Interference reflection microscopy
Encyclopedia
Interference reflection microscopy or IRM is an optical microscopy technique that utilizes polarized light 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 in contrast to TIRF microscopy
.
.
The technique was refined and the qualitative and quantitative aspects of the technique were later described by several researchers in the 70s and 80s: Bereiter-Hahn and his colleagues correlated the technique with electron microscopy, showing that different mammalian cell lines adhere to the glass substrate in specific focal adhesion sites.
is passed through a polarizer
. This linear polarized light is reflected by a beam splitter
towards the objective
, which focuses the light on the specimen. The glass surface is reflective to a certain degree and will reflect the polarized light. Light that is not reflected by the glass will travel into the cell and be reflected by the cell membrane. Three situations can occur:
The reflected light will travel back to the beam splitter and pass through a second polarizer, which eliminates scattered light, before reaching the detector (usually a CCD camera
) in order to form the final picture. Note that the polarizers can increase the efficiency by reducing scattered light, however in a modern setup with a sensitive digital camera, they're not required.
, according to the following rule:
Reflectivity
is a ratio of the reflected light intensity (
) and the incoming light intensity (
):
Using typical refractive indices for glass (1.50-1.54, see list), water (1.31, see list), the cell membrane (1.48) and the cytosol (1.35), one can calculate the fraction of light being reflected by each interface. The amount of reflection increases as the difference between refractive indices increases, resulting in a large reflection from the interface between the glass surface and the culture medium (about equal to water: 1.31-1.33). 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. Because the cone of light focused on the sample gives rise to different angles of incident light, there is a broad range of interference patterns. When the patterns differ by less than 1 wavelength (the zero-order fringe), the patterns converge, resulting in increased intensity. This can be obtained by using an objective with a numerical aperture greater than 1.
The light source needs to produce high intensity light, as a lot of light will be lost by the beam splitter and the sample itself. Different wavelengths result in different IRM images; Bereiter-Hahn and colleagues showed that for their PtK 2 cells, light with a wavelength of 546nm resulted in better contrast than blue light with a wavelength of 436nm. There have been many refinements to the basic theory of IRM, most of which increase the efficiency and yield of the image formation. By placing polarizers and a quarter wave plate
between the beam splitter and the specimen, the linear polarized light can be converted into circular polarized light
and afterwards be converted back to linear polarized light, which increases the efficiency of the system. The circular polarizer article discusses this process in detail. Furthermore, by including a second polarizer, which is rotated 90° compared to the first polarizer, stray light can be prevented from reaching the detector, increasing the signal to noise ratio (see Figure 2 of Verschueren).
and cell migration
.
in chromaffin cells
. When imaged using DIC, chromaffin cells appear as round cells with small protrusions. When the same cell is imaged using IRM, the footprint of the cell on the glass can be clearly seen as a dark area with small protrusions (see Figure 2 in the gallery below). When vesicles fuse with the membrane, they appear as small light circles within the dark footprint (Figure 2, bright spots in the top cell in the right panel). An example of vesicle fusion in chromaffin cells using IRM is shown in movie 1. 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. Another benefit of the lack of fluorescent labels is reduced phototoxicity.
Luminous intensity
In photometry, luminous intensity is a measure of the wavelength-weighted power emitted by a light source in a particular direction per unit solid angle, based on the luminosity function, a standardized model of the sensitivity of the human eye...
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 in contrast to TIRF microscopy
Total internal reflection
Total internal reflection is an optical phenomenon that happens when a ray of light strikes a medium boundary at an angle larger than a particular critical angle with respect to the normal to the surface. If the refractive index is lower on the other side of the boundary and the incident angle is...
.
History
The method was first used for the studying of thin films of oil. In 1964, the first application of the technique in cell biology was introduced by Curtis to study embryonic chick heart fibroblasts. 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 pseudopodiaPseudopod
Pseudopods or pseudopodia are temporary projections of eukaryotic cells. Cells that possess this faculty are generally referred to as amoeboids. Pseudopodia extend and contract by the reversible assembly of actin subunits into microfilaments...
.
The technique was refined and the qualitative and quantitative aspects of the technique were later described by several researchers in the 70s and 80s: Bereiter-Hahn and his colleagues correlated the technique with electron microscopy, showing that different mammalian cell lines adhere to the glass substrate in specific focal adhesion sites.
Theory
In order to form an image of the attached cell, light of a specific wavelengthWavelength
In physics, the wavelength of a sinusoidal wave is the spatial period of the wave—the distance over which the wave's shape repeats.It is usually determined by considering the distance between consecutive corresponding points of the same phase, such as crests, troughs, or zero crossings, and is a...
is passed through a polarizer
Polarizer
A polarizer is an optical filter that passes light of a specific polarization and blocks waves of other polarizations. It can convert a beam of light of undefined or mixed polarization into a beam with well-defined polarization. The common types of polarizers are linear polarizers and circular...
. This linear polarized light is reflected by a beam splitter
Beam splitter
A beam splitter is an optical device that splits a beam of light in two. It is the crucial part of most interferometers.In its most common form, a rectangle, it is made from two triangular glass prisms which are glued together at their base using Canada balsam...
towards the objective
Objective (optics)
In an optical instrument, the objective is the optical element that gathers light from the object being observed and focuses the light rays to produce a real image. Objectives can be single lenses or mirrors, or combinations of several optical elements. They are used in microscopes, telescopes,...
