Interferometric reflectance imaging
One specific form of photometric biosensing technique developed by researchers at Boston University is interferometric reflectance imaging. Using optical interference techniques, imaging of antibodies was successfully performed. This was achieved without altering the antibody structure or using bio-markers such as fluorescent proteins. The basis of this technique stems solely from optical interference. By using a reflective substrate such as silicon, light reflected from proteins will interfere with light reflected from the substrate. In result, interference patterns are generated that alter the intensity of the reflected light. This phenomenon is measurable by a camera.
Proteins have indices of refraction based on their concentration. When light is shined on the proteins, a portion of the light is transmitted through the molecules and reflected off the silicon's surface. The interference of the light initially reflected off the proteins and the light reflected off the surface of the silicon will have a relative phase difference (after being transmitted back through the protein) contributing to a wavelength-dependent sinusoidal variation in the total amount of reflected light (captured by the imaging device).
The Interferometric Reflectance Imaging Sensor (IRIS) was developed by the Unlu research group at Boston University for the purpose of label-free biosensing. Using simple lenses and low-powered, coherent LEDs, the device offers “exquisite sensitivity and reproducibility” and is able to image with remarkable resolution “beyond the classical diffraction limit.” [1] This relatively cheap solution also presents minimal hazards when compared to a laser illumination source.
The IRIS operates solely on optical reflection. The ability for it to image with extremely high spatial resolution stems from the integration of a diffuser into the design of the microscope. The diffuser randomizes the directionality of the light from a single LED source (called Köhler illumination) which allows for sharp focusing of incident light without back-imaging the source in the image projection.
Practical uses of this device include the detection of bacterial and viral infections in underdeveloped countries. When pathogen specific growth factors are introduced into a microarray, only spots with the targeted pathogens will grow and increase in concentration. In turn, this dictates a change in the reflected intensity compared to pre-growth. Thus, by measuring how reflectance changes over time, unknown pathogens and their growth rates can be easily characterized and identified.