CFU/100 cm2) and fungi (detection threshold > 10 CFU/100 cm2) which meet the pre-flight and in-flight microbial mapping microbial monitoring requirements as defined by the International Space Station Medical Operations Requirements Documents (ISS MORD). Potential NASA Applications (Limit 1500 characters, approximately 150 words): Numerous NASA applications benefit from ensuring proper disinfection of surfaces, particularly habitat protection applications. This technology would enable in-situ measurement of spacecraft, lander, rover, and instrument cleanliness. Potential Non-NASA Applications (Limit 1500 characters, approximately 150 words): A fluorescence imager has multiple applications in the medical, defense, and industrial markets. This technology could be applied to ultraviolet (UV) light disinfection systems used in hospitals to reduce healthcare-associated infections (HAIs) to ensure proper disinfection and identify pathogens in the hospital. This technology could also be used for bio-agent sensing for defense applications. Duration: 24