Microscopic Techniques
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What is 1 wavelength?
the distance from a point in a cycle to the corresponding point on the next cycle
What form of radiation is light and what type of light does optical microscopy make use of?
• electromagnetic • visible
What is frequency?
the number of vibrations of a given wavelength in one second
What are the units of frequency?
Hz * 1 Hz = 1 wave completed per second
What is the link between wavelengths and frequency?
longer wavelengths vibrate fewer times so the longer the wavelength the lower the frequency
What are the properties of waves?
• light travels in a straight line from a source and reaches a definite and constant speed in any given homogenous medium or material • in vacuum, all waves in the electromagnetic spectrum travel at the same speed • not all waves slow down to the same speed outside a vacuum • as any wave enters a material from a vacuum it slows down
What formula relates the three properties of waves?
velocity (c) = frequency (f) x wavelength (λ)
What is absorption?
when a photon of light enters a material, but doesn not exit again * results in thermal, electrical or chemical changes * depends on colour (dark or light)
What is reflection?
the light ray is turned back into the incident material instead of travelling on into the new material * dependant on colour, texture (rough or smooth)
What is refraction?
the light ray’s path is bent when it passes from one transparent material to another transparent material where its velocity changes * if there is total refraction then nothing is reflected * the bend at which the ray moves is representative of the material and a change in velocity
What factors affect refraction?
• the material involved • the angle of the incident ray of light • the wavelength of the incident ray
How is the refractive index of the two substances related to refraction?
• degree to which the light ray bends • direction it bends
How does wavelength relate to refraction?
increasing the wavelength (changing the colour) changes the amount of refraction
How are the light ray angles and refractive indices related to each other?
by Snell’s Law
What is Snell’s Law? equation
sinθ1/sinθ2 = n21 = n2/n1 * n = refractive indices * θ1 = angle of incidence * θ2 = angle of refraction
What is resolution?
• ability to distinguish between 2 points on the specimen • understanding focussing lenses are key to improve resolution
What are the key specifications of a light microscope?
• resolution • depth of focus • field of view
What is depth of focus?
• ability to maintain focus over a range of depths within the specimen • comparatively low - gets worse with higher magnifications
What is field of view?
• size of specimen that can be imaged at the same time • good
What is focussing lenses?
• the ability of the lens to resolve details of a sample is influenced by the quality of the lens but is limited by diffraction • as light passes through a circular aperture, it is focussed to a point. The spot size is given by the diameter of the airy disk which is dependent on the wavelength of the light
What is the equation for spot size in focussing lenses?
d = 1.22 x λ x focal length/lens diameter * d = spot size * λ = wavelength/resolution
What defines the lens resolution?
• spot size • λ
How can the optimum resolution be improved to 200 nm?
use blue light and special objectives
What is the optimum resolution for a light microscope?
~ 1 um