Remember, in lecture that we talk about the fact that shorter wavelengths (blue range of visible light or even shorter UV light or electrons, which have an even shorter wavelength than light) and larger numerical aperture provide the greatest resolution. So, even without using the formula, you can deduce that a decrease in wavelength would increase resolution. However, you can also see this mathematically by playing with the formula:
For example:
RP = 400 nm = 160 nm 400 nm is the wavelength of light in the violet range and 1.25 is the
2 x 1.25 numerical aperture of the 100x objective lens.
RP = 200 nm = 80 nm 200 nm is the wavelength of light in the UV range and 1.25 is the
2 x 1.25 numerical aperture of the 100x objective lens.
Also, remember that nanometers (nm) are very small units of measurement, below micrometers
(µm), millimeters (mm), meters (m), etc. We usually measure
microbes in micrometers (µm).
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Microscope: a device for magnifying objects that are too small to be seen with the naked eye.
- Simple microscope: single lens magnifier
- Compound microscope: employs two or more lenses
Parfocal: the objective lenses are mounted on the microscope so that they can be interchanged without having to appreciably vary the focus.
Resolving power or resolution: the ability to distinguish objects that are close together. The better the resolving power of the microscope, the closer together two objects can be and still be seen as separate.
Magnification: the process of enlarging the size of an object, as an optical image.
Total magnification: In a compound microscope the total magnification is the product of the objective and ocular lenses (see figure below). The magnification of the ocular lenses on your scope is 10X.
Objective lens X Ocular lens = Total magnification For example: low power: (10X)(10X) = 100X high dry: (40X)(10X) = 400X oil immersion: (100X)(10X) = 1000X
Immersion Oil: Clear, finely detailed images are achieved by contrasting the specimen with their medium. Changing the refractive index of the specimens from their medium attains this contrast. The refractive index is a measure of the relative velocity at which light passes through a material. When light rays pass through the two materials (specimen and medium) that have different refractive indices, the rays change direction from a straight path by bending (refracting) at the boundary between the specimen and the medium. Thus, this increases the image’s contrast between the specimen and the medium.
One way to change the refractive index is by staining the specimen. Another is to use immersion oil. While we want light to refract differently between the specimen and the medium, we do not want to lose any light rays, as this would decrease the resolution of the image. By placing immersion oil between the glass slide and the oil immersion lens (100X), the light rays at the highest magnification can be retained. Immersion oil has the same refractive index as glass so the oil becomes part of the optics of the microscope. Without the oil the light rays are refracted as they enter the air between the slide and the lens and the objective lens would have to be increased in diameter in order to capture them. Using oil has the same effect as increasing the objective diameter therefore improving the resolving power of the lens.