New true direct methods of sensitive real-time recording of molecular reactions on a surface and detection of bio- and chemical agents have been developed. The methods are based on measuring changes of thickness of a sensor layer due to binding reactions. A transparent plate or a gap between two surfaces of optical materials is used as the sensor layer. The methods allow employment as biochips of microscopic glass slips without deposition of any metal or dielectric films. Alternatively, direct pumping of liquid samples through the sensing gap with deposited recognition layers can be used. For label-free biosensing, different optical schemes were realized to record thickness changes due to receptor-ligand bindings with picometer-scale resolution. Biosensors named one-dimensional PicoscopeTM and AffinoscopeTM have been developed for real-time detection of several biological agents by different recognition spots or wells with specific receptors on the biochip surface. The devices have been successfully employed for detection of food pathogens, for investigation of pharmaceutical substances, for epitope mapping of different monoclonal antibodies and immunotherapy research, for monitoring of bacteriocin production, etc. Application of the devices can be as wide as that of optical microscopes as they provide standard lateral resolution and, in addition, offer much more comprehensive information with outstanding real-time resolution in depth, e.g., for measuring molecular binding kinetics, monitoring of assembling in molecular structures, etc. The Picoscope technology significantly increases power of research instruments for bio-, nano- and pico-technologies.