Lecture 4: Microscopic techniques Flashcards
Wavelength
The distance from a point in the cycle to the corresponding point in the next cycle
Frequency
The number of vibrations of a given wavelength in one second
How does wavelength relate to frquency?
Longer wavelengths vibrate fewer times so the longer the wavelength, the lower the frequency.
What are the frequencies for visible light?
Red: 4.3 x 10 15 Hz
Yellow: 5.4 x 1015 Hz
Violet: 7.5 x 1015 Hz
How does light travel in a homogenous material?
Light travels in a straigth line from a source and reaches a definite and constant speed in any given homogenous medium or material
What happens to waves in a vacuum?
In a vacuum, all waves in the electromagnetic spectrum travel at the same speed as nothing is interacting with it.
3 x 108 m/s-1
What happens when a wave enters a material after a vacuum?
As any wave enters a material from a vacuum it slows down. Whilst it’s slowing down, as long as it’s in the same material, it will stay at the same speed.
What is the equation for Velocity?
Velocity (c) = Frequency (f) x Wavelength (λ)
What is absorption?
When a photon of light comes from one material and hits the interface of a material but does not exit again as it’s all absorbed.
What can you learn from absorption?
The colour of the matieral as different colours absorb different amounts of light.
Darker materials absorb more light.
What is reflection?
The light ray is turned back into the incident material instead of travelling on into the new material.
What is specular reflection?
Specular reflection is when a light path hits a material and all of it is turned backed (reflected away).
Nothing travels through the material and nothing is absorbed.
What happens when you change the wavelength in specular reflection?
- If you change wavelength (changing the colour) changing the angle means the incidence reflected angle matches exactly the reflected angle on the same side, they remain the same.
- This is the case for perfect specular reflection however few materials actually have this.
What happens when you increase surface roughness is specular reflection?
- If you increase surface roughness, multiple colours begin to show and broaden out.
- It comes of at different angles and you begin to get diffuse reflection.
What happens as you increase surface roughness in specular reflection?
70-80% surface roughness
As you increase surface roughtness, diffuse reflection increase and specular reflection decreases.
Once you get to a surface roughness of around 70-80% you’ll get virtually no specular reflection and all is diffusely reflected in multiple directions
What is refraction?
Transparent material
- The light ray’s path is bent when it passes from one transparent material to another transparent material where its velocity changes.
- Light passes through at an angle, the angle is representative of the material and as a result of a change in velocity.
The difference in refractive index…
The difference in refractive index defines how much refraction occurs.
What does Snells law tell us?
The relationship between two materials and the ratio between them.
Equation for n21
sinθ1 / sinθ2
n2/n1 = ?
n2/n1 = v1 / v2
sinθ1/sinθ2 = ? = ? = ?
sinθ1/sinθ2 = n21 = n2 / n1 = v1 / v2
What is resolution?
Ability to distinguish between 2 points on the specimen.
What is the resolution on a light microscopy?
Can reach values close to 200nm
What defines the lens resolution?
Spot size (& hence λ) defines the lens resolution.
Increased diameter of spot size =
increased diameter = increased magnification + increased angle aperture
For a light microscope the optimum resolution is on the order of ?
For a light microscope the optimum resolution is on the order of ~1μm
What do lenses have that affect the resolution?
Spherical and chromic abberations
What is depth of focus?
- Ability to maintain focus over a range of depths within the specimen.
- Depth of focus is how much of what we’re looking at remains in focus at the same time.
- How many different focal planes you can pick out?
How is depth of focused affected when magnication increases?
In a light micrscope depth of focus decreases when magnification increases
What is the depth of focus on a light micrscope?
Comparatively low
What is field of view?
The Size of the specimen that can be imaged at the same time.
What are lenses used for in optical microscopy?
In optical microscopy, lenses are used to focus (refract) incoming light (from a sample) to a point.
What influences the ability of the lens to resolve details of a sample?
The ability of the lens to resolve details of a sample is influenced by the quality of the lens but is ultimately limited by diffraction.
What is the diameter of the airy disk depedant on?
The spot size (d) is given by the diameter of the Airy disk which is dependent on the wavelength of the light, focal length and lens diameter.
Increasing the lens diameter…
Increasing the lens diameter increases the angle aperture therefore increasing magnification.
How does wavelength affect resolution
Shorter wavelengths you can get an improved resolution.
What affect does using a single wavelength have?
Often if you’re changing down to a single of wavelength you’ll have fewer photons and much less light going through. This makes it more difficult to see.
Equation for spot size (d)
d = 1.22 x λ x focal length / lens diameter
Higher magnification…
Higher magnification requires more complicated objective systems in order to combat aberrations.
What do more lenses allow you to do and what is the drawback of this?
- More lenses allow you to focus and move closer and control the light in order to improve resolution.
- More lenses = Higher cost
What is the stereoscopic microscope good for examining?
- Good for bulky artefacts
- Great first step when looking at physical features of trace evidence
How do we obtain magnification images of small samples?
We use visible light and a system of lenses to obtain magnification images of small samples
What is absorption?
When a photon of light comes from one material and hits the interface of a material but does not exit again as it’s all absorbed.
What does absorption result in?
Results in thermal, electrical or chemical changes as the energy in the light has to go somewhere.
What materials absorb more light?
Darker materials
What happens when you get to 70-80% surface roughness?
- Once you get to a surface roughness of around 70-80% you’ll get virtually no specular reflection and all is diffusely reflected in multiple directions
- This varies, diffuse reflection will vary by wavelength going in.
What is refraction afffected by?
It is affected by the materials involved, the angle of the incident ray of light and the wavelength of the incident ray.
What is the angle of incidence?
This is the angle the light is coming in at.