Principle of interference reflection microscopy (IRM) showing interference effect on reflected waves and the result on the final image intensity. Dark purple wave represents the light from the light source. The light purple waves are the reflections from the cell membrane and from the glass surface. Upon hitting the glass surface, the reflected waves are shifted half a wavelength. When the membrane is very close to the glass, the reflected light will be reflected out of phase with the reflected beam from the glass. This will cause destructive interference (see red line), resulting in a dark pixel. If there is more distance between the membrane and the glass, the returning waves will be less shifted and will cause constructive interference (see red line), resulting in a brighter pixel in the final image. Indicated are the typical refractive indices of the glass, medium and the cell membrane, which determine the amount of reflection.
Interference reflection microscopy (IRM), also called Reflection Interference Contrast Microscopy (RICM) or Reflection Contrast Microscopy (RCM) depending on the specific optical elements used, is an optical microscopy technique that leverages thin-film interference effects to form an image of an object on a glass surface. The intensity of the signal is a measure of proximity of the object to the glass surface. This technique can be used to study events at the cell membrane without the use of a (fluorescent) label as is the case for TIRF microscopy.