, which focuses the light on the specimen. The glass surface is reflective to a certain degree and will reflect the polarized light. Light that is not reflected by the glass will travel into the cell and be reflected by the cell membrane. Three situations can occur:
- When the membrane is close to the glass, the reflected light from the glass is shifted half of a wavelength, so that light reflected from the membrane will have a phase shift compared to the reflected light from the glass phasesPhase (waves)Phase in waves is the fraction of a wave cycle which has elapsed relative to an arbitrary point.-Formula:The phase of an oscillation or wave refers to a sinusoidal function such as the following:...
and therefore cancel each other out (interference). This interference results in a dark pixel in the final image (the left case in the figure). - 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).
- 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 reflected light will travel back to the beam splitter and pass through a second polarizer, which eliminates scattered light, before reaching the detector (usually a CCD camera
Charge-coupled device
A charge-coupled device is a device for the movement of electrical charge, usually from within the device to an area where the charge can be manipulated, for example conversion into a digital value. This is achieved by "shifting" the signals between stages within the device one at a time...
) in order to form the final picture. Note that the polarizers can increase the efficiency by reducing scattered light, however in a modern setup with a sensitive digital camera, they're not required.
Mathematical theory
Reflection is caused by a change in the refraction index, so on every boundary a part of the light will be reflected. The amount of reflection is given by the reflection coefficient, according to the following rule:Reflectivity
is a ratio of the reflected light intensity () and the incoming light intensity ():Using typical refractive indices for glass (1.50-1.54, see list), water (1.31, see list), the cell membrane (1.48) and the cytosol (1.35), one can calculate the fraction of light being reflected by each interface. The amount of reflection increases as the difference between refractive indices increases, resulting in a large reflection from the interface between the glass surface and the culture medium (about equal to water: 1.31-1.33). 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. Because the cone of light focused on the sample gives rise to different angles of incident light, there is a broad range of interference patterns. When the patterns differ by less than 1 wavelength (the zero-order fringe), the patterns converge, resulting in increased intensity. This can be obtained by using an objective with a numerical aperture greater than 1.
Requirements
In order to image cells using IRM, a microscope needs at least the following elements:- a light source, such as a halogen lamp
- an optical filter (which passes a small range of wavelengths)
- a beam splitter (which reflects 50% and transmits 50% of the chosen wavelength)
The light source needs to produce high intensity light, as a lot of light will be lost by the beam splitter and the sample itself. Different wavelengths result in different IRM images; Bereiter-Hahn and colleagues showed that for their PtK 2 cells, light with a wavelength of 546nm resulted in better contrast than blue light with a wavelength of 436nm. There have been many refinements to the basic theory of IRM, most of which increase the efficiency and yield of the image formation. By placing polarizers and a quarter wave plate
Wave plate
A wave plate or retarder is an optical device that alters the polarization state of a light wave travelling through it.- Operation :A wave plate works by shifting the phase between two perpendicular polarization components of the light wave. A typical wave plate is simply a birefringent crystal...
between the beam splitter and the specimen, the linear polarized light can be converted into circular polarized light
Circular polarization
In electrodynamics, circular polarization of an electromagnetic wave is a polarization in which the electric field of the passing wave does not change strength but only changes direction in a rotary type manner....
and afterwards be converted back to linear polarized light, which increases the efficiency of the system. The circular polarizer article discusses this process in detail. Furthermore, by including a second polarizer, which is rotated 90° compared to the first polarizer, stray light can be prevented from reaching the detector, increasing the signal to noise ratio (see Figure 2 of Verschueren).
Biological applications
There are several ways IRM can be used to study biological samples. Early examples of uses of the technique focused on cell adhesionCell adhesion
Cellular adhesion is the binding of a cell to a surface, extracellular matrix or another cell using cell adhesion molecules such as selectins, integrins, and cadherins. Correct cellular adhesion is essential in maintaining multicellular structure...
and cell migration
Cell migration
Cell migration is a central process in the development and maintenance of multicellular organisms. Tissue formation during embryonic development, wound healing and immune responses all require the orchestrated movement of cells in particular directions to specific locations...
.
Vesicle fusion
More recently, the technique has been used to study exocytosisExocytosis
Exocytosis , also known as 'The peni-cytosis', is the durable process by which a cell directs the contents of secretory vesicles out of the cell membrane...
in chromaffin cells
Chromaffin cell
Chromaffin cells are neuroendocrine cells found in the medulla of the adrenal gland and in other ganglia of the sympathetic nervous system. They are modified post-synaptic sympathetic neurons that receive sympathetic input...
. When imaged using DIC, chromaffin cells appear as round cells with small protrusions. When the same cell is imaged using IRM, the footprint of the cell on the glass can be clearly seen as a dark area with small protrusions (see Figure 2 in the gallery below). When vesicles fuse with the membrane, they appear as small light circles within the dark footprint (Figure 2, bright spots in the top cell in the right panel). An example of vesicle fusion in chromaffin cells using IRM is shown in movie 1. Upon stimulation with 60 mM potassium
Potassium
Potassium is the chemical element with the symbol K and atomic number 19. Elemental potassium is a soft silvery-white alkali metal that oxidizes rapidly in air and is very reactive with water, generating sufficient heat to ignite the hydrogen emitted in the reaction.Potassium and sodium are...
, 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
Fluorescence microscope
A fluorescence microscope is an optical microscope used to study properties of organic or inorganic substances using the phenomena of fluorescence and phosphorescence instead of, or in addition to, reflection and absorption...
and TIRF microscopy to study protein dynamics together with vesicle exocytosis and endocytosis. Another benefit of the lack of fluorescent labels is reduced phototoxicity.