Abstract
By taking advantage of combinations of the many rich properties of photons, new forms of optical microscopy can now be used to visualize features of samples beyond thickness and density variations. We are now within reach of viewing the motions, orientations, binding kinetics and specific transient associations of previously 'submicroscopic' cellular structures and single molecules.
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Glossary
- Dipole orientation
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Incident light of an appropriate colour can be absorbed by a fluorophore, but the absorption is most efficient if the light is polarized along a particular axis that is fixed relative to the fluorophore, known as the absorption dipole orientation. Fluorescence (emitted as the fluorophore returns to the ground state) is also polarized, generally along an axis that is fixed relative to the fluorophore, known as the emission dipole orientation.
- Interference
-
If two laser beams from the same laser, split by a mirror or prism, are made to intersect, in some places in the region of intersection the sine-wave crests of one beam will always add to the crests of the other, and likewise for the troughs. These places will experience larger electric field oscillations than either beam separately, and they will have enhanced brightness. Other places will have crests of one beam arriving at the same time as troughs of the other and these places will experience a cancellation of the electric field, leading to relative darkness. The pattern of bright and dark is known as an interference pattern. Only beams of the same polarization will interfere. If the two beams are broad and collimated, the interference pattern will be a series of dark and bright stripes or planes, the spacing of which is determined by the relative angle of intersection. Typically, the spacing is measured by the distance from one minimum (a dark region) to the next.
- Photobleaching
-
A fluorophore in the excited state is typically less chemically stable than one in the ground state. It is more easily attacked by oxygen and also more likely to break on its own. Photochemical reactions that involve excited molecules can generate by-products that can even destroy nearby fluorophores in the ground state. The result is a progressive and permanent loss of viable fluorophores and a dimming of fluorescence, which is known as photobleaching.
- Plane of incidence
-
When light encounters a planar interface, some reflects and some refracts (for sub-critical angle incidence) or forms an evanescent field (for super-critical angle incidence). The incident beam, the reflected beam and the refracted beam all lie in the same plane of incidence.
- Polarization
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A coherent laser beam can be viewed as a travelling sine wave of electric field with the field pointing one way in the crests of the sine wave and the opposite way in the troughs, and always perpendicular to the direction of propagation. The electric field direction is referred to as the polarization.
- p-polarization
-
If the incident beam's polarization lies in the plane of incidence, the reflected beam and refracted beam (or evanescent field) are also polarized in that plane; this is known as p-polarization (p-pol).
- Resolution
-
This is the minimum distance that two points of light on the sample must be separated so that their two images can be distinguished from each other. The classic Raleigh criterion is usually used: 0.61 multiplied by (the wavelength of light) divided by (the numerical aperture of the objective). Modern image analysis can improve on this and distinguish separate points that are considerably closer together than specified by the Raleigh criterion.
- Saturation
-
If the excitation light is bright, fluorophores will become re-excited shortly after they briefly return to the ground state, thereby spending most of their time in the excited state. This situation, which is known as saturation, depletes the number of fluorophores in the ground state that are available for further excitation.
- s-polarization
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If the incident beam's polarization is perpendicular to the plane of incidence, then so are the reflected and refracted beams (or evanescent field); this is known as s-polarization (s-pol).
- Total internal reflection, evanescent field and critical angle
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A light beam that obliquely (that is, non-perpendicularly) approaches an interface with a less dense medium (such as water) can totally internally reflect if the angle of incidence (measured from a line that is perpendicular to the surface) is larger than a well defined critical angle. However, an evanescent field does penetrate into the less dense medium and propagates along the surface. The evanescent-field intensity decays exponentially in the direction perpendicularly away from the interface, generally with a characteristic distance of tens to hundreds of nanometers, depending on the angle of incidence. Fluorophores that reside in the evanescent field emit photons at a rate that exponentially decays with distance from the interface.
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Axelrod, D., Omann, G. Combinatorial microscopy. Nat Rev Mol Cell Biol 7, 944–952 (2006). https://doi.org/10.1038/nrm2062
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DOI: https://doi.org/10.1038/nrm2062
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