What Is Refraction?
If we look at refraction in the context of microscopy, it is perhaps the most important behavior that light exhibits. The underlying principle of the microscope is that lenses refract light, which allows for magnification.
In simple terms, refraction is the bending of light as it travels from one medium to another of different densities. The bending of that light rays gives rise to an angle of refraction or the degree of bending.
For Example, if you placed a pencil partially in the water, the underwater portion of the pencil appears to bend at the water surface. This is due to the bending of light rays as they move from water to the air.
The refraction of light can be seen in many places in our everyday life. It makes objects under a water surface appear closer than they really are.
It is what optical lenses are based on, allowing for instruments such as glasses, cameras, binoculars, microscopes, and the human eye. Refraction is also responsible for some natural optical phenomena including rainbows and mirages.
As we discussed Light refract when it moves from one medium to another but why is that happen? and its importance in the microscope.
Why Light Refract When It Travel In Different Medium?
A correct explanation of refraction involves two separate parts, both a result of the wave nature of light.
1) Slowing Of Light In A Medium
Light slows as it travels through a medium other than vacuum (such as air, glass, or water). This is not because of scattering or absorption.
Rather it is because, as an electromagnetic oscillation, light itself causes other electrically charged particles such as electrons, to oscillate.
The oscillating electrons emit their own electromagnetic waves which interact with the original light. The resulting “combined” wave has wave packets that pass an observer at a slower rate.
The light has effectively been slowed. When light returns to a vacuum and there are no electrons nearby, this slowing effect ends and its speed returns to c.
2) Bending Of Light As It Enters And Exits A Medium
When light enters, exits, or changes the medium it travels in, at an angle, one side or the other of the wavefront is slowed before the other. This asymmetrical slowing of the light causes it to change the angle of its travel.
What Is Index Of Refraction Or Refraction Index
As we discussed Different transparent materials transmit light at different speeds; thus, light can change speed when passing from one material to another. This change in speed usually also causes a change in direction (refraction), with the degree of change depending on the angle of the incoming light.
The extent to which a material slows transmission speed relative to empty space is called the Refractive Index of that material.
Refractive Index (Index of Refraction) is a value calculated from the ratio of the speed of light in a vacuum to that in a second medium of greater density. The refractive index variable is most commonly symbolized by the letter n or n’ in descriptive text and mathematical equations.
In optical microscopy, the refractive index is an important variable in calculating numerical aperture, which is a measure of the light-gathering and resolving power of an objective.
In most instances, the imaging medium for microscopy is air, but high-magnification objectives often employ oil or a similar liquid between the objective front lens and the specimen to improve resolution. The numerical aperture equation is given by:
NA (numerical aperture) = n × sin(θ)
where n is the refractive index of the imaging medium and θ is the angular aperture of the objective. It is obvious from the equation that increasing the refractive index by replacing the imaging medium from the air (refractive index = 1.000) with a low-dispersion oil (refractive index = 1.515) dramatically increases the numerical aperture.
Refractive index of some transparent substances
| Substance | Refractive index | Speed of light in substance (x 1,000,000 m/s) | The angle of refraction if incident ray enters substance at 20º |
| Air | 1.00 | 300 | 20 |
| Water | 1.33 | 226 | 14.9 |
| Glass | 1.5 | 200 | 13.2 |
| Diamond | 2.4 | 125 | 8.2 |
How Does Refraction Cause Magnification?
The curved surface, unlike a flat surface, bends the light as it comes out from the water, and causes this magnification effect. This effect is very similar to how a microscope or magnifying glass works, except instead of a curved drop of water, the lens is made of a curved piece of glass.
For example, When light crosses a boundary into a material with a higher refractive index, its direction turns to be closer to perpendicular to the boundary (i.e., more toward a normal to that boundary). This is the principle behind lenses.
We can think of a lens as an object with a curved boundary, that collects all of the light that strikes it and refracts it so that it all meets at a single point called the image point (focus).
A convex lens can be used to magnify because it can focus at a closer range than the human eye, producing a larger image. Concave lenses and mirrors can also be used in microscopes to redirect the light path.
As we can see in Figure the focal point (the image point when light enters the lens is parallel) and the focal length (the distance to the focal point) for convex and concave lenses.
Artificial lenses placed in front of the eye (microscopic lenses) focus light before it is focused (again) by the lens of the eye, manipulating the image that ends up on the retina (e.g., by making it appear larger).
Images are commonly manipulated by controlling the distances between the object, the lens, and the screen, as well as the curvature of the lens.
For example, for a given amount of curvature, when an object is closer to the lens, the focal points are farther from the lens.
As a result, it is often necessary to manipulate these distances to create a focused image on a screen. Similarly, more curvature creates image points closer to the lens and a larger image when the image is in focus. This property is often described in terms of the focal distance, or distance to the focal point.
How Dose Refraction affect Microscope Resolution?
As the refractive index increases, the speed of the light passing through a medium is slower. As light slows down the wavelength gets shorter and yields better resolution. Objective lenses are manufactured that allow imaging in immersion oil which has a refractive index of 1.51 and substantially shortens light waves.
One of the most important factors in determining the resolution of an objective is the angular aperture, which has a practical upper limit of about 72 degrees (with a sine value of 0.95).
When combined with the refractive index, the product “n(sin(θ))” is known as the numerical aperture (abbreviated NA), and provides a convenient indicator of the resolution for any particular objective.
Numerical aperture is generally the most important design criteria (other than magnification) to consider when selecting a microscope objective.
Values range from 0.1 for very low magnification objectives (1x to 4x) to as much as 1.6 for high-performance objectives utilizing specialized immersion oils.
As numerical aperture values increase for a series of objectives of the same magnification, we generally observe a greater light-gathering ability and increase in resolution.
