Phase Contrast Microscopy - Biomedical Optical Phase Microscopy and Nanoscopy - page 3

Refractive index of a medium is defined as the ratio of speed of light in a vacuum to the
speed of light in a medium.

n 5 c vac =c medium (1.1)

A vacuum therefore has a refractive index of 1.0000, air 1.0003, water 1.33, cytosol 1.35,
and the glass most commonly used to make optics 1.52. The refractive index of a medium
is an aspect of the interaction between the light and the electrical and magnetic
susceptibilities of the medium. Overall the cell has a different refractive index than the
surrounding medium. Finer scale differences within the cell provide observable subcellular
resolution: different constituents such as lipids, proteins, and nucleic acids and different
concentrations of these in an organelle or region produce local changes in refractive index.
In a transmitted light image, some of the light will have passed through a cell and will have
interacted with this complex refractive index topology and have been affected. Both the
occurrence of diffraction of the light and the refractive index differences produce phase
shifts which can be exploited to produce contrast.

This and the consequent means of exploiting the effects in a phase contrast microscope are
most easily explained pictorially with a series of annotated diagrams.

Figure 1.2 shows schematically a phase shift by a sample. Reducing the sample to a gray
box that represents an object, such as a cell, of a different refractive index to its
surroundings and having a propensity to diffract and deviate some of the light we see light

An object,
such as a
cell

Amplitude is unchanged by
the object

The phase of the wave is
shifted by the sample

Figure 1.2
Phase shift introduced by a sample.