Interference reflection microscopy

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.

In 1964, Adam S. G. Curtis coined the term Interference Reflection Microscopy (IRM), using it in the field of cell biology to study embryonic chick heart fibroblasts.

[1][2] He used IRM to look at adhesion sites and distances of fibroblasts, noting that contact with the glass was mostly limited to the cell periphery and the pseudopodia.

[1] In 1975, Johan Sebastiaan Ploem introduced an improvement to IRM (published in a book chapter[3]), which he called Reflection Contrast Microscopy (RCM).

[4] The improvement is to use a so-called anti-flex objective and crossed polarizers to further reduce stray light in the optical system.

Today, this scheme is mainly referred to as Reflection Interference Contrast Microscopy (RICM),[5][6] the name of which was introduced by Bareiter-Hahn and Konrad Beck in 1979.

The multiplicity of names used to describe the technique has caused some confusion, and was discussed as early as 1985 by Verschueren.

[8] To form an image of the attached cell, light of a specific wavelength is passed through a polarizer.

This interference results in a dark pixel in the final image (the left case in the figure).

Second, when the membrane is not attached to the glass, the reflection from the membrane has a smaller phase shift compared to the reflected light from the glass, and therefore they will not cancel each other out, resulting in a bright pixel in the image (the right case in the figure).

Third, when there is no specimen, only the reflected light from the glass is detected and will appear as bright pixels in the final image.

The polarizers can increase the efficiency by reducing scattered light; however in a modern setup with a sensitive digital camera, they are not required.

This means that without a cell the image will be bright, whereas when the cell is attached, the difference between medium and the membrane causes a large reflection that is slightly shifted in phase, causing interference with the light reflected by the glass.

Because the amplitude of the light reflected from the medium-membrane interface is decreased due to scattering, the attached area will appear darker but not completely black.

[8] In order to image cells using IRM, a microscope needs at least the following elements: 1) a light source, such as a halogen lamp, 2) an optical filter (which passes a small range of wavelengths), and 3) a beam splitter (which reflects 50% and transmits 50% of the chosen wavelength).

[7] There have been many refinements to the basic theory of IRM, most of which increase the efficiency and yield of the image formation.

When vesicles fuse with the membrane, they appear as small light circles within the dark footprint (bright spots in the top cell in the right panel).

Upon stimulation with 60 mM potassium, multiple bright spots begin to appear inside the dark footprint of the chromaffin cell as a result of exocytosis of dense core granules.

Because IRM doesn't require a fluorescent label, it can be combined with other imaging techniques, such as epifluorescence and TIRF microscopy to study protein dynamics together with vesicle exocytosis and endocytosis.

Principle of interference reflection microscopy (IRM)
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.
Two chromaffin cells imaged with DIC (left) and IRM (right).
Vesicle fusion visualized using IRM. Scale bar represents 2 μm.